Advances in Microbial Physiology Volume 33

Advances in

MICROBIAL PHYSIOLOGY

This Page Intentionally Left Blank

Advances in

MICROBIAL PHYSIOLOGY Edited by

A. H. ROSE School of Biological Sciences Bath University, U K

Volume 33

ACADEMIC PRESS Harcourt Brace Jovanovich, Publishers London San Diego New York Boston Sydney Tokyo Toronto

ACADEMIC PRESS LIMITED 24-28 Oval Road London NWl 7DX US Edition published by ACADEMIC PRESS INC. San Diego CA92101

Copyright 0 1992 by ACADEMIC PRESS LIMITED This book is printed on acid-free paper

AN Rights Reserved

No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers British Library Cataloguing in Publication Data

Advances in microbial physiology. Vol. 33 1. Micro-organisms-Physiology 1. Rose, A. H. 576’.11 OR84 ISBN & I 2 4 2 7 7 3 3 4 ISSN 0065-291 1

Typeset by J&L Composition Ltd, Filey, North Yorkshire Printed in Great Britain at the University Press, Cambridge

Contributors L. Adler Department of General and Marine Microbiology, University of Goteborg, Carl Skottsbergs Gata 22, 413 19 Goteborg, Sweden V. A. Bankaitis Department of Microbiology, University of Illinois, Urbana, Illinois, USA A. Blomberg Department of General and Marine Microbiology, University of Goteborg, Carl Skottsbergs Gata 22, 413 19 Goteborg, Sweden A. E. Cleves Department of Microbiology, University of Illinois, Urbana, Illinois, USA M. D. Manson Department of Biology, Texas A&M University, College Station, Texas 77843-3258, USA P. Messner Zentrum fur Ultrastrukturforschung und Ludwig BoltzmannInstitut fur Ultrastrukturforschung, Universitat fur Bodenkultur, A-1180 Wien, Austria U. B. Sleytr Zentrum fur Ultrastrukturforschung und Ludwig BoltzmannInstitut fur Ultrastrukturforschung, Universitat fur Bodenkultur, A-1180 Wien, Austria M. Stratford AFRC, Institute of Food Research, Colney Lane, Norwich, Norfolk NR4 7UA, UK

This Page Intentionally Left Blank

Contents Contributors

V

Yeast Flocculation: A New Perspective MALCOLM STRATFORD 1. Introduction

11. 111. IV. V. VI . VII . VIII . IX . X.

Industrial flocculation: the art of brewing Measurement of flocculation Basic physiology of flocculation Physics of cell-cell interactions Mechanism of flocculation Onset of flocculation Genetics of flocculation Concluding remarks Acknowledgements References

2 4 9 13 23 43 53 60 62 63 63

Secretory Pathway Function in Saccharomyces cerevisiae ANN E . CLEVES and VYTAS A. BANKAITIS

I. Introduction 11. The yeast secretory pathway 111. Protein transport from the cytoplasm into the endoplasmic reticulum IV . Protein trafficking from the endoplasmic reticulum to the Golgi complex V. The Golgi complex as a secretory organelle VI . Fusion of Golgi complex-derived vesicles with the plasma membrane VII . Summary VIII. Acknowledgements References

73 75 77 88 111

132 139 140 140

Physiology of Osmotolerance in Fungi ANDERS BLOMBERG and LENNART ADLER

I. Introduction 11. The thermodynamic state of water 111. Osmotolerance IV. Initial osmotic response V. Osmoregulation VI . Osmotic hypersensitivity VII. Cellular factors involved in determining Yminvalues VIII. Conclusion IX. Acknowledgements References

146 148 155 161 167 190 197 204 206 206

Crystalline Bacterial Cell-Surface Layers PAUL MESSNER and U W E B. SLEYTR

I. Introduction 11. Structure and morphogenesis of S-layers 111. Chemistry, genetics and biosynthesis of S-layers

IV . V. VI . VII .

Functional aspects of S-layers Application potential of S-layers Concluding remarks Acknowledtements References

213 227 237 251 257 260 262 262

Bacterial Motility and Chemotaxis MICHAEL D. MANSON

I. 11. 111. IV. V. VI . VII .

Introduction Flagella Motility Chemoreception Chemotactic signal transduction Conclusion Acknowledgements References

Corrigendum Author index Subject index

277 280 287 296 310 334 334 335 347 349 377

Yeast Flocculation: A New Perspective MALCOLM STRATFORD AFRC Institute of Food Research. Colney Lane. Norwich. Norfolk NR4 7UA. U K

1. Introduction . A . Definitions

. . . . . . . . . . I1. Industrial flocculation: the art of brewing . . . . . A . Flocculation and attenuation . . . . . . B . Flocculation and fermenter design . . . . . C . Classification of brewing strains . . . . . . I11. Measurement of flocculation . . . . . . . . IV . Basic physiology of flocculation . . . . . . . A. Historical perspective . . . . . . . . B . Inorganic-ion effects . . . . . . . . C . Inhibition bysugars . . . . . . . . D . pH value and temperature effects . . . . . E . Effect of protein denaturation . . . . . . F. Mitochondrialinvolvementinflocculation . . . G . Heterologous flocculation . . . . . . . V . Physics of cell-cell interactions . . . . . . . A . YeastsuspensionsandBrownianmotion . . . B . Mechanicalagitationandflocculationrate . . . C . Collision frequency . . . . . . . . D . Energy of collision . . . . . . . . E . Extent of flocculation and equilibrium . . . . F. Morphology of flocculation . . . . . . G . Flocculation without agitation . . . . . . H . Floccompression byagitationandgravity . . . 1. Cascade theory and fractal flocculation . . . . VI . Mechanism of flocculation . . . . . . . . A . The yeast cell wall . . . . . . . . . B . The calcium-bridging hypothesis . . . . . C . The lectin hypothesis . . . . . . . . D . Two lectin mechanisms: Flol and NewFlo phenotypes E . Receptors of flocculation . . . . . . . F . Surfacelectinsofotheryeastsandbacteria . . . VII . Onset of flocculation . . . . . . . . . A.

B. C. D.

. .

. .

. .

. .

. .

Control by inhibition . . . Control by activation or exposure Control by synthesis or secretion Premature flocculation . .

ADVANCES IN MICROBIAL PHYSIOLOGY. VOL . 33 ISBN 0-124277334

. . . .

. . . .

. . . .

. . . .

. . . .

. . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . .

. . .

. . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . .

. . .

. . .

. . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

.

. . .

.

. . .

.

2 3 4 4 6 7 9 13 13 14 16 17 18 19 20 23 23 25 26 28 30 33 36 36 38 43 43 44 47 48 49 51 53 55 57 57 58

Copyright0 1992. by Academicpress Limited All rights of reproductionin any form reserved

2

M. STRATFORD

Genetics of flocculation . . . . . . . A . EarlygeneticsanddiscoveryoftheFLOgenes B. Flocculation instabilityand suppression . . C. Regulatory nature of FLO genes . . . IX. Concludingremarks . . . . . . . X. Acknowledgements . . . . . . . . References . . . . . . . . . .

VIII.

. .

. .

. .

. .

. .

. .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . .

.

.

.

.

.

.

60

60 61 61 62 63 63

I. Introduction We live in a universe composed of particles and forces. Particles, of whatever size, tend to be attracted towards one another. Since attractive forces have not, as yet, resulted in total mass aggregation, it is obvious that attractive forces are often balanced by repulsive forces. For example, there is the balance of gravitational attraction and orbital momentum between the Earth and Moon. Similarly, small particles, attracted by van der Waals forces, may be repelled by electrostatic repulsion, steric hindrance or hydrophobic effects. Whilst the size of particles and the nature of forces may be entirely different, there are often striking parallels between different systems. This demonstrates that interaction of forces with particles (masses) often has similar results, entirely irrespective of scale. Aggregation of micro-organisms is a very widespread phenomenon. Many aggregations are sexual in nature, with agglutination of cells into clumps preluding exchange of genetic material between cells. The reasons for other microbial aggregations, including yeast flocculation, are less immediately obvious and have given rise to much speculation as to their evolutionary survival value. Aggregation can be regarded as an intermediary stage between single- and multicelled organisms. Cells can agglutinate into cellular masses which have no cohesive united existence, no recognition of position within the group, and no consequential differentiation of cellular function o r form, as are normally found in multicellular organisms. Until some semblance of these properties is developed, as in slime moulds of the genus Dictyostelium (Newell, 1977), aggregated cells must be regarded as collections of individual and discrete entities. An alternative method of formation of cellular masses is a consequence of cell division and subsequent continued attachment of mother to daughter cell. This more probable origin of multicellular organisms is observed in tissue-culture growth of plant and animal cells. In yeasts this is limited to formation of short chains of cells in “chain-forming’’ strains of Saccharomyces cerevisiae, o r as pseudomycelial structures in some yeasts (species of Candida o r Trichosporon). These extensive structures blur the distinction between yeasts and mycelial fungi. However, microbial cellular

YEAST FLOCCULATION: A NEW PERSPECTIVE

3

aggregation was defined by Calleja (1987) as the gathering together of units to form larger units, aggregation being the process of gathering. This implies movement of cells, which were initially single, into aggregations and, as a consequence, this definition excludes aggregates of cells formed by cell division of growth. Recent reviews of microbial aggregation in general and of yeasts specifically are by Calleja (1984) and Calleja (1987), respectively. In this review, one particular form of yeast aggregation is examined in detail-that of flocculation, usually ascribed to brewer’s yeast. However, many aspects of flocculation, especially physical aspects, are equally applicable to any particulate system involving interparticle bonding. The physics of aggregation of sticky particles are universal, and independent of mechanisms of bonding or nature of the “glue” employed. Yeast flocculation is a phenomenon normally associated with brewing strains of Sacch. cerevisiae and Sacch. carlsbergensis. Recent studies have shown that flocculation is also observable in Schizosaccharomyces pombe (Johnson et al., 1988), Kluyveromyces bulgaricus (Hussain et al., 1986), Kl. marxianus (Bajpai and Margaritis, 1986) and. Saccharomycodes ludwigii (M. Stratford, unpublished observations). It seems probable that flocculation may be a more common yeast property than was originally assumed. Species within other yeast genera will probably be revealed as capable of flocculation, once careful examination is carried out. A . DEFINITIONS

There are three mechanisms that give rise to clumps of yeast cells. These are formation of mating aggregates, chain formation and flocculation. Yeast flocculation (Fig. 1) has been defined as “the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended” (Stewart et al., 1976). This lucid and useful definition needs further refinement since, as it stands, it also encompasses mating aggregation within its definition. In practice, both mating aggregates and yeast flocs are dispersed by proteases but flocs may be distinguished by their dispersal by EDTA (Johnson et al., 1988) and subsequent reflocculation following calcium-ion addition. Yeast flocculation may also be revealed by specific inhibitions by sugars but, since sugar inhibitions vary with strain and genus, this property is less useful for definition. Chain-formed aggregates (common in brewery strains), after prolonged growth, are often found up to 100 cells in size and, in media, appear indistinguishable from small flocs. However, chain aggregates are insensitive to EDTA but can be broken mechanically. After breakage, chainforming cells cannot reaggregate spontaneously as do flocculent cells. As a quick practical test, flocs, if allowed to settle under gravity, rapidly fuse to

4

M. STRATFORD

FIG. 1. A micrograph of flocculated cells of Succhuromycescerevisiue, strain NCYC 1364. Individual cells are approximately 5 pm in diameter.

form single, flat flocculated masses, whereas chain-aggregate particles do not fuse but remain indefinitely as sandy deposits of discrete grains. To conclude, flocculation may be defined as the property of certain yeast strains to adhere into clumps, dispersible by EDTA or specific sugars, and the subsequent removal of these clumps from the medium.

11. Industrial Flocculation: The Art of Brewing A. FLOCCULATION AND ATTENUATION

The importance of yeast flocculation in brewing over the centuries cannot be overemphasized in its role of media clearance and formation of a bright conditioned product. In the modern brewery, this role is less essential since cloudy beer may be filtered or centrifuged, but yeast flocculation remains a most cost-effective method of clearing beer. Given its importance in brewing, it is surprising that yeast flocculation is not widely employed in production of other beverages. Wine fermentations were in the past generally conducted annually, using yeasts indigenous to the grape surface and generally smaller fermentation vessels than those used in the brewing industry. Small vessel size and leisurely fermentation allow

YEAST FLOCCULATION: A NEW PERSPECTIVE

5

sufficient time for even non-flocculent yeasts to sediment. Any flocculent properties of yeasts could not be passed on to subsequent fermentations, which would ferment using a new population of grape yeasts. However, examination of wine yeasts by Thornton and Bunker (1989) showed that a high proportion possessed some degree of flocculation. Future wine fermentations may well employ flocculent yeasts, whether naturally occurring or introduced by genetic techniques (Thornton, 1985). Non-flocculent powdery yeast was preferred for use by distilleries and only slightly flocculent yeast for production of pressed baker’s yeast (Jansen, 1958). Nonflocculent yeast strains are selected by bakers to enable homogeneous mixing of yeast into dough. Curtis (1956) stated that brewers were concerned with attenuation and final yeast counts and not with flocculation. While a more enlightened approach to pure science by brewers may have developed, this statement does accurately reflect concern by brewers on the effects of flocculation rather than in yeast flocculation per se. Yeast is pitched into sweet wort primarily to convert wort sugars into alcohol (attenuation), and the rate at which this occurs (fermentation velocity) depends on the concentration of suspended yeast (De Clerck and van Roey, 1951; Kijima, 1963). If flocculation occurs early, removing suspended yeast, attenuation will cease and result in a hung fermentation containing residual sugar. Such premature flocculation has been widely reported as being responsible for poor attenuation (Hartong, 1951; Curtis, 1956; Devreux, 1962; Yoshida, 1962; Rainbow, 1966; Gyllang and Martinson, 1972). Flocculation was considered by St Johnston (1953) as the chief factor in termination of fermentation. While this is probably an effect of mass yeast removal by flocculation, Windisch (1968) suggested that this could also be influenced by the small effective surface area of flocs; small suspended flocs have a smaller area over which to exchange sugars and ethanol. Premature flocculation has been reported as being brought on by the presence of high molecular-weight wort constituents (Fujino and Yoshida, 1976; Axcell et al., 1986). This whole concept will be discussed in detail in a later section (VI1.D). The brewery has, over the centuries, acted as a very powerful selection mechanism for flocculent strains. Flocculent cells accumulated in convenient masses and were used for pitching subsequent fermentations, whereas non-flocculent suspended cells were discarded with other wild yeasts and bacteria. Thus, flocculent strains became established in breweries at a far higher frequency than in “wild” populations of yeasts. However, flocculation is an unstable property (Lund, 1951; Chester, 1963; Stewart, 1975) and non-flocculent yeasts are able to outgrow flocculent strains (Davis and Parnham, 1989). Without positive selection pressure, flocculation is easily lost from strains. Devreux (1962) described yeast degeneration with

6

M . STRATFORD

successive uses, where selection increased yeast flocculation and attenuation declined. Similar effects were noticed by Gyllang and Martinson (1972) but were attributed to selection ofpetite mutants unable to ferment maltotriose. However, Curtis (1956) suggested that progressive yeast changes with repitching were concerned with copper accumulation and toxicity. Progressive flocculation loss can still occur, however, despite selection. Curtis and Wenham (1958) showed that a contaminant non-flocculent strain could be mutually flocculated by a flocculent culture yeast, and was thus able progressively to increase in proportion. B . FLOCCULATION AND FERMENTER DESIGN

Flocculent brewery strains are usually described as top- or bottomfermenting. Lager yeasts, now recognized as Sacch. pastorianus (ex. Sacch. carlsbergensis), are traditionally bottom-fermenting, thus settling to the bottom of fermenting vessels. British ale yeast strains, Sacch. cerevisiae, may be top- or bottom-fermenting but, customarily, flocs adhere to gas bubbles and are carried up to form a yeasty foam, up to a metre in depth. At the end of fermentation, this foam subsides to form a few centimetres of thick yeasty scum, (Fig. 2) which is skimmed off for repitching.

FIG. 2. Thick yeasty foam formed by a top-fermenting ale strain, in the final stages of a British brewery ale fermentation. Folds of foam are approximately 6 cm across.

YEAST FLOCCULATION: A NEW PERSPECTIVE

7

Fermenter design has in the past evolved to suit the particular characteristics of the yeast employed. Very flocculent top-fermenting strains were used to advantage in Yorkshire stone-square fermenters (Lloyd Hind, 1943) while the Burton Union system was developed to make use of non-flocculent powdery yeasts (Curtis, 1956). These fermenters are no longer in general use, but open-vessel top-skimming ale fermentations are still widespread (Fig. 2). However, there is a modern trend towards use of moderately flocculent yeasts in closed, vertical cylindroconical vessels (Stewart, 1977). Yeast flocs collect after fermentation in the steeply conical bases of fermenters, and can be easily removed for washing and repitching. Recently, there has been much interest in continuous fermentation in variants of the tower fermenter (Greenshields etal., 1972), originally by the brewing industry but latterly for fuel ethanol production. Media are pumped into the base of fermenters and pass up through very high concentrations of highly flocculent yeast (Comberbach and Bu’lock, 1984; Kuriyama et al., 1985; Raghav et al., 1989). Non-flocculent yeast and bacterial contaminants are washed out of the top of the system (Jones et al., 1984; Netto el al., 1985) with the converted media. Normally flocculating brewery strains, which only flocculate in the stationary phase of growth, are obviously unsuitable for use in tower fermenters, since single exponentially growing cells would be washed out. In these fermenters, only strains that are highly flocculent under all normal circumstances can be used (Prince and Barford, 1982). C. CLASSIFICATION OF BREWING STRAINS

Brewery yeast strains have been classified by flocculation characteristics in a number of ways. The widely recognized method of Gilliland (1951, 1957) used brewery characteristics of flocculation, those of timing of flocculation and attenuation. Physiological groupings were made by Hough (1957) using flocculation in buffers containing calcium ions, with sugar and ethanol selectors. Porter and Macauley (1965) also used pH value and calciumion effects to distinguish three groupings. Campbell and his colleagues (Campbell and Brundzynski, 1966; Campbell et al., 1968) attempted to use antibody-serological tests on flocculent yeasts, while Stewart et al. (1975a) and Stewart and Russell (1981) classified flocculation by the composition of the media in which flocculation occurred. Until there is a better understanding of the mechanism of yeast flocculation, meaningful attempts to classify strains will be fraught with difficulty. Many secondary effectors may be mistakenly included in the classification. For example, if flocculation occurs when a strain lacks compound X, another strain of identical flocculation characteristics may be misclassified if it is unable to transport or take up compound X.

8

M. STRATFORD

The Gilliland classification benefits at least from simplicity. Class-I strains are non-flocculent and described as powdery. Wort is attenuated fully by them, but is slow to clear. Class-I1 strains flocculate towards the end of fermentation into small flocs of up to 1000 cells. These are regarded as archetypal good ale strains, both attenuating fully and separating well. Class-111 strains are similar but more so. Large flocs of many thousands of cells separate rapidly from the wort, and can leave residual wort sugars. Class-IV yeasts are chain formers, generally unsuitable for brewing due to early sedimentation of chains, leaving a sweet wort. Lager yeasts may be similarly classified, although Watson (1964) reported difficulty in application of the Gilliland tests. In essence, the Gilliland system recognizes non-flocculation, two strengths of flocculation by arbitrary line, and chain formation; it is a classification of pre-eminent value to practical brewers, but less so scientifically. Class-IV chain formers, despite their regarded unsuitability for brewing, were found to be fairly common in a survey of ale strains (Stratford and Assinder, 1991). It is possible that, as these weak chain formers formed aggregates of only 2&50 cells, attenuation was less affected and these strains were not previously recognized as being of class IV. Chain formation is known to be independent of flocculation and under genetic control (Gilliland, 1951, 1957). However, normal non-flocculent, non-chain-forming yeast can be induced to form chains by starvation of magnesium ions (Jansen and Mendlik, 1951), biotin (Dunwell et al., 1961; Duffus et al., 1982) or inositol (Smith, 1951; Ghosh et af., 1960; Lewin, 1965; Dominguez et af., 1978). If chain formation is truly independent of flocculation, as seems probable, then both non-flocculent and flocculent strains would be expected to form chains. A recent study of brewing strains (Stratford and Assinder, 1991) has shown this to be the case. Many strains were found to form chains throughout exponential growth and become truly flocculent in the stationary phase of growth. These were in addition to strains which were only flocculent or chain-forming or neither. Another related but independent characteristic of flocculent yeasts is foaming and the categorization of strains into top and bottom fermenters. Foaming is usually caused by the presence of surface-active compounds. Foaming by wine-producing strains has been shown to be caused by hydrophobic yeast-surface proteins (Kasahara et af., 1974; Dittrich and Wenzel, 1976; Thornton, 1978). The presence of dominant genes FRO2 and FRO2 confer the foaming property on strains. Flocculation of yeast also involves formation of specific proteins (Miki et al., 1982a), which at first sight suggests a relationship between flocculation and foaming, especially since (a) Jansen and Mendlik (1953) suggested a close relationship between flocculation and media surface-active agents, and (b) Naji et al. (1987) demonstrated that antifoaming agents decrease flocculation.

9

YEAST FLOCCULATION: A NEW PERSPECTIVE

However, flocculation and foaming are probably independent, involving different surface proteins, since most foaming strains are non-flocculent (Eddy, 1958a) and bottom-fermenting flocculent yeast strains do not foam. In addition, top-fermenting strains, possessing both foaming and flocculent properties, are found to express them separately at different times during growth (St Johnston, 1953). It remains a possibility that, since both foaming and flocculation involve surface proteins, yeast strains with secretory abnormalities would be simultaneously altered in both properties. To conclude, yeast aggregation appears to be influenced by three independent properties, namely flocculation, chain formation and foaming. Given three categories for flocculation (Gilliland classes I, I1 and 111), and the presence or absence of foaming and chain formation, this results in 12 distinct possible yeast groupings (Table 1). Any further subdivisions of either property would, of course, greatly increase possible yeast groupings. Perhaps this accurately reflects the picture of complexity, strain differences and apparent contradictions found within the field of yeast flocculation. TABLE 1 . Three independent properties influencing the phenomenon of yeast aggregation. Yeast strains possess one attribute from each column, e.g. a purely foaming strain lacks the ability to flocculate and form chains but causes foaming Flocculation

Gilliland class

Foaming

Chain formation

Absence Absence Presence Presence Three types

x

Two types x

Two types = 12 groups

111. Measurement of Flocculation

The search for quantifiable methods of measuring flocculation has been pursued for a great many years without any consensus, either in details of the method or even of the relevant parameters requiring measurement. Researchers have tended to develop individual variations of methods which further complicate comparisons of results from one laboratory to another. Results obtained prior to the realization of the effects of agitation on flocculation (Stratford and Keenan, 1987) must be treated with some caution. It is all too easy to envisage a scientist, in all innocence, picking

10

M. STRATFORD

up and shaking a flask of flocculent yeast in which there is particular interest, and finding a higher degree of flocculation. While early results may fairly report the presence or absence of flocculation, quantification and comparison of flocculations may be questionable unless conditions of agitation were scrupulously repeated. Under brewery conditions, measurement of flocculation becomes simpler, since measurements are required largely to determine whether a yeast is flocculent or not. Concepts such as sedimentation percentage or bond strength are generally less relevant to brewing than the final concentration of yeast suspended in the beer. To this end, an excellent, quick and simple test was devised by Burns (1937, 1941) that is still used in a modified form by many breweries today. The Burns test, developed from earlier tests by Schonfeld (1909,1910) and McCandlish and Hagues (1929), is a measure of the volume of yeast sedimented within a short time. A known quantity of washed, pressed yeast is resuspended in tap water to form a 5% (w/v) solution in a 10 ml graduated tapering test tube. After standing for 10 minutes, the volume of flocculated sediment is measured. Non-flocculent or poorly flocculent strains form little deposit, less than 0.5 ml, in this time. This method was modified and improved by Helm et al. (1953) by substitution of buffer at pH 4.5 containing calcium salts for tap water. Exact

-------

Equilibrium position

I

I ,I

I

Initial rate

Time

-

FIG. 3. A diagram illustrating the basis of measurement of flocculation. Flocculation is a process where, under agitation, the total cell concentration, all initially single, progressively forms into two fractions, those of flocculated and single cells. Measurements may be taken of the initial rate of flocculation, and of the final equilibrium ratio between single and flocculated fractions.

YEAST FI.OCCUI.ATION: A NEW PERSPECTIVE

11

values recorded in this test do not have any deep scientific meaning; they were not designed to. This is an excellent and valuable indication of flocculation to the brewer, and no more than this. In essence, flocculation is an ongoing process where a yeast cell population, initially consisting entirely of single cells, is transformed into two fractions, flocculated and single-cell (Fig. 3 ) . By good fortune, floc-size distribution is bimodal (Davis and Hunt, 1986). This means that there are single cells and flocs, not a whole spectrum of sizes of miniflocs with no clear dividing line between single and flocculated fractions. In order to quantify flocculation, it is necessary to measure any two of the free-cell fraction, the flocculated fraction or the total cell concentration (Fig. 3 ) : Free-cell fraction

+ Flocculated fraction = Total cell count.

From this equation, knowledge of any two parameters will yield the third. Single measurements of sedimentation o r flocculated percentage are, in isolation, relatively meaningless, since flocculation is an ongoing process. However, several such measurements can be used to obtain scientifically meaningful values of the rate and extent of flocculation. The Dahlem conference, held in 1984 and which discussed aggregation in general, proposed that aggregates may be quantitatively characterized by: (a) strength, (b) morphology, (c) extent and (d) rate (Calleja et al., 1984). These latter two qualities, named in this far-sighted report, are easily measured in yeast flocculation. The initial rate of flocculation depends upon agitation, the square of the cell concentration (Stratford and Keenan, 1988) and probably particle mass. Given that these values are all constant, the rate will depend on interparticle repulsion, which is a composite of charge repulsion, steric hindrance and hydrophobic effects. It is probably little affected by flocculation mechanism or bond strength, since the rate is limited by collisions of sufficient energy. However, the minimum agitation threshold is an alternative measure of cell-cell repulsion that has the advantage of being technically much easier to measure. The extent of flocculation is best portrayed as a ratio of flocculated to free cells that is eventually achieved under continuous agitation. This ratio is independent of cell concentration, and can be regarded as an equilibrium constant. It has the practical advantage of being stable over many hours at a constant rate of agitation. This equilibrium is a balance of forces attempting to destroy flocs and the intercell bond strength holding them together. Thus, for given agitation conditions, the position of this equilibrium may be used as a direct indication of flocculent bond strength. Cell-cell bond strength is probably one of the most meaningful concepts

12

M. STRATFORD

in flocculation (Curtis, 1973). Flocculent cells are attached together by bonds, the nature of which will be discussed later (Section VI). Overall bond strength can be varied by alteration of numbers of bonds between cells, i.e. an increasing number of bonds per unit area, or by changing the nature of those bonds. Bond strength is the strength of the weakest link or part. It is possible that the linking mechanism may be stronger than the anchorage into the cell wall. This is unlikely in practice since flocs dispersed by extreme agitation show undiminished flocculation after the agitation rate is lowered. Given a single flocculation mechanism, it is therefore probable that overall bond strength is an indication of the numbers of bonds formed between cells. Size and morphology of flocs are widely recognized to be determined by the rate of agitation imposed on them (Tambo and Hozumi, 1979; Brohan and Mcloughlin, 1984; Stratford et al., 1988) and by the interparticle bond strength. There is also a strong correlation between floc size and the size of the free-cell fraction (Stratford and Keenan, 1988). However, while floc size and morphology may be good visual guides to bond strength, the practical difficulties of accurately measuring floc size or cell content may preclude these as routine measurements. The methods by which flocculation is generally assessed have been examined and reviewed recently by Geilenkotten and Nyns (1971), Calleja and Johnson (1977) and Calleja (1984, 1987). Briefly, visual estimation of flocculation emerges as a worthy measure and usually takes into account clarity of media, floc size and floc number. Flocculated cell fractions may be assessed by floc volume or inferred from the size of free-cell fractions, which may be measured by haemocytometer counting (Calleja and Johnson, 1977) or using spectrophotometry (Kato and Nishikawa, 1957; Kijima, 1963). A total cell count may be made after floc dispersal by proteinases (Calleja and Johnson, 1977) or EDTA (Beavan et al., 1979). Several authors appear to confuse rate of flocculation with rate of sedimentation, these being two entirely distinct parameters (Woof, 1962; Williams and Wiseman, 1973a). Sedimentation rate is a physical phenomenon concerning particle size, aspect and mass, and medium viscosity. Very different approaches to measurement of yeast flocculation have been made by other researchers. Gilliland (1951) determined the point in fermentation that yeast became flocculent; Eddy (1955~)titrated sugar solutions to disperse flocs; Stahl et al. (1983) similarly used EDTA; while Taylor and Orton (1975) determined the temperature required to “melt” flocs. These are certainly measures of something, but is it necessarily flocculation? For example, it is possible that the “floc points” determined by Gilliland (1951) may reflect the rate of removal of inhibiting sugars rather than the development of flocculation.

YEAST FLOCCULATION: A NEW PERSPECTIVE

13

IV. Basic Physiology of Flocculation A. HISTORICAL PERSPECTIVE

While flocculation has been in use in brewing probably for millennia, it was not until 1876 that this phenomenon was first noted scientifically (Pasteur, 1876). In the years that followed, there was a good deal of interest in flocculation, resulting in an extensive early literature. These reports must now be regarded with considerable caution, since there is often little indication whether observed aggregation was due to true flocculation, chain formation, chemical coprecipitation, or sedimentation due to adsorbed material. The term “flocculation” in the early literature was used to encompass all causes of aggregation and sedimentation. The importance of media components in flocculation was apparent from the earliest studies. Seyffert (1896) demonstrated the importance of lime and water softness, while Kusserow (1897) and Lange (1899) reported on the effects of peptones on flocculation. The symbiotic theory of flocculation was developed from the work of Barendrecht (1901), where aggregation was caused by the presence of other micro-organisms. In this study, a bacterium (then known as a Leuconostoc sp., now Lactococcus agglutinans) caused yeast agglutination under aerobic conditions. This appeared to be the result of adhesion brought about by mucilage produced by the bacterium (Beijerinck, 1908). Lactic acid bacteria from molasses have also been demonstrated to flocculate yeast (Malkow, 1934). This theory has received more recent support (Momose et al., 1969) with further evidence of yeast aggregation by lactobacilli. Furthermore, it has been shown that flocculent yeasts are able to bind to bacteria in the genera Hafnia, Lactobacillus and Pediococcus (Stewart and Russell, 1987) and that yeast walls are bound by mannose-specific lectins from walls and pili of Escherichia coli (Eshdat et al., 1981; Firon et al., 1982). However, from as long ago as 1908, it was recognized that this could not be the sole cause of flocculation and that flocculation of uninfected yeast cultures required a different explanation (Beijerinck, 1908). It is comforting to believe that flocculation in a modern brewery is not brought about by gross bacterial contamination. A protein precipitation theory, accounting for flocculation, was suggested by Lange (1907), where wort acidity caused precipitation of gummy proteins, which adhere to cells and thereby induce flocculation. Nonflocculent yeasts were not so affected, being richer in surface peptidase and able to break down precipitated protein. This theory was later expanded to involve calcium phosphate precipitation (Schonfeld and Krumhaar, 1918a,b) and received further support by Stockhausen (1927). Whilst this

14

M . STRATFORD

theory has since fallen from favour, the involvement of wort proteins in flocculation of some yeast strains has recently been suggested by Stewart (1972, 1975). The colloidal theory of flocculation was a natural consequence of the development of classic colloid chemistry. Colloidal particles are maintained in stable suspension by Brownian motion. If particles become aggregated into large flocs they will fall out of suspension since Brownian movements are far smaller in large particles. Colloidal particles are attracted by van der Waals forces but repelled by surface electric charges. If the concentration of ions in solution is raised, so as to mask surface charge, repulsion between particles becomes negligible and particles rapidly coagulate (Kruyt, 1952; Overbeek, 1952). Early workers on yeast flocculation anticipated that flocculation occurred when yeast surface charge was sufficiently lowered or neutralized (Koch, 1928). Work on yeast surface charge by Geys (1922) suggested that the charge was reversed several times throughout fermentation. Later studies showed that cells are normally negatively charged with charge size declining with p H value (Winslow and Fleeson, 1926; Jansen and Mendlik, 1951; Eddy and Rudin, 1958a,b,c). The colloidal aspects of flocculation were combined with surface protein effects by Stockhausen (1927), where flocculation occurred following surface protein coagulation at p H 4.7-4.8. Later work suggested involvement of yeast gum (Stockhausen and Silbereisen 1933, 1935). The colloidal theory of flocculation has now been largely discredited by several pieces of evidence. Firstly, the results of Wiles (1951) and Jansen and Mendlik (1951) demonstrated that yeast never loses its negative surface charge during fermentation. Secondly, when yeast surface charge is neutralized either by p H adjustment to the isoelectric point (Fisher, 1975) or by growth in medium containing a low concentration of phosphate (Eddy and Rudin, 1958b), yeast does not rapidly coagulate but remains dispersed. Thirdly, while some positively charged ions promote flocculation, other ions, such as sodium and potassium ions, antagonize it (Stewart and Goring, 1976), and the concentrations of calcium ions required to promote flocculation are sufficiently low as to preclude any surface-charge neutralization. Fourthly, two independent papers presented at the European Brewery Convention (Gilliland, 1951; Thorne, 1951a,b) clearly demonstrated the genetic basis of flocculation. Undoubtedly, flocculation is influenced by surface charge, but it is not a colloidal precipitation phenomenon. B . INORGANIC-ION EFFECTS

The presence of inorganic salts was one of the first effectors of flocculation to be recognized (Seyffert, 1896; Hayduck and Schiickling, 1908; Schonfeld

YEAST FLOCCULATION: A NEW PERSPECTIVE

15

and Krumhaar, 1918a). This promotion of flocculation, especially by salts of calcium and magnesium, undoubtedly contributed to early colloidal theories of flocculation. Later work by Burns (1937), Gilliland (1951) and Jansen and Mendlik (1951) demonstrated the effectiveness of chlorides and sulphates of calcium, magnesium, sodium and potassium. Flocculated cells of some yeast strains may be dispersed into single cells by repeated washing in distilled water (Jansen and Mendlik, 1951). After addition of low concentrations of calcium , magnesium or barium ions, flocculation was restored. Using different yeast strains, Eddy (1955a) was unable to disperse flocs by washing with distilled water. This matter was later resolved in that some yeast strains were deflocculated by water alone, while other strains were dispersed by ion chelation with EDTA (Taylor and Orton, 1973; Stewart, 1975; Stewart and Goring, 1976; Stewart etal., 1976). After EDTA dispersal, cells were rapidly reflocculated by addition of further calcium, manganese or magnesium ions. Isolated cell walls of flocculent yeasts have been found not only to retain their flocculent character (Masschelein and Devreux, 1957) but also to respond to stimulation by salts (Mill, 1966). This clearly demonstrates a role for salts in activation of flocculation at the cell-wall level rather than in flocculation derepression or secretion. The reported higher calcium-binding capabilities of flocculent cell walls (Masschelein and Devreux, 1957; Lyons and Hough, 1971) may well be a consequence of flocculation activation. Saturable calcium-binding sites on flocculent cells were also demonstrated by Williams and Wiseman (1973a). While there is a general consensus that calcium ions promote flocculation of dispersed cells (Lindquist, 1953; Eddy, 195%; Harris, 1959; Mill, 1964b; Lyons and Hough, 1970a, 1971; Taylor and Orton, 1973, 1975; Stewart et al., 1975a; Stewart and Goring, 1976; Jayatissa and Rose, 1976; Miki et al., 1982a; Nishihara et al., 1982) and are generally reckoned to be the most effective cation influencing flocculation, there are many reports of flocculation brought about by other cations, especially of magnesium (Jansen and Mendlik, 1951,1953; Lindquist, 1953; Eddy, 195%; Porter and Macauley, 1965; Patel and Ingledew, 1975; Stewart, 1975; Stewart and Goring, 1976; Miki et al., 1982a) but also by dipositive ions of transition metals such as manganese, iron, cobalt, nickel, zinc, copper and cadmium. Response to cations appears to be yeast-strain variable (Porter and Macaulay, 1965; Stewart and Goring, 1976) and is influenced by pH value (Porter and Macaulay, 1965; Miki et al., 1982a). There is an increasing body of evidence that calcium ions specifically are required for flocculation. Group-2a calcium-ion analogues, namely strontium and barium ions, competitively antagonize calcium ion-induced flocculation (Taylor and Orton, 1973; Nishihara et al., 1982; Stratford,

16

M. STRATFORD

1989c) and sodium ions, of similar crystal ionic radius to calcium ions, also competitively inhibit flocculation (Mill, 1964b; Stewart and Goring, 1976; Nishihara et al., 1982; Stratford, 1989~).Trace amounts of calcium M) were found sufficient to induce flocculation (Taylor and Orton, 1975). The chelating agent, EGTA, with a higher affinity for calcium over magnesium ions, can be used (Stratford, 1989c) to demonstrate that flocculation does not occur in the presence of free magnesium ions. Magnesium ions and other cations caused release of cellular calcium and thus indirectly caused flocculation. The strain variability observed by Porter and Macauley (1965) in respect of calcium-ion requirement and pH value is probably a result of strain variations in calcium-ion leakage rather than in flocculation. Many salts are reported to cause inhibition of flocculation. This can be by competition with calcium ions, as previously described. But, at higher concentration, many salts show an inhibition of flocculation (Calibo et al., 1989; Stratford and Brundish, 1990). Partially inhibitory concentrations of different salts were additive in effect and were exacerbated by prolonged incubation and pH extremes. It was concluded (Stratford and Brundish, 1990) that this non-specific salt inhibition was chaotropic in nature and caused surface-protein distortion both by binding high charge-density cations and dehydration effects. Smaller and more highly charged ions were most effective inhibitors. At concentrations below inhibitory levels, salts were found to enhance flocculation. This appeared to be both by lowering yeast surface charge, and also by modifying surface proteins in a manner similar to that described as “salting in and salting out” (Lehninger, 1975) for protein solubility. Finally, in addition to direct effects of salts in flocculation activation and inhibition, there are probably salt effects on the development of flocculation. Eddy and Rudin (1958b) demonstrated phosphate requirement for flocculation expression, while Nishihara et al. (1976a) prevented flocculation development by growth in media lacking magnesium ions. Amri et al. (1982) suggested that media (Ca2+:K+ion ratios affected flocculation expression. To conclude, flocculation development is influenced by media salt content. After flocculation expression, there is a specific “activation” by calcium ions which may be competitively inhibited by calcium-ion analogues. Other cations activate flocculation indirectly by causing calcium-ion leakage or release. Non-specific chaotropic effects cause flocculation enhancement at low salt concentrations, and inhibition at high concentrations. C. lNHlBlTlON BY SUGARS

Early observations by Lindner (1901) suggested that the presence of sugars prevented yeast flocculation. This was confirmed by Burns (1937) and later

YEAST FLOCCULATION: A NEW PERSPECTIVE

17

by Lindquist (1953), Eddy (1955a,b), Mill (1964b) and Fujino and Yoshida (1976). It was found that maltose and mannose were most effective inhibitors and that sucrose and glucose were less effective. Sugars such as galactose and fructose were ineffective; flocs were dispersed by pyranose and not furanose sugars (Kamada and Murata, 1984~).However, contrary results (Taylor and Orton, 1978; Miki et al., 1982a; Lipke and HullPillsbury, 1984) suggested that flocculation was inhibited specifically by mannose and derivatives. Data presented by Kihn et al. (1988a) showed that one yeast strain was inhibited only by mannose and that two strains were additionally affected by maltose and glucose. A comprehensive survey of yeast strains (Stratford, 1989b) showed two distinct groupings of strains: the Flol phenotype, inhibited only by mannose, and the NewFlo phenotype, inhibited by several sugars. In view of the fact that sugars inhibiting flocculation were all metabolized by yeast, the question was raised (Calleja, 1984) as to whether this was a direct effect, or merely a result of stimulation of yeast metabolism; that is, a stepping-up effect (Brohan and McLoughlin, 1985). Recent evidence demonstrates that sugars directly affect the flocculation mechanism. Yeast suspensions, metabolically crippled by low temperature (4°C) or heat killing (60°C for five minutes), still retain flocculation inhibition by sugars (Stratford, 1989b; Stratford and Assinder, 1991). Flocculent bond strength at 4°C is progressively lowered by increasing mannose concentration (Stratford et al., 1988). 2-Deoxy-~-glucose,a potent glycolytic inhibitor, was found to be a strong inhibitor of flocculation (Stratford and Assinder, 1991). The mechanism by which sugars act on flocculation is discussed in Section V1.C. In addition to direct inhibition of flocculation, sugars, in particular glucose, are necessary for development of flocculation (Nishihara et al., 1976b). Possibly this acts via production of metabolic energy, vital at all stages of the secretory pathway (Novick et al., 1981). D.

pH

VALUE AND TEMPERATURE EFFECTS

Since flocculation is normally expressed late in brewing fermentations, at a stage when the pH value of wort has fallen, it was once widely believed that pH value was important in promoting flocculation, and considerable research was carried out upon this factor (De Clerck, 1930; Malkow et al. , 1933; Malkow, 1934; Hennig and Ay, 1938). The currently fashionable colloidal theory suggested that surface charge was lowered by increased hydrogen-ion concentration, while other factors coinciding with onset of flocculation, such as changes in ethanol or sugar concentrations, were conveniently ignored. However, the pH value at which flocculation occurs was not constant, even for a given strain of yeast (Koch, 1928) but appeared

18

M. STRATFORD

to reflect the initial p H value of the wort. Flocculation does not happen at one particular p H value, but can occur between pH 1.5 and 9 (Stratford et af., 1988) with an optimum between pH 3.5 and 4.6 (St Johnston, 1953; Mill, 1964b; Williams and Wiseman, 1973a). pH value is now recognized as not being a determining factor of flocculation under brewery conditions (Gilliland, 1951;Jansen, 1958). However, pH value does undoubtedly affect surface charge and, while this does not cause flocculation, cell-to-cell contact becomes progressively easier as surface charge is lowered (Stratford and Keenan, 1987). The overall result of this lowering of surface charge is an increase in the rate of flocculation. Temperature may influence flocculation development or expression. Mill (1964b) first demonstrated that flocs were dispersed by high temperatures (5040°C). This was described as a “melting” of flocs that was readily reversible on cooling. Thermal deflocculation was confirmed by Taylor and Orton (1975), Hussain et af. (1986) and the temperature at which flocs dissociated (TF) was used as a measure of flocculation (Taylor and Orton, 1975). This temperature range was considered by Mill (1964b) to indicate that flocs were bound together by a process involving hydrogen bonding. At physiological temperatures (15-32°C) there is generally little influence of temperature on flocculation (Stewart et af., 1975b). However, several reports suggest that flocculation development in some strains is temperaturesensitive (Lund, 1951; Lindquist, 1953; Holmberg and Kielland-Brandt, 1978) with flocculation suppressed at 25°C. Since the yeast secretory pathway is prone to temperature-sensitive mutations, and was originally studied using such mutants (Novick et al., 1980), it is tempting to suggest that temperature-sensitive flocculation may be caused by such mutations. E. EFFECT OF PROTEIN DENATURATION

The involvement of cell-surface proteins in flocculation was discovered by Eddy and Rudin (1958a), who showed that papain treatment of flocs caused irreversible flocculation loss. Other proteolytic enzymes, namely pronase E, proteinase K, trypsin, chymotrypsin and pepsin, together with chemical protein modifiers, including mercaptoethanol, urea and guanidine, were all found similarly to disable flocculation irreversibly (Stewart et al. , 1973; Nishihara et af.,1977,1982; Miki et af.,1982a; Watari ef af.,1987; Stratford and Assinder, 1991). Proteases have also been used to distinguish different flocculation types (Hodgson et al., 1985; Stratford and Assinder, 1991). Other degradative enzymes, namely lipase, RNase, DNase and lysozyme, had no effect on flocculation (Nishihara et af., 1982). Trypsin-digested flocculent cells, in addition to losing flocculence, are reported to yield a substance, YCSS, that agglutinates yeast cells (Kamada

YEAST FLOCCULATION: A NEW PERSPECTIVE

19

and Murata, 1984a). This substance appeared to be a mannoprotein, and agglutination was calcium ion-dependent and dispersed by maltose (Kamada and Murata, 1984b). A similar cell-surface protein, showing EDTA- and calcium ion-effected agglutination, was extracted from flocculent cells by Kijima (1964). Simultaneous loss of flocculence and surface mannoproteins may also be caused by osmotic shock (Williams and Wiseman, 1973b). This evidence suggests that not only do surface proteins play a vital role in flocculation (Nishihara et a f . , 1977), but that the active protein may be readily removed from the cell surface. Its continued activity suggests a multivalent activity since, in order to release the protein, the original linkage to the cell wall must have been broken. F. MITOCHONDRIAL INVOLVEMENT IN FLOCCULATION

Prolonged fermentation by a yeast culture, either by continuous fermentation (Stewart, 1977) or by batch culture with repitching (Gyllang and Martinson, 1972), leads to loss of attenuative power and a progressive increase in the frequency of petite strains in cultures. These mitochondrial mutants are reported to have lost the ability to ferment maltotriose (Gyllang and Martinson, 1972) but also may affect attenuation, by sedimenting faster (Silhankova et a f . ,1970; Cowan et al., 1975), i.e. becoming more flocculent or chain-forming. However, most reports state that induction of petite strains causes diminishment or loss of flocculation in yeast strains (Stewart et al., 1976; Stewart and Russell, 1976, 1977; Holmberg, 1978; Evans et af., 1980) while a survey of 125 petite strains found that 109 had diminished flocculation characteristics. Egilsson et a f .(1979) demonstrated that carcinogens caused loss of flocculence as a consequence of mitochondrial toxicity, and antibiotic-resistant mitochondrial mutants had lost flocculence (Spencer et al., 1981). A revertant strain regained the power to flocculate. The genetic basis for the observed link of mitochondria with flocculation has been researched by Esser et al. (1987) and Hinrichs et a f . (1988). Two sites in the mitochondrial genome were implicated, namely olif and oxi2, simultaneous deletion of both causing loss of flocculence. This is supported by observations that cytochrome oxidase activity formed from oxi2 is influenced by the presence of unsaturated fatty acids (Getz et al., 1981). This factor has also been shown to control expression of flocculation (Lands and Graff, 1981). Attempts to transform a non-flocculent strain to flocculent, using these genes, were unsuccessful (Hinrichs et al. , 1988), suggesting perhaps that the non-flocculent strain was not necessarily non-flocculent for lack of these genes. What then is the nature of the mitochondrial effect on flocculation?

20

M. STRATFORD

Nishihara et al. (1976b) concluded, after work using mitochondrial inhibitors, that mitochondrial function was important rather than synthesis of mitochondrial proteins. There is now some evidence to suggest that mitochondria affect the secretion process rather than acting directly on flocculation. As already mentioned, Gyllang and Martinson (1972) noted loss of maltotriose fermentation by petite strains, while Spencer et al. (1981) described simultaneous loss of flocculation and extracellular glucoamylase activity caused by mitochondrial mutation. Wilkie and Nudd (1981) reported a mitochondrial effect on wall enzymes and permeases. Mitochondria have also been implicated in formation of surface proteins in Schiz. pombe and in protein secretion by Schwanniomyces alluvius (Calleja, 1973; Calleja et al., 1986, respectively). It thus seems probable that mitochondria influence protein secretion in general rather than flocculation in particular. This may also be supported by evidence of aerobic-anaerobic effects on flocculation. Studies generally find aeration stimulatory to flocculation (Piendl, 1969a,b; Miki et al., 1982b; Ramirez and Boudarel, 1983), although there are reports to the contrary (Masschelein and Devreux, 1957). Aerobiosis has been found similarly necessary for protein secretion (Calleja et al., 1986).

G . HETEROLOGOUS FLOCCULATION

Most yeast strains that express flocculation are able to flocculate alone, without the presence of any other yeasts, bacteria or additional material, particulate or amorphous. This is described here as homologous flocculation but has also been termed self-flocculation (Johnson et al., 1988) or autoflocculation. In addition, there are many reports of flocculation between unlike microbial particles, both yeast with unlike yeast, with bacteria, and with particulate material and bacterial slime, which may be given the umbrella title of heterologous flocculation. These various heterologous flocculation types are summarized in Fig. 4. Yeast cells may be non-specifically aggregated by fining agents or by mucilage (Beijerinck, 1908), usually bacterial in origin (Kurane etal., 1986). Alternatively, cells may be flocculated by adhesion with other particulate matter. Particles, whether other yeasts, bacteria or inanimate debris, can effectively act as bridges linking cells together into aggregates. Bacteria have been demonstrated to adhere to yeasts both by adhesins on the bacterial surface (Firon et al., 1982; Sharon et al., 1983) or on flocculent yeasts (White and Kidney, 1979; Stewart and Russell, 1987). Yeast aggregation has also been caused by inert powders (Weeks et al., 1983).

21

YEAST FLOCCULATION: A N E W PERSPECTIVE

A

B

FIG. 4. A diagram showing possible heterologous flocculation. Yeast cells are aggregated by adhesion to bridging material. This may be (A) fining agents or bacterial mucilage, (B) bacterial cells or (C) dissimilar yeast cells.

A process described as mutual flocculation (Eddy, 1957,1958a) was found to occur when pairs of yeast strains, which individually were weakly flocculent or non-flocculent, rapidly formed large aggregates when mixed. Strains were classified as types I and I1 and mutual flocculation occurred when strains of opposite types were mixed. This phenomenon was discovered shortly afterwards in brewery strains (Hough, 1957) and was later shown to be calcium ion-dependent and proteinase-sensitive (Stewart and Garrison, 1972; Stewart et al., 1973, 1975a). Mutual flocculation, here termed coflocculation, occurred when cell types were mixed within ratios of 9:1 in either direction (Stewart, 1972). This can be regarded as evidence that there are two parts to the flocculent bond (Rainbow, 1966), namely an

22

M. STRATFORD

Non-flocculent cell lacking adhesins and receptors

Non-flocculent cell, with adhesins only

Non-flocculent cell, with receptors only

Flocculent cell,with receptors and adhesins

FIG. 5. Four potential combinations of adhesins and receptors on cell surfaces of Saccharornyces cerevisiae. Cell A lacks adhesins and receptors, cell B has adhesins only, cell C has receptors only, while cell D has both. Mutual flocculation can occur between cells of types B and C, whereas coflocculation can occur between flocculent cells D and cells of either B or C types.

YEAST FLOCCULATION: A NEW PERSPECTIVE

23

adhesin and a receptor, one of which is found on each strain of mutually flocculating pairs. Coflocculation has also been used to describe a similar phenomenon, where non-flocculent cells are sedimented by adhesion of flocculent yeast (Curtis and Wenham, 1958; Miki etal., 1981,1982a). Here, one strain, able to self-flocculate, having both adhesins and receptors, adheres to nonflocculent cells bearing only receptors (Miki et af., 1981, 1982a). The non-flocculent cells are not “trapped” in flocs (Docherty et af., 1986) but adhered to (Miki etal., 1982a) since Schiz. pombe strains, lacking receptors, were not coflocculated. Receptors for flocculation do not appear to be universal on all non-flocculent strains, as was originally suggested by Miki et af. (1982a). There is some variation in non-flocculent strains, in the ability to be coflocculated with the same flocculent strain. Some non-flocculent cells which were normally unable to be coflocculated (Nishihara and Toraya, 1987) were able to do so following mercaptoethanol treatment, which appeared to expose receptors. To conclude, yeast strains appear to be of four types (Fig. 5). There are non-flocculent strains, lacking both receptors and adhesins (type A), strains with adhesins but lacking receptors (type B) and strains with receptors only (type C ) . Strains able to self-flocculate have both adhesins and receptors (type D). Mutual flocculation will occur between types B and C, neither of which is flocculent alone. Coflocculation can occur between flocculent type D and either type B or C. Coflocculation has been used (Stewart, 1972, 1975) to describe mutual flocculation but mutual flocculation takes precedence by priority (Eddy, 1957, 1958a).

V. Physics of Cell-Cell Interactions A. YEAST SUSPENSIONS AND BROWNIAN MOTION

Yeast cells are frequently described as suspended or being in suspension in liquid. This term, borrowed from colloidal science, indicates that the cells are being held up or suspended. But what precisely is holding them up? A classical colloidal suspension consists of very small particles in a medium, all of which are of course attracted by gravity but are sufficiently small to remain suspended by Brownian motion. Brownian motion, first noticed by Robert Brown in 1828, describes the irregular motion of microscopic suspended particles, a “random-walk pathway”, which are in kinetic equilibrium with the molecules of the medium (Kruyt, 1952). Thus, colloidal particles attempting to fall are maintained suspended by random movements induced by asymmetric impacts from molecules of the surrounding medium.

24

M. STRATFORD

Since particles are in kinetic equilibrium with the surrounding molecules (Kruyt, 1952), it is obvious that Brownian motion must depend on particle mass and size. Larger particles receiving identical molecular impacts move less than do smaller particles, since the overall energy change is identical. Furthermore, particle motion is caused by asymmetric distribution of impacts and asymmetric impacts on larger particles occur with decreased probability (Einstein, 1906; von Smoluchowski, 1906). Thus, as particle size and mass increase, Brownian motion becomes rapidly less significant both in size and frequency of movements. This means that colloidal suspension must be restricted only to small particles. In the early years of this century, with colloidal science in its infancy, colloidal particles were conveniently regarded as those that could pass through a 0.45 pm pore-size filter. This rule-of-thumb has obviously been since modified, but it gives an approximate figure for maximum colloidal particle size. Colloidal particles are attracted to one another by van der Waals’ forces but repelled by like electrostatic charges on the particles. Raising the ionic concentration of media causes a neutralization of charge repulsion and particles undergo rapid coagulation (Tuorila, 1927; Overbeek, 1952) where

2o 18 16

14

12 10

Rate of falling (mm h-I) 8 6 4

2

0

[:

I 1

2

3

4 5 6 7 8 9 1 0 1 1 1 2 Aggregation number (cells floc -l )

FIG. 6. Histogram showing rate of sedimentation of small yeast flocs in water. Measurements were obtained by microscopic examination of clumps falling in a vertically positioned haemocytometer slide, 0.2 mm in depth. Times taken for clumps to pass across the haemocytometer gradations were recorded, together with numbers of cells comprising the clump. At least 12 observations (+ SD) were made for each size of aggregate (M. Stratford, unpublished observations).

YEAST FLOCCULATION: A NEW PERSPECTIVE

25

the rate of aggregation is limited only by collision frequency. Aggregates rapidly build up; newly formed large particles are too large to be maintained in Brownian suspension and sediment rapidly. Large particles are also able to fall more rapidly, having less drag in proportion to their mass. Yeast cells are not colloidally suspended in liquid media. Firstly, yeast cells at approximately 5 pm in diameter are obviously much larger, being some 1000-fold greater in volume than colloidal particles and of higher density due to their thick cell walls. Secondly, Fig. 6 shows that yeast cells in water are not suspended but are falling out of the media at 4-5 mm h-'. Falling was observed to be smooth, without irregular movements caused by Brownian motion, thereby confirming that yeast cells are too large to be so affected. Undoubtedly, individual constituent parts of cells may be undergoing micro-Brownian vibration (Kuhn, 1938) but, as a whole, do not show macro-Brownian motion. Figure 6 also demonstrates progressively faster sedimentation with increased cell numbers in the flocs. In this experiment, small microflocs of up to 12 cells were measured to be falling at 16 mm h-' ;large flocs of many millions of cells obviously move considerably faster. B. MECHANICAL AGITATION AND FLOCCULATION RATE

Yeast flocculation requires that cells are brought into intimate contact and that any repulsion between cells is overcome. Having shown that yeast cells are barely affected by Brownian motion, which has long been assumed responsible for bringing about cell collisions, it is surprising that the profound effects of mechanical agitation on flocculation were discovered entirely by chance (Stratford and Keenan, 1987). Experiments designed to tear non-flocculent cells away from flocs by hard shaking showed entirely the reverse behaviour. The harder flocculent yeasts were agitated, the better they flocculated. More systematic investigation showed that, without mechanical agitation, flocculation did not occur (Fig. 7) and that flocculent yeast cells failed to clump but sedimented slowly as single cells. Agitation caused rapid and progressive flocculation but, if at any time agitation ceased, flocculation stopped. The whole process was arrested and single cells in suspension remained therein, after rapid sedimentation of flocs already formed. Experimentally, this was very fortuitous since flocculation could be followed with measurements taken while the process was halted, simply by switching off the shaker. The theory of mechanical-agitation effects has its roots in the early colloidal theory of flocculation. Yeast cells are undoubtedly electrostatically charged (Brohult, 1951; Wiles, 1951; Jansen and Mendlik, 1951, 1953;

26

)-PO-

I I I

I

0

1

2

3

4

5

6

Time (mid FIG. 7. Time-course of flocculation of Saccharomyces cerevisiae S6461B. Flocculation was initiated at zero time by addition of calcium ions to cell suspensions previously dispersed with EDTA. Suspensions were either left standing (O), and then shaken at the time indicated by the arrow at 80 r.p.m. (O), or shaken continuously at 80 r.p.m. ( O ) , or for just one minute )(. Data are reproduced from Stratford and Keenan (1987).

Amory et al., 1988; Bowen and Cooke, 1989). The isoelectric point, where the overall yeast-cell charge is neutral, is between pH 2 and 3.2 (Fisher, 1975;Jayatissa and Rose, 1976;Beavan et al., 1979) with increasing negative charge at higher pH values. This charge is a result of charged phosphate (Eddy and Rudin, 1958a,b) and protein groups in the wall. Yeasts are kept dispersed by repulsion between like-charged cells (Rose, 1984). This charge repulsion may be regarded as an energy barrier preventing cells approaching sufficiently close to bond together (Unz, 1987). Mechanical agitation gives cells sufficient momentum to overcome this energy barrier and physically collide, enabling bonds to form between cells. C. COLLISION FREQUENCY

Theoretical expressions of collision frequency under Brownian motion, laminar-flow and turbulent-flow regimes have been calculated (von

YEAST FLOCCULATION: A NEW PERSPECTIVE

27

Smoluchowski, 1917; Camp and Stein, 1943). These expressions demonstrate the importance of particle size in determining the effectiveness of Brownian motion. When the diameter of at least one of the colliding particles is greater than 1 pm, agitation (orthokinesis) becomes more important than Brownian motion (perikinesis) in causing collisions. This value is in reasonable agreement with that of 0.45 pm used by early colloid scientists to delimit colloidal particle size, by being too large to remain suspended by Brownian motion. Based solely on collision frequency, yeast cells (about 5 pm in diameter) are predicted to be strongly influenced by mechanical agitation. These theories of collision frequency were developed with classic colloidal systems in mind, such as aqueous suspensions of gold, quartz or clay (Tuorila, 1927; Yusa, 1977). Here, after charge neutralization by media ions, rapid coagulation occurs where, since there is no repulsion, the rate of flocculation is limited only by particle-collision frequency (Overbeek, 1952), which is in turn affected by particle size. Such mathematically perfect flocculation was experimentally demonstrated by Tuorila (1927). Regrettably, biological particles do not obey the rules and are very much more complex. Even after surface charge has been neutralized, there are still a number of factors which cause repulsion between cells: (a) Large protruding surface glycoproteins may hinder surface-to-surface contact unless pushed aside. This is commonly termed steric hindrance or steric repulsion (Greig and Jones, 1976). (b) Most surface groups on biological particles are hydrophilic in nature, and would be expected to resist water displacement caused by contact with another cell and the presence of a hydrophobic environment within the contact. (c) Electrostatically charged surface groups, generally negative, attract oppositely charged ions from the media, thereby forming an electric double layer. In addition, zwitterionic water molecules are attracted, forming a cushioning water shell. Even if the overall charge on cells has been neutralized, the water shells and ionic atmosphere remain. These result in repulsion between cells since, in order to achieve contact, considerable energy is needed to push these atmospheres aside. Thus, even after repulsion between cells is diminished by charge neutralization, other repulsive forces remain. Mathematical expressions of rapid coagulation were developed, assuming collision frequency to be rate-limiting, given negligible repulsion. But, given considerable repulsion, it is probable that force of collision rather than frequency is rate-limiting.

28

M. STRATFORD

h c

0

20

40 60 Agitation (r.pm.)

80

100

120

FIG. 8. Effect of agitation on initial rate of flocculation of Saccharomyces cerevisiae NCYC 1195, at pH 4.5, from an initial cell density of 6 mg dry wt ml-’. No flocculation was detected below the minimum agitation threshold, 40 r.p.m. The broken line indicates the predicted increase in cell-cell collision frequency with agitation, indicated by n=ct’”, where n is the number of collisions,cis a constant and t is the shaking speed. Initial rates were determined by sampling at 10 second intervals. D. ENERGY OF COLLISION

The observed intense promotion of flocculation by mechanical agitation could be the result of increased frequency of collisions, or of the force with which they collide, o r both. While some reports of the effects of mechanical agitation on flocculation clearly regard agitation as causing increased collision frequency (Amory et al., 1988; Kihn et al., 1988a,b), more careful examination reveals this not to be so, due to the following considerations: (a) There is a minimum threshold of agitation for flocculation to occur (Stratford and Keenan, 1987). If flocculent yeast cells are shaken gently, below the minimum agitation threshold, no flocculation occurs (Fig. 8). Agitation harder than this threshold induces rapid flocculation, which increases dramatically in rate with yet more vigorous agitation. If agitation increased collision frequency, one

YEAST FLOCCULATION: A NEW PERSPECTIVE

29

would predict a progressive increase in flocculation with agitation but starting from the origin. The presence of a minimum agitation threshold suggests that gentle collisions from minimal shaking are insufficient to overcome cell-cell repulsion. (b) The number of collisions between particles in a fluid system can be calculated (Chapman and Cowling, 1970) from a complex function. The effect of alteration of energy input, temperature or agitation can be resolved from the function n = ct“’, where n is the number of collisions, c is a constant and t is temperature or agitation. This function is graphically illustrated in Fig. 8. Numbers of collisions increase sharply with agitation from zero but level off in a parabolic curve. If flocculation rate depended on collision frequency, it would be predicted to follow this curve, which it manifestly does not. (c) If the minimum threshold of agitation is the result of particle collisions needing to overcome repulsion between cells, it would follow that increasing repulsion by increasing surface charge would cause an increase in the minimum threshold of agitation. Raising the p H value has been shown (Fig. 9) to increase surface charge, while minimum agitation thresholds increased substantially with pH value. (d) For a single rate of agitation, at which the collision frequency is constant, for example 80 r.p.m. (Fig. 9), the rates of flocculation at various pH values are substantially different. Since pH value has been shown for this strain (Stratford et al., 1988) to have little effect on bond strength, the frequency of collisions would seem to play little part in flocculation, its rate being substantially determined by the energy of collision. Identical conclusions were drawn on the influence of temperature on the rate of chemical reactions. While temperature does slightly increase collision frequency, it is the energy of collision that determines whether a chemical reaction occurs. In a chemical reaction, it is customary to regard this minimum collision energy as an energy barrier or “activation energy” (Fig. 10) where raising the temperature increases the kinetic energy of molecules and increases the proportion of collisions with enough energy to overcome this barrier (Barrow, 1973). Activation energy may be quantified by Arrhenius plots of the logarithm of the rate constant against the reciprocal of temperature. The similarity of flocculation to chemical reaction was further demonstrated in that Arrhenius plots of the logarithm of the flocculation-rate constant against the reciprocal of the agitation rate resulted in straight lines (Stratford and Keenan, 1987), enabling the activation energy of flocculation to be determined. This activation energy was least at pH 2-3, increasing with

M. STRATFORD

Agitation (r.p.m.) FIG. 9. Interaction of agitation and pH value on the rate of flocculation of Succhurornyces cerevisiue NCYC 1195, from an initial cell density of 6 mg dry wt ml-'. pH 2.0 is indicated by (0), pH 7.0 (0)and pH 9.0 ( 0 ) .At 80 r.p.m. agitation (broken line), rates of flocculation differ with pH value, despite identical collision frequencies.

pH value and closely reflecting yeast-surface charge (Jay atissa and Rose, 1976; Beavan et af., 1979). This is further evidence that agitation energy is used to overcome charge repulsion, but it also demonstrates that, even at the yeast isoelectric point of pH 2-3, activation energy is still needed and therefore some repulsion remains. Presumably this residue is the proportion of repulsion caused by hydrophilic effects, steric hindrance and ionic displacements. E. EXTENT OF FLOCCULATION AND EQUILIBRIUM

Under continuous agitation, flocculation proceeds from a high initial rate that rapidly declines to an eventual steady state when flocculation has apparently ceased (Stratford and Keenan, 1988). Flocculation, by analogy to chemical reactions, does not go to completion but leaves a small quantity of single yeast cells in suspension. The time taken to reach this steady state varies, depending on the degree of agitation. With vigorous agitation

YEAST FLOCCULATION: A NEW PERSPECTIVE

31

Repulsion

Energy

FIG. 10. Activation energy of flocculation is required to overcome mutual repulsion between like-charged yeast cells, enabling cellkell contact and flocculent bond formation. Repulsion may also be caused by steric hindrance, hydrophobic effects and the need to displace ionic atmospheres.

this time may be brief but, in practice, several hours are required for confirmation that flocculation has entirely ceased. It has been suggested that this steady state is in fact a dynamic equilibrium (Kihn et af.,1988a; Stratford and Keenan, 1988). The evidence for this is as follows: (a) The single-cell fraction is flocculent. If cells are removed and concentrated by centrifugation, flocculation occurs similarly as in the original suspension. (b) If the single-cell fraction is removed carefully from an equilibrated flocculent yeast suspension and replaced with buffer, cells are rapidly released from the jostling flocs, thereby restoring the single-cell fraction (Stratford ef al., 1988). (c) The single-cell fraction is a constant proportion of the total yeast count (Stratford et af., 1988). There is a constant ratio of single to flocculated cells for a given agitation and, if more cells are added, the single-cell fraction rises in proportion. This would therefore appear to be a dynamic equilibrium (Fig. 11). While overall flocculation has ceased, there is a rapid exchange, a balance, between cells lost by jostling flocs and cells adhering to flocs. This is essentially similar to dynamic equilibria formed by chemical reactions, the

32

M. STRATFORD

0 I

0

0

0 0 00 FIG. 1 1 . Diagram showing how the steady state of flocculation is a dynamic equilibrium, where the rate of cell loss from flocs by erosion or floc fracture is equalled by the rate of cell aggregation into new flocs, and single-cell adhesion onto existing flocs.

ratio of two sides of the equilibrium being in constant proportion. This ratio may thus be expressed as an equilibrium constant for any given agitation, which remains independent of changes in cell concentration. Agitation, however, does have profound effects on this equilibrium. While flocculation under more vigorous agitation is much more rapid and reaches equilibrium sooner, the proportion of single cells is found to increase (Stratford et al., 1988) and the equilibrium constant altered. In addition, floc particles become noticeably smaller with increased agitation. It appears that flocs increase until reaching a maximum size permitted by the rate of agitation. Shear forces are known to increase as the square of the particle radius (Curtis, 1973) and, if particles exceed a size limitation defined by bond strength, shear forces decrease particle size, either by floc fracture or by erosion of pieces (cells) from the surface (TeKippe and Ham, 1971; Parker et al., 1972). If the agitation conditions are sufficiently extreme, flocs may be entirely dispersed into single cells. Under normal conditions of flocculation, such agitation would be rarely experienced but, if flocculation bond strength were lowered by sugars or low p H values, flocs may be dispersed by relatively gentle agitation (Stratford et al., 1988). While increased agitation generally tends to promote rapid flocculation, strains with a weak or inhibited bond strength may be dispersed by such treatment.

YEAST FI.OCCUI.ATION: A NEW PERSPECTIVE

33

F. MORPHOLOGY OF FLOCCULATION

Another very noticeable effect of agitation on flocculation concerns the eventual morphology and size of flocs. While different yeast strains, with different floc bond strengths, may vary as to exactly at which agitation rate the different morphologies occur, the overall pattern and sequence of morphology are strain-independent. At very low agitation rates, barely sufficient to cause flocculation, flocs fall from suspension and congeal together with flat slabs of yeast, several centimetres across. As the agitation rate is increased, these slabs begin sliding gently around the bottom of flasks, becoming smaller, more rounded and with raised rims. These raised edges are formed by collisions between slabs, causing upward movement of cells, which eventually results in thickened rims. Higher agitation rates are associated with slabs becoming very much thicker and the raised rim occupying a large proportion of the area; flocs frequently resemble lozenges (Fig. 12(a)). At about 100 r.p.m. agitation, a number of more strongly flocculent yeasts form flocs that are perfect cubes (Fig. 12(b)). This wildly improbable phenomenon occurs at slightly higher agitation rates than those at which lozenges are found. Cubes are usually identical in size within a flask and can be between 3 and 8 mm across. The flatness of sides was obviously maintained by cubes gently rubbing against each other and the bottom of the flask, but the perfect symmetry of these cubical flocs suggested that there might perhaps be a “crystallization” of flocculated yeast cells into ordered arrays within flocs. An ordered structure, e.g. cubic close packing, would tend to be reflected in the large-scale floc morphology. Cubical yeast flocs have previously been reported by Ault and Newton (1968). At higher agitation rates, spherical flocs tend to be formed which roll rather than slide over the flask bottom (Fig. 12(c)). At very high agitation rates, flocs are kept in suspension in the liquid by the force of agitation and are found to become progressively smaller (less than 1 mm in diameter; and generally flattened, spherical o r disc-like in shape 1 ~ 1 0 , 0 0 cells) 0 (Fig. 12(d)). The effect of gravity, pushing flocs against flat flask bottoms, would seem to play a major part in formation of some of these bizarre floc shapes. This was confirmed by examination of flocs formed in caesium chloride solutions. High concentrations of this salt, insufficient to inhibit flocculation, increased media density to the extent that flocs were neutrally buoyant and did not sink after formation. In such solutions, at low agitation rates, large generally spherical flocs were formed, which were more irregular in size and shape than those formed at the bottom of flasks.

34

M. STRATFORD

FIG. 12. Morphology of yeast flocs resulting from different degrees of agitation. At low rates of agitation (80 r.p.m., (a)) large lozenge-shaped flocs are formed with pronounced raised rims. As agitation increases, cubical flocs (100 r.p.m., (b)) give way to spheres (120 r.p.m., (c)), finally forming small spheroidal particles at high rates of agitation (150 r.p.m., (d)). Flocs are all of Saccharomyces cerevisiae NCYC 1195.

YEAST FLOCCULATION: A NEW PERSPECTIVE

35

36

M. STRATFORD

G. FLOCCULATION WITHOUT AGITATION

Flocculation has been observed in many situations, noticeably breweries, where the medium is not agitated and appears relatively still. However, considerable local agitation is generated by passage of bubbles of carbon dioxide through the medium, generated by the fermentation. The word fermentation itself describes excitement, agitation and tumult, and suggests considerable natural agitation in unstirred vessels. Perhaps flocculation in these instances should be described as occurring without artificial agitation. In the complete absence of any form of agitation, flocculation does not occur (Stratford and Keenan, 1987). Instead, flocculent cells sediment very slowly as single cells, and eventually build up as a deposit on the bottom of the vessel. Non-flocculent powdery yeasts form soupy unconsolidated deposits (Windisch, 1968) but flocculent yeasts anneal together to form consolidated masses, in other words flocs. While, without agitation, flocs are not formed in the body of the liquid, gravity enables cells to be pressed together and to overcome mutual repulsion. This may be either the effect of yeast falling at 5 mm h-' impacting on other cells at the bottom, or an effect of the pressure of accumulation of a large depth of yeast cells. While of academic interest, floc formation by gravity is of little practical use since the rate of clearing of cells from media is no faster than with non-flocculent yeasts.

H.

FLOC COMPRESSION BY AGITATION AND GRAVITY

Unlike the morphology of flocs formed by agitation to equilibrium, flocs found in breweries are much lighter in structure, and have a feathery appearance. The word flocculate itself, derived from the Latin JEoccus, meaning a tuft of wool, implies an open structure (Stewart and Russell, 1986, 1987), very different from the dense floc pellets formed after hours of agitation. Loose, fluffy flocs, similar to those found in brewery fermentations, are formed after very limited periods of agitation. This suggests that, in the brewery context, agitation may be limiting and that a greater extent of flocculation and floc compaction would result from further agitation. The process of floc compaction is called syneresis, and describes not only the progressive stages in formation of denser flocs but also the consequential mass exudation of liquid from flocs (Stevenson, 1972; Yusa, 1977). This process is commonly found after flocculation of inorganic hydroxides or clays, and is influenced yet again by agitation and bond strength. In the first few moments of agitated flocculation, cells, by impacting rapidly into one another, form extremely loose, nebulous, interlinked and branched structures. Cells in these structures are linked to relatively few other cells, with

YEAST FLOCCULATION: A NEW PERSPECTIVE

37

the result that these structures are energetically unstable (Calleja et al., 1984). Rolling movements (Fig. 13) of cells around one another, while remaining firmly attached, result in cell A coming into direct contact with cell C which had previously only been indirectly linked by cell B. The overall effect is one of greatly increasing numbers of cell contacts and of the stability of the structure. Such movements will, of course, shorten chains of cells and cause floc compression.

FIG. 13. Diagram showing rolling movements of flocculent cells after initial contact, leading to floc compaction and consequent exudation of liquid, known as syneresis. Strains forming very strong bonds between cells tend to resist syneresis and remain as open structures.

High bond strength will generally resist attempts to roll cells around one another into contact with other cells, and will require more time or energy input. Since yeast strains with low bond strength will be easily compacted and compressed, these low bond-strength strains will tend rapidly to form dense structures and fall out of suspension. This means that yeast strains forming thin diffusive flocs, often described as “weak”, contrary to expectation, may possess exceptionally strong flocculation bonds, able to resist pressures to crush and condense their diaphanous structures. The energy for floc compression, as before, may come from agitation or from gravity. Immediately after formation, flocs occupy a large volume but, with progressive agitation with time, these flocs are continuously compressed until a limit is reached where cells are close packed (Stratford,

38

M . STRATFORD

1989a, and unpublished observation). Standing under gravity similarly compresses flocs. Strains with a high bond strength require considerably greater agitation to promote compression and may never be fully compressed by gravity. Centrifugation may be required for full compaction. It is possible that some instances of mutual flocculation (coflocculation) between pairs of strains may be explained by these effects. Instances where one of the mutually flocculating strains is described as weak diffusively flocculent may in fact be an uncompressed very strongly flocculent strain (Fig. 14). Cells of another, non-flocculent strain could interpenetrate the diffusive structure, filling the gaps left by the strongly flocculent strain. In this way, the combination of two strains would result in compact flocs, easily discerned as sedimenting. It is not suggested here that the behaviour of all mutually and coflocculent strains is attributable to mixing high and low bond-strength strains, but only that some cases might have been misidentified, especially if one strain was diffusively flocculent alone.

.. + .. 0 . 0 0

O . 0

Strongly flocculent strain forming loose, open structures

Non-llocculentstrain with receptors

Mutual flocculation with a close-packed structure

FIG. 14. Possible mechanism for mutual flocculation between a diffusively flocculent and a non-flocculent strain. Strongly flocculent strains form loose openstructured flocs which sediment slowly. Non-flocculent cells, with flocculation receptors, are able to interpenetrate these, binding to flocculent cells and thus filling the structure. These close-packed structures are able to sediment rapidly. I . CASCADE THEORY AND FRACTAL FLOCCULATION

This theory concerns the pattern by which single yeast cells accumulate into flocs of millions and therefore largely involves the first few moments of flocculation. This is in effect the flocculation “Big Bang”, but the ramifications of cascade theory extend right through yeast flocculation and into other forms of particle interaction. Our normal concept of flocculation is one of progressive accretion, in which numbers of small flocs are continuously formed and progressively build up in size by single-cell adhesion. Large flocs become larger targets for contact by single cells, and can be regarded as sweeping through the medium, gathering all single cells from their paths. This gathering was

YEAST FLOCCULATION: A NEW PERSPECTIVE

39

implied by Calleja (1987) in his definition of aggregation, as a process of addition. However, this view of flocculation leaves unexplained several pieces of evidence concerning the gathering of cells: (a) The frequency of floc particle sizes is bimodal in distribution (Davis and Hunt, 1986; M. Stratford, unpublished observation). In other words, there are lots of big flocs and single cells but very few mediumsized or small flocs. (b) The rate of flocculation increases in proportion to the square of the cell concentration (Stratford and Keenan, 1987). Flocculation thus behaves as a perfect second-order chemical reaction (Barrow, 1973). This means that the rate-limiting step in flocculation is the meeting of two particles, whereas flocs contain millions of particles. (c) Examination of the floc size-density relationship for single strains showed that, as floc size increased, floc density diminished. Larger flocs have an increased proportion of empty space within them (Brohan and McLoughlin, 1984; Davis and Hunt, 1986), and are therefore more loosely packed. This effect, also found in inorganic aluminium flocculation (Tambo and Watanabe, 1979), is hard to reconcile with accretion by cell addition. (d) Visual examination of single cells and small flocs falling under gravity showed that large fast-moving flocs did not collide with single cells in their path but swept them aside in mass flows of liquid ahead of the particles. This is analogous to the bow-wave effect ahead of moving ships. The only collisions observed, and subsequent adhesion, were between floc particles of approximately similar size (Fig. 15). In the light of this evidence, a new model of flocculation is proposed, namely a cascade theory where particles only succeed in colliding with particles of similar size. Single cells combine to form doublets which combine to form groups of four, and on to eight, 16,32,64, etc. (Fig. 16). All other collisions between unlike-sized particles are relatively rare due to the observed bow-wave effect, sweeping smaller particles aside. Flocs then rapidly build up in size by combining with similar-sized flocs until they reach the maximum size limit imposed by shear forces of agitation. The rate-limiting step in flocculation is the combination of single cells to form doublets. This explains point (b), namely that flocculation behaves as a second-order reaction where rate is limited by collisions between pairs of particles. Subsequent collisions between pairs of increasingly larger particles are energetically easier and are therefore not rate-limiting. The explanation for this is that repulsive forces, such as surface charge, which form the activation energy, remain constant. Particle or floc mass is doubled at each stage, and thus the energy of collision (imv’) is also progressively

40

M. STRATFORD

0

0 0

0

0 0 0

0

0 0

0

0

0

0

0

0

0

FIG. 15. Diagram showing observed sedimentation of small flocs and single cells under gravity. Rapidly moving flocs do not contact any of the single cells, but sweep them aside in the flow of liquid around the moving particle. Impacts are thus prevented by the "bow waves" of larger particles.

FIG. 16. Diagram illustrating the cascade theory of floc formation. It is proposed that flocs are built by the meeting of similar-sized particles. This schematic diagram shows progressive doublings of floc size, leading to the building of loose, open agglomerates where the structures of component clusters are maintained. Eventual floc structure is one of clusters of clusters.

YEAST FLOCCULATION: A NEW PERSPECTIVE

41

increased. This means that, as particle mass increases, repulsive forces become increasingly easily overwhelmed by force of collisions. This has been experimentally demonstrated (M. Stratford and P. Wilson, unpublished observation) using a strain that was both chain-forming and flocculent. Single batches of cells were harvested and divided into two aliquots. One was gently dispersed using EDTA to give chain structures of 20-30 cells; the other was mechanically disrupted using EDTA to give single cells. The minimum agitation threshold was then determined for each suspension. Chains of 20-30 cells required very little agitation to flocculate compared with single cells, and were described as superflocculent. The larger particle mass obviously caused sufficiently energetic collisions to overcome repulsion, even at very low agitation. This, perhaps, might explain why so many brewery flocculent strains are also found to be chain formers (Stratford and Assinder, 1991) since flocculation could still then occur under the mild agitation of brewery conditions. The bimodal distribution of floc-particle sizes can also be explained by this effect. Under agitation, a number of small .flocs are initiated which immediately double and redouble into large flocs. This will leave a small population of single cells which will initiate new flocs only slowly since the cell concentration is now low. The overall population will then consist of single cells and large flocs, with small flocs being found rarely since the transition time to redouble into large flocs is brief. As a consequence of rapid formation of large flocs, little time for relocation and floc compression elapses, and cells remain within their small groupings within the large floc structure. These then become clusters of clusters and superclusters of superclusters, very much on the lines of current theories of galactic structures. At each level of clustering, empty spaces are left in the structure on a similar scale to cluster size, such that large flocs built of large clusters incorporate very large spaces. The overall effect is that flocs become progressively less dense and incorporate more empty space as size increases (Brohan and McLoughlin, 1984; Davis and Hunt, 1986). A floc, built up from clusters of clusters, is a form of fractal structure (Fig. 17). Fractals, a recent development in the physical sciences, may be defined as structures showing self-symmetry, repeated at different enlargements and scales. Thus, fractal objects look the same, whether viewed from nearby or far away (Mandelbrot, 1990). Snowflakes are a common example of fractals, where the crystalline patterns are repeated on an ever smaller scale within the flake. Here, in flocculation (Fig. 16), we have a form of fractal structure, repeating cluster formations within clusters. The fractal dimension measured for yeast flocs by examination of plots of the floc size-density relationship (Davis and Hunt, 1986) was found to be 1.79. This figure confirms the cluster and supercluster nature of floc structure (Weitz et al., 1984; Meakin et al.,

42

M. STRATFORD

FIG. 17. A fractal triangle formed from clusters of clusters. This demonstrates iteration and reiteration of pattern at different spacial scales within the structure, a universal feature of fractals, and also an increasing proportion of void space with increased scale. This results in a decrease of fractal-particle density with increasing size. Yeast flocs are observed to decrease in density with increased size in a manner consistent with a clusterkluster fractal structure.

1985) and also indicates that the structure was formed by a diffusion-limited process (Schaefer, 1989). These fractal floc structures are, as previously mentioned, transitory phenomena. Cell relocation and floc compaction, powered by agitation-induced collisions, eventually result in close-packed floc pellets. At this later stage, floc density is very much higher and independent of floc size. To conclude this section, mechanical agitation is fundamentally involved at all stages of the flocculation process. Agitation causes cells to collide with sufficient energy to overcome repulsion and adhere together. Small flocs thus formed rapidly combine with similar-sized particles in a cascade reaction, eventually forming floc fractal structures comprised of clusters of clusters. Further agitation causes floc compaction and compression, eventually resulting in close packing. After prolonged agitation, a dynamic equilibrium is set up between the single-cell and flocculated fractions, with the number of cells lost from flocs being balanced by floc formation by single cells.

YEAST FLOCCULATION: A NEW PERSPECTIVE

43

VI. Mechanism of Flocculation A . THE YEAST CELL WALL

Flocculation, once developed, is an intrinsic property of the cell wall. Isolated walls retain the ability to flocculate (Masschelein and Devreux, 1957; Eddy and Rudin, 1958c) in a calcium ion-dependent manner (Mill, 1966). The cell wall of Sacch. cerevisiae is a complex carbohydrate structure, some 100-200 nm thick, that surrounds the periplasmic space and plasma membrane. It is composed largely of glucans and mannan, together with smaller proportions of chitin and protein (Griffin and MacWilliam, 1969). Exact composition and proportions are found to vary with yeast strain and culture conditions (McMurrough and Rose, 1967; Griffin and MacWilliam, 1969). P-Glucan constitutes an inner skeletal-structural layer that is arranged in a network of crystalline fibrils (Kopecka et al., 1974). This may be exposed after the outer amorphous layer is removed by treatment with cell wall-degrading enzymes (Kopecka et al., 1974). It is formed of glucose residues in P-(1+3)-linked linear chains with occasional P-(1+6) side branches (Duffus et al., 1982). Chitin is composed of P-(1-+4)-linked N-acetylglucosamine residues in linear chains. It may be a very minor component of cell walls (Tronchin et al., 1981) but is normally found in primary septa and bud scars (Cabib and Bowers, 1971). Wall mannan and protein are now considered to coexist as mannoproteins, since together they are inextricably linked in formation, secretion and also physically in covalent complexes. Secreted proteins (FarkaS, 1989), including wall enzymes (Lampen, 1968), are all glycosylated, and it would appear that glycosylation is a consequence of secretion (Eylar, 1965). Secreted proteins are gradually released from the periplasmic space (Pastor et al., 1982) or fixed into the wall structure (Zlotnik et al., 1984; Margui et al., 1985). It is thus possible to regard the mannan outer layer of the cell wall as a coating of secreted mannoproteins that is fixed onto a P-glucan framework. While the P-glucan and chitin structural elements are synthesized at the plasma membrane (FarkaS, 1989), mannan is laid down onto protein precursors deep within the cell, during the endoplasmic-reticulum and Golgi stages of secretion (Linnemans et al., 1977; Esmon et al., 1981; Haselbeck and Tanner, 1983). Ballou and Raschke (1974) recognized two classes of wall mannoproteins, namely enzymes containing 50-70% mannan and structural mannoproteins containing 90% (Frevert and Ballou, 1985). The structure of the mannan moiety of wall glycoproteins has been examined in detail (Ballou, 1976; Cohen et al., 1982; Fleet, 1990). An inner core of 11mannose residues is attached via an N-acetylglucosamine residue

44

M. STRATFORD

to asparagine residues in the protein moiety (Cohen et al., 1982). To the inner core is linked an outer chain of up to 150 mannose residues. The basic mannan structure consists of an a-(1+6)-linked backbone to which are attached short side-chains by a-( 1-2) linkage (outer chain), or a-( 1-2) and a-( 1+3) linkage (inner core). Side-chains are one to three residues in length and are linked by a-(1-2)- and a-(1-3)-glycosidic bonds. Phosphate has been shown to be present in yeast mannan in low proportions (Northcote and Horne, 1952; Lindquist, 1953; Eddy, 1958b) but it has a major influence over yeast-surface charge (Eddy and Rudin, 1958a,b,c; Amory et al., 1988). Phosphate was found to be attached to C-6of certain mannose residues (Mill, 1966) and was later shown by Ballou (1976) to form (1-6) linkages for attachment of certain side-chains. Mutations of mnn4 interfered with this linkage (Ballou and Raschke, 1974). Wide variations in mannan phosphate content have been found in different strains (Griffin and MacWilliam, 1969; Lyons and Hough, 1970b). Apart from inner-core and outer-chain mannan, there exist short manno-oligosaccharide chains that are directly attached to serine and threonine residues present in proteins (FarkaS, 1989), but which represent a minor proportion of wall mannan. This brief resume is intended to review components of the cell wall that could possibly participate in flocculent bond formation. These, largely chemical, studies convey little as to the three-dimensional structure of the wall. We may envisage an inner glucan fibrillar layer covered with more amorphous mannoproteins, but the fine architecture (Rose, 1984) of this layer must allow for complex functions, such as access of substrates to wall enzymes, passage of very large molecules through the wall, e.g. proteins and nucleic acids, both into and out of cells, or signal transfer caused by binding of large molecules, e.g. killer toxins or mating factors, to the surface. Clearly, the yeast cell wall is a physiologically complex organelle and cannot be regarded as a mere shell of carbohydrate and protein. More detailed reviews of wall structure and carbohydrate metabolism are by FarkaS (1989) and Fleet (1991). B . THE CALCIUM-BRIDGING HYPOTHESIS

The calcium-bridging hypothesis is based on observations of specific calcium requirement for flocculation. This theory was formally proposed by Mill (1964b), and extended earlier speculations by Harris (1959). It was suggested that bonds between flocculent cells consisted of salt bridges formed by calcium atoms, its two valencies enabling formation of bonds between combining sites on different cells (Fig. 18(a)). Cell-surface groups capable of combining with calcium ions at pH 4.6 were considered to be

YEAST FLOCCULATION: A NEW PERSPECTIVE

45

00 p04@& - - -

04p

LBCtin proteins

Sugar residua

FIG. 18. Diagram showing suggested mechanisms of yeast flocculation. (a) Divalent calcium ions form bridges between surface carboxyl groups on different cells. These are supported by hydrogen bonding between mannan hydroxyl groups and hydrogen moieties. (b) Phosphate groups of wall mannan form the attachment groups for calcium bridging. (c) Surface proteins with lectin properties specifically bind to sugar residues in the wall mannan of neighbouring cells. Calcium ions are required to maintain the lectins in active conformations.

carboxyl, phosphate and sulphate groups. The observed effect of pH value on flocculation suggested carboxyl groups as the most likely combining sites (Mill, 1964b; Fig. 18(a)). Since such salt bridges would be largely dissociated at pH 4.6, it was proposed that they would be stabilized by hydrogen bonding between complementary carbohydrate hydrogen atoms and hydroxyl groups in the cell surface. The floc-dissociation temperature

46

M . STRATFORD

(5040°C) was regarded as consistent with a structure maintained by hydrogen bonding. The involvement of carboxyl groups is supported by the observed irreversible inhibition of flocculation by 1,2-epoxypropane (Mill , 1964b; Jayatissa and Rose, 1976), which esterifies carboxyl groups. In addition, onset of flocculation in the stationary phase of growth was shown to be accompanied by an increase in density of wall carboxyl groups (Beavan et af., 1979). Carboxyl groups are probably associated with protein components of wall mannoproteins and this theory is therefore consistent with the observed loss of flocculation caused by proteases and proteindenaturing agents (Eddy and Rudin, 1958a; Stewart et af., 1973; Nishihara et a f . , 1977, 1982). An alternative combining site for calcium bridging, suggested by Lyons and Hough (1970a,b, 1971), are phosphodiester groups present in yeast cellwall phosphomannan (Fig. 18(b)). The evidence for this was essentially a correlation of increased wall phosphate content with flocculation observed despite considerable variation between yeast strains. Griffin and MacWilliam (1969) failed to find any such correlation after examining 10 strains. Flocculation of K f . bufgaricus was similarly correlated with an increased content of wall phosphate by Al-Mahmood et af. (1987) but not by Teixeira et af. (1989). Evidence against phosphodiester group involvement comes from electrophoretic studies (Jayatissa and Rose, 1976; Beavan et af., 1979). These demonstrated a lack of correlation of wall phosphate content with flocculation onset. Furthermore, Amory et af. (1988) showed a decline in phosphate content at this time. Hydrofluoric acid treatment of flocculent yeast (Jayatissa and Rose, 1976), removing phosphodiester groups, did not decrease flocculation. Binding of Alcian blue, which stains compounds containing phosphodiester linkages, did not correlate with flocculation (Stewart et af., 1976). Recent evidence supporting involvement of phosphodiester groups (Kihn et af., 1988a) has been questioned by Stratford and Assinder (1991), since the observed inhibition of flocculation by mannose 6-phosphate under extremely acidic conditions could not be repeated in buffer at pH 4.0. There is little direct evidence against the calcium-bridging hypothesis. However, the proposed role for divalent calcium-ion bridging is a difficult concept from a chemical point of view. Calcium metal may fairly be regarded as divalent and could form bonds with two structures. Calcium ions, however, are no longer divalent since they have already reacted, losing electrons to various anions. The calcium ion in solution has the electron configuration of a noble gas, namely argon; moreover, it has a net positive charge held by the nucleus and is therefore as unlikely to bridge between two combining sites as the singly charged sodium ion. Calcium ions are thus usually described as dipositive rather than divalent. Another criticism of the

YEAST FLOCCULATION: A NEW PERSPECTIVE

47

calcium-bridging hypothesis concerns the lack of any explanation for inhibition of flocculation by sugars. This omission eventually led to a new theory in which sugar inhibitions figure prominently and calcium ions assume a lesser role. C. THE LECTIN HYPOTHESIS

The lectin, o r “lectin-like” hypothesis proposes that specific surface proteins of flocculent cells bind mannose residues intrinsic to mannan in other yeast cell walls, thus forming bonds between the cells (Fig. 18 (c)). The proposed function of calcium ions is that of maintaining the surface proteins, lectins, in an active conformation. Protein-to-carbohydrate bonding was first suggested by Taylor and Orton (1978) as a result of specific inhibition of flocculation by mannose. This was later fully expounded in the “lectinlike” hypothesis (Miki et al., 1981, 1982a,b). In many respects, the “lectinlike” hypothesis is a logical extension of the bridging hypothesis. Both recognize the importance of wall proteins and mannan carbohydrates and agree that flocculation ultimately occurs by hydrogen bonding. The major difference concerns calcium ions, from a leading role in calcium bridging to one of support for lectin proteins. The evidence for the lectin-like hypothesis can be summarized as follows: (a) Two parts to the flocculation bond were recognized by Eddy and Rudin (1958a), namely adhesin and receptor, in view of mutual flocculation by pairs of strains. This can be regarded as each of these strains possessing either receptors or adhesins, not both. (b) Adhesins, found only on flocculent cells, are sensitive to proteases and protein denaturing agents and are therefore probably surface proteins (Miki etal., 1982a). Antibodies to proteins on flocculent cells have been raised by Nagarajan and Umesh-Kumar (1990). (c) The observed calcium-ion specificity and EDTA dispersal of flocculation (Taylor and Orton, 1975; Stratford, 1989c) is consistent with lectin behaviour. Early studies (Sharon and Lis, 1972) showed that all lectins then examined contained calcium and manganese. This has since been modified with classification of calcium-dependent (C-type) and thiol-dependent (S-type) lectins in animals (Drickamer, 1988). X-Ray studies have shown calcium and manganese ions located within lectin structures, close to saccharide-binding sites (Blake, 1975; Einspahr et al., 1988), and necessary for correct lectin conformation. (d) Receptors, found on flocculent and non-flocculent cells, are not proteins (Miki, 1982a) but are sensitive to blocking with concanavalin

48

M . STRATFORD

A or periodate attack, which indicates the presence of residues of a-gluco- or mannopyranose carbohydrate (Miki et al., 1982a; Nishihara and Toraya, 1987). Involvement of wall a-mannan is also suggested by specific sugar inhibition by mannose (Taylor and Orton, 1978) and the lack of coflocculation with Schiz. pombe (Miki et al., 1982a), a yeast known to lack mannan in its cell wall (Tkacz et al., 1971; Barkai-Golan and Sharon, 1978). Lectins are proteins, frequently glycoproteins, that specifically bind sugars (Boyd and Shapleigh, 1954). They have in the past been defined as having multiple sugar-binding sites (Goldstein et al., 1980; Barondes, 1981) and, in consequence, were able to agglutinate plant or animal cells bearing suitable sugar receptors. This definition excludes many proteins, including toxins, with single binding sites. Flocculation was therefore described as “lectin-like” in view of uncertainty as to binding-site numbers. However, structural homology between lectins and one binding-site proteins has led to lectin redefinition as “carbohydrate binding proteins other than enzymes or antibodies” (Barondes, 1988). This definition obviously encompasses the known description of flocculation surface proteins. While flocculation probably involves surEace lectins, there are clearly matters still to be resolved. One such matter is the observed increase in wall carboxyl groups at the onset of flocculation (Beavan et al., 1979). This may well indicate formation of a carboxyl-rich surface lectin. Carboxyl groups have been implicated in the binding of galactose to yeast lectins (Basu et al., 1986; Kundu et al., 1987). This matter will only be resolved by isolation and characterization of surface proteins. There are several reports that indicate that intact, or at least active, floc proteins may be released from cell walls, either by Zymolyase treatment and osmotic shock (Williams and Wiseman, 1973b) or by trypsin treatment (Kamada and Murata, 1984a,b). Attempts have been made to determine which surface protein correlated with active flocculation, either by using isogenic strain pairs differing only in flocculation (Holmberg, 1978) or by examining a single strain under conditions of non-flocculence and flocculation expression (Stewart et al., 1976; Miki et al., 1982b). Proteins with a molecular weight of 13,000 (Holmberg, 1978) or 37,000 (Stewart et al., 1976) and appearance of no new protein (Miki et af., 1982b) were found to correlate with flocculation. This discrepancy may reflect strain variation or perhaps different degrees of glycosylation remaining after protein extraction. D. TWO LECTIN MECHANISMS:

FlOl

AND

NewFlo

PHENOTYPES

Careful examination of the physiology of a large number of flocculent strains, both laboratory and brewery, showed that there were two distinct

YEAST FLOCCULATION: A NEW PERSPECTIVE

49

groupings of yeast strains (Stratford, 1989a; Stratford and Assinder, 1991). One group was termed the Flol phenotype since it contained all strains bearing Flol genes, and showed constitutive flocculence, flocculating in all media examined at all stages of growth. The other group, the NewFlo phenotype, was predominant in brewery ale strains and flocculated in the stationary phase of growth. These NewFlo strains flocculated only in undefined wort or yeast extract-containing media. NewFlo strains appeared very similar to a group of ale strains requiring an inducer from wort/yeast extract-peptone-gelatin in order to flocculate (Stewart, 1972; Stewart and Garrison, 1972; Stewart et al., 1975a, 1976). Flol and NewFlo phenotypes were further distinguished (Stratford and Assinder, 1991) by being inhibited by high concentrations of salt and low pH values and by selective protease sensitivity. These could be regarded as further examples of strain variability in flocculation but correlated exactly with specific differences in sugar inhibitions. Lectins are customarily defined by sugar inhibition and thus, by inference, by the specific sugar residues to which they bind. Flol phenotype-strain lectins were mannose-specific whereas NewFlo phenotype lectins were less specific, binding gluco- and mannopyranoses. The sugar specificities were clearly defined, and not a matter of degree of inhibition. Sugar specificity of lectins is similar to that described for enzymes and substrates. There appears to be structural homology between unrelated sugar-binding proteins (Quiocho, 1986). Sugar-binding sites are located in deep clefts between globular protein domains. Detailed sugar specificity relies on hydrogen bonding to all of the individual sugar hydroxyl groups which must be present and at the correct orientation for sugar recognition and bonding (Quiocho and Vyas, 1984; Quiocho, 1986). Any alterations to the sugar structure by removal of hydroxyl groups, reorientation or substitution results in non-recognition of structure. In many instances, substrate specificity of lectins is found to be comparable with or exceed that of antibodies (Kabat, 1978; Doyle and Slifkin, 1989). It is therefore probable that the different sugar specificities of Flol and NewFlo phenotypes indicate two distinct lectin-like mechanisms of yeast flocculation. E. RECEPTORS OF FLOCCULATION

The generalized identification of wall a-mannan as the flocculation receptor has come about through the specific inhibition of flocculation by mannose (Taylor and Orton, 1978) and later by mannan blocking and chemical modification (Miki et al., 1982a; Nishihara and Toraya, 1987). However, a-mannan has a complex structure (Ballou, 1976), many parts of which are potential flocculation receptors. In 1920, Landsteiner published a technique

50

M. STRATFORD

YEAST FLOCCULATION: A NEW PERSPECTIVE

51

for identification of antibody receptors. This involves examination of inhibition, and the degree of inhibition of binding by simple molecules, enabling a picture of the ideal receptor to be established. It assumes that most potent inhibitors will most closely mimic the true receptor. Using this method, Stratford and Assinder (1991) concluded that flocculation lectins, both Flol and NewFlo, bound preferentially to the non-reducing termini of a-(1-+3)-linkedmannan side branches, two or three mannopyranose residues in length (Fig. 19). Since mannan-containing cell walls are generally reckoned to be universal in strains of Saccharomyces spp. , it could be assumed that all cells, flocculent and non-flocculent, possess receptors for flocculation. However, this is not so. Mutual flocculation (Eddy, 1958a; Rainbow, 1966) between pairs of strains shows that some flocculent strains lack the necessary receptors. While most non-flocculent yeasts can be adhered to by flocculent cells (coflocculation-Miki et al. , 1982a), some non-flocculent strains are unable to do so (Nishihara and Toraya, 1987). Strains may also vary as to the extent to which an external lectin, such as concanavalin.A, is bound (Evans et al., 1980). Figure 20 demonstrates the range of extent to which non-flocculent strains may be coflocculated by an identical flocculent culture. This suggests that, while the gross chemical composition of mannans is similar, there are subtle differences in mannan structure between strains. Ballou (1976) suggested that mannans of individual yeast strains differed in the arrangements and lengths of side branches. Since these same side branches are probable flocculation receptors, it would appear that flocculation reception depends on the availability or particular arrangement of these side branches. Chemical modification of mannan can expose hidden flocculation receptors (Nishihara and Toraya, 1987), demonstrating that not only do cells have to possess suitable mannan side branches but that the fine architecture of their arrangement is crucial for flocculation reception. F. SURFACE LECTINS OF OTHER YEASTS AND BACTERIA

Surface lectins have been implicated in flocculation of KI. marxianus (Bajpai and Margaritis, 1986; Teixeira et al., 1989) and Kl. bulgaricus (Hussain et al., 1986). Flocculation was calcium ion-dependent and was mediated by galactose- and fucose-specific lectins that were also found released into the media (Al-Mahmood et al., 1987, 1988; Giumelly et al., 1989). Conceptual FIG. 19. Molecular model of a portion of the outer-chain mannan of Saccharomyces cerevisiae based on the work of Ballou (1976). Side branches, one to three rnannose residues in length, are attached to a vertically oriented backbone of (1-+6)-linked residues. Side branches are (1+2) and ( 1 h 3 ) linked.

52

:

M. STRATFORD

Percent cells coflocculated

40

20

0

Yeast Strain

0

>

Y

,-

r

.

.

.

Y0

Y0

0

0

0

0

0

0

z

z

>

z

>

z

>

z

FIG. 20. Histogram showing variations in coflocculation of different non-flocculent yeasts with the flocculent Saccharornyces cerevisiae NCYC 1195. Cells washed with EDTA were mixed in a 1:l ratio at 1 x lo8 cells ml-', prior to addition of calcium ions. After agitation to equilibrium the proportion of non-flocculent cells coflocculated was calculated.

difficulties arise from the fact that this yeast does not contain galactose or fucose residues within the cell wall. Explanations for this could involve low levels of wall receptors or use of galactose and/or fucose-containing bridging agents. The adhesion of Cundida spp. to host cells, thus determining virulence, also appears to occur using surface lectins (Douglas, 1985; Critchley and Douglas, 1987a,b). In this case, surface adhesins are directed not to yeast cell-wall receptors but to receptors of host cells. There appear to be several lectin mechanisms of adhesion, inhibited by fucose, mannose or N-acetylglucosamine. Lectin use by infecting organisms could be seen to have two roles, firstly in recognizing suitable target cells by their specific

YEAST FLOCCULATION: A NEW PERSPECTIVE

53

receptors, and secondly by bonding to the target, thereby preventing infecting cells being swept away. Many bacterial infections are now recognized to be mediated by bacterial surface lectins (Read, 1989). Bacterial adhesion to host cells and subsequent infection may be inhibited by suitable sugars, demonstrated with mannose and derivatives on infections caused by E. coli (Aronson et al., 1979; Firon et al., 1982; Sharon et al., 1983). A majority of bacteria associated with infections were found to possess mannose-specific surface lectins (Sharon et al., 1981). Infection by viruses is similarly aided in host recognition and adhesion by surface lectins (Markwell et al., 1981; Markwell, 1986; Weis et al., 1988). Lectins of infectious micro-organisms are frequently associated with surface spike structures. These may be termed pili or fimbriae; especially well known are the type 1 fimbriae of E. coli, which are associated with mannose-specific lectins (Pearce and Buchanan, 1980; Eshdat et al., 1981; Firon et al., 1982). Viral lectins are similarly associated with spikes (Markwell, 1986). These structures are believed to aid infection by allowing easier cell-cell contact despite repulsive forces (Heckels et al., 1976; Ofek and Beachey, 1980;Stratford and Wilson, 1990). It is interesting to note that similar fimbriae have been associated with flocculation activity in Sacch. cerevisiae (Day et al., 1975).

VII. Onset of Flocculation

Flocculation is regarded by many as a series of complex phenomena influenced by many and varied factors differing according to yeast strain. These complex phenomena can be resolved into a series of interacting processes. Each is simple in itself, but exerts control over the whole. Layers of control are thus formed, which in combination present an impenetrable array of facts and effectors. Here, the whole flocculation process is resolved into just such a series (Fig. 21): (a) Flocculation involves yeast surface proteins. Therefore, there are structural genes coding for these proteins within cells. The presence of such genes is essential for flocculation. (b) Genes must be transcribed and translated into protein. Flocculation may be effected by regulatory genes acting on the structural gene since flocculation of brewing yeasts is normally only expressed in the stationary growth phase. Regulation may be by repression or activation. (c) The structural gene product needs to be secreted. The yeast secretory

54

M. STRATFORD

pathway has been studied in detail for invertase (Esmon et al., 1981; Novick et al., 198l), which is found in the cell wall and periplasmic space. Cell-wall external enzymes are heavily glycosylated (Lampen, 1968), containing up to 50% mannan (Rodriguez et al., 1980). The glycosylation step is blocked by tunicamycin (Gallili and Lampen, 1977). Flocculation development is blocked by tunicamycin and cycloheximide (Baker and Kirsop, 1972; M. Stratford, unpublished results), therefore the structural protein is probably glycosylated. This may occur as proteins are inserted from ribosomes into the endoplasmic reticulum as with invertase (Novick et al., 1981). “Chaperones” may possibly be used to ensure correct folding (Ellis and Hemmingsen, 1989). Proteins are then transferred to the Golgi complex, packaged into vesicles and secreted. Novick et al. (1980) demonstrated that this process involves at least 23 complementation groups and metabolic energy. There is obviously scope for flocculation to be influenced by factors acting on any of the genes involved in secretion. (d) Following secretion through the plasmalemma into the periplasmic space, the protein must pass into the cell wall and be fixed in place. It has been demonstrated that invertase turnover from the periplasmic space is rapid (Pastor et al., 1982) but the enzyme fixed into the cell wall is stable with a slow turnover. (e) Flocculation proteins may require activation which could involve proteolytic cleavage, addition of cofactors, removal of inhibitors, or alteration of wall structure and architecture to allow physical access to the protein. (f) Finally, for flocculation to be expressed, receptor groups must be available on other yeast walls to allow bonding by the flocculation protein. Formation of receptors probably involves a whole series of stages which for simplicity are condensed into this one. Flocculation requires the presence of surface proteins and mannan receptors. If these are not available, or masked, blocked or generally inhibited or denatured, flocculation cannot occur. Onset of flocculation is an aspect of the subject in which there is greatest commercial interest but about which relatively little is known. The ideal brewing yeast remains in suspension as fermenting single cells until the end of fermentation (Gilliland, 1951; Geilenkotten and Nyns, 1971) when sugars in media are depleted, and only then flocculates out rapidly. What signals the onset of flocculation, and does it affect adhesidreceptor synthesis, exposure, activation or relief from inhibition?

YEAST FLOCCULATION: A NEW PERSPECTIVE

COfaCtOrS

5. Activation by

55

6. Surface receptors

Proteolytkdeavage Physlcal exposure

4. Incorporation into cell wall

I

3. Secretion and glycosylation

I

(2. Transcription and translation

I

I

I . Lectin structural gene

I

FIG. 21. Flow diagram of the possible stages in processing, secretion and activation of flocculation lectins following synthesis. This indicates the various points at which flocculation may be controlled o r inhibited, by internal or external effectors.

A . CONTROL

BY INHIBITION

Flocculation is inhibited by several fermentable sugars, including maltose, mannose, sucrose and glucose (Eddy, 1955a, Mill, 1964b). Maltose in particular is present in high concentration in wort (Stewart and Russell, 1979). Given high maltose concentrations, potent inhibition of flocculation by maltose and ideal coincidence of flocculation onset with sugar depletion, it is probable that flocculation is developed early by brewing yeasts. It is then inhibited by maltose until sugar depletion by metabolism enables cells to flocculate (Baker and Kirsop, 1972). This can be demonstrated under laboratory conditions as shown in Fig. 22. Here flocculation was detected by removing portions of culture and allowing flocculation to take place in buffer. While flocculation in the original medium was not visible until 32 hours, cells were competent to flocculate from 10 hours. Clearly, flocculation onset is under at least two levels of control; these are signals governing production of the flocculation mechanism, and relief from inhibition by medium components.

56

M. STRATFORD

- 100 - 80 -60

F-I

0

.u

- 40 c

- 20

s

0)

a a0 0

8

24

16

32

40

Time (h)

FIG. 22. Time-course of growth (0) of Saccharomyces cerevisiae NCYC 1224 in yeast base media containing 4% maltose. Yeast cells flocculated in the growth medium after 32 hours are indicated by (0).Washed cells were competent to flocculate in calcium-containing buffer from eight hours (0). M. Stratford (unpublished results).

Not all yeast strains are subject to the same sugar inhibition. Strains of the Flol phenotype (Stratford, 1989c; Stratford and Assinder, 1991) are unaffected by maltose and are only inhibited by mannose, a sugar rarely encountered at high concentrations in commercial fermentations. Flol phenotype strains may be regarded as practically immune to sugar inhibition. This can, in part, account for observations that these strains flocculated throughout growth. Almost certainly, strains used in tower fermenters are of the Flol phenotype. Here, highly flocculent yeast strains are required to flocculate under all normal circumstances, forming self-immobilized yeast pellets in the presence of very high concentrations of sucrose or glucose (Greenshields et al., 1972; Prince and Barford, 1982; Comberbach and Bu’lock, 1984; Kuriyama et al., 1985). Under these conditions, NewFlo strains would certainly be dispersed and washed out. Other media factors conceivably inhibiting flocculations are low pH values or ethanol. Low pH values certainly inhibit flocculation (Stratford et al., 1988) but such extremes are unlikely to be encountered in commercial

YEAST FLOCCULATION: A NEW PERSPECTIVE

57

fermentations. While ethanol at high concentration may induce non-specific agglutination of non-flocculent cells (Mill, 1964b), there are several reports of yeast strains requiring low concentrations of ethanol in order for flocculation expression (Eddy, 195%; Shieh and Chen, 1986; Amory et al., 1988). These strains are sufficiently unusual, and ethanol concentrations sufficiently low, for ethanol not to be considered a controlling factor in the onset of flocculation. B . CONTROL BY ACTIVATION OR EXPOSURE

Another mechanism by which flocculation could be controlled concerns activation or exposure of surface proteins. It has been suggested that there is a correlation between flocculation and wall mannan content (Masschelein and Devreux, 1957; Masschelein et al., 1963). Here, it was predicted that the wall components involved in flocculation were normally covered or masked by mannan, but exposed in flocculent yeasts with low mannan content. However, other researchers (Griffin and MacWilliam, 1969) have found that wall mannan increased in thickness in the stationary phase of growth, when strains became flocculent. The possibility that flocculation proteins require unmasking still remains, since the discovery (Beavan et al., 1979) that the presence of a-mannosidase, an enzyme with the potential to modify wall mannan, correlated with flocculation. This of course also could indicate simultaneous secretion of flocculation proteins and a-mannosidase, or a role for a-mannosidase in insertion of secreted Po proteins into the wall structure. C. CONTROL BY SYNTHESIS OR SECRETION

The development of flocculation probably requires de novo protein synthesis and secretion. This is indicated by the action of inhibitors of protein synthesis and secretion, cycloheximide and tunicamycin, which prevent flocculation development but have little effect if added after flocculation has occurred (Baker and Kirsop, 1972; Stewart et al., 1973; Nishihara et al., 1976b). Wall enzyme and mannan synthesis are similarly inhibited (Lampen, 1968; Elorza and Sentandreu, 1969; Elorza etal., 1976). Signals indicating derepression of flocculation are probably derived from media components and concentration. Experiments by Mill (1964a) indicated that these signals were probably due to nutrient depletion and not to accumulation of waste products. Yeast nutrients can be broadly divided into carbon and nitrogen sources, vitamins and trace elements. Carbon sources, principally sugars, are unlikely to be involved since flocculation develops in the presence of sugars.

58

M . STRATFORD

Nitrogen-source depletion is, however, a strong candidate for inducing flocculation derepression. The assimilable nitrogen content of brewer’s wort is generally the limiting factor in yeast growth (Gilliland, 1981). Worts containing high assimilable nitrogen lead to delayed and diminished flocculation (Devreux, 1962; Stewart et al., 1975b; Baker and Kirsop, 1972) and altered foaming by top-fermenting yeasts (Oldfield et al., 1951). Nitrogen-deficient worts led to early flocculation (Gilliland, 1951). This effect has been narrowed down to ammonium salts or basic amino acids (Mill, 1964a; Stewart et al., 1973) causing prolonged exponential growth and delayed flocculation. Depletion of assimilable nitrogen-containing compounds would thus seem to be implicated in the onset of flocculation, at least in the brewery context. Another nutrient possibly implicated in the onset of flocculation is inositol. Early flocculation occurred in inositol-deficient media (Nishihara et al., 1976b), which was supported by results presented by Amri et al. (1982). These results must be regarded with caution since inositol deficiency has also been widely reported to cause aggregation by chain formation (Smith, 1951; Ghosh et al., 1960; Challinor et al., 1964; Lewin, 1965; Dominguez et al., 1978; Duffus et al., 1982). It is, however, equally possible that some reports of chain formation may be due to flocculation. Finally, it is probable that the onset of flocculation depends not only on the concentration of signal nutrients, assimilable nitrogen, inositol or other undiscovered factors, but on the rate and/or efficiency of use by yeast and also on the concentration ratio of signal nutrient to sugar. Flocculation may only be expressed in the presence of sugar (Mill, 1964a; Nishihara et al., 1976b). Were the signal nutrient to be depleted after depletion of the sugar, flocculation would not occur. Thus, the ratio of concentration of signal nutrient to sugar may conceivably influence flocculation. D. PREMATURE FLOCCULATION

Premature flocculation is regarded as one of the major causes of hung or stuck fermentations (St Johnston, 1953; Stewart, 1975; Stewart et al., 1976). In these situations, yeast cells flocculate out, leaving unattenuated wort containing much residual sugar. This is immediately curious, since it is known that yeast develops flocculation ability early on and remains as single cells due to sugar inhibition of flocculation (Baker and Kirsop, 1972). It therefore appears that sugar inhibition of flocculation is being overcome, rather than being an effect of early development of flocculation. This could come about by an alternative bonding mechanism, by lectins insensitive to the wort sugars (e.g. the Flol phenotype) or by out-competition of wort sugars. There are numbers of reports of premature flocculation being instigated

YFAST FLOCCULATION

59

A NEW PERSPFCTIVF

by high molecular-weight wort factors. Fractionation of wort has revealed a chemically mixed group of compounds, including polysaccharides from sixrowed barley (Kudo, 1952, 1969) such as treberin, a wort humic acid, barmigan (Kudo and Kijima, 1960), wort araboxylan-glycoprotein fractions (Morimoto et al., 1975) and acidic polysaccharides (Fujino and Yoshida, 1976). Some worts appear especially prone to premature flocculation, in particular those made from Japanese six-rowed barley. High molecularweight wort components are very unlikely to be transported into yeast cells, but they have been shown to adhere to yeast cell walls (Fujino and Yoshida, 1976; Bowen and Cooke, 1989). A novel approach to this phenomenon was taken by Herrera and Axcell (1989) who investigated the possibility that premature flocculation could be attributed to the presence of barley lectins in wort. A barley lectin was isolated but was found to be specific for N-acetylglucosamine and did not induce premature flocculation when added to worts. Barley lectins also could not account for the known characteristics of flocculent strains, since exogenous lectins would tend t o precipitate all yeasts, flocculent and nonflocculent, except those lacking receptors. Multivalent polysaccharides

a

n

A

A A iz

A

a

D

a a

n

V

a

A

n

FIG. 23. Proposed mechanism by which premature flocculation may be caused by the large multivalent polysaccharides present in certain worts. Yeast cells are competent to flocculate for much of the fermentation but are prevented from

flocculation by inhibition of surface lectins by wort sugars. Polysaccharides often contain side-chains, three or more residues in length, with which lectins have many times greater affinity than for simple sugars. Multivalent polysaccharides would be predicted to bind to lectins and form bridges between cells, thus overcoming sugar inhibition and causing premature flocculation.

60

M. STRATFORD

A common factor, however, in all of the diverse fractions causing premature flocculation is high molecular-weight polysaccharide. Flocculent yeast cells have been shown to develop flocculence (surface lectins) at an early stage in flocculation (Fig. 22) and to be held dispersed by media-sugar inhibition. Large polysaccharides almost certainly have a far higher affinity for lectins than simple sugars (Stratford and Assinder, 1991), as has been found in lectins from E. cofi (Firon et a f . , 1982) and concanavalin A (So and Foldstein, 1968). Given that flocculent yeast is inhibited by sugars, and has a high affinity for polysaccharides, it is probable that premature flocculation is caused by large polysaccharide fractions bridging between cells (Fig. 23).

VIII. Genetics of Flocculation A . EARLY GENETICS AND DISCOVERY OF T H E

FLO

GENES

Flocculation is generally recognized to involve surface proteins. Since these proteins are unlikely to be exogenous, such as barley lectins (Herrera and Axcell, 1989), it follows that, within the yeast, there are genes coding for these proteins. In addition to these specific structural genes, flocculation undoubtedly involves the use of many other gene products, in particular for protein secretion and wall location (Novick et a f . , 1980; Pastor et af., 1982; Wen and Schlesinger, 1984). The genetic basis of flocculation was recognized by many early researchers (Pomper and Burkholder, 1949; Gilliland, 1951; Thorne, 1951a,b; Roman et a f . , 1951; Roman and Sands, 1953) but their work must be regarded with caution since it is not always clear whether flocculation or other aggregation phenomena were being examined (Calleja, 1987). With this in mind, flocculent was recognized as dominant to non-flocculent with flocculation under the control of multiple gene pairs. Using strains of brewery origin (Johnston and Lewis, 1974; Lewis and Johnston, 1974; Lewis et a f . , 1976), three genes conferring flocculation were recognized. Genes designated FLOl and FL02 were dominant with a high degree of linkage, whileflo3 was recessive. Flocculation occurred in strains containing either dominant gene and no additive effect resulted when strains contained more than one allele. Shortly afterwards, a fourth gene was discovered (Stewart et a f . ,1976; Stewart and Russell, 1977). This dominant gene, FL04, mapped onto chromosome I, 37 cM from the adel marker, on the far side of the centromere. Further research demonstrated that FLOl, FL02 and FL04 were in fact allelic, and were consolidated into the new FLOI locus on chromosome I (Russell et af., 1980). FL0.5 is a non-allelic dominant gene conferring strong flocculation (Johnston and Reader, 1982,

YEAST FLOCCULATION: A NEW PERSPECTIVE

61

1983) whilePo6 andPo7 have been described (Johnston and Reader, 1982) as semidominant genes which may be alleles of FLOl that are subject to suppression. FLO8, a dominant gene mapping onto chromosome VIII with loose linkage to arg4, was discovered by Yamashita and Fukui (1983). Expression of FL08 was suppressed, however, in heterozygous diploids of mating types a/a and ala. Overall to date, flocculation may be conferred by three dominant genes, FLOl, FL05 and FL08, and one recessive gene, flo3. B . FLOCCULATION INSTABILITY AND SUPPRESSION

Yeast-strain instability with respect to flocculation has often been reported by the brewing industry. Often it is impossible to determine whether this is a result of changes in wort composition or a true change in yeast characteristics, However, flocculation loss by strains has also been demonstrated under laboratory conditions (Thorne, 1951b; Chester, 1963; Stewart, 1975; Lewis et al., 1976), the apparent mutation rate being as high as 5-7% per generation. Chester (1963) suggested that this was not mutation but a gradual loss of flocculation, modified by cytoplasmic elements. Suppressor genes have been identified, however, including fsul , which was originally termed sufl , and fsu2 (Holmberg and Kielland-Brandt, 1978; Holmberg, 1978; Stewart and Russell, 1981). These semidominant genes have been shown to suppress flocculation of FL04 strains. It has recently been proposed (Stratford, 1992) that suppression of flocculation, both in this context and by mitochondria1 mutation, could be the result of secretory modification. This is supported by evidence linking flocculation with secretion of other glycoproteins, such as glucoamylases (Spencer et af.,1981; Yamashita and Fukui, 1984). It is proposed that many suppressions of flocculation are due to general secretory mutations rather than to flocculation specifically. C. REGULATORY NATURE OF

FLO

GENES

In addition to changes involving flocculent strains becoming non-flocculent, there are also rare instances of non-flocculent strains becoming flocculent (Thorne, 1951b; Lewis et af., 1976). This has been regarded as a consequence of suppressed-flocculent strains regaining flocculence (Docherty et af.,1986). Recently, however, a series of seven complementation groups has come to light (Carlson et al., 1984) pleiotropically causing flocculation and constitutive invertase secretion in previously non-flocculent strains. In addition, many independently isolated mutations of the TUPI and CYC8 loci were all found to cause yeast flocculation (Lemontt, 1977; Lemontt

62

M. STRATFORD

et a f . ,1980; Rothstein and Sherman, 1980; Stark et a f . ,1980; Trumbly, 1986; Thrash-Bingham and Fangman, 1989; Fujita et a f . ,1990). Other mutations, namely abs, W a f , PD7 and SFLI, have also been reported to cause flocculation (Hansche, 1975; Lange, 1979; Manney e t a f . ,1983; Fujita etal., 1989). Disruption of the CKAZ gene, which codes for casein kinase I1 and is essential for cell proliferation, also causes intense flocculation (Padmanabha et a f . ,1990). Mutations obviously cannot generate genes coding for surface lectins; therefore, it follows that these non-flocculent yeasts already possessed lectin genes and expressed them following mutation. In view of the large numbers of independently isolated mutants, it is probable that most, possibly all, non-flocculent strains contain these structural flocculation genes. These are then expressed following regulatory gene mutation such as of the TUPl or CYC8 loci (Schultz and Carlson, 1987; Trumbly, 1988). Examination of the flocculation physiology of tupl or cyc8 mutants showed great similarity between these and FLOl genotype strains (Lipke and Hull-Pillsbury, 1984; Stratford and Assinder, 1991). Furthermore, strains containing the flocculation genes FLOl, FL04, FL0.5 and FL08 were physiologically similar to each other and to tupl and cyc8 mutant strains. All expressed the Flol phenotype and mannose-specific flocculation. This has two important consequences. Firstly, this means that nothing is at present known about the genetics of NewFlo phenotype flocculation, perhaps an indication of the practical difficulties of flocculating these strains. Secondly, this means that dominant FLO genes are probably positive regulators of flocculation rather than being structural genes in themselves (Stratford, 1991).

IX. Concluding Remarks

So far, this review has been totally concerned with how yeasts flocculate, which is generally a factual matter. Why yeasts flocculate remains largely a matter for speculation. Most microbial aggregations are sexual in nature (Calleja, 1984,1987) whereas flocculation in Sacch. cerevisiae appears to be entirely distinct from mating (Johnson et a f . , 1988). Possibly flocculation could represent a means of protection during adverse environmental conditions (B. F. Johnson, personal communication to Stewart and Russell, 1981). Certainly, yeast cells within flocs are almost indefinitely immune to damage by ultraviolet radiation (M. Stratford, unpublished observation). This is probably the result of shielding of cells within flocs by exterior cells. However, a great majority (90%) of micro-organisms bearing surface lectins have been recognized to have pathogenic potential (Mirelman and

YEAST FLOCCULATION: A NEW PERSPECTIVE

63

Ofek, 1986). Microbial lectins are generally used aggressively to recognize and bind host cells. Yeast flocculation does not fall into this category since surface lectins are targeted to the yeast cell wall, rather than to a potential host. However, similarity between yeast-surface lectins and viral-coat proteins resulted in speculation that flocculation could be the result of surface expression of yeast virus lectins (Stratford, 1992). Viral proteins, and whole particles, are known to be secreted through the classical secretory pathway (Bergmann et al., 1981) in a similar manner to yeast external enzymes. Lectins of a yeast virus would be expected to target the yeast cell wall. Such a viral association with flocculation could be “fossil” in nature, a relic of a virus integrated within the yeast genome, or a portion of viral genome left after viral secretion from cells. Viral coat-protein genes are commonly found abandoned in mammals following retrovirus infection (Lerner et al., 1976). However, an association between K O 1 phenotype flocculation and the presence of killer L virus (Stratford, 1990), known to carry coat protein genes (Young, 1987), suggests active rather than fossil viral involvement. It is thus possible that flocculation is either the accidental result of viral gene expression, or perhaps may be a deliberate mechanism of viral transfer and or infection.

X. Acknowledgements

I wish to acknowledge gratefully my colleagues, Dr Andrew Carter and Dr Kenneth Westlake, for their assistance with this manuscript, and the Ministry of Agriculture, Fisheries and Food who funded my research. REFERENCES ACMahmood, S., Giumrnelly, P. and Bonaly, R. (1987). Applied Microbiology and Biotechnology 26, 462. Al-Mahrnood, S . , Giummelly, P . , Bonaly, R . , Delrnotte, F. andMonsigny, M. (1988). Journal of Biological Chemistry 263, 3930. Amory, D. E., Rouxhet, P. G . and Dufour, P. J . (1988). Journal of the Institute of Brewing94, 19. Amri, M. M., Bonaly, R . , Duteurtre, B . and Moll, M. (1982). Journal of General Microbiology 128, 2001. Aronson, M., Medalia, O., Schori, L., Mirelrnan, D., Sharon, N. and Ofek, I . (1979). Journal of Infectious Diseases 139, 329. Auk, R. G . and Newton, R. (1968). Journal of the Institute of Brewing 7 4 , 418. Axcell, B . C., Tulej, R . and Mulder, C. J. (1986). Proceedings of the 19th Convention of the Institute of Brewing, Australia and New Zealand Section, p. 63. Bajpai, P. and Margaritis, A. (1986). Biotechnology and Bioengineering 28, 283. Baker, D. A . and Kirsop, B . H. (1972). Journal of the Institute of Brewing 7 8 , 454. Ballou, C . E. (1976). Advances in Microbial Physiology 14, 93. Ballou, C . E. and Raschke, W. C. (1974). Science 181, 127.

64

M. STRATFORD

Barendrecht, H. P. (1901). Zentralblatt fur Bakteriologie, Parasitenkunde und Infektionskrunkheiten, Abteilung 2 , 7, 623. Barkai-Golan, R. and Sharon, N. (1978). Experimental Mycology 2, 110. Barondes, S. H. (1981). Annual Review of Biochemistry 50, 207. Barondes, S. H. (1988). Trends in Biochemical Sciences 13, 480. Barrow, G. M. (1973). “Physical Chemistry”, 3rd ed. McGraw-Hill Kogakusha, Tokyo. Basu, J., Kundu, M., Mukherjee, K. and Chakrabarti, P. (1986). Biochemicaland Biophysical Research Communications 136, 596. Beavan, M. J., Belk, D. M., Stewart, G. G. and Rose, A. H. (1979). Canadian Journal of Microbiology 25, 888. Beijerinck, M. W. (1908). Zentralblatt fur Bakteriologie, Parasitenkunde, und Infektionskrunkheiten, Abteilung 2 20, 137. Bergmann, J. E., Tokuyasa, K. T. and Singer, S. J. (1981). Proceedings of the Nutional Academy of Sciences of the United States of America. 78, 1746. Blake, C. C. F. (1975). Nature 255, 278. Bowen, W. R. and Cooke, R. J. (1989). Biotechnology and Bioengineering 33, 706. Boyd, W. C. and Shapleigh, E. (1954). Science 119, 419. Brohan, B. and McLoughlin, A. J. (1984). Applied Microbiology and Biotechnology 20. 16. Brohan, B. and McLoughlin, A. J. (1985). Irish Journalof FoodScienceand Technology 9,43. Brohult, S . (1951). Proceedings ofthe European Brewery Convention Congress, Brighton. p . 3. Burns, J. A. (1937). Journal of the Institute of Brewing 43, 31. Burns, J. A. (1941). JournaL of the Institute of Brewing 47, 10. Cabib, E. and Bowers, B. (1971). Journal of Biological Chemistry 246, 152. Calibo, R. L., Matsumura, M. and Kataoka, H. (1989). Journal of Fermentation und Bioengineering 67, 40. Calleja, G. B. (1973). Archives of Biochemistry and Biophysics 154, 382. Calleja, G. B. (1984). “Microbial Aggregation”. CRC Press, Boca Raton. Calleja, G. B. (1987). In “The Yeasts” (A. H. Rose and J. S. Harrison, eds), 2nd edn, vol. 2. p. 165. Academic Press, London. Calleja, G. B. and Johnson, B. F. (1977). Canadian Journal of Microbiology 23, 68. Calleja, G. B., Atkinson, B., Garrod, D. R . , Hirsch, P., Jenkins, D., Johnson, B. F.. Reichenbach, H., Rose, A. H., Schink, B., Vincent, B. and Wilderer, P. A. (1984). In “Microbial Adhesion and Aggregation” (Life Science Research Report No. 31) (J. C. Marshall, ed.). Dahlem Konferenzen, p. 303. Springer Verlag, Berlin. Calleja, G. B., Nasim, A , , Lusena, C. V. and Levy-Rick, S. (1986). Biochemistry Internutiond 12, 81. Camp, T. R. and Stein, P. C. (1943). Journal ofthe Boston Society of Civil Engineers 30, 219. Campbell, I . and Brudzynski, A. (1966). Journal of the Institute of Brewing 72, 556. Campbell, I . , Robson, F. 0. and Hough, J. S. (1968). Journal of the Institute of Brewing 74. 360. Carlson, M., Osmond, B. C., Neigeborn, L. and Botstein, D. (1984). Genetics 107, 19. Challinor, S. W., Power, D. M. and Tonge, R. J. (1964). Nature 203, 250. Chapman, S. and Cowling, T . G. (1970). “The Mathematical Theory of Non-uniform Gascs“. 3rd edn. Cambridge University Press, Cambridge. Chester, V. E. (1963). Proceedings of the Royal Society, Series B, 157, 223. Cohen, R. E., Zhang, W. and Ballou, C. E. (1982). Journalof Biological Chemistry 257.5730. Comberbach, D. M. and Bu’Lock, J. D. (1984). Biotechnology Letters 6, 129. Cowan, W. D., Hoggan, J. and Smith, J. E. (1975). Master Brewers Associationofthe Americas Technical Quarterly 12, 15. Critchley, I. A. and Douglas, L. J. (1987a). Journal of General Microbiology 133, 629. Critchley, I . A. and Douglas, L. J. (1987b). Journal of General Microbiology 133, 637. Curtis, A. S. G . (1973). In “Progress in Biophysics and Molecular Biology” (J. A. V. Butler and D. Nobel, eds), vol. 27, p. 317. Pergamon Press, Oxford. Curtis, N. S. (1956). Brewer’s Guild Journal 42, 175. Curtis, N. S. and Wenham, S. (1958). Journal of the Institute of Brewing 64,421. Davis, R. H. and Hunt, T. P. (1986). Biotechnology Progress 2, 91.

YEAST FLOCCULATION: A NEW PERSPECTIVE

65

Davis, R. H. and Parnham, C. S . (1989). Biotechnology and Bioengineering 33, 767. Day, A. W., Poon, N. H. and Stewart, G. G. (1975). Canadian Journal of Microbiology 21, 558. De Clcrkc, J. (1930). Bulletin de I’Association des Anciens El2ves de I’Ecole SupCrieure de Brasserie de I‘UniversitC de Louvain 30, 1. Dc Clcrck, J. and van Roey, G. (1951). Proceedings of the European Brewery Convention Congress, Erighton, p. 120. Dcvreux, A. (1962). Journal of the Institute of Brewing 69, 266. Dittrich, H. H. and Wenzel, K. (1976). Wein-Wissenschaft 31, 263. Docherty, J., dc-Zoysa, P. and Johnston, J. R . (1986). Yeast 2, S93. Domingucz, A, , Villanucva, J. R. and Sentandreu, R. (1978). Antonie van Leeuwenhoek 44, 25. Douglas, L. J. (1985). Microbiological Sciences 2, 243. Doyle, R. J. and Slifkin, M. (1989). American Society for Microbiology News 55, 655. Drickamcr, K . (1988). Journal of Biological Chemistry 263, 9557. Duffus, J. H., Levi, C. and Manner, D. J. (1982). Advances in Microbial Physiology 23, 151. Dunwcll, J. L., Ahmad, F. and Rose, A. H. (1961). Biochimica et Biophysica Acta 51, 604. Eddy, A. A. (1955a). Journal o f t h e Institute of Brewing 61, 307. Eddy, A. A. (195%). Journal o f t h e Institute of Brewing 61, 313. Eddy, A. A. (195%). Journal of the Institute of Brewing 61, 318. Eddy, A. A. (1957). Chemistry and Industry, p. 99. Eddy, A. A. (1958a). Journal of the Institute of Brewing 64,143. Eddy, A. A. (1958b). Proceedings of the Royal Society, Series B 149, 425. Eddy, A. A. and Rudin, A. D. (1958a). Journal of the Institute of Brewing 64, 19. Eddy, A. A. and Rudin, A. D. (19%). Journal of the Institute of Brewing 64,139. Eddy, A. A. and Rudin, A . D. (1958~).Proceedings of the Royal Society, Series B 148, 419. Egilsson, V., Evans, I . H. and Wilkic, D. (1979). Molecular and General Genetics 174, 39. Einspahr, H . , Parks, E. H., Phillips, S. R. and Suddath, F. L. (1988). In “Lectins, Biology, Biochemistry, Clinical Biochemistry” (Proceedings of the 9th lcctin meeting, Cambridge, July 1987) (T. C. Bog-Hanscn and D. L. J . Freed, cds), vol. 6, p. 245. Sigma Chemical Company, St Louis. Einstein, A. (1906). Annalen der Physik 19, 371. Ellis, R. J. and Hemmingscn, S . M. (1989). Trends in Biochemical Sciences 14, 339. Elorza, M. V. and Sentandreu, R. (1969). Biochemical and Biophysical Research Communications 36, 741. Elorza, M. V., Lostau, C. M., Villanueva, J . R. and Scntandreu, R. (1976). Biochimica et Biophysica Acta 454, 263. Eshdat, Y., Spcth, V. and Jann, K. (1981). Infection and Immunity 34,980. Esmon, B. E., Novick, P. and Schekman, R. (1981). Cell 25, 451. Esser, K., Hinrichs, J . and Kucs, U. (1987). In “Flocculation in Biotechnology and Separation Systems” (Y. A. Attia, cd.), p. 383. Elscvicr, Amsterdam. Evans, I. H., Diala, E. S . , Earl, A. and Wilkic, D. (1980). Biochimica et Biophysica Acta 602, 201. Eylar, E. H. (1965). Journal of Theoretical Biology 10, 89. FarkaS, V. (1989). In “The Yeasts” (A. H. Rose and J. S. Harrison, eds), 2nd edn, vol. 3, p. 317. Academic Press, London. Firon, N., Ofek, I. and Sharon, N. (1982). Biochemical and Biophysical Research Communications 105, 1426. Fisher, D . J. (1975). Journal of the Institute of Brewing 81, 107. Fleet, G . H. (1991). In “The Yeasts” (A. H. Rose and J . S. Harrison, eds), 2nd edn, vol. 4, p. 199. Academic Press, London. Frevert, J. and Ballou, C. E. (1985). Biochemistry 24, 753. Fujino, S. and Yoshida, T. (1976). Report of the Research Laboratories of the Kirin Brewery Company 19, 45. Fujita, A,, Kikuchi, Y., Kuhara, S . , Matsumoto, S. and Kobayashi, H. (1989). Gene 85, 321.

66

M. STRATFORD

Fujita, A,, Matsumoto, S., Kuhara, S., Misumi, Y. and Kobayashi, H. (1990). Gene 89,93. Gallili, G. and Lampen, J. 0. (1977). Biochimica et Biophysica Acta 475, 113. Geilenkotten, I . and Nyns, E. J. (1971). Brewer’s Digest 46, 64. Getz, G. S., Tung, B., Cottrell, S., Unger, E. R., Levin, B., King, L., Banerjee, S., Jakovcic, S. and Rabinowitz, M. (1981). In “Current Developments in Yeast Research. Advances in Biotechnology” (G. G. Stewart and I. Russell, eds), p. 317. Pergamon Press, Toronto. Geys, K. (1922). Zeitschrift fur das Gesamte Brauwesen 45, 51. Ghosh, A , , Charalampous, Y., Sison, Y. and Borer, R. (1960). Journal ofBiologica1 Chemistry 235, 2522. Gilliland, R. B. (1951). Proceedings of the European Brewery Convention Congress, Brighton, p. 35. Gilliland, R. B. (1957). Wallerstein Laboratories Communications 20, 41. Gilliland, R. B. (1981). In “Brewing Science ” (J. R. A. Pollock, ed.), p. 1. Academic Press, London. Giumelly, P., Separi, F., Suptijah, P. and Bonaly, R. (1989). Yeast 5 , S309. Goldstein, I. J., Hughes, R. C., Monsigny, M., Osawa, T. and Sharon, N. (1980). Nature285, 66. Greenshields, R. N., Yates, J., Sharp, P. andDavies,T. M. C. (1972). JournaloftheInstituteof Brewing 78, 236. Greig, R. G. and Jones, M. N. (1976). Journal of Theoretical Biology 63, 405. Griffin, S. R. and MacWilliam, I. C. (1969). Journal of the Institute of Brewing 7 5 , 355. Gyllang, H. and Martinson, E. (1972). Proceedings of the European Brewery Convention Congress, Estoril, p. 265. Hansche, P. E. (1975). Genetics 79, 661. Harris, J. 0. (1959). Journal o f t h e Institute of Brewing 65, 5. Hartong, B. D. (1951). Proceedings of the European Brewery Convention Congress, Brighton, p. 110. Haselbeck, A. and Tanner, W. (1983). FEBS Letters 158, 335. Hayduck, F. and Schuckling, K. (1908). Wochenschriftfur Brauerei 25, 241. Heckels, J. E., Blackett, B., Everson, J. S. and Ward, M. E. (1976). Journal of General Microbiology 96, 359. Helm, E., N@hr,B. and Thorne, R. S. W. (1953). Wallerstein Laboratories Communications 16, 315. Hennig, K. and Ay, H . (1938). Biochemische Zeitschrift 299, 123. Herrera, V .E . and Axcell, B. D. (1989). American Society ofBrewingChemists’Journal47,29. Hinrichs, J., Stahl, U. and Esser, K. (1988). Applied Microbiology and Biotechnology 29,48. Hodgson, J. A., Berry, D. R. and Johnston, J. R. (1985). Journal of General Microbiology 131, 3219. Holmberg, S. (1978). Carlsberg Research Communications 43, 401. Holmberg, S . and Kielland-Brandt, M. C. (1978). Carlsberg Research Communications 43,37. Hough, J . S . (1957). Journal o f t h e Institute of Brewing 63, 483. Hussain, T., Salhi, O., Lematre, J., Charpentier, C. and Bonaly, R. (1986). Applied Microbiology and Biotechnology 23, 269. Jansen, H. E. (1958). In “The Chemistry and Biology of Yeasts” (A. H. Cook, ed.), p. 635. Academic Press, New York. Jansen, H. E . and Mendlik, F. (1951). Proceedings of the European Brewery Convention Congress, Brighton, p. 59. Jansen, H . E. and Mendlik, F. (1953). Proceedings of the European Brewery Convention Congress, Nice, p. 143. Jayatissa, P. M. and Rose, A. H. (1976). Journal of General Microbiology 96, 165. Johnson, B. F., Walker, T., Calleja, G. B. and Seligy, V. L. (1988). Canadian Journal of Microbiology 34, 1105. Johnston, J. R. and Lewis, C. W. (1974). In “Second International Symposium on the Genetics of Industrial Microorganisms” (K. D . MacDonald, ed.), p. 339. Academic Press, London. Johnston, J. R. and Reader, H . P. (1982). Proceedings of the Zlth International Conference on Yeast Genetics and Molecular Biology, Montpeleier, p. 124.

YEAST F1,OCCULATION: A NEW PERSPECTIVE

67

Johnston, J . R. and Reader, H. P. (1983). In “Yeast Genetics, Fundamental and Applied Aspects” (J. F. T. Spencer, D. M. Spencer and A. R. W. Smith, eds), p. 205. SpringerVerlag, New York. Jones, S. T., Korus, R. A., Admassu, W. and Heimsch, R. C. (1984). Biotechnology and Bioengineering 26, 742. Kabat, E. A. (1978). Journal of Supramolecular Structure 8, 79. Kamada, K. and Murata, M. (1984a). Agricultural and Biological Chemistry 48, 2423. Kamada, K. and Murata, M. (1984b). Nippon Nogeikagaku Kaishi 58, 977. Kamada, K. and Murata, M. (1984~).Nippon Nogeikagaku Kaishi 58, 983. Kasahara, H., Ouchi, K., Kurata, K., Ishido, T. and Nunokawa, Y. (1974). Journal of Fermentation Technology 52, 348. Kato, S. and Nishikawa, N. (1957). Bulletin of Brewing Science, Tokyo 3, 39. Kihn, J. C., Masy, C. L. and Mestdagh, M. M. (1988a). Canadian Journalof Microbiology 34, 773. Kihn, J. C., Masy, C. L., Mestdagh, M. M. and Rouxhet, P. G . (1988b). Canadian Journal of Microbiology 34, 779. Kijima, M. (1963). Report of the Research Laboratories of the Kirin Brewery Company 6, 35. Kijima, M. (1964). Report of the Research Laboratories of the Kirin Brewery Company 7, 7. Koch, R. (1928). Wochenschrift fur Brauerei 45, 175. Kopecka, M., Phaff, H. J. and Fleet, G. H. (1974). Journal of Cell Biology 62, 66. Kruyt, H. R. (1952). In “Colloid Science. Irreversible Systems” (H. R.Kruyt, ed.), vol. I, p. 1 . Elsevier, Amsterdam. Kudo, S. (1952). Proceedings of the Japan Academy 28, 93. Kudo, S. (1959). Report of the Research Laboratories of the Kirin Brewery Company 2, 1 1 . Kudo, S. and Kijima, M. (1960). Report of the Research Laboratories of the Kirin Brewery Company 3, 33. Kuhn, W. (1938). Angewandte Chemie 51, 640. Kundu, M., Basu, J., Ghosh, A. and Chakrabarti, P. (1987). Biochemical Journal 244, 579. Kurane, R., Takeda, K. and Suzuki, T. (1986). Agricultural and Biological Chemistry 50, 2301. Kuriyama, H., Seiko, Y., Murakami, T., Kobayashi, H. and Sonoda, Y. (1985). Journal of Fermentation Technology 63, 159. Kusserow, R. (1897). Brennerei-Zeitung 14, 318. Lampen, J. 0. (1968). Antonie van Leeuwenhoek 34, 1. Lands, W. E. M. and Graff, G. (1981). In “Current Developments in Yeast Research. Advances in Biotechnology” (G. G . Stewart and I. Russell, eds), p. 331. Pergamon Press, Toronto. Landsteiner, K. (1920). Biochemische Zeitschrift 104, 280. Lange, H. (1899). Wochenschrift fur Brauerei 16, 49. Lange, H. (1907). Journal of the Institute of Brewing 14, 110. Lange, P. (1979). Molecular and General Genetics 173, 211. Lehninger, A. L. (1975). “Biochemistry. The Molecular Basis of Cell Structure and Function”, 2nd edn. Worth, New York. Lemontt, J. F. (1977). Mutation Research 43, 179. Lemontt, J. F., Fugit, D. R. and MacKay, V. L. (1980). Genetics 94, 899. Lerner, R. A, , Wilson, C. B., Del Villano, B. C., McConahey, P. J. and Dixon, F. J . (1976). Journal of Experimental Medicine 143, 151. Lewin, L. M. (1965). Journal of General Microbiology 41, 215. Lewis,C. W. and Johnston, J . R. (1974). Proceedingsofthesocietyfor GeneralMicrobiology I , 73. Lewis, C. W., Johnston, J . R. andMartin, P. A. (1976). Journalof the Instituteof Brewing82, 158. Lindner, P. (1901). Jahrbuch der Versuchs und Lehranstalt fur Brauerei, Berlin 4, 309. Lindquist, W. (1953). Journal of the Institute of Brewing 59, 59. Linnemans, W. A. M., Boer, P. and Elbers, P. F. (1977). Journal of Bacteriology 131, 638. Lipke, P. N. and Hull-Pillsbury, C. (1984). Journal of Bacteriology 159, 797.

68

M. STRATFORD

Lloyd-Hind, H. (1943). “Brewing Science and Practice”. Chapman and Hall, London. Lund, A. (1951). Proceedings of the European Brewery Convention Congress, Brighton, p. 10. Lyons, T. P. and Hough, J. S. (1970a). Brewers’ Digest 45, 52. Lyons, T. P. and Hough, J. S. (1970b). Journal of the Institute of Brewing 76, 564. Lyons, T. P. and Hough, J. S. (1971). Journal of the Institute of Brewing 77, 300. McCandlish, D. and Hagues, G. (1929). Journal of the Institute of Brewing 35, 61. McMurrough, 1. and Rose, A. H. (1967). Biochemical Journal 105, 189. Malkow, A. (1934). Zentralblattfur Bakteriologie, Parasitenkunde, und Infektionskrankheiten, Abteilung 2 90, 212. Malkow, A , , Petina, A. and Zwetkowa, N. (1933). Zentralblatt fur Bakteriologie, Parasitenkunde, und Infektionskrankheiten, Abteilung 2 88, 193. Mandelbrot, B. B. (1990). New Scientist 127 (1734) 38. Manney, T. R., Jackson, P. and Meade, J. (1983). Journal of Cell Biology 96, 1592. Margui, A., Elorza, V. and Sentandreu, R. (1985). Biochimica et Biophysica Acta 841,215. Markwell, M. A. K. (1986). In “Microbial Lectins and Agglutinins: Properties and Biological Activity” (D. Mirelman, ed.), p. 21. Wiley, New York. Markwell, M. A. K., Svennerholm, L. and Paulson, J. C. (1981). Proceedings of the National Academy of Sciences of the United States of America 78, 5406. Masschelein, C . A. and Devreux, A. (1957). Proceedings of the European Brewery Convention Congress, Copenhagen, p. 194. Masschelein, C . A, , Jeunehomme-Ramos, C., Castiau, C. andDevreux, A. (1963). Journalof the Institute of Brewing 69, 332. Meakin, P., Chen, Z.-Y. and Deutch, J. M. (1985). Journal of Chemical Physics 82, 3786. Miki, B. L. A, , Poon, N. H . , James, A. P. and Seligy, V. L. (1981). In “Current Developments in Yeast Research. Advances in Biotechnology” (G. G. Stewart and I. Russell, eds), p. 165. Pergamon Press, Toronto. Miki, B. L. A, , Poon, N. H., James, A. P. and Seligy, V . L. (1982a). Journal of Bacteriology 150, 878. Miki, B. L. A., Poon, N. H. and Seligy, V. L. (1982b). Journal of Bacteriology 150, 890. Mill, P. J. (1964a). Journal of General Microbiology 35, 53. Mill, P. J. (1964b). Journal of General Microbiology 35, 61. Mill, P. J . (1966). Journal of General Microbiology 44, 329. Mirelman, D . and Ofek, I. (1986). In “Microbial Lectins and Agglutinins: Properties and Biological Activity” (D. Mirelman, ed.), p. 1 . Wiley, New York. Momose, H., Iwano, K. andTonoike, R. (1969). Journalof GeneralandApplied Microbiology 15, 19. Morimoto, K . , Shimazu, T., Fujii, T. and Horie, Y. (1975). Report of the Research Laboratories of the Kirin Brewery Company 18, 63. Nagarajan, L. and Umesh-Kumar, S. (1990). Journal of General Microbiology 136, 1747. Naji, B., Pawiroharsono, S . , Bonaly, R . , Tonetti, F., Chasseboeuf, C. and Richter, J. P. (1987). Applied Microbiology and Biotechnology 27, 174. Netto, C. B., Destruhaut, A. and Goma, G. (1985). Biotechnology Letters 7, 355. Newell, P. C. (1977). Endeavour ( N S ) 1 , 63. Nishihara, H. and Toraya, T. (1987). Agricultural and Biological Chemistry 51, 2721. Nishihara, H., Toraya, T . and Fukui, S. (1976a). Journal of Fermentation Technology 54,351. Nishihara, H . , Toraya, T . and Fukui, S. (1976b). Journal of Fermentation Technology 54,356. Nishihara, H., Toraya, T . and Fukui, S. (1977). Archives of Microbiology 115, 19. Nishihara, H., Toraya, T. and Fukui, S. (1982). Archives of Microbiology 131, 112. Northcote, D. H. and Horne, R. W. (1952). Biochemical Journal 51, 232. Novick, P., Field, C. and Schekman, R. (1980). Cell 21, 205. Novick, P., Ferro, S. and Schekman, R. (1981). Cell 25, 461. Ofek, I. and Beachey, E. H. (1980). In “Bacterial Adherence” (E. H . Beachey, ed.), Receptors and Recognition, series B, vol. 6 , p. 3. Chapman and Hall, London. Oldfield, A. I., St Johnston, J. H. and Taylor, A. E. (1951). Proceedings o f t h e European Brewery Convention Congress, Brighton, p . 98.

YEAST FLOCCULATION: A NEW PERSPECTIVE

69

Overbeek, J. Th. G. (1952). In “Colloid Science. Irreversible Systems” (H. R. Kruyt, ed.), vol. 1, p. 278. Elsevier, Amsterdam. Padmanabha, R., Chen-Wu, J. L.-P., Hanna, D. E. and Glover, C. V. C. (1990). Molecular and Cellular Biology 10, 4089. Parker, D. S., Kaufman, W. J. and Jenkins, D. (1972). Journal of the Sanitary Engineering Division, American,Society of Civil Engineers 98, 79. Pasteur, L. (1876). “Etudes sur la Biere”, p. 196. Gauthier-Villars, Paris. Pastor, F. I. J., Herero, E. and Sentandreu, R. (1982). Archives of Microbiology 132, 144. Patel, G. B. and Ingledew, W. M. (1975). Journal of the Institute of Brewing 81, 123. Pearce, W. A. and Buchanan, T. M. (1980). In “Bacterial Adherence” (E. H. Beachey, ed.), Receptors and Adherence, series B, vol. 6, p. 289. Chapman and Hall, London. Piendl, A. (1969a). Proceedings of the European Brewery Convention Congress, Stockholm 12 381. Piendl, A. (1969b). Tageszeitung fur Brauerei 85/86, 553. Pomper, S. and Burkholder, P. R. (1949). Proceedings of the National Academy of Sciences of the United States of America 35, 456. Porter, A. M. and Macaulay, R. J . (1965). Journal of the Institute of Brewing 71, 175. Prince, I. G. and Barford, J . P. (1982). Biotechnology Letters 4, 621. Quiocho, F. A. (1986). Annual Review of Biochemistry 55, 287. Quiocho, F. A. and Vyas, N. K. (1984). Nature 310, 381. Raghav, R., SivaRaman, H., Gokhale, D. V. and SeetaramaRao, B. (1989). Biotechnology Letters 11, 739. Rainbow, C. (1966). Process Biochemistry 1, 489. Ramirez, A. and Boudarel, M. J. (1983). Biotechnology Letters 5, 659. Read, R. (1989). New Scientist 124 (1688) 38. Rodriguez, L., Ruiz, T., Elorza, M. V., Villanueva, J . R. and Sentandreu, R. (1980). Biochimica et Biophysica Acta 629, 445. Roman, H. and Sands, S. M. (1953). Proceedings of the National Academy of Sciences of rhe United States of America 39, 171. Roman, H., Hawthorne, D. C. and Douglas, H. C. (1951). Proceedings of the National Academy of Sciences of the United States of America 37, 79. Rose, A. H. (1984). In “Microbial Adhesion and Aggregation” (Life Sciences Research Report, No. 31) (K. C. Marshall, ed.) Dahlem Konferenzen, p. 323. Springer-Verlag, Berlin. Rothstein, R. J. and Sherman, F. (1980). Genetics 94, 871. Russell, I., Stewart, G . G., Reader, H. P., Johnston, J . R. andMartin,P. A. (1980). Journalof the Institute of Brewing 86, 120. St Johnston, J . H. (1953). Wallerstein Laboratories Communications 16, 39. Schaefer, D. W. (1989). Science 243, 1023. Schonfeld, F. (1909). Wochenschrift fur Brauerei 26, 521. Schonfeld, F. (1910). Wochenschrift fur Brauerei 27, 541. Schonfeld, F. and Krumhaar, H. (1918a). Wochenschrift fur Brauerei 35, 302. Schonfeld, F. and Krumhaar, H. (1918b). Wochenschrift fur Brauerei 35, 342. Schultz, J. and Carlson, M. (1987). Molecular and Cellular Biology 7, 3637. Seyffert, H. (1896). Zeitschrift fur das Gesamte Brauwesen 19, 318. Sharon, N. and Lis, H . (1972). Science 177, 949. Sharon, N., Eshdat, Y., Silverblatt, F. J . and Ofek, I. (1981). CibaSymposium Series80, 119. Sharon, N . , Firon, N. and Ofek, I. (1983). Pure and Applied Chemistry 55, 671. Shieh, W. and Chen, L. (1986). Applied Microbiology and Biotechnology 25, 232. Silhankova, L., Mostek, J., Savel, J. and Solinova, H. (1970). Journal of the Institute of Brewing 76, 289. Smith, R. H. (1951). Journal of General Microbiology 5, 772. So, L. L. and Goldstein, 1. J. (1968). Journal of Biological Chemistry 243, 2003. Spencer, J. F. T., Miller, R., Spencer, D. M. and Wilkie, D. (1981). 1n“Current Developments in Yeast Research. Advances in Biotechnology”, (G. G. Stewart and I. Russell, eds), p. 33. Pergamon Press, Toronto.

70

M. STRATFORD

Stahl, U., Kues, U. and Esser, K. (1983). European Journal of Applied Microbiology and Biotechnology 17, 199. Stark, H. C., Fugit, D. and Mowshowitz, D. B. (1980). Genetics 94, 921. Stevenson, D . G. (1972). Journal of the Institution of Water Engineers, London 26, 155. Stewart, G. G. (1972). Master Brewers Association of the Americas Technical Quarterly 9.25. Stewart, G. G. (1975). Brewer’s Digest 50, 42. Stewart, G. G . (1977). Master Brewers Association ofthe Americas Technical Quarterly 14,l. Stewart, G. G. and Garrison, I. F. (1972). Proceedings o f t h e American Society of Brewing Chemists, p. 118. Stewart, G. G. and Goring, T. E. (1976). Journal o f t h e Institute of Brewing 82, 341. Stewart, G. G. and Russell, I. (1976). Proceedings of the 5th International Fermentation Symposium, Berlin, p. 187. Stewart, G. G. and Russell, I. (1977). Canadian Journal of Microbiology 23, 441. Stewart, G. G. and Russell, I. (1979). Proceedings of the European Brewery Convention Congress, Berlin, p. 475. Stewart, G. G. and Russell, I. (1981). In “Brewing Science” (J. R. A. Pollock, ed.), vol. 2, p. 61. Academic Press, London. Stewart, G. G. and Russell, I. (1986). Journal o f t h e Institute of Brewing 92, 537. Stewart, G. G. and Russell, I. (1987). In “European Brewery Convention Monograph-XII” (E.B.C. Symposium on Brewer’s Yeast, Vuoranta (Helsinki), Finland. November 1986). p. 53. Carl (Brauwelt-Verlag), Nurnberg. Stewart, G. G., Russell, I. and Garrison, I. F. (1973). Proceedings ofthe American Society of Brewing Chemists, p . 100. Stewart, G. G., Russell, I. and Garrison, I. F. (1975a). Journalofthe Institute ofBrewing81, 248. Stewart, G. G., Russell, I. and Goring T. (1975b). Proceedings o f t h e American Society of Brewing Chemists 33, 137. Stewart, G. G., Garrison, I. F., Goring, T. E., Meleg, M., Pipasts, P. and Russell, I. (1976). Kemia-Kemi 3, 465. Stockhausen, F. (1927). Wochenschrift fiir Brauerei 44, 121. Stockhausen, F. and Silbereisen, K. (1933). Wochenschrift fur Brauerei 50, 349. Stockhausen, F. and Silbereisen, K. (1935). Wochenschrift fiir Brauerei 52, 145. Stratford, M. (1989a). Yeast 5, s97. Stratford, M. (1989b). Yeast 5, s441. Stratford, M. (1989~).Yeast 5 , 487. Stratford, M. (1992). Yeast 8 , in press. Stratford, M. and Assinder, S. (1991). Yeast 7, in press. Stratford, M. and Brundish, H. M. (1990). Yeast 6 , 77. Stratford, M. and Keenan, M. H. J. (1987). Yeast 3 , 201. Stratford, M. and Keenan, M. H. J . (1988). Yeast 4 , 107. Stratford, M. and Wilson, P. D. G. (1990). Letters in Applied Microbiology 11, 1. Stratford, M., Coleman, H. P. and Keenan, M. H. J. (1988). Yeast 4 , 199. Tambo, N. and Hozumi, H. (1979). Water Research 13, 421. Tambo, N. and Watanabe, Y. (1979). Water Research 13, 409. Taylor, N. W. and Orton, W. L. (1973). Journal o f t h e Institute of Brewing 7 9 , 294. Taylor, N. W. and Orton, W. L. (1975). Journal o f t h e Institute of Brewing 81, 53. Taylor, N. W. and Orton, W. L. (1978). Journal o f t h e Institute of Brewing 84, 113. Teixeira, J . , Goncalves, M. H., Gama, F. M., Moradas-Ferreira, P. and Mota, M. (1989). Biotechnology Letters 11, 579. TeKippe, R. J . and Ham, R. K. (1971). Journalofthe American Water Works Association 63, 439. Thorne, R. S . W. (1951a). Compte Rendu des Travaux du Laboratoire de Carlsberg, Serie Physiolonigue 25, 101. Thorne, R‘: S. W. (1951b). Proceedings of the European Brewery Convention Congress, Brighton, p . 21. Thornton, R. J . (1978). FEMS Microbiology Letters 4 , 207.

YEAST FLOCCULATION: A NEW PERSPECTIVE

71

Thornton, R. J. (1985). American Journal of Enology and Viticulture 36,47. Thornton, R. J. and Bunker, A. (1989). Journal o f t h e Institute of Brewing 95, 181. Thrash-Bingham, C. and Fangman, W. L. (1989). Molecular and Cellular Biology 9, 809. Tkacz, J. S., Cybulska, E. B. and Lampen, J. 0. (1971). Journal of Bacteriology 105, 1. Tronchin, G., Poulain, D., Herbaut, J. and Biguet, J. (1981). European Journal of Cell Biology 26, 121. Trumbly, R. J. (1986). Journal of Bacteriology 166, 1123. Trumbly, R . J. (1988). Gene 73,97. Tuorila, P. (1927). Kolloidchemische Beihefte 24, 1. Unz, R. F. (1987). In“Floccu1ation in Biotechnology and Separation Systems ” (Y. A. Attia, ed.), p. 351. Elsevier, Amsterdam. von Smoluchowski, M. (1906). Annalen der Physik 21, 756. von Smoluchowski, M. (1917). Zeitschrift fur Physikalische Chemie 92, 129. Watari, J., Takata, Y., Nishikawa, N. and Kamada, K. (1987). Proceedings of the European Brewery Convention Congress, Madrid, p. 537. Watson, D. H. (1964). Brewers Guardian 93,17. Weeks, M. G., Munro, P. A. and Spedding, P. L. (1983). Biotechnology and Bioengineering 25, 687. Weis, W., Brown, J. H., Cusack, S., Paulson, J. C., Skehel, J . J. and Wiley, D . C. (1988). Nature 333,426. Weitz, D. A,, Huang, J. S . , Lin, M. Y. and Sung, J. (1984). Physical Review Letters 53, 1657. Wen, D. and Schlesinger, M. J . (1984). Molecular and Cellular Biology 4,688. White, F. H . and Kidney, E . (1979). Proceedings of the European Brewery Convention Congress, Berlin, p. 801. Wiles, A. E. (1951). Proceedings of the European Brewery Convention Congress, Brighton, p . 84. Wilkie, D. and Nudd, R. C. (1981). In “Current Developments in Yeast Research. Advances in Biotechnology” (G. G. Stewart and 1. Russell, eds), p. 345. Pergamon Press, Toronto. Williams, N. J. and Wiseman, A . (1973a). Biochemical Society Transactions 1, 291. Williams, N. J. and Wiseman, A. (1973b). Biochemical Society Transactions 1, 1301. Windisch, S. (1968). Brewers Digest 43,62. Winslow, C. E. A. and Fleeson, E. H . (1926). Journal of General Physiology 8 , 195. Woof, J. B. (1962). Journal o f t h e Institute of Brewing 68, 315. Yamashita, I. and Fukui, S. (1983). Agricultural and Biological Chemistry 47,2889. Yamashita, I. and Fukui, S. (1984). Agricultural and Biological Chemistry 48, 131. Yoshida, S. (1962). Bulletin of Brewing Science, Tokyo 8 , 37. Young, T. W. (1987). In “The Yeasts” (A. H . Rose and J. S. Harrison, eds), 2nd edn, vol. 2, p. 131. Academic Press, London. Yusa, M. (1977). International Journal of Mineral Processing 4,293. Zlotnik, H., Fernandez, M. P., Bowers, B. and Cabib, E . (1984). Journalof Bacteriology 159, 1018.

This Page Intentionally Left Blank

Secretory Pathway Function in Saccharomyces cerevisiae ANN E. CLEVES and VYTAS A . BANKAITIS Department of Microbiology, University of Illinois, Urbana, Illinois, USA

I. Introduction . . . . . . . . . . . . . . . 11. The yeast secretory pathway . . . . . . . . . . . . A. Elucidation of form . . . . . . . . . . . . . . 111. Protein transport from the cytoplasm into the endoplasmic reticulum A. The paradigms: mammalian andprokaryoticmodels . . . . . B. Genetic analyses . . . . . . . . . . . . . C. In vitro systems . . . . . . . . . . . . . IV. Protein trafficking from the endoplasmic reticulum to the Golgi complex . . A. The mammalian paradigm . . . . . . . . . . . B. Reconstructionoftheyeastprocessinvitro . . . . . . . C. Molecular analysis of genes whose products stimulate transport from the endoplasmic reticulum to the Golgi complex . . . . . . D. The retention problem. . . . . . . . . . . . V. TheGolgicomplexasasecretoryorganelle . . . . . . . . A. Functional compartmentalization of the yeast Golgi complex . . . B. Involvementof aphospholipid-transferprotein . . . . . . C. Theretentionproblem: theroleofclathrin . . . . . . . D. Coupling of Golgi-complex and actin-cytoskeleton functions . . . . . VI . Fusion of Golgi complex-derived vesicles with the plasma membrane A. Involvement of a GTP-binding protein . . . . . . . . B. Other gene products that potentiate GTP-binding protein function . VII. Summary . . . . . . . . . . . . . . . . VIII. Acknowledgements . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

73 75 75 77 77 79 86 88 89 91 94 103 111 112 117 127 129 132 132 137 139 140 140

I. Introduction

The eukaryotic secretory pathway plays an essential role in maintaining the cellular requirements for biochemical compartmentation, and it represents a major aspect of intracellular protein traffic within the eukaryotic cell. It is ADVANCES IN MICROBIALPHYSIOLOGY, VOL. 33 ISBN &l2-0277324

Copyright 0 1992. by Academic Press Limited All rightsofreproduction inany form reserved

74

A . E. CLEVES AND V. A. BANKAITIS

defined by a set of biochemically and morphologically distinct membraneenclosed organelles that house activities associated with catalysis of protein transport, and sorting of proteins from the cytoplasm to various intracellular organelles and the cell surface. Deservedly, the study of intracellular protein transport now commands a great deal of scientific effort and represents a major discipline in cell biology. The basic form of the eukaryotic secretory pathway was elucidated in a series of studies performed by Palade and his coworkers (Palade, 1975). Proteins are synthesized on cytoplasmic ribosomes, inserted into the lumen of the endoplasmic reticulum (ER), and subsequently transported to the Golgi complex (a distinct organelle). Delivery of proteins to the cell surface is the end result of fusion of Golgi complex-derived secretory vesicles, or secretory granules, to the plasma membrane. It is now appreciated that intercornpartmental protein traffic (and, in the case of the Golgi complex, intracompartmental protein traffic) is driven by the budding and specific fusion of transitional vesicles from donor compartments to specific acceptor organelles (Pfeffer and Rothman, 1987). An appreciation of the mechanisms by which these transport vesicles bud from donor organelles and specifically target to (and fuse with) acceptor membranes is a necessary component of an understanding of secretory pathway function. Superimposed upon these issues are considerations of maintenance of organelle identity in the face of a massive bulk flow of protein and lipid from the ER, movement of phospholipids between organelle membranes, protein sorting, and secretory polarity. The definitive demonstration of the basic form of the yeast secretory pathway, and its direct analogy to that of mammalian cells, was provided by the pioneering work of Schekman and his colleagues (Novick et al., 1980; Schekman, 1985). The tractability of Saccharamyces cerevisiae t o genetic, molecular, biochemical and cell biological analyses has made this organism an increasingly attractive experimental system with which to study various aspects of secretory pathway function. A number of fundamental insights concerning the molecular aspects of eukaryotic secretory pathway function have already been forthcoming from this system, with the promise of many more to follow. In this review, we will consider only those topics that are immediately relevant to biosynthetic protein transport through the yeast secretory pathway in Sacch. cerevisiae with some consideration of protein retention within organelles and secretory polarity. Detailed considerations of signal-sequence function, protein glycosylation, protein sorting from the secretory pathway to the vacuole, and aspects of vacuolar biogenesis are discussed in recent reviews (Ballou, 1982; Wickner and Lodish, 1985; Klionsky et al., 1990).

SECRETORY PATHWAY FUNCTION IN S A C C H A R O M Y C E S CEREVISIAE

75

11. The Yeast Secretory Pathway A . ELUCIDATION OF FORM

The basic scheme of the yeast secretory pathway is directly analagous to that of higher eukaryotes, namely E R + Golgi complex + vesicles + ultimate compartment (i.e. the plasma membrane or vacuole). Elucidation of this pathway involved the isolation and characterization of yeast mutants elaborating temperature-sensitive ( t s ) defects at specific stages of the secretory pathway. These defects exhibited the dual properties of being both recessive and thermoreversible traits. As such, these sec mutants identified gene products whose activity was required to stimulate secretory pathway function in yeast (Schekman, 1985). The isolation of such sec mutants was greatly facilitated by a density-enrichment strategy that took advantage of the fact that sec mutants became dense, with respect to wild-type cells, upon imposition of the secretory block (Novick and Schekman, 1979; Novick, et al., 1980). Two classes of secretion-defective mutants were obtained. Class A sec mutants exhibited intracellular accumulation of active invertase (a secretory protein) under the restrictive condition whereas class B sec mutants did not accumulate active invertase at 3TC, even though protein synthesis was not affected. The class B mutants defined two complementation groups, SEC.53 and SEC59, whose products were somehow involved in glycosylation of proteins that had been inserted into the E R lumen (FerroNovick et al., 1984; Feldman et al., 1987). Subsequent molecular and biochemical analyses have identified SEC.53 as the structural gene for phosphomannomutase, the enzyme that converts mannose 6-phosphate to mannose 1-phosphate (Kepes and Schekman, 1988). Thus, in sec53 mutants, production of the sugar donor (GDP-mannose) for synthesis of dolichol-linked oligosaccharides is blocked. The SEC.59 gene product encodes a hydrophobic 59 kDa polypeptide that also plays some role in transfer of mannose to the dolichol-linked oligosaccharide (Bernstein et al., 1989). Consequently, class B mutants were not remarkably informative with respect to mechanisms of secretory pathway function. Genetic analysis of class A mutants revealed that these fall into 23 complementation groups whose products are involved in secretory protein transport through defined stages of the secretory pathway (Fig. 1). The latter conclusion was reached by electron-microscopic visualization of the terminal phenotypes of such mutants (Novick et al., 1980). These experiments permitted classification of the mutants into several classes: (i) those that were blocked in protein traffic from the E R to the Golgi complex (these ER-blocked mutants defined nine complementation groups, SEC12, SEC13, SEC16, SECl7, SEC18, SEC20, SEC21, SEC22 and SEC23);

76

A . E. CLEVES AND V. A. BANKAlTlS

FIG. 1 . Diagram illustrating the yeast secretory pathway. Yeast secretory glycoproteins are synthesized in the cytoplasm and are transported into the lumen of the endoplasmic reticulum (ER) where they undergo N-linked core glycosyl modification. After passage through the Golgi complex, where the remodelling of glycosyl chains is completed, glycoproteinsare delivered to their final destinations,either the cell surface or the vacuole. The involvement of 21 of the 23 original SEC gene products is indicated (Novick et al., 1980). (ii) those that were blocked in protein traffic through the Golgi complex (i.e. sec7 and secl4 mutants); (iii) those mutants that were defective for fusion

of Golgi complex-derived secretory vesicles to the plasma membrane (10 complementation groups, S E C l , SEC2, SEC3, SEC4, SECS, SEC6, SEC8, SEC9, SEClO and SECI.5). Mutations in two complementation groups, SECI1 and SEC19, exhibited noteworthy terminal phenotypes. The secll mutants failed to exaggerate any organelle membranes whereas the secl9 mutants exaggerated all of them. That these class A mutants affected the function of the same pathway was confirmed by epistasis analyses where the appropriate sec double-mutant combinations were genetically constructed and, upon imposition of the 37°C block, visualized for terminal phenotype. Novick et al. (1981) found that the ER-blocked mutations were epistatic to the Golgi complex-block and vesicle-block lesions, and that Golgi-complex blocks were epistatic to the secretory vesicle blocks. This is consistent with the concept of a linear pathway (ER -+ Golgi complex + vesicles) as opposed to a concept of independent parallel pathways. Moreover, these epistasis analyses established the relative order of the first execution points of these SEC gene products on the pathway, and demonstrated the relative order of organelle involvement. This temporal order of organelle involvement on the secretory pathway was further

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

77

supported by the studies of Esmon et af. (1981), who showed that assembly of oligosaccharide chains on secretory glycoproteins in yeast occurred in defined stages. Glycoproteins blocked at the E R possessed immature (i.e. core) oligosaccharide chains and Golgi complex-blocked glycoproteins had either more extensively (but nonetheless incompletely) modified oligosaccharide chains (i.e. the case of sec7), or fully modified sugar chains (i.e. s e c l l ) . Glycoproteins blocked at the secretory vesicle stage appeared uniformly to exhibit fully matured glycosyl chains (Esmon et al., 1981). Thus, class A sec mutants provided the means by which the general form of the yeast secretory pathway could be demonstrated. Furthermore, these mutants provided a convenient starting point for biochemical and genetic strategies aimed at determining the mechanisms that underlie stage-specific secretory protein transport. Much of this review is devoted to a discussion of what the second-generation experiments with these mutants have revealed with respect to protein-transport mechanisms.

111. Protein Transport from the Cytoplasm into the Endoplasmic Reticulum

The biochemical identity of intracellular organelles is in part a function of the unique sets of proteins that reside within each of them. At one level, the organelle membranes serve as impermeable barriers to diffusion of such resident proteins (although the problem of protein retention is still a very real one; see below). This barrier function for organelle membranes is not compatible with the first event that signifies entry of a polypeptide into the secretory pathway, that is, export of proteins from their cytoplasmic site of synthesis into the E R lumen. The problem of protein translocation across the first membrane is one that is faced by all cells, and it is one that has received a great deal of experimental scrutiny in both mammalian and prokaryotic systems. A. T H E PARADIGMS; MAMMALIAN AND PROKARYOTIC MODELS

Protein export from the cytoplasm can be considered to involve four conceptually distinct events: (a) the initial sorting event where proteins destined for export are recognized by a cytoplasmic machinery by virtue of N-terminal signal peptides that are elaborated by precursor forms of such secretory proteins, (b) delivery of the engaged secretory precursor polypeptides to the target membrane, (c) translocation of the polypeptides across the membrane (possibly through a proteinaceous pore), and (d) proteolytic processing, or maturation, of the translocated proteins (Blobel and Dobberstein, 1975).

78

A. E. CLEVES AND V. A . BANKAlTlS

In mammalian systems, it is the signal recognition particle (SRP) that is involved in serving as a cytoplasmic adapter between nascent secretory polypeptide chains and the protein translocation apparatus of the ER membrane (Fig. 2; for a review, see Walter and Lingappa, 1986). The SRP is a ribonucleoprotein particle that sediments at 1Is in sucrose velocity gradients, has an affinity for ribosomes, and consists of a 300-nucleotide RNA component (termed 7SL RNA) that is complexed with six unique polypeptides of 9,14,19,54,68 and 72 kDa (Siege1 and Walter, 1985). The SRP experiences a dramatic increase in its binding affinity for those ribosomes, presenting an emerging nascent polypeptide chain that exhibits a signal sequence. Subsequent to binding of the signal peptide by the 54 kDa SRP subunit, the SRP-nascent chain-ribosome complex is targeted to the E R membrane. This targeting is realized on the basis of the direct interaction of the 68 kDa/72 kDa SRP subunits with a heterodimeric integral membrane protein of the E R membrane, the SRP receptor. Translocation

FIG. 2. Diagram illustrating the signal hypothesis. This diagram describes thc cotranslational export of a secretory protein from the cytoplasm into the endoplasmic reticulum (ER) lumen. The basic tenets of this scenario have been established by elegant studies in mammalian cell-free systems (Walter and Lingappa, 1986). Nascent polypeptide chains are bound by the signal-recognition particle (SRP) to form a soluble SRP-nascent chain-ribosome complex that is targeted to ER membranes by virtue of the interaction of the SRP with a heterodimeric membrancbound SRP receptor (docking protein). The signal peptide then engages the signalsequence receptor and the SRP is released into the cytosol for participation in another round of ER targeting. Translocation of the nascent polypeptide ensues in a manner that is not at all understood, and signal-peptidecleavage occurs either during or immediately after the translocation step.

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVlSlA E

79

of the nascent secretory polypeptide is believed to ensue in a cotranslational, but otherwise undetermined, fashion. It is not known whether translocation proceeds through a proteinaceous pore or through the membrane bilayer itself. Finally, the signal sequences of secretory polypeptides are proteolytically removed by a signal peptidase, an enzymic activity associated with a complex composed of six non-identical polypeptide chains (Evans et af., 1986). In Escherichia coli, the basic features of the signal hypothesis for protein export are also fulfilled (Bankaitis et af., 1986a). Although there is still some question as to whether there exists an obvious direct analogue to the SRP in E. cofi, the cytoplasmic adapter function appears to be provided by a peripheral membrane protein, the secA gene product (abbreviated to SecAp; Oliver and Beckwith, 1982). This gene product appears to interact with both signal sequence and mature regions of precursor proteins, and exhibits an ATPase activity that is stimulated by acidic phospholipids (Lill et af., 1990). Maximal stimulation of the SecAp ATPase activity also requires the SecYp, an integral protein of the cytoplasmic membrane in E. cofi(Crooke etal., 1988). Thus, one can broadly consider the SecYp to act as the SecAp receptor. It is also clear that proteins can be exported from E. coli in either co- or post-translational modes in vivo (Randall, 1983) and that cytoplasmicfactors, termed chaperonins, are involved in the maintenance of polypeptides in a translocation-competent (i.e. less tightly folded) state, a consideration that is of particular importance to the latter mode of protein export (Collier et al., 1988). While the mechanistic details of the translocation step are entirely unclear, the signal peptidase in E. cofi has been shown to be a 36 kDa polypeptide that is essential for viability of the bacterium (Date, 1983). Conditional defects in signal peptidase function lead to the general accumulation of precursors for exported proteins that have, nonetheless, been translocated across the cytoplasmic membrane (Dalbey and Wickner, 1985). B. GENETIC ANALYSES

Unlike our current understanding of the scheme for protein translocation across the cytoplasmic membrane in E. cofi, which is built upon both powerful in vivo and in vitro evidence, the mamma,lian paradigm is based solely upon in vitro arguments, compelling and elegant though these be. Thus, Sacch. cerevisiae has provided a flexible experimental system that has been amenable to the application of both in vivo and in vitro approaches directed at the study of protein transport from the cytoplasm into the E R lumen. A classic genetic approach to the understanding of such protein transport in yeast required the isolation of mutants unable to export proteins

80

A . E. CLEVES A N D V. A . BANKAITIS

into the E R lumen. The initial sec mutant hunt failed to produce such mutants. Fortunately, the insights acquired from genetic selections designed to generate the analogous export mutants in bacteria yielded proven strategies for the positive selection for yeast mutants defective in protein export from the cytoplasm into the E R lumen. 1. The HOL'

Selection

The strategy for isolating mutants of Sacch. cerevisiae that were defective in the cellular machinery that translocates secretory precursors from the cytoplasm into the E R lumen was described by Deshaies and Schekman (1987) and extended by Rothblatt et al. (1989). This selection was analogous to the one devised by Oliver and Beckwith (1982) that yielded the first secA" mutants of E. coli.Briefly, the normally cytoplasmic HIS4 gene product (the HIS4p) was fused to an N-terminal signal peptide provided by the prepro-afactor, the precursor of the secreted a-factor pheromone. This chimera targeted to the E R lumen and, in yeast strains that were otherwise mutant for HZS4, rendered cells incapable of growing on minimal media supplemented with histidinol (rather than histidine). This hol- phenotype presumably reflected the inability of histidinol to gain access into the ER lumen so that the HIS4p chimera (which retained HIS4p enzymic activity) could convert the histidinol to histidine (Deshaies and Schekman, 1987; Rothblatt et al., 1989). A positive selection for HOL+ mutants failing to translocate the HIS4p chimera into the E R lumen was thus generated. By selecting for HOL' derivatives, a number of ts mutants were recovered that exhibited conditional defects in protein translocation from the cytoplasm into the E R lumen.

2. Genes Involved in Protein Translocation Three genes (SEC61 , SEC62 and SEC63) were identified by the HOL" selection (Deshaies and Schekman, 1987; Rothblatt et al., 1989). Thermosensitive alleles of each of these three genes appeared to block protein translocation at the same point in the pathway, that is, prior to translocation into the E R lumen and signal-peptide cleavage. Such mutants were found to accumulate non-glycosylated precursor forms of three secretory proteins (prepro-a-factor, acid phosphatase and invertase) and a vacuolar protein (carboxypeptidase Y). It should be noted, however, that sec62 mutants were only partially defective for invertase transport. Subcellular fractionation experiments indicated that, while the precursor forms exhibited an association with intracellular membranes, these precursors had clearly not been translocated into the E R lumen. This was indicated by the susceptibility of

SECRETORY PATHWAY FUNCTION IN S A C C H A R O M Y C E S C E R E V l S l A E

81

the precursors to proteases that had access to the cytoplasmic surfaces, but not lumenal aspects, of intracellular membranes (Rothblatt et al., 1989). Finally, it was observed that the sec6Zfssec63" double-mutant combination resulted in loss of cell viability, whereas the sec6Zfssec62" and sec62" sec63" double-mutant combinations exhibited exaggerated ts growth and translocation defects. On the basis of such genetic interactions, Rothblatt et al. (1989) suggested that these three gene products act along the same functional pathway. Genomic clones of the SEC62 and SEC63 genes have been recovered and characterized. The nucleotide sequence of SEC62 suggested that the SEC62p was a 283-residue polypeptide, predicted to be of 32,381 Da and, for yeast proteins, an unusually basic isoelectric point of 10.7 (Deshaies and Schekman, 1989). O n the basis of the inferred SEC62p primary sequence, Deshaies and Schekman (1989) speculated that this polypeptide did not contain an N-terminal signal peptide that would serve to direct insertion of the SEC62p into E R membranes. Nevertheless, these workers suggested that the SEC62p was an integral membrane protein of the E R , one that spanned the membrane bilayer twice. These suggestions rested on the identification of two potential membrane-spanning domains in the inferred SEC62p primary sequence, and the fact that E R functions were affected (both in vivo and in vitro) under conditions of SEC62p dysfunction (Rothblatt et al., 1989; Deshaies and Schekman, 1989). The idea that the SEC62p is an integral membrane protein of the E R is an attractive one as it would be entirely consistent with the proposed role of the SEC62p as a component of the E R translocation machinery. The demonstration that this protein is exclusively localized in E R membranes, as would also be predicted, still awaits the appropriate fractionation and immunolocalization experiments. In any event, the SEC62p clearly plays a unique and essential cellular function in yeast as sec62 disruption mutations represented recessive, haploid-lethal events (Deshaies and Schekman, 1989). The SEC63 gene was similarly essential for vegetative growth of Sacch. cerevisiae, as reported by Sadler et al. (1989), who obtained genomic clones of SEC63. Interestingly, Sadler et al. (1989) were engaged in a molecular analysis of cellular components (such as the NPLZ gene product) that facilitated import of proteins from the cytoplasm into the yeast nucleus. Mutants conditionally defective in NPLZ gene function were unable to support efficient nuclear-protein transport under restrictive conditions, and the allelism of NPLZ and SEC63 was unambiguously established by genetic criteria. Sequence analysis of DNA has established that SEC63 potentially encodes a 663-residue polypeptide that exhibits three potential membranespanning regions (Sadler et al., 1989). The most interesting aspect of the inferred primary sequence was that a region of 72 amino-acid residues of the

82

A . E. CLEVES AND V . A. BANKAITIS

SEC63p, which accounted for most of the domain bounded by the second and third potential transmembrane segments, exhibited a 43% identity with the N-terminus of the DnaJ protein in E. coli, a member of the heat-shock family in this bacterium. At this point, it is unclear whether the SEC63p region of DnaJ homology is exposed to the yeast cytosol or whether it is exposed to the lumenal E R surface. In either event, this homology suggests that the SEC63p may be involved in maintaining the translocation competence of polypeptides (i.e. a chaperonin function) or may serve to nucleate formation of some sort of translocation complex. The dual function of the SEC63p in E R and nuclear transport still needs to be reconciled, however. One possibility is that the SEC63p serves as a common translocator component for both E R and nuclear membranes with compartment specificity being determined by some other component(s). Alternatively, defects in the SEC63p may cause general dysfunction of the E R and nuclear membranes, which are contiguous, thereby affecting E R and nuclear transport processes indirectly. It is of interest to note that sec6Z and sec62 mutations failed to cause nuclear-transport defects (Sadler et al., 1989). Localization of the SEC63p in an intracellular compartment should help resolve the question of whether the primary function of this gene product is most directly relevant to E R processes, nuclear processes, or both.

3. The Cytosolic Factors Involved in Translocation The HOL’ selection, while yielding mutants that are likely to be defective for the translocation step in protein transport into the E R lumen, failed to generate mutants defective in the earliest steps of this process, namely the recognition and membrane-targeting events that precede the translocation step (see Section 1II.A). The mammalian and prokaryotic paradigms for this process, however, laid some of the groundwork for fruitful “wreck and check” approaches. These strategies have provided key insights into the role for chaperonins in intracellular protein transport, and have revealed promising avenues for further investigation with respect to SRP-like activities in yeast. Deshaies et al. (1988) investigated whether the yeast 70 kDa heat-shock protein (HSP70) homologues found in Sacch. cerevisiae served as molecular chaperones for polypeptides destined for insertion into the E R lumen. The SSA subgroup of the nine HSP70 homologues in Sacch. cerevisiae consists of four genes (SSAZ-SSAQ) whose products execute interchangeable functions in vivo (Werner-Washburne et al., 1987). Although these genes are functionally redundant (and therefore invisible to classical loss-of-function mutant screens), ssal ssa2 double-mutant strains are ts for growth whereas ssal ma2 ssal triple mutants are inviable. Thus, Deshaies et al. (1988)

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVlSlAE

83

engineered galactose-inducible expression of SSAl in the ssal ssa2 ssal triple mutant background and asked whether depletion of SSAlp (induced by glucose challenge of such cells) had any specific consequence for protein transport. Depletion of the SSAlp in vivo resulted in the accumulation of precursor forms of the soluble vacuolar proteinase carboxypeptidase Y (CPY) and the secretory prepro-a-factor. These precursors were blocked in transport at a point prior to their insertion into the E R lumen. The evidence came from their accessibility to exogenously added proteases in cell-free lysates, and their comigration in sodium dodecyl sulphate-polyacrylamidegel electrophoresis (SDS-PAGE) systems with the unmodified forms of the corresponding preproteins obtained from sec61 and sec62 mutants. This precursor accumulation was considered to be specific for two reasons. First, cells ceasing to grow due to glucose-dependent depletion of at least two other essential gene products failed to show a secretory defect. Second, the SSAlp by itself was capable of restoring translocation activity to SSAlpdeficient extracts in vitro (Deshaies et al., 1988; Chirico et al., 1988). These data clearly showed an involvement of the SSA .gene products in protein transport. That this involvement was not limited to E R transport was dramatically demonstrated by the concomitant in vivo defects in transport of the mitochondrial F,ATPase p-subunit into the mitochondrial matrix under SSA-deficient conditions (Deshaies et al., 1988). Given the central role that the SRP occupies in the mammalian scheme for ER protein transport, some effort has been directed at identifying genes whose products could potentially be involved in SRP-like activities in yeast. The two components that have received the most attention to date include the 7SL RNA component of the SRP and the 54 kDa SRP subunit that is thought to be the direct mediator of signal-peptide recognition. Ribes et al. (1988), as well as Brennwald et al. (1988) and Poritz et a f . (1988), described the identification of a 7SL RNA in Schizosaccharomyces pombe, reported the cloning of the structural gene, and found significant structural and primary sequence homologies between the Schiz. pornbe and human 7SL RNA species. Both Ribes et al. (1988) and Brennwald et al. (1988) employed gene-disruption experiments to show that 7SL RNA was essential for vegetative growth of Schiz. pombe. That the 7SL RNA in Schiz. pombe might assemble into an SRP-like particle in vivo was suggested by several biochemical experiments. First , the 7SL RNA was recovered from post-ribosomal supernatants in an 11sparticle. Second, the 7SL RNA from this yeast bound to canine SRP proteins under stringent conditions. Third, the canine SRP proteins of 19 kDa and the 68 kDa/72 kDa heterodimer footprinted to homologous regions of both the mammalian and Schiz. pombe RNAs (Poritz et al., 1988). Interestingly, although the dimorphic yeast Yarrowia lipolytica also contains a 7SL RNA that showed essentially

84

A . E. CLEVES AND V. A . BANKAlTlS

the same biochemical properties as that from Schiz. pombe (Poritz et al., 1988), Sacch. cereuisiae was devoid of the 7SL species (Brennwald et al., 1988).Thus, the Schiz. pombe system appears to be the eukaryotic model of choice for dissecting 7SL function in uiuo. The current challenge is to determine whether or not 7SL function is relevant to secretory function in Schiz. pombe. To investigate further the role of SRP polypeptide subunits in ER transport, cDNA clones for the mammalian 54 kDa SRP subunit (i.e. the signal peptide-binding subunit SRP54) were identified and characterized (Bernstein et al., 1989;Romisch et al., 1989). The inferred primary sequence of the SRP54 revealed two domains of interest; an N-terminal domain that contained a potential GTP-binding motif and a C-terminal rnethionine-rich domain that was suggested to form a "methionine-bristle" structure that could serve as a signal peptide-binding groove. Comparisons of the SRP54 primary sequence with available protein data bases revealed several homologies of interest, but the most relevant homology for the purposes of this review was that observed between SRP54 and a 48 kDa protein from E. coli that associates with a 4.5s RNA, in an SRP-like particle, and has been named FFH (fifty-four hornologue) (Bernstein et al., 1989; Romisch et al., 1989; Ribes et al., 1990). Both SRP54 and FFH were significantly homologous over their entire length, and FFH exhibited the putative GTPbinding and methionine-bristle sequence motifs. This similarity suggested the presence of SRP-like subunit polypeptides in organisms that did not produce 7SL RNAs. Hann et al. (1989) took advantage of the highly conserved regions in the GTP-binding domains of mammalian SRP54 (SRP54mam)and FFH to design PCR oligonucleotides for amplification of genomic sequences in both Sacch. cereuisiae and Schir. pombe. While initial attempts to amplify DNA from Sacch. cereuisiae were equivocal, the reactions of Schiz. pombe were productive and ultimately yielded an SRP54 homologue (SRP54"P) of this yeast. Nucleotide-sequence analysis indicated that SRP54'P (the designation given to SRP54 from Schiz. pombe) was composed of 522 amino-acid residues of 57 kDa and a predicted isoelectric point of 9.9 (Hann et al., 1989). Moreover, comparison of the inferred SRP54""" and SRP54sp primary sequences revealed additional homologies that ultimately yielded a productive strategy for identification and cloning of SRP54", the Sacch. cerevisiae SRP54 homologue. This homologue was predicted to be a basic protein (pZ9.5) of 541 residues of 60 kDa. The yeast and mammalian SRP54 species were notably homologous over their entire primary sequence, and both yeast SRP54 homologues exhibited recognizable GTP-binding and methionine-bristle domains. That at least SRP54" played an essential in uiuo function was indicated by the demonstration that disruption of the

SECRETORY PATHWAY FUNCTION IN SACCHANOMYCES CEREVISIAE

85

SRP54" structural gene is a haploid-lethal event in Sacch. cerevisiae. It is anticipated that the same conclusion will be reached for SRP54'P. Further delineation of SRP54 function in vivo awaits construction of the appropriate conditional mutants defective in SRP54"" and SRP54'P function, and the test of whether such defects lead to dysfunction in transport of proteins into the ER lumen. However, it is tempting to believe it likely that such will be the case. Hann and Walter (1990) found that SRP54'P was associated in vivo with 7SL RNA in Schiz. pombe, in direct analogy to the mammalian SRP, whereas SRP.54" associated in vivo with RNA in Sacch. cerevisiae of some 600 bp in size and sedimented as a 16s ribonucleoprotein particle. 4. Signal-Peptide Processing

Transport of a secretory protein into the E R lumen is marked by cleavage of the N-terminal signal peptide by a highly specific endoprotease, the signal peptidase. As already noted, vertebrae signal peptidases are multisubunit complexes. Mammalian signal peptidase consists of six unique polypeptide subunits whereas the signal peptidase from the hen oviduct consists of two unique polypeptide subunits (Evans et al., 1986; Baker and Lively, 1987). The signal peptidase in E. coli is an ectopic 36 kDa protein of the cytoplasmic membrane (Wolfe et al., 1983). The signal peptidase is an important component of the transport process as the rate of protein export from the E R is greatly hastened by signal peptide cleavage, even though translocation into the E R lumen does not depend upon the processing event. Finally, the processing event displays remarkable fidelity and conserved specificity. Prokaryotic and eukaryotic signal peptidases are able to process correctly and efficiently precursors of either prokaryotic or eukaryotic origin in vitro (Bankaitis et al., 1986a). Haguenauer-Tsapis and Hinnen (1984) and Schauer et al. (1985) demonstrated that secretory proteins which fail to be processed exhibit dramatically lower rates of transit through the yeast secretory pathway. These results suggested that signal-peptidase mutants would, as a result of the processing dysfunction, exhibit a general delay in protein secretion, rather than a stagespecific secretory block. Interestingly, Bohni et al. (1988) discovered that seclltS mutants exhibited the appropriate behaviour expected for signalpeptidase mutants. Note that secll mutants were the only sec mutants that failed to exaggerate an intracellular organelle when challenged with the restrictive condition (see above). At the non-permissive temperature, pulseradiolabelled polypeptides accumulated as core-glycosylated species that had entered the E R lumen in secll" strains. However, these coreglycosylated species retained their signal peptides. Bohni et d. (1988) recovered SECIl clones, and found that the SECII nucleotide sequence

86

A. E. CLEVES A N D V. A. BANKAlTlS

predicted an 18.8 kDa polypeptide containing a putative N-terminal signal peptide and one potential asparagine-linked glycosylation site. As a result, the SECl l p exhibited some superficial similarity with the 22-24 kDa henoviduct signal-peptidase glycoprotein subunit and the two canine signalpeptidase glycoprotein subunits of 22 kDa and 23 kDa. These vertebrate glycoprotein subunits uniformly contained a polypeptide backbone of 19 kDa, but there was no significant primary sequence homology detected between the S E C l l p and the canine glycoprotein subunit (Deshaies et al., 1989). It remains to be determined whether or not the yeast signal-peptidase exists in a multisubunit complex.

c. In Vitro SYSTEMS Although “wreck and check” approaches have shown some promise, it is desirable to have functional assays at one’s disposal for a biochemical dissection of protein transport. To this end, powerful in vitro systems for monitoring protein transport into the E R have been developed. The current efforts with these systems follow the classical resolution and reconstitution strategy. The systems for in vitro translocation of yeast secretory proteins into the ER lumen were nearly simultaneously developed by three research groups (Rothblatt and Meyer, 1986a; Hansen et af., 1986; Waters and Blobel, 1986). The basic features of these similar systems involved generation of radiochemically pure precursor substrates by specifically programmed in vitro translation systems, incubation of yeast microsomes with the labelled substrate, and determination of whether translocation into the microsome lumen had occurred. Three operational criteria are generally applied for signifying translocation: (a) core glycosylation of substrate, (b) acquisition by the substrate of resistance to exogenously provided protease, and (c) processing of the substrate by signal peptidase. Under optimal conditions, it was found that some 40% of the total input prepro-a-factor substrate could be translocated into E R microsomes. A truncated form of secretory invertase could also be faithfully translocated into E R microsomes in vitro (Rothblatt and Meyer, 1986a). One of the interesting findings that was forthcoming from characterization of the in vitro system was that, in contrast to mammalian in vitro translocation systems, the prepro-a-factor was capable of being translocated in an entirely post-translational manner in the yeast system, and that the efficiency of the post-translational mode of transport was similar to that of transport measured in the cotranslational reaction (Hansen et al., 1986; Waters and Blobel, 1986). The ability to undergo the post-translational reaction was substrate-specific, however (Rothblatt et al., 1987; Hansen and Walter, 1988). As an illustration of this,

SECRETORY PATHWAY FUNCTION IN S A C C H A R O M Y C E S CEREVISIAE

87

prepro-carboxypeptidase Y (CPY) and a truncated pre-invertase were efficient post-translational translocation substrates, while full-length preinvertase could be translocated in vitro only in cotranslational reactions (Hansen and Walter, 1988). Regardless, the ability to translocate certain substrates post-translationally effectively uncoupled transport from protein synthesis, and permitted a strict analysis of the energy requirements for transport. It is clear that ATP was required to support transport, perhaps by fuelling an ATP-driven translocase activity (Waters and Blobel, 1986; Hansen et al., 1986; Rothblatt and Meyer, 1986b). Non-hydrolysable analogues of ATP were unable to support transport, while maintenance of a membrane potential was not required for translocation. The process was refractory to challenge by a number of ionophores or other uncouplers (Hansen et al., 1986; Waters and Blobel, 1986). The availability of the in vitro translocation system in Sacch. cerevisiae permitted the pursuit of several different lines of investigation directed at addressing the question of whether the E R transport scheme in yeast operates via the same mechanism as the one described by the mammalian paradigm. One line of investigation involved an attempt to resolve the in vitro system by methods that were successful in resolving key aspects of the mammalian reaction. Another approach has been to characterize the in vitro defects of the appropriate sec mutants in the transport reaction. The first strategy was employed by Rothblatt and Meyer (1986a), who tested whether washing yeast microsomes with solutions containing high concentrations of salt, or proteolytic treatment of such microsomes with elastase, compromised the translocation competence of microsomes. Unfortunately, neither did so. It should be noted that the microsomal salt wash fractionated the mammalian SRP, whereas elastase treatment of microsomal membranes yielded an assay for the mammalian SRP receptor (reviewed in Walter and Lingappa, 1986). However, in contrast to the negative results obtained with these treatments, the experiments of Hansen et al. (1986) demonstrated a requirement for microsomal membrane proteins in the transport reaction. Pretreatment of microsomes with the sulphydryl alkylating agent Nethylmaleimide rendered the microsomes unable to support translocation. The identities of the essential components that are sensitive to the alkylating agent remain to be determined. A combination of the “wreck and check” and in vitro resolution strategies holds considerable potential. It will be of great interest to determine whether depletion of SRP54” from the in vitro reaction has any deleterious consequences for translocation, and whether such consequences are specific for proteins that employ exclusively the cotranslational mode of transport. The in vitro translocation system has also permitted a more complete characterization of mutants that were defective in their ability to translocate

88

A. E. CLEVES AND V. A. BANKAlTlS

proteins from the cytoplasm into the E R lumen. In particular, since the cytosolic components of the reaction are readily separated from the microsomal components of the reaction, it has been possible to assign the relevant translocation defects to one of the two specific components. For instance, the sec62, sec62 and sec63 mutants were defective in ER translocation, while nucleotide sequences of the SEC62 and SEC63 gene products suggested that both of these were integral membrane proteins of the E R (see Section III.B.2). Confirmatory evidence has been obtained from in uitro analysis of the sec62 and sec63 mutants. Rothblatt et al. (1989) demonstrated that, while cytosol fractions prepared from sec63 cells were competent to support translocation of prepro-a-factor into wild-type microsomes, even a short pre-incubation of sec63 microsomes at the nonpermissive temperature markedly decreased the efficiency of prepro-afactor translocation, even with wild-type cytosol. Similar results were reported for sec62 with the added caveat that sec62 microsomes were generally defective in the translocation assay (Deshaies and Schekman, 1989). Chirico et al. (1988) fractionated the cytosolic component of the reaction into two distinct activities, one sensitive and the other resistant to Nethylmaleimide. Both activities were required for efficient post-translational transport of prepro-a-factor into E R microsomes. The N-ethylmaleimideresistant factor consisted of the two constitutively expressed 70 kDa heatshock proteins of yeast, the SSAlp and SSA2p. Deshaies et al. (1988) similarly reproduced in uitro the in uiuo secretory defect associated with SSAp depletion, and demonstrated reconstitution of the translocationsustaining activity of the depleted cytosol by addition of the SSAlp. That the SSA proteins function to maintain the prepro-a-factor translocation substrate in a transport-competent state was suggested by the finding that pretreatment of the substrate with urea increased the translocation rate (Chirico et al., 1988). The cytosolic N-ethylmaleimide-sensitive translocation factor has not yet been suitably resolved for biochemical analysis.

IV. Protein Trafficking from the Endoplasmic Reticulum to the Golgi Complex

Once a polypeptide has been translocated into the E R lumen, it has essentially overcome the major topological obstacle. That is, the polypeptide no longer has to traverse a lipid bilayer en route to its ultimate destination. As a result (sorting and retention considerations aside), an analysis of further progression through the secretory pathway becomes a matter of understanding: (a) the biogenesis of transport vesicles on the donor

SECRETORY PATHWAY FUNCTION IN M C C H A R O M Y C E S C E R E V l S l A E

89

compartment surface, (b) the targeting of the transport vesicles to the correct acceptor membrane, and (c) fusion of the transport vesicles to the acceptor membrane. Within the broad framework of such a model, one can envisage two general types of components. Firstly, there are those that are commonly employed by various compartments for vesicle metabolism. Secondly, there are those that play compartment-specific roles in vesicular traffic. A . THE MAMMALIAN PARADIGM

A general diagram depicting the prevailing view of steps involved in vesiclemediated intercompartmental protein transport is shown in Fig. 3. The basic form of a single round of vesicle transport was determined by elegant biochemical and morphological studies of protein transport between Golgicomplex compartments in vitro (Orci et af.,1989; Rothman and Orci, 1990). Rothman and his colleagues (for a review, see Rothman and Orci, 1990) proposed that transport vesicles budding from the donor membrane are driven by an ATP-dependent assembly of vesicle-coat protein subunits that ultimately cause pinching off of a coated transport vesicle. Upon docking to the target membrane, the vesicle is uncoated and allowed to enter the fusion pathway. Biochemical studies have revealed that ATP, cytosolic factors, and long-chain fatty-acyl-CoA esters are required for both vesicle budding and for fusion of uncoated vesicles to their target membranes. An N-ethylmaleimide-sensitivefactor (NSF), which is a 76 kDa ATP-binding protein that functions as a tetramer, is required for fusion. The function of this factor requires participation of other protein cofactors, some of which belong to a family of three 36 kDa polypeptides that promote attachment of the factor to membranes known as the soluble NSF attachment proteins (a-, b- and y-SNAPS). Uncoating of transport vesicles occurs at the target membrane and involves GTP-binding proteins, as shown by the finding that a non-hydrolysable analogue of GTP (i.e. GTPyS) inhibited the uncoating reaction. The cytosolic factors identified by resolution of the in vitro transport reaction in the Golgi complex may well represent factors that are commonly employed in budding-fusion reactions. The NSF is already known to be required for fusion of Golgi complex-derived vesicles to Golgicomplex compartments (Malhotra et af., 1988), fusion of ER-derived vesicles to Golgi-complex membranes (Beckers et a f . , 1989) and fusion of endocytic vesicles (Diaz et al., 1989). This will likely prove to be true for the SNAPS as well. That there has been a basic functional conservation in the components that drive intercompartmental protein transport has been demonstrated by Dunphy et af. (1986). Yeast cytosol can substitute for the cytosolic requirement in reactions involving mammalian membranes. As a

90

A . E. CLEVES AND V. A. BANKAlTlS

Acceptor Compartment

I I I

Donor Compartment

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

91

result, yeast provides a facile system with which to study the intercompartmental protein transport problem. A detailed biochemical dissection of transport from the E R to the Golgi complex in mammalian cells has been carried out by Balch and his colleagues (Beckers et al., 1990). These workers have established that transport can be divided into two stages. The first is considered to involve passage of a secretory protein through an early step that requires ATP, NSF, and GTP-binding proteins. It is thought that this stage describes the budding of transport vesicles from the E R surface, subsequent passage of secretory proteins through a still poorly understood intermediate compartment that is operationally defined as the site at which protein transport is blocked in mammalian cells by challenge with cold temperatures (lS0C), docking of transport vesicles to cis-Golgi-complex membranes, and subsequent uncoating of these docked transport vesicles. The second stage is thought to describe late events associated with fusion of vesicles to the cis-Golgicomplex membranes. Calcium ions were cofactors for this stage. Moreover, a role for GTP-binding proteins in fusion of uncoated vesicles to cis-Golgicomplex membranes was suggested by the sensitivity of this late stage to inhibition by a synthetic peptide homologous to the effector domain of rub proteins, a family of small GTP-binding proteins (Beckers et af., 1990; Pliitner et af., 1990). B . RECONSTITUTION OF THE YEAST PROCESS

In Vitro

I. Characterization of the Assay Reconstitution of transport from the E R to the Golgi complex in vitro was independently developed in the laboratories of Randy Schekman (Baker et af., 1988) and Susan Ferro-Novick (Ruohola et al. , 1988). The development of these in vitro systems rested on two technological advances. First, there FIG. 3 Diagram illustrating a single round of vesicular transport. The paradigm showing how proteins are shuttled from one compartment to another in a transport vesicle-dependent manner has been established by both biochemical and morphological criteria (Rothman and Orci, 1990). The cycle involves an ATP- and longchain acyl-CoA-dependent recruitment of cytosolic transport factors (CF) that catalyse formation and targeting of a transport vesicle. Upon engagement of the transport vesicle with the acceptor membrane, an uncoating reaction occurs in a manner that involves GTP-binding protein function, as shown by the inhibition of this particular step by a non-hydrolysable analogue of GTP (i.e. GTPyS), and vesicle-associated factors are liberated into the cytosol for another round of vesicle transport. Finally, fusion of uncoated vesicles to acceptor membranes requires the function of an N-ethylmaleimide (NEM)-sensitivefusion factor, with ATP and longchain fatty-acyl-CoA acting as cofactors.

92

A . E. CLEVES A N D V. A . BANKAlTlS

was the demonstration that radiochemically pure prepro-a-factor was an efficient post-translational translocation substrate (see above), which yielded an unambiguous method for following transport of labelled substrate from the ER to the Golgi complex. Second, a method for preparing “semi-intact” yeast cells by gentle osmotic lysis of sphaeroplasts was developed. These “semi-intact” cells were essentially intact from a cellular standpoint in that the major organelles remained structurally unperturbed and organized. However, the cytoplasm had been released from the cells through perforations in the plasma membrane. Thus, one could now prepare “semi-intact” yeast cells, add radiolabelled prepro-a-factor as a translocation substrate which had access to the cell interior, and subsequently follow transport of the substrate. In this system, glycosylation was again used as a criterion by which to establish that transport occurred. Core glycosylation of the 19 kDa prepro-a-factor resulted in conversion to a 26 kDa form, and this was evidence for transport into the ER. Conversion of the ER form to a heterogeneous higher molecular-weight form that was recognized by antibodies specifically directed against a-( 1+6)-mannose linkages indicated that transport from the ER to the Golgi complex occurred. Incubation of substrate, washed “semi-intact” cells (i.e. membranes), yeast cytosol, an ATP-regenerating system and GDP-mannose (the sugar donor) resulted in conversion of some 25% of the substrate into an antia-( 1+6)-Man serum-precipitable (i.e. Golgi complex) form. This was evidence for an efficient transport of substrate from the ER to the Golgi complex in the in vitro reaction since only 50% of the input substrate entered the ER and was therefore available for transport from the ER to the Golgi complex (Baker et al., 1988; Ruohola et al., 1988). Further characterization of the in vitro system revealed that low-temperature (10°C) incubation did not significantly affect translocation of substrate into the ER, but did effectively preclude transport from the ER to the Golgi complex (Baker el al., 1988). This was an important finding since it permitted analysis of transport as a two-stage reaction. This involved incorporation of substrate into the ER lumen in a 10°C first-stage reaction, subsequent isolation of the donor compartment in a form that was substantially free from cytosolic factors, followed by a warming of the reaction in the presence of fresh cytosol so that the second-stage ER-to-Golgi complex leg of the reaction could be completed. This effectively uncoupled the translocation reaction from the ER-to-Golgi complex transport reaction, and paved the way for a strict analysis of the requirements for the latter. These can be summarized as follows (Baker et al., 1988). First, ATP was required for transport from the ER to the Golgi complex, as apyrase treatment of the second reaction stage completely inhibited the

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

93

ability of substrate to acquire precipitability with a-(l+6)-Man antibodies. Second, GDP-mannose stimulated the reaction but was not required to support it. Third, pretreatment of membranes with the non-ionic detergent saponin completely destroyed transport activity, indicating that organelle integrity was necessary for transport. Fourth, transport was stimulated some six-fold by addition of cytosol. This reflected the involvement of cytosolic proteins, as indicated by the finding that treatment of cytosol with N-ethylmaleimide, heat (15 minutes at 95°C) or trypsin destroyed the stimulatory activity. Fifth, transport was greatly inhibited by addition of GTPyS ( K i 5 p ~ to ) the reaction, and this inhibition was overcome by concomitant addition of excess GTP, suggesting the involvement of GTPbinding proteins in the process. Finally, E G T A was found to inhibit a late step in transport from the E R to the Golgi complex, signifying the requirement for calcium ions as a necessary cofactor in the reaction (Baker et a f . , 1990). Thus, transport from the E R to the Golgi complex in Sacch. cerevisiae exhibited similar requirements to those possessed by the mammalian reaction. That the yeast in vitro reaction faithfully reconstituted bonafide transport from the E R to the Golgi complex was indicated by two independent lines of evidence. First, the Golgi-complex form of the substrate was sequestered within a compartment that could only be sedimented at 100,OOOg. In contrast, the core-glycosylated ER form of the substrate was recovered only in membranes that sedimented at much lower gravitational forces. Furthermore, the E R marker, NADPH4ytochrome-c reductase, was almost quantitatively recovered with the rapidly sedimenting membranes (Baker et af., 1988). These data provided a strong indication of intercompartmental transport of the substrate. Second, it was demonstrated that lysates prepared from sec mutants that were conditionally defective for transport from the E R to the Golgi complex in vivo failed to support such transport in vitro (Baker et a f . , 1988; Ruohola et af.,1988). In secl2 and secl8 mutant lysates, transport was defective at all temperatures. In lysates of secl2 mutants, addition of wild-type cytosol failed to have any restorative effect (as might be expected when membranes are defective) whereas, in lysates of secl8 mutants, supplementation with wild-type cytosol exhibited a rather mild stimulatory effect. The behaviour of sec23 mutant lysates in the in vitro reaction was especially noteworthy. The ts defect in transport from the E R to the Golgi complex observed for sec23‘”mutants in vivo was reproduced in vitro (Baker et al., 1988; Ruohola et a f . , 1988). This in vitro defect was complemented by wild-type cytosol (indicating that the SEC23p was a cytosolic factor) and provided a biochemical complementation assay by which the functional SEC23p was partially purified (Hicke and Schekman, 1989). It was found that the SEC23p exibited a monomeric molecular mass

94

A . E. CLEVES AND V. A . BANKAlTlS

of 85 kDa and copurified with a 105 kDa polypeptide in a large complex (approximately 400 kDa) (R. Schekman, personal communication). 2. Isolation of Transitional Vesicles that Execute Transport from the Endoplasmic Reticulum to the Golgi Complex

Biochemical characterization of the transport vesicles that shuttle between the E R and the Golgi complex has been hindered by their elusive nature. Groesch etal. (1990) took advantage of the observation that these transport vesicles were released from “semi-intact” cells under transport-competent conditions, and could be captured prior to their engagement with acceptor Golgi-complex membranes. Operationally, these vesicles were first identified as a population of substrate molecules that had escaped from cell membranes into the supernatant fraction (Ruohola et al., 1988). Subsequent characterization indicated that the released substrate was pelleted upon centrifugation (around 100,000g) and was resistant to digestion by exogenous protease in the absence (but not the presence) of detergent (Groesch et al., 1990). These data indicated sequestration of released substrate in vesicles. That the vesicles were of a homogeneous population was indicated by equilibrium centrifugation experiments that demonstrated recovery of the vesicles in a single, sharp peak corresponding to the 40% sucrose region of the gradient (1.1764 g ~ m - (Groesch ~ ) et al., 1990). The density of the putative transport vesicles was considerably different from that of the heavier E R membranes, thereby distinguishing the two compartments. Moreover, the yeast E R marker enzyme hydroxymethylglutarylCoA reductase (isozyme 1) was not enriched in the vesicle fraction. This finding eliminated the possibility that such vesicles arose by adventitious fragmentation of the ER. Finally, these vesicles behaved as true transport intermediates by yet another criterion. They were competent to undergo fusion with acceptor Golgi-complex membranes in an ATP- and cytosoldependent manner. Detailed characterization of these transport vesicles now awaits their purification in sufficient quantities so that the protein components that define the vesicle surface can be identified and their precise functions determined. C. MOLECULAR ANALYSIS OF GENES WHOSE PRODUCTS STIMULATE TRANSPORT FROM THE ENDOPLASMIC RETICULUM TO T H E GOLGl COMPLEX

As indicated above, transport from the E R to the Golgi complex can be categorized into three steps: (a) budding of transport vesicles from the cell surface, (b) targeting of the vesicles to the Golgi complex, and (c) fusion of the transport vesicles to the acceptor membrane. A complementary

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVlSlAE

95

approach to an in vitro resolution of the various factors that govern such events has been the detailed analysis of sec mutants that are defective in the transport process in vivo. Nine sec mutants (secl2,secl3, secl6, secl7, secl8, sec20, sec21, sec22 and sec23) and two bet mutants exhibit such a defect. A genetic analysis of these mutants has yielded key insights into which of the three events associated with intercompartmental protein transport is likely to be defective. 1. Early SEC Gene Products Define Functions Involved in Vesicle Budding

and Fusion Kaiser and Schekman (1990) undertook a careful morphological analysis of intracellular membrane structure in wild-type Sacch. cerevisiae and ERblockedsectsmutants of this yeast. The aim was to look for subtle differences in the terminal phenotypes of these mutants to assess the nature of their block in transport from the E R to the Golgi complex. It was found that these nine sec mutants could be categorized into two general categories on the basis of the terminal phenotype. The class-I mutants (secl2,secl3, secl6 and sec23) exhibited an exaggeration of the E R , but did not exhibit any significant proliferation of small vesicles. Class-I1 mutants (secl7, secl8, sec20, sec21 and sec22), in addition to the exaggerated E R phenotype, also exhibited an approximately five-fold increase in the average number of cytoplasmic vesicles with respect to the average number of such vesicles measured in wild-type and class-I mutant strains. The mean diameter of these vesicles was approximately 50 nm, in contrast to the dimensions of Golgi complex-derived secretory vesicles that measured some 80 nm in diameter. The terminal phenotypes were not allele-specific for the gene in question, suggesting that the 50 nm-vesicle proliferation was a gene-specific property. Moreover, class-I phenotypes were epistatic to class-I1 (i.e. vesicle-proliferating) terminal phenotypes. These epistatic relationships suggested that the class-I sec gene products acted at an execution point that preceded the execution point of the class-I1 gene products. On the basis of these data, Kaiser and Schekman (1990) suggested that the class-I sec blocks were exerted at the level of vesicle formation on the E R surface, whereas the class-I1 sec blocks were exerted at the level of transport-vesicle consumption. It should be noted, however, that vesicle proliferation was not as exaggerated for the sec20 and sec21 mutants compared with that observed for the other class-I1 mutants. The distinction between class-I and class-I1 ER-blocked sec mutants on the basis of morphological criteria was reflective of a fundamental difference between the execution points of the corresponding gene products. Construction of a number of haploid sec" double mutants indicated that combinations

96

A. E. CLEVES A N D V . A . B A N K A l T l S

of class-I sec" and class-I1 sectsmutations in haploids did not compromise cell viability and did not cause any exaggerated ts phenotypes. As a general rule, however, haploid cells were unable to tolerate double-mutant combinations restricted to class-I or class-I1 sectsalleles (Kaiser and Schekman, 1990). This intolerance was usually manifested by non-viability but, in a few instances, caused a significant depression in restrictive temperature and general sickliness of the double mutants. In the latter cases, the general morbidity of the double mutants was directly correlated to dramatic decreases in the efficiency of protein transport from the E R to the Golgi complex in vivo. Thus, these synthetic lethalities revealed a set of genetic interactions that redefined precisely the same two sect.* mutant classes that were established on morphological grounds. These findings suggested the occurrence of intraclass functional interactions between members of the class-I gene products and between members of the class-I1 gene products. An extreme extension of this reasoning leads to the suggestion that the intraclass interactions are of a direct nature and reflect the formation of a transport vesicle-budding and fusion machinery, respectively. The two bet mutants (betl and bet2) were isolated via a ['Hlmannose suicide selection and found to exhibit ts defects in protein transport in vivo from the E R to the Golgi complex, and to exaggerate the E R when challenged with restrictive conditions (Newman and Ferro-Novick, 1987). Subsequent characterization of the relationship between BETI and early SEC gene function revealed a pattern of genetic interactions that suggested a direct role for the B E T l p in transport from the E R to the Golgi complex. The relevant findings are summarized as follows (Newman et al., 1990). Genetic crosses revealed that betl sec22 double-mutant haploids were nonviable. That this synthetic lethality was reflective of some real functional relationship between these genes was further supported by the observation that overproduction of the BETI product suppressed the sec22'" defect. Overproduction of the B E T l p was also noted to have a weak suppressive effect on sec2ItS. Another novel gene ( B O S I ) was identified by virtue of its high dosage-mediated suppression of betl'", but not lethal betl null, mutations. Furthermore, BOSlp overproduction also suppressed se~22'~ (Newman et al., 1990). Deciding whether or not BET1 and SEC22 gene products interact directly awaits co-immunoprecipitation experiments. Regardless, the observed genetic interactions of BETl with a class-I1 SEC gene implies a class-I1 designation for BETl as well.

2. Characterization of Early SEC Gene Products A detailed molecular analysis has been carried out for three of the nine SEC genes whose products stimulate transport from the E R to the Golgi

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVlSlAE

97

complex; these are SEC12, SEC23 and SEC18. In at least one case, such an analysis has provided a penetrating insight into the role of the SEC gene product in the transport process. Yeast genomic SECI2 clones were obtained by complementation of the sec12-4'' mutation, and verified by integrative genetic mapping strategies (Nakano et af., 1988). Nucleotide-sequence analysis demonstrated that SECZ2 contained an open reading frame with the potential to encode a 471residue hydrophilic protein, and that the SEC12p exhibited one potential membrane-spanning domain located towards the C-terminus of the protein. Unfortunately, protein similarity searches have not provided any meaningful homologies that might provide some insight into the function of the SEC12p. Nonetheless, the SEC12p plays an essential role in yeast vegetative growth as shown by the lethality associated with secl2 null mutations (Nakano et af., 1988). Immunodetection of the SEC12p has revealed some interesting features concerning biogenesis of this protein (Nakano et af.,1988). First, the protein could only be detected in yeast cells that were overproducing it. These data indicated that the SEC12p was not an abundant protein, and this conclusion was consistent with the estimate that there were only two to four SEC12 mRNA transcripts in each haploid yeast cell. Second, the observed SEC12p molecular mass (70 kDa) was considerably larger than the one predicted from the SEC12 nucleotide sequence (52 kDa). This discrepancy was due to glycosyl modification of the gene product at one or two N-linked sites, and additional glycosylations of presumably the O-linked variety. Interestingly, even though the SEC12p exhibited both types of glycosyl modification at short times after synthesis (as a 65 kDa form), the protein experienced a slow but progressive glycosylation with time until it finally achieved its mature apparent molecular mass of 70 kDa some three hours after synthesis. This slow maturation did not involve acquisition of N-linked oligosaccharide chains, was blocked by cycloheximide treatment (thereby revealing a role for ongoing protein synthesis) and was also blocked by the secZ8'' defect, suggesting participation of the Golgi complex in the slow terminal maturation process. A role for the Golgi complex in SEC12p biogenesis was further suggested by immunoelectron-microscopy experiments that demonstrated a specific labelling of exaggerated sec7 Golgi-complex stacks (see below) with gold-labelled SEC12p antibodies. The significance of this finding was somewhat diluted, however, by the inability to visualize the gene product in wild-type cells. Therefore it remains unclear as to what fraction of the total cellular SEC12p is localized in the Golgi complex at any given time. Finally, subcellular fractionation experiments indicated an integral membrane location for the SEC12p, and that the product was predominantly localized in a rapidly sedimenting membrane fraction that contained the bulk of the

98

A . E. CLEVES AND V. A . BANKAlTlS

cellular E R (Nakano et al., 1988). The finding that the SEC12p is an integral membrane protein was consistent with the in vitro data which indicated that secZ2 membranes were defective in transport from the E R to the Golgi complex (Section 1V.B). However, the class I designation for secZ2 mutants is more readily incorporated into a scenario where the SEC12p is found in ER rather than exclusively Golgi-complex membranes. It should be noted that the SEC12p could be functionally localized in both Golgi-complex and ER membranes, a situation that might be indicative either of a recycling of the gene product between the E R and Golgi-complex membranes or the existance of multiple execution points with only the first one being phenotypically apparent. This last point is treated in more detail later in this review. Genomic clones of SEC23 have also been obtained and characterized (Hicke and Schekman, 1989). Sequence analysis demonstrated that SEC23 had the potential to encode a hydrophilic protein of 768 amino-acid residues (M,85,400) that did not exhibit any obvious candidate signal peptides or membrane-spanning regions. While protein similarity searches failed to reveal any informative homologies, it was noted that the SEC23 sequence was identical with that of NUCZ, a yeast gene identified by mutants defective in nuclear transport. While the complete ramifications of this identity remain unclear, some general remarks to this effect were made for the analogous SEC63-NPLZ identity (see Section III.B.2). In any event, the SEC23p was essential for yeast vegetative growth, as shown by the lethality associated with sec23 null mutations (Hicke and Schekman, 1989). During the course of these gene-disruption experiments it was noted that the heterozygous sec23'"l~ec23~ condition caused death of diploid cells, even though the mutant protein exhibited a wild-type abundance. On the basis of this intriguing observation, Hicke and Schekman (1989) suggested that the SEC23p was a limiting factor for cell growth. Immunodetection of the SEC23p confirmed the basic inferences that were forthcoming from the SEC23 nucleotide-sequence data. The protein was found to be unglycosylated with a monomeric molecular mass of some 84 kDa. Although subcellular fractionation experiments indicated that the SEC23p from both wild-type and mutant strains sedimented with membranes at 100,000g in p H 6.5 buffer, subsequent analyses demonstrated that the association of the SEC23p with intracellular membranes was of a peripheral nature (Hicke and Schekman, 1989). First, the SEC23p was completely digested by protease in cell-free lysates where the integrity of intracellular organelles was maintained. These data indicated a cytoplasmic location for the SEC23p. Second, the SEC23p was rendered at least partially soluble by treatment of the lysate with 2.5 M urea, 0.5 M potassium acetate, 25 mM EDTA or buffer (pH 7.5). Release of the SEC23p under these

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVlSlAE

99

conditions was also consistent with the behaviour of a peripheral, rather than integral, membrane protein. While the precise biochemical function of the SEC23p is presently unresolved, on the basis of purification it seems likely that it functions in a multicomponent complex (Section IV.B.1), perhaps at the level of stimulation of transport-vesicle formation (i.e. SEC23 is a class-I gene: see Section IV.C.1). While molecular analysis of the SEC12 and SEC23 genes has not yet yielded remarkable insights into SEC12p and SEC23p function, molecular analysis of SECZ8 has converged with in vitro studies of intercompartmental protein transport in mammalian systems to provide several key findings relating to the enzymology of transport-vesicle metabolism (Rothman and Orci, 1990). The SECI8gene was characterized and its product identified by Eakle et af. (1988). This gene was found to encode potentially for an 84 kDa polypeptide that was essential for cell growth, as shown by the haploid-lethal nature of secZ8 null mutations. Immunoprecipitation experiments utilizing SEC18p-specific antisera revealed two cross-reacting species in yeast lysates. One exhibited a molecular mass of 84 kDa whereas the other was an 82 kDa polypeptide. These SEC18p species appeared to arise from differential translation-initiation events rather than heterogeneity in transcription-start sites or some other sort of processing events. Both SEC18p species fractionated identically in subcellular fractionation experiments; these forms exhibited essentially equal distributions between the 100,OOOg pellet and supernatant fractions. Thus, the SEC18p also behaved as a peripheral membrane protein. Although initial homology searches failed to provide any insight into SEC18p function (Eakle et af.,1988), such an end was realized by Rothman and his colleagues who undertook a molecular characterization of the structural gene for the mammalian NSF (Wilson et al., 1989). The nucleotide sequence data predicted a hydrophilic polypeptide of some 83 kDa, which was consistent with the monomeric molecular mass of the NSF as determined by SDS-PAGE (76 kDa). The striking finding was that the NSF and SEC18p shared a 48% identity and 63% similarity (allowing for conservative substitutions) over their entire primary sequence. This homology suggested that the NSF and SEC18p exhibited similar biochemical activities and, on the basis of several criteria, this was conclusively established by Wilson et af. (1989). First, both the NSF and SEC18p were shown to be ATP-binding proteins, and it was determined that both proteins were stabilized by ATP. Second, overproduction of the SEC18p in yeast cells resulted in a concomitant elevation of NSF activity in yeast lysates. Finally, secl8‘” mutants failed to produce any NSF activity that could be assayed in vitro. Taken together, these data indicated that the SEC18p was the yeast NSF,

100

A . E. CLEVES A N D V. A . RANKAlTlS

and these data demonstrated that the SEC18p was involved in fusion of ER-derived transport vesicles to the acceptor Golgi complex. It should be noted that this conclusion was entirely consistent with the general interpretation of class-I1 sec mutants, namely that those mutants were defective in consumption of ER-derived transport vesicles (Kaiser and Schekman, 1990). The discovery that the SEC18p is the yeast NSF has yielded significant insight into the biochemical function of at least one other SECgene product. As already noted (Section IV.A), NSF function required participation of other protein cofactors, most notably the three SNAPS (a, P and y) which promoted association of the NSF with Golgi-complex membranes. Clary et a f .(1990) found that yeast cytosol elaborated SNAP activity and, moreover, that yeast SNAP was responsive to the SEC18p but not to the mammalian NSF. A functional in vitro assay for yeast SNAP activity was devised and cytosol preparations from wild-type secl6", secl 7 ts ,sec2@, sec21ts,sec22" and sec23" strains were screened. The sec17" cytosol was uniformly defective in in vitro SNAP activity at all temperatures, and could be complemented by wild-type cytosol in a SEC18p-independent fashion. Moreover, purified bovine a-SNAP corrected the transport defect associated with secl7" cytosol. It was noteworthy that inclusion of purified P-SNAP or y-SNAP failed to complement the secl7" defect in the transport reaction. Although it has not yet been proven that SECZ7is the structural gene for the yeast a-SNAP, it is tempting to believe that this will prove to be correct. The biochemical data are in striking agreement with the genetic arguments for a functional interaction of the SEC18p with the SEC17p, and with the class-I1 morphological categorization of these two activities as being required for consumption of transport vesicles at the acceptor membrane (Section 1V.C.l). Thus, the present data argue strongly for an adapter role for the SEC17p in delivery of the SEC18p to Golgi-complex membranes, perhaps as a crucial event in the building of a transport-vesicle "fusion machine". The finding that the SEC18p is the yeast NSF, and that the SEC17p probably represents the yeast analogue of a-SNAP, is indicative of one final point. This is that many (and perhaps all) of the SECgene products assigned to the ER-to-Golgi complex stage of transport participate at many steps along the secretory pathway and therefore represent general factors involved in transport-vesicle metabolism. Assignment of these gene products to an early stage of the secretory pathway was based upon an analysis that could only reveal the first execution point (Novick e t a f . ,1980). In this regard, we note that the SEC23p and the SEC18p are also required for fluid-phase endocytosis in yeast (Riezman, 1985).

SECRETORY PATIIWAY FUNCTION IN SAC‘CHAROMYCES CEREVISIAE

101

3. GTP-Binding Proteins

To date, three GTP-binding proteins have been implicated in transport from the ER to the Golgi complex in Sacch. cerevisiae; these are the SA R l , Y P T l and ARF gene products. The data suggest that at least this stage of the secretory pathway requires the action of multiple GTP-binding proteins. The SARI gene was initially obtained in attempts to clone the SEC12 gene, where it was recognized as a multicopy suppressor of secl2‘” (Nakano etal., 1988). Overproduction of the S A R l p was not capable of suppressing the lethality of secl2 null mutations, indicating that increased S A R l p function diminished, but did not bypass, the need for SEC12p function (Nakano and Muramatsu, 1989). Interestingly, as little as a presumed twofold increase in SARl gene dosage was sufficient for efficient suppression of secl2”. Nucleotide-sequence analysis suggested that the SARlp was a polypeptide of 190 residues that exhibited significant homology to the ras family of proteins, while gene-disruption experiments demonstrated that SARl was essential for cell growth and viability. To test whether the function of S A R l p was relevant to secretory pathway function (as implied by the genetic interaction with secl2‘”), Nakano and Muramatsu (1989) engineered SARI expression that was under G A L promoter control, and investigated the consequences of depletion of the S A R l p in cells. These workers found that S A R l p deficiency was specifically manifested by a secretory defect. In particular, core-glycosylated forms of the secretory prepro-a-factor and vacuolar proCPY were found to accumulate intracellularly. Genetic interactions of SARl with SEC12 implicate a class-I (i.e. transport-vesicle formation) execution point for the SARlp. A more precise understanding of the function of the S A R l p awaits localization of the protein to some clearly definable membrane structure. The gene Y P T l was originally identified by Gallwitz et al. (1983) as an open reading frame situated between the actin and j3-tubulin structural genes. Interest in Y P T l was stimulated by virtue of the finding that the inferred 207-residue YPTlp exhibited significant sequence identity with the human ras proto-oncogene products. Subsequent gene-disruption experiments revealed Y P T l to be essential for yeast-cell viability (Schmitt et al., 1986; Segev and Botstein, 1987), and the study of temperatureconditional mutants (both ts and cs) indicated that loss of YPTlp function had profound effects on secretory pathway function, calcium-ion metabolism, microtubule organization, and nuclear metabolism (Schmitt et a l . , 1988; Segev et al., 1988). Schmitt et al. (1988) proposed that the primary defect in yptl‘” strains was related to impaired homeostasis of calcium ions, in part because the yptl‘” growth phenotype was calcium-ion remedial and uptake of these ions was greatly increased in these mutants. Segev et al. (1988),

102

A. E. CLEVES A N D V. A . BANKAlTlS

however, argued for a primary role for the YPTlp in stimulating secretory pathway function. This hypothesis was supported by their findings that: (a) the YPTlp was located in the yeast Golgi complex in indirect immunofluorescence experiments utilizing wild-type cells, (b) a mammalian YPTlplike protein was also associated with the Golgi complex in mouse L-cells, (c) loss of YPTlp function resulted in a proliferation of yeast Golgi-complex membranes reminiscent of that observed for Golgi complex-blocked sec7’” mutants, and (d) abnormally high intracellular pools of secretory invertase were accumulated under restrictive conditions. Curiously, the secretory defect was only partial and it was noted that the secreted invertase was remarkably undermodified, regardless of whether restrictive or permissive conditions were imposed. T o resolve some of the major questions regarding YPTlp function, and the role of calcium-ion metabolism, Bacon et al. (1989) and Baker et al. (1990) analysed the dependence of the in vitro ER-to-Golgi complex transport reaction (Section 1V.B) on the function of the YPTlp. It was found that the system was dependent on this function, as shown by lack of transport detected in cell lysates prepared from yptl mutants and the inhibitory effect of addition of anti-YPTlp Fab fragments to the reaction mixture. More detailed fractionations revealed that it was the acceptor Golgi-complex membranes that were defective for transport in the yptl mutants. Titration of the free calcium ions present in the in vitro reactions, over a wide range of concentrations, failed to remedy yptl defects, and consumption of the calcium ion-requiring intermediate was not inhibited by anti-YPTlp Fab fragments. These data indicated that YPTlp and calcium ions acted at functionally independent execution points (Baker et al., 1990). Thus, it seems unlikely that YPTlp functions primarily to control intracellular concentrations of calcium ions. Rather, the YPTlp functions essentially to stimulate protein transport through the secretory pathway. At present, the YPTlp is considered to be involved in protein transport through the Golgi complex, largely on the inference that the undermodified invertase elaborated by yptl mutants possessed early Golgi-complex carbohydrate modifications (for a discussion, see Bacon et al., 1989). It remains less clear whether the YPTlp is also involved in fusion of ERderived vesicles to the Golgi complex. This problem will be resolved once the nature of the secretory block associated with depletion of the functional YPTlp in vivo is determined. The yeast ARFl gene product was recognized as a homologue to the mammalian ADP-ribosylation factor (ARF), a ubiquitous and highly conserved 21 kDa GTP-binding protein that is related to both ras and GTPbinding protein a-subunit families (Sewell and Kahn, 1988). The gene ARFl was shown not to be essential for vegetative growth because of a redundant

SECRETORY P A I H W A Y FUNCTION IN SACCHAROMYCES CEREVISIAE

103

function that was expressed at low levels from the ARF2 gene, which encodes a polypeptide that exhibits 96% primary sequence identity with the ARFlp (Stearns et al., 1990). Whereas arfl" arf2' double mutants were inviable, arfl" mutants exhibited a number of phenotypes, including cold sensitivity for growth. Analysis of the secretory competence of arfl" yeast strains revealed that they were partially impaired in secretion ability at both permissive and restrictive temperatures, and that the accumulated and secreted invertase populations were uniformly undermodified. These invertase-undermodificationand partial secretory block phenotypes mimicked the defects seen in yptl strains. Stearns et al. (1990) also analysed the consequences of depleting cells of all A R F activity. The data showed an ERto-Golgi complex secretory block in those cells. Finally, immunocytochemistry demonstrated a Golgi-complex localization of the mammalian ARF in NIH3T3 cells, and it appeared that there may have been some enrichment of A R F on the cytoplasmic face of cis-Golgi-complex membranes. Bourne (1988) proposed a mechanism for the involvement of GTPbinding proteins in regulation of vesicular transport. The basic tenets of this model are discussed in Section V1.A. D. THE RETENTION PROBLEM

The E R provides the proteins and phospholipids that sustain bulk secretory flow through the secretory pathway. As a result, the E R experiences a massive efflux of proteins and phospholipids of such magnitude that Wieland et al. (1987) estimated that the lumenal E R volume is turned over within approximately 10 minutes in mammalian cells. That the integrity of the ER is maintained under these conditions is testimony to the remarkably efficient mechanisms for retention of resident E R proteins in the face of such massive protein efflux from the reticulum, and retrieval of phospholipids back to the ER. The latter point is discussed in the following section of this review (Section V.C). Finally, the E R also plays a quality-control function in the sense that it is within this compartment that unfolded or malfolded polypeptides are withheld until the appropriate conformations are achieved and further transit through the secretory pathway is permitted.

I . Retention of Incompletely Assembled Polypeptides

How does the E R monitor the assembly state of polypeptides within its lumen? Although polypeptide properties that determine their ability to be assembled remain mysterious, studies on mammalian cells have identified a resident E R protein termed BiP (binding protein) that is intimately involved in recognition of the state of polypeptide assembly, or perhaps even catalysis

104

A. E. CLEVES AND V. A. BANKAlTlS

of the assembly process itself (Munro and Pelham, 1986; Gething et al., 1986; Bole et al., 1986). As such, BiP fulfills some general criteria associated with a sorting function. That is, it sorts polypeptides not yet ready to leave the ER from those that have assembled and are therefore competent to enter the default pathway for exit (Pfeffer and Rothman, 1987; Rothman, 1989). Saccharomyces cerevisiae has a BiP homologue and it is encoded by the KAR2 gene (Rose et al., 1989). Originally, KAR2 was identified in a search for mutants that failed to undergo karyogamy (i.e. nuclear fusion) during the mating process (Polaina and Conde, 1982). The recognition that KAR2 encodes yeast BiP came from a molecular analysis of KAR2 and studies of its transcriptional regulation. Authentic KAR2 clones were recovered by Rose et al. (1989) and the nucleotide sequence was determined. The KAR2 gene was inferred to encode a 682-residue protein that exhibited a 67% identity with mammalian BiP, and similar homologies to members of the yeast, amphibian, insect and bacterial HSP70 family. Several other structural features were noted that were suggestive of equivalence of BiP and the KAR2p. These included the findings that: (a) the KAR2p (like BiP) contained a functional signal peptide that directed its entry into the secretory pathway in a SEC6lp-dependent fashion, a feature not exhibited by any of the other HSP70 cognates, and (b) the KAR2p contained a Cterminal H D E L sequence that functions in retention by the E R in yeast (Section IV.D.2), implying a lumenal E R location for this gene product (as with BiP in mammalian cells). This latter point was confirmed by immunofluorescence experiments, coupled with protease protection experiments that independently assigned a lumenal location for the protein. Finally, the demonstration that KAR2 is an essential gene (Rose et al., 1989; Normington et al., 1989), coupled with the genetic demonstration that KAR2 was not allelic to SSCZ (the sole member of the eight previously identified yeast HSP70 cognate-gene family that is essential for growth; Craig et al., 1987), identified KAR2 as a ninth member of the yeast HSP70 family. The inability of the other HSP70 cognates to supply redundant functions is probably related to the unique compartmentalization of the KAR2p within the E R lumen. Mammalian BiP and the KAR2p were also found to exhibit several common features with respect to their transcriptional regulation (Rose et al., 1989; Normington et al., 1989). Challenging of yeast cells with either 2deoxyglucose or tunicamycin, a drug that inhibits addition of N-linked glycosyl chains to secretory proteins, resulted in a five- to 10-fold elevation in the level of KAR2 mRNA. Treatment with the calcium ionophore A23187 resulted in a slight elevation. These same conditions induce expression of the mammalian BiP gene, and are specific for KAR2 as these fail to induce expression of other HSP70 cognates. However, heat shock also induced

SECRETORY PATHWAY FUNCTION IN SACCHAR0MYCI.S CEREVISIAE

105

KAR2 expression. This induction occurred rapidly, as a measurable increase in the levels of the KAR2 message was apparent after some five minutes of heat shock, and maximal induction (about seven-fold over basal level) was reached some 30 minutes after imposition of heat shock. Interestingly, this induced level of expression was not sustained. It decayed rapidly after achievement of peak induction, and basal levels of KAR2 expression were re-established after one hour of heat shock. It is similarly noteworthy that BiP synthesis is not heat-shock inducible. To investigate further the role of the KAR2p in vivo, Vogel et al. (1990) generated a number of kar2" alleles and characterized the tightest of these, kar2-159, in detail. Imposition of non-permissive conditions onto kar2-159 cells led to a rapid and irreversible loss of viability. The particular defect associated with the kar2-159 lesion involved defects in bud emergence and growth, suggestive of defects in the secretory pathway. Analysis of the biogenesis of two secretory proteins (invertase and prepro-a-factor), and a vacuolar proteinase (carboxypeptidase Y), yielded a surprising result. This was that these polypeptides failed to be translocated into the E R lumen under conditions of the kar2-159 block. This was surprising because the kar2-159 defect was observed for events involving the cytosolic face of the ER, whereas the KAR2p is a lumenal E R component. This translocation defect was reproduced when functional KAR2p was depleted in yeast, confirming that this defect was the result of loss of KAR2p function rather than some sort of disruptive action of the defective kar2-159 gene product. It was also noteworthy that nuclear fusion could be uncoupled from translocation using these mutants. Thus, the karyogamy defect associated with kar2159 occurred under conditions that were entirely permissive for E R translocation. The kar2-1 lesion also lacked secretory effect, even though nuclear fusion was unconditionally defective in these strains. The unanticipated E R translocation defect in kar2-159 strains is not completely understood. Vogel et al. (1990) proposed two general models for KAR2p function that seek to account for this unexpectedly early execution point. First, the KAR2p may be required to maintain polypeptides in an unfolded state during the translocation process. In strains unable to synthesize the KAR2p, an unscheduled and premature folding of polypeptides in transit might somehow inactivate the secretion machinery, resulting in an ER translocation defect. A broadly similar function has been alluded to with the SEC63 gene product (Section III.B.2). The second model suggests that the process of protein translocation through the E R consumes some component of the secretion machinery, perhaps by a stable association of such a component with the secretory polypeptide during transit. In this scenario, the KAR2p would function in a dissociation event that permits recycling of the secretion-machinery component. Further

106

A. E. CLEVES AND V . A. BANKAITIS

experimentation is required to determine which, if either, of these possibilities is correct. 2. Retention of Resident Endoplasmic-Reticulum Proteins

Two basic mechanisms have been considered for retention of resident ER proteins, namely a receptor-mediated mechanism and a mechanism that involves a structural exclusion of resident proteins from packaging into transport vesicles. In the former mechanism, it is considered that resident ER proteins are sorted from the secretory pathway by means of a retention signal that is recognized by a specific receptor. In the mammalian system, Pelham and others have shown that a C-terminal peptide sequence (KDEL) is necessary and sufficient for retention in the E R (Munro and Pelham, 1987; Pelham, 1988; Ceriotti and Colman, 1988). Interestingly, the KDELdependent retention system appears to involve an efficient retrieval from a compartment of the secretory pathway located after the E R (Pelham, 1988; Ceriotti and Colman, 1988). Pelham et al. (1988) have shown that yeast also exhibits an analogous sorting system. The KAR2p has a C-terminal HDEL sequence that closely resembles the mammalian KDEL retention signal, although these signals are not interchangeable. Attachment of the FEHDEL sequence directly to the C-terminus of secretory invertase failed to cause retention of the tagged secretory protein within the cell. However, when an appreciable linker (55 residues) was engineered onto the C-terminus of invertase, followed by FEHDEL, efficient retention was observed (Pelham et al., 1988). The results with prepro-a-factor were more convincing. Engineering of an 11residue linker, followed by the FEHDEL sequence, to the C-terminus of prepro-a-factor resulted in efficient retention of the secretory protein within the cell (Dean and Pelham, 1990). Efficient retention required that the HDEL sequence be at the extreme C-terminus of the protein. An FEHDELS sequence was a poor retention signal, albeit a signal nonetheless (Pelham et al., 1988). Furthermore, efficient retention required a low rate of tagged secretory protein synthesis, suggesting some saturability in the retention system. This saturability was confirmed in experiments in which it was demonstrated that high-level synthesis of HDEL-tagged prepro-afactor not only resulted in its own secretion, but also secretion of an endogenous HDEL-containing protein (i.e. the KAR2p) (Dean and Pelham, 1990). The saturable nature of the retention system suggested the existence of an HDEL receptor, the identity of which has probably been determined (see below). Does HDEL-dependent retention of proteins involve a true retention mechanism or does it reflect an efficient retrieval system? Current data

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

107

strongly suggest the latter. Subcellular fractionation experiments have indicated that the retained HDEL-tagged secretory protein was localized in the yeast E R on the basis of its sedimentability with membranes at 10,000g and cosedimentation in sucrose gradients with core-glycosylated proCPY (used as a marker for E R lumen; Dean and Pelham, 1990). This was a pleasing result as it was consistent with retention of the tagged secretory protein in the appropriate intracellular compartment. However, analysis of the glycosyl modifications of the tagged secretory protein revealed that the glycosyl chains were not of the core variety but, rather, had been more extensively modified. Specifically, the HDEL-tagged secretory proteins had acquired at least some mannose residues in a-(1+6) linkage, an early Golgicomplex modification, in a SEC18p-dependent manner. However, the HDEL-containing reporter protein did not acquire late Golgi-complex glycosyl modifications (i.e. mannose residues in a-(1-+3) linkage). A discussion of N-glycosylation and its compartmentalization in yeast is presented in Section V.A.2. Taken together, these data were incorporated into a model in which H D E L is recognized by a specific receptor that functions in retrieval of resident E R proteins from an early Golgi-complex compartment (Dean and Pelham, 1990). The ability of H D E L to confer E R retention on secretory invertase provided a basis for direct selection for mutants that fail to retain tagged invertase but, rather, secrete it. This selection was based on a general scheme that was first employed to isolate mutants that were defective in targeting of proteins to the yeast vacuole (Bankaitis et af.,1986b). A total of 50 such erd (ER retention defective) mutants were isolated, and two well-defined complementation groups (i.e. E R D l and ERD2) identified (Hardwick et af., 1990). Cells lacking either functional E R D l p or ERD2p secreted significant amounts of HDEL-tagged invertase and native HDELcontaining polypeptides (i.e. the KAR2p) (Hardwick et al., 1990; Semenza et af., 1990). However, the rate of KAR2p secretion was very slow in erdl strains (the half-life was seven hours as opposed to less than three minutes for normal secretory proteins), and these strains exhibited normal intracellular levels of the KAR2p as well. This homeostasis was possibly the result of elevated KAR2p synthesis in erdl cells. A further, an puzzling, curiosity associated with erdl mutants was that their erd phenotypes were nutritionally remedial, in that growth in medium containing low concentations of sulphate suppressed the KAR2p secretion phenotype (Hardwick et af., 1990). Clones of E R D l were recovered by Hardwick and his coworkers (1990) and studied in some detail. The nucleotide sequence revealed a single long open reading frame that had the potential to encode a 362-residue polypeptide whose primary sequence was not homologous with that of any

108

A. E. CI.EVES AND V. A . BANKAlTlS

other known protein. Furthermore, the ERDlp was inferred to be an integral membrane protein since it exhibited several candidate transmembrane domains. Some indirect support for this conclusion was forthcoming from gene-fusion experiments demonstrating that the first 101 residues of the ERDlp were sufficient to permit transfer of invertase into the ER lumen. Unfortunately it was not determined whether or not the ERDlpinvertase fusion polypeptide was an integral membrane protein. Gene-disruption experiments revealed ERDl to be a non-essential gene, and all erdl phenotypes were reproduced in the null mutant. Interestingly, erdl null mutants exhibited severe defects in Golgi complex-dependent modifications of N-linked oligosaccharide chains for at least three glycoproteins, namely CPY, invertase and a polypeptide that exhibited immunological cross-reactivity to invertase. Taken together, the data suggested a Golgi-complex defect in erdl mutants. However, neither sorting of CPY to the vacuole (a late Golgi-complex event) nor protein transport from the Golgi complex to the cell surface appeared to be affected. Furthermore, loss of ERDlp function did not result in morphologically aberrant Golgi complexes. At present, it seems most likely that erdl defects result in a general perturbation of Golgi-complex function. Although the collective data imply an indirect effect of the E R D l p on the HDEL-dependent ER retention system, they are nevertheless strongly supportive of the idea that the HDEL system involves an efficient retrieval of protein from the Golgi complex back to the ER. Given such a scenario, it seems probable that the ERDlp will be found to be in the Golgi complex. Current evidence strongly suggests that the ERD2p is the HDEL receptor. Other than the initial observation that erd2 mutants fail to retain HDEL-containing polypeptides, there are two additional lines of evidence that identify the ERD2p as the HDEL receptor. First, levels of ERDZ expression are directly proportional to the capacity of the HDEL-retention system. Overproduction of the ERD2p suppressed secretion of HDELtagged prepro-a-factor or the endogenous KAR2p, caused by high levels of synthesis of the tagged species (Semenza et al., 1990). Second, the specificity of the retention system was determined by the ERD2p. The budding yeast Kluyveromyces lactis apparently exhibits two ER retention signals, namely HDEL and DDEL, and Sacch. cerevisiae is incapable of recognizing DDEL. Exchange of ERDZ from K . lactis to Sacch. cerevisiae for the native ERD2 conferred upon the recipient cells the ability to recognize both HDEL and DDEL (Lewis et al., 1990). Cloning of ERDZ revealed an open reading frame of 219 residues that could encode a hydrophobic 26 kDa polypeptide, an inference that was confirmed by identification of the ERD2p as a 26 kDa integral membrane protein. Immunofluorescence studies indicated that the ERD2p was not in the ER but rather in some other cytoplasmic organelle

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

109

that may represent the Golgi complex. In any event, disruption of ERD2 proved to be a lethal event, although depletion experiments indicated that arrested cells remained viable for extended periods of time. The primary defect associated with depletion of the ERD2p appeared to be at the level of Golgi-complex function, as with dysfunction of the E R D l p . This erd2 Golgi-complex dysfunction was manifested by likely defects in transport of vacuolar CPY from the late Golgi complex, and by abnormal elaboration of intracellular membranes, although it remains unclear as to whether these proliferating membranes were Golgi-complex derived (Lewis et at., 1990). Thus, as anticipated, the H D E L receptor appears to act at the level of the Golgi complex, most likely by a retrieval mechanism. It remains unclear, however, as to why erd2 defects affect late events in the Golgi complex (i.e. transport of CPY) when previous data suggested that retrieval occurred from an early Golgi-complex compartment (see above). Perhaps the ERD2p has multiple functions, only one of which is involved in HDELdependent retrieval of polypeptides from the Golgi complex back to the E R (Lewis et at., 1990).

3. Speculations on the Role of Calcium Ions in Endoplasmic-Reticulum Retention

A second independent mechanism for retaining resident proteins in the ER could involve their exclusion from those areas of the reticulum that participate in formation of transport vesicles. Sambrook (1990) suggested a model for how calcium-ion sequestration within the E R lumen could participate in such an exclusion mechanism. This model was based on two lines of evidence. The first of these was the observation that treatment of mammalian cells with the calcium ionophore A23187 resulted in a transient secretion of resident E R proteins (i.e. BiP, protein disulphide isomerase and CRP55) and a striking dispersion of the reticulum itself (Booth and Koch, 1989). Since the E R lumen serves as the major intracellular reservoir for calcium ions, these data suggested a role for elevated lumenal calcium-ion concentrations in retention of resident E R proteins. The second line of evidence was obtained from a study of yeast mutants defective in a Ca2+-ATPase pump, the product of the PMRI gene, which probably functions in mobilization of calcium ions into the ER lumen (Rudolph et al., 1989). Interestingly, this gene was originally identified in a search for mutants that exhibited more efficient secretion of heterologous proteins engineered for expression in yeast, and was termed SSCl (Smith et al., 1985). This gene is not to be confused with the structural gene for the mitochondria1 HSP70 (Section 1V.D). Detailed analyses of wild-type and pmrl strains revealed

110

A . E. CLEVES AND V. A. BANKAITIS

that native secretory proteins, although efficiently secreted from pmrl strains, were remarkably underglycosylated in a manner similar to that observed foryptl mutants (Section IV.C.3). Moreover, P M R l was found to be a non-essential gene while p m r l null mutants grew poorly under conditions of calcium-ion stress (Rudolph et al., 1989). That loss of Ca2+-ATPase function could alleviate secretory difficulties experienced by heterologous proteins engineered for secretion by yeast implied an unscheduled export of protein from the E R , a point that was consistent with the underglycosylation of secretory proteins in p m r l mutants. However, it remains to be determined whether p m r l mutants exhibit true erd phenotypes. Nevertheless, the yeast prnrl data were at least broadly consistent with the conclusions concerning calcium ions and E R retention in mammalian cells. How then might high levels of calcium ions in the E R lumen contribute to retention of resident ERproteins? Sambrook (1990) proposed that the ERis loaded with proteins (such as BiP) having reasonable calcium ion-binding capacities. These could form an extensive calcium-co-ordinated matrix involving interactions with each other and with the appropriate anionic phospholipid head groups in the inner leaflet of the E R membrane. Transport-vesicle formation at the E R surface might involve fluxes of calcium ions across the E R membrane, and resident E R proteins would largely be excluded from being packaged into such transport vesicles because of their being immobilized in the lumenal calcium-ion-protein matrix. Any residents escaping such an immobilization would be retrieved from the Golgi complex via the receptor-mediated H D E L system, while wholesale depletion of calcium ions from the E R lumen, induced by either the presence of A23187 in mammalian cells or absence of a functional Ca2+-ATPase in yeast, would lead to disintegration of the calcium-ion-protein matrix and subsequent disorganization and vesicularization of the E R . It is this disruption of the calcium-ion-protein matrix that is thought to lead to unscheduled secretion of resident E R proteins (Sambrook, 1990). A final point regarding the effect of p m r l null mutations on yeast secretory function deserves comment. Rudolph et al. (1989) observed that pmrl null mutations suppressed conditional lethal yptl defects. These workers then speculated that loss of Ca2+-ATPasefunction may bypass the YPTlp requirement. This was an interesting suggestion since the YPTlp had, at one point, been considered to play a role in calcium-ion homeostasis in yeast (Schmitt etal., 1988). Given our present understanding, however, it seems unlikely that bypass of the YPTlp is a consequence of defects in P M R l . Rather, p m r l null mutations more likely cause yeast to fail to sequester calcium ions in the E R lumen. This may lead to an elevated

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

111

cytosolic calcium-ion concentration that exerts a remedial effect on a labile Yptlcs activity (see Section IV.C.3).

V. The Golgi Complex as a Secretory Organelle It has recently become appreciated that the Golgi complex occupies a unique position in the cellular scheme for protein-traffic control. This organelle plays a fundamentally important role in regulating several aspects of protein and membrane movement throughout the cell. For example, the Golgi complex is the compartment for sorting of proteins from the constitutive secretory pathway to the lysosomal and regulated secretory pathways. Furthermore, the Golgi complex is involved in regulation of membrane recycling from the cell surface and in communication with aspects of the endocytic pathway (Griffith and Simons, 1986). Finally, it must be stressed that the Golgi complex represents a segmentally structured and inherently compartmentalized organelle across which secretory protein traffic must flow. In order to understand fully how the Golgi complex functions, two basic informational requirements must be met. First, one must appreciate the architecture of the organelle from the perspective of knowing the identity and function of the polypeptides that are indigenous to the complex. Second, one must know the identity and function of polypeptides that drive, or otherwise regulate, Golgi-complex secretory functions. Although significant progress has been made on both counts, our understanding of each remains limited. Electron microscopy has indicated a conservation of Golgi-complex morphology across the eukaryotic kingdom. This organelle consists of a set of some five o r more cisternae that exhibit flattened centres and dilated rims (Farquhar, 1978; Tartakoff, 1980). The aspect of the Golgi complex that lies in apposition to the transitional ER has been designated as the cis-face while the aspect oriented towards the plasma membrane has been designated as the trans-face (Ehrenreich et al., 1973). The intermediate cisternae lying between cis- and trans-Golgi-complex aspects define the medial aspect of the Golgi-complex. The observations of Bergmann and Singer (1983) and Saraste and Hedman (1983), which indicated a vectorial transport of glycoproteins across the Golgi complex in the cis- to trans-direction, suggested that the complex was not composed simply of repeating units of compositionally identical cisternae but, rather, was organized in a functionally asymmetric fashion. Some of the key advances in Golgi-complex research in recent years have confirmed demonstrations of the compartmental organization of the Golgi complex. More specifically, four lines of research have converged to a

112

A. E. CLEVES AND V. A . BANKAlTlS

point where it is now clear that the three regions of the complex are biochemically distinct, and that operational criteria can be applied to which cisternae constitute cis-, medial or trans-aspects. These lines of investigation included: (a) classical histochemical studies, (b) analysis of oligosaccharideproduct distribution within the Golgi complex, (c) subfractionation of biochemically distinct Golgi-complex membranes, and (d) in situ localization of defined marker enzymes (or undefined polypeptide antigens) to distinct regions of the complex (reviewed in Dunphy and Rothman, 1985). As already discussed (Section IV.A), Rothman and his colleagues devised a cell-free system which reconstituted intercisternal protein transport through the Golgi complex with the aim of identifying compartment-specific transport components by the classical strategy of resolution and reconstitution. While the biochemical approach has yielded rich dividends in identifying the basic concepts involved in transport-vesicle biogenesis and consumption, the transport components resolved to date (i.e. the NSF and the SNAPS) probably represent general fusion components involved in a number of stages on the secretory pathway (see Section IV.C.2). With the isolation and characterization of sec mutants, an independent means for recognizing compartment-specific functions has become available. These mutants permitted identification of two genes, SEC7 and SECII, whose products are essential for secretory function by the Golgi complex. Analysis of the roles of these two gene products in the function of the Golgi complex has yielded substantial insights into its organization and secretory function. In particular, characterization of the SEC14p has provided new perspectives on several long-standing issues in cell biology. A . FUNCTIONAL COMPARTMENTALIZATION OF THE YEAST GOLGI COMPLEX

I . Identification of the Organelle Saccharomyces cerevisiae appears to exhibit only a constitutive secretory pathway that transports proteins from the E R to the cell surface extremely rapidly. This characteristic, coupled with the high density of cytoplasmic ribosomes in this yeast, has obscured all but the largest organelles (i.e. nucleus, vacuoles and mitochondria) from a consistent view by electron microscopy. As a result, cells of this yeast do not display the morphologically correct Golgi complex typical of most other eukaryotic cells. Thus, identification of yeast Golgi complexes by cytochemistry remains somewhat problematic. The first evidence for Golgi complex-like structures in yeast was obtained from thin-section electron-microscopic analysis of the terminal phenotypes exhibited by sec7’’ and s e c l B strains. These mutants revealed a proliferation

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

113

of toroid membaneous structures or, under appropriate nutritional conditions (low concentrations of glucose), stack-like structures reminiscent of mammalian Golgi-complex membranes (Novick et al., 1980). That such membranes represented defective forms of a functional Golgi complex was indicated by the structure of the N-linked oligosaccharides of the secretory glycoproteins trapped within them (see below). A second cytochemical method for identification of yeast Golgi complexes has recently been developed; this is indirect immunofluorescence microscopy using the KEX2p as a Golgi-complex marker. The KEX2p is an integral membrane endoprotease involved in proteolytic maturation of the yeast a-factor mating pheromone. Based on the a-factor precursor species that accumulated within sects mutants at 37”C, Julius et al. (1984) assigned the KEX2p to the yeast Golgi complex, and it now appears that the KEX2p functions in a late Golgi-complex compartment (Franzusoff and Schekman, 1989). Indirect immunofluorescence experiments employing affinity-purified anti-KEX2p primary sera revealed a punctate cytoplasmic staining of some four to six bodies in each focal plane of a cell (Franzusoff etal., 1991). This staining was quite similar to that observed for the YPTlp (Section IV.C.3). At present, yeast Golgi complexes can be most readily detected in wildtype cells by immunofluorescence microscopy, a low-resolution method. Visualization of yeast Golgi complexes by high-resolution electronmicroscopy methods unfortunately requires imposition of secretory blocks that may well distort Golgi-complex structure to an extent such that structural inferences gleaned from such exaggerated organelles may be inaccurate. Thus, cytochemical methods have failed to provide any solid evidence for a segmental and functionally compartmentalized organization for the yeast Golgi complex. Clearly, methods for morphological detection of Golgi-complex bodies in wild-type strains of yeast need to be vastly improved before the full complement of direct evidence to this effect can be obtained. 2. Compartmental Organization of the Yeast Golgi Complex Presently, the notion that the yeast Golgi complex posseses a functional compartmentalization akin to that in mammalian Golgi complexes has been based on evidence that the oligosaccharide modifications experienced by yeast glycoproteins in transit through the Golgi complex are executed in a sequential and biochemically separable manner. This evidence is somewhat analogous to localization of defined carbohydrate-assembly reactions to distinct regions of the mammalian Golgi complex (reviewed by Dunphy and Rothman, 1985). To extend analyses of carbohydrate assembly to the yeast

114

A.

E. CLEVES AND V . A . BANKAlTlS

Golgi complex, it was necessary to know the precise structure of the N-linked oligosaccharides on yeast glycoproteins. The core oligosaccharides of Sacch. cerevisiae were identical in composition with those of mammalian cells (Li et al., 1978; Lehle, 1980). However, in contrast to the complex carbohydrate of mammalian glycoproteins, it was demonstrated that the yeast outer-chain carbohydrate consisted entirely of a-D-mannose residues in a-(1+6), a-(1+2) and a-(1+3) linkages together with a limited number of mannobiosylphosphate groups in diester linkage (Ballou, 1976). Furthermore, the order of events that occur in conversion of the core oligosaccharide to fully matured N-linked glycans has been partially deduced (Flores-Carreon, 1990). Raschke et al. (1973) isolated mutants (termed mnn) that exhibited alterations in the oligosaccharide-residue composition of the yeast cell-wall mannoprotein. Further analyses of these mnn mutants revealed that specific linkages of mannose residues were absent from the outer chain and some cell-free extracts derived from mnn strains were deficient in particular mannosyltransferase activities. For example, mnnl cells did not possess terminal a-(1+3)-mannose residues in their cell-wall glycoproteins. This defect correlated directly with the absence of a-(1+3)-mannosyltransferase activity from mnnl cell extracts (Nakajima and Ballou, 1975). It was subsequently established that the mnnl locus encoded an a-(1-3)mannosyltransferase (Ballou, 1982). However, this enzyme has not yet been localized to a specific subcellular compartment. Although these studies provided a detailed structural analysis of the yeast outer-chain mannoprotein, no evidence for the compartmentalized assembly of oligosaccharides was obtained. The first evidence for the compartmentalization of mannosyltransferase activities in yeast was provided by Esmon et al. (1981), who examined the oligosaccharide modifications of secretory invertase that had accumulated in the appropriate intracellular compartments in particular sec mutants. A comparison of the electrophoretic mobilities of the invertase retained in the ER-blocked, Golgi complex-blocked and late secretory vesicle-blocked mutants showed that modification of N-linked oligosaccharides occurred in at least two distinct compartments. A t 37"C, the ER-blocked mutants contained intracellular invertase with immature glycosyl chains, while the Golgi complex-blocked and vesicle-blocked mutants accumulated invertase species that migrated on non-denaturing gels with a profile similar to the invertase obtained from wild-type strains. To verify that the differences in the electrophoretic mobilities of invertase from the sec mutants were due solely to differences in carbohydrate content, it was shown that endoglycosidase H treatment converted the various forms of invertase into a single 61 kDa polypeptide species.

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVlSlAE

115

Some additional evidence for functional compartmentation of the yeast Golgi complex was obtained by Franzusoff and Schekman (1989), who characterized the glycosylation states of the invertase, CPY and a-factor precursors accumulated in secF strains at 37°C. These studies employed antisera raised against mannose residues of specific linkages and which could be used, in conjunction with electrophoretic mobilities, to characterize more precisely the outer-chain composition of yeast glycoproteins. The intracellular invertase obtained from secF mutants represented three populations that differed only in the number and linkage type of mannose residues added to the core oligosaccharide. One species of the accumulated invertase migrated with the same mobility on SDS gels as the coreglycosylated form and was not precipitable with anti-a-(1-+3)-Man or antia-(1+6)-Man antisera. A second population displayed a migration that was intermediate between the core-glycosylated protein and invertase from an mnn9 mutant. The mnn9 strain failed to mature completely the outer chain of glycoproteins, resulting in N-linked carbohydrates with only four or five mannose residues added to each core oligosaccharide. The third invertase population migrated similarly to fully matured invertase. However, these species lacked the terminal a-(1+3)-mannose residues. The p l (i.e. core-glycosylated) form of proCPY contains four Winked core oligosaccharide units, and its conversion to the p2 form is dependent upon addition of outer-chain and terminal a-(1+3)-mannose residues. It was previously reported that, after one hour at 37”C, 88% of the CPY in sec7‘ strains was in the p l form (Stevens et al., 1982). Following further analyses of the proCPY accumulated in sec7” mutants, it was shown that, in addition to p l CPY, a new species with a slightly lower electrophoretic mobility than p l CPY was also present. The thought was that this novel intermediate had experienced addition of a few outer-chain a-(1-6)mannose residues to the core oligosaccharide chains on the basis of the following observations. Both p2 CPY and mCPY from wild-type yeast were precipitable with anti+( 1-6)-Man and anti-a-( 1+3)-Man sera, while p l CPY was recognized by neither of these antisera. The new proCPY species found in the secF strain at 37°C was precipitated by anti-a-(1+6)-Man sera but not anti-a-(1+3)-Man sera. In addition to invertase and CPY, immature forms of the yeast matingpheromone a-factor accumulated in the sec7’ strains (Julius et al., 1984). Normal maturation of a-factor involves specific glycosylation and proteolytic processing events. As with invertase, a-factor was accumulated in various populations of precursor forms, one of which was heterogeneously glycosylated. The relevant aspect was that these species were precipitable with anti-a-( 1+6)-Man sera but not with anti-a-(1+3)-Man sera. Taking into account the variety of incompletely glycosylated precursors

116

A. E. CLEVES A N D V. A . BANKAlTlS

I

CELL SURFACE

I

SECRETORY VESICLES

Asn-Core t 6 M C [ 6 M C 6 M C 6 M + 6 M ] n + 6 M

Asn - Core 1‘M

(

ER Core Glycosylation

FIG. 4. Diagram illustrating compartmental organization of the yeast Golgi complex. Franzusoff and Schekman (1989) proposed this general model for biochemical compartmentation of the yeast Golgi complex-associated mannosyltransferase activities. Although this model enjoys considerable experimental support (see text), Franzusoff and Schekman (1989) point out that the depiction of one activity in each cisterna is rather arbitrary. The illustration of the yeast Golgi complex as an organelle that physically resembles a mammalian Golgi complex is merely convenient, and has not been unambiguously established by any direct means. Asn indicates the asparagine residue which is linked to the glycosyl chains. Mannose residues (M) in the outer glycosyl chain are indicated. Numbers indicate the linkage through the a-1 carbon atom to the preceding mannose residue in the outer-chain carbohydrate.

that accumulated in secF strains at 37”C, Franzusoff and Schekman (1989) proposed the model shown in Fig. 4 for compartmentation of the yeast Golgi complex. This model suggests a sequential, compartment-specific assembly

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

117

of N-linked oligosaccharides based on the previously determined structure of the yeast outer chain. Also, assignment of multiple execution points for the SEC7p accounted for the pleiotropic defects observed in s e c P mutants at 37°C. The suggestion of a-( 1+6)-mannose-residue addition occurring prior to a-( 1+3)-mannose-residue addition was further supported by in vitro ER+Golgi-complex assays that monitored glycosylation of the prepro-a-factor substrate (Section 1V.B.1). Transported prepro-a-factor acquired a-(l-+6)-mannoseresidues but not a-( 1+3)-mannose residues. These data suggested that this system reconstituted transport to the early Golgi-complex compartments but not subsequent early Golgi-complex+late Golgi-complex transport events. The SEC7 gene has been characterized at the nucleotide level and shown to be essential for yeast vegetative growth (Achstetter et af., 1988). The SEC7 gene was found potentially to encode a 2008 amino acid-residue hydrophilic protein predicted to be of some 230 kDa. In accordance with the nucleotide-sequence data, anti-SEC7p serum was used to probe extracts from radiolabelled yeast and precipitated a polypeptide that migrated on SDS gels with an apparent molecular mass of 227 kDa (Franzusoff et af., 1991). Fractionation experiments demonstrated that 60% of the total SEC7p was soluble while the remaining 40% was associated with pellet fractions. Sedimentable SEC7p was solubilized from pellet fractions by treatment with urea or alkaline sodium carbonate, but not by treatment with non-ionic detergents. Thus, the SEC7p behaved as a peripheral membrane protein. Indirect immunofluorescence microscopy revealed a punctate staining pattern of four to six particles in each cell and focal plane for the SEC7p. That at least some of these structures represented authentic yeast Golgi complexes was supported by two lines of evidence. First, sec24" cells, known to accumulate Golgi complex-like cisternae at 37"C, displayed a temperature-dependent exaggeration of the punctate structures that were seen in cells grown at the permissive temperature. Second, in doublelabelling experiments, some 58% of the structures that stained with antiSEC7p serum also stained with anti-KEX2p serum. Conversely, 81% of the KEX2p-positive structures were also SEC7p-positive. Taken together, the data suggested that the SEC7p was a peripheral membrane protein of the yeast Golgi complex that exhibited multiple execution points in intercisternal Golgi-complex transport. As yet, there are no obvious clues as to the biochemical function of the SEC7p although, interestingly, it has been found to be a phosphoprotein (Franzusoff et af., 1991). B . INVOLVEMENT OF A PHOSPHOLIPID-TRANSFER PROTEIN

Whereas the SEC7p is considered to be involved in a number of execution points in protein transport through the Golgi complex, current data suggest

118

A. E. CIEVES AND V. A. BANKAITIS

that the SEC14p is specifically involved in stimulating protein transport from a late Golgi-complex compartment. This inference is based on two lines of evidence. First, it has been observed that the secretory invertase accumulated in sec14-ltsstrains was quantitatively immunoprecipitated with anti-a(1+3)-Man serum, indicating that the intracellular invertase had experienced a complete maturation of its N-linked glycosyl chains (Franzusoff and Schekman, 1989). Moreover, the kinetics of a-( 1+3)-mannose acquisition '~ under non-permissive conditions appeared to by invertase in ~ e c l 4 - lstrains exhibit wild-type rates (A. E. Cleves and V. A. Bankaitis, unpublished data). Second, analysis of biogenesis of CPY in se~14-1~'mutants also indicated that the secl4 block was exerted at the step at which the ultimate proCPY precursor form (a late Golgi-complex species) was processed to the mature vacuolar form (Stevens et al., 1982). Thus, the SEC14p appears to represent a truly compartment-specific transport factor. A study of the SEC14p has revealed a precise biochemical activity for this polypeptide, and some penetrating insights into the role of the SEC14p in stimulation of yeast Golgi-complex secretory function have been obtained. In particular, these studies have indicated an active role for phospholipids in the secretory process, a concept that is generally ignored in current models of intercompartmental protein transport.

I . The SEC14p is the Yeast PhosphatidylinositoNPhosphatidylcholineTransfer Protein The yeast SECl4 gene and its gene product have been extensively characterized by Bankaitis and his coworkers (Bankaitis et al., 1989, 1990; Salama et al., 1990; Cleves et al., 1991). Nucleotide-sequence analysis of the SECl4 revealed a 304-codon open reading frame that was interrupted between the third and fourth codons by a 156-nucleotide intron. The primary sequence of the inferred SEC14p indicated a hydrophilic 35 kDa polypeptide that exhibited no remarkable hydrophobic character, no canonical sites for addition of N-linked oligosaccharide chains, and a general acidic character ( p l 5.3) (Bankaitis et al., 1989). These basic conclusions were confirmed by immunoprecipitation of the SEC14p from cell-free yeast lysates. The protein was found to be a non-glycosylated polypeptide with an apparent molecular mass of some 37 kDa. Subcellular fractionation experiments demonstrated that, as expected, the SEC14p was predominantly a cytosolic species. Some 60% of the total SEC14p was recovered from the 100,OOOg supernatant fraction. However, significant fractions of the total SEC14p were also found to be associated with membranes that pelleted at 12,000gand lOO,OOOg(Bankaitisetal.,1989). Although it wasnot initially clear that these membrane associations were of any physiological

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES C E R E V I S M E

119

relevance, subsequent experiments demonstrated that such was indeed so (Cleves et al., 1991). Treatment of permeabilized yeast cells with fluorescein-labelled SEC14p antibodies revealed a staining of four to eight ovoid cytoplasmic bodies in each cell that were superimposed upon a diffuse cytoplasmic staining. That the SEC14p-positive structures represented yeast Golgi complexes was confirmed by double-label immunofluorescence experiments that showed colocalization of the punctate SEC14p with the yeast Golgi-complex marker KEX2p. Independent corroboration of the cytochemical data was obtained from quantitative subcellular fractionation experiments. It was demonstrated that sedimentable SEC14p codistributed with the KEX2p throughout a rigorous fractionation regimen. The collective data indicated a remarkable enrichment of sedimentable SEC14p to yeast Golgi-complex membranes, a minor class of intracellular membrane, thereby establishing a direct link between the SEC14p and the organelle that is dysfunctional in secZ4'" mutants. Taken together, the available evidence suggested that the SEC14p was a cytosolic factor directly required for yeast Golgi-complex secretory function. The essential nature of the SEC14p was revealed by gene-disruption experiments that demonstrated a recessive lethality of secl4 disruption mutations (Bankaitis et al., 1989). Although initial protein similarity searches failed to provide any meaningful homologies between the SEC14p and other known protein sequences (Bankaitis et al., 1989), subsequent searches provided the first clues as to the biochemical nature of SEC14p function. Salama et al. (1990) discovered a significant homology between the SEC14p and the human retinaldehydebinding protein (HRBP) at the primary sequence level. The SEC14p exhibited a 25% identity with the HRBP over an uninterrupted stretch of 219 amino-acid residues, which was statistically significant. On the basis of this homology, Salama et al. (1990) proposed that the SEC14p served as a carrier that delivered a hydrophobic ligand to yeast Golgi-complex membranes. This hypothesis was confirmed by the finding that the SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein (Bankaitis et al., 1990). This discovery represented a convergence of work by Bankaitis and his colleagues on SEC14p function and the independent analysis of phospholipid-transfer protein function by Dowhan and his coworkers (Aitken et al., 1990). It was noted that the N-terminal30 residues of the purified yeast PI/PC-transfer protein corresponded exactly to the SEC14p N-terminus (inferred from the nucleotide sequence) when the initiator methionine residue was excluded, and that the DNA sequence of the 5' end of the transfer-protein structural gene (PZTZ) was also identical with that of SECZ4 (intron inclusive). The ultimate proof of identity of SECZ4 and PIT1 involved completion of the PITI nucleotide

120

A. E. CI.FVES AND V. A. BANKAlTlS

sequence and the finding that it was identical with the published SEC14 sequence. Phospholipid-transfer proteins (PL-TPs) are cytosolic factors that catalyse exchange of phospholipids between natural or synthetic membranes in vitro (reviewed by Helmkamp, 1986). These proteins exhibit a range of substrate specificities. Some are quite substrate non-specific whereas others are specific for a very few, or even a single, phospholipid species. The SEC14p catalyses transfer to both PI and P C in vitro, but exhibits a marked preference for PI (Daum and Paltauf, 1984). Although PL-TPs have been detected in the cytoplasm of every eukaryotic cell type tested, and have been the subject of intense biochemical and biophysical characterization over the past 25 years, the relevance of their in vitro activities with respect to in vivo function has remained unclear. This has not only been due to the lack of an in vivo experimental system, but also due to the peculiar properties of the in vitro transfer activities that these proteins catalyse. For instance, they generally catalyse an in vitro exchange of phospholipids, rather than a net vectorial transfer of phospholipid. Second, they have the curious ability to utilize essentially any natural or synthetic membrane as a phospholipid donor or acceptor in vitro. It has been difficult to propose any useful model for in vivo function for such seemingly indiscriminate activities. Yet, the data of Cleves et al. (1991), which demonstrated a clear enrichment of the SEC14p in yeast Golgi-complex membranes, indicates an in vivo specificity for membrane targeting that is not apparent in the in vitro system. The clear implication from this work is that the current notions of PL-TP function are fundamentally incomplete. As a result, the discovery that the SEC14p is the yeast PI-PC-transfer protein had a dual significance. First, it assigned a biochemical activity to a compartment-specific transport factor. Second, it provided the first experimental system with which the function of a PL-TP could be studied in vivo. 2. SEC14p Controls the Phosphatidylinositol: Phosphatidylcholine Ratio of Yeast Golgi-Complex Membranes The identification of S E C l 4 as the structural gene for the yeast PI-PCtransfer protein raised the possibility that the primary biochemical defect in secl4-1‘”strains might correlate with a phospholipid transfer defect in v i m . Bankaitis et al. (1990) tested this hypothesis by assaying the PI-PC-transfer activities in cytosol prepared from secZ4-Z‘” strains. Cytosols prepared from either SECZ4 or ~ecl4-1‘~’ strains displayed comparable PI-transfer activities when assayed at 25°C. Pre-incubation of wild-type cytosol at 3 T C , followed by assay at 37”C, had no effect on PI-transfer activity. However, the secZ4-Z‘”cytosol exhibited no measurable PI-transfer activity under the same

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

121

conditions. Thus, the PI-transfer activity of secZ4-Z'" strains was thermolabile in vitro. This thermolability was completely relieved by incorporation of SECZl on a single-copy centromere plasmid into secZ4-Z'" strains, while the PI-transfer activity of secZ4-1'" strains was partially thermoreversible in vitro. It was noted that the biochemical properties of the PI-transfer activity displayed bysecl4-Z'" strains correlated quite well with the in vivo behaviour of secZ4-1'" mutants. Interestingly, PC-transfer activity was absent from the cytosol of secZ4-1'" mutants, regardless of assay temperature, even though this activity was readily detected in wild-type cytosol preparations (Bankaitis et al., 1990). Taken together, these data provided a direct correlation between the in vivo ~ e c l 4 - 1 'defect ~ and the in vitro defect in phospholipid transfer. Moreover, the data indicated that the SEC14p represented by far the major, if not the only, PI-PC-transfer activity in the yeast cell, Bankaitis et al. (1990) proposed two distinct models for SEC14p function in vivo. First, they considered the possibility that the in vitro PI-PC-transfer activity of the SEC14p might accurately reflect SEC14p function in vivo. Within the framework of this model, the SEC14p would transfer PI and/or PC among intracellular membranes. The second model considered a scenario in which the in vitro PI-PC-transfer activity of the SEC14p might be an artifact of PI or PC binding by the SEC14p as a means for allosterically regulating some unrecognized SEC14p biochemical activity (much like guanine nucleotides regulate the effector functions of GTP-binding proteins, for example). To distinguish between these two models, Cleves et al. (1991) undertook a detailed analysis of SEC14p function in vivo and concluded that the SEC14p does indeed catalyse PI and/or PC transfer in vivo. Current evidence suggests that the in vivo function of the SEC14p is to generate and maintain an appropriate P1:PC ratio in late Golgi-complex membranes. The salient evidence to this effect is now summarized. Key insights into the role that the SEC14p plays in stimulation of yeast Golgi-complex function came from an analysis of mutations that suppressed the secZ4-Z'" defect (Cleves et al., 1991). These suppressor mutations were unlinked to the secZ4-Z'" locus and led to the indentification of six genes. Two of these genes, BSDl and BSD2, were defined by dominant mutations whereas the other four (SACZ, BSR2, BSR3 and BSR4) were defined by recessive mutations. Mutations in all of these genes were capable of efficiently suppressing normally lethal secl4 null mutations without activation of some cryptic PI-PC-transfer activity. Furthermore, secretory pathway function was fully restored in these suppressor mutants, and secretion involved all of the appropriate compartments, as shown by: (a) the proper glycosyl modifications experienced by secretory glycoproteins in these strains, and (b) a requirement for the 10 late-acting SECgene products

122

A. E. CLEVES AND V. A. BANKAlTlS

for secretion to occur. The cumulative data indicated that these suppressor mutations rendered yeast cells independent of the requirement for an essential PL-TP, and did so without elaboration of some other PTPCtransfer activity. Moreover, suppression was essentially limited to secZ4 defects (Cleves et al., 1991). The sacl mutations were exceptional in this regard as they also suppressed certain actin defects and several other lateactingsecdefects (Clevesetal., 1989). TheSACZ gene, its product, andsacl suppressor properties are discussed in detail later in this review (Section V.D). To date, the most penetrating insights into SEC14p function in vivo have been derived from analysis of the recessive suppressors, in particular BSR gene products. Molecular analysis of BSR4 revealed its identity to a previously recognized gene, CKI, the structural gene for yeast choline kinase (Cleves et al., 1991). This is the enzyme that catalyses the first step in synthesis of PC from free choline via the CDP-choline pathway (Carman and Henry, 1989). The identity of BSR4 with CKI was an intriguing result that linked biosynthesis of a SEC14p ligand to SEC14p function in vivo. Moreover, cki null mutations were found to suppress efficiently normally lethal secZ4 null alleles, a result that was in complete agreement with the recessive nature of the original bsr4 alleles. That suppression was a general consequence of dysfunction of the CDP-choline pathway was indicated by: (a) the observation that disruptions of CPTZ, the structural gene for the enzyme that catalyses the ultimate step in biosynthesis of PC via the CDP-choline pathway (i.e. choline phosphotransferase), exhibited bsr phenotypes (Cleves et al., 1991), and (b) the finding that bsr2 and bsr3 mutants failed to incorporate in vivo choline into PC, thereby indicating that these mutants were also defective in functioning of the CDP-choline pathway. Current data suggest that BSR2 and CPTZ are allelic (M. K. Fung and V. A. Bankaitis, unpublished observation). Thus, the absence of PC biosynthesis by the CDP-choline pathway bypassed the normally essential SEC14p requirement. In marked contrast, lack of PC synthesis by the only other alternative mechanism available to yeast, the methylation pathway (Carman and Henry, 1989), in no way relieved the cellular requirement for SEC14p function (Cleves et al., 1991). The demonstration that the SEC14p requirement could be specifically and efficiently bypassed by PC biosynthetic defects argued strongly for a phospholipid equilibration function for the SEC14p. Moreover, the work of Cleves et al. (1991) provided the first link between biosynthetic protein transport, intracellular phospholipid transport and phospholipid biosynthesis. Cleves et al. (1991) proposed two general models to reconcile the suppression data, in particular the striking feature that inactivation of the CDP-choline pathway for PC synthesis resulted in a bypass of the SEC14p

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAL

123

whereas inactivation of the methylation pathway did not; these are illustrated in Fig. 5. Both of these models assumed that the SEC14p catalyses mobilization of phospholipids in vivo and, in doing so, maintains an appropriate P1:PC ratio in Golgi-complex membranes that is a critical feature of the secretory competence of these membranes (Fig. 5(a)). The compartmentalized PC-synthesis model suggests that PC synthesis by the CDP-choline pathway occurs in the Golgi complex whereas PC synthesis by the methylation pathway does not. The resulting net PC synthesis in the Golgi complex would lower the P1:PC ratio of Golgi-complex membranes below some critical level if it were not countered by removal of PC from the complex. In this model, the SEC14p is proposed to function in such a removal of PC (Fig. 5(b)). Bypass of the SEC14p would then be the expected result of inactivation of the CDP-choline pathway. Clearly, localization of enzymes of the CDP-choline pathway in specific membranes will provide a crucial test of this hypothesis. The second model, the regulation model, suggests that inactivation of the CDP-choline pathway results in an altered balance of P1:PC in bulk membranes, in particular the E R membranes where PC synthesis by the methylation pathway would be expected to occur. Thus, an appropriate P1:PC ratio would be imposed upon the Golgi complex by bulk-membrane flow from the E R (Fig. 5(c)). In this scenario, the SEC14p would raise the P1:PC ratio of Golgi-complex membranes above that of bulk membranes, and the achievement of an appropriate P1:PC ratio in the E R would obviate the need for SEC14p function. The key prediction of the regulation model is that bsr mutants will have elevated P1:PC ratios in bulk-membrane fractions relative to the P1:PC ratio of bulk membranes from wild-type cells. The various predictions of these two models are testable, and provide a precise experimental framework for a further investigation of SEC14p function in vivo. Outstanding questions still remain and must be addressed. These include: (a) is there a SEC14p receptor in Golgi-complex membranes that imposes a specificity of membrane interaction, (b) does the SEC14p catalyse phospholipid exchange or net phospholipid transfer in vivo and, if net transfer, which phospholipid is the ligand, and (c) which membranes serve as partners in the transfer reaction in vivo? Finally, in vivo analyses of SEC14p function were informative from the perspective of the models for SEC14p function that were rejected by the data. One such example has already been discussed. Another important model rejected by the data dealt with the role of PL-TPs in recycling of bulk intracellular membrane. Wieland et al. (1987) had argued that the rate of phospholipid removal from the E R by bulk-membrane flow was far greater than the rate at which E R phospholipid could possibly be regenerated by synthesis. A convincing argument was made for phospholipid retrieval back

124

A

F. C I E V E S AND V A BANKAITIS

(a) Golgi complex (inactive 1

Golgi complex (active)

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

125

to the E R as a necessary mechanism to compensate for this imbalance, and preserve the integrity of the E R . Wieland et a f .(1987) proposed that PL-TPs were the determining participants in such a retrieval, and Rothman (1990) offered a highly speculative model for the manner in which the SEC14p might be involved in such a bulk phospholipid retrieval in yeast. Current data do not support this hypothesis. Salama et a f .(1990) demonstrated that, in wild-type cells, the SEC14p was present at levels some 10-fold in excess of those required for efficient Golgi-complex secretory function. This was not immediately consistent with a bulk phospholipid movement function for the SEC14p. Moreover, the findings of Cleves et al. (1991) that indicated a bypass of SEC14p function could occur without activation of some cryptic PIPC transfer activity, coupled with the fact that the SEC14p ligands (PI and PC) represent the two most abundant phospholipids in yeast, rendered such a bulk phospholipid mobilization model for the SEC14p entirely untenable. Other mechanisms for phospholipid retrieval must operate in vivo.

3. Conservation of SEC14p Structure and Function The ubiquity of PI/PC transfer-protein activities in eukaryotic cells appears to be accompanied by a conservation of transfer-protein structure across wide evolutionary distances. Cytosolic fractions prepared from various mammalian, insect, amphibian, reptilian and avian cells all harbour polypeptides of some 35 kDa that exhibit an immunological cross-reactivity with the bovine PI/PC-transfer protein ( M , 36 kDa) (Wirtz, 1982).

FIG. 5. Diagram illustrating a phospholipid mobilization model for the SEC14p. (a) Cleves et al. (1991) proposed that the SEC14p elevates the phosphatidylinositol: phosphatidylcholine(P1:PC) ratio in yeast Golgi-complexmembranes above that of bulk membranes, a function that is critical for maintaining the secretory competence of Golgi-complex membranes. Two models were offered in an attempt to reconcile the observation that mutations in the CDP-choline pathway, but not the methylation pathway, for PC synthesis bypass the SEC14p requirement: (b) PC synthesis by the CDP-cholinepathway is localized in the Golgi complex whereas PC synthesis via the methylation pathway is not. As the SEC14p functions to remove PC from the Golgi complex, a block in PC synthesis by the CDP-choline pathway results in bypass of SEC14p function. (c) Neither PC biosynthetic pathway operates in the Golgi complex, but inactivation of the CDP-choline pathway results in an altered balance of PI:PC in the endoplasmic reticulum (ER) (the most likely biosynthetic compartment for PI and PC). This favourable PI:PC ratio is then imposed upon the Golgi complex by bulk membrane flow in a SEC14p-independent fashion. CDP-DG, CDPdiacylglycerol; PDME, phosphatidyldimethylethanolamine; PE, phosphatidylethanolamine; PMME, phosphatidylmonomethylethanolamine.

126

A . E. CLEVES AND V. A. BANKAlTlS

Moreover, Helmkamp and his colleagues reported hybridization of rat PI/PC transfer-protein cDNA to genomic sequences of a number of higher eukaryotic organisms including Drosophila melanogaster (Dickeson et al., 1989). Whether these structural homologues exhibit a functional relatedness in vivo is still an open question. Recent studies in the yeast system have firmly established that the SEC14p is structurally and functionally conserved in lower eukaryotes that are separated by vast evolutionary distances. Bankaitis et al. (1989) identified SEC14p-immunoreactive polypeptides in cell-free lysates prepared from two widely divergent yeasts, namely Kluyveromyces lactis and Schiz. pombe. These SEC14p homologues had apparent molecular weights (as judged by SDS-PAGE) that were similar to, but not identical with, that of the SEC14p in Sacch. cerevisiae. The functional identity of these SEC14p species was unambiguously established by recovery of genomic clones from K. lactis and cDNA clones from Schiz. pombe that complemented secI4 defects in Sacch. cerevisiue, and the demonstration that these clones encoded the corresponding SEC14p structural homologues. The SECI4 gene (SEC14KL)in K . lactis and its product were characterized by Salama et al. (1990). It was shown that the SEC14pKL could functionally substitute for the SEC14p (SEC14pSC) in Sacch. cerevisiae, as shown by the patently wild-type kinetics for protein secretion and vacuolar protein biogenesis exhibited by strains that carried a normally lethal secl4 null mutation and SEC14KL in an ectopic single-copy configuration. Moreover, the level of the SEC14pKL in such strains was very much decreased relative to normal intracellular levels of the SEC14pSC, thereby arguing for a functional identity for these SEC14p species. Nucleotide sequence analysis of SEC14KLrevealed a single, uninterrupted 301-codon open reading frame that encoded a polypeptide with an inferred molecular weight of 34,165. The SEC14pKLexhibited a 77% identity at the primarysequence level with the SEC14pSC,a result that was immediately consistent with the structural and functional homology between these two polypeptides. Clones of cDNA from the SECl4 gene from Schiz. pombe (SEC14SP) were recovered by H . B. Skinner and V. A . Bankaitis (unpublished observation). The SEC14pSP exhibited a functional identity with the SEC14pSCon the basis of the ability of the former to complement fully the growth and secretory defects associated with normally lethal secl4 null mutations. The SEC14SP nucleotide sequence has been determined and inferred to encode a 284-residue polypeptide ( M , 32,788) that displayed almost a 60% identity with the SEC14pSC at the primary sequence level. This striking structural and functional relatedness to the SEC14pSCwas even the more remarkable when one considers that Sacch. cerevisiae and Schiz. pombe exhibit as wide a phylogenetic divergence from each other as each of

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVlSlAE

127

these yeasts exhibits from mammals (Russell and Nurse, 1986). Taken together, the data strongly suggest that the corresponding in vivo roles for the SEC14pKLand SEC14pSPwill be very similar to that of the SEC14pSC, namely a role in Golgi-complex secretory function. It will be of singular interest to determine the subcellular localization of the SEC14pSPin Schiz. pombe and in the heterologous Sacch. cerevisiae. Finally, it is of some interest to note that these three yeast SEC14p species exhibit a significant relatedness to the human retinaldehyde-binding protein (HRBP), a carrier for the hydrophobic ligands 11-cis-retinol and ll-cisretinaldehyde (Salama et al., 1990). Thus, it appears likely that the molecular architecture of these carrier proteins may share some common ancestry, even though the SEC14p and HRBP ligands bear little similarity. It is interesting to note that the SEC14p shows no significant homology to its mammalian biochemical counterpart, the rat PI/PC-transfer protein. This lack of relatedness is especially intriguing in light of the possible conservation of P I P C transfer-protein structure in higher eukaryotes (see above). The very real possibility that the higher eukaryotic PI/PC-transfer proteins serve fundamentally different in vivo functions than do yeast SEC14ps must still be considered seriously in the absence of any direct evidence to the contrary. C. THE RETENTION PROBLEM: THE ROLE OF CLATHRIN

Like the E R , the Golgi complex also contains unique resident proteins that must be retained in face of the massive bulk flow of biosynthetic secretory traffic. The KEX2p is an example of such a Golgi-complex resident in yeast, while Fuller et al. (1989) have shown that the C-terminal cytosolic tail of the KEX2p is required for its retention in the Golgi complex. Although the signals for retention remain undefined, it appears that yeast clathrin plays an entirely unanticipated role as a critical component of a yeast Golgi-complex retention machinery. Clathrin coats are polyhedral lattices that are thought to function in protein sorting, the clustering of receptors and formation of vesicles from the plasma membrane and trans-Golgi-complex network in mammalian cells (Goldstein etal., 1985; Griffiths and Simons, 1986). In yeast and mammalian cells, the major components of clathrin cages are units of three cytosolic clathrin heavy-chain ( M , about 180 kDa) and three clathrin light-chain ( M , about 30-40 kDa) polypeptides (Pearse, 1976; Kirchhausen and Harrison, 1981; Mueller and Branton, 1984; Lemmon et al., 1988) which can selfassemble into basket-like structures in vitro (Keen et al. , 1979; Woodward and Roth, 1979; Lemmon et al., 1988). In order to determine more precisely the role of clathrin in intracellular

128

A. E. CLEVES AND V . A . BANKAITIS

protein traffic in vivo, studies of clathrin function were initiated in Sacch. cerevisiae. Using antibodies raised against yeast-clathrin heavy chain, clones of the gene encoding this polypeptide, CHCl , were recovered (Payne and Schekman, 1985; Lemmon and Jones, 1987). Haploid cells bearing a deletion of the CHCl gene were sickly, but nonetheless viable, and exhibited essentially wild-type rates of secretion. Furthermore, no clathrin heavy-chain homologues were identified in strains with a deletion in chcl, designated Achcl , by low-stringency DNA hybridization or antibody crossreactivity (Payne and Schekman, 1985). These data suggested that yeast clathrin plays no role in protein transport to the cell surface. Also, because protein secretion in yeast was virtually unaffected in the absence of the clathrin heavy chain, and yeast showed no obvious substitute for the CHClp, models that invoked an essential role for clathrin in protein transport were rejected. It should be pointed out, however, that the nonessential nature of chcl null mutations remains controversial. Lemmon and Jones (1987) found such lesions to be lethal in other yeast genetic backgrounds. Nevertheless, the ability to generate Achcl mutants, in at least some genetic backgrounds, has yielded startling clues as to the role of clathrin in yeast. Further insights into the role of clathrin in yeast were obtained by Payne and Schekman (1989) who observed that Achcl cells secreted a precursor form of a-factor that had not been proteolytically processed. This precursor comigrated on SDS gels with the unprocessed form of a-factor secreted by cells that lacked the KEX2p endoprotease that is required for a-factor maturation. It was subsequently demonstrated that secretion of a-factor precursor in Achcl strains was due to a failure of such strains to retain the KEX2p in the Golgi complex, the normal location of this protease. It was found that the KEX2p could be specifically radio-iodinated in Achcl but not CHCl cells under conditions of exclusive surface labelling. Moreover, the KEX2p enzymic activity could be measured at the cell surface of Achcl but not CHCl cells. About 75% of the total KEX2p was estimated to be mislocalized to the cell surface of Achcl cells at the steady-state level (Payne and Schekman, 1989). Based on these data, it was proposed that clathrin serves to: (a) retain certain proteins within the Golgi complex, or (b) retrieve escaped resident proteins from the plasma membrane to the complex. Localization of clathrin to a specific subcellular compartment should discriminate between these two general possibilities. In either case, clathrin is a critical component of the mechanism by which organelle identity is maintained in yeast. This retention model for clathrin function will be further supported if other resident Golgi-complex proteins are found to be mislocalized in clathrindeficient yeast.

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVlSlAE

129

D. COUPLING OF GOLGI-COMPLEX AND ACTIN-CYTOSKELETON FUNCTIONS

Cells of Sacch. cerevisiae show a polarized mode of secretion that is an essential feature of cell growth and division in this organism. Golgi complexderived secretory vesicles are targeted almost exclusively to the growing bud. As a result, the bud grows whereas the mother cell largely does not. Based on the structural changes exhibited by actin during the yeast cell cycle and the phenotypes displayed by actin-deficient mutants, it was proposed that the polarized mode of cell growth is mediated by the actin cytoskeleton (Kilmartin and Adams, 1984; Adams and Pringle, 1984; Novick and Botstein, 1985). Fluorescence-microscopy analyses revealed a yeast filamentous actin cytoskeleton comprised of two basic components: (a) actin cables oriented along the mother-bud axis, and (b) membrane-associated cortical patches preferentially distributed to the bud in regions associated with cell-surface growth (Kilmartin and Adams, 1984; Adams and Pringle, 1984). Thus, the orientation of the actin cytoskeleton correlated with the polarity of cell growth. Analysis of the terminal phenotypes of mutants bearing conditionally lethal mutations in ACT1 (the essential, single-copy structural gene for yeast actin) provided confirmatory evidence for involvement of actin in organization of the late stages of the yeast secretory pathway. Under non-permissive conditions, the actlts mutants contained increased intracellular pools of fully glycosylated secretory invertase, a proliferation of intracellular membranes that appeared to represent Golgi complex-derived secretory vesicles and, in the case of actl-lfS,Golgicomplex bodies, and a wholesale disorganization of cell-surface growth. This last characteristic was shown by the arrest of growth at the nonpermissive temperature of actl" mutants as swollen unbudded cells and their aberrant deposition of cell-wall chitin (Novick and Botstein, 1985). Taken together, these data argued for an actin involvement in the late stages of the secretory pathway and in the establishment and maintenance of correct secretory polarity. Recent work has indicated that the yeast cell co-ordinates Golgi-complex secretory and actin cytoskeleton function, and some insights have been obtained with regard to the identity of the cellular machinery by which such a coupling might be achieved. These findings represented the convergence of two independent lines of inquiry, one involving a study of mutations that suppress actin defects and the other a study of mutations that bypass the normally essential SEC14p requirement for Golgi-complex function (Section V.B). The SAC1 gene product is an excellent candidate for a component of a cellular machinery that couples the activities of the late stages of the secretory pathway and those of the actin cytoskeleton. This gene was independently identified in suppressor analyses of the nctl-1" and secl4-1'" defects, respectively. Novick et al. (1989) attempted to identify gene

130

A. E. CLEVES AND V. A. BANKAITIS

products that interact with yeast actin in vivo by isolating recessive mutations that suppressed the actl-l'" defect and, additionally, exhibited a new cs phenotype. The phenotypic properties of sacZCSmutants fulfilled several genetic criteria that might be expected of a mutant that is defective in an actin-binding protein. First, sacZcssuppressed actl'" defects and did so in an allele-specific manner. That is, while sacZCSsuppressed actl-Z'", the combination of sadCSand actl-2'" was a haploid-lethal event. Allele-specific interactions of this sort are often used as genetic diagnoses for interactions between the corresponding gene products (Botstein and Maurer, 1982). Second, the terminal phenotypes of sacZCS mutants at the restrictive temperature of 14°C redisplayed some of the actZ-ItSterminal phenotypes. At 14"C, sacZCSmutants failed to exhibit visible actin cables and showed a randomization of actin cortical patches between mother and bud. An aberrant pattern of chitin deposition was also strikingly apparent. Novick et al. (1989) recovered SACZ clones and demonstrated that the s a d null phenotype was cold-sensitive for growth. As discussed in Section V.C.3, extragenic suppressors of secZ4-Z'" were isolated and characterized. The rsdZ complementation group was recognized amongst these suppressors as a class of recessive mutations that uniformly exhibited an unselected cs phenotype (Cleves et al., 1989, 1991). The terminal rsdl phenotypes were characterized at 14°Cand were found to coincide exactly with those observed for sucZcsmutants. Interestingly (even though rsdl" suppressed sec14-Zts),rsdl'" mutants failed to exhibit any significant secretory defects or any significant exaggeration of intracellular membranes. Because the actin cytoskeleton was thought to be involved in block was most the later stages of the secretory pathway, and the ~ecZ4-Z'~ likely exerted at the level of a late Golgi-complex compartment, Cleves et al. (1989) considered the possibility that RSDZ and SACZ were allelic. From several lines of genetic evidence this allelism was proven and the sad nomenclature was adopted. Strikingly, sacZCSalleles, generated as suppressors of actl-l'", were also shown to suppress secZ4-Z'". In fact, sacZCSalleles bypassed the SEC14p requirement altogether as sacla suppressed normally lethal secl4 null mutations (Cleves et al., 1989). To determine if the sacZCSmutants exhibited genetic interactions with any of the other sects mutations, the appropriate double mutants were constructed and analysed for growth at a variety of temperatures. The s a ~ Z - 6 ~ ~ allele was chosen because it had been shown to suppress actZ-Z'" and se~Z4-Z~". Interestingly, s a ~ Z - 6exhibited ~~ a partial suppression of sec6" and sec9" as well. Since sec6'" and sec9" represented defects at the level of Golgi complex-derived secretory vesicle function, and actin had been implicated in the later stages of the secretory pathway, it was suggested that the suppression data provided additional evidence for a late execution point

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

131

for the SEC14p, perhaps at the level of secretory protein exit from the Golgi complex (Cleves et al., 1989). The SAC1 gene and its gene product have been characterized (Cleves et al., 1989; A . E. Cleves and V. A . Bankaitis, unpublished observation). Sequence analysis of the DNA revealed a 623-codon open reading frame that potentially encoded a polypeptide with a molecular mass of 71,132. Inspection of the SAClp primary sequence revealed several features. First, a run of 23 uncharged amino-acid residues, which constituted a good candidate for a membrane-spanning region, divided the SAClp into a large 521-residue N-terminal domain and a small 79 residue C-terminal domain. Second, there were no obvious signal-peptide structures that might direct the SAClp into the secretory pathway. Finally, the C-terminal 95-residue region of the SAClp identified a very basic domain (PI 9.8). Immunoprecipitation experiments using anti-SAClp monoclonal antibodies showed the SAClp to be an unglycosylated polypeptide with an apparent molecular mass of some 65 kDa. The inconsistency between the predicted molecular mass and the value estimated from the mobility on SDS-PAGE gels is considered to be most likely due to the basic character of the SAClp. In subcellular fractionation experiments, the SAClp was quantitatively recovered in pellet fractions and was not extracted from membranes by treatment with 0.1 M sodium carbonate (pH 11.5), indicating the SAClp to be an integral membrane protein. Protease protection experiments employing lysates from gently lysed sphaeroplasts, in which the integrity of small organelles was maintained, suggested that the SAClp was oriented such that the large N-terminal domain was lumenally disposed while the small C-terminal domain was exposed to the cytosol. Preliminary subcellular fractionation and immunofluorescence microscopy data suggest a localization of the SAClp to the Golgi complex and the ER. In summary, the discovery that mutations in a single gene, SACZ, could suppress secretory and cytoskeletal defects provided the first genetic link between actin-cytoskeleton assembly and secretory pathway activities, in particular late Golgi-complex function. How might the SAClp co-ordinate such seemingly disparate activities as Golgi-complex and yeast actin function? We can now offer a speculative model. It has recently been demonstrated that the actin-binding proteins profilin and gelsolin also bind the phospholipid phosphatidylinositol4,5-bisphosphate(PIP2) with a higher affinity than that which these proteins exhibit for actin, and these proteins have been proposed to cluster PIP2 on the cytoplasmic leaflet of membranes (Yin et al., 1988; Hartwig et al., 1989; Goldschmidt-Clermont et al., 1990). Since the C-terminal tail of the SAClp, which appears to be disposed to the cytosol , exhibits some primary-sequence homology to several known actin-binding proteins, the question of whether the SAClp binds

132

A. E. CLEVES AND V. A. BANKAITIS

phospholipids becomes relevant. The fact that: (i) the SEC14p is the PI-transfer protein (Bankaitis et a f . , 1990), (ii) the SEC14p appears to function in modulating the P1:PC ratio of yeast Golgi-complex membranes (Cleves et a f . , 1991), and (iii) PI is the precursor to PIP2, provides an intriguing possibility as to how the SAClp might influence both SEC14p and actin function. Perhaps the SAClp exerts these comodulatory effects by a phospholipid (PI, PIP or PIP2)-sequestration mechanism. Clearly, in vitro phospholipid-binding analyses should provide some further insight. In any event, we note that the idea of some sort of coupling of actin and Golgicomplex function is an attractive one. If actin cables do indeed direct secretory vesicles to the bud, it seems that regulating aspects of actin assembly in the immediate vicinity of the late Golgi-complex membranes, from which such transport vesicles will be derived, is a reasonable strategy. VI. Fusion of Golgi Complex-Derived Vesicles with the Plasma Membrane

Ten genes whose products participate in targeting and/or fusion of Golgi complex-derived secretory vesicles with the plasma membrane have been identified in yeast (Novick et al., 1980). Mutations in SECZ, SEC2, SEC3, SEC4, SECS, SEC6, SEC8, SEC9, SECIO and SECIS result in the accumulation of these vesicles. Molecular and genetic analyses of these lateacting SEC gene products have categorized these genes into two classes, namely those that exhibit genetic interactions with SEC4 and those that do not. Little is known about the latter class of gene products and these will not be discussed in this review. A study of the yeast SEC4p, a ras-like GTPbinding protein, has yielded valuable insights into how vesicular transport is regulated. In fact, the work of Novick and his colleagues represented the first direct demonstration of GTP-binding protein involvement in protein transport, and has spawned a great deal of current effort, in numerous laboratories, directed at analysing how such proteins regulate secretory processes. As the subject of GTP-binding protein function has been exhaustively reviewed elsewhere (Gilman, 1987; Bourne, 1988; Hall, 1990), we will limit our treatment of this subject to those topics that relate directly to secretion in yeast. A. INVOLVEMENT OF A GTP-BINDING PROTEIN

The initial demonstration that a small GTP-binding protein plays a role in secretory pathway function was provided by Novick and his colleagues who extensively characterized the SEC4 gene product (Salminen and Novick, 1987; Goud et af., 1988; Walworth et af., 1989; Kabcenell et a f . , 1990).

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

133

Nucleotide sequence analysis of SEC4 revealed a 215-codon open reading frame that had the potential to encode a hydrophilic polypeptide with an apparent molecular mass of 23.5 kDa (Salminen and Novick, 1987). Inspection of the inferred SEC4p primary sequence was immediately informative since the SEC4p primary sequence displayed a 32% and a 48% homology with the human H-ras and yeast YPTZ gene products, respectively. Moreover, sec4 disruption alleles proved to represent haploid-lethal events. Thus, there is no redundancy in SEC4p function in yeast. This is in contrast to yeast RAS function, which is genetically redundant (Kataoka et al., 1984). Also, in spite of the extensive homology between the SEC4p and the YPTlp, these polypeptides exhibited no detectable functional overlap (Salminen and Novick, 1987). That the SEC4p is indeed a GTP-binding protein was established by Goud et a f . (1988) who employed a ligandblotting assay to demonstrate that, although the SEC4p bound GTP in this assay, the sec4-8" gene product did not. However, as pointed out by these workers, these data did not unambiguously indicate that the ~ec4-8~' mutation directly affects GTP binding by the SEC4p. The demonstration that GTP binding was required for SEC4p function was provided by Walworth et al. (1989), who created mutant Sec4p derivatives that failed to bind GTP and showed that such mutant proteins were non-functional in vivo. Using the combined techiques of differential centrifugation and densitygradient fractionation, Goud et al. (1988) determined the subcellular localization of the SEC4p in both wild-type and secretory vesicleaccumulating yeast strains. In wild-type cells, the SEC4p was located predominantly in a 10,OOOg pellet fraction, while a smaller amount was recovered from a 100,OOOgfraction, and some 10% of the total SEC4p was found in the cytosolic fraction. In marked contrast, after imposition of the secretory-vesicle block, the majority of the SEC4p recovered from a sec6" yeast strain was recovered from the 100,000gpellet fraction. Only some 10% of the SEC4p sedimented at 10,OOOg under these conditions. Subsequent sucrose density-gradient fractionations demonstrated that the SEC4p recovered from the 10,OOOg pellets in both wild-type and sec6'' lysates cofractionated with a plasma-membrane marker. The SEC4p recovered from the 100,000g pellet in the sec6" lysate cofractionated with secretory invertase, a lumenal secretory vesicle marker under those conditions. It is presumed that the minor SEC4p fraction found in the 100,OOOg pellet from wild-type cells also represented secretory vesicle-associated material. Unfortunately, there do not as yet exist any secretory vesicle markers in wild-type cells that can be used to establish this point unambiguously. Nevertheless, Goud et a f . (1988) further demonstrated that the membranebound SEC4p was exposed to the yeast cytosol, as judged by its sensitivity to

134

A . E. CLEVES AND V. A. BANKAITIS

exogenous protease challenge of intact secretory vesicles, and that the SEC4p could be chased from the plasma membrane to secretory vesicles upon imposition of the sec6" block. These data indicated a redistribution of the SEC4p and raised the possibility that the SEC4p recycled from the plasma membrane to secretory vesicles, perhaps via a soluble intermediate. The SEC4p primary sequence indicated a hydrophilic protein that was devoid of significant hydrophobic character (Salminen and Novick, 1987). Yet, the kinetics of the association of the SEC4p with membranes in vivo were extremely rapid, exhibiting a half-life of less than one minute (Goud et al., 1988). To investigate further the nature of the association of the SEC4p with membranes, Goud et al. (1988) attempted to extract the SEC4p from membranes by a variety of treatments. The results clearly showed that the SEC4p behaved as an integral membrane protein. It was not liberated by treatment with an alkaline sodium carbonate solution and it partitioned into the detergent phase following solubilization in an aqueous solution of Triton X-114. Overproduction of the SEC4p specifically increased the soluble pool of this protein, however, and this soluble pool partitioned into the aqueous phase following Triton X-114 solubilization. Thus, it seems that the SEC4p post-translationally acquires a hydrophobic modification that promotes membrane attachment, much like the farnesyl and palmityl modifications that are experienced by ras and other GTP-binding proteins, and that SEC4p attachment to membranes is specific and saturable. Inspection of the SEC4p primary sequence reveals that, as with the YPTlp, the polypeptide terminates with two cysteine residues (Salminen and Novick, 1987). This sequence resembles the canonical CAAX box that is characteristic of the C-termini of GTP-binding proteins. Molenaar et al. (1988) showed that these terminal cysteine residues were required for YPTlp function. To determine if the same was true for the SEC4p, Walworth et al. (1989) constructed a SEC4 allele from which the terminal cysteine residues were deleted (sec4-CCA).Gene-replacement experiments showed that sec4-CCA did not encode a functional protein as the deletion failed to complement the sec4-8" growth defect and represented a recessivelethal mutation. Subcellular fractionation experiments indicated that the Sec4p-CCA was a stable protein whose GTP-binding properties remained intact, but which was located exclusively in the cytosol. These data demonstrated that the C-terminus of the SEC4p was involved in determining the membrane-binding capacity of the gene product, and that membrane binding was required for SEC4p function. A direct demonstration of a posttranslational modification of SEC4p is, however, not yet available. Based on the collective data, Walworth et al. (1989) and Bourne (1988) argued that SEC4p function can be viewed as being analogous to that of the translation elongation factor EF-Tu in E. coli (Kaziro, 1978). An adaptation

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVlSlAE

135

Nucleotide

FIG. 6. Diagram showing a Cycling model for function of the SEC4p. Soluble SEC4p exchanges GDP for GTP and binds to a newly formed secretory vesicle, thereby rendering the labelled vesicle competent for interaction with a plasmamembrane effector protein. Engagement of the vesicle with the effector triggers the fusion event. Guanine nucleotide hydrolysis by the SEC4p results in disengagement of the SEC4p.GDP from the effector so that both components can be re-utilized for another round of secretory vesicle targeting and fusion. From Walworth etaf.(1989).

of this model is shown in Fig. 6. In this scheme, G D P is exchanged for GTP by soluble SEC4p. This SEC4p-activation step may require an ancillary guanine-nucleotide exchange activity. The SEC4p.GTP then engages a newly formed secretory vesicle, and renders the “labelled” vesicle competent for docking with the plasma membrane. Such a docking event may require participation of an effector protein that recognizes the SEC4p. Following the subsequent fusion event, release of the SEC4p is coupled to a conformational change induced by GTP hydrolysis. The released

136

A. E. CLEVES AND V. A. BANKAITIS

SEC4peGDP is then available for participation in another round of secretory vesicle docking and fusion. This model provides a satisfactory explanation for the need for the SEC4p in secretory vesicle consumption. It also makes a powerful prediction, namely that an irreversibly activated SEC4p would serve as a potent inhibitor of the vesicle-fusion process by virtue of its progressive and irreversible occupation of the effector. Important support for this model, in particular from the standpoint of the last prediction, is forthcoming from a study of sec4-Ife133. This allele was designed to mimic an oncogenic H-ras mutation that resulted in constitutive activation of RAS function in the absence of any GTP binding (Walworth et a f ., 1989; Walter et af.,1986). Expression of sec4-ZfeZ33 resulted in a specific and dose-dependent block at the level of secretory vesicle fusion to the ' ~ ~ a dominant plasma membrane in vivo. At low levels, the S e ~ 4 p " " caused ts phenotype that correlated with a late-acting vesicle block in secretion. At high levels, the S e ~ 4 p " " caused ' ~ ~ a dominant-lethal secretory block. These secretory blocks were uniformly characterized by accumulation of secretory vesicles within the yeast cell (Walworth et a f . , 1989). Deletion of the ~~ suppressed its terminal cysteine residues of the S e ~ 4 p " " 'completely dominant-negative effects on secretion without destabilizing the protein. Thus, one can incorporate the S e ~ 4 p ' " ' ~ data ~ directly into the model of Walworth et a f . (1989) (Fig. 6). The S e ~ 4 p " most ~ ' ~ ~likely represents a protein that is permanently locked into the active state, and is thereby liberated from the guanine-nucleotide switch that normally regulates SEC4p function. In this scenario, the dominant-negative S e ~ 4 p " " 'catalyses ~~ the first round of secretory vesicle fusion, but fails to disengage from the effector once fusion has occurred. The net consequence of this behaviour would be a dose-dependent and irreversible occupation of the effector, thereby precluding participation of the effector in more than a single round of S e ~ 4 p " ~ ' ~ ~ - c a t a l yvesicle s e d fusion. We note that the inhibitory role of GTPdS in a number of intercompartmental transport systems in v i m is also very easily incorporated into such a scenario. Evidence obtained from mammalian systems indicates that small GTPbinding proteins similar to the SEC4p are likely to be involved in regulating vesicular traffic at several stages of interorganelle transport. Chavrier et af. (1990) identified a number of novel GTP-binding proteins (termed rab proteins) that appear to be involved in endocytic as well as exocytic transport (Chavrier et a f . , 1990). At this point, it seems likely that the current model for SEC4p function can be extended to the function of other small GTP-binding proteins in secretion, and serve as a paradigm for describing one level of regulation of vesicular traffic in the eukaryotic secretory pathway.

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

B. OTHER GENE PRODUCTS THAT POTENTIATE

137

GTP-BINDING PROTEIN FUNCTION

The SEC4p has been purified and its biochemical properties that relate to nucleotide binding and GTP hydrolysis examined (Kabcenell et al., 1990). This polypeptide was found to have high affinities for GTP and GDP, but release of GTP from the SEC4p (0.0012 min-') was exceedingly slow relative to that of GDP (0.21 min-'). Given these kinetics, an unregulated SEC4p would be expected to exist predominantly in a GTP-bound state, a condition that would not be favourable for sustained secretory vesicle fusion to the plasma membrane. Thus, auxiliary factors must participate in regulation of nucleotide binding, exchange and hydrolysis by the SEC4p. Moreover, there remains the question of the identity of the effector through which the SEC4p operates. Elucidation of the identities of these auxiliary factorsand of the effector will be the next step in attaining a more complete understanding of the SEC4p cycle. The remaining nine late-acting S E C gene products are good candidates for factors that either modulate or respond to SEC4 function in vivo. This view was strongly supported by the observation that SEC4 exhibited striking genetic interactions with several of the other late-acting S E C genes (Salminen and Novick, 1987). Duplication of SEC4 elicited a clear suppression of the seclSS,sec2" and sec8" defects, a weaker suppression of seclts,sec5" and secl@' defects, and no detectable suppression of the ~ e c 3 ' ~ , sec6" and secP defects. With respect to this suppression, it is of interest to note that SEC4 clones were originally recovered in attempts to recover SECl5 clones (Salminen and Novick, 1987). These suppression data suggested a functional relationship between the SEC4p and the late gene products whose defects were dosage compensated by the SEC4p. Further support for a functional interaction between the SEC4p and a subset of the late SEC gene products was obtained by an independent line of evidence. The sec4-8" mutation exhibited a synthetic lethality when combined with either of the sec2", sec3", secF, secSts,s e c l p and ~ e c l.5mutations. '~ Thus, SEClS, SEC2 and SEC8 exhibited consistent genetic interactions with SEC4 whereas the significance of the SEC4 interactions with S E C l , SEC3, SECS and SEClO are less clear. There are, at present, no genetic grounds for considering SEC6 or SEC9 gene function to have any particular relationship to SEC4 function. As a result of the particularly strong genetic interactions between SEC4, SEC2 and SECl.5, the latter two genes have been a primary focus of study. The SECZS gene has been characterized at the molecular level and shown to be an essential gene that could potentially encode a hydrophilic polypeptide of 911 amino-acid residues (Salminen and Novick, 1989). The SEC15p has been identified and shown to exhibit an apparent molecular

138

A . E. CLEVES AND V. A . BANKAlTlS

mass of 116 kDa that was consistent with the predicted molecular mass of 105 kDa. Subcellular fractionation experiments revealed that, in spite of its hydrophilic character, the SEC15p behaved as a peripheral membrane protein which could be extracted from membranes with salt or urea or at increased pH values. Although the SEC15p was not detected in wild-type cells by indirect immunofluorescence without resorting to SEC15p overproduction (Salminen and Novick, 1989), subsequent fractionation experiments indicated that some 23% of the total gene product was associated with the plasma membane while the remainder fractionated as a high molecularmass soluble species (1000-2000 kDa) (Bowser and Novick, 1990). Further insight into SEC15p function was obtained by analysing yeast cells that overproduced the SEC15p (Salminen and Novick, 1989). Increased amounts of the SEC15p interfered with secretory vesicle fusion with the plasma membrane. Indeed, secretory vesicles aggregated into a striking patch within the growing bud. That this patching was a manifestation of some physiologically relevant aspect of SEC15p function was indicated by the findings that: (a) overproduction of the Sec15pt”failed to induce vesiclepatch formation, (b) overproduction of the SEC15p resulted in a coincident patching of the SEClSp, as judged by indirect immunofluorescence, and (c) SEC15p patch formation was dependent on the SEC4p and SEC2p. On the basis of these data, Salminen and Novick (1989) suggested that one interpretation of the data was that the SEC15p may facilitate docking of SEC4p-labelled secretory vesicles to the plasma membrane. Patching would simply reflect incorporation of the SEClSp onto vesicles, thereby resulting in a target-membrane identity crisis which causes inappropriate vesiclevesicle docking reactions (i.e. patching). The data also suggest that the SEC2p and SEC4p act upstream of the SEC15p execution point in the secretory vesicle fusion pathway, although the order of action of the SEC2p and SEC4p is not revealed by these data. At present, the collective SEC15p data suggest an involvement for this protein in effector function. It will be of great interest to determine whether the SEC4p exhibits a direct physical interaction with the SEC15p in vivo. Clones of the SEC2 gene have also been recovered and characterized (Nair et al., 1990). The SEC2p was inferred to be a hydrophilic polypeptide of 759 amino-acid residues ( M , 84 kDa), which gene-disruption experiments have indicated to be essential for yeast viability. Homology searches have revealed that the N-terminal one-third of the SEC2p exhibited a 25% identity with the rod region of the myosin heavy chain, and similar homologies to other cytoskeletal proteins. Nair et al. (1990) suggested that these homologies reflect the presence of an a-helical, coiled-coil domain (a general feature of the rod region of the myosin heavy chains) in the SEC2p, rather than some cytoskeletal association of the SEC2p. This suggestion is

SECKETOKY PATHWAY FlJNLTION IN SACCHAROMYCES CEREVISIAE

139

supported by subcellular fractionation experiments that indicated a predominantly cytosolic localization of the SEC2p in a high molecular-mass complex (500-700 kDa). A striking confirmation for the coiled-coil domain of the SEC2p playing a crucial role in the SEC2p function was forthcoming from analysis of sec2" mutations. Two such alleles were determined to be nonsense mutations, specifically opal mutations. These data suggested that the C-terminus of the SEC2p was at least partially dispensible, and this idea was further supported by gene-disruption experiments. Truncation of the last 251 SEC2p residues, a removal of one-third of the primary sequence, did not detectably affect SEC2p function. Truncation of the last 368 residues rendered SEC2p function thermosensitive. Deletion of the N-terminus of the SEC2p was a haploid-lethal event (Nair et al., 1990). Clearly, the means now exist for a detailed study of how the function of the SEC15p and SEC2p, in concert with the SEC4p, regulate secretory vesicle fusion to the plasma membrane. It remains less clear, at this time, what roles the other late-acting SEC gene products play at this step. Continued molecular analysis of these genes should eventually permit an incorporation of those corresponding gene products into a model that fully describes secretory vesicle targeting to, and fusion with, the plasma membrane.

VII. Summary A genetic analysis of secretory pathway function in yeast was initiated some 12 years ago in the laboratory of Randy Schekman. These mutants held great promise in terms of providing an experimental system with which molecular participants of secretory pathway function could be investigated. This early promise has not failed. For the last five years, analysis of yeast secretory pathway function has been at the cutting edge of our understanding of the mechanisms by which proteins travel between intracellular compartments. In some cases, Sacch. cerevisiae has provided a valuable in vivo corroboration of the concepts derived from biochemical studies of mammalian intercompartmental protein transport in vitro. In other cases, studies conducted in the yeast system have defined previously unanticipated involvements for known catalytic activities in the secretory process. It is clear that yeast will continue to play a major role in setting the pace of research directed towards a detailed molecular understanding of protein secretion. Since it is now apparent that the basic strategies that underlie secretory pathway function have been conserved among eukaryotes, further exploitation of the powerful and complementary yeast and mammalian experimental systems guarantees that the next decade will see even greater progress towards

140

A. E. CLEVES AND V. A. BANKAlTlS

our understanding of protein secretion in eukaryotic cells than did the first.

VIII. Acknowledgements

We wish to thank all of our colleagues for their cooperation in providing us with results in advance of publication. The expert assistance of Sandy Henson in the preparation of this manuscript is also greatly appreciated. Work in the authors’ laboratory was supported by grants from the National Science Foundation (DCB-9003750) and the American Heart Association Illinois Affiliate (880752).

REFERENCES Achstetter, T., Franzusoff, A., Field, C. and Schekman, R . (1988). Journal of Biological Chemistry 263, 11711 . Adams, A. E. M. and Pringle, J. R. (1984). Journal of Cell Biology 98, 934. Aitken, J. F., van Heusden, G . P. H., Temkin, M. and Dowhan, W. (1990). Journal of Biological Chemistry 265, 471 1. Bacon, R. A, , Salminen, A , , Ruohola, H., Novick, P. and Ferro-Novick, S. (1989). Journalof Cell Biology 109, 1015. Baker, D., Hicke, L., Rexach, M., Schleyer, M. and Schekman, R. (1988). Cell 54, 335. Baker, D., Wuestehube, L., Schekman, R., Botstein, D. and Segev, N. (1990). Proceedingsof the National Academy of Sciences of the United States of America 87, 355. Baker, R. K. and Lively, M. 0. (1987). Biochemistry 26, 8561. Ballou, C. (1976). Advances in Microbial Physiology 14, 93. Ballou, C. E. (1982). In “The Molecular Biology of the Yeast Saccharomyces”, (J. N. Strathern, E. Jones and J. Broach, eds), pp. 335-360. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Bankaitis, V. A., Altman, E . and Emr, S. D. (1986a). “Bacterial Outer Membranes as Model Systems”, pp. 75-116. Wiley, New York. Bankaitis, V. A,, Johnson, L. M. and Emr, S. D. (1986b). Proceedings of the National Academy of Sciences of the United States of America 83, 9075. Bankaitis, V. A,, Malehorn, D. E., Emr, S. D. and Greene, R. (1989). Journalof Cell Biology 108, 1271. Bankaitis, V. A,, Aitken, J. R., Cleves, A. E. and Dowhan, W. (1990). Nature 347, 561. Beckers, C. J. M., Block, M. R., Glick, B. S . , Rothman, J . E. andBalch, W. E. (1989). Nature 339, 397. Beckers, C. J. M., Plutner, H., Davidson, H. W. and Balch, W. E. (1990). JournalofBiological Chemistry 265, 18298. Bergman, J. E . and Singer, S. J. (1983). Journal of Cell Biology 97, 1777. Bernstein, H. D., Rapoport, T . A. and Walter, P. (1989). Cell 58, 1017. Blobel, G. and Dobberstein, B. (1975). Journal of Cell Biology 67, 835. Bohni, P. C., Deshaies, R. J. and Schekman, R. (1988). Journal of Cell Biology 106, 1035. Bole, D. G., Hendershot, L. M. and Kearney, J. R. (1986). Journalof Cell Biology 102,1558. Booth, C. and Koch, G . L. E . (1989). Cell 59, 729. Botstein, D. and Maurer, R. (1982). Annual Review of Genetics 16, 61. Bourne, H. R. (1988). Cell 53, 669.

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVlSlA E

141

Bowser, R. and Novick, P. (1990). Journal of Cell Biology 111, 206a. Brennwald, P., Liao, X., Holm, K., Porter, G. and Wise, J. A. (1988). Molecularand Cellular Biology 8, 1580. Carman, G. M. and Henry, S. A. (1989). Annual Review of Biochemistry 58, 635. Cenotti, A. and Colman, A. (1988). EMBO Journal 7 , 633. Chavrier, P., Vingron, M., Sander, C., Simons, K. and Zerial, M. (1990). Molecular and Cellular Biology 10, 6578. Chirico, W. J., Waters, G . and Blobel, G. (1988). Nature 332, 805. Clary, D. O., Griff, I. C. and Rothman, J. E. (1990). Cell 61, 709. Cleves, A. E., Novick, P. J. and Bankaitis, V. A. (1989). Journal o f c e l l Biology 109,2939. Cleves, A. E., McGee, T . P., Whitters, E. A , , Champion, K. M., Aitken, J . R., Dowhan, W., Goebl, M. and Bankaitis, V. A. (1991). Cell 64,789. Collier, D. N., Bankaitis, V. A , , Weiss, J. B. and Bassford, P. J. (1988). Cell 53, 273. Craig, E . A., Kramer, J. and Kosic-Smithers, J. (1987). Proceedings ofthe National Academy of Sciences of the United States of America 84, 4156. Crooke, E., Guthrie, B., Lecker, S., Lill, R. and Wickner, W. (1988). Cell 54, 1003. Dalbey, R. and Wickner, W. (1985). Journal of Biological Chemistry 260, 15925. Date, T. (1983). Journal of Bacteriology 154, 76. Daum, G. and Paltauf, F. (1984). Biochimica et Biophysica Acta 794, 385. Dean, N . and Pelham, H. R. B. (1990). Journal of Cell Biology 111, 369. Deshaies, R. J. and Schekman, R. (1987). Journalof Cell Biology 105, 633. Deshaies, R . J. and Schekman, R. (1989). Journal of Cell Biology 109, 2653. Deshaies, R. J., Koch, B. D., Werner-Washburne, M., Craig, E. A. and Schekman, R. (1988). Nature 332, 800. Deshaies, R . J., Kepes, F. and Bohni, P. C. (1989). Trends in Genetics 5 , 87. Diaz, R., Mayorga, L. S., Weidman, P. J., Rothman, J . E. and Stahl, P. D. (1989). Nature339, 398. Dickeson, S. K., Lim, C. N., Schuyler, G . T., Dalton, T . P., Helmkamp, G . M. and Yarbrough, L. R. (1989). Journal of Biological Chemistry 264, 16557. Dunphy, W. G . and Rothman, J. E. (1985). Cell 42, 13. Dunphy, W. G., Pfeffer, S. R., Clary, D. O., Wattenberg, B. W., Glick, B. S. and Rothman, J. E. (1986). Proceedings of the National Academy of Sciences of the United States of America 83, 1622. Eakle, K. A,, Bernstein, M. and Emr, S. D. (1988). Molecular and Cellular Biology 8,4098. Ehrenreich, J . H., Bergeron, J. J. M., Siekevitz, P. and Palade, G. E. (1973). Journalof Cell Biology 59, 45. Esmon, B., Novick, P. and Schekman, R . (1981). Cell 25, 451. Evans, E., Gilmore, R. and Blobel, G . (1986). Proceedings of the National Academy of Sciences of the United States of America 83, 581. Farquhar, M. G. (1978). Journal of Cell Biology 77, R35. Feldman, R. I., Bernstein, M. and Schekman, R. (1987). Journal of Biological Chemistry 262, 9332. Ferro-Novick,S., Novick, P., Field, C. and Schekman, R. (1984). Journal of Cell Biology 98,35. Flores-Carrkon, A., Hixson, S. H., Gomez, A., Shao, M.-C., Krudy, G., Rosevear, P. R. and Wold, F. (1990). Journal of Biological Chemistry 265, 754. Franzusoff, A. and Schekman, R. (1989). EMBO Journal 8, 2695. Franzusoff, A , , Redding, K., Crosby, J., Fuller, R. S. and Schekman, R. (1991). Journalof Cell Biology 112, 27. Fuller, R. W., Brake, A. J. and Thorner, J. (1989). Science 246, 482. Gallwitz, D., Donath, C. and Sander, C. (1983). Nature 306, 704. Gething, M. J., McCammon, K. and Sambrook, J . (1986). Cell 46, 939. Gilman, A. G. (1987). Annual Review of Biochemistry 56, 615. Goldschmidt-Clermont, P. J., Machesky, L. M., Baldassare, J. J. and Pollard, T. D. (1990). Science 247, 1575. Goldstein, J . L., Brown, M. S., Anderson, R. G . W., Russel, D. W. and Schneider, W. J. (1985). Annual Review of Cell Biology 1, 1.

142

A . E. CLEVES AND V. A . BANKAlTlS

Goud, B., Salminen, A , , Walworth, N. C . and Novick, P. J. (1988). Cell 53, 753. Griffiths, G. and Simons, K. (1986). Science 234, 438. Groesch, M., Ruohola, H., Bacon, R., Rossi, G . and Ferro-Novick, S. (1990). Journalof Cell Biology 11 1, 45. Haguenauer-Tsapis, R. and Hinnen, A. (1984). Molecular and Cellular Biology 4, 2668. Hall, A. (1990). Science 249, 635. Hann, B. C. and Walter, P. (1990). Journal of Cell Biology 111, 386a. Hann, B. C., Poritz, M. A. and Walter, P. (1989). Journal of Cell Biology 109, 3223. Hansen, W. and Walter, P. (1988). Journal of Cell Biology 106, 1075. Hansen, W., Garcia, P. D. and Walter, P. (1986). Cell 45, 397. Hardwick, K. G. , Lewis, M. J., Semenza, J., Dean, N. and Pelham, H. R. B. (1990). EMBO Journal 9, 623. Hartwig, J. H., Chambers, K. A. and Stossel, T. P. (1989). Journal of Cell Biology 108,467. Helmkamp, G. M. (1986). Journal of Bioenergetics and Biomembranes 18, 71. Hicke, L. and Schekman, R. (1989). EMBO Journal 8, 1677. Julius, D. , Schekman, R. and Thorner, J. (1984). Cell 36, 309. Kabcenell, A . K., Goud, B., Northup, J. K. and Novick, P. J. (1990). Journal of Biological Chemistry 265, 9366. Kaiser, C. and Schekman, R. (1990). Cell 61, 723. Kataoka, T., Powers, S., McGill, C : , Fasano, O., Strathern, J., Broach, J. and Wigler, M. (1984). Cell 37, 437. Kaziro,'Y. (1978). Biochimica et Biophysica Acta 505, 95. Keen, J. H., Willingham, M. C . and Pastan, I. H. (1979). Cell 16, 303. Kepes, F. and Schekman, R. (1988). Journal of Biological Chemistry 263, 9155. Kilmartin, J. V. and Adams, A. E. M. (1984). Journal of Cell Biology 98, 922. Kirchhausen, T. and Harrison, S. C. (1981). Cell 23, 755. Klionsky, D. J . , Herman, P. K. and Emr, S. D. (1990). Microbiological Reviews 54, 266. Lehle, L. (1980). European Journal of Biochemistry 109, 589. Lemmon, S. K. and Jones, E. W. (1987). Science 238, 504. Lemmon, S. K., Lemmon, V. P. and Jones, E. W. (1988). Journalof Cellular Biochemistry36, 329. Lewis, M. J., Sweet, D. J. and Pelham, H. R. B. (1990). Cell 61, 1359. Li, E . , Tabas, I. and Kornfeld, S. (1978). Journal of Biological Chemistry 253, 7762. Lill, R., Dowhan, W. and Wickner, W. (1990). Cell 60, 271. Malhotra, V . , Orci, L., Glick, B. S . , Block, M. R. and Rothman, J. E. (1988). Cell 54,221. Molenaar, C., Pranger, R . and Gallwitz, D. (1988). EMBO Journal 7, 971. Mueller, S. C. and Branton, D . (1984). Journal of Cell Biology 98, 341. Munro, S. and Pelham, H . R. B. (1986). Cell 46, 291. Munro, S. and Pelham, H . R. B. (1987). Cell 48, 899. Nair, J., Miiller, H., Peterson, M. and Novick, P. (1990). Journal of Cell Biology 110, 1897. Nakajima, T. and Ballou, C. E. (1975). Proceedings of the National Academy of Sciences of the United States of America 72, 3912. Nakano, A. and Muramatsu, M. (1989). Journal of Cell Biology 109, 2677. Nakano, A,, Brada, D. and Schekman, R. (1988). Journal of Cell Biology 107, 851. Newman, A . P. and Ferro-Novick, S. (1987). Journal of Cell Biology 105, 1587. Newman, A. P., Shim, J. and Ferro-Novick, S. (1990). Molecular and Cellular Biology 10, 3405. Normington, K., Kohno, K., Kozutsumi, Y., Gething, M. J. and Sambrook, J. (1989). Cell57, 1223. Novick, P. and Botstein, D. (1985). Cell 40, 405. Novick, P. and Schekman, R. (1979). Proceedings of the National Academy of Sciences of the United States of America 76, 1858. Novick, P . , Field, C. and Schekman, R. (1980). Cell 21, 205. Novick, P., Ferro, S. and Schekman, R. (1981). Cell 25, 461. Novick, P., Osmond, B. C . and Botstein, D. (1989). Genetics 121, 659. Oliver, D. B. and Beckwith, J. (1982). Cell 30, 311.

SECRETORY PATHWAY FUNCTION IN SACCHAROMYCES CEREVISIAE

143

Orci, L., Malhotra, V., Amherdt, M., Serafini, T . and Rothman, J. E. (1989). Cell 56, 357. Palade, G. (1975). Science 189, 347. Payne, G. S. and Schekman, R. (1985). Science 230, 1009. Payne, G. S. and Schekman, R. (1989). Science 245, 1358. Pearse, B. M. F. (1976). Proceedings of the National Academy of Sciences of the United States of America 21, 1255. Pelham, H. R. B. (1988). EMBO Journal 7, 913. Pelham, H. R. B., Hardwick, K. G . and Lewis, M. J. (1988). EMBO Journal 7, 1757. F'feffer, S. R. and Rothman, J. E. (1987). Annual Review of Biochemistry 56, 829. Pliitner, H., Schwaninger, R., Pind, S. and Balch, W. E. (1990). EMBO Journal 9, 2375. Polaina, J. and Conde, J. (1982). Molecular and General Genetics 186, 253. Poritz, M. A., Siegel, V., Hansen, W. and Walter, P. (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 4315. Randall, L. L. (1983). Cell 33, 231. Raschke, W. C., Kern, K. A., Antalis, C. and Ballou, C. E. (1973). Journal of Biological Chemistry 248, 4660. Ribes, V., Dehoux, P. and Tollervey, D. (1988). EMBO Journal 7, 231. Ribes, V., Romisch, K., Giner, A , , Dobberstein, B. and Tollervey, D. (1990). Cell 63, 591. Riezman, H . (1985). Cell 40, 1001. Romisch, K., Webb, J., Herz, J., Prehn, S., Frank, R., Vingron, M. and Dobberstein, B. (1989). Nature 340, 478. Rose, M. D., Misra, L. M. and Vogel, J. P. (1989). Cell 57, ,1211. Rothblatt, J. A. and Meyer, D. I. (1986a). Cell 44, 619. Rothblatt, J. A. and Meyer, D. 1. (1986b). EMBO Journal 5, 1031. Rothblatt, J. A., Webb, J. R., Ammerer, G. and Meyer, D. I. (1987). EMBOJournal6,3455. Rothblatt, J. A, , Deshaies, R. J., Sanders, S. L., Daum, G . and Schekman R. (1989). Journal of Cell Biology 109, 2641. Rothman, J. E. (1989). Cell 59, 591. Rothman, J. E. (1990). Nature 347, 519. Rothman, J. E. and Orci, L. (1990). FASEB Journal 4, 1460. Rudolph, H. K., Antebi, A , , Fink, G. R., Buckley, C. M., Dorman, T. E., LeVitre, J., Davidow, L. S., Mao, J. and Moir, D. T . (1989). Cell 58, 133. Ruohola, H., Kabcenell, A. K. andFerro-Novick, S. (1988). Journalof Cell Biology 107,1465. Russell, P. and Nurse, P. (1986). Cell 45, 781. Sadler, I., Chiang, A., Kurihara, T., Rothblatt, J., Way, J. and Silver, P. (1989). Journal of Cell Biology 109, 2665. Salama, S. R., Cleves, A. E., Malehorn, D. E., Whitters, E. A. and Bankaitis, V. A. (1990). Journal of Bacteriology 172, 4510. Salminen, A. and Novick, P. (1987). Cell 49, 527. Salminen, A. and Novick, P. J. (1989). Journal of Cell Biology 109, 1023. Sambrook, J. F. (1990). Cell 61, 197. Saraste, J . and Hedman, K. (1983). EMBO Journal 2, 2001. Schauer, I., Emr, S., Gross, C. and Schekman, R. (1985). Journal of Cell Biology 100, 1664. Schekman, R. (1985). Annual Review of Cell Biology 1 , 115. Schmitt, H. D., Wagner, P., Pfaff, E. and Gallwitz, D. (1986). Cell 47, 401. Schmitt, H. D., Puzicha, M. and Gallwitz, D. (1988). Cell 53, 635. Segev, N. and Botstein, D. (1987). Molecular and Cellular Biology 7, 2367. Segev, N., Mulholland, J. and Botstein, D. (1988). Cell 52, 915. Semenza, J. C., Hardwick, K. G., Dean, N. and Pelham, H. R. B. (1990) Cell 61, 1349. Sewell, J . L. and Kahn, R. A. (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 4620. Siegal, V. and Walter, P. (1985). Journal of Cell Biology 100, 1913. Smith, R. A., Duncan, M. J . and Moir, D. T . (1985). Science 229, 1219. Steams, T., Willingham, M. C., Botstein, D. and Kahn, R. A. (1990). Proceedings of the National Academy of Sciences of the United States of America 87, 1238. Stevens, T., Esmon, B. and Schekman, R. (1982). Cell 30, 439.

144

A. E. CLEVES AND V. A. BANKAlTlS

Tartakoff, A. (1980). International Review of Experimental Pathology 22, 227. Vogel, J . P., Misra, L. M. and Rose, M. D. (1990). Journal of Cell Biology 110, 1885. Walter, M., Clark, S. and Levinson, A. (1986). Science 233, 649. Walter, P. and Lingappa, V. R. (1986). Annual Review of Cell Biology 2 , 499. Walworth, N. C., Goud, B., Kabcenell, A. K. and Novick, P. J. (1989). E M B O Journal 8, 1685. Waters, M. G. and Blobel, G . (1986). Journal of Cell Biology 102, 1543. Werner-Washburne, M . , Stone, D. E. and Craig, E. A . (1987). Molecular and Cellular Biology 7, 2568. Wickner, W. and Lodish, H. F. (1985). Science 230, 400. Wieland, F. T., Gleason, M. L., Serafini, T . A. and Rothman, J. E. (1987). Cell 50, 289. Wilson, D. W., Wilcox, C. A,, Flynn, G . C., Chen, E., Kuang, W., Henzel, W. J., Block, M. R., Ullrich, A. and Rothman, J. E. (1989). Nature 339, 355. Wirtz, K. W. A. (1982). In “Lipid-Protein Interactions” (P. C. Jost and 0. H. Griffith, eds), pp. 151-231. Wiley-Interscience, New York. Wolfe, P. B., Wickner, W. and Goodman, J. M. (1983). Journalof Biological Chemistry 258, 12073. Woodward, M. P. and Roth, T. F. (1979). Journal of Supramolecular Structure 11, 237. Yin, H. L., Iida, K. and Janmey, P. A . (1988). Journal of Cell Biology 106, 805.

Physiology of Osmotolerance in Fungi' ANDERS BLOMBERG and LENNART ADLER Department of General and Marine Microbiology. University of Goteborg. Carl Skottsbergs Gata 22. 413 19 Goteborg. Sweden

I . Introduction . . . . . I1. The thermodynamic state of water

. . The concept of water potential .

. . .

. . .

. . . . . . . . . . . . A. . . . . . . B . Componentsofthewaterpotentialofcells . . . . . . 111. Osmotolerance . . . . . . . . . . . . . . A . Cardinal water potentialsof growth . . . . . . . . . IV . Initial osmotic response . . . . . . . . . . . . A . Microscopic observations . . . . . . . . . . B . Boyle-van't Hoff plots and non-osmotic volumes . . . . C . Water loss in relation to cell-wall elasticity and initial turgor pressure V . Osmoregulation . . . . . . . . . . . . . A . Compatible solutes . . . . . . . . . . . B . Inorganic ions . . . . . . . . . . . . . C . Solute compartmentation . . . . . . . . . . D . Regulation of polyol accumulation . . . . . . . . VI . Osmotic hypersensitivity . . . . . . . . . . . A . Osmotichypersensitivitydeterminants . . . . . . . B . Physiological overlap . . . . . . . . . . . values . . . . . VII . Cellular factors involved in determining ymin A . Generation of energy (ATP) at low water potentials . . . .

B . Costofmaintenanceatlowwaterpotentials . . C. Ion transport and accumulation . . . . . D . Production and accumulation of a compatible solute VIII . Conclusion . . . . . . . . . . . IX . Acknowledgements . . . . . . . . . References . . . . . . . . . . .

.

. . . . .

.

. . . . .

.

. . . . .

.

. . . . .

146 148

148 151 155 156 161 161 163 164 167 167 182 185 186 190 193 196 197 198 199 202 202 204 206 206

'

This review is dedicated to Professor Birgitta Norkrans who introduced us to this field of science.

ADVANCESIN MICROBIALPHYSIOLOCY. VOL . 33 ISBN &I24277395

Copyright 0 195'2 .by Academic Press Limited All rights of reproduction in any form reserved

146

A . BLOMBERG A N D L. A D I E R

I. Introduction

The cell is, in all its wealth of forms, encapsulated by a membrane, which by its semipermeable properties bestows the phenomenon of osmosis upon the cell. The water molecule permeates the cell membrane more freely than other cellular constituents, implying for most instances a thermodynamic water equilibrium (or quasi-equilibrium) of the cell with its environment. Since active cellular processes occur in water solutions, the physiology of cells inevitably has to cope with these osmotic phenomena and appropriately adjust the cell for survival and growth. Early reports on the relations of fungi to high concentrations of sugar or salt mainly focused on specific effects of the solutes. These studies were of primary economic importance in helping to define water-deficit conditions that extend the shelf life of stored foods. In fact, this interest has followed humans through history. Corry (1987) mentions in her review that grain was preserved in ancient Egypt by drying and stored in granaries (Genesis 41: 3 5 4 ) . However, micro-organisms have frequently been at variance with this strive of humankind for a safe and well-supplied future, which has certainly been a most important incentive for continued studies of fungal relations to solute-rich environments. With the review by Scott (1957), the focus was shifted from the implications of the solute molecules per se to the indirect effect these additives had on water availability, quantitatively expressed as water activity. This unifying way of viewing the effects of high concentrations of solutes directed interest towards the intracellular physiology of cells growing under conditions of low water activity, which led Brown and Simpson (1972) to introduce the concept of “compatible solute”. The concept and its fundamental implications on cell physiology were experimentally solidified by work on yeast (Brown, 1976, 1978), and has today gained general acceptance by scientists working in a wide variety of biological fields (Yancey et af., 1982). However, more recently Pitt (1989) pointed out that water activity as an important factor governing life was outweighed in a number of articles by those relating cellular activities to temperature, pH value and oxygen. He concluded that water activity remained the neglected parameter in ecology as well as physiology. In Fig. 1 the results of a recent database search for articles relating to water-osmosis and fungi are illustrated. Articles published during the period 1987-89 were classified into the indicated categories; studies involving yeasts have been separately plotted. As is evident, the vast majority of articles in the field of physiology have been conducted on yeasts while work on filamentous fungi is mainly directed towards growth-

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

147

Number of articles

FIG. 1. Results of a computer search in the BIOSIS database for articles published during 1987-89 concerning water-osmosis and fungi. Eighty-nine articles (25% of the total) addressed this overlap in their title, and were subsequently classified according to their main focus into the categories indicated in the figure. Solid bars indicate data on filamentous fungi, shaded bars those on yeasts.

survival-related studies. This bias in available information is necessarily reflected in the present review, although efforts have been taken to include physiological information about filamentous fungi whenever relevant studies to our knowledge exist. We are convinced that studies on the fungal physiological response to a changing water availability and the exploration of homeostatic mechanisms that fine tune the cell physiology to its water environment will yield fruitful information relevant to many fields of cell science. Fungi are a heterogeneous group of organisms and many types of vegetative as well as sexual life-cycles are represented, giving the scientist a vast repertoire to choose within, including many species highly amenable for experimentation. Little interest has so far been directed towards genetic studies of fungal water relations (Fig. 1). We believe this avenue with its unparalleled opportunity for applying powerful molecular genetic methods to yeasts and fungi will bring within the range of experimental resolution many of the fundamental mechanisms in the molecular biology of fungal osmotolerance, the physiology of that phenomenon being the main subject of this review.

148

A. BLOMBERG AND 1.. ADLER

11. The Thermodynamic State of Water

A consequence of the small size of the water molecule is its unusually high concentration in solutions. Pure water has a concentration at 20°C of 55.4 M and, even in what is regarded as a highly concentrated solution of any other molecule, the water concentration is not significantly altered, e.g. in a 1 M solution of sodium chloride at 20°C the water concentration is 54.4 M. The water molecules in a solution, however, are “associated” with the dissolved solute, which instigates a change in the state of water, thus giving rise to a dramatic decrease in the amount of thermodynamically available water for living organisms. The state of water can be described by different physicochemical parameters, e.g. osmotic pressure, osmolality, water potential, chemical potential of water, osmotic potential and water activity, their use often being related to the field of science by historical or practical reasons. It should be kept in mind, however, that all of these parameters are strictly related (Wyn Jones and Gorham, 1983).The diversity also holds true for the units of the parameters, where atmospheres (atm), millimetres of mercury (mmHg or torr), bars and pascals (Pa) are in use. A more uniform usage of these parameters and units is certainly recommended and would facilitate communication among scientists in interrelated fields. In this review, water potential (w) will be used because of the mechanistic advantages of its additive nature in describing the overall state of water in the cell. Water potential is the term most frequently in use by plant physiologists and, even though the concept of osmotic or turgor pressure could equally well be applied to most microbes, the generality of water potential because of its gravitational term (see below) should not be overlooked in mycology. Thisis especially valid for those fungi that transport water vertically over great distances, which has been reported for vegetative mycelia of Sepufu lacrimans (reviewed by Jennings, 1983b, 1987). The unit used in this review is the pascal (1 MPa = 10 bars), the recommended unit in the SI system.

A.

THE CONCEPT OF WATER POTENTIAL

The term “chemical potential of water” (p,), by analogy with the chemical potential of any other compound, is defined as follows:

p,

=

p+

+ R T In a, + V,.P + m,gh,

where denotes the chemical potential of water in its standard state (that can be arbitrarily chosen), R and Tare the gas constant and the temperature (K), respectively, a, is the thermodynamic activity of water, V,. the partial molal volume of water and P the hydrostatic pressure. The gravitational term is described by m,gh, where m, denotes the mass per mole of water, g

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

149

acceleration due to gravity and h the height. The gravitational term is of insignificant importance for single-cell micro-organisms, but will, as already stated, be involved in water flow in vertical mycelia since the water potential increases by 0.01 MPa per metre of height. Equation (1) differs from the general formula for the chemical potential of any compound ‘i” by omitting the term ZiFE representing the effect of electrical potential on the chemical potential, since water carries no charge (2, = 0). The expression p W - - ~ has . proven to be of considerable importance for studies on the water relations of plants. It represents the thermodynamic work involved in moving 1 mol of water from some point in a system to a pool of pure water (Nobel, 1974). This chemical potential difference divided by V,. is defined as the water potential and given the symbol w. The osmotic potential (n); (equal in magnitude but opposite in sign to the osmotic pressure) is defined as 71:

= R T In aw/Vw.,

(2)

which transforms the water potential relation into

y~ = n

+ P + 6,gh,

(3)

where 6 , (density of water) equals rn,lV,.. The use of osmotic potential instead of osmotic pressure in evaluating equation (3) is, in our view, preferable since, in that way, the thermodynamic expression of the state of water will have the same sign inside and outside of the cell (see below). Equation (3) thus becomes generally applicable to solutions as well as cells. Sometimes a matrix potential term is included in the expression to account for water bound to surfaces. It is believed, however, that this term is negligible under quite a range of water potentials, and becomes significant only under severe cellular dehydration or in extremely dry soil (Harris, 1981). The validity of a separate matrix term has even been questioned, and it was argued that the term should principally be included in R or P (Passioura, 1980). The water potential of pure water has been given the value zero, and the term will accordingly decrease with increased concentration of a solute, e.g. sea water with a molality of about 0.5 will have a water potential of about -2.5 MPa while a high water-potential basal-growth medium for yeast corresponds to a water potential of roughly -0.4 MPa. The term “water potential” has received little attention by microbiologists over the years, and instead the thermodynamic activity of water (a,) has been used (especially in the field of food preservation). The water activity is experimentally determined as the vapour pressure of the solution divided by the vapour pressure of pure water. Furthermore, the term “osmolality” (Osm) is sometimes used as a concept relating any solution to its “ideal” equivalents

150

A . RLOMBERG A N D L. ADLER

by an osmotic coefficient (+) (see the appendix in Harris, 1981). All these physicochemical parameters describing the state of water in a solution are strictly related (if pressure and gravitational terms are excluded from y~), as can be seen in equation (5):

w

=

x

=

R T In awlVw.= RT6, Osmll000,

(5)

which at 20°C yields

y

=

135 In a,

or y~ = -2.43 Osm.

The water potential is a colligative property of a solution, and is related to other colligative properties such as depression of freezing point, depression of vapour pressure and elevation of boiling point. A solution that is 1 Osm at

0

I

I

I

I

I

I

0.2

0.4

0.6

0.8

1.0

1.2

Particle molality ( M 1 FIG. 2. The water potential of solutions in relation to solute “particle” molality, either calculated from data given by Robinson and Stokes (1959) on osmotic coefficients at 25°C (open symbols) or as estimated from the freezing-point depression and chemical data at 20°C (solid symbols) given by Wolf etal. (1979). The solutions were adjusted with sodium chloride (circles) or sucrose (squares), or represent an ideal solution either at 20°C (. . . .) or at 25°C (--).

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

151

its freezing point has a freezing-point depression of 1.86“C. However, as pointed out by Wyn Jones and Gorham (1983), measuring the freezing-point depression strictly only yields the osmolality at the freezing point, since 4 is temperature-dependent. This temperature dependence is most notable for high concentrations and at temperatures near the freezing point (Harned and Owen, 1958). For most practical purposes in experimental physiology, however, the water-potential value of a solution of low particle molality will give essentially the same result irrespective of the method used and will even adhere closely to the ideal van’t Hoff relation (Fig. 2). The two sets of data presented have been recorded at different temperatures. However, a 5°C decrease of a 1 Osm solution will lower the y-value by not more than 0.04 MPa. Most values of the water potential given in this review are calculated for 20°C from the data of Wolf et al. (1979). The water potential of any solution is, at low concentrations where the solution can be regarded as ideal, a function of the concentration of solute “particles”, as stated in the van’t Hoffrelation (e.g. Brown, 1976). It should be noted, however, that this relation is not only applicable to ideal solutions but, furthermore, only for very dilute ideal solutions since it is a special case of the more general equation (2) for osmotic potential (osmotic pressure) (Thain, 1967). The nearly ideal behaviour of dilute solutions breaks down at increased concentrations, where the type of solute molecule becomes increasingly important. Generally, sugars like sucrose or glycerol contribute more (lower water-potential values) than ions at equal solute “particle” molalities (Fig. 3). B. COMPONENTS OF T H E WATER POTENTIAL OF CELLS

Since water is extremely permeable to biological membranes, cells are in most instances in thermodynamic water equilibrium with the solution in their environment, i.e. the water potential of the environment equals the water potential of the cell: yen”= ycell.The water potential of the environment is almost always a function of the osmotic-potential component (including matrix phenomena). Fungi, however, are confined by a more or less rigid cell wall, which restricts swelling of the cytoplasm. The total water potential of the cell is thus the sum of both the osmotic potential of the cytoplasm and the turgor pressure implied by the cell wall. 1 . Osmotic Potential of the Cell

By dissolving the wall of Saccharomyces cerevisiae by treatment with the gut juice of the snail Helix pomatia, Eddy and Williamson (1957) prepared protoplasts which were stable for several hours in solutions of 0.5 M

152

A . BLOMBERG AND L. ADLER

-251 0

I

I

I 3.0

1

I

I 6 .O

I

I

I 9.0

Particle rnolality ( M )

FIG. 3. Water potential of solutions at 20°C in relation to solute “particle” molality, calculated from data on freezing-point depression given by Wolf et al. (1979). The solutions were adjusted with sodium chloride (solid symbols) or glycerol (open symbols), or represent an ideal solution (--).

rhamnose. Increased concentrations of the sugar decreased the size of the protoplasts while, in more dilute media, the protoplasts became swollen. These results indicate that the cytoplasmic “particle” concentration is of the order of 0.5 M and of an osmotic potential around -1.5 MPa. By applying the same approach a similar value was obtained for protoplasts of Neurospora crassa (Bachmann and Bonner, 1959) and a somewhat lower value of -3 MPa for Zoophthora radicans (Glare et al., 1989). A particle molarity value of around 0.5 M for Sacch. cerevisiae was confirmed by Conway and Armstrong (1961) by use of the microcryoscopic technique, which determines the degree of freezing-point depression. They found a total intracellular molarity of stationary-phase cells corresponding to - 1.5 MPa. However, metabolically active cells fermenting glucose displayed a decreased intracellular osmotic potential of -2.2 MPa. This type of study was extended to other fungi by use of thermocoupled psychometry, where mycelia were growing on solid media to obtain easily material free

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

153

from the solute used, thereby eliminating the need for washing prior to cell disruption (Adebayo et al., 1971). In a high water-potential medium of -0.6 MPa, Mucor hiemalis and Aspergillus wentii exhibited osmotic potentials of -1.1 and -2.1 MPa, respectively. It was found for both fungi that the osmotic potential decreased when the water potential of the growth medium was lowered, finally resulting in an osmotic potential in both species of around -4.3 MPa at an external water potential of -3.1 MPa. Luard (1982a) used the same technique to manifest a decreased osmotic potential for Penicillium chrysogenum and Chrysosporium fastidium proportional to a decreased external water potential. An indirect method for determining the osmotic potential of the cell involves chemical analysis of cell constituents such as ions, sugars, amino acids and other metabolites, in conjunction with measurements of the cytoplasmic osmotic volume. This enables calculations of the osmotic potential as the sum of all of the individual contributions of the cellular constituents. A problem with this approach is that some abundant cell compound might be overlooked in the analysis, resulting in a too high (less negative) value for the osmotic potential. The calculated osmotic potential for P. chrysogenum was found almost to coincide with that experimentally observed by psychrometry for glucose-adjusted media to - 10 MPa, while potassium chloride-adjusted cultures to the same water potential deviated from the calculated value by about 40% (Luard, 1982a). It should be stressed, however, that chemical analysis of cells will yield important information on the balance between different osmotically active substances during growth at different water potentials (Burke and Jennings, 1990; Larsson etal., 1990; Luard, 1982a) (see Section V). A combination of these two techniques is preferable, since a lower osmotic potential obtained by the psychrometric technique will indicate the presence of constituents not analysed in the chemical analysis.

2. Turgor Pressure of the Cell Different means to measure the turgor pressure ( P ) have been applied, most of them being indirect measurements where turgor is estimated from measurements of the osmotic potential of the cell (n) and information on the external water potential (w) ( P = w- n). The requirement for a positive turgor pressure for growth and hyphal extension has been proposed by several authors (e.g. Lockhart, 1965; Reed, 1984), while an alternative hypothesis implicates microtubule-mediated extension. However, inhibitors of microtubule and microfibril function, like colchicine and vinblastine, had no significant effect on hyphal extension in Serpula lacrimans (Jennings, 1983b). Evidence against turgor as a prerequisite for growth comes from a

154

A . BLOMBERG AND L. ADLER

study by Walsby (1980) on a square bacterium isolated from saturated brine pools, with an apparently non-existent turgor pressure. Controversy exists as to whether cells regulate turgor, volume or some other unknown parameter. However, from experimental data, species can be classified as either turgor- or volume-regulating (Reed, 1984). From a general point of view, volume seems a more universal variable than turgor, considering the infinitesimally small turgor of wall-less cells. Furthermore, mechano-sensitive ion channels have been identified in the yeast plasma membrane, the activity of the channels reportedly being regulated by the tension in the plane of the membrane (Gustin et al., 1988), which is related to the cell volume and lipid content but not presumably to turgor. Regulation of cell volume has even been attributed to a role in the cell cycle of yeast, as an essential signal governing the start point (Pringle and Hartwell, 1981). However, the turgor pressure is an essential part of the total water potential of the cell, and thus has to be measured in order to understand fully the cellular response to fluctuations in the water status of the environment. Its fundamental importance in growth, however, merits further substantiation. Thermocouple psychrometry has been used to study the water-potential components of fungal mycelia, and it was found that the turgor ranged from 0.4 to 1.1 MPa for Mucor hiemalis (at different external water potentials), and between 1.2 and 1.8 MPa for Aspergillus wentii (Adebayo et al., 1971). While turgor of M . hiemalis increased with a decrease in water potential, the turgor of A . wentii remains relatively constant. Luard and Griffin (1981) extended studies on turgor of fungi to include nine fungal species, representing a wide range of tolerance to growth at low water potentials. It was found that most fungi slightly increased their turgor in response to lower water potentials of the medium, e.g. Phytophthora cinnamomi and Phellinus noxius increased their turgor from 0.9 to 1.4 MPa as a result of a water-potential decrease of the medium from -0.5 to -3.5 MPa. In a recent study on the marine yeast Debaryomyces hansenii, however, the turgor pressure dropped with decreased external water potential. In high waterpotential basal medium, turgor was kept at 2.2 MPa and decreased to just 0.5 MPa during growth in 1.35 M sodium chloride (Larsson et al., 1990). In that study, cells were grown in a chemostat and it was reported that turgor was not significantly altered by rate of growth, at the water potentials examined. The studies reported above indicate complex modes of turgor regulation among fungi during cultivation at low water potentials, some regulating to a constant pressure while yet others tolerate or adjust to deflecting turgor values (higher or lower compared to growth at high water potentials). Using a probe is the only way of directly measuring the turgor of

PHYSIOLOGY OF OSMOTOLEKANCE IN FUNGI

155

eukaryotic cells (Zimmermann, 1978). The probe is an oil-filled microcapillary linked to a pressure transducer, which can be applied to cells with a diameter of more than 20 pm. Besides giving values on the turgor, the technique can be used to determine the elastic modulus of the cells, the hydraulic conductivity of the cell surface and the permeability coefficient of the membrane for any solute. The only study to our knowledge applying the pressure probe for fungal cells is a study on the water relations of the sporangiophores of Phycomyces blakesleeanus (Cosgrove et al., 1987), the study being possible because of the large size of the structure (about 100 pm in diameter and many centimetres in length). The sporangiophore was found to have an average turgor of 0.4 MPa, a value which remained the same irrespective of sporangiophore development. Another direct technique for turgor measurement is the one applied to prokaryotes with gas vesicles, these gas vesicles being used as pressure probes. An externally applied pressure will collapse the vesicles, thereby changing the turbidity of the culture, which by measurements on turgid and plasmolysed cells will yield the turgor pressure of the cell without any assumptions about the cell volume or concentration of constituents (Walsby, 1986). By applying that technique to cells of Ancylobacter aquaticus, it was even possible to measure the turgor of individual cells by observing the gas vesicles of cells in the phase microscope while increasing the pressure. It was found that turgor significantly differed among cells, with a standard deviation of 0.04 MPa and a mean individual turgor of around 0.3 MPa during growth at high water potentials (Pinette and Koch, 1987). The deviating individual turgor pressures in these asynchronous cultures of A . aquaticus could reflect cell cycle-specific turgor values. Experimental data indicating such turgor fluctuations in Sacch. cerevisiae during the cell cycle have been presented (Brown, 1990).

111. Osmotolerance

Different terms are used in the literature to describe the overall response of organisms to low availability of water, such as “osmotolerance”, “xerotolerance”, “halophilism”, “osmophilism” and “desiccation tolerance” (Onishi, 1963; Pitt, 1975; Brown, 1976, 1978; Tilbury, 1980; Corry, 1987; Hoekstra and Van Roekel, 1988). As far as fungi are concerned, the first two terms are the most generally used. Attempts have been made to classify species according to their ability to grow below some defined threshold water potential. It should be pointed out, however, that the water relations of organisms distribute over a range rather than being restricted to two main classes such as tolerant and non-tolerant. Such a classification is thus

156

A . BLOMBERG AND L. ADLER

prone to be artificial, but can of course be used as a relatively qualitative indicator. To determine experimentally the degree of osmotolerance, different approaches have been applied, growth-related studies being the most frequent. However, Koppensteiner and Windisch (1971), besides studying growth, included both survival criteria and a study of gas production, and found that the investigated yeasts exhibited different critical water potentials depending on the method used. Generally, the survival criteria gave the lowest y-values while growth was reported to be the most osmosensitive feature. Similarly, respiration and fermentation by yeasts isolated from the sea were reported by Norkrans (1968) to be generally more tolerant than growth to low water potentials. A.

CARDINAL WATER POTENTIALS OF GROWTH

Since the ultimate manifestation of life is multiplication, the rate of the increase in biomass seems to be a relevant parameter for studies on how much the cell is affected by water-potential change. Several growth-related investigations have appeared over the years. Most of these studies present results which are basically similar to those reported by Scott (1957) and Anand and Brown (1968). The latter authors measured growth rates of 16 yeasts at 30°C in media adjusted to different water potentials with polyethylene glycol 200. Examples of osmotolerant yeasts included in the study were Zygosaccharomyces rugosus, Zygosacch. rouxii, Torulopsis halonitratophila and Saccharomyces mellis, while the non-osmotolerant isolates were either Sacch. fragilis o r different strains of Sacch. cerevisiae. All strains more or less fitted the generalized graph depicted in Fig. 4, where yoptand yminfor growth. The graph one can identify values for ymax, indicates that these yeast strains grew faster at high water potentials, with their yoptvalues being closer to ymax than ymin. Thus, these strains merely tolerated low water potentials, which is why the term “osmotolerant” is a more preferable description of the water relations of these organisms than “osmophilic”. 1 . Values for ymax

For all of the yeasts but one used in the study by Anand and Brown (1968), the ymax value was close to 0 MPa, with this limit probably determined by the availability of carbon and energy sources in extremely dilute solutions and not by the high water potential per se. However, some fungi are unable to grow in high water-potential basal media, and seem to require addition of some solutes to lower the y-value. Two representatives for this seemingly

157

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

/ 80

' f

'\

8

u

f

3

/

60

/

.4-

/

0

a, *

0 [L

/ /

40

0 -25

/ I/ /

- 20

I -15

I -10

I -5

1 0

Water potential ( MPa 1

FIG. 4. Generalized graph of the relative rate of growth in relation to the water potential of the growth medium. Indicated are the cardinal water potentials of growth, namely Wrnax, Wept and Wrnin.

osmophilic response are Xeromyces bisporus and Chrysosporiumfastidium, with reported vmax values at 25°C of -5.6 and -2.8 MPa, respectively (Pitt and Hocking, 1977). The osmophilic character of these species determined at 25°C may, however, change at lower temperatures since not only is the water potential of a solution dependent on the temperature but the water relations of organisms are also affected. This was pointed out by Onishi (1960b), who reported isolation from old soy-mash of Torulopsis halonitratophila, which at first sight seemed to be an obligate halophilic yeast. The halophilic phenotype was, however, lost by decreasing the temperature from 30 to 20°C since, at the lower temperature, the strain grew in high water-potential medium. Inversely, the cardinal temperatures for growth, namely Tmin,Toptand T,, for an organism, are dependent on the water potential of the medium. For osmotolerant yeasts, a decrease in the water potential from -1.5 to -15 MPa by addition of glucose resulted in about a 5°C increase in all three cardinal temperatures (Jermini and Schmidt-Lorentz, 1987). The interrelation between water potential and temperature has been manifested

158

A. BLOMBERG AND L. ADLER

also for temperature-conditional mutants auxotrophic for amino acids and nucleotides, which with a decreased water potential in the medium at the restrictive temperature reverted to a prototrophic phenotype (osmotic remedial mutants; Hawthorne and Friis, 1964). The increase in the cardinal temperatures, as well as the permissive temperature of conditional mutants, is possibly brought about by an increased accumulation of compatible solutes during growth at low water potentials (see Section V.A). These intracellular solutes have been shown to stabilize proteins against thermal denaturation in vitro (Back et al., 1979), thus opposing the detrimental effects on the cells of raised temperatures. Thus, species unable to grow at high water potentials might suffer from denaturation of proteins at the prevailing temperature, and growth will be made possible either by a decreased water potential or a diminished temperature. An alternative physiological explanation for an osmophilic response comes from a mutant of Zygosacch. rouxii unable to grow at high water potentials. This defect seemed to be caused by a decrease in the mechanical strength of the cell wall (Koh, 1975). 2. Values for yopt

Anand and Brown (1968) reported values that differed for different strains of yeasts but were around -1 to -5 MPa in media adjusted with polyethylene glycol, with the osmotolerant yeasts exhibiting a broader optimum range; for example, the rate of growth for one of the strains of Zygosacch. rouxii was nearly unaffected by water potentials between -1.0 and -5.6 MPa. The non-osmotolerant strains, however, generally displayed sharper optima at water potentials around -1.5 MPa. In his studies on the fungus Eurotium amstelodami, Scott (1957) postulated that the optimum water potential for growth of osmotolerant fungi was independent of the predominant solute of the medium. This hypothesis has subsequently been substantiated by work by Pitt and Hocking (1977), Luard and Griffin (1981) and Andrews and Pitt (1987), extending the study to include at least 20 species of fungi. Among those species adhering to the hypothesis were: Eurotium chevalieri, yoptabout -7 MPa; Aspergillus jlavus, yopt-3.5 MPa (Pitt and Hocking, 1977); Phytophthora cinnamomi, yopt - 1.2 MPa; Aspergillus restrictus, yopt -5 MPa (Luard and Griffin, 1981); Aspergillus ventii, Vopt -7 MPa; Exophiala werneckii, yopt-7 MPa (Andrews and Pitt, 1987). Some other species exhibited more specialized solute tolerance, exemplified by Basipetospora halophila for which the yoptvalue in glycerol- or glucose-fructose-adjusted media was - 14 MPa, while in sodium chloridecontaining media the voptvalue was -21 MPa (Andrews and Pitt, 1987). As

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

159

an approximate rule of thumb, however, Scott's idea hypothesis of a soluteindependent yoptvalue seems to be valid. Despite the solute independence on yoptvalues, Scott (1957) noted that the absolute rate of growth was related to the nature of the solute. This growth-rate discrepancy seems to be maximal at or around the yoptvalue, e.g. the rate of radial growth of Xeromyces bisporus at yopt-22 MPa was in glycerol-containing media reported to be about 20 pm h-', while in glucose-fructose-containing media that brought about the same yoptvalue the growth rate was increased three-fold (Pitt and Hocking, 1977). Even though the yoptvalue seems to be relatively unaffected by the type of stress solute, it is reasonable to believe that it should be related to growth temperature; detrimental effects by increased temperature will be opposed by increased accumulation of a compatible solute. Thus, water potential and temperature have appropriately to balance each other for optimum growth to occur. This was shown for Wallemia sebi, which at 20°C exhibited a yopt value of -5.5 MPa while, at 34"C, the value decreased substantially to -18 MPa (Wheeler et al., 1988). Another interesting and apparently general finding by Anand and Brown (1968) was that the most osmotolerant yeasts had a maximal growth rate substantially lower than that of the non-osmotolerant strains. This was shown to be an intrinsic property of the organisms since it was not caused specifically by polyethylene glycol, but the trend still persisted in solutions of glycerol or sucrose. The mean generation times for the osmotolerant species were about four to eight hours while those of the non-osmotolerant species were in the range of 1.5-2 hours. No relevant explanation has been given so far for this interesting finding.

3. Values for v/min Among filamentous fungi and yeasts, there are representatives with an extreme capacity for growth at low water potentials. In an overview of microbial tolerance to low water potentials (including bacteria, fungi and algae), the majority capable of growth below -25 MPa were fungi (Harris, values below -22 MPa (a, 0.85) and 1981). Pitt (1975) listed fungi with ymin found that only 11 genera of filamentous fungi were represented, all being ascomycetes. The ascomycetes constitute a large group of fungi with roughly 3000 genera. Clearly, a very small number of highly specialized variants with high osmotolerance have evolved. Some representative species with ymin values below -45 MPa are Aspergillus conicus, Chrysosporium fastidium, Eurotium amstelodami and Xeromyces bisporus. Three species of yeast were listed, namely Deb. hansenii, Sacch. (now Zygosacch.) bailii and Zygosacch. rouxii. Barnett et al. (1983) reported in their taxonomic

160

A . BLOMBERG AND L. ADLER

classification of 469 species of yeasts data on the growth response of yeasts in glucose media adjusted to either -11 or -15 MPa. It was found that about 56% of the species were unable to grow at water potentials below -11 MPa, 28% grew at -11 MPa but not at -15 MPa and the remaining 16% had ymin values lower than -15 MPa. It can thus be concluded that, even if both filamentous fungi and yeasts encompass extremely osmotolerant species, the majority of fungal species exhibit a moderate tolerance to growth at low water potentials. It has furthermore been shown that the water potential of a solution is sometimes not the single parameter determining the response of an organism, but that solute-specific properties have to be taken into account. This statement can be exemplified by the study of growth of yeasts in media containing fructose or polyethylene glycol 200 (Anand and Brown, 1968). The osmotolerant strains had a ymin value in high concentrations of fructose of at least -36 MPa while the non-osmotolerant strains ceased growing at roughly - 12 MPa. This great difference in ymin value among strains during growth in the presence of fructose was substantially diminished when the strains were grown in polyethylene glycol-adjusted media, where growth was restricted to water potentials above about - 10 MPa, with no real evident difference in yminvalues between osmotolerant and non-osmotolerant strains. The solute-specific effect in this case might be related to uptake of the stress solute to various extents by the different organisms. Polyethylene glycol 200 seems readily to penetrate the plasma membrane of some yeasts, since Rose (1975) found that the cell volume of some strains did not respond to increased concentrations of this solute. However, polyethylene glycols of higher molecular weight are sometimes used to impose matrix waterpotential stress on organisms, assuming that these bulky compounds cannot penetrate the cell wall (Adebayo and Harris, 1971; Smith et a f . , 1990). Andrews and Pitt (1987) found ymin values for some filamentous fungi to values for be solute-invariant (e.g. Aspergifluspenociffoides),while the ymin others like Exophiafa werneckii and Geomyces sp. were more solutedependent. For species exhibiting different growth responses depending on the stress solute applied, more solute-specific terms like “salt tolerance” or “sugar tolerance” are sometimes used instead of osmotolerance. However, even these terms are found to be too simplistic since different salts exhibit marked differences in growth inhibition (Onishi, 1957a). One of the extreme cases reported was the difference between sodium chloride and lithium chloride, the latter salt inhibiting growth completely at 0.5 M (about -2.5 MPa) while growth was sustained in the presence of sodium chloride even at 3 M (-15 MPa). We believe that the term “osmotolerance” can still be used as an overall description of the phenomenon, since all media will inevitably have an

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

161

osmotic effect on the cells, an effect that usually extends to the whole growth period but may differ in magnitude depending on solute-specific interactions with organisms. However, the specific effects should never be overlooked, and any phenomenon being classified as “osmotic” in nature should have been shown to apply for both salts and sugars. Many studies have reported that fungi show greatest tolerance to low water potentials when other environmental conditions are close to their high water-potential optimum for the species (Corry, 1987). Ayerst (1969) values for Aspergillus chevalieri to be lowest at Topt.It was showed ymin furthermore shown by Onishi (1957b) that pH value strongly influenced the ability of the cells to grow at low water potentials. While Zygosacch. rouxii grew well over a wide range of p H values at high water potentials (pH 3-7), the pH range allowing growth was narrowed to p H 4-5 in media adjusted with sodium chloride to roughly - 15.0 MPa. In conclusion, if cardinal water potentials for growth are to be reported, care should be taken in accurately describing the side factors involved, e.g. ymin-20.0 MPa (sodium chloride, 3WC, pH 4.5). For further discussions about cellular factors involved in determining the yminvalue, see Section VII. IV. Initial Osmotic Response A . MICROSCOPIC OBSERVATIONS

Direct microscopic observations on the initial response of yeast cells subjected to low water-potential media revealed cell shrinkage for both the osmotolerant Zygosucch. rouxii and the less osmotolerant Candidu utilis. This decrease in cell size was reported to be a result of non-isotropic shrinkage, the short axis being the major axis of decrease in length (Corry, 1976). This type of study was extended to include species of the yeast genera Candida, Cryptococcus, Kluyveromyces, Pichia, Rhodosporidium, Rhodotorulu, Saccharomyces and Schizosaccharomyces, which all were shown to respond qualitatively similarly to 2 Osm sodium chloride (-5.0 MPa), that is, by non-isotropic cell shrinkage. A quantitative measure of the non-isotropicity was also given for Sacch. cerevisiae, with the long axis being decreased by 11Yo while the short axis was decreased by 23% (Morris et al., 1986). The decrease in cell volume was shown to be osmotic in nature and not just an electrochemical effect of concentrated solutions on the structure of the cell wall, since treatment of intact yeast cells with Triton X-100 (a procedure that will impair the semipermeability of the plasma membrane) failed to cause a reduction in cell volumes even in 4 Osm sodium chloride (-10.0 MPa). The response of the cell size to changes in water potential was reported to

162

A . BLOMBERG AND L. ADLER

be reversible, since osmotically dehydrated cells of Sacch. cerevisiae resuspended in high water-potential media immediately increased in volume. This cycle of dehydration-hydration could be repeated without loss in cellular osmotic responsiveness (Morris et af., 1986). When exponentially growing Sacch. cerevkiae (basal medium, -1.2 MPa) was transferred to media adjusted to -2.8 MPa by addition of glycerol or sorbitol, cells immediately decreased in volume to about 60% of their pretransfer size (Niedermeyer et al., 1977). These shrunken cells were studied by freeze-fracture electron microscopy, which revealed that large depressions of the plasma membrane appeared in response to cellular dehydration. These depressions were always associated with the typical plasma-membrane invaginations seen on cells during growth at high water potentials. In contrast to the plasma membrane of intact cells, however, that of protoplasts retained its normal ultrastructure in response to dehydration, and depressions could not be seen. Vacuoles of Sacch. cerevisiae were reported to decrease in size in vivo in response to cellular dehydration (Niedermeyer et a f . , 1976; Morns et a f . , 1986). In contrast to whole cells, the tonoplast response was reported to be irreversible; cells once shrunk could not attain their original vacuolar volume upon cellular rehydration at high water potentials (Niedermeyer et a f . ,1976). This irreversibility could also be mimicked in vitro for isolated tonoplasts, and it was furthermore proposed that the degree of elasticity of the tonoplast membrane was dependent on the protein components of the membranes (Niedermeyer, 1976). The alga Dunaliefla safina exhibited organelle shrinkage upon osmotic cell dehydration. The surface area of the endoplasmic reticulum increased, however, leading to the suggestion that it acted as a reservoir for membrane material present in temporary excess when the organelles shrink (Einspahr et af., 1988). The effect of osmotic stress on mitochondria1 structures in yeast was studied by using the fluorescent probe rhodamine 123, the pattern of staining being severely altered only at water potentials below - 14.5 MPa (Morris et a f . , 1986). Taken together, not only does the total cell volume respond to, but also the organelles of the cell will be affected by, dehydration. A well-known and well-characterized phenomenon in plant cells is plasmolysis, which is the physical separation of the plasma membrane from the cell wall as a consequence of substantial protoplast shrinkage. By definition, the turgor pressure at the point of plasmolysis is zero. For microbial cells, this phenomenon is less well documented, and conflicting data and views appear in the literature as to its occurrence. Older articles and textbooks describe the phenomenon, while in more recent publications the information is absent. However, in Salmonella typhimurium, substantial plasmolysis has been documented both by electron-microscopic examination and solute-distribution measurements (Stock et a f ., 1977).

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

163

Recent reports on severe osmotic dehydration of Sacch. cerevisiae have not been able microscopically to identify plasmolysed cells (Morris et al., 1986; Meikle et al., 1988). Saccharomyces cerevisiae, stained with saffranin and subsequently osmotically dehydrated by treatment with 2.6 M sodium chloride, revealed no plasmolysis but the cell wall appeared rough (A. Blomberg, unpublished result). This is in accordance with studies on cells dehydrated by slow freezing resulting in extensive cell-volume decrease, where plasmolysis could not be observed either in the electron microscope (Bank, 1973; Bank and Mazur, 1973) or in the light microscope (Morris et al., 1988). The reported reduction in cell volume at water potentials of -10 MPa and lower is a puzzle (Morris et al., 1986), since turgor should at that point be zero and the cell wall be in its most relaxed state, i.e. constant cell size. However, the above-discussed depressions of the plasma membrane upon cell dehydration might indicate plasma-membrane anchorage with restricted protoplast shrinkage beyond incipient plasmolysis. A consequence of this might be protoplast-driven total cell-volume shrinkage, with the wall attaining a “negative” tension and the whole cell being subjected to a negative turgor. A negative turgor pressure has been proposed for Escherichia coli as a transient state prior to the plasmolysed state, resulting in a contraction of the cell wall below its most relaxed state (Koch and Pinette, 1987). The initial cell-shrinkage of Sacch. cerevisiae in response to low water potentials is a rapid process and, at 24”C, dehydration is completed in less than a minute, irrespective of the final water potential (Morris et al., 1986). The rapid response is a consequence of the high water permeability of biological membranes, the water permeability coefficient being around 2X cm s-l for the osmotolerant alga D.salina (Degani and Avron, 1982), human erythrocytes (Shporer and Civan, 1975) and artificially produced vesicular lipid membranes (Lipschitz-Farber and Degani, 1980). The permeability for solutes is generally much lower, as reflected in the permeability coefficient for sugars and sodium ions of around lo-’ and lo-” cm s-l, respectively (Stein, 1986). B. BOYLE-VAN’T

HOFF PLOTS AND NON-OSMOTIC VOLUMES

The Boyle-van’t Hoff relation states that nV,,,, is constant, where V,,, denotes the osmotically active volume. On rearrangement one obtains the more usually applied form for plotting volume-related data, which states that, for non-turgid cells, volume is inversely related to the external water potential. The non-osmotic volume of cells is extrapolated out of the plot as the intercept with the y-axis. By use of the Coulter-counter technique in determining average cell volume and, by plotting the data in conjunction

164

A. BLOMBERG AND L. ADLER

with the Boyle-van’t Hoff relation, the non-osmotic volume (V,,) of cells of Sacch. cerevisiae was reported to be 35% of the original protoplast volume (Meikle et al., 1988). Whole cells had a larger V,, value than protoplasts (48%), indicating that some of the V,, value consists of cell-wall material. The nature of the remaining part of the V,, value might be organelles and storage granules or even accumulated glycerol, as indicated by the increased V,, values during growth at low water potentials (Reed et al., 1987). Even though incipient plasmolysis could not be visually observed as already discussed, it could be extracted from the Boyle-van? Hoff plot as the external water potential instigating non-linear osmotic behaviour of cells, and was reported to be -2.48 MPa (corresponding to roughly 1 Osm) (Meikle et al., 1988).

C. WATER LOSS IN RELATION TO CELL-WALL ELASTICITY AND INITIAL TURGOR PRESSURE

The amount of water leaving the cell, as a response to a decrease in the extracellular water potential, is not only related to the water potential difference but also a function of the elastic properties of the cell wall and the initial turgor pressure. The constant relating the relative change in cell volume to the pressure generated is called the volumetric elastic modulus (E):

AP

= E

AVIV,,

where Vdenotes the total cell volume and V, the reference volume, which is usually the volume at incipient plasmolysis (Dainty, 1976). Thus, the responses of walled and wall-less cells to a perturbation of the external osmotic potential may be considered as formally identical, the relative changes in the pressure and volume components being related via the volumetric elastic modulus (Wyn Jones and Gorham, 1983). The yeast cell wall is fairly elastic (De Bruijne and Van Steveninck, 1970) and reported to have an &-valueof 2-5 MPa (Levin, 1979; Meikle et al., 1988), with stationary phase cells being most elastic (Meikle et al., 1988). A consequence of this cell-wall elasticity is a 20% decrease in total cell size before the point of incipient plasmolysis. Meikle et al. (1988) obtained an &-value that was constant over the turgor range studied. Pressure-probe studies of plant cells have indicated that the value of E is strongly dependent on turgor and cell volume at low pressure, whereas at high pressures E seems to approach a constant (Steudle et al., 1977). A theoretical treatment of cellular water loss subsequent to a sudden exposure to low water potentials is depicted in Fig. 5. Cells being absolutely

PHYSIOLOGY OF OSMOTOLERANCE IN FUNtil

165

rigid ( E = w ) will in principle not lose water until the point of incipient plasmolysis (Fig. 5(a)). On the contrary, cells with elastic walls will face a decreased water content even after minor fluctuations in the water potential of the environment, the magnitude of the water loss being related to the &-value.Furthermore, the water potential at incipient plasmolysis (yplasm) will be altered, giving rise to steadily decreasing yplasm values for more elastic cells. Thus, for the theoretical cell with the lowest &-value(3 MPa) in is -6 MPa. To minimize water loss in an environment with Fig. 5(a), yplasm only slightly fluctuating water potentials, a fairly rigid cell wall would be beneficial. However, if plasmolysis is to be avoided during sudden exposure to low water potentials, the cell should have an elastic wall. At water potentials below yplasm, however, the amount of water lost is independent of E. An alternative strategy for minimizing the water loss would be decreased intracellular osmotic potential (x), which results in an increased turgor pressure that will cushion the cell against water loss in a fluctuating environment (Harris, 1981). In Fig. 5(b) are depicted three cell types with different turgor pressures at y = 0, all cells having the same &-value. Even more pronounced than a decreased water loss in relation to initial turgor is its consequence on yplasm, cells with highest initial turgor not being plasmolysed until a water potential of -18 MPa. Thus, in order to avoid plasmolysis or to retain a positive turgor pressure upon severe dehydration, cells ought to adjust their intracellular osmotic potential to enhance turgor. As reported by Meikle et ul. (1988), Sacch. cerevisiae is weakly buffered against water loss on sudden osmotic dehydration, since, at a water potential of -10 MPa (sodium chloride), only about 10% of the initial V,,, value remained. Similar results were presented by Rose (1975) for osmotic dehydration in sucrose solutions, where the cell volume of Sacch. cerevisiae at -17 MPa almost exclusively consisted of V,,,. On the contrary, it was found that the highly osmotolerant species Zygosacch. rouxii subsequent to a sudden stress at -17 MPa retained almost 77% of its V,,, value. A conservative osmotic response was also exhibited by the marine yeast Deb. hansenii, which at -13 MPa retained about 40% of the osmotically active volume (Norkrans and Kylin, 1969). Thus, the osmotolerant species were not only able to grow at low water potentials but also resisted severe dehydration on sudden exposure. The low degree of water loss might partly be explained by both a low n-value and a high &-value.However, in order to understand the remarkable resistance of Zygosacch. rouxii, additional factors have to be included like rapid solute penetration or some kind of cellwall imposed negative turgor (see above).

166

A . BLOMBERG AND L. ADLER

-10

I -8

I -6

I

-4 Water potential (MPa)

I -2

1 0

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

167

V. Osmoregulation

By osmoregulation is understood the ability of cells to adjust their intracellular solute content in the face of an external water stress. The term has been considered misleading (Cram, 1976; Reed, 1984) in not recognizing the importance of cellular control of turgor or volume in this process. It is also argued that the term “regulation” should be reserved for a stricter meaning than is usually the case in biology. Although it appears logical that turgor or volume is the controlled parameter, evidence for this is still only indirect. We therefore adhere to the more commonly adopted term “osmoregulation”, which will be used here to describe the ability of the cell to adjust the total number of intracellular solute molecules, without making any assumptions concerning the mechanisms by which the process is controlled. The discussion will be concerned with the types of solutes used by fungi to adjust their intracellular osmotic potential during either adaptive osmoregulation, by which the cells respond to a changed water potential by increasing or decreasing their internal solute . content, or steady-state osmoregulation, by which the growing cells maintain an intracellular solute content which is appropriate to the prevailing water potential.

A . COMPATIBLE SOLUTES

A general mechanism by which micro-organisms counteract the dehydration effects of diverse and fluctuating external solute compositions is by excluding the stress solute and by compensating through intracellular accumulation of one o r more specific solutes called compatible solutes (Brown and Simpson, 1972) or osmolytes (Yancey et al., 1982). These compounds can be accumulated by endogenous production or by uptake from the medium to high concentrations without giving rise to appreciable enzyme inhibition or inactivation (Brown, 1976, 1978, 1990). Thus, this FIG. 5. Theoretical predictions of the initial decrease in the osmotic volume upon a sudden osmotic dehydration at indicated water potentials. Calculations have been based on equation (8) and the Boyle-van’t Hoff relation. The value of E was considered to be independent of pressure and volume. (a) Initial water loss in relation to cell-wall elasticity. Values are depicted for a model fungal cell with n = -2 MPa and P = 2 MPa at an external y-value of 0 MPa, but with different elasticities: (A)E = 3 MPa, (0)E = 10 MPa and (W) E = m MPa (rigid cell wall). (b) Initial water loss in relation to cell turgor when y = 0 MPa. Values are depicted for a model fungal cell with E = 3 MPa, but with various P-values at y = 0 MPa: (0) P = 1 MPa, (A)P = 2 MPa and (W) P = 3 MPa. In all cases a V,, of 40% has been used. Arrows indicate predicted water potentials for the model cell at incipient plasmolysis.

168

A . BLOMBERG AND L. ADLER

allows cellular processes to operate at low intracellular osmotic potentials without the requirement for structural modification of sensitive cellular enzymes or structures. Some compatible solutes, sometimes called osmoprotectants, are not only highly innocuous to protein structure and function but also alleviate some of the inhibitory effects of high ionic strength (Pollard and Wyn Jones, 1979; Yancey et al., 1982). Only a limited number of organic compounds are used as compatible solutes. These compounds can be considered to fall into two main groups: (a) polyhydroxy compounds and (b) amino acids or amino-acid derivatives (Yancey et al. , 1982). As will be discussed below, glycerol and other polyhydroxy alcohols (polyols) are the main organic compatible solutes in fungi. The biophysical and biological properties of compatible solutes have been discussed by Wyn Jones and Pollard (1982), Yancey et al. (1982) and Low (1985). They all share an ability to raise the denaturation temperature and lower the solubility of codissolved globular proteins. Density measurements by Timasheff and his coworkers have demonstrated that glycerol (e.g. Gekko and Timasheff, 1981) is excluded from the vicinal hydration sphere of proteins in protein-glycerol solutions. This property promotes minimal protein-solvent interactions, which accounts for observed consequences such as subunit association and stabilization against denaturation. The preferential hydration and the concomitant solute exclusion from the immediate vicinity of the proteins appear to be general mechanisms by which compatible solutes stabilize proteins (see Low, 198.5, and references therein).

I . Polyols Polyols are widely distributed in fungi (Lewis and Smith, 1967; Rast and Pfyffer, 1989). Onishi (1960a, b) surveyed 119 strains of yeasts for polyol production and found that most species produced glycerol and arabinitol, and a few of them also small amounts of erythritol. Analysis of the pattern of polyol production by some 450 fungal species for the purpose of chemotaxonomy (Pfyffer et al., 1986, 1990; Rast and Pfyffer, 1989) detected polyols (the main polyols being glycerol, threitol, erythritol, ribitol, arabinitol, xylitol, sorbitol, mannitol and galactitol) in all fungi examined except among the Oomycetes. All other fungal taxa fell into two groups with respect to polyol production: (a) those that contained various polyols except mannitol (Zygomycetes and Hemiascomycetes) and (b) those that contained mannitol as well as other polyols (Chytidriomycetes, Euascomycetes, Basidiomycotina and Deuteromycotina). Work by Onishi and his coworkers, reviewed by Onishi (1963), showed that high concentrations of sugars or salts in the growth medium changed the

PIIYSIOLOGY OF OSMOTOLERANCE IN FUNGI

169

pattern of fermentation in many osmotolerant yeasts, resulting in increased production of polyols. An intracellular role for these polyols was indicated by Brown and his colleagues, who demonstrated that osmotolerant yeasts, in contrast to non-tolerant species, contained arabinitol as a major intracellular component when grown in dilute basal medium (Brown and Simpson, 1972). The significance of polyols in yeast-water relations was further emphasized by the observation that the intracellular polyol content in Zygosacch. rouxii and Sacch. cerevisiae increased when the external water potential was lowered by addition of polyethylene glycol 200, glucose (Brown, 1974) or sodium chloride (Brown, 1978) and that the internal concentrations at low water potentials could reach molal levels. Similar conclusions were reached by Norkrans who emphasized the role of glycerol in the salt relations of yeasts. It was first reported that the total salt content in the marine yeast Deb. hansenii did not balance the water potential of the growth medium when the cells were cultured at high salinities (Norkrans and Kylin, 1969). Glycerol was suggested as an osmoregulator (Gezelius and Norkrans, 1970) and subsequent studies confirmed that the intracellular level of glycerol in growing Deb. hansenii increased in parallel with the external concentration of sodium chloride (Gustafsson and Norkrans, 1976; Gustafsson, 1979). It was later demonstrated that isotonic concentrations of sodium chloride, potassium chloride and sucrose promoted glycerol accumulation to similar levels, while return of cells to more dilute media resulted in rapid release of glycerol in amounts that were proportional to the decrease of external solute concentration (Andre et al., 1988). This work also provided more direct evidence for a role for glycerol in osmoregulation by the finding that externally supplied glycerol re-established ability to grow in the presence of high salinity for a mutant of Deb. hansenii with a lowered capacity to produce glycerol (see Section VII1.D). The importance of glycerol as a compatible solute in yeast has been further confirmed by natural-abundance 13C nuclear magnetic resonance (NMR) spectroscopy, which allows identification and quantification of all classes of organic compounds that attain a significant concentration within the intact cell (e.g. Norton, 1980). Analysis by NMR of yeasts grown in media containing concentrations of sodium chloride from 0 to 0.86 M showed that glycerol was the major osmoresponsive organic solute in exponentially growing Sacch. cerevisiae, Zygosacch. rouxii and Deb. hansenii (Reed et al., 1987). Simultaneous measurements of the non-osmotic volume allowed an estimate to be made of the intracellular glycerol concentrations, which were calculated to counterbalance up to 95% of the total external osmotic pressure. In a more detailed NMR study by Bellinger and Larher (1987), glycerol, arabinitol and the disaccharide trehalose were detected as the main internal organic solutes in Hansenula anornala, while glycerol and trehalose

170

A . BLOMBERG AND L. ADLER

prevailed in Sacch. cerevisiae. Of these solutes only glycerol responded to growth-medium salinity. One of the problems involved in evaluating the importance of polyols in yeast osmoregulation is the dynamics of the individual pol yo1 concentrations during the growth cycle in batch culture. In saline media, the glycerol content of Deb. hansenii increases during the lag and early exponential phases and decreases again during the late exponential phase of growth. The level of arabinitol increases slightly but progressively during exponential growth and reaches its highest value in the early stationary phase (Adler and Gustafsson, 1980). On a water-potential basis, this increase does not compensate for the decrease in glycerol. Thus, there has been a requirement for studies using continuous culture to establish an osmotic budget of cells grown under steady-state conditions and to separate clearly the effects of the external water potential from those due to changes in growth rate, pH value and nutrient composition. Larsson et af. (1990) examined the intracellular solute composition of Deb. hansenii in glucose-limited chemostat cultures containing 0, 0.7 (-3 MPa) and 1.4 M (-6.5 MPa) sodium chloride. To determine intracellular solute concentrations, the osmotic volume of the cells was determined by a radiotracer technique. The results showed that arabinitol responded little to salinity but appeared to be slightly adjusted relative to growth rate, at high salinity, as the levels increased with decreased rate of growth (Fig. 6). Glycerol, on the other hand, behaved as a

(b)

(a)

0.04 0.0 6 0.08 0.10 0 12 0 14

.. .

-

I

-

I

I.

-

. I

1

-

.

0 04 0 0 6 0.08 0.10 0.12 0 14

1

Dilution rate ( h - ' )

. . .. .... .. n riu. 0. inrraceiiuiar concenrrarions OT \a) araoiniroi ana (0)glycerol in ueoaryornyces hansenii grown in a glucose-limited chemostat at four different dilution rates in media containing 4 mM (triangles), 0.7 M (squares) and 1.4 M (circles) sodium chloride. Redrawn from Larsson et al. (1990). -I-

I

r

" , \

I

,

,

I

\

.

I

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

171

typical osmoresponsive solute; the contents rose dramatically with medium salinity and also appeared adjusted relative to arabinitol such that the overall polyol content remained relatively constant at the various growth rates. The total polyol pool counteracted about 75% of the external water potential in media containing 0.7 and 1.4 M sodium chloride. It was concluded from this work and a similar study by Burke and Jennings (1990) that Deb. hansenii adjusted to the prevailing water potential using primarily potassium chloride at low external salinity and glycerol and sodium chloride at high salinity. Nobre and DaCosta (1985b) followed the content of arabinitol in Deb. hazsenii during growth in batch culture adjusted to low water potential by various stress solutes. Low water potential promoted an up to two-fold increase in the arabinitol content of stationary phase cells, but the final content was more influenced by the type of stress solute used than by its concentration. The authors concluded that osmotic control of the arabinitol level appears imprecise and that the primary control may be exerted by other factors. Similarly, Moran and Witter (1979) observed a solute-specific increase in arabinitol accumulation in Zygosacch. rouxii. The cells increased their content of arabinitol when grown in the presence of increased concentrations of glucose, but not with increased sucrose concentrations. Van Zyl and Prior (1990) followed polyol accumulation in Zygosacch. rouxii during growth in continuous culture at lowered water potential, and demonstrated that glycerol was the principal compatible solute in cells grown in media adjusted with sodium chloride. When sodium chloride was substituted by isotonic concentrations of polyethylene glycol 400, the intracellular glycerol concentration remained essentially similar to that reached in the saline medium, while the intracellular retention of arabinitol increased several-fold, so that the concentration of this polyol became, on a molar basis, similar to that of glycerol. Thus, arabinitol, which is normally an only slightly osmoresponsive polyol, may in response to specific stress solutes be used as an osmoregulatory compound. Evidence that polyols play an important role in water relations of filamentous fungi has accumulated during the last decade. Adler etal. (1982) examined the pol yo1 content in vegetative mycelium of Penicillium chrysogenum and Aspergillus niger grown in concentrations of sodium chloride ranging from 0 to 2 M. Both organisms contained glycerol, erythritol, arabinitol and mannitol and the total polyol contents increased strongly in response to raised salinity; glycerol and erythritol became increasingly predominant in the highly saline media (Fig. 7). Using published values for dry weight:fresh weight ratios at low water potential (Luard, 1982a; Beever and Laracy, 1986), the intracellular polyol concentrations can be estimated to counteract 40-60% of the external water potential in media containing 1.4 and 2.1 M sodium chloride. Luard (1982a) reported that glycerol was

172

A . BLOMBERG AND L. ADLER

increasingly accumulated in P . chrysogenum and the osmotolerant species Chrysosporium fastidium as the water potential was decreased by adjustment with potassium chloride or glucose. The higher polyols identified (erythritol, arabinitol and mannitol) did not change consistently and were for this reason, and from behaviour in osmotic-shock experiments (Luard, 1982b), not considered to be of importance in osmotic adjustment. From analysed intracellular solute levels, glycerol accounted for 1540% of the calculated osmotic potential after growth on media adjusted with potassium chloride or glucose to -10 MPa. Similar observations that glycerol is the major osmoresponsive compatible solute within mycelia exposed to low water potential (generated by various stress solutes) have since been reported by Hocking and Norton (1983), who used natural-abundance I3C NMR to examine four osmotolerant and one non-tolerant species, by Gadd et al. (1984) for Penicillium ochro-chloron and by Beever and Laracy (1986) for Aspergillus nidulans. Although there appears to be a clear emphasis on glycerol production and accumulation under conditions of water stress in filamentous fungi, glycerol is not, as stressed by Jennings (1984, 1986), the solely osmoresponsive polyol. The type of polyol accumulated appears to be dependent on culture age, nutrient conditions, type of stress solute used and the organism studied.

1

I 1.4

1,

I 2.1

Concentration of sodium chloride ( M )

FIG. 7. Intracellular polyol contents of Aspergillus niger grown in media containing 0, 0.7, 1.4 or 2.1 M sodium chloride. For every salt concentration each set of open columns describes, from left to right, contents of glycerol, erythritol, arabinitol and mannitol, respectively. The filled-in columns describe the total content of polyols. Redrawn from Adler et al. (1982).

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

173

Hocking (1986a) followed the time-course of glycerol accumulation in five fungi grown at low water potential and demonstrated, as observed for yeasts (e.g. Adler and Gustafsson, 1980; Meikle et al., 1988), that culture age influences the glycerol content. The glycerol content remained high during vegetative growth, but decreased as the culture senesced and started spore formation. When the marine fungus Dendryphiella salina was grown in media in which the water potential was adjusted by addition of various electrolytes, the total soluble polyol concentration increased with decreased water potential (Wethered et al. ,1985). Within that pool, the proportions of the individual polyols differed depending upon the salt used. Highest glycerol values were found in media containing sodium chloride and the lowest when sodium sulphate was included in media. In the presence of 0.44 M sodium chloride (- 1.9 MPa), the polyols accounted for 30% of the calculated intracellular osmotic potential. The content of arabinitol and mannitol changed little with increasingly saline media but increased, on the other hand, when non-growing mycelium was exposed to saline media (Jennings, 1973). Although glycerol was the major osmoresponsive polyol in the species of Penicillium and Aspergillus examined, erythritol showed the behaviour of an osmoregulatory compound; it progressively increased with increasing external solute concentration (Adler et al., 1982; Gadd et al. , 1984; Beever and Laracy, 1986) and decreased after hypo-osmotic shock (Beever and Laracy, 1986). Furthermore, Da Costa and Niederpruem (1982) presented evidence for an osmoregulatory role for arabinitol in Geotrichum candidum, and Al-Hamdani and Cooke (1987) found that both mannitol and glycerol were the carbohydrates that increased most at decreased water potential in sclerotia of Sclerotinia sclerotiorum. Thus, though available evidence indicates that, in growing cells of yeasts and filamentous fungi, glycerol is the predominant osmoresponsive polyol it cannot, at least in filamentous fungi, be singled out as the exclusive osmolyte. It is probably significant, however, that in some instances where glycerol was found to give little contribution to cellular osmotic relations (Moran and Witter, 1979; Da Costa and Niederpruem, 1982; Al-Hamdami and Cooke, 1987) the organisms were washed before extraction under conditions which probably resulted in significant loss of this highly osmoresponsive solute. As pointed out by Pitt (1989), glycerol is the smallest polyhydroxy compound that can be used as a compatible solute. Therefore, from the point of carbon economy, glycerol production represents an effective way to osmoregulate. This may be the reason why glycerol is a preferred osmolyte among growing fungi while, as the organisms enter a non-growing stage, glycerol disappears and higher polyhydroxy compounds become the predominant intracellular organic compounds. These compounds, which are less permeable than glycerol and thus easier to retain,

174

A. RLOMBERG AND L. ADLER

may not fully substitute for glycerol in terms of contribution to the osmotic potential. However, maintained turgor may be irrelevant to the resting cell, and the higher polyols may, nevertheless, serve to protect cellular structures against dessication stress. Many of these polyols, such as arabinitol in osmotolerant yeasts and mannitol in many filamentous fungi, are present in high concentrations irrespective of the external water potential, and could be regarded as constitutive compatible solutes in accordance with the terminology of Harris (1981). Hence, they give significant contribution to the internal osmotic potential, and thereby the turgor, also in growing cells under non-stress conditions, and their presence will tend to buffer such cells against dehydration effects by a fluctuating external water potential as discussed in Section 1V.C. Recovery of turgor-volume and growth after exposure to osmotic stress does not necessarily involve endogenous synthesis of compatible solutes. Uptake and accumulation of the external stress solute represent an alternative option, certainly when the external osmoticum has the characteristics of a compatible solute. Nobre and Da Costa (1985b) observed that, when Deb. hansenii was grown in medium containing 1 M sodium chloride in which glucose was substituted by erythritol as the carbon source, only slight intracellular accumulation of glycerol occurred and instead glycerol was replaced by erythritol as the internal compatible solute. Likewise, intracellular arabinitol was replaced by mannitol when mannitol served as the carbon source. When D.salina was furnished with the non-metabolizable sugar 3-O-methylglucose, it was intracellularly concentrated and the cell responded by converting intracellular mannitol into glycogen and preserved in this way a constant intracellular sugar concentration (Jennings and Austin, 1973). It is also apparent from numerous observations (e.g. Luard, 1982a; Gadd et al., 1984; Wethered et al., 1985; Meikle et al., 1988; Smith et al., 1990) that solutes used to change the water potential of a medium can to various extents be accumulated in yeast cells or in fungal hyphae. However, the contribution of the stress solute to the intracellular solute composition is usually difficult to determine experimentally, because a small amount within the cells has to be separated from a large amount in the medium. Beever and Laracy (1986) commented on these difficulties, and they observed in their study of A . nidulans that the external osmotica (sodium chloride, potassium chloride or glucose) played only a transient role in cellular osmoregulation after a hyperosmotic shock. There was an increased influx of the stress solute after such a shock, but the level decreased as endogenously produced glycerol and erythritol increased. When, on the other hand, glycerol was used as the stress solute, the glycerol content was maintained high and the intracellular rise of erythritol was depressed.

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

175

2. Other Roles for Polyols

Apart from serving as compatible solutes, polyols may have other roles in fungi. Lewis and Smith (1967) suggested, in their review, that polyols, beside having a potential role in osmoregulation, could serve as carbohydrate reserves, translocatory compounds and a storage for reducing power. There is still not much information as to whether polyols are stored as carbon reserves, but mannitol is a most conspicuous candidate, since this compound is often found in mycelia, sclerotia, fruiting bodies and spores in large quantities (McCullough et al., 1986; Rast and Pfyffer, 1989). Such a role would not necessarily be in conflict with a function as an osmotic buffering agent as already discussed. Jennings (1987) discussed translocation of solutes in filamentous fungi. Available evidence points to trehalose as the main carbohydrate translocated (Hammond and Nichols, 1976; Brownlee and Jennings, 1981) and there is little to support a role for polyols in long-distance transport of carbon compounds. Turning to the role of a redox sink, a maintained cell metabolism is dependent upon a continuous supply of cytosolic NAD+ to sustain oxidation of substrates. Polyols are more reduced than sugars, and polyol formation represents a way of disposing of reducing equivalents to re-oxidize NADH. It is well established that glycerol formation in Sacch. cerevisiae is a means by which the cells oxidize NADH during alcoholic fermentation (Section V.A.4). Holligan and Jennings (1972) demonstrated that the type of nitrogen-containing compound included in media affects the relative amounts of arabinitol and mannitol in mycelia of Dendryphiella salina. It was suggested that the quest for NAD(P)H in reduction of the nitrogen source was responsible for the amount of the two polyols formed. Reduction of an aldose or ketose to the corresponding polyol has also been considered as a means of removing hydrogen ions from the cytoplasm (Jennings, 1984). Although the overall reaction for diversion of sugars to polyols is not proton-consuming, polyol production may be integrated in cellular p H regulation simply by constituting a neutral end-product. Experimental evidence for a role for polyols in pH control has yet to be produced.

3. Other Candidates: Trehalose and Amino Acids Trehalose is a disaccharide containing glucose residues which is used as a compatible solute in bacteria (Giaever et al., 1988). It is widely distributed in fungi and is normally accumulated during conditions of decreased growth, particularly during periods of starvation and differentiation (Thevelein, 1984). Although stationary phase cells of Sacch. cerevisiae contain higher

176

A. ELOMBERG AND L. ADLER

trehalose contents when grown in media of decreased water potential (Meikle et al., 1988), trehalose seems to have little role in osmoregulation during steady-state growth. In chemostat experiments, the trehalose content in Succh. cerevisiae grown in the salinity range of M . 8 6 M sodium chloride increased with elevated salinity ( 0 = 0 . 1 4 . 2 h-'), but the intracelM a r concentration never exceeded 50 mM (K. Larsson and R. Olz, personal communication). Likewise, Gadd et al. (1984) observed an insignificant contribution of trehalose to the intracellular solute composition of Penicillium ochro-chloron grown at concentrations of sodium chloride and copper sulphate up to 0.5 M. However, trehalose is implicated in osmotic-shock tolerance (see Section VI.3) and as a desiccation protectant of stationary phase cells exposed to harsh water-stress conditions such as during drying in air or on lyophilization (Van Laere, 1989; Wiemken, 1990). Many organisms use amino acids as compatible solutes (Yancey et al., 1982). In yeasts, the content of free amino acids does not appear to be osmoresponsive, as shown from 13C NMR measurements (P.-A Jovall and L. Adler, unpublished observations) and conventional analysis. In Deb. hansenii, the total amino-acid content was slightly lower in cells growing in medium containing 2.7 M sodium chloride than in dilute basal medium (Adler and Gustafsson, 1980) while Brown and Stanley (1972) reported that sodium chloride up to 0.5 M had no observable effect on the free amino-acid content and pool composition in Sacch. cerevisiae and Zygosacch. rouxii when grown in chemostat cultures. Malaney et ul. (1988) observed an increase in the proportion of basic amino acids and a strong relative increase in the content of citrulline in baker's yeast grown in medium containing 0.6 M sodium chloride. These changes were, however, insignificant in terms of their contribution to the osmotic potential of the cytoplasm. In sporangia of the fungi Thraustochytrium aureum and T. roseum, on the other hand, the content of the amino acid proline increased in a linear fashion as the salinity of the medium was adjusted to that of sea water (Wethered and Jennings, 1985). Although proline was the major organic solute in these sporangia, ions made by far the greatest contribution to the intracellular osmotic potential. It was not clear from the experiments whether proline was synthesized by the fungi or taken up from the medium. Luard (1982~) observed that proline accumulated in Phytophthora cinnarnomi to a concentration of 0.4-0.5 M as the external water potential was adjusted to -2 MPa with sucrose. It was suggested that proline serves as the main compatible solute in this organism which belongs to the Oomycetes, which are noted for their inability to produce polyols (Pfyffer et al., 1986). It is noteworthy, however, that Luard (1982~)observed the presence of low levels of arabinitol in P. cinnamomi and even higher amounts (arabinitol and mannitol) in Pythium debaryanum grown at -1.5 MPa.

177

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

4. Yolyol Metabolism

Because of the importance of polyols as compatible solutes in fungi, a brief overview of polyol metabolism is presented in the following section. For more detailed information, the reader is referred to the thorough review by Jennings (1984). In Sacch. cerevisiae, biochemical evidence indicates that glycerol is formed from dihydroxyacetone phosphate which, as a first step, is reduced by an NAD+-linked glycerol-3-phosphate dehydrogenase (GPD, EC 1. l . 1.8; Gancedo et al., 1968). This reaction produces glycerol 3-phosphate which is subsequently dephosphorylated by a seemingly specific phosphatase (Tsuboi and Hudson, 1956) to yield glycerol. The same pathway is responsible for glycerol formation in Zygosacch. rouxii and Deb. hansenii (see Fig. 8), as indicated by enzyme studies (Edgley and Brown, 1983; Adler et al., 1985; Nilsson and Adler, 1990), although GPD in Zygosacch. rouxii is dependent on NADP+ rather than NAD+ (Verachtert and Dooms, 1969; Brown and Edgley, 1980). Glycerol is a mandatory end-product when yeasts ferment sugar to ethanol. Production of ethanol from glucose is namely a redox-neutral process, while biosynthesis of cell material is an overall oxidative process. To maintain redox balance, dihydroxyacetone phosphate Glucose

16-phospate

-/+Gu l cose

c Fructose6- phosphote

DHAP-

14

A

7

+

6 -Phosphogluconate Erythrose 4- phosphate

GAP Ribose5-phosphate

I

FIG. 8. Metabolic pathways for glycerol and arabinitol production in Debaryornyces hansenii. The scheme is based on information in Adler et al. (1985) and Jovall et al. (1990). 1, indicates the glycerol-transport system; 2, glycerol kinase; 3, mitochondria1 glycerol-3-phosphate dehydrogenase; 4, NAD+-specific glycerol-3phosphate dehydrogenase; 5, phosphatase. D H A P indicates dihydroxyacetone phosphate; GAP, glyceraldehyde phosphate.

178

A . BLOMBERG A N D L. ADLER

is reduced to glycerol (Lagunas and Gancedo, 1973; Nordstrom, 1966, 1968). Therefore, Sacch. cerevisiae which maintains a mainly fermenting metabolism under aerobic conditions, produces glycerol (about 5 mmol per gram of yeast (dry weight) formed) when cultured in a basal glucosecontaining medium, while the respiratory yeast Deb. hansenii does not. In filamentous fungi, this pathway for glycerol formation appears to occur in Neurospora crassa, as judged from the enzyme studies by Viswanath-Reddy et a f . (1977). Legisa and Mattey (1986) detected, on the other hand, high activity of a NADP+-specific glycerol dehydrogenase in crude extract of Aspergillus niger, and have suggested that glycerol formation in this organism is by reduction of dihydroxyacetone to glycerol. Growth on glycerol as the sole source of carbon was demonstrated for 89% of the 469 yeast strains described by Barnett et af. (1983). By isolating mutants defective in glycerol utilization, Sprague and Cronan (1977) provided genetic evidence that glycerol utilization in Sacch. cerevisiae involves phosphorylation by a glycerol kinase (EC 2.7.1.30) followed by oxidation by a mitochondria1 GPD (EC 1.1.99.5) to dihydroxyacetone phosphate. In Sacch. cerevisiae these enzymes are repressed by glucose. The structural gene (GUTZ) for glycerol kinase has been cloned by functional complementation of the corresponding gut mutant and Northern-blot analysis has confirmed the occurrence of glucose repression at the transcriptional level (B. Ronnow and M. Kielland-Brandt, personal communication). Enzyme and mutant studies have shown this so-called phosphorylative pathway for glycerol catabolism to be operative also in Deb. hansenii (Adler et a f . , 1985; Fig. 8) and it seems to be a common pathway for glycerol utilization in yeasts (De Koning et al., 1987). Biochemical and genetic evidence has been presented showing that the phosphorylative pathway is the main route for glycerol dissimilation in N. crussa (Courtright, 1975a,b; Denor and Courtright, 1982) and Aspergillus nidufans (Arst et a f . , 1990; Hondmann et al., 1991). The phosphorylative pathway is induced by glycerol in these fungi (Courtright, 1975b;Hondmann et al., 1991), as appears to be the situation also in Candida utilis (Gancedo et a f . , 1968). Based on enzyme-activity measurements, an additional route in which glycerol is utilized by an NADP+-dependent oxidation to glyceraldehyde has been suggested to operate in N. crassa (Tom et al., 1978), but the enzyme activities are considerably lower than those on the phosphorylative pathway (Denor and Courtright, 1982). A third alternative may occur in Aspergillus japonicus in which glycerol induces glycerol oxidase, an enzyme that oxidizes glycerol to glyceraldehyde and hydrogen peroxide in the presence of molecular oxygen (Uwajima et a f . , 1980). Glycerol catabolism through direct oxidation of glycerol was proposed for Schiz. pombe due to lack of a detectable glycerol kinase activity, the

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

179

presence of an NAD+-linked glycerol dehydrogenase (EC 1.1.1.6) and a dihydroxyacetone kinase (May and Sloan, 1981; May et al., 1982). The operation of this so-called oxidative pathway in Schiz. pombe has received support from studies with mutants lacking the enzymes of the pathway (Gancedo et al. , 1986; Kong et al., 1987). Mannitol formation in fungi is believed to occur by an NAD+-dependent reduction of fructose 6-phosphate to mannitol 1-phosphate by mannitol-lphosphate dehydrogenase (EC 1.1.1.17) followed by dephosphorylation to mannitol by mannitol-l-phosphatase (EC 3.1.2.22) (Wang and Le Tourneau, 1972), while mannitol is considered to be dissimilated by an NADP+dependent oxidation to fructose by mannitol dehydrogenase followed by phosphorylation by hexokinase (McCullough et al., 1986). A cyclic pathway involving interconversion of fructose and mannitol for transfer of reducing equivalents from NADH to NADPH, has been proposed to occur in fungi (Hult and Gatenbeck, 1978). The presence of the enzymes constituting the mannitol cycle has been demonstrated in a number of fungi (Hult et al., 1980). However, doubts have been raised concerning the importance of the cycle for NADPH generation (McCullough et al., 1986; Singh et al., 1988). Spencer et al. (1956) determined the labelling pattern of arabinitol formed by an unidentified osmotolerant yeast from glucose labelled in position 1or 2. They concluded that the pentitol was formed by activities of the nonoxidative and oxidative branch of the pentose phosphate pathway. By using specifically labelled substrates, similar conclusions were reached for arabinito1 production in Dendryphiella salina (Lowe and Jennings, 1975) in which the final step in arabinitol synthesis involved reduction of xylulose (Holligan and Jennings, 1972). The situation is more unclear regarding the precursor for arabinitol in Zygosacch. rouxii. Blakely and Spencer (1962) suggested a direct conversion of xylulose to arabinitol according to the labelling pattern of arabinitol formed from xylulose labelled in position 5 , whereas Ingram and Wood (1965) found a labelling pattern from [6-'4C]glucose and enzymic activities which suggested formation through dephosphorylation of ribulose 5-phosphate and an NADP+-dependent reduction of ribulose to arabinitol. Using NMR spectroscopy, Jovall et al. (1990) examined the labelling pattern of arabinitol formed from [1-I3C]- and [6-'3C]glucose by Deb. hansenii. The almost exclusive labelling of C-5 of arabinitol from [6-'3C]glucose suggested formation via ribulose 5-phosphate (Fig. 8). Catabolism of arabinitol in yeasts appears to be initiated by NAD(P)+linked dehydrogenase to give the corresponding pentulose which may then be phosphorylated and catabolized by the pentose phosphate cycle (Barnett, 1976). In yeast, erythritol is believed to be formed by the action of transketolase on fructose 6-phosphate, which leaves a four-carbon fragment that is

180

A . BLOMBERG AND L. ADL.ER

reduced and dephosphorylated to give erythritol (Spencer and Spencer, 1978). Utilization of erythritol was found to depend on synthesis of an NAD+-linked erythritol dehydrogenase in Schizophyllum commune (Braun and Niederpruem, 1969). A constitutive NADPf-dependent erythrose reductase activity was also present. Many polyol dehydrogenases have a low degree of specificity towards the carbohydrate substrate. Hence, a single enzyme may act on pentitols and pentoses as well as hexitols and hexoses. The NADP+-dependent alditol dehydrogenase (EC 1.1.1.21) which has been purified from Pichia querquum by Suzuki and Onishi (1975) is an example of a well-characterized dehydrogenase with a wide substrate specificity.

5 . Polyol Transport A transport system with high specificity for glycerol has been identified in Deb. hansenii (Adler et al., 1985; Lucas et al., 1990). The system is constitutive and the K , value remains relatively constant at about 0.5-1 mM at external concentrations of sodium chloride ranging from 0 to 3 M. Cells starved for potassium and sodium ions transported glycerol by passive diffusion while addition of sodium or potassium gave transport according to Michaelis-Menten kinetics (Lucas et al., 1990). Addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) induced collapse of the glycerol gradient. Since the levels to which the transport system could accumulate glycerol were correlated with the extracellular concentration of sodium chloride, it was suggested that glycerol was transported by a glycerol-sodium symporter and that the transmembrane sodium-ion gradient which drives the glycerol uptake is maintained by an Na+/H+ antiporter. Potassium ions were accepted as a cosubstrate when the sodium-ion concentration was low. This system is so far the only one characterized in fungi, but genetic evidence has been presented for a glycerol-transport system in Aspergillus nidulans (Visser et al., 1988). Less is known about the mechanism by which higher polyols are taken up. Observations by Canh et al. (1975) led them to suggest that erythritol and a number of pentitols and hexitols crossed the plasma membrane of Sacch. cerevisiae by passive diffusion. However, mannitol uptake in Sacch. cerevisiae was shown to have the characteristics of an energy-dependent carrier-mediated transport system (Maxwell and Spoerl ,1971). Kloppel and Hofer (1976) demonstrated that Rhodotorula gracilis possesses a constitutive transport system for mannitol, xylitol and arabinitol, and an inducible uptake system with lower polyol affinity by which the polyol uptake was associated with proton absorption. In Schizophyllum commune, an uptake

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

181

system for mannitol and arabinitol was demonstrated which appeared to be both specific and inducible (Aitken and Niederpruem, 1972). 6. Membrane Permeability An intriguing fact that is difficult to reconcile with intracellular accumulation of glycerol is the reported high permeability of glycerol through lipid bilayers (e.g. Stein, 1986). However, membrane permeability to glycerol may vary greatly. Collander (1937) observed 1000-fold differences in permeability coefficients for glycerol with different types of cells, and more recent NMR studies of glycerol permeability by Brown et al. (1982) yielded a cm s-', while permeability coefficient for phospholipid vesicles of about the corresponding value for the osmotolerant algae Dunaliella salina was exceptionally low (lo-" cm s - l ) . Expressed in another way, egg phosphatidylcholine vesicles permitted glycerol leakage with half-lives of seconds, whereas the half-life for glycerol leakage through the cell membrane of D . salina was more than 400 hours. The structural and molecular background for such a low permeability to glycerol is not known. The general observations are, however, that factors which increase order in the hydrocarbon region decrease membrane permeability to polyols (Cullis et al., 1985). By introducing double bonds into phospholipid fatty-acyl groups, and by decreasing the fatty-acyl chain length, glycerol permeability through artificial membranes is increased (De Gier et al., 1968). There is little information as to whether plasma membranes of osmotolerant cells are intrinsically different from those of non-tolerant cells, or to the extent to which a decreased water potential induces changes in membrane-lipid composition. It is commonly found, however, that a decreased water potential causes a decrease of the polyenoic CI8acids (usually c 1 8 : 2 ) and an approximately corresponding increase in the content of oleic acid ( C 1 8 : 1 ) residues in fungal phospholipids (Adler and Liljenberg, 1981; Hocking 1986b; Tunblad-Johansson and Adler, 1987; Watanabe and Takakuwa, 1988). Saccharornyces cerevisiae is a notable exception to this rule; it lacks polyunsaturated acids and, in this yeast, increased salinity was observed to cause a slight decrease in the proportion of C l hacids and a corresponding small increase in the proportion of CI8acids (Tunblad-Johansson and Adler, 1987). The ability of sterols to induce a significantly more ordered acyl-chain region is reflected in decreased permeability properties to glycerol and erythritol across artificial lipid membranes (De Gier et al., 1968; D e Kruyff et al., 1973). A high level of free sterols may therefore contribute to the osmotolerance of fungi. Tunblad-Johansson et al. (1987) noted that the molar ratio of stero1:phospholipid decreased but was still maintained high

182

A . BLOMBERG A N D I.. ADLER

(1:3) in the osmotolerant yeast Deb. hansenii when cultured in the presence of 2.7 M sodium chloride. Thus, there is meagre information to substantiate acclimations in fungal membrane-lipid compositions related to, and relevant to, membrane permeability and osmotic stress. The studies cited clearly suffer from the limitation of having analysed only the overall lipid composition of the cellular array of membranes and membrane organelles. Detailed studies on the composition of lipid molecular species performed on isolated plasmamembrane preparations are still awaited. In bacteria, the high apparent permeability for glycerol seems to be associated with facilitated diffusion, involving membrane proteins (Ingraham et a f . ,1983). The glycerol transport facilitator in Escherichia coli is described as a channel in the plasma membrane allowing passage of polyols but not charged molecules like glycerol 3-phosphate and dihydroxyacetone phosphate (Heller et al., 1980). The lipid composition of the membrane also affects the osmotic stability of the yeast plasma membrane. Alterthum and Rose (1973) observed that sphaeroplasts of Sacch. cerevisiae enriched in linoleic or y-linolenic acid residues, rather than oleic acid residues, were more sensitive to lysis in hypotonic solutions. It was also observed that sphaeroplasts enriched with phosphatidylethanolamine were more resistant to osmotic lysis than those enriched in phosphatidylcholine (Hossack et al., 1977). Likewise, sphaeroplasts enriched with ergosterol or stigmasterol were more stable than those enriched in cholesterol, campesterol or 7-dehydrocholesterol (Hossack and Rose, 1976). It was suggested that stigmasterol and ergosterol, the latter being a predominant sterol in yeasts, conferred better stability by more effectively restricting the mobility of the fatty-acyl chains, which increases as the membrane is stretched. Qualitative alterations of the endogenously produced sterols, as observed in nystatin-resistant mutants of Sacch. cerevisiae, did not, on the other hand, affect the resistance to osmotically induced membrane stretching (McLean-Bowen and Parks, 1982).

B . INORGANIC IONS

Inorganic ions play an important role in the osmotic responses of bacteria (Csonka, 1989), algae (Hellebust, 1976) and plants (Munnsetal., 1983). The role of these ions in the osmotic relations of fungi is less clear, although it is known that adjustments of the major intracellular cations do occur in response to osmotic stress. The following section considers the inorganic ion relations of fungi on exposure to low water potential.

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

183

I . Intracellufar Levels The intracellular levels of sodium and potassium ions in Sacch. cerevisiae can vary depending on p H value, the availability of substrate and concentration of electrolytes in the medium. (Rothstein, 1963, 1974; Borst-Pauwels, 1981). In resting cells, levels of these ions are about equal while, on resumption of growth, sodium-ion level decreases and potassium-ion level inreases rapidly (Jones et al., 1965). In chemostat experiments, Watson (1970) observed that Sacch. cerevisiae grown in medium lacking or containing 1 M sodium chloride, contained the same amount of potassium ions (about 0.3 M) while the internal sodium-ion concentration was increased from about 0.05 M at low to about 0.13 M at high external salinity. Similar chemostat studies of Deb. hansenii grown in concentrations of sodium chloride ranging from 0.004 to 1.4 M demonstrated an increase in the internal sodium-ion potassium-ion level from about 0.4 M in basal medium to about 0.7 M at the highest salinity. Concomitantly, internal K+:Naf ratios decreased from >10 at low salinity to about 0.5-in the presence of high salinity (Larsson et al., 1990). Experiments in which the salinity was progressively increased by feeding the chemostat with strongly saline medium (Burke and Jennings, 1990) showed that Deb. hansenii adjusted on a short-term basis by a marked influx of sodium ions, which contrasted with the adjustment under steady-state growth, which involved decreased levels of sodium ions and increased glycerol content. It is also evident from the data of Hobot and Jennings (1981) and Burke and Jennings (1990) that Deb. hansenii grown in the presence of high concentrations of sodium chloride admits more sodium ions in alkaline media (concentrations above 1 M were reported) than at lower p H values, In the marine fungus Dendryphiella salina, ionic content is kept around 0.1 M potassium and 0.2 M sodium and chloride ions in organisms grown in basal medium (Jennings, 1983a). When salinity was adjusted to 0.8 Osm with sodium chloride or sulphate, sodium-24 efflux studies showed that the content of sodium ions changed slightly to 0.19 and 0.26 M, respectively, while the potassium-ion content reached a value of about 0.09 M (Wethered et al., 1985). While the organisms discussed so far admit sodium ions to a certain extent, it was observed that this ion is effectively excluded by Penicillium ochro-chloron (Gadd et al. , 1984) and Aspergillus nidulans (Beever and Laracy, 1986), and that potassium ions remain the predominant cations in these fungi also when cultured in media containing sodium chloride. In cases where the water potential of the growth medium has been amended with a non-ionic solute, only small adjustments of the internal concentration of potassium and sodium ions occur, as observed for P. chrysogenum,

+

184

A . BLOMBERG AND L. ADLER

Chrysiporium fastidium (Luard, 1982a), D . salina (Wethered et al., 1985) and A . nidulans (Beever and Laracy, 1986). An osmotically induced increase in the cation content requires an equivalent increase of anions to maintain electroneutrality. Wethered et al. (1985) made an attempt to balance cations and anions in D . salina grown in media adjusted to low water potential with different osmotica. The authors did not achieve charge balance and discussed the difficulties in this task. It is generally observed that, in media amended with salts, an increased uptake of the cation is accompanied by an increased, although not equivalent, uptake of the anion. The ability to absorb the prevalent chloride anion appears to vary. While Sacch. cerevisiae seems to exclude this ion (Conway and Armstrong, 1961), other fungi admit it to varying extents (Shere and Jacobson, 1970; Luard, 1982a,c; Wethered and Jennings, 1985; Wethered et al., 1985).

2. Transport The plasma membrane H+-ATPase from fungal and yeast cells has been thoroughly characterized (Slayman, 1987; Serrano, 1989). This enzyme generates a proton electrochemical gradient which provides the primary driving force for transport of nutrients and inorganic ions across the membrane. In the best documented example, that in Neurospora crassa, a high-affinity transport system that carries potassium ions inward in cotransport with protons has been identified (Rodriguez-Navarro et al., 1986; Blatt and Slayman, 1987). Although potassium-ion transport is mechanistically coupled to proton uptake, parallel operation of the proton pump yields net proton export (Kf/Hf exchange). There is evidence that potassium-ion uptake is driven by the electrogenic proton pump also in Sacch. cerevisiue (Pefia 1975; Eddy, 1982). The transport systems responsible for potassiumion uptake in N . crassa and Sacch. cerevisiae are known to have dual affinities (Rodriguez-Navarro and Ramos, 1984; Ramos and RodriguezNavarro, 1985) and a gene encoding a plasma-membrane protein (TRKI) necessary for high-affinity uptake of potassium ions in Sacch. cerevisiae was recently cloned and sequenced (Gaber et al., 1988). Sodium ions are believed to enter the yeast cell through the potassium-ion carriers and probably also by sodium-ion substrate symporters (Roomans et al., 1977; Borst-Pauwels, 1981). Mutations in the H O L l of Sacch. cerevisiue confer increased uptake of sodium ions and sensitivity to a number of cations (Gaber et al., 1990). The H O L l protein was genetically distinct from the potassium-ion transporters and it was suggested that H O L l might encode an endogenous sodium-ion transporter. Sodium-ion efflux from yeasts has been proposed to be mediated by an Na+/Hf antiporter, which transports

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

185

sodium ions out of the cell in exchange for protons (Eddy, 1978). The presence of such a mechanism has received some experimental support (Rodriguez-Navarro and Sancho, 1979; Rodriguez-Navarro and Ortega, 1982). By using patch-clamp electrical recording technique on sphaeroplasts of Sacch. cerevisiae, voltage-gated potassium-ion channels were demonstrated in the plasma membrane (Gustin et u f . , 1986). These channels were outwardly conducting and favoured the passage of potassium over sodium ions by more than 20-fold. Later Gustin et af. (1988) identified mechanosensitive ion channels in the yeast plasma membrane. These channels differed from the potassium-ion channels in inhibitory sensitivity, conductance and were permeable to both cations and anions. The strain energy that regulated the channels showed a dependence on cell size that indicated regulation by membrane tension rather than membrane pressure. The mechanosensitive ion channels suggest a molecular mechanism by which stretching of the membrane elicits permeability changes. C. SOLUTE COMPARTMENTATION

Subcellular compartmentation is an important aspect of solute accumulation. The presence of vacuoles in yeasts and filamentous fungi offers the organisms a possibility of segregating ions into this rather inert organelle and, in this way, to lower the ion concentration in the cytoplasm. In higher plant halophytes (Flowers and Lauchli, 1983; Munns et u f . , 1983) and the halophilic alga Dunafieffusafina (Hajibagheri et af., 1986) sodium and chloride ions are kept low in concentration in the cytoplasm and accumulated in the vacuole under saline conditions. In yeast, the vacuolar solution is isotonic with that of the cytoplasm but has a different composition (Diirr et af., 1975); the vacuole is considered as a storage compartment for cationic solutes (calcium ions, arginine, lysine, etc.). Several transport systems have been identified in the vacuolar membrane of Succh. cerevisiue, including an H+-ATPase that generates a proton-motive force across the membrane (Ohsumi and Anraku, 1981) and solutelnH' antiporters that transport calcium ions (Ohsumi and Anraku, 1983) and amino acids (Sato etuf.,1984). Ion channels that displayed a broad specificity for cations showed voltagedependent gating and required calcium ions to be opened were reported by Wada et a f . (1987). There is little to sustain a selective confinement of sodium or potassium ions to the vacuole in fungi exposed to saline environments. Ortega (1988) provided some evidence for a preferential distribution of sodium ions to the vacuole after exposure of Sacch. cerevisiae to a sodium-rich medium. However, in growing cells of the marine fungus Dendryphieffasafina,X-ray

186

A . BLOMBERG AND L. ADLER

micro-analysis demonstrated that the vacuole, which constitutes as much as 22% of the protoplasmic volume (Clipson et al., 1989), did not accumulate ions when cultured under saline conditions up to sea-water salinity (Clipson et al., 1990). Evidence for a selective distribution of polyols between the vacuolar and cytoplasmic compartments is to our knowledge entirely lacking. D. REGULATION OF POLYOL ACCUMULATION

As is clear from the foregoing, the generalized scheme for osmoregulation in fungi, when growth occurs in dilute media, involves ions (potassium ions in yeasts) as the main contributor to osmotic relations. Following a hyperosmotic shock, there is a rapid osmotic adjustment resulting in loss of turgor and volume, which is followed by a longer phase characterized by a compensatory and selective accumulation of polyols. The steady-state level of individual polyols and ions appears dependent on the stress solute used and the organism studied. Accumulation of polyols in response to an osmotic challenge can result from an increased production, a decreased dissimilation or efflux, or from uptake of the polyol. In the following sections the strategies for polyol accumulation are described for organisms in which the process has been studied, and regulation of the process is discussed.

I . Debaryomyces hansenii The osmotolerant marine yeast Deb. hansenii adjusts to low water potential by increasing its content glycerol (Section V.A.l; Fig. 6). Production of glycerol is osmotically induced, but is not achieved by any marked induction or repression of the glycerol-metabolizing enzymes. At most, two-fold changes in specific activities were observed for these enzymes when the salinity of the growth medium was increased to 1.4 M (Adler et al., 1985; Nilsson, 1988). As judged from the low activity of the glycerol kinase, there is little turnover of glycerol under saline conditions. Glycerol kinase might even be feed-back inhibited by fructose, 1,6-bisphosphate under physiological conditions (Adler et al., 1985), although such inhibition could not be confirmed with the enzyme in its purified state (Nilsson et al., 1989). Glycerol-3-phosphate dehydrogenase is sensitive to high concentrations of salt when assayed with chlorides or sulphates; the enzyme maintains less than 10% of its control activity when the ionic strength exceeds 0.6 M (Nilsson and Adler, 1990). However, when assayed in the presence of sodium glutamate, the enzyme maintains an about two-fold increased activity still at 0.9 M ionic strength. Glutamate is a major organic anion in

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

187

Deb. hansenii (Adler and Gustafsson, 1980; Jovall et a f . , 1990) and the characteristics of GPD in the presence of glutamate are obviously a key feature in explaining glycerol production of osmotically dehydrated cells, although control of glycerol production must involve additional regulatory mechanisms. Following transfer of cells to a highly saline medium, there is an immediate admission of sodium ions followed by a period of sodium-ion release and glycerol accumulation. It is of interest that Commerford et af. (1985) detected an ATPase activity with an alkaline pH optimum in Deb. hansenii grown at high salinity, which was not present in cells cultured in basal medium. Induction of additional ATPase activity under saline conditions might facilitate expulsion of cytoplasmic sodium ions in this yeast. Since the glycerol-transport system is responsive to the sodium-ion gradient (Lucas et a f . ,1990), the magnitude of this gradient will also affect the capacity for glycerol accumulation. The importance of the glyceroltransport system in determining the magnitude of the glycerol gradient across the membrane is presently not known. Chemostat experiments show that a 500&10,000-fold glycerol gradient is maintained during steady-state growth in the presence of 0.68 and 1.35 M sodium chloride (Larsson et a f . , 1990). It is also reported that Deb. hansenii accumulates glycerol to appropriate levels when sugars are used as external osmotica (Nobre and daCosta, 1985b), under which conditions the glycerol transport would lack its driving sodium-ion gradient. These authors also made the interesting observation that erythritol takes over the role of glycerol as the main compatible solute in cells cultured in saline media containing erythritol. It is likely that erythritol is taken up by a transport system which is different from that of glycerol, since glycerol uptake was not inhibited by a 25-fold excess of erythritol over glycerol (Adler et a f . , 1985), and that erythritol either represses glycerol production or causes the glycerol to be released from the cell. The information required to distinguish between these two possibilities is lacking but would allow predictions on the mechanisms for osmotic regulation of glycerol production in Deb. hansenii. The second polyol produced by Deb. hansenii, arabinitol, is accumulated to relatively high intracellular concentrations (0.3-0.5 M), irrespective of external water potential, thereby contributing to the relatively high turgor of these cells (Larsson et af., 1990) under non-stress conditions.

2. Zygosaccharomyces rouxii This yeast produces glycerol and arabinitol by the same general scheme as outlined for Deb. hansenii in Fig. 8 (Spencer, 1968). However, in Zygosacch. rouxii, glycerol is not osmotically induced but is produced constitutively, and the cells regulate their intracellular glycerol content by altering the

188

A. BLOMBERG AND L. ADLER

proportion retained within the cell (Brown, 1978; Van Zyl and Prior, 1990). In agreement with such a regulation, Brown and Edgley (1980) noted only slight changes in specific activities of GPD and a few other enzymes of carbohydrate metabolism as the growth-medium salinity was increased. It is not quite clear by which mechanisms the cells regulate their retention of glycerol. The glycerol concentration ratio across the membrane of cells grown in glucose-limited chemostat in the presence of 1.2 M sodium chloride (-5.5 MPa) ranged from 150- to 900-fold (Van Zyl and Prior, 1990). An active glycerol uptake was suggested by radiotracer experiments (Brown, 1974). Conceivably, such a system would not be driven by sodium ions in Zygosacch. rouxii since, in their chemostat studies, Van Zyl and Prior (1990) observed a high retention of glycerol irrespective of whether sodium chloride or polyethylene glycol 400 was used as external osmoticum. In the latter medium, the cells responded not only by increasing retention of glycerol but also of arabinitol. Although no measurements on electrolyte contents were performed, it is likely that the cells increased their retention of arabinitol to compensate for lower contents of sodium ions when sodium chloride was exchanged for a non-ionic external osmoticum. Although most workers have found that arabinitol production responds but slightly to osmotic stress, induction of enzymes on the arabinitol pathway may occur under certain conditions. Moran and Witter (1979) reported an enhanced intracellular arabinitol accumulation by cells grown in media containing a high concentration of glucose and noted that such cells showed an enhanced participation of the pentose phosphate pathway and an increased arabinitol dehydrogenase activity (about three-fold at 60% (w/v) glucose).

3. Saccharomyces cerevisiae This yeast produces no polyol other than glycerol, and responds to water stress by increasing its production of this polyol and by losing increasing amounts to the surrounding medium. During growth on glucose, there appears to be no reutilization of glycerol since the glycerol-dissimilating enzymes are effectively repressed (Section V.A.4). In cells cultured at high salinity there was an about two-fold increased specific activity of phosphofructokinase (Brown and Edgley, 1980) and an up to 30-fold increased activity of GPD (Edgley and Brown, 1983). Estimations of the flux-control coefficient suggested a high controlling power for GPD in glycerol production, indicating a key role for this enzyme in formation of this polyol (Blomberg and Adler, 1989). Since the osmotically induced increase in specific activity was prevented by cycloheximide, de novo synthesis of the

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

189

enzyme seemed likely. This was recently confirmed by Western-blot analysis (AndrC et a f . ,1991) using antibodies raised against GPD as purified by Chen et al. (1987). Using a probe that was deduced from the N-terminal aminoacid sequencing of GPD, clones were selected from a plasmid library. Limited DNA analysis of one of the clones showed about 54% identity of the predicted amino-acid residues with residues 1-80 of the corresponding mouse enzyme. This cloned DNA hybridized to an mRNA transcript that was salt induced (AndrC, 1990). Although further confirmation is required, the results strongly suggest that osmoregulation in Sacch. cerevisiae involves control at the gene level. In glycerol formation, dihydroxyacetone phosphate provided by glycolysis is not the only essential substrate; cytoplasmic NADH is required in equimolar amounts. During adaptive osmoregulation, the enhanced requirement for NADH is met by a decreased reduction of acetaldehyde to ethanol and an increased oxidation of this intermediate to acetate. This metabolic shift is reflected in an about two-fold decrease in the specific activity of alcohol dehydrogenase (EC 1.1.1.1) and an about equally large increase in that of aldehyde dehydrogenase (EC 1.2.1.5). The observed increase in the rate of acetate production did, however, not fully compensate for the increased quest for reducing equivalents in glycerol formation (Blomberg and Adler, 1989). The kinetics for glycerol uptake in Sacch. cerevisiae has been interpreted to indicate transport by passive diffusion (Gancedo et a f . , 1968; Brown, 1974). However, glycerol efflux from the cell does not appear to be uncontrolled. The glycerol that is produced under non-stress conditions (Section V.A.4) is quantitatively released to the surroundings, whereas cells which, by treatment with cycloheximide, are forced to maintain the same glycerol production rate under osmotic stress start accumulating glycerol (Blomberg and Adler, 1989). This observation indicates a degree of control at the level of the membrane. Membrane channels which are activated by membrane stretching are described in Sacch. cerevisiae (Section V.B .2). Since these channels readily pass both cations and anions, they could serve as general down-regulators of osmotic stress by permitting solute efflux from the cell. Specific membrane-stretching channels which selectively release glycerol when optimum turgor is adjusted would explain why glycerol is quantitatively released in the absence of osmotic stress but accumulated when cells are subjected to dehydration stress. In enterobacteria, the intracellular concentration of potassium ions acts as a signal which controls induction of a transport system for uptake of the compatible solute betaine (Sutherland et al., 1986). There is no evidence for a corresponding potassium-ion dependence of glycerol production in Sacch. cerevisiae. By manipulation of the composition of the growth

190

A . RLOMBERG AND L. ADLER

medium, it was established that glycerol accumulation occurs on exposure to osmotic stress independent of the potassium-ion concentration in the cell (Meikle et al., 1991; I. Tunblad-Johansson and L. Adler, unpublished observations). Meikle et al. (1988) demonstrated that protoplasts of Sacch. cerevisiae increased the intracellular level of glycerol when subjected to osmotic stress (high-glucose media). The protoplasts produced more glycerol than did whole cells under corresponding conditions and, on continued incubation, the protoplasts increased in size and developed large vacuoles. These observations seem to indicate that turgor (rather than cell volume) mediates some degree of control of the osmotic adjustment. 4. Phycomyces blakesleeanus

Little is known of the control of polyol formation in response to osmotic stress in filamentous fungi. From the number of participating polyols, regulation appears more complex than in yeasts. However, it was suggested by Jennings (1984) that the salinity-induced increase in synthesis of both glycerol and erythritol in Aspergillus niger and Penicillium chrysogenum (Adler et al., 1982) might be due to the involvement of an unspecified dehydrogenase. An NADP+-linked polyol dehydrogenase with wide substrate specificity has been described in P. chrysogenum (Chiang and Knight, 1959). The most relevant studies on the water relation of polyol regulation have been conducted on germinating spores of Phycomyces blakesleeanus (Van Schaftingen and Van Laere, 1985; Van Laere and Hulsmans, 1987). During germination, the spore has to generate a low intracellular osmotic potential to mediate turgor and swelling. Interestingly, spores of P. blakesleeanus transiently produce large amounts of glycerol after induction of germination (Van Schaftingen and Van Laere, 1985). This glycerol is largely formed from trehalose by activation of trehalase and glycerol-3-phosphatase. The activity of glycerol-3-phosphatase was rapidly but transiently about 10-fold activated during early germination. Since this activation was mimicked in vifro by addition of CAMP,protein phosphorylation is probably involved in glycerol formation. These results are particularly interesting, indicating a CAMP-dependent phosphorylation cascade and a transient covalent modification might be involved in regulation of glycerol synthesis in fungi. VI. Osmotic Hypersensitivity Only a minor proportion of exponentially growing cells of Sacch. cerevisiae taken from cultures in basal medium and plated onto low water-potential

191

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

media had the capacity to form colonies, even when the water potential of the plating medium was well above ymin. This phenomenon was qualitatively described by MacKenzie et al. (1986) and given the name “water stress plating hypersensitivity”. Here we will use the term “osmotic hypersensitivity” for the phenomenon. Either sodium chloride or glucose included as an osmotic agent in the plating media manifested the osmotic hypersensitivity of the cells. However, the water potential of the medium had to be below a critical threshold, as shown in Fig. 9, for the phenomenon to be observed. Saltcontaining media will give a somewhat higher value for the critical threshold compared with glucose-adjusted media (yh,,(NaCI) = -4 MPa; A. Blomberg, unpublished result). By culturing cells in salt, their subsequent degree of osmotic hypersensitivity was drastically decreased. At salt concentrations above 0.35 M , no hypersensitivity to 48% (wh) glucose was observed. Similarly, short-time conditioning (60 minutes) in media

a

100

--8 u

w

’OF

0)

5

P In .-0)

-5 I F 0

0

0.1

0.01 -12

I -10

I

-8

I -6

I -4

I -2

I 0

Water potential (MPa)

FIG. 9 Colony-forming capacity of a culture of Saccharomyces cerevisiae at indicated water potentials of glucose-adjusted media, expressed as the percentage of colonies formed relative to that on high water-potential medium (w = 0.5 MPa). The water potential was calculated from data given by Norrish (1966) at 25°C. For an explanation of v h y p , see the text. Redrawn from Mackenzie et a[. (1986).

192

A. BLOMBERG AND L. ADLER

containing 0.7 M sodium chloride transformed the whole population into a resistant state (Blomberg and Adler, 1989). Different species of yeast were tested for the osmotic hypersensitivity response, of which only Sacch. cerevisiae and Candida krusei exhibited the phenomenon at 48% (w/v) glucose (MacKenzie et al., 1986). It should be noted, however, that the existence of a osmotic hypersensitivity threshold (Whyp) instigates a conditional character to the phenomenon, implying that those species-strains that did not display the osmotic hypersensitive phenotype at a particular y-value might do so at a lower y-value. This has been shown to be true for Deb. hansenii, which did not display the phenomenon in the presence of 48% (wh) glucose but did so with 2.8 M sodium chloride, with less than 1% of the mid-exponential culture being viable at this salt concentration. The v h y p value for Deb. hansenii seems to be about -10 MPa, since no plating discrepency was observed at 2.1 M sodium chloride (Larson, 1990). The importance of the physiological state of the culture in determining the degree of osmotic hypersensitivity was substantiated by a study in which growth, on either glucose or ethanol as the carbon source, was monitored by the use of a microcalorimeter (Blomberg et al., 1988). Exponentially growing cells in either medium were hypersensitive to an osmotic shock as only 0.01% of the culture formed colonies on 1.5 M sodium chloridecontaining medium, while cells from the stationary phase or the transition phase between respirofermentative and respiratory growth were more tolerant. Furthermore, the phenomenon was not restricted to plating, since the osmotic hypersensitivity of exponentially growing cells was also reflected in its long lag phase (more than 100hours) in media containing 1.5 M sodium chloride compared to 15 hours for cells from the transition phase. The physiological state of the culture has also been shown to be of the utmost importance for the resistance of Sacch. cerevisiae to a number of environmental factors such as heat (Schenberg-Frascino and Moustacchi, 1972) and chemical mutagens (Parry et al. , 1976). The phenomenon of osmotic hypersensitivity is probably a reflection of lethal effects on the cells during the initial dehydration, since a culture of Sacch. cerevisiae rapidly lost roughly 50% of its viability as a response to a sudden exposure to lower water potentials (Morris et al., 1986). Viability measurements were performed after 15 minutes of incubation in low water-potential media, and it was reported that prolonged incubation for up to about two hours did not significantly alter viability, i.e. the viability decrease was a rapid process. The response was shown to be quantitatively similar for both sodium chloride and glycerol at equal water potentials, while methanol at concentrations up to 6 Osm (about -15 MPa) did not lower cell viability. Since methanol even at high concentrations did

’

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

193

not induce any changes in cell volume, while both sodium chloride and glycerol did, it was concluded that the decreased viability was a result of irreversible damage caused by osmotically induced cell shrinkage. The viability decrease of yeast during exposure to low water potential is not a new phenomenon, and was reported by Onishi (1959) to occur for the osmotolerant yeast Zygosacch. rouxii. An important observation by Onishi (1959) was that Zygosacch. rouxii, when exposed to 3 M sodium chloride (-14.9 MPa) in a phosphate buffer at pH 4.8 without an energy source, died off quite rapidly, and a 10,000-fold decrease in viability was observed after two days of incubation. However, if 5 % glucose was added, no major change in viability occurred over five days incubation in the salt solution. This implies that not only the magnitude of the osmotic stress but also the additional nutritional constituents will be of importance in determining the initial cellular response and the subsequent viability of the cells. A. OSMOTIC HYPERSENSITIVITY DETERMINANTS

1. Osmoregulation and Osmotic Hypersensitivity

The resistance to ultraviolet radiation of exponentially growing cells of Sacch. cerevisiae is dependent on active DNA repair mechanisms (Parry et al., 1976). By analogy, the most osmotolerant cells to respond to a sudden exposure to water potentials below v h y p could be those with a functional and active osmoregulatory system. The main features of this osmoregulatory system in yeast seem to be glycerol production and accumulation. The rate of glycerol production in Sacch. cerevisiae appears to be controlled by the amount of the enzyme GPD (Blomberg and Adler, 1989). If a high rate of glycerol production would promote osmotic-shock tolerance, induced levels of GPD should be a favourable osmotolerance feature for cells. The activity of GPD seems at first glance to be a good candidate for an osmotolerance factor, since both transition-phase, stationary phase (Blomberg et al., 1988) as well as osmotically conditioned cells (Blomberg and Adler, 1989) exhibit enhanced specific activities. The absolute specific activity of GPD, however, cannot be the determining factor per se, since ethanol-grown cells have high specific activities of GPD but no increased resistance to osmotic shock (Blomberg et al., 1988). It has been proposed that compatible solutes that accumulate intracellularly during growth in basal medium act as an osmotic buffer to minimize water loss from cells subsequent to osmotic shock (Harris, 1981). Debaryomyces hansenii, displaying a lower v h y p value than Sacch. cerevisiae, has been shown to produce constitutively and accumulate arabinitol (Adler and Gustafsson, 1980). In a study on Deb. hansenii, only minor variations in

194

A. BLOMBERG AND L. ADLER

the intracellular pools of polyols were reported during growth, exhibiting no correlation with fluctuations in osmotic hypersensitivity (Larsson, 1990). It was furthermore shown by Blomberg and Adler (1989) that osmotically conditioned cells, washed free of glycerol, only partly increased their osmotic hypersensitivity. This precludes glycerol (polyols) as the general factor determining the degree of osmotic hypersensitivity, while leaving polyols as a candidate in setting the absolute value of V h y p in different species. 2. Proteins

In the study by Blomberg and Adler (1989), it was shown that the transition of Sacch. cerevisiae into an osmotolerant state (not displaying osmotic hypersensitivity) was dependent on protein synthesis. Addition of cycloheximide, which blocked protein synthesis, almost completely inhibited acquisition of osmotolerance, in spite of the fact that the cells accumulated glycerol during the conditioning. These findings indicate that the state of resistance to a sudden osmotic shock is protein mediated. The specific activity of some enzymes has been reported to be modulated during adaptation and growth to water stress. Brown and Edgley (1980) reported enhanced activities of phosphofructokinase, as well as of some key enzymes in the pentose phosphate cycle during growth of Sacch. cerevisiae at low water potentials. Similarly, the increased rate of acetate production during osmotic conditioning was reflected in the decreased specific activity of alcohol dehydrogenase and an increased specific activity of acetaldehyde dehydrogenase (Blomberg and Adler, 1989). In order to get a view of the overall shift in protein synthesis during osmotic conditioning, proteins were labelled with [35S]methionine, and subsequently separated by two-dimensional gel electrophoresis. It was found that strain Y41 of Sacch. cerevisiae increased expression of about 20 proteins more than 10-fold during osmotic conditioning, differential synthesis of the majority of proteins not being significantly altered. Interestingly, another strain (SKQ2n), which was found to exhibit a lower Vhyp compared with strain Y41 and thus was more resistant to a sudden osmotic shock, displayed a constitutive high expression at high water potentials of many of these osmoresponsive proteins found in strain Y41 (A. Blomberg, unpublished result). 3. Trehalose The maintenance of a sufficient quantity of intracellular trehalose has been attributed a key role in cellular resistance towards dehydration at water i.e. for cells not displaying osmotic hypersensitivity potentials above ymin,

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

195

(MacKenzie et al. ,1988). These authors claimed that the threshold value for a single cell of Sacch. cerevisiae to be protected from the deleterious effects was 0.15 pmol (mg dry wt)-', which roughly corresponds to 75 mM (if cell water is taken as 2 pl (mg dry wt)-'). A correlation was found between the level of trehalose and the viability of the culture on low water-potential medium, trehalose approaching the threshold value concomitant to ethanol depletion and onset of the stationary phase. Additional support for the trehalose-threshold hypothesis comes from studies on a mutant lacking the ability to dephosphorylate trehalase, keeping the enzyme in its trehalosedegrading form. This mutant produces and accumulates trehalose to a lesser extent than the wild type (three-fold less) and exhibits no recovery from osmotic hypersensitivity in the stationary phase of growth (MacKenzie et al., 1988). Furthermore, dinitrophenol induced breakdown of trehalose in the wild type, eventually rendering the culture more sensitive to osmotic dehydration. Additional information on factors involved in cellular survival subsequent to dehydration comes from work on cellular desiccation. Desiccation could either be effected by freeze drying (Gadd et al., 1987) or incubation in a desiccator of filter-spread cells (Hottiger et al. ,1987a). The physicochemical principles underlying desiccation are qualitatively not different from osmotic dehydration in a concentrated solution, but will usually differ in the degree of stress applied and the kinetics of the dehydration process. For example, osmotic dehydration in a saturated solution of sodium chloride (about 35% (w/v), -30 MPa) is regarded as a major stress and found generally to be rapid. Cellular desiccation, on the other hand, usually proceeds in an atmosphere of not more than 10% relative humidity (a, = 0.10, w = - 315 MPa) which is a 10-fold decrease compared with saturated sodium chloride, and y-equilibrium, especially in the desiccator, will be slowly achieved. Desiccation should thus by all means be regarded as an extreme stress situation for the cell, which in nature can be properly dealt with only by a small number of organisms. Among these resistant forms are some fungal spores (Sussman and Lingappa, 1959), macrocysts of Dictyosreliurn spp. (Clegg, 1965) and dry baker's yeast (Payen, 1949). In all of these organisms and structures capable of surviving complete dehydration (the so-called anhydrobiotic organisms), trehalose is accumulated to as much as 20% of the dry weight. The function of trehalose in protection of cells to desiccation seems to be its unique capacity to substitute for water by hydrogen bonding of the hydroxyl groups of carbohydrates and the polar head groups of phospholipids (Crowe et al., 1984). This interaction is believed to protect the integrity of dry membranes during cellular desiccation. The work by Gadd et al. (1987) and that of Hottiger et al. (1987a) supports the role of trehalose as

196

A. BLOMBERG AND L. ADL.ER

an inducer of desiccation tolerance in Sacch. cerevisiae, demonstrating increased resistance for cells in the stationary phase of growth which are reported to contain a high concentration of trehalose. Externally supplied trehalose (500 mM) during freeze drying dramatically increased resistance for both stationary phase and exponential-phase cells (Gadd ef al., 1987). These authors also applied a threshold hypothesis for intracellular trehalose to be effective, in this case being 120 mM trehalose. Furthermore, trehalose accumulation seems to be induced by a number of treatments besides glucose exhaustion, such as exposure to ethanol, copper sulphate or hydrogen peroxide (Attfield, 1987) and a rise in temperature (Hottiger et al., 1987a), all treatments which inhibit growth. Heat-induced trehalose accumulation was reported to increase the desiccation tolerance of Sacch. cerevisiae (Hottiger et al., 1987a). Heat conditioning of heat-sensitive exponential-phase cells has been shown to transform the culture into a state of acquired thermotolerance (Hottiger et al., 1987a; Trollmo et al., 1988), the state of tolerance being proposed either as being mediated by proteins (McAlister and Finkelstein, 1980) or trehalose (Hottiger et al., 1987a). Surprisingly, not only was one of the enzymes involved in trehalose synthesis, trehalose-6-phosphate synthase, rapidly induced by heat treatment and correlated to trehalose accumulation, but the degrading enzyme, trehalase, was also induced. This is believed to reflect not just an increased need for trehalose as such, but more importantly that the trehalose path might be a futile cycle acting as a sink for ATP over-production (Hottiger et al., 1987b). It was reported by Trollmo et al. (1988) that exponential cells of Sacch. cerevisiae submitted to heat conditioning did not improve their osmotic hypersensitivity, even though the cells became thermotolerant, concluding that the supposed heat-induced trehalose accumulation (unfortunately not measured) did not confer a higher degree of resistance towards osmotic This conclusion is further substantiated dehydration above the value for vmin. by trehalose analysis of osmoresistant cells exponentially growing in medium containing 0.86 M sodium chloride, with trehalose levels well below 50 mM (K. Larsson and R. Olz, personal communication). In summary, the role of trehalose in protecting cells from the lethal effects of desiccation at wvalues far below vmin seems to rest on a solid basis, while the involvement of the carbohydrate as a factor governing the osmotic hypersensitivity at water has to be further substantiated. potentials above vmin B . PHYSIOLOGICAL OVERLAP

The physiological overlap in Sacch. cerevisiae between tolerance to heat and osmotic stress was shown to be unidirectional; osmotically conditioned cells

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

197

(0.7 M sodium chloride for 60 minutes) became thermotolerant while heatconditioned cells (37°C for 60 minutes) did not improve their osmotic hypersensitive state (Trollmo et al. , 1988). It was furthermore shown that the thermotolerance acquired by osmotic conditioning was not a response of the cellular dehydration per se, since thermal resistance was not imposed by suspending non-conditioned cells in 0.7 M sodium chloride during heat treatment in accordance with results earlier reported (Beuchat, 1981). A decreased water potential during heat treatment has been reported to generally alter thermotolerance (Van Uden, 1984), but microbial species and solute considerations have to be taken into account (Beuchat, 1981), sucrose being more generally effective as a thermoprotector than sodium chloride. In comparison with sucrose or sorbitol, glycerol exhibited a low protective effect on heat denaturation of pure enzyme solutions (Back et al., 1979). In accordance with this, only a slight decrease in thermotolerance resulted from a two-fold decrease in the glycerol content of osmotically conditioned cells (brought about by washing; C. Trollmo, unpublished result). The thermotolerant state is generally believed to rely on production of so-called heat-shock proteins. By use of two-dimensional gel electrophoresis, it was shown that some heat-shock proteins also display increased synthesis during the osmotic conditioning (A. Blomberg, unpublished result).

VII. Cellular Factors Involved in Determining yminValues Superficially, it might seem a trivial pursuit to search for cellular factors values since, at extremes of water potentials, any limiting growth at ymin factor might be the cellular “Achilles heel” for a particular organism. At high concentrations of sodium chloride, for example, some membranebound proteins could suffer from ionic strength-mediated conformation changes inhibiting their activity and thus precluding growth. Thus, according to this line of thought, allocation of the limiting factor at yminin one species would not reveal any of the general cellular mechanisms of osmotolerance, but merely be a consequence of evolutionary chance. However, the physiological consensus between organisms in their response to the water potential of the environment, namely production of a compatible solute, mode of osmoregulation, growth response and osmotic hypersensitivity, indicates that the principles determining ymin values in one species might well be universally applicable and not species-specific. The simplest model for cellular osmotolerance is, of course, that there is only one cellular factor involved. Plant-breeding efforts over the years indicate, however, that there will be no single gene product which

198

A . BLOMBERG AND L. ADLER

determines salt tolerance (Cheeseman, 1988). Even if no single gene might confer tolerance, a mutation in one gene might confer intolerance. Two loci involved in sensitivity to osmotic stress in Sacch. cerevisiae have been identified and named O S M l and O S M 2 (Singh and Sherman, 1978). The locus O S M 2 mapped close to the A R 0 7 locus, and it was later shown that AR07and O S M 2 are allelic (Ball e t a f . ,1986). LocusAR07is thestructural gene for chorismate mutase and the enzyme catalyses the first step from chorismate to prephenate on the phenylalanine and tyrosine pathway. The molecular basis behind the osmotic sensitivity of the aro7 mutant is not known, and furthermore, the transcriptional level of the AR07gene was not affected by alterations in the water potential of growth media. will be In the following discussion, factors involved in determining ymin treated individually. Information is scanty on the relative importance that values for a fungal species, and it these factors have in determining ymin might well be that the sum of two or more of them will co-operate in defining the ultimate limit for growth in relation to the water potential. A.

GENERATION OF ENERGY (ATP) AT LOW WATER POTENTIALS

Norkrans (1968) investigated the effect of a decreased water potential on respiration and fermentation for yeasts isolated from the sea. The main species under study were Deb. hansenii and Sacch. cerevisiae, their water relations being described in an earlier work (Norkrans, 1966). She found respiration and fermentation of both strains almost unaffected by a minor increase in sodium chloride from 0 to 0.68 M . A further decrease in water potential, however, considerably lowered the respiratory and fermentative value for Sacch. cerevisiae (about 1.9 M sodium chloride values. At the ymin both fermentation and respiration rates decreased to about 20% of the high water-potential values. The initial inhibition on these ATP-generating reactions was similar in magnitude for both yeasts, and thus not related to their degree of osmotolerance. These declining values were recorded for cell suspensions with externally supplied glucose, which contrasted with the almost constant values for endogenous respiration-fermentation (no glucose supplied). The unaffected endogenous rates suggest a minor impact of lowered water potential on central metabolic paths involved in fermentation as well as respiration. Rather, the sharply decreased activity at concentrations above 0.68 M sodium chloride in the case of externally supplied glucose indicates a major detrimental impact on the transport system for glucose. This hypothesis was later investigated in studies on the effect of sodium chloride on the K , and V,,, values for the glucose-transport system, by use of D-glucosamine as substrate (Lindman, 1981). Although Sacch. cerevisiae

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

199

displayed a much higher V,,, value than Deb. hansenii, it was more strongly affected by an increased concentration of sodium chloride. Debaryomyces hansenii exhibited a two-component system for glucose transport, with two different affinities for D-glucosamine. The K , value for the high-affinity system was increased from about 1 to 24 mM when cells were grown in the presence of 2.7 M sodium chloride compared to non-saline medium, while the V,,, value was just slightly decreased. It was concluded that the transport system for glucose in Deb. hansenii was affected but still functional at this high concentration of sodium chloride. The transport system was further characterized and found to demonstrate an energy-dependent uptake. Since uptake was neither affected by p H value nor by alkalization upon sugar addition, a proton gradient was rejected as the driving force (Lindman, 1981). This is in contrast to the situation in the marine fungus Dendryphiella salina for which a proton symport of glucose has been reported (Davies et al., 1990). The glucose-transport system in both Deb. hansenii and Sacch. cerevisiae displayed increased K , values at lower water potentials, a feature which will considerably diminish the scavenging capacity for an energy source in an energy-poor environment. In summary, studies on respiration-fermentation and transport indicate diminished rates of ATP generation by decreased water potentials. B . COST OF MAINTENANCE AT LOW WATER POTENTIALS

A cellular complication to growth at low water potentials is the reported increased cost of maintenance, as demonstrated in chemostat experiments with a respiration-deficient mutant (petite) of Sacch. cerevisiae (Watson, 1970). The uptake rate of an energy-yielding substrate specifically used for maintenance (me)was shown to increase by a factor of 10, from 0.2 to 2 pmol glucose (mg dry wt)-' h-', when cells were cultured in medium containing 1.0 M sodium chloride compared with growth in the absence of salt. By studying the carbon dioxide production, it was shown that an increased fraction of the glucose used for maintenance was, in sodium chloridecontaining medium, diverted into non-ATP-generating activities. This was clearly demonstrated to be a function of the increased maintenance production of glycerol, the me value in salt being 1 pmol (mg dry wt)-' h-', i.e. 50% of the glucose maintenance value. Even in medium lacking salt, glycerol was formed but production was in this case independent of growth rate and strictly related to biomass production (see Section V.A.2). Thus, under growth at low water potentials, glycerol production was included in the cost of maintenance. The use of respiratory deficient mutants enabled calculations of the amount of ATP generated per unit of glucose utilized, care being taken of

200

A. BLOMBERG AND L. ADLER

the glycerol effect. It was accordingly shown that the ATP yield, regardless of culture media, was 11 mg dry wt (mol ATP)-’, indicating a constant cost of ATP in biosynthetic reactions irrespective of the water potential of the media. However, the me value for ATP increased four-fold from 0.52 to 2.2 pmol (mg dry wt)-’ h-’ in media lacking or containing 1.0 M sodium chloride, respectively. Taken together, the result by Watson (1970) clearly states that, during water-potential stress, Sacch. cerevisiae deviates an increased amount of energy source into maintenance reactions, partly as glycerol production and partly as an increased expenditure of ATP. The degree of increase in ATP utilized for maintenance seems to match the energy dispensed on enhanced ion pumping (Norkrans and Kylin, 1969; Watson, 1970). It has also been found that the increased catabolic energy expenditures during growth in medium containing 0.68 M sodium chloride, was about four times larger for Sacch. cerevisiae compared with that of Deb. hansenii. This value was calculated from measurements on heat dissipation from growing cultures, care being taken to subtract the “glycerol effect” (Gustafsson and Larsson, 1990). Increased maintenance cost has also been reported for growth at low pH values for Sacch. cerevisiae, the rate of maintenance being proportional to the proton concentration (Verduyn et al., 1990). Similarly, Gustafsson and Larsson (1990) found an increased maintenance expenditure for Deb. hansenii at low pH values, and it was concluded that the maintenance requirement at p H 3 was of the same order of magnitude as for growth in the presence of 1.0 M sodium chloride. This might explain the earlier discussed ymindependence on pH value (see Section III.A.3), additional environmental factors besides low water potentials adding an extra load onto the total maintenance cost of the cells. Maybe more significant from an ecological point of view than the saltinduced initial decrease in energy generation (Norkrans, 1968) is the fact that many natural environments, e.g. the sea, are nutrient limited, so that energy generation per cell and hour is determined mainly by supply from the environment, provided efficient cellular nutrient-transport systems exist. This implies that the report by Watson (1970) indicating an increased cost of maintenance in the presence of salt probably has long-ranging implications on the ecology and evolution of organisms. A simple model for this is depicted in Fig. 10, where the rate of maintenance cost is assumed to increase, and the energy-generating capacity of an organism predicted to decrease, in response to a decreased water potential of the environment. Note that maintenance will be a function of the physicochemical environment and not related to energy supply. A decreased water potential will both decrease the rate of ATP generation and increase maintenance, the two

201

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI I

1

1

1

I

Rich J

al 0 C

C

0 .-a-

m C

h

al

a-

a, C

C

: 5 1-

0)

cn

a I-

-I

Lc

0

-4-

0 al

al

a-

m

w

m U

a:

Water potential (MPa)

FIG. 10. A simple model showing how the rate of maintenance (. . .) and rate of energy generation (-) will influence the value of ymin.Indicated are two environments, energy-rich and energy-poor, and arrows indicate the different ymin values obtained.

processes eventually being equal at ymin and growth no longer possible. An appropriate example of the rich habitat might be the conical flask in the laboratory, supplemented with a high concentration of easily accessible nutrients and energy sources. In the other situation, when energy supply is poor, ymin will be reached at a much higher water-potential value, since the energy supply is not sufficient for the increased cost of maintenance at lower y-values. This might represent the situation sea-living organisms have to face and which will be important components of their evolutionary pressure: both genetic adaptation to effective energy scavenging and to low-cost maintenance at -2.6 MPa (0.5 M salt). The high osmotolerance of, for example, Deb. hansenii as measured in the laboratory (growth in the presence of 4.1 M sodium chloride) (Norkrans, 1966) might thus not be an indication of adaptation to these extremes of water potentials, but merely a consequence of a high degree of genetic adaptation in minimizing its cost spent on osmoregulation. Thus, at the water potential of the sea, minimizing the cost of maintenance spent on osmoregulation might be an important selection pressure.

202

A. BLOMBERG AND L. ADLER

C. ION TRANSPORT AND ACCUMULATION

In order to maintain appropriate activities of cellular enzymes, fairly low internal levels of sodium ions are accepted. It was shown for both Sacch. cerevisiae and Deb. hansenii that the rate of sodium influx was proportional to the external sodium-ion concentration (Norkrans and Kylin, 1969). Both yeasts selectively accumulated potassium ions in preference to sodium ions, which was reflected in the high intracellular K+:Na+ratio of around 100-300 during growth in basal medium, Deb. hansenii consistently displaying higher ratio values than Sacch. cerevisiae. The competition for the same ion carrier (see Section V.B.2) might explain the decreased ratio of about unity during growth in the presence of 2.7 M sodium chloride (-13.4 MPa), the competition theory being supported by an almost constant proportion between the intracellular K+:Na+ ratio and the extracellular K+:Na+ ratio, irrespective of medium salinity. The salt-tolerant yeast Deb. hansenii exhibited a higher capacity for sodium-ion efflux than Sacch. cerevisiae (Norkrans and Kylin, 1969). The sodium-ion extrusion is reported to be coupled to proton import (RodriguezNavarro and Ortega, 1982). The driving force, the proton gradient, is produced by the plasma-membrane ATPase which is an electrogenic proton pump. The ATPase gene has been cloned (Serrano et al., 1986), and was by site-specific deletion shown to be essential for growth. By modifying the promoter region of the ATPase gene, Vallejo and Serrano (1989) were able to construct transformants with altered expression of the ATPase gene. One of these constructs with a low ATPase content was shown to be unaffected in its osmotolerance as demonstrated at 1.15 M sodium chloride (-5.7 MPa, pH 6.0), under which conditions the mutant grew at the same rate as the wild type. No results were reported for growth of the mutant in media closer to the ymin value of Sacch. cerevisiae. Whether active and efficient sodium-ion extrusion can explain the differences in yminmerits further investigation. Among the ATPase mutants of Sacch. cerevisiae obtained by McCusker et al. (1987) some, but not all, were reported to display an osmosensitive phenotype. It was later shown that, in one of the osmosensitive strains, the mutated ATPase interacted with a voltage-gated potassium-ion channel (Ramirez et al. , 1989). This might indicate a central role for potassium-ion transport in osmotolerance, which is supported by studies of potassium iontransport mutants (isolated by Gaber et al. 1988,1990) which displayed high ymin values (A. Blomberg, unpublished result). D. PRODUCTION AND ACCUMULATION OF A COMPATIBLE SOLUTE

Substantial indirect evidence for the importance of accumulation of a compatible solute for growth to occur at low water potentials comes from the

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

203

vast literature on polyol accumulation (see Section V.A.la). A more direct proof of the prerequisite for a compatible solute came from studies on a mutant of Deb. hanseriii with impared glycerol production, the rate of glycerol production being about four-fold lower than for the wild type in the presence of 1.4 M sodium chloride (Andre etul., 1988). The requirement for glycerol accumulation at low water potentials was demonstrated by an experiment in which addition of 0.5 mM glycerol completely restored the colony-forming capacity of the mutant in the presence of 2.4 M sodium chloride, a concentration that is normally inhibitory for the mutant but not for the wild type. No colonies appeared o n plates lacking glycerol o r on media where glycerol was substituted by 1 mM concentrations of other wellknown osmoprotectants (Yancey et al., 1982) like mannitol, arabinitol, proline, trimethylaminoxide and betaine. It was furthermore shown that the mutant at high salt concentrations rapidly accumulated externally supplied glycerol to wild-type intracellular levels. This clearly indicates that accumulation of sufficient intracellular levels of a compatible solute was required to permit growth at low water potentials. Further demonstration of the importance of appropriate regulation of production and accumulation of a compatible solute during growth at low water potentials comes from the isolation of osmosensitive mutants of Zygosacch. rouxii (Yagi and Tada, 1988). Twenty mutants unable to grow in the presence of 2 M sodium chloride were isolated and classified into physiological groups by investigating their glycerol production and accumulation at permissive water potentials. For most of the mutants, the glycerol criteria explained their growth response, glycerol being produced but not retained or production per se being meagre. For some mutants, however, additional unknown factors seemed to be involved in their osmosensitivity. value of Sacch. cerevisiae is much A possible explanation why the ymin higher than for Zygosacch. rouxii has been substantiated by work by Brown and his coworkers (Brown, 1978). It was found that Sacch. cerevisiue responded much more vigorously in its total glycerol production to increased concentrations of sodium chloride compared to Zygosacch. rouxii with roughly 30 mol% of the consumed glucose being converted into the polyol at 1.4 M sodium chloride compared to 5 mol% in basal medium. Zygosaccharomyces rouxii, on the other hand, showed a much higher “unstressed” production of around 15 mol% but, in contrast to Sacch. cerevisiae, the production was kept almost constant over the range of 0-3 M sodium chloride. However, as salinity increased, Zygosucch. rouxii retained an increased proportion of the glycerol produced (see Section V.D.3). The explanation for the high cost of glycerol maintenance in Sacch. cerevisiue is an apparent lack of a transport system for glycerol, glycerol retention thus being set by the balance between rate of production and

204

A. BLOMBERG AND L. ADLER

leakage. The theoretical maximum of glycerol produced is 1 mol of glycerol from each mole of consumed glucose, since the cell cannot divert all triose phosphates produced in the early glycolytic steps to glycerol, but has to channel half of them into pyruvate production to generate NADH, which is needed as the reducing moiety in conversion of DHAP into glycerol 3phosphate (see Section V.A.4). A figure close to this theoretical optimum was reported for well aerated cultures containing 1.35 M sodium chloride (Larsson and Gustafsson, 1987), for which it was found that 68 mol% of the consumed glucose ended up as glycerol. A more glucose-conserving mode of glycerol production would be NADPH-dependent reduction of DHAP into glycerol 3-phosphate, resulting in a theoretical value of two units of glycerol formed per unit of glucose consumed. This is the mode of glycerol production proposed for Zygosacch. rouxii (Brown and Edgley, 1980). The conclusion is that the absolute value of ymin of Sacch. cerevisiae is mainly dictated by its poor retention and wasteful mode of production of its compatible solute glycerol. Presumably, the existence of a transport system for the compatible solute in possibly both Deb. hansenii and Zygosacch. rouxii (see Section V.A.3) allows the glycerol maintenance of these species to be significantly smaller. The part of the total ATP maintenance involved in energy expenditure on the uphill transport of glycerol during growth at low water potentials, however, will in these species probably be relatively high. VIII. Conclusion

The response of a fungus to osmotic stress involves the integrated function of many components of cell metabolism (Fig. 11). An important mechanism by which the dehydration stress is countered entails accumulation of polyols, primarily glycerol, to achieve an internal environment that is conducive for enzyme function and growth under water stress. The changes in the composition of the cytoplasm are controlled by systems for biosynthesis of polyols and for transport of inorganic ions. The detailed regulation of these systems is little understood, but evidence points to regulation at gene as well as protein level. The intracellular retention of glycerol is controlled at the level of glycerol efflux and by systems for glycerol uptake. The osmoregulatory processes require energy to drive transport and carbon supply for polyol formation. The capacity for substrate uptake under osmotic stress and the efficiency by which the osmoregulatory processes are operated are important in setting the limits for growth at low water potentials. The fungal response to changes in the external water potential must involve sensing as well as transduction of the received signal. Are

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

205

Hf

FIG. 11. A schematic representation of cellular functions involved in osmoregulation in fungi. 1 indicates glucose uptake; 2, glycerol production;3, glycerol uptake; 4, glycerol efflux; 5 , energy supply; 6, efflux of sodium ions; 7 , influx of potassium ions.

mechanosensation (e.g. turgor sensing) and phosphorylation cascades involved in this response? To what extent and in what way does the signal affect gene expression? It appears that combined genetic and physiological analysis is required for a deeper understanding of fungus-water relations. Analysis at this level has revealed sequential induction of osmotically controlled genes in enteric bacteria and given exciting insights in signal transduction and regulation of the process (see the review by Csonca, 1989). The experimental tractability and developed genetics of many fungal species makes these organisms equally attractive eukaryotic experimental systems for exploration of fundamental mechanisms in osmoregulation and osmotolerance.

206

A. BLOMBERG AND L. ADLER

IX. Acknowledgements The compilation of data and the writing of this review were made possible by a grant from NFR (B-Bu4645-302). We greatly appreciate the efficiency of the staff at the Botanical Library, University of Goteborg, with a special thanks due to Helena G r a v b for her technical help with the computer search in the BIOSIS database. The everlasting patience and support from our families during the period of writing are deeply acknowledged. REFERENCES Adebayo, A. A. and Harris, R. F. (1971). Proceedings of the Soil Science Society of America, 35, 465. Adebayo, A. A., Harris, R. F. and Gardner, W. R. (1971). Transactions of the British Mycological Society 57, 145. Adler, L. and Gustafsson, L. (1980). Archives of Microbiology 124, 123. Adler, L. and Liljenberg, C. (1981). Physiologia Plantarum 53, 368. Adler, L., Pedersen, A. and Tunblad-Johansson, 1. (1982). Physiologia Plantarum 56, 139. Adler, L., Blomberg, A. and Nilsson, A. (1985). Journal of Bacteriology 162, 300. Aitken, W. B. and Niederpruem, D. J. (1972). Archiv fur Microbiologie 82, 173. Al-Hamdani, A. M. and Cooke, R. C. (1987). Transactions of the British Mycological Society 89, 51. Altherthum, F. and Rose, A. H. (1973). Journal of General Microbiology 77, 371. Anand, J. C. and Brown, A. D. (1968). Journal of General Microbiology 52, 205. Andrews, S . and Pitt, J. I. (1987). Journal of General Microbiology 133, 233. Andre, L. (1990). Ph.D. Thesis: University of Goteborg, Sweden. AndrC, L., Nilsson, A. and Adler, L. (1988). Journal of General Microbiology 134, 669. AndrC, L., Hemming, A. and Adler, L. (1991). FEBS Letters 286, 13. Arst, H . N., Hondmann, D. H. A. and Visser, J. (1990). Molecular and General Genetics223, 134. Attfield, P. V. (1987). FEBS Letters 225, 259. Ayerst, G. (1969). Journal of Stored Products Research 5 , 127. Bachmann, B. J. and Bonner, D. M. (1959). Journal of Bacteriology 78, 550. Back, J . F., Oakenfull, D. and Smith, M. B. (1979). Biochemistry 18, 5191. Ball, S . G., Wickner, R. B., Cottarel, G., Schaus, M. and Tirtiaux, C. (1986). Molecular and General Genetics 205, 326. Bank, H. (1973). Cryobiology 10, 157. Bank, H. and Mazur, P. (1973). Journal of Cell Biology 57, 729. Barnett, J . A. (1976). Advances in Carbohydrate Chemistry and Biochemistry 32, 125. Barnett, J . A., Payne, R. W. and Yarrow, D. Y. (1983). “Yeasts: Characteristics and Identification.” Cambridge University Press, Cambridge. Beever, R. E. and Laracy, E. P. (1986). Journal of Bacteriology 168, 1358. Bellinger, Y . and Larher, F. (1988). Canadian Journal of Microbiology 34, 605. Beuchat, L. R. (1981). Journal of Food Protection 44,765. Blakely, E. R. and Spencer, J. F. T. (1962). Canadian Journal of Biochemistry and Physiology 40, 1737. Blatt, M. and Slayman, C. L. (1987). Proceedings of the National Academy of Sciences ofthe United States of America 84, 2737. Blomberg, A. and Adler, L. (1989). Journal of Bacteriology 171, 1087. Blomberg, A,, Larsson, C. and Gustafsson, L. (1988). Journal of Bacteriology 170, 4562. Borst-Pauwels, G. W. F. H. (1981). Biochimica et Biophysica Acta 650, 88. Braun, M. L. and Niederpruem, D. J. (1969). Journal of Bacteriology 100, 625.

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

207

Brown, A. D. (1974). Journal of Bacteriology 118, 769. Brown, A. D. (1976). Bacteriological Reviews 40, 803. Brown, A. D. (1978). Advances in Microbial Physiology 17, 181. Brown, A. D. (1990). “Microbial Water Stress Physiology. Principles and Perspectives”. Wiley, Chichester. Brown, A. D. and Edgley, M.A. (1980). In “Genetic Engineering of Osmoregulation” (D. W. Rains, R. C. Valentine and A. Hollander, eds), pp. 75-90. Plenum Press, New York. Brown, A. D. and Simpson, J. R. (1972). Journal of General Microbiology 72, 589. Brown, C. M. and Stanley, S. 0. (1972). Journal of Applied and Chemical Biotechnology 22, 363. Brown, F. F., Sussman, I., Avron, M. and Degani, H. (1982). Biochimica et Biophysica Acta 690, 165. Brownlee, C. and Jennings, D. H. (1981). Transactions of the Brirish Mycological Society 77, 615. Burke, R. M. and Jennings, D. H. (1990). Mycological Research 94, 378. Canh, D. S., Horhk, J., Kotyk, A. and Rihova, L. (1975). Folia Microbiologica 20, 320. Cheeseman, J. M. (1988). Plant Physiology 87, 547. Chen, S.-M., Trumbore, M. W., Osinchak, J. E., and Merkel, J. R. (1987). Preparative Biochemistry 17, 435. Chiang, C. and Knight, S. G. (1959). Biochimica et Biophysica A d a 35, 454. Clipson, N. J. W., Jennings, D. H. and Smith, J. L. (1989). New Phytologkt 113, 21. Clipson, N. J. W., Hajibagheri, M. A. and Jennings, D. H. (1990). Journal of Experimental Botany 41, 199. Clegg, J. S. (1965). Comparative Biochemistry and Physiology 14, 135. Collander, R. (1937). Transactions of the Faraday Society 33, 985. Cornerford, J. G., Spencer-Phillips, P. T. N. and Jennings, D.H. (1985). Transactions of the British Mycological Society 85, 431. Conway, E. J. and Armstrong, W.M. (1961). Biochemical Journal 81, 631. Corry, J. E. L. (1976). Journal of Applied Bacteriology 40, 269. Corry, J. E. L. (1987). In “Food and Beverage Mycology” (L. R. Beuchat, ed.), 2nd edn, pp. 51-100. Van Nostrand Reinhold, New York. Cosgrove, D. (1986). Annual Review of Plant Physiology 37, 377. Cosgrove, D. J., Ortega, J. K. E. and Shropshire Jr, W. (1987). Biophysical Journal51,413. Courtright, J. B. (1975a). Archives of Biochemistry and Biophysics 167, 21. Courtright, J. B. (1975b). Journal of Bacteriology 124, 497. Cram, W. J. (1976). In “Encyclopedia of Plant Physiology” (U. Liittge and M. G. Pittman, eds), vol. 2A, pp. 284-316. Springer-Verlag, Berlin. Crowe, J. H., Crowe, L. M. and Chapman, D. (1984). Science 223, 701. Csonca, L. N. (1989). Microbiological Reviews 53, 121. Cullis, P. R., Hope, M. J., de Kruijff, B., Verkleij, A. J. and Tilcock, C. P. S. (1985). In “Phospholipids and Cellular Regulation” (J. F. Kuo, ed.), pp. 1-59. CRC Press, Boca Raton. Da Costa, M. S. and Niederpruem, D. J. (1982). Archives of Microbiology 131, 283. Dainty, J. (1976). In “Encyclopedia of Plant Physiology” (U. Luttge and M. G. Pittman, eds), vol. 2A, pp. 12-35. Springer-Verlag, Berlin. Davies, J. M., Brownlee, C. and Jennings, D. H. (1990). Journalof Experimental Botany41, 449. De Bruijne, A. W. and Van Steveninck, J. (1970). Biochimica er Biophysica Acta 196, 45. De Gier, J., Mandersloot, J. G. and Van Deenen, L. L. M. (1968). Biochimica et Biophysica Acta 150, 666. De Koning, W., Harder, W. and Dijkhuizen, L. (1987). Archives of Microbiology 148, 314. De Kruyff, B., De Greef, W. J., Van Eyk, R. V. W., Demel, R. A. and Van Deenen, L. L. M. (1973). Biochimica et Biophysica Acta 298, 479. Degani, H. and Avron, M. (1982). Biochimica et Biophysica Acta 690, 174. Denor, P. F. and Courtright, J. B. (1982). Journal of Bacteriology 151, 912. Diirr, M., Boller, T. and Wiemken, A . (1975). Archives of Microbiology 105, 319.

208

A . BLOMBERG A N D L. ADLER

Eddy, A. A. (1978). Current Topics in Membranes and Transport 10, 279. Eddy, A. A. (1982). Advances in Microbial Physiology 23, 1. Eddy, A. A. and Williamson, D. H. (1957). Nature 179, 1252. Edgley, M. A. and Brown, A. D. (1983). Journal of General Microbiology 129, 3453. Einspahr, K. J., Maed, M. and Thompson, G . A. Jr, (1988). Journal of Cell Biology 107, 529. Flowers, T. J. and Lauchli, A. (1983). In “Encylopedia of Plant Physiology, Inorganic Plant Nutrition” (A. Lauchli and R. L. Bielski, eds), vol. 15, pp. 651-681. Springer-Verlag, Berlin. Gaber, R. F., Styles, C. A. and Fink, G. R. (1988). Molecular and Cellular Biology 8, 2848. Gaber, R. F., Kielland-Brandt, M. C. and Fink, G. R. (1990). Molecularand CelfufarBiology 10, 643. Gadd, G. M., Chudek, J. A,, Foster, R. and Reed, R. H. (1984). Journal of General Microbiology 130, 1969. Gadd, G. M., Chalmers, K. and Reed, R. H. (1987). FEMS Microbiology Letters 48, 249. Gancedo, C., Gancedo, J. M. and Sols, A. (1968). European Journal ofBiochemistry 5,165. Gancedo, C., Llobell, A,, Ribas, J.-C. and Luchi, F. (1986). European JournalofBiochemistry 159, 171. Gekko, K. and Timasheff, S. N. (1981). Biochemistry 20, 4667. Gezelius, K. and Norkrans, B. (1970). Archivfiir Microbiologie 70, 14. Giaever, H. M., Styrvold, 0. B., Kaasen, I. and Strom, A. R. (1988). Journal ofBacteriology 170, 2841. Glare, T. R., Milner, R. J. and Chilvers, G. A. (1989). Mycological Research 92, 477. Gustafsson, L. (1979). Archives of Microbiology 120, 15. Gustafsson, L. and Larsson, C. (1990). Thermochimica Acta 172, 95. Gustafsson, L. and Norkrans, B. (1976). Archives of Microbiology 110, 177. Gustin, M. C., Martinac, B., Saimi, Y., Culbertson, M. R. and Kung, C. (1986). Science 233, 1195. Gustin, M. C., Zhou, X.-L., Martinac, B. and Kung, C. (1988). Science 242, 762. Hajibagheri, M. A., Collins, J. C. and Flowers, T. J . (1986). Journal ofExperimenta1 Botany 37, 1725. Hammond, J. B. W. and Nichols, R. (1976). Journal of General Microbiology 93, 309. Harned, H. S. and Owen, B. B. (1958). “The Physical Solutions of Electrolyte Solutions”, p. 492, Reinhold, New York. Harris, R. F. (1981). In “Water Potential Relations in Soil Microbiology” (Soil Science Society of America Special Publication No. 9) (J. F. Parr, W. R. Gardner and L. F. Elliott, eds), pp. 23-96. Soil Science Society of America, Madison, Wisconsin. Hawthorne, D. C. and Friis, J. (1964). Genetics 50, 829. Hellebust, J. A. (1976). Annual Review of Plant Physiology 27, 485. Heller, K. B., Lin, E. C. C. and Wilson, T. H. (1980). Journal of Bacteriology 144, 274. Hobot, J. A. and Jennings, D. H. (1981). Environmental Mycology 5 , 217. Hocking, A. D. (1986a). Journal of General Microbiology 132, 269. Hocking, A. D. (1986b). Ph.D. Thesis: University of New South Wales, Australia. Hocking, A. D. and Norton, R. S. (1983). Journal of General Microbiology 129, 2915. Hoekstra, F. A. and Van Roekel, T. (1988). Plant Physiology 88, 626. Holligan, P. M. and Jennings, D. H. (1972). New Phytologist 71, 583. Hondmann, D. H. A,, Busink, R., Witteveen, C. F. B. and Visser, J. (1991). Journal of General Microbiology 137, 629. Hossack, J. A. and Rose, A. H. (1976). Journal of Bacteriology 127, 67. Hossack, J. A., Sharpe, V. J. and Rose, A. H. (1977). Journal of Bacteriology 129, 1144. Hottiger, T., Boller, T. and Wiemken, A. (1987a). FEES Letters 220, 113. Hottiger, T., Schmutz, P. and Wiemken, A. (1987b). Journal of Bacteriology 169, 5518. Hult, K. and Gatenbeck, S. (1978). European Journal of Biochemistry 88, 607. Hult, K., Veide, A. and Gatenbeck, S. (1980). Archives of Microbiology 128, 253. Ingraham, J . L., Maaloe, 0. and Neidhardt, F. C. (1983). “Growth of the Bacterial Cell”. Sinauer Associates Inc., Sunderland, Mass. Ingram, J. M. and Wood, W. A. (1965). Journal of Bacteriology 89, 1186.

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

209

Jennings, D. H. (1973). In “Ion Transport in Plants” (W. P. Anderson, ed.) pp. 323-335. Academic Press, London. Jennings, D. H. (1983a). Biological Reviews 58, 423. Jennings, D. H. (1983b). In “The Ecology and Physiology of the Fungal Mycelium” (D. H . Jennings and A. D. M. Rayner, ed.), pp. 143-164. Cambridge University Press, Cambridge. Jennings, D. H. (1984). Advances in Microbial Physiology 25, 149. Jennings, D. H. (1986). In “The Biology of Marine Fungi” (S. T. Moss, ed.), pp. 81-89. Cambridge University Press, Cambridge. Jennings, D. H. (1987). Biological Reviews 62, 215. Jennings, D. H . and Austin, A. (1973). Journal of General Microbiology 75, 287. Jermini, M. F. G. and Schmidt-Lorentz, W. (1987). Journal of Food Protection 50, 473. Jones, W. B. G., Rothstein, A,, Sherman, F. and Stannard, J. N. (1965). Biochimica et Biophysica Acta 104, 310. Jovall, P.-A., Tunblad-Johansson, I. and Adler, L. (1990). Archives ofMicrobiology 154,209. Kloppel, R. and Hofer, M. (1976). Archives of Microbiology 107, 329. Koch, A. L. and Pinette, M. F. S. (1987). Journal of Bacteriology 169, 3654. Koh, T. Y. (1975). Journal of General Microbiology 88, 101. Kong, Y.-C., Marshall, J. H. and May, W. (1987). FEMS Microbiology Letters 43, 71. Koppensteiner, G. and Windisch, S. (1971). Archiv fur Microbiologie 80, 300. Lagunas, R. and Gancedo, J. M. (1973). European Journal of Biochemistry 37, 90. Larsson, C. (1990). Ph.D. Thesis: University of Goteborg, Sweden. Larsson, C. and Gustafsson, L. (1987). Archives of Microbiology 147, 358. Larsson, C., Morales, C., Gustafsson, L. and Adler, L. (1990). Journal of Bacteriology 172, 1769. Legisa, M. and Mattey, M. (1986). Enzyme and Microbial Technology 8, 607. Levin, R. L. (1979). Journal of Membrane Biology 46, 91. Lewis, D. H. and Smith, D. C. (1967). New Phytologist 66, 143. Lindman, B. (1981). Ph.D. Thesis: University of Goteborg, Sweden. Lipschitz-Farber, C. and Degani, H. (1980). Biochimica et Biophysica Acta 600, 291. Lockhart, J. A. (1965). Journal of Theoretical Biology 8, 264. Low, P. S. (1985). In “Transport Processes, Iono- and Osmoregulation” (R. Gilles and M. Gilles-Baillien, eds), pp. 467477. Springer-Verlag, Berlin. Lowe, D. A. and Jennings, D. H. (1975). New Phytologist 74, 67. Luard, E. J . (1982a). Journal of General Microbiology 128, 2563. Luard, E. J. (1982b). Journal of General Microbiology 128, 2575. Luard, E. J . (1982~).Journal of General Microbiology 128, 2583. Luard, E. J. and Griffin, D. M. (1981). Transactions ofthe British Mycological Society 76,33. Lucas, C., Da Costa, M. and Van Uden, N. (1990). Yeast 6, 187. McAlister, L. and Finkelstein, D. B. (1980). Biochemical and Biophysical Research Communications 93, 819. McCullough, W . , Roberts, C. F . , Osmani, S. A. and Scrutton, M. C. (1986). In “Carbohydrate Metabolism in Cultured Cells” (M. J. Morgan, ed.), pp. 287-355. Plenum Press, New York. McCusker, J. H . , Perlin,D. S. andHaber, J . E. (1987). MolecularandCellularBiology7,4082. MacKenzie, K. F., Blomberg, A. and Brown, A. D. (1986). Journalof General Microbiology 132, 2053. MacKenzie, K. F., Singh, K. K. and Brown, A. D. (1988). Journalof General Microbiology 134, 1661. McLean-Bowen, C. A. and Parks, L. W. (1982). Lipids 17, 662. Malaney, G. W . , Tanner, R. D., Shipp, E. A. and Slonim, A. E. (1988). Journal of Microbiological Biotechnology 3, 1 . Maxwell, W. A. and Spoerl, E. (1971). Journal of Bacteriology 105, 753. May, J. W. and Sloan, J. (1981). Journal of General Microbiology 123, 183. May, J. W . , Marshall, J. H. and Sloan, J. (1982). Journal ofGeneral Microbiology 128, 1763. Meikle, A. J., Reed, R. H. and Gadd, G. M. (1988). Journalof General Microbiology 134, 3049. Meikle, A. J., Reed, R. H. and Gadd, J. M. (1991). FEMS Microbiology Letters 78, 89.

210

A. BLOMBERG AND L. ADLER

Moran, J. W. and Witter, L. D. (1979). Journal of Bacteriology 138, 823. Morris, G. J., Coulson, G. E. and Clarke, K. J. (1988). Cryobiology 25, 471. Morris, G. J., Coulson, G. E. and Leeson, E. A. (1985). Journal of Cell Science 76, 145. Morris, G. J., Winters, L., Coulson, G. E. and Clarke, K. J. (1986). Journal of General Microbiology 132, 2023. Munns, R., Greenway, H. and Kirst, G. 0. (1983). In “Encyclopedia of Plant Physiology. Physiological Plant Ecology 111. Responses of the Chemical and Biological Environment” (0.L. Lange, P. S. Nobel, S. B. Osmond and H. Ziegler, eds), vol. 12, pp. 59-135. SpringerVerlag, Berlin. Niedermeyer, W. (1976). Cytobiologie 13, 380. Niedermeyer, W., Parish, G. R. and Moor, H. (1976). Cytobiologie 13, 364. Niedermeyer, W., Parish, G. R. and Moor, H. (1977). Protoplasma 92, 177. Nilsson, A. (1988). Ph.D. Thesis: University of Goteborg, Sweden. Nilsson, L. A., Thomson, K. S. and Adler, L. (1989). Biochimica et Biophysica Acta 991,296. Nilsson, L. A., Thomson, K. S. and Adler, L. (1990). Biochimica et Biophysica Acta 1034,180. Nobel, P. S. (1974). “Biophysical Plant Physiology”, p. 44. W. H. Freeman, San Fransisco. Nobre, M. F. and Da Costa, M. S. (1985a). Canadian Journal of Microbiology 31, 467. Nobre, M. F. and Da Costa, M. S. (1985b). Canadian Journal of Microbiology 31, 1061. Norkrans, B. (1966). Archiv fur Mikrobiologie 54, 374. Norkrans, B. (1968). Archiv fur Mikrobiologie 62, 358. Norkrans, B. and Kylin, A. (1969). Journal of Bacteriology 100, 836. Nordstrom, K. (1966). Acta Chimica Scandinavica 20, 1016. Nordstrom, K. (1968). Journal of the Institute of Brewing 74, 429. Norrish, R. S. (1966). Journal of Food Technology 1, 25. Norton, R. S. (1980). Bulletin of Magnetic Resonance 3, 29. Ohsumi, Y. and Anraku, Y. (1981). Journal of Biological Chemistry 256, 2079. Ohsumi, Y. and Anraku, Y. (1983). Journal of Biological Chemistry 258, 5614. Onishi, H. (1957a). Bulletin of the Agricultural and Chemical Society of Japan 21, 137. Onishi, H. (1957b). Bulletin of the Agricultural and Chemical Society ofJapan 21, 143. Onishi, H. (1959). Bulletin of the Agricultural and Chemical Society of Japan 23, 332. Onishi, H. (1960a). Bulletin of the Agricultural and Chemical Society ofJapan 24, 131. Onishi, H. (1960b). Bulletin of the Agricultural and Chemical Society of Japan 24, 226. Onishi, H. (1963). Advances in Food Research 12, 53. Ortega, M. D. (1988). Microbiologia 4, 61. Parker, K. R. and von Borstel, R. C. (1987). Mutational Research 189, 11. Parry, J. M., Davies, P. J. and Evans, W. E. (1976). Molecular and General Genetics 146.27. Passioura, J . B. (1980). Journal of Experimental Botany 312, 1161. Payen, R. (1949). Canadian Journal of Research 27, 749. Pefia, A. (1975). Archives for Biochemistry and Biophysics 167, 397. Pfyffer, G. E.. Pfyffer, B. U. and Rast, D. M. (1986). Sydowia 39, 160. Pfyffer, G. E., Boraschi-Gaia, C., Weber, B., Hoesch, L., Orpin, C. G. and Rast, D. M. (1990). Mycological Research 94, 219. Pinette, M. F. S. and Koch, A. L. (1987). Journal of Bacteriology 169, 4737. Pitt, J. E. (1975). In: “Water Relations of Foods” (R. B. Duckworth, ed.), pp. 273-307. Academic Press, London. Pitt, J. 1. (1989). Australian Microbiologist 10, 93. Pitt, J. I. and Hocking, A. D. (1977). Journal of General Microbiology 101, 35. Pollard, A. and Wyn Jones, R. G. (1979). Planta 144, 291. Pringle, J. R. and Hartwell, L. H. (1981). In “The Molecular Biology of the Yeast Saccharomyces” ( J . S . Strathein, E. W. Jones and J. R. Broach, eds), vol. 2, pp. 97-142. Cold Spring Harbor Laboratory, New York. Ramirez, J. A., Vacata, V., McCusker, J. H., Haber, J. E., Mortimer, R. K., Owen, W. G. and Lecar, H. (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 7866. Ramos, J. and Rodriguez-Navarro, A. (1985). Biochimica et Biophysica Acta 815, 97. Rast, D. M. and Pfyffer, G. E. (1989). Botanical Journal of the Linnean Society 99, 39.

PHYSIOLOGY OF OSMOTOLERANCE IN FUNGI

211

Reed, R. H. (1984). Piant, Cell and Environment 7, 165. Reed, R. H., Chudek, J. A,, Foster, R. and Gadd, G. M. (1987). Appliedand Environmental Microbiology 53, 2119. Robinson, R. A. and Stokes, R. H. (1959). “Electrolyte Solutions”, 2nd edn. Butterworths, London. Rodriguez-Navarro, A. and Ortega, M. D. (1982). FEBS Letters 138, 205. Rodriguez-Navarro, A. and Ramos, J. (1984). Journal of Bacteriology 159, 940. Rodriguez-Navarro, A. and Sancho, E. D. (1979). Biochimica et Biophysica Acta 522, 322. Rodriguez-Navarro, A., Blatt, M. R. and Slayman, C. L. (1986). Journal of General Physiology 87, 649. Roomans, G. M., Blasco, F. and Borst-Pauwels, G. W. F. H. (1977). Biochimica et Biophysica Acta 467, 65. Rose, D. (1975). Journal of Applied Bacteriology 38, 169. Rothstein, A. (1963). In “The Cellular Function of Membrane Transport” (J. F. Hoffman, ed.), pp. 2 S 3 9 . Prentice-Hall, Englewood Cliffs. Rothstein, A. (1974). Journal of General Physiology 64, 608. Sato, T., Ohsumi, Y. and Anraku, Y. (1984). Journal of Biological Chemistry 259, 11505. Scott, W. J. (1957). Advances in Food Research 7, 83. Schenberg-Frascino, A. and Moustacchi, E. (1972). Molecular and General Genetics 1 15, 243. Serrano, R. (1989). Annual Review of Plant Physiology and Plant Molecular Biology 40,61. Serrano, R., Kielland-Brandt, M. C. and Fink, G. R. (1986). Nature 31’9, 689. Shere, S. M. and Jacobson, L. (1970). Physiologia Plantarum 23, 51. Shporer, M. and Civan, M. (1975). Biochimica et Biophysica Acta 385, 81. Singh, A. and Sherman, F. (1978). Genetics 89, 653. Singh, M., Scrutton, N. S. and Scrutton, M. C. (1988). Journalof General Microbiology 134, 643. Slayman, C. L. (1987). Journal of Bioengineering and Biomembranes 19, 1 . Smith, S. N., Ince, E . and Armstrong, R. A. (1990). Mycological Research 94, 71. Spencer, J. F. T. (1968). In “Progress in Industrial Microbiology” (D. J. D. Hockenhull, ed.) vol. 7, pp. 1 4 2 . Churchill, London. Spencer, J. F. T. and Spencer, D . M. (1978). In “Economic Microbiology” (A. H. Rose, ed.), pp. 393-424. Academic Press, London. Spencer, J. F. T., Neish, A. C., Blackwood, A. C. and Sallans, H. R. (1956). Canadian Journal of Biochemistry and Physiology 34, 495. Sprague, G . F. and Cronan Jr, J. E. (1977). Journal of Bacteriology 129, 1335. Stein, W. D. (1986). “Transport and Diffusion Across Cell Membranes”. Academic Press, London. Steudle, E., Zimmerman, U. and Liittge, U. (1977). Plant Physiology 59, 285. Stock, J. B., Rauch, B. and Roseman, S. (1977). Journal of Biological Chemistry 252,7850. Sussman, A. S. and Lingappa, B. T. (1959). Nature 130, 1343. Sutherland, L., Cairney, J., Elmore, M. J., Booth, I. R. and Higgins, C. F. (1986). Journalof Bacteriology 168, 805. Suzuki, T. and Onishi, H. (1975). Agricultural and Biological Chemistry 39, 2389. Thain, J. F. (1967). “Principles of Osmotic Phenomena”. The Royal Institute of Chemistry, London. Thevelein, J . M. (1984). Microbiological Reviews 48, 42. Tilbury, R. H. (1980). In “Biology and Activities of Yeasts” (F. A. Skinner, S. M. Passmore and R. R. Davenport, eds), vol. 9, pp. 153. Academic Press, London. Tom, G. D., Viswanath-Reddy, M. and Howe, H. B., Jr (1978). Archivesof Microbiology 117, 259. Trollmo, C., Andre, L., Blomberg, A. and Adler, L. (1988). FEMS Microbiology Letters 56, 321. Tsuboi, K . K. and Hudson, P. B. (1956). Archives of Biochemistry and Biophysics 61, 197. Tunblad-Johansson, I. and Adler, L. (1987). FEMS Microbiology Letters 43, 275. Tunblad-Johansson, I . , Andrt, L. and Adler, L. (1987). Biochimica et Biophysica Acta 921, 116.

212

A . BLOMBERG AND L. ADLER

Uwajima, T., Akita, H., Ito, K., Mihara, A. Aisaka, K. and Terada, 0. (1980). Agricultural and Biological Chemistry 44,399. Vallejo, C. G. and Serrano, R. (1989). Yeast 5 , 307. Van Laere, A. (1989). FEMS Microbiology Reviews 63,201. Van Laere, A. J. and Hulsmans, E. (1987). Archives of Microbiology 147,257. Van Schaftingen, E. and Van Laere, A. J. (1985). European Journal ofBiochemistry, 148,399. Van Uden, N. (1984). Advances in Microbial Physiology 25, 195. Van Zyl, P. J. and Prior, B. A. (1990). Applied Microbiology and Biotechnology 33, 12. Verachtert, H. and Dooms, L. (1969). Antonie van Leeuwenhoek 35, 19. Verduyn, C., Postma, E., Scheffers, W. A. and Van Dijken, J. P. (1990). Journalof General Microbiology 136,405. Visser, J., Van Rooijen, R., Dijkema, C., Swart, K. and Sealy-Lewis, H. M. (1988). Journalof General Microbiology 134,655. Viswanath-Reddy, M., Bennett, S. N. and Howe, H. B. (1977). Molecular and General Genetics 153,29. Wada, Y., Ohsumi, Y., Tanifuji, M., Kasai, M. and Anraku, Y. (1987). Journal ofBiologica1 Chemistry 262, 17260. Walsby, A . E. (1980). Nature 283,69. Walsby, A. E. (1986). FEMS Microbiology Reviews 39, 45. Wang, S.-Y. C. and Le Tourneau, D. (1972). Archivfiir Mikrobiologie 81, 91. Watanabe, Y. and Takakuwa, M. (1988). Journal of Fermentation Technology 66, 461. Watson, T. G. (1970). Journal of General Microbiology 64, 91. Wethered, J. M. and Jennings, D. H. (1985). Transactionsofthe British Mycological Society85, 439. Wethered, J. M., Metcalf, E. C. and Jennings, D. H. (1985). New Phytologkt 101,631. Wheeler, K. A,, Hocking, A. D. and Pitt, J. I. (1988). Transactions ofthe British Mycological Society 90, 365. Wiemken, A. (1990). Antonie van Leeuwenhoek 58,209. Wolf, A. V., Brown, M. G. and Prentiss, P. G. (1979). In “CRC Handbook of Chemistryand Physics”, 59th edn, pp. D219-D271. CRC Press, West Palm Beach. Wyn, Jones, R. G. and Gorham, J. (1983). I n “Encyclopediaof Plant Physiology. Physiological Plant Ecology 111. Responses of the Chemical and Biological Environment” (0. L. Lange, P. S. Nobel, C. B. Osmond and H. Ziegler, eds), vol. 12c, pp. 35-58. Springer-Verlag, Berlin. Wyn Jones, R. G. and Pollard, A. (1982). In “Biophysics of Water” (F. Franks, ed.), pp. 335-339. Wiley, New York. Yagi, T. and Tada, K. (1988). FEMS Microbiology Letters 49,317. Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D. and Somero, G. N. (1982). Science 217, 1214. Zimmermann, U. (1978). Annual Review of Plant Physiology 29, 121.

Crystalline Bacterial Cell-Surface Layers PAUL MESSNER and UWE B . SLEYTR Zentrum fur Ultrastrukturforschung und Ludwig Boltzmann-Institut fur Molekulare Nanotechnologie. Universitat fur Bodenkultur. A-1 180 Wien. Austria

I . Introduction . . . . . . . . . . . TI . Structure and morphogenesis of S-layers . . . . . . . . . . . A . Location and ultrastructure B . Self-assembly and morphogenesis . . . . . 111. Chemistry,geneticsandbiosynthesisof S.layers . . . A . Chemicalanalyses . . . . . . . . . B . Geneticstudies . . . . . . . . .

. . . . .

. . . . . . . . . . . . . . .

. . . . . . . . . . . . . . C . Biosynthesis . . . . . . . . . . . . IV . Functional aspects of S-layers . . . . . . . . . A . S-layem related to pathogenicity . . . . . . . B . Interaction between S-layers and other bacteria or bacteriophages . C . Shape-maintainingfunctionofS-layers . . . . . . . . D . S-Layers as molecular sieves . . . . . . . . . . E . RelevanceofchargedgroupsonS-layers . . . . . . . F . RelevanceofglycosylationofS-layerproteins . . . . . . V . Application potential of S-layers . . . . . . . . . . . A . S-Layers as isoporous ultrafiltration membranes . . . . . . B . S-Layers as support for covalent attachment of macromolecules . . C . S-Layers as support for Langmuir-Blodgett films . . . . . . D . S-Layers as carriers for artificial antigens; vaccine development . . E . Secretion of S-layer proteins, a model for producing extracellular proteins . . . . . . . . . . . . . . . VI . Concluding remarks . . . . . . . . . . . . . VII . Acknowledgements . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

213 227 227 231 237 237 244 248 251 251 253 253 254 255 256 257 257 258 259 259 260 260 262 262

.

I Introduction

In the course of evolution. prokaryotic cells have developed several different ways in which to present their surfaces to the environment . One of the most remarkable features of many Gram-positive and Gram-negative ADVANCES IN MICROBIALPHYSIOL0GY.VOL . 33 ISBN C-124277324

Copyright@ 1992. by Academic Press Limited All rights of reproductionin any form reserved

214

P. MESSNER AND U. B. SLEYTR

eubacteria and archaebacteria is the presence of a regularly ordered protein or glycoprotein layer as the outermost component of the cell envelope. This layer is called the surface layer or S-layer (Sleytr, 1978). Apart from the S-layer the crystalline arrays have also been termed the RS layer (regular surface layer; Beveridge and Murray, 1974), and with regard to specific organisms as the T-layer (tetragonal layer) in Bacillus sphaericus (Aebi et al., 1973), the A-layer (additional layer) in Aeromonas safmonicida (Udey and Fryer, 1978), the HPI layer (hexagonally packed intermediate layer) in Deinococcus rudiodurans (Baumeister and Kubler, 1978), the MWP and OWP (middle- and outer-wall proteins) in Bacillus brevis (Yamada et al., 1981), the RA (regular array) in Cfostridium dificile (Kawata et al., 1984), the CSG (cell-surface glycoprotein) in Halobacterium hafobium (Lechner and Sumper, 1987) the SPA (surface-protein antigen) in Rickettsiaprowazekii (Carl and Dasch, 1989), and the SAP (surface-array protein) in Campyfobacter fetus (Blaser and Gotschlich, 1990). At the Second International Workshop on S-Layers, held in Vienna in 1987, it was agreed to use the abbreviation “S-layer” for two-dimensional crystalline arrays of proteinaceous subunits forming surface layers on prokaryotic cells (Sleytr et a f . , 1988b). The first crystalline S-layer to be described was observed by Houwink (1953) in a study of a Spiriffumspecies by electron microscopy. Since then, S-layers have been reported on hundreds of different species of nearly every taxonomic group of walled eubacteria and represent an almost universal feature of archaebacterial cell envelopes (Sleytr and Messner, 1983,1988a) (Table 1). Based on comparisons of molecular structures and sequences a new taxonomy of organisms has been recently introduced, suggesting that life above the level of kingdoms be divided into new taxons called domains. The terminology Archaea, Bacteria and Eucarya are now used instead of the common classification into the kingdoms Archaebacteria, Eubacteria and Eukaryotes (Woese et a f . ,1990). Nevertheless, in this review we still use the older terminology. S-Layers can be considered as the simplest biological membranes developed during evolution (Sleytr, 1981; Sleytr and Messner, 1983, 1989). They are usually composed of a single molecular species, protein or glycoprotein in nature, and are endowed with the ability to assemble into two-dimensional crystalline arrays by an entropy-driven process. Since S-layers, particularly in eubacteria, often tend to be lost during prolonged cultivation of the organisms under optimal laboratory conditions, it was only a few years ago that their widespread occurrence in fresh isolates was appreciated (Sleytr and Messner, 1983, 1988a). Due to the fact that S-layers possess a high degree of structural regularity, and that they are the most abundant of all bacterial cellular proteins, they

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

TABLE 1 .

215

Crystalline surface layers o n bacteria"

Organism

Characterization (latticeb, spacing', M,d (reference))

Section 1. Spirochaetes Spirochaeta plicatilis Spirochaeta stenostrepta 21 Spirochaeta zuelzera Spirochaeta litoralis R1 Spirochaeta aurantia JI Treponema pallidum Nichols Treponema phagedenis, biotype Reiter Treponema refringens Treponema minutum (CIP 5162) Treponema calligyrum (CIP 6441) Treponema genitalis VDRL-2 Treponema microdentium Treponema microdentium (four strains) Treponema sp. E-21

P; -; - (Blakemore and Canale-Parola, 1973) P; -; - (Holt and Canale-Parola, 1968) H; -; - (Hovind-Hougen, 1976) P; -; - (Hespell and Canale-Parola, 1970) P; -; - (Breznak and Canale-Parola, 1969) P; -; - (Jackson and Black, 1971) P; -; 69 (Masuda and Kawata, 1986a) H; -; - (Hovind-Hougen, 1976) H; 8-10; - (Hovind-Hougen, 1974) H; -; - (Hovind-Hougen, 1974) H; -; - (Hovind-Hougen, 1975) P; -; - (Listgarten and Socransky, 1964) H; -10; - (Hovind-Hougen, 1974) H; 16.3; 62 (Masuda and Kawata, 1982)

Section 2. Aerobichnicroaerophilie,motile, helicallvibroid Gram-negative bacteria S; 8; 75,80 (Smith and Murray, 1990) Aquaspirillum sinuosum' H; 18; 130 H; 14.5; 140 (Buckmire and Murray, 1970,1976; Aquaspirillum serpens VHA Glaeser et al., 1980; Koval and Murray, 1981, 1985; Dickson et al., 198d) H; 16; 150,125 (Stewart and Murray, 1982; Aquaspirillum serpens MW5' Kist and Murray, 1984) Aquaspirillum serpens MW6 H; 22.5; - (Murray, 1968) Aquaspirillum putridiconchylium (ATCC 15279) 0; 11.8/7.8; 156 (Beveridge and Murray, 1974; Stewart et al., 1980) Aquaspirillum metamorphum (ATCC 15280)' S ; 5/10; - (Beveridge and Murray, 1975) H: 20; Aquaspirillum "Ordal"' S; 12; - (Beveridge and Murray, 1976b; Holt H; 20; - and Beveridge, 1982) H; 12-14; - (Houwink, 1953) Aquaspirillum sp. H;-;98(McCoyetal., 1975;Winteretal., 1978) Campylobacter fetus 23D Campylobacter fetus (several strains) P; 8.8; 97-149 (Dubreuil et al., 1988; Pei et al., 1988) H; 24; 94,98 (Fujimoto et al., 1989) Campylobacter fetus TK H; -; - (Sleytr and Messner, 1988b) Campylobacter pylori Section 4. Gram-negative aerobic rods and cocci Pseudomonadaceae Pseudomonas putida Pseudomonas acidovorans (46 strains) Pseudomonas acidovorans (seven strains) Pseudomonas acidovorans (three strains) Pseudomonasacidovorans (Comamonas acidovorans) Pseudomonas delafieldii

S; -; - ( D u ~ o Y1973) , S; 6.5; - (Lapchine, 1976, 1979) H; 8; - (Lapchine, 1976, 1979) S:. 12:, - (Laochine. 1976. 1979) S ; I 1 ; 130(32) (Chalcroft et al., ,1986'; GerbCRieger et al., 1988g) s; 6.5; - (Lapchine, 1976, 1979)

~.

216

P. MESSNER AND U. B. SLEYTR

TABLE 1. Continued Organism Pseudomonas facilis Pseudomonas avenue (NCPPB 1011) Pseudomonas-like bacterium strain EU2 Azotobacteriaceae Azotobacter vinelandii Azomonas agilis Azomonas insigne Methy lococcaceae Methylomonas albus Methylomonas albus BG8 Neisseriaceae Acinetobacter sp. MJTIFSII99A Acinetobacter sp. MJT/F5/5 Other genera Thermus aquaticus Thermus thermophilus HB8 (ATCC 27634) Thermomicrobiumroseum (ATCC 27502) Bordetella pertussis Tohama 111 Lampropedia hyalina' (punctate layer) Lampropedia hyalina" (perforate layer) Section 5. Facultatively anaerobic rods Vibrionaceae Aeromonas salmonicida A450

Aeromonas salmonicida V75193 Aeromonas salmonicida Aeromonas hydrophila Aeromonas sobria Other genera Cardiobacterium hominis

Characterization (latticeb, spacing', M,d (reference))

S; 6.5; - (Lapchine, 1976, 1979) S; 6.7; -(Wells et al., 1983) S ; 13.2; 55 (Austin et al., 1990) S; 12.5; 65 (Schenk and Earhart, 1981; Bingle et al., 1984, 1986, 19876,b) H; 10; - (Houwink, 1963a) H; 12.5; - (Glauert, 1962) H;-;-(Haubold, 1978;Jeffriesand Wilkinson, 1978) G,H; -; - (Fassel et al., 1990) S ; 618; 65 (Sleytr and Thornley, 1973; Thornley, 1975) H; 11; - (Glauert and Thornley, 1971; Sleytr et al., 1974) S; -; - (Heinen and Heinen, 1972) H; 24; 100 (Caston et al., 1988; Faraldo et al., 1988) H ; -; 75 (Ramaley et al., 1978; Merkel et al., 1980) T; 8.5; 40 (Kessel et al., 1988a)g H; 25.6; 240,60 (Austin et al., 1989; Austin and Murray, 1990; Chapman et al., H; 14.6;31.5 1963; Pangborn and Starr, 1966)

S; 12.5; 49 (Udey and Fryer, 1978; Phipps etal., 1983; Kay et al., 1984; Dooley el a[., 1989') S ; 10; 54 (Evenberg and Lugtenberg, 1982; Evenberg et al., 1982, 1985) S; -; 50-53 (Griffiths and Lynch, 1990) S; 12.0; 52 (Al-Karadaghi et al., 1988'; Murray et al., 1988; Paula et al., 1988) S ; -; 44-46 (Paula et al., 1988) S; 5.5; - (Reyn et al., 1971)

Section 6. Anaerobic Gram-negative straight, curved and helical rods Bacteroidaceae P; -; - (Haapasalo et al., 1985) Bacteroides buccae (ATCC 33574) H; 21.5; - (Sjogren et al., 198qg) Bacteroides buccae Bacteroides nodosus (six strains) H; 6-7; - (Every and Skerman, 1980)

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

217

TABLE 1. Continued Organism

Bacteroides capillus (ATCC 33690, ATCC 33691) Bacteroides pentasaceus NP333 and WPH61 Bacteroides forsythus (ATCC 43037) Bacteroidesforsythus Bacteroides heparinolyticus Bacteroides sp. ES42 and ES57 Bacteroides sp. Wolinella recta Wolinella recta Wolinella recta (several strains) Selenomonas palpitans Section 9. Rickettsiae and chlamydiae Rickettsiaceae Rickensia prowazekii Rickettsia prowazekii

Rickettsia typhi Rickettsia rickensii Rickettsia akari Chlamydiaceae Chlamydia trachomatis TE55 Chlamydia psittaci

Characterization (lattice*, spacing', M,d (reference)) P; -; - (Haapasalo et al., 1985) P; -; - (Haapasalo et al., 1985) 0; -10; - (Tanner et al., 1986; Kerosuo, 1988) 0; 8.0/6.8; - (A. Sjogren, unpublished data) H; 20; - (Kerosuo et al., 1988) P; -; - (Tanner et al., 1986) H; -; - (Ranta el al., 1983) H; 20; - (Lai et al., 1981) H; 21 .O; - (Dokland et al., 1988f) P: -; 138-159 (Borinski and Holt, 1990) H; 13.5; -(Van Iterson, 1956)

S; 13; -(Palmer et al., 1974a) Biochemical evidence; -; 120 (Dasch and Bourgeois, 1981) Biochemical evidence; -; 120 (Carl and Dasch, 1989) Biochemical evidence; -; 155 (Carl and Dasch, 1989) S; 13; -(Palmer ef al., 1974b) H; 17.5; 40 (Chang ef al., 1982r) H; 18; 14-44 (Manire and Wyrick, 1979)

Section 12. Gram-positive cocci

Deinococcus radiodurans Sark Deinococcus radiodurans R1

Peptostreptococcus asaccharolyticus (ATCC 14963) Peptostreptococcus magnus AHC 5155

H; 16.5; 115 (Lancy and Murray, 1977; Thompson et al., 1982) H ; 18; 104 (Work and Griffiths, 1968;SIeytr etal., 1973;Baumeister and Kubler, 1978;Kubler etal., 1980; Baumeister et al., 1982, 1986') H; 14.2; 91 (Messner and Buckel, 1988) P; -; - (Lounatmaa et al., 1988)

Section 13. Endospore-formingGram-positive rods and c m i S; 7.5; - (Howatson and Russel, 1964) Bacillus subtilis H; 10-12; - (Yoshii, 1966) Bacillus alvei 183 0; 10.0/7.9; 127-140 (Sbra et al., 1990b; Altman Bacillus alvei (CCM 2051) et al., 1991) H; 7-10; - (Gerhardt, 1967; Holt and Bacillus anthracis Leadbetter, 1969; Doyle et al., 1986) H; (14.5); 104 (Yamada et al., 1981; Tsuboi Bacillus brevis 47' (outer wall) et al., 1982, 1986, 1989') H; 18.3; 115 Bacillus brevis 47' (middle wall)

218

P. MESSNER AND U. B. SLEYTR

TABLE 1. Continued 0rgan ism Bacillus brevis HPD31, HPD52, HP033 Bacillus brevis S1 Bacillus brevis (CCM 1463)' Bacillus cereus (ATCC 4342) Bacillus cereus (several strains) Bacillus circulans (CCM 1084) Bacillus circulans (CCM 2048) Bacillus coagulans E38-66 Bacillus fastidiosus Bacillus licheniformis NM 105 Bacillus megaterium Bacillus polymyxa (NCIB 4747)

Bacillus polymyxa (CCM 1459) Bacillus schlegelii (DSM 2000) Bacillus sphaericus (NTCC 9602, wild type) Bacillus sphaericus (NTCC 9602 lmw, spontaneous variant) Bacillus sphaericus P-1 Bacillus sphaericus (CCM 2120) Bacillus sphaericus (several strains) Bacillus sphaericus (several strains) Bacillus stearothermophilus (39 strains) Bacillus stearothermophilus (four strains) Bacillus thuringiensis ssp. galleriae (NRLL 4045) Bacillus aneurinolyticus type I (seven strains) Bacillus aneurinolyticus type I1 (13 strains) Bacillus macroides A and D' Bacillus psychrophilus W16A Bacillus sp. (CIP 76-1 11) Bacillus sp. KLl Bacillus sp. KL8 Bacillus sp. M3198 Bacillus sp. Clostridium aceticum (DSM 1496) Clostridium aceticum (DSM 14%) Clostridium aminovalericum T2-7

Characterization (latticeb, spacing', M,d (reference)) H; 17.4-18.4; 110-150 (Gruber et al., 1988) H; 17; 129 (Abe et al., 1983) H; 17.0; 118 (Sara el al., 1990b) 0;11.0/8.0; 102 S; 9-10; - (Ellar and Lundgren, 1967) Biochemical evidence; -; - (Goderdzishvili et al., 1989) 0;9.6/8.0; 117 (Sara et al., 1990b) S; 8.6; 120 (Sara et al., 1990b) 0;9.4/7.4; 100 (Pum et al., 1989a) S; 12-13.5; - (Leadbetter and Holt, 1968; Holt and Leadbetter, 1969) 0; 11.6; 98 (Tang et al., 1989) S; 19; - (Sawatake, 1966) S; 10.0; - (Nermut and Murray, 1967; Finch et al., 1967; Goundry et al., 1967; Burley and Murray, 1983) S; 10.0; 142 (Sara et al., 1990b) S; 9; - (Schenk and Aragno, 1979) S; 13; 142 (Hastie and Brinton, 1979) S; 13; 120 (Hastie and Brinton, 1979) S; 13; 140 (Howard and Tipper, 1973; Aebi el al., 1973;Lepault and Pitt, 1984;Lepault etal., 1986') S; 11.2; 120 (Sara etal., 1990b) S; 13; - (Holt and Leadbetter, 1969; Sleytr, 1970; Sleytr and Glauert, 1975) S; -; 127-133 (Word el al., 1983; Lewis et al., 1987) S,O,H; 5.2-17.9; 83-170 (Sleytr el al., 1967; Messner et al., 1984) S,O,H; 8.5-22.5; 93-170 (Sleytr et al., 1986) 0;8.517.2; 91.4 (Luckevichand Beveridge, 1989) S; 9.6; 129 (Abe and Kimoto, 1984) S; 9.6; 114 (Abe and Kimoto, 1984) S; S6;- (Holt and Leadbetter, 1969) s; %lo; S; 15-16; - (Holt and Leadbetter, 1969) H; 11; 255 (Leduc et al., 1977) P; -; - (Wahlberg et al., 1987) H; 9-10; - (Kari er al., 1990) 0;9.5; - (Haapasalo et al., 1988) P; -; 107,103 (Haikara et al., 1985) S; 10; - (Braun et al., 1981) S; 12; 120 (Woodcock et al., 198d) H; 18.5; 110 (W. Buckel and P. Messner,

CRYSTALLINE BACTERIAL. CELL-SURFACE LAYERS

219

TABLE 1. Continued Organism

Clvstridium botulinum Clvstridium difficile GA10714 Clostridium difFcile GA14131 Clvstridium difFcile (nine strains) Clvstridium formicoaceticum (DSM 912) Clvstridium novyi Clostridium polysaccharolyticum Clvstridium sporogenes Clostridium symbiosum HB25 Clostridium tetani Clvstridium thermoautotrophicum Clostridium thermohydrosulfuricum (13 strains) Clostridium thermohydrosulfuricum L111-69 Clostridium thermohydrosulfuricum (DSM 568) Clostridium thermosaccharolyticum (three strains) Clostridium thermosaccharolyticum D 12C-70

Clostridium thermosaccharolyticum (DSM 571) Clostridium thermosaccharolyticum Clostridium thermolacticum (DSM 2911) Clvstridium tyrobutyricum (ATCC 25755 and three strains) Clvstridium lentoputrescens (ATCC 17791) Clostridium tartarivorum (two strains) Clostridium xylanvlyticum (ATCC 49623) Clostridium sp. EM1 Desulfotomaculum nigrifcans (several strains) Desulfotomaculum nigrijicans (four strains) Sporosarcina ureae (ATCC 13881)

Section 14. Regular, non-sporing, Gram-positive rods Lactobacillus acidophilus (ATCC 4357) Lactobacillus acidophilus (ATCC 4356) Lactobacillus acidophilus (several strains) Lactobacillus helveticus (ATCC 10797)

Characterization (lattice’, spacing‘, M,d (reference)) unpublished data) P; 12.5; - (Takagi et al., 1965) S; 8.2; 45,32 (Masuda and Kawata, 1986b) S; -8; 38,42 (Masuda et al., 1989) S; -; 46-32 (Kawata et al., 1984) S; 10; - (Sleytr and Messner, 1988b) P; 10; - (Schallehn and Wecke, 1974; Wecke et al., 1974) P; -; - (Van Gylswyk et al., 1980) S: -; - (Betz, 1970) S: -17; 140 (Konig et al., 1985; Messner et al., 1990) P; 14.5; - (Takagi et al., 1965) S; -; - (Wiegel et al., 1981) H; -14; - (Sleytr et al., 1968; Hollaus and Sleytr, 1972; Sleytr and Glauert, 1975) H; 14; 120 (Sleytr and Thorne, 1976; Crowther and Sleytr, 1977) H; 16.0; - (Cejka et al., 1986) S; 8-9.5; - (Sleytr et al., 1968; Hollaus and Sleytr, 1972) S; 11; 115 (Sleytr and Glauert, 1976; Sleytr and Thorne, 1976; Crowther and Sleytr, 1977; Altman et al., 1990) S; 11; - (Cejka and Baumeister, 1987’) S; -; - (Van Rijssel and Hansen, 1989) P; 7; - (LeRuyet ef al., 1985) Biochemical evidence; -; 110-125 (Hayes et al., 1984; Bergkre et al., 1986) 0; 11; 140 (Sleytr and Messner, 1988b) S; 10; - (Hollaus and Sleytr, 1972; Sleytr and Glauert, 1975) 0; 6.6h.3; 180 (Rogers and Boecker, 1991; G. M. Rogers and P. Messner, unpublished data) H; -; - (Antranikian et al., 1987) S; 8-12; - (Houwink, 1963b; Sleytr el al., 1969) S,O; 9.0-14.6; 85-131 (Sleytr et al., 1986) S; 12.8; 150 (Holt and Leadbetter, 1969; Beveridge, 1979; Stewart and Beveridge, 1980; Engelhardt et al., 1986)

P; -; P; -; P; -; P; -;

43 (Kawata, 1981) -,46 (Ray and Johnson, 1986) 42-59 (Johnson et al., 1987) 51 (Kawata, 1981)

220

P. MESSNER AND U. B. SLEYTR

TABLE 1. Continued Organism

Lactobacillus helveticus, biotype jogurti (ATCC 521) Lactobacillus helveticus (15 strains) Lactobacillus casei Lactobacillus brevis (ATCC 8287)

Characterization (latticeb, spacing', M,d (reference)) P; -;

- (Masuda

and Kawata, 1983)

0; 4.1-5.4B.9-11.0; 51-58 (M. Sara, unpublished data) H; -; - (Barker and Thome, 1970) 0; 7.0/4.5; 51 (Masuda and Kawata, 1978, 1980)

Lactobacillus brevis (YIT 0017) Lactobacillus buchneri (ATCC 4005) Lactobacillus buchneri (YIT 0040) Lactobacillus fermentum (NCTC 7230) Lactobacillus bulgaricus (YIT 0045) Lactobacillus sp. Enteric lactobacilli (several strains)

P; -; - (Masuda and Kawata, 1983) H; 6; 55 (Masuda and Kawata, 1981) P; -; - (Masuda and Kawata, 1983) 0; 9.6/6.2; 52 (Kawata et al., 1974; Kawata, 1981) P; -; 51.5 (Masuda and Kawata, 1983) S; 10.7; - (Wang et al., 1986) Biochemical evidence; -; 40-60 (Reniero et al., 1990)

Section 15. Irregular, non-sporing, Gram-positive rods Corynebacterium diphtheriae C4' S; 5.3; - (Kawata and Masuda, 1972) s; 3.5; P; -; - (Blom and Heltberg, 1986) Group JK bacteria (coryneform rods) H; 15.7; - (Wang et al., 1986) Eubacterium tenue P; -; - (Margaret and Krywolap, 1986) Eubacterium yurii ssp. yurii S; 10.6; - (Kerosuo et al., 1988; Sjogren Eubacterium yurii ES4C et al., 1988') P; -; - (Lounatmaa et al., 1988) Eubacterium lentum AHP 6099 H ; 15.7; - (Lounatmaa et al., 1988; Eubacterium sp. AHN 990 Sjogren et al., 1988/) 0;-12/8; - (Mayer et al., 1977) Acetobacterium woodii WBl H; -; - (Wiegel and Ljungdahl, 1981) Thermoanaerobacter ethanolicus Section 16. Mycobacteria Mycobacterium bovis BCG (five strains) Section 18. Anoxigenic phototrophic bacteria Purple bacteria Chromatiaceae Chromatium okenii Chromatium weissei Chromatium warmingii Chromatium buderi

Chromatium gracile Thiocapsa jloridana 9314 Amoebobacter bacillosus Ectothiorhodospiraceae Ectothiorhodospira mobilis 8112

0; -5.5/5.5; - (Lounatmaa and Brander, 1989)

G,H; 19; - (Hageage and Gherna, 1971) G,H; 19; - (Hageage and Gherna, 1971) G,H; -30; - (Hageage and Ghema, 1970) G,H; -35; - (Remsen et al., 1970; Remsen and Triiper, 1973) H;5; - (Remsen et al., 1970) S; 8; - (Takacs and Holt, 1971) H; 4; - (Cohen-Bazire et al., 1969) S; 6-8; - (Remsen et al., 1968)

221

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

TABLE 1 . Continued Organism Ectothiorhodospira halochloris BN9850 Rhodospirillaceae Rhodospirillum rubrum Rhodospirillum molischianum RhodospiriNum safexigens (DSM 2132) Rhodopseudomonas palustris Rhodopseudomonas acidophila Green bacteria Pelodictyon sp. Chlorochromatium aggregatum Seetion 19. Oxygenic photosynthetic bacteria Cyanobacteria Chroococcales Synechococcus sp. GC Cyanothece minewah Aphanothece halophyticah (ATCC 29534) Gloeocapsa alpicola Aphanocapsa rivularis Aphanocapsa sp. Merismopedia sp. Synechocystis sp. PCC 6803 Synechocystis aquatilis Synechocystis aquatilis Synechocystis aquatilis f. salina Synechocystis fuscopigmentosa Synechocystis sp. CB3 Synechocystis sp. CLII Synechocystis sp. Microcystis firma Microcystis incerta Microcystis marginatah Microcystis sp. Chrmccacean cyanobacteria (22 strains) Pleurocapsales Chroococcidiopsis sp.

Characterization (latticeb, spacing, M,d (reference))

P; -;

- (Imhoff and Triiper,

1977)

H; 16.5; - (Salton and Williams, 1954; Kuhn and Holt, 1972) S; 10; - (Giesbrecht, 1969) H; -; 68 (Evers et al., 1984) H; 16; - (Giesbrecht, 1969) H; -; - (Tauschel and Hoehniger, 1974) P; -; P; -;

- (Caldwell and Tiedje, 1975) - (Caldwell and Tiedje, 1975)

P; -; - (Vaara, 1982) S; -; - (Gromov et al., 1986; Smarda, 1991) P; -; 200 (Simon, 1981; Smarda, 1991) P; -; - (Jensen and Sicko, 1972h; Smarda, 1991) H; -; - (Smarda, 1991) P; -; - (Smarda, 1991) P; -; - (Smarda, 1991) P; -; - (Vaara, 1982) H; 15.4; - (Smarda et al., 1979) H; -; - (Schiewer and Jonas, 1977; Smarda, 1991) H; -; - (Smarda, 1988) P; -; - (Smarda, 1988) H; 15.5; - (Lounatmaa el al., 1980) H; 15.2; I00 (Karlsson et al., 1983’) P; -; - (Smarda, 1991) P; -; - (Vaara, 1982) H; 11.3; - (Smarda, 1988) H; -14; - (Kessel, 1978) P; -; - (Smarda, 1991) H; -; - (Vaara, 1982)

0; 2-3; - (Biidel and Rhiel, 1985; Smarda, 1991)

Section 20. Chemolithotrophic bacteria and associated organisms Nitrifying bacteria H; 15; - (Watson and Remsen, 1969) Nitrosomonas sp. H; -; 35-42 (Martikainen et al., 19899) Nitrosospira sp. XI01

222

P. MESSNER AND U. B. SLEYTR

TABLE 1. Continued Organism Nitrosocystis oceanus‘ Colourless sulphur bacteria Thiobacillus kabobis Section 21. Budding andor appendaged bacteria Prosthecate bacteria Hyphomicrobium type-halophilic microorganism Pedomicrobium sp. Caulobacter crescentus CB15 Caulobacter sp. Caulobacter sp. Non-prosthecate bacteria Planctomyces sp. ES “Planctomyces gracilis” Hortobigyi 1965

Characterization (latticeh, spacing‘, M,d (reference)) H; 12; - (Remsen et al., 1967; S; 7.5; - Watson and Remsen, 1970) P; -; - (Reynolds et al., 1981)

T; 9.0; - (Kessel et al., 1985) P; 18.5; - (Ghiorse and Hirsch, 1979) H; 23.5; 130 (Smit et al., 1981; Smit and Agabian, 1982) P; 15; - (De Boer and Spit, 1964) H; -; - (MacRae and Smit, 1991) H; 12; - (Schmidt, 1978) H; -; - (Starr et al., 1984)

Section 23. Non-photosynthetic, non-fruiting gliding bacteria Cytophagaceae G,H; -; - (Pate and Chang, 1979; Cytophaga johnsonae (ATCC 17061) Burchard, 1981) Other genera G,H; -; - (Pate and Chang, 1979; Flexibacter columnaris Burchard, 1981) G,H; 25; 1S80 (Ridgway and Lewin, Flexibacter polymorphus (ATCC 27820) 1973; Ridgway, 1977; Pate and Chang, 1979; Burchard, 1981) Section 24. Myxobacteria Myxococcus xanthus DK1050 Genus not yet classified: Acetogenium kiwi (ATCC 33488) Acetogenium k i w i (DSM 2030) Pelobacter carbinolicus (DSM 2909) Thermotoga maritima (DSM 3109) Nitriloacetate-utilizing bacteria (several strains) Putrescine-degrading bacterium (strain NorPutl) Section 25. Archaebacteria Methanogenic archaebacteria Methanobacteriales Methattobacterium sp. G2R Methattorhermus fervidus V24S (DSM 2088)

Biochemical evidence; -; 74 (Maeba, 1986) H; 20; - (Leigh et al., 1981) H 19; -90 (Rasch et al., 1984; Baumeister et al., 198d) H; 22; - (Dubourguier et al., 1986) T; 12.4; 42 (Huber et al., 1986; Rachel et al., 19909) H; -16; - (Wehrli and Egli, 1988) P; -; - (Matthies et al., 1989)

P; -; - (Sprott et al., 1984) H; 10.8; 93 (Stetter et al., 1981; Sleytr and Messner, 1988b)

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

223

TABLE 1. Continued Organism Methanothermus sociabib KFl-Fl

Methanococcales Methanococcus vannielii SB (DSM 1224)

Characterization (latticeb, spacing', M,d (reference)) H; 19.2; - (Lauerer et al., 1986; Sleytr and Messner, 1988b)

H; 10.8; 60 (Jones et al., 1977; Konig and Stetter, 1986) Methanococcus voltae PS (DSM 1537) H; -10; 76 (Konig and Stetter, 1986; Koval and Jarrell, 1987) Methanococcus thermolithotrophicus SNl (DSM 2095) H; 9.8; 83 (Huber etal., 1982; Konig and Stetter, 1986; Nusser and Konig, 1987) H; 10.5; 90 (Jones et al., 1983; Konig and Stetter, Methanococcus jannaschii JAL-1 (DSM 2661) 1986; Nusser and Konig, 1987) H; 12.3; - (Sleytr and Messner, 1988b) Methanococcus aeolicus PL- 15/H H; -; - (Burggraf et al., 1990a) Methanococcus igneus Kol 5 (DSM 5666) Methanomicrobiales P; -; - (Paynter and Hungate, 1968; Sleytr Methanomicrobium mobile BP and Messner; 1988b) H; 15.3; 155 (Rivard et al., 1984; Zellner Methanolacinia paynteri (DSM 2545) et al., 1989a) Methanospirillum hungatei JFI (ATCC 27890), sheath 0; 2N5.6; 24 (Beveridge et al., 1985, 1988; Stewart et al., 1985) Methanospirillum hungatei GPl (DSM 1101). sheath 0; 23/23; - (Shaw et al., 1985') Methanospirillum hungatei GPl (DSM 1101), septum H; 18.2; H; 14; 117 (Romesser et al., 1979; Zabel Methanogenium cariaci JR1 (DSM 1497) et al., 1984) H; -; 138 (Romesser et al., 1979; Zabel Methanogenium marisnigri JRI (DSM 1498) et al., 1984) H; -; 130 (Zabel et al., 1985) Methanogenium thermophilicum (DSM 2640) H; -; 120 (Zabel et al., 1984) Methanogenium tationis (DSM 2702) H; 15.4; 118 (Zellner et al., 1990) Methanogenium liminatans (DSM 4140) P; -; - (Aldrich et al., 1986) Methanosarcina mazei (DSM 2053) Methanosarcina acetivorans C2A (DSM 2834, ATCC 35395) P; -; - (Sowers et al., 1984) H; 11.2; 156 (Konig and Stetter, 1982, 1986) Methanolobus tindarius T3 (DSM 2278) Methanolobus siciliae T4/M (DSM 3028) P; -; - (Sleytr and Messner, 1988b) 0; 2.U5.6; - (Zehnder et al., 1980; Konig Methanothrix soehngenii (DSM 2139), sheath and Stetter, 1986) 0;2.U5.6; - (Beveridge et al., 1986a, b) Methanothrix concilii, sheath Methanococcoides methylutens TMA-I0 (ATCC P; -; - (Sowers and Ferry, 1983) 33938) H; 14.0; 143 (Wildgruber et al., 1982) Methanoplanus limicola M3 (DSM 2279) H; 14.3; 90 (Zellner et al., 1987, 1989c) Methanocorpusculum parvum XI1 (DSM 3823) H; 15.8; 92 (Zellner et al., 1989c) Methanocorpusculum sinense (DSM 4274) H; 16.0; 94 (Zellner et al., 1989c) Methanocorpusculum bavaricum (DSM 4179) Archaebacterial sulphate reducers H; 17.5; - (Stetter et al., 1987; Kessel et al., Archaeoglobus fulgidus, strain VC-16 (DSM 4304) 1996)

224

P. MESSNER AND U. 8 . SLEYTR

TABLE 1. Continued Oreanism

Characterization (latticeb, spacing‘, M,d (reference))

H; 17.5; 132 (Zellner et al., 1989b) Archaeoglobus fulgidus, strain Z (DSM 4139) P; -; - (Burggraf et al., 1990b) Archaeoglobus profundus AV 18 (DSM 5631) Extremely halophilic archaebacteria Halobacterium salinarium (DSM 668, ATCC 19700) H; -; 200 (Mohr and Larsen, 1963; Mescher et al., 1974; Mescher and Strominger, 1976) H; 13-16; 200 (Houwink, 1956; Stoeckeniusand “Halobacterium halobium” (several strains) Rowen, 1967; Kirk and Ginzburg, 1972; Robertson el al., 1982; Wieland et al., 1982; Lechner and Wieland, 1989) H; 15-16.5; 200 (Kushner et al., 1964; D’Aoust “Halobacterium cutirubrum” and Kushner, 1972; Sleytr and Messner, 1988b) P; -; 220 (Sleytr and Messner, 1988b) Halobacterium saccharovorum (DSM 1137) P; -; - (Sleytr and Messner, 1988b) Halobacterium sp. r-4 (DSM 1411) H; -; - (Steensland and Larsen, 1969) Halobacterium sp. 5 H; 13-14; 190 (Mullakhanbhaiand Larsen, 1975; Haloferax volcanii DS2 (ATCC 29605) Cohen et al., 1983a, b) P; -; 185 (Sleytr and Messner, 1988b) Haloferax volcanii (CCM 3361) H; 15.5; 200 (Kessel et al., 1986, 1988b) Haloferax volcanii Extremely thermophilic So metabolizers P; -; - (Zillig et al., 1983b) Thermococcus celer (DSM 2476) H; 12.5; - (Fiala and Stetter, 1986) Pyrococcus furiosus Vcl (DSM 3638) H;31,4;325(Zilligetal., 1981;KonigandStetter, Thermoproteus tenax Kra 1 (DSM 2078) 1986; Messner et al., 1986a; Wildhaber and Baumeister, 1987’) H; 30.3; - (Fischer et al., 1983; Messner Thermoproteus neutrophilus Hw24 (DSM 2338) et al., 1986a) H; 27.7; - (Sleytr and Messner, 1988b) Thermoproteus autotrophicus H; -; - (Fischer el a/., 1983) Thermoproteus sp. H3 H; -; - (Zillig et al., 1983a) Thermojilum pendens Hw3 (DSM 2475) S; 17.5; - (Zillig et al., 1982; Sleytr and Desulfurococcus mucosus (DSM 2162) Messner, 1988b) S; 18; 50 (Zillig et al., 1982; Wildhaber et al., Desulfurococcus mobilis (DSM 2161) 1987’) H; -; - (Fiala et al., 1986) Staphylothermus marinus F1 (DSM 3639) H; 20.5; 172 (Stetter, 1982; Stetter e t a / . , 1983; Pyrodictium occultum PL-19 (DSM 2709) Sleytr and Messner, 1988b) H; 23.1; 150 (Stetter et al., 1983; Sleytr and Pyrodictium brockii S1 (DSM 2708) Messner, 1988b) P; -; - (Fischer et al., 1983) Thermodiscus maritimus S2 H; 22; 140-170 (Michel etal., 1980; Taylor etal., Sulfolobus acidocaldarius 98-3 (DSM 639) 1982; Deatherage et al., 198v) H; 20; - (Weiss, 1974) Sulfolobus acidocaldarius (ATCC 27360) H; 20; 100,40 (Inatomi et a/., 1983) Sulfolobus acidocaldarius 7 H; 20; -(Millonig et a/., 1975;Zillig et al., 1980; Sulfolobus solfataricus-“Caldariella acidophila” Priischenk and Baumeister, 1987’) (DSM 1616, DSM 1617)

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

225

TABLE 1. Continued Organism Acidianus infernus So4a (DSM 3191) Acidianus brierleyi (DSM 1651)

Sfygiolobus azoricus FC6 (DSM 6296) Genus not yet classified: “Square bacterium” (halophilic) Hyperihermus butylicus Methanohalophilus oregonense WALl (DSM 5435) Pyrobaculum islandicum GE03 (DSM 4184) Pyrobaculum organotrophum H1V Thermophilic archaebacterium, strain ES1

Characterization (latticeb, spacing‘, M,d (reference)) H; 12.4; - (Segerer et al., 1986; Sleytr and Messner, 1988b) H;20.0; 123(Zilligetal., 1980;KonigandStetter, 1986; Segerer et al., 1986; Sleytr and Messner, 1988b) H; -; - (Segerer et al., 1991)

H,S; 14-23;-(Walsby, 1980;Stoeckenius, 1981; Kessel and Cohen, 1982) H;25.8; - (Baumeister et al., 1996; Zillig et al., 1990) P; -; - (Liu et al., 1990) H; 29.9; - (Phipps et al., 1996) H; 20.0; - (Baumeister et al., 1989) P; -; - (Pledger and Baross, 1989)

Classification according to “Bergey’s Manual of Systematic Bacteriology” (vols 1 4 ) (Holt, 1984, 1986, 1989a,b). Unit cell symmetry (latticesymmetry): H, hexagonal (p6);T, trimeric (p3); S, square (p4); 0,oblique (p2); P, periodic structure not further characterized (e.g. evidence from thin sections). Centre-to-centre spacing (nm). Molecular mass of S-layer protein (kDa). ‘Two superimposed S-layers. Three-dimensional image reconstruction has been accomplished. Crystalline outer-membrane protein (cOmp, romp) (Kerosuo et al., 1987; Baumeister et al., 1988). * Taxonomic classification may not be correct. a

are ideal model systems for studying the dynamic process of assembly of a supramolecular structure during cell growth (Sleytr and Messner, 1989).It is particularly the surface location of S-layers which indicates their biological significance. Since prokaryotic organisms possessing S-layers have been shown to be ubiquitous in the biosphere (Table l), the crystalline surface layers must have evolved as a consequence of most diverse interactions between cells and their environment. Although only a few precise functional aspects have yet been elucidated it appears that S-layers can provide organisms with a selection advantage by functioning as protective coats, molecular sieves and molecule and ion traps, as well as promotors for cell adhesion and surface recognition and frameworks, determining and

226

P. MESSNER AND U. B. SLEYTR

FIG. 1 . Electron micrographs of freeze-etched preparations of intact cells of (a) Cfostridium therrnohydrosuffuricum L110-69 showing a hexagonal (p6) lattice, (b) Desuffotomacufum nigrijkans B20CL71 showing a square (p4) lattice and (c) Lactobaciffusacidophifus SH1 showing an oblique (p2) lattice. Excess S-layer material locally leads to double-layer formation. Bars indicate 100 nm.

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

227

maintaining cell shape or envelope rigidity (for reviews, see Sleytr and Messner, 1983, 1988a; Smit, 1987; Koval, 1988; Baumeister et al., 1988). This review summarizes available knowledge about the structure, chemistry, genetics, synthesis, morphogenesis and function of S-layers. A final section is devoted t o the application potential of two-dimensional protein crystals.

11. Structure and Morphogenesis of S-Layers A. LOCATION AND ULTRASTRUCTURE

Crystalline cell-surface layers have been identified as outermost cellenvelope components in prokaryotic cells by electron microscopy of thin-sectioned, freeze-etched, freeze-dried and shadowed or negatively

SA ........

--SA I . . .

S

FIG. 2. Schematic illustrations of the major classes of prokaryotic cell envelopes containing S-layers. (a) Cell-envelope structure of Gram-negative archaebacteria with crystalline S-layers as an exclusive cell-wall component. (b) S-Layers with an additional regularly arranged sheath. (c) The cell envelope as observed in Gram-

positive eubacteria and archaebacteria (containing peptidoglycan or pseudomurein, respectively). (d) S-Layers in the cell envelope of Gram-negative eubacteria. S indicates the crystalline surface layer, SA the location for possible additional S-layer, and SH a sheath. Modified from Sleytr and Messner (1989).

228

P. MESSNER AND U. B. SLEYTR

stained preparations. Freeze-etched preparations of whole bacterial cells, in particular, have shown that S-layers cover the cell surface completely (Fig. l ) , leaving no gaps during cell growth and cell division (Sleytr and Glauert, 1975; Sleytr, 1978; Sleytr and Messner, 1989). Although there are variations in the complexity and structure of prokaryotic cell envelopes considering the fundamental structural and functional principles derived from electron microscopy and from biochemical and biophysical analysis, three main categories of cell-envelope structures can presently be distinguished (Fig. 2). In many archaebacteria, S-layers represent the only wall component (Fig. 2(a)) outside the cytoplasmic membrane (Kandler and Konig, 1985; Konig, 1988). In bacteria in the genera Methanospirillurn and Methanothrix, where cells occur in chains forming filaments, external to the S-layer is a tubular sheath which consists of proteinaceous subunits in a two-dimensional crystalline arrangement (Fig. 2(b)). In Gram-positive eubacteria, S-layers are associated with the peptidoglycan-containing rigid layer (Fig. 2(c)) and, in Gram-positive archaebacteria, the crystalline arrays are attached to the analogous pseudomurein sacculus (Fig. 2(c)). In the complex cell envelope in Gram-negative eubacteria (Fig. 2(d)) S-layers adhere to or even partly penetrate into the matrix of the outer membrane (for reviews, see Beveridge, 1981; Sleytr and Glauert, 1982; Sleytr and Messner, 1983, 1989). Our present knowledge of the ultrastructure of S-layers is based primarily on results obtained by high-resolution electron microscopy of negatively stained preparations (Sleytr et al., 1988a). Resolution down to 1 nm has been reported (Rachel et al., 1986) but, more usually, values of about 2 nm are obtainable (Stewart, 1986; Hovmoller et a f . ,1988). Both two- and threedimensional (computer) image reconstruction techniques have been applied for studying S-layers (Baumeister and Engelhardt, 1987; Baumeister et al., 1988; Engelhardt, 1988; Hovmoller et al., 1988), but in most instances information obtained on the mass distribution of the crystalline arrays is limited by structural changes such as drying and staining artefacts induced by the preparation procedure (for reviews, see Kellenberger and Kistler, 1979; Robards and Sleytr, 1985; Hovmoller et al., 1988; Sleytr et al., 1988a; Messner and Sleytr, 1991a). For comparison of structures, the suggestion was made to classify S-layer lattices according to space groups, unit cell size and the position of protomers and pores relative to the symmetry elements (Sjogren et al., 1985; Hovmoller, 1986; Saxton and Baumeister, 1986; Hovmoller et al., 1988). Electron-microscopical studies revealed (Fig. 1) that most S-layers of archaebacteria have hexagonal (p6) symmetry. So far, S-layers with hexagonal, square (p4) and oblique (p2) lattices have been identified in

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

oblique

square

229

hexagonal

FIG. 3. Schematic drawing of the most common types of S-layer lattices observed

on prokaryotic cells. eubacteria (Fig. 3 and Table 1). Although theoretically possible, p l and p3 lattices have not yet been convincingly demonstrated. The morphological units of oblique, square and hexagonal lattices consist of two, four and six monomers, respectively, and have lattice constants ranging from around 3 to 35 nm (Table 1). From freeze-etched, freeze-dried and shadowed preparations as well as three-dimensional models of the mass distribution derived from negatively stained preparations, it is also known that the S-layer surface exposed to the external environment is frequently smoother than the surface adhering to the underlying cell envelope layer (Hovmoller et al., 1988). Some bacteria produce more than one S-layer on their surface (Fig. 2). These superimposed crystalline arrays can exhibit identical or different crystallographic lattice types (for reviews, see Sleytr and Messner, 1983, 1988a; Table 1). Due to their crystalline nature and because they are composed of identical subunits, S-layers exhibit uniform pore morphologies, whereby individual lattices can display more than one type of pore (Baumeister et al., 1988; Hovmoller et al., 1988; Sleytr et al., 1988b). The present limitations in resolution and structural fidelity of electronmicroscopic preparations do not allow an accurate prediction of the pore size of S-layers. On the other hand, permeability studies on isolated S-layers (see Sections IV and V) have provided useful information on the permeability properties and molecular exclusion limits. Comparative studies on the distribution and uniformity of S-layers have shown that individual strains of a species can exhibit a remarkable degree of heterogeneity regarding the geometry and the lattice constants of the

230

P. MESSNER AND U . B. SLEYTR

crystalline array and the molecular weight and the degree of glycosylation of the constituent subunits (see also Section 1II.A). Detailed comparative studies have been performed on S-layers of different strains of members of the Bacillaceae (Messner et al. , 1984; Sleytr et al., 1986; Lewis et al., 1987) and Aeromonas salmonicida (Evenberg et al., 1982; Kay et al., 1984). Data indicating a diversity of S-layers on strains within a species or the absence or presence of S-layers, meanwhile, have been provided for many other organisms (for a review, see Sleytr and Messner, 1988a). Thus, it appears that, in many eubacteria, S-layers can be considered as a strain-specific taxonomical feature only. The observed diversity even among strains of the same species resembles clearly the nonconservative character of other bacterial cell-surface components such as exopolysaccharides or lipopolysaccharides (Sutherland, 1977; Osborn, 1979). Genetic approaches, such as sequence-homology studies (see Section 1II.B) ,will provide a more detailed insight into the taxonomical significance of S-layers and will also help to elucidate the relationship of S-layer protomers and other pore-forming proteins such as the regularly arranged outer-membrane proteins of Gram-negative bacteria (Kerosuo et al. , 1987; Smit, 1987; Baumeister et al., 1988). Crystalline two-dimensional arrays of

FIG. 4. Schematic representation of possible interactions of crystalline arrays of proteins with the cytoplasmic membrane (CM) in Gram-negative archaebacteria (ax), and the outer membrane (OM) in Gram-negativeeubacteria (d-f). S-Layers may be superficially attached to (a, d) or penetrate into the lipid bilayer to various extents (b, c, e). Regular crystalline arrays of outer-membrane proteins (cOmp, romp) (e) may represent structurally and/or functionally “intermediate states” between S-layers and porins (see also Fig. 2(a) and (d)). Modified from Sleytr and Messner (1989).

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

231

proteins (cOmp, crystalline outer-membrane protein; r o m p , regular outermembrane protein) observed as components of the outer membrane in Gram-negative bacteria (Fig. 4) apparently require a lipid bilayer for assembly and structural integrity. From the limited structural and biochemical data available, it appears that, in archaebacteria depending on the amphipathic structure of the S-layer subunits, the crystalline arrays may only be associated peripherally with the plasma membrane (Fig. 4(a)) or penetrate into the lipid matrix (Fig. 4(b, c)). By analogy, S-layers observed in Gram-negative eubacteria may, depending on the extension of their hydrophobic domains, either only be attached to (Fig. 4(d)), or partially or completely (Fig. 4(e)) penetrate, the matrix of the outer membrane. Such a type of molecular incorporation will closely resemble porins (Fig. 4(f)), which generate aqueous channels through the outer membrane (Engel et al., 1985; Hancock, 1987; Singer et al., 1987; Sleytr and Messner, 1989). S-Layers associated with the plasma membrane in archaebacteria or with the outer membrane of Gramnegative eubacteria can be expected to interact specifically with individual molecular components of these layers, leading to functionally “co-operative assemblies”. B . SELF-ASSEMBLY AND MORPHOGENESIS

Various procedures have been developed for the detachment of S-layers from Gram-positive and Gram-negative eubacteria and archaebacteria and for disintegration of the crystalline arrays into their protomeric units (Sleytr, 1978, 1981; Koval and Murray, 1984; Messner and Sleytr, 1988~). Most commonly, a complete disintegration of S-layers can be achieved using a high concentration of chaotropic agents (e.g. urea, guanidine hydrochloride) or by lowering or by raising the pH value (Sleytr, 1976, 1981). Some S-layers, particularly from Gram-negative bacteria, may also disintegrate by applying metal-chelating agents (e.g. EDTA, EGTA) or cation substitution (Beveridge and Murray, 1976 a,c). Although considerable differences with respect to the mechanical stability have been demonstrated between S-layers of individual organisms, data indicate that the constituent subunits in most S-layers are held together and onto the supporting envelope layer (Fig. 2) by non-covalent forces including hydrophobic interactions and hydrogen bonds, as well as ionic bonds involving divalent cations or direct interaction of polar groups. Experiments involving S-layer extraction and disintegration further suggest that the bonds holding the lattice subunits together are stronger than those binding S-layer lattices to the underlying layer (for reviews, see Sleytr, 1978, 1981; Masuda and Kawata, 1980; Sleytr and Messner, 1983, 1988a; Koval and

232

P. MESSNER A N D U. 9. SLEYTR

Murray, 1984; Smit, 1987). Some archaebacterial S-layers are highly resistant to common denaturing agents, indicating that covalent bonds are stabilizing the crystalline arrays (Konig and Stetter, 1986; Messner et al., 1986a). Isolated S-layer subunits from numerous Gram-positive and Gramnegative eubacteria and archaebacteria have the ability to assemble into crystalline arrays with the same lattice dimensions as those observed in intact cells upon removing the disrupting agents used for their isolation. Data on this entropy-driven self-assembly process of S-layers from members of the Bacillaceae indicate that there is a rapid initial phase in which oligomeric precursors consisting of 12-16 unit cells are formed, which then recrystallize in a slower second process to give larger arrays (Jaenicke et af., 1985). Depending on the specific bonding properties of the S-layer subunits and their mass distribution, protomers may aggregate into flat sheets, openended cylinders (Fig. 5) or closed vesicles (for reviews, see Sleytr, 1981; Sleytr and Messner, 1983,1989). Changing the assembly conditions (e.g. pH value, temperature, ionic strength, presence or absence of divalent cations) may favour one or other assembly route. In addition to monolayers, double and multilayers are also often formed. In double layers, the two constituent monolayers can face each other either with their inner or their outer side (Messner et al., 1986b; Pum et al., 1989a). Fragments of self-assembly products obtained by mechanical disruption, or S-layer fragments detached from cell envelopes, have shown a strong tendency to fuse and to recrystallize (Sleytr and Plohberger, 1980; Sleytr, 1981). Most recently, it has been shown that the 55 kDa S-layer protein, isolated from the Pseudomonas-like strain EU2 using a low concentration of

FIG. 5. Electron micrographs of negatively stained preparations of the most common types of S-layer self-assembly products. (a) Sheet-like assemblies of Desulfotomaculum nigriJicansNCIB 8706 and (b) cylindrical assemblies of Bacillus stearothermophilus NRS 2004/3a. Bars indicate 100 nm.

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

233

guanidine hydrochloride re-assembled into “multilamellar” planar sheets upon removal of the chaotropic agent. Interestingly, these assemblies gave some indications of formation of three-dimensional crystals (Austin et al., 1990). If such crystals, amenable to X-ray diffraction, could be obtained, this would open the way for elucidating S-layer structures down to an atomic resolution. The S-layer self-assembly system can also be studied in the presence of supporting layers. Isolated S-layer subunits or fragments have shown the ability to re-attach to cell surfaces, from which they had been removed (homologous re-attachment), to those of other organisms (heterologous re-attachment) (Sleytr, 1975,1976) or to other “pattern-neutral’’ charged or uncharged surfaces such as carbon layers or polymer films (Messner et al., 1986b; Pum et al., 1989a). Detailed studies have also been performed elucidating the dynamic process of assembly of S-layers during cell growth (for reviews, see Sleytr, 1981; Sleytr and Messner, 1989). A coherent S-layer on an average-sized prokaryotic cell consists of about 5 ~ 1 monomers. 0 ~ This implies that, at a generation time of 20 minutes, at least 400 copies of a single polypeptide species with a molecular weight generally larger than 100,000 have to be synthesized per second, translocated to the cell surface and incorporated in the S-layer lattice. In most organisms studied so far, the rate of synthesis of S-layer subunits appears to be strictly controlled since only minor amounts of S-layer proteins can be detected in the growth medium. Few organisms produce and shed a considerable excess of these proteins into the surrounding medium (Tsukagoshi et al., 1984). From the in vitro selfassembly studies, it became evident that the information required for this assembly and recrystallization process is entirely contained within the individual S-layer subunit (Sleytr, 1975; Sleytr and Plohberger, 1980). With the exception of those Gram-negative archaebacteria in which the S-layer is directly associated with the cytoplasmic membrane (Fig. 2(a)), the protomeric units have to pass through intermediate envelope layers (Fig. 2(c,d)) before reaching the sites of lattice-assembly growth. In Gram-positive archaebacteria or eubacteria, S-layer protomers or oligomeric assemblies will have to penetrate and/or diffuse laterally within the polymer meshwork of the rigid wall component (Fig. 2(c)). In Gramnegative eubacteria (Fig. 2(d)), this will be the periplasmic space and the outer membrane (Belland and Trust, 1985; Smit, 1987). Detailed studies have not so far been performed. However, preliminary studies on selected organisms have provided no indication for pools of S-layer proteins in the cytosol (see Section 1II.C). In some Gram-positive eubacterial organisms it was shown that, upon

234

P. MESSNER AND U. B. SLEYTR

FIG. 6. Electron micrographs of ultrathin sections of intact cells of (a) Bacillus

stearothermophilus NRS 106/1b2 possessing a thin peptidoglycan layer (PG) and (b) Lactobacillus acidophilus 145 with an extremely thick peptidoglycan layer. Interestingly, inner and outer regions of the peptidoglycan reveal different staining behaviour. (c) A thin-sectioned cell-wall preparation of Bacillus sphaericus CCM 2120 with S-layers (S) on both surfaces of the peptidoglycan. The outer S-layer (0s) exists on intact cells whereas the inner S-layer (is) is formed by excess S-layer subunits upon removal of the cytoplasmic membrane (CM). Bars indicate 50 nm.

removal of the cytoplasmic membrane (e.g. in the course of cell breakage), an additional S-layer can assemble on the inside of the peptidoglycancontaining layer (Fig. 6), indicating that a surplus of S-layer subunits is present in the peptidoglycan meshwork (Sleytr and Glauert, 1976; Sleytr, 1978; Masuda and Kawata, 1981). Several organisms (see Table 1) assemble double S-layers. In Aquaspirillum serpens MW5 (Stewart and Murray, 1982; Kist and Murray, 1984), Bacillus brevis 47 (Tsuboi et al., 1982; Sara et al. , 1990b), Aquaspirillum sinuosum (Smith and Murray, 1990) and Nitrosocystis oceanus (Watson and Remsen, 1970), the two superimposed crystalline arrays are composed of different subunit species. The morphogenesis of such a self-assembly system will require different but highly specific structural interaction between the constituent subunits generating the monolayers. In A . serpens, a detailed study has clearly shown that the inner S-layer assembles entropically by itself on the surface of the outer membrane whereas the outer S-layer required the inner layer as a template for assembly (Kist and Murray, 1984). Recently published reports assume that archaebacteria are also able to synthesize double S-layers (Baumeister et al., 1989; Bonch-Osmolovskaya et al. , 1990) but, to answer this question definitely, more detailed analyses are necessary.

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

235

Freeze-etching preparations of rod-shaped cells generally reveal a characteristic orientation and a good long-range order of the S-layer lattice on the cylindrical part of the cell. To maintain such good order during cell growth and division, S-layers must have the ability to recrystallize on the supporting envelope layer to assume a structure with a low free energy (Sleytr and Glauert, 1975; Sleytr, 1978). Only a few studies have been performed aimed at localizing the incorporation sites of new subunits into the dynamically growing closedsurface crystals. Labelling experiments with indirect fluorescent-antibody techniques (Howard et al., 1982; Gruber and Sleytr, 1988) and the protein A-colloidal-gold marker method (Smit and Todd, 1986; Gruber and Sleytr, 1988) indicate that different patterns of S-layer growth may exist for Grampositive and Gram-negative bacteria. With the Gram-positive bacteria Bacillus sphaericus and B. stearothermophilus, the major area of S-layer incorporation was a band at the sites of an incipient cell division. The two cell poles generated by the subsequent split have been shown to maintain static lattice domains. Growth of the S-layer lattice primarily occurs over the remaining cylindrical part of the cell by insertion at multiple bands or helically arranged bands of S-layer (Howard et al., 1982; Gruber and Sleytr, 1988). A somewhat different pattern of S-layer extension was observed on the Gram-negative bacterium Caufobacter crescentus (Smit and Agabian, 1982). Over the main body of this rod-shaped stalk-forming organism, incorporation of new subunits occurs at diffuse sites. Regions of incipient cell division and the stalk region reveal entirely new S-layer material. Very little information is available on S-layer growth in those archaebacteria in which the crystalline arrays represent the only wall component and probably play an important role in determining and maintaining cell shape (see Section 1V.C). In the hexagonal S-layer lattice of Thermoproteus tenax, a rod-shaped extremely thermophilic archaebacterium (Fig. 7), the demonstration of six five-fold vertices (wedge disclinations) at each of the cell poles provided strong evidence that these lattice faults are incorporation sites for S-layer material required for elongation growth of the cylindrical part of the cell (Messner et al., 1986a; Wildhaber and Baumeister, 1987). Most archaebacteria (e.g. halobacteria, thermo-acidophiles and methanogenic bacteria) which possess S-layers as the exclusive wall component (see Table 1) show a highly lobed morphology. In freeze-etching preparations of these morphologically less well-defined cells, both dislocations and disclinations have been demonstrated as lattice faults in the hexagonal S-layer lattice. However, their function as incorporation sites for S-layer growth has not yet been proven by high-resolution labelling experiments (for a review, see Sleytr and Messner, 1989). The necessity for five- and

236

P. MESSNER

A N D U. B. SLEYTR

FIG. 7. Electron micrographs of freeze-dried and metal-shadowed preparations of envelopes of Thermoproteus tenax. Non-labelled envelopes (a) reveal a smooth outer-surface and a rough inner-surface topography. Labelling with polycationic ferritin (b) indicates positions of net negatively charged regions on the crystalline array. One marker molecule binds to each morphological unit of the S-layer lattice. Bars indicate 200 nm.

seven-fold vertices for producing the hexagonally ordered, highly lobed envelope structure of archaebacteria, with S-layers as the exclusive wall component, has been discussed in detail by Deatherage et al. (1983). In theoretical papers on lattice faults in two-dimensional crystals, Harris (1977, 1978) also drew attention to the fact that fission of cells can be explained by models in which the essential process is movement of a -360" disclination on a closed-surface crystal. By this mechanism, a closed-surface crystal can be divided, leading to two separate cells without breaking the surface (Harris, 1977). Most recently, we have provided evidence for this model by using high-resolution freeze-etching preparations from Methanocorpusculum sinense (Sleytr and Messner, 1989; Pum et al., 1991).

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

237

111. Chemistry, Genetics and Biosynthesis of S-Layers A. CHEMICAL ANALYSES

Comparison of amino-acid analyses of hydrolysates of S-layer (glyco)proteins, isolated from organisms of different taxonomic origin, have shown that the crystalline arrays are usually composed of weakly acidic proteins (Sleytr and Messner, 1983; Table 2). The content of hydrophobic amino-acid residues is in the range of 40-50% and the cysteine- or methionine-residue content is generally low (Sleytr and Messner, 1983; Dooley et al., 1988; Dubreuil et al., 1988). Exceptions are the S-layer glycoproteins of Methanotherrnusfevridus and Mt. sociabilis, which possess eight cysteine residues per S-layer subunit (Brockl et al., 1991). Ultrastructural and chemical analyses have revealed that most bacteria are entirely covered by a single S-layer. However, examples are known where more than one chemically different S-layer covers the cell surface (Fig. 3 and Table 1). For most S-layers, sodium dodecyl sulphate-polyacrylamide-gel electrophoresis (SDS-PAGE) revealed molecular masses for the constituent subunits in the range of 30 to around 220 kDa. Even higher molecular weights have been suggested for S-layer subunits from mass-distribution determinations by high-resolution electron microscopy (Baumeister et al., 1988). The estimated molecular weights of S-layer glycoproteins derived from SDS-PAGE are too high due to the presence of covalently linked carbohydrates (Lechner and Sumper, 1987; Sumper et al., 1990). This was confirmed by removal of the carbohydrate residues from the glycoprotein by treatment with deglycosylating agents revealing the molecular weight of the deglycosylated protein (Messner and Sleytr, 1988b, 1991b). Recently, several S-layer-like structures with low molecular-mass subunits (around 30 kDa) were found on Gram-negative eubacteria and archaebacteria (Kerosuo et al., 1987; Baumeister et al., 1988; Kessel et al., 1988a; Rachel et al., 1990) which were considered as crystalline or regular outer-membrane proteins (cOmp, romp) (Kerosuo et al., 1987; Baumeister et al., 1988). They were distinguished from S-layer proteins by the fact that they were predominantly hydrophobic membrane proteins, presumably at least partially embedded in the hydrophobic region of the membrane lipid bilayer (see also Section 1I.B and Fig. 4). Consequently, aggregation of these molecules will only take place within the lipid layer (Hovmoller et al., 1988). Sequence-homology studies may show that there is no sharp evolutionary borderline between S-layers and crystalline outer-membrane proteins (Fig. 4). Further, specific functional studies, like that carried out on Bordetella pertussis (Kessel et al., 1988a), are required to substantiate their distinction from S-layers on a functional basis.

TABLE 2. Characterization of Selected S-layers Number of aminoacid residues per

Organism

subunit (signalsequence) is given M, in parentheses) (mature) p l

A. By analysis of the secondary structure" Aeromonas hydrophila TF7 520' Campylobacter fetus VCll9 1304' 577' Azotobacter vinelandii UWl

52,000' 4.6 131,000' 6.4 60,200' 4.4

B. With known nucleotide sequence of the Slayer gened Bacillus brevis 47 (OWP) 1004 (24) Bacillus brevis 47 (MWP) 1053 (23) Halobacterium halobium R I M , 852 (34) Deinococcus radiodurans Sark 1036 (n.d.) Bacillus sphaericus 2362 1176 (30) 762 (26) Acetogenium kivui Bacillus brevis HPD31 1087 (53 or 23) Campylobacter fetus 84-32 (23D) 933 1612 Rickettsia pro wazekii 828 (34) Haloferax volcanii DS2 593 (22) Methanothermus fervidus V24S Methanothermus sociabilis KFI-FI 593 (22) Aeromonas salmonicida A450 502 (21)

103,745 114,830 86,538 98,000' 122,189 80,046 123,465 96,758 169,874 81,732 65,000 65,000 50,826

Determined by circular-dichroism measurements. complete DNA sequence. n.d. not determined.

a

3.8 3.7 n.d. n.d. 4.8 n.d. n.d. 4.6 5.8 n.d. 8.4 8.4 4.8

Secondary structure

(YO)

a-Helix

P-Sheet

0-Turn Random

19 28 -

43 36 35

12 5 -

25 31 35

4 5 n.d. 7 n.d. 12 n.d. 39 n.d. n.d. 7 7 25

26 27 n.d. 31 n.d. 33 n.d. 46 n.d. n.d. 44 44 26

44 44

26 24 n.d. n.d. n.d. n.d. n.d. 8 n.d. n.d. n.d. n.d. 43

n.d. 11 n.d. 8 n.d. 7 n.d. n.d. n.d. n.d. 7

References Dooley et al. (1988) Dubreuil et al. (1988) Bingle et al. (1986) Tsuboi et al. (1986) Tsuboi et al. (1988) Lechner and Sumper (1987) Peters et al. (1987) Bowditch et al. (1989) Peters et al. (1989) Ebisu et al. (1990) Blaser and Gotschlich (1990) Carl et al. (1990) Sumper et al. (1990) Brockl et al. (1991) Brockl et al. (1991) Chu et al. (1991)

Determined from amino-acid analysis. Determined by SDS-PAGE.

Predicted from

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

239

Over the past few years, considerable progress was made in the implementation of straightforward analytical and preparative purification techniques for S-layers (e.g. high-performance liquid chromatography, affinity chromatography) (Dooley et al., 1988; Dubreuil et al., 1988; Pei et al. , 1988). Using highly purified S-layer preparations, biochemical, biophysical and immunological characterizations were performed. For example, Austin and Murray (1990) were able to separate by hydroxyapatite chromatography the two superimposed, hexagonally ordered but structurally different S-layers of Lampropedia hyalina. Immunolabelling was used to demonstrate the involvement of at least two polypeptides (240 and 60 kDa) in the assembly process of the outer, so-called “punctate layer” of this strain. Assembly studies indicated that a 66 kDa polypeptide is responsible for attachment of the layer to the inner “perforate layer”. The protein of the inner layer had a molecular mass of 31.5 kDa (Austin and Murray, 1987). To predict the secondary structure of S-layer proteins, circular dichroism spectra were analysed. By this method, conformational changes introduced during the isolation and purification procedure would become apparent. For example, no major changes either at low or neutral pH values were observed when the S-layer protein of Campylobacter fetus was removed by treatment with a low-pH reagent (0.2 M glycine hydrochloride, pH 2.2; Dubreuil et al., 1988). Under denaturing conditions (low pH value, presence of sodium dodecyl sulphate) , however, drastic changes occurred in the secondary structure of proteins on Aeromonas salmonicida and A . hydrophila (Dooley ef al. , 1988).The a-helix content increased considerably to values of approximately 40%. Data available on the secondary structure of Slayers of selected strains of archaebacteria and eubacteria are summarized in Table 2. Another remarkable feature of both archaebacteria and eubacteria is their ability to glycosylate S-layer proteins. For several years it was assumed that glycosylation is restricted to archaebacteria (Mescher, 1981; Kandler, 1982), but detailed chemical and structural analyses have shown that S-layers of eubacteria can also be glycosylated (for a review, see Messner and Sleytr, 1988a, 1991b). In several previous studies, a positive periodic acid-Schiff staining reaction was considered sufficient proof for the existence of a glycosylated S-layer protein. Later, it turned out that a negative-staining reaction is not an absolute confirmation for the absence of carbohydrate residues. Thus, for a conclusive assignment, detailed chemical analyses are required. Kandler and Konig (1985) listed chemical compositions of isolated S-layer glycoproteins from several Gram-negative archaebacteria (e.g. species of Thermoproteus, Sulfolobus and Halobacterium). Based on gas-liquid chromatography and amino-acid analyses, sugar contents were

240

P. MESSNER A N D U. B. SLEYTR

TABLE 3. Glycan structures of S-layer glycoproteins Halobacterium halobium RIMl (Lechner and Wieland, 1989)

I

2

Ala-NH2

so42>>fb

I

I

+3)-GalNAc-( 1+4)-GlcNAc-( 1+4)-GalA-( 1+3)-GalNAc 6 3

t

-(l+N)-Asn

t

1 3-OMe-GalA

I I Ser I

Ala

1 Galf

n

I I X I

GlcA-( 1+4)-IdA-( 1-+4)-GlcA-(1+4)-P-~-Glc-(1+N)- Asn

1 3

so42-

Thr/Ser

I

I

a-D-Glc-(1+2)-Gal-( I+O)-Thr

I Haloferax volcanii DS2 (Sumper et al., 1990) N-Glycans, structure not analysed

I I

Glc-(1+2)-Gal-( l+O)-Thr

Bacillus stearothermophilus NRS 2004I3a (Christian et al., 1986; Messner et al., 1987; Messner and Sleytr, 1988b)

I

+2)-a-~-Rhap-(1+2)-a-~-Rhap-(1+3)-P-~-Rhap-( 1 4

and

1

I I

Rha-(l+N)- Asn n-50

241

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

Clostridiurn thermohydrosulfuricurn Ll ll-69 (Christian et al., 1988)

+4)-a-~-Manp-( 1+3)-a-~-Rhap-(1+

1

possibly an 0-glycosidic bond n=60

Clostridium thermosaccharolyticurn D120-70 (Altman et al., 1990) I

I

II

1

-3)-p-D-Manp-( 1+4)-a-~-Rhap-(1+3)-a-~-Glcp-(1+4)-a-~-Rhap-(1+ 6 2

t

t

1 a-D-Galp

1 P-D-GIc~

and

I +4)-P-~-GlcpNAc-(1+3)-~-~-ManpNAc-(l+ 1 In Bacillus alvei CCM 2051 (Altman et al., 1991)

I

+3)-P-~-Galp-( 1+4)-P-~-ManpNAc-(16

t

1

a-D-GICp

n=40

Clostridium symbiosum HB25 (Messner et al., 1990)

+6)-a-~-ManpNAc-(1+4)-P-~-GalpNAc-(1+3)-a-~-BacpNAc-(1 4 ) - a - D GalpNAc-(I+PO3H)+

1

n=15

Abbreviations: Glcp, glucose (pyranose form); GalJ”, galactose (furanose form); Man, mannose; Rha, rhamnose; GINAc, N-acetylglucosamine (2-acetamido-2-deoxyglucose); GalNAc, N-acetylgalactosamine; ManNAc, N-acetylmannosamine; BacNAc, NGlcA, glucuronic acid; acetylbacillosamine (2-acetamido-4-amino-2,4,6-trideoxyglucose); GalA, galacturonic acid; ManA, mannuronic acid; IdA, iduronic acid; 3-OMe-GalA, 3-0methylgalacturonic acid; SO,*-, sulphate; Asn, asparagine; Ser, serine; Thr, threonine; Ala, alanine; X, interchangeable amino-acid residue.

242

P. MESSNER AND U. B. SLEYTR

found in the range of approximately 1-20%. Generally, the content of neutral hexose residues was high, but also residues of rhamnose, a 6-deoxy sugar and amino sugars were found in significant amounts, whereas pentose residues were only present in traces. The most detailed structural and biosynthetic studies were performed on the S-layer glycoprotein from Halobacterium halobium (for reviews, see Sumper, 1987; Lechner and Wieland, 1989). The structures of the three different types of covalently linked oligosaccharides are shown in Table 3. The cell-envelope glycoprotein from Hb. halobium (safinarium)was the first glycoprotein from a prokaryote to be described (Mescher and Strominger, 1976). Just recently, Sumper et al. (1990) reported a partial chemical characterization of the S-layer glycoprotein of Haloferax volcanii. The molecular weight of this glycoprotein is different from that of Hb. halobium (see Table 2) but both N - and 0-glycosidically linked carbohydrates were detected. The N-glycans were not analysed in detail in this study but, obviously, the 0-glycans are identical to those from the glycoprotein of Hb. halobium (Lechner and Sumper, 1987). Preliminary chemical data indicate major differences in the structure of the N-glycosidically linked saccharides of the glycoprotein on Huloferax vofcanii compared with those on Hb. halobium. Nusser et al. (1988) analysed the S-layer glycoprotein of Methanothermus fervidus and estimated a sugar content of 17 mol% . The predominant sugar residues were mannose (7.8 mol%) and 3-0-methylglucose (7.0 mol%) whereas the hexosamine-residue content was low (1.6 mol%). Interestingly, 3-0-methylsugar residues (see Section III.C), which were also found in other bacterial oligosaccharides such as the S-layer glycoprotein of Hb. halobium (Lechner and Wieland, 1989), or the cellulosome of Clostridium thermocellum (Gerwig et al., 1989), were present in the mature glycoprotein (Hartmann and Konig, 1989; Konig et al., 1989a). Some 15 years ago, Sleytr and Thorne (1976) reported the first analyses of the S-layer glycoproteins of Clostridiurn thermohydrosulfuricum and Cl. thermosaccharolyticurn. Their data indicated that eubacteria are also able to glycosylate proteins. In the course of the characterization of the S-layer glycoprotein of Bacillus stearothermophifus NRS 2004/3a, we obtained convincing evidence that one of the carbohydrate chains (Table 3) is linked by an N-glycosidic linkage to the protein (Kupcu et al., 1984). After isolation of the linkage region by treating the polyrhamnan (Christian et al., 1986) with hydrofluoric acid, residues of rhamnose and asparagine were shown to be constituents of a novel type of N-glycosidic linkage (Messner and Sleytr, 1988b). Interestingly, most recently this type of linkage was also observed in sheaths of Methanothrix soehngenii (Pellerin et a f . , 1990).

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

243

S-Layer glycoproteins with different carbohydrate contents (1-15%) were detected in strains of the thermophilic species B. stearothermophilus and Desulfotomaculum nigriJicans(Sleytr et a f . ,1986). In these organisms, heterogenity was also observed in the molecular weight of the constituent protein subunits and the ultrastructure of the S-layers (Messner et al., 1984). All S-layer glycoproteins characterized so far are summarized in Table 3. It became evident that both the composition as well as the carbohydrate-protein linkages differ significantly from glycoproteins found in eukaryotic organisms (Kornfeld and Kornfeld, 1980; Kobata, 1984). The glycan chains of all S-layer glycoproteins of members of the Bacillaceae investigated so far resemble to a high degree the O-antigens of lipopolysaccharides from Gram-negative bacteria (Osborn, 1979). The linear or branched sugar chains contain up to 150 monosaccharide residues, showing apparent molecular weights in gel-filtration experiments in the range of 15,000-25,000. In Hb. hafobium, only the glycosaminoglycan-like carbohydrate chain consists of repeating units, whereas the sulphated oligosaccharides and the O-linked sugar residues possess only short chain-length carbohydrate residues (Lechner and Wieland, 1989). Identical O-linked glycan chains are present in the S-layer on Haloferax volcanii (Sumper et al., 1990). Small carbohydrate contents were found in the S-layers of Aquaspiriflum serpens (Buckmire and Murray, 1973), Bacillus sphaericus (Word et al., 1983; Lewis et al., 1987), Deinococcus radiodurans (Peters et al., 1987) and Acetogenium k i w i (Peters et al., 1989). Particularly for D . radiodurans, the results have to be substantiated because of the possibility of contamination originating from the complex cell-envelope structure of this bacterium. For example, detailed studies on purified S-layer preparations of C. fetus (Dubreuil et al. , 1988) could not confirm the presence of carbohydrate as originally reported by Winter et al. (1978). Obviously, in the earlier study, the S-layer preparation was contaminated with lipopolysaccharide remnants from the outer membrane. Structural elucidation of the glycan chains is usually performed on glycopeptides after exhaustive proteolytic degradation of the purified S-layer glycoprotein by a combination of degradation experiments (e.g. periodate oxidation, Smith degradation), methylation analysis, mass spectrometry and 'H and 13C nuclear magnetic-resonance measurements. Unambiguous evidence for potential glycosylation sites can also be obtained from nucleotide-sequence analysis of the gene coding for the S-layer glycoprotein. For N-glycosidic linkages, sequons with Asn-XSer(Thr) are typical (Kornfeld and Kornfeld, 1985) and O-glycosidic linkages are usually indicated by high contents (clusters) of serine or threonine residues (see Section 1II.B). The presence of glycans has

244

P. MESSNER AND U. B. SLEYTR

then to be confirmed by sequence analysis of proteolytically derived glycopeptides. B. GENETIC STUDIES

Molecular cloning and characterization of the genes coding for crystalline Slayer (g1yco)proteins are essential for elucidation of both the precise properties of these macromolecules and the mechanisms involved in their biosynthesis, translocation across membranes, and assembly at the external cell surface. In 1984Tsukagoshi, Udaka and their coworkers reported for the first time cloning of DNA fragments of the protein-producing Bacillus brevis 47 into Escherichia coli and Bacillus subtilis (Tsukagoshi et al., 1984). Bacillus brevis 47 contains two S-layer proteins, namely the outer-wall protein OWP and the middle-wall protein MWP (see Table 1).Portions of the structural genes for these cell-wall proteins were cloned into the bacteria described where they directed synthesis of polypeptides which reacted with antisera against the cell-wall proteins. Such probes were then used for determination of the complete nucleotide sequence of the outer-wall protein gene whose sequence was determined first (Tsuboi et al., 1986). Interestingly, analysis of transcripts in B. brevis 47 suggested that the genes for the MWP and OWP constitute a cotranscriptional unit (cwp (cell-wall protein gene) operon). After analysis of the DNA sequence of the promoter region (Yamagata et al., 1987), the complete nucleotide sequence of the cwp operon was determined (Tsuboi et al., 1988). Both genes code for precursor proteins with common signal sequences. After cleavage of the signal peptides, mature S-layer proteins were obtained with molecular weights of 114,830 (MWP) and 103,745 (OWP), respectively (Table 2). Primer extension assay of cwp operon transcripts showed the existence of several tandemly arranged promoters in the 5’ region of the cwp operon. These different promoters were used in B . brevis 47 at different stages of growth and play distinct roles in growth phase-specific expression of the cell-wall proteins (Adachi et al., 1989). The middle-wall protein gene has two tandemly located translation iniation sites. Both of them can be utilized to start translation in B. brevis 47 resulting in two different leader sequences (Adachi et al., 1990). Today, the S-layer protein genes of B. brevis 47 represent the genetically best studied S-layer system. So far, the sequences of the S-layers of the archaebacteria Halobacterium halobium (Lechner and Sumper, 1987), Haloferax volcanii (Sumper et al., 1990), Methanothermus fervidus and Mt. sociabilis (Brockl et al., 1991) and the eubacteria Deinococcus radiodurans (Peters et al. , 1987), Bacillus sphaericus (Bowditch et al., 1989), Acetogenium kivui (Peters et al., 1989),

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

245

B. brevis HPD31 (Ebisu et al., 1990), Campylobacter fetus (Blaser and Gotschlich, 1990), Rickettsia prowazekii (Carl et a f . , 1990), Aeromonas salmonicida (Chu et a f . 1991) and Bacillus stearothermophifus (B. Kuen, personal communication) are known. The cell-surface glycoprotein of Hb. halobium was the first S-layer glycoprotein whose complete nucleotide gene sequence was determined (Lechner and Sumper, 1987). Three different types of glycan chains (Table 3) are attached to the polypeptide chain of molecular weight 86,538 (Table 2). The contribution to the molecular mass coming from all covalently linked glycan chains is approximately 30 kDa. Together this would result in a total molecular mass for one S-layer glycoprotein subunit of about 110-120 kDa. Therefore, values in the range of 200 kDa, estimated by SDS-PAGE for the cell-surface glycoprotein of Hb. halobium and other halobacterial S-layer glycoproteins (see Table l ) , are obviously too great. This aberrant migration behaviour on sodium dodecyl sulphatepolyacrylamide gels is most probably caused by the unusual composition of the covalently linked glycan chains of these glycoproteins. Twelve potential N-glycosylation sites (sequons Asn-X-Ser/Thr; X not being Asp or Pro) were found on the polypeptide chain. Apparently, most of them are indeed linked to saccharide moieties (one repetitive pentasaccharide at residue Am2 and about 10 sulphated oligosaccharides). Approximately 20 0-linked disaccharides (Table 3) occur in a cluster near a highly hydrophobic stretch of amino-acid residues which is only three amino-acid residues away from the C-terminus (Lechner and Sumper, 1987). Sequence analysis of the S-layer gene of H . vofcanii showed that the gene encoded a polypeptide of 794 amino-acid residues preceded by a 34 amino-acid residue signal sequence (Sumper et a f . , 1990). The predicted amino-acid residue sequence contains seven potential Nglycosylation sites distributed throughout the polypeptide chain and a cluster of hydroxyamino-acid residues near a highly hydrophobic stretch close to the C-terminus. This hydrophobic region is considered to serve as the membrane anchor. Sequencing of the gene encoding for the hexagonally packed intermediatelayer protein of Deinococcus radiodurans revealed that the gene encoded for 1036 amino-acid residues (Peters et a f . , 1987). Since the N-terminus was unaffected by Edman degradation, evidence for the presence of a signal sequence was deduced both from proteolytic modifications of the hexagonally packed intermediate layer in situ and molecular-weight determinations of the hexagonally packed intermediate-layer polypeptide expressed in E . cofi.The protein contained at least two disulphide bridges, as well as residues of tightly bound reducing sugars and fatty acids (Peters et a f . , 1987).

246

P. MESSNER A N D U. B. SLEYTR

The surface-layer protein (rsaA) gene of Caulobacter crescentus was cloned and the sequence of the first third of the gene reported (Fisher et al., 1988). Resequencing of the entire segment revealed that, in contrast to the previous report (Fisher et al., 1988), cysteine residues were absent (J. Smit, personal communication). Caulobacter crescentus is able to transport flagellar proteins without cleaved signal sequences across membranes (Ohta et al., 1985). Presumably, a similar process is possible with the S-layer protein (Fisher et al., 1988). Vectors incorporating the transcription-translation-initiation regions of the rsaA gene were constructed (Bingle and Smit, 1990). These vectors can be introduced into Cb. crescentus by electroporation at high frequency, or by conjugal transfer (Gilchrist and Smit, 1991). A 4251 bp DNA fragment containing the gene for the S-layer glycoprotein of B. sphaericus 2362 was cloned into E. coli encoding for a protein with 1176 amino-acid residues (Bowditch et al., 1989). This protein is the precursor of the 122 kDa S-layer protein. From Ochterlouny immunodiffusion experiments, and from the sequence identity of the N-termini of these molecules, as well as the antibody reaction, it was concluded that the S-layer protein, which is formed during vegetative growth, serves as a precursor of the 110 kDa larvicidal protein, appearing during sporulation of B. sphaericus 2362 (Bowditch et al., 1989). The gene coding for the S-layer polypeptide of Ac. kivui was cloned in E. coli on two overlapping fragments using plasmid pUC18 as the vector. It was expressed under control of a cloned promoter from Ac. kivui or the lucZ gene (Peters et al., 1989). The mature protein contains 736 amino-acid residues, is weakly acidic and contains a relatively high proportion of hydroxyamino-acid residues, including two clusters of serine and threonine residues. Chemical analyses have revealed significant amounts of residues of glucose, N-acetylglucosamine and N-acetylgalactosamine. These data indicate that the S-layer protein is heterogeneously glycosylated. Sequence homology with the middle-wall protein of B. brevis 47 (Tsuboi et al., 1988) was ascertained for an N-terminal region of about 200 residues. The 98 kDa S-layer protein of an alkaline phosphatase secretion-deficient mutant (NM 105) of Bacillus licheniformis 749/C was found on exponentially growing cells. In the stationary phase of growth, it was overproduced and secreted into the growth medium (Tang et al., 1989). Using h-phage EMBL3, the gene was cloned into E. coli JM 539 and B. subtilis MI112. As already observed by Tsukagoshi et al. (1984) with B. brevis, transformed cells of E. coli JM 539 produced only truncated polypeptide (75 kDa) while the S-layer protein expressed in B. subtilis MI112 had the same molecular weight as the authentic protein purified from B. licheniformis NM 105 (Tang et al., 1989).

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

247

Conserved structures of cell-protein genes in different S-layer proteinproducing B. brevis strains were investigated to examine the complex organization of the regulatory regions of these genes (Ebisu et a f . , 1990). Bacillus brevis HPD31 contains a hexagonally arranged S-layer protein (HWP). The gene coding for this protein was cloned and sequenced. Analysis of the DNA sequence revealed that there is an open reading frame encoding for a polypeptide of 1087 amino-acid residues with a molecular weight of 123,456. The deduced amino-acid sequence of the protein showed a high degree of homology (78%) with that of B. brevis 47. For aminoacid residues 1-548, the homology was 90%. This finding was confirmed by immunological analysis using antisera against both S-layer proteins of 8. brevis 47 (Gruber et a f . , 1988). The structural gene for the surface-array protein of C. fetus strain 84-32 (23D) was cloned into E. cofiY1090 using bacteriophage h g t l l as a vector. An open reading frame was found to encode a 933 amino-acid residue polypeptide calculated to be of 96,758 Da (Blaser and Gotschlich, 1990). The first 20 residues exactly match those determined from N-terminal sequencing, indicating that this protein is secreted without a cleaved leader sequence. An identical phenomenon was described with the S-layer protein on Cb. crescentus (Fisher et al., 1988). Recently, the sequence of the spaP gene coding for the surface-protein antigen (SPA) of R. prowazekii was reported (Carl et af., 1990). Although the gene encodes a protein with a molecular weight of 169,874, the R. prowazekii-derived S-layer was estimated to be of 120 kDa. This decrease in the observed molecular weight can only be explained by further processing of the spaP gene product by the rickettsiae (Carl et a f . , 1990). Cloning and sequencing of the genes coding the S-layer glycoproteins of the extremely thermophilic methanogens Mt. fervidus and Mt. sociabifis revealed that the nucleotide sequences were highly homologous, differing in only nine positions (Brockl et af., 1991). Both genes encode for a precursor for the S-layer protein, comprising 593 amino-acid residues with a signal sequence of 22 residues. From the deduced protein sequences, 20 sequon structures for N-glycosylation (Asn-X-Ser/Thr) as possible carbohydratebinding sites are present. As observed with Hb. hafobium (Lechner and Sumper, 1987) the sugar content of 17 molo/o caused a changed migration behaviour on sodium dodecyl sulphate-polyacrylamide gels. Instead of a calculated molecular mass of about 76 kDa for the mature glycoprotein, two bands with apparent molecular masses of 92 and 60 kDa were obtained (Nusser et af., 1988). The isoelectric point (computer determination) of 8.4 is much higher than that of other S-layer proteins (Table 2). Unusual for S-layer proteins is also the high content of eight cysteine residues (Brockl et al., 1991).

248

P. MESSNER AND U. B. SLEYTR

The best described S-layer in terms of ultrastructure, biological activity as well as export and assembly is the A-layer of the fish-pathogenic bacterium A. salmonicida (Kay et al., 1984; Kay and Trust, 1991). The species-specific structural gene for the A-layer was cloned in bacteriophage hgtll (Belland and Trust, 1987) and cosmid pLA2917. The cloned gene was shown to code for a polypeptide of 502 amino-acid residues with a 21-residue signal sequence and a molecular weight of the mature protein of 50,826 (Chu et al., 1991). Genomics Southern-blot analysis revealed that this gene was in a single copy on the chromosome and was conserved in a wide range of A. salmonicida strains (Belland and Trust, 1985). Trypsin cleavage provided evidence for formation of two structural domains. A 37 kDa polypeptide comprising the N-terminus and a 16.7 kDa fragment of the C-terminus were formed. The larger polypeptide appeared to be completely resistant to trypsin while the smaller peptide was ultimately degraded in the presence of trypsin:protein ratios of 1:100. It was attempted to correlate these data with the three-dimensional organization (Dooley et al., 1989) of the native A-layer protein. Most recently, the gene coding for the non-glycosylated, hexagonally arranged S-layer of B. stearothermophilus PV72 was cloned and sequenced (Kuen et al., 1988; B. Kuen, personal communication). To determine homology between S-layer (g1yco)proteins of different bacteria and also with other functionally or chemically related proteins, sequence comparisons were performed. Usually, with the exceptions already described for closely related strains (Kay et al., 1984; Peters et al., 1989; Ebisu et al., 1990), there is relatively little overall homology of either primary or secondary structure with the characterized S-layer proteins (Table 2). These findings confirm the diversity of S-layers observed by biochemical and ultrastructural investigations (Sleytr and Messner, 1983; Hovmoller et al., 1988; Table 1) and provide further support for the non-conservative character of these important bacterial cell-surface macromolecules.

C. BIOSYNTHESIS

Over the past few years, substantial information has been gathered on biosynthesis and excretion of S-layer (g1yco)proteins (Belland and Trust, 1985;Sleytr et al., 1988b). Deeper insights into the molecular basis of S-layer biosynthesis came from cloning and sequencing studies (see the previous section). With few exceptions (Fisher et al., 1988; Blaser and Gotschlich, 1990; Carl et al., 1990), all S-layer polypeptides analysed so far were synthesized as precursors with an N-terminal extension known as the signal

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

249

peptide (Table 2) that is cleaved during translocation of the polypeptide chain across the cytoplasmic membrane. Although there are differences in the primary structure of the signal peptides, all of them share common structural features. Predominantly, they consist of hydrophobic amino-acid residues and have basic residues near the N-terminus. Though many S-layer (g1yco)proteins are synthesized as secretory precursors (with signal peptides), the mature proteins are not usually excreted into the surrounding medium but are assembled into regular arrays through interaction with each other and also with the underlying cell-envelope component (Fig. 2) (Sleytr, 1978, 1981). The exceptions are those strains in which excess material is growth phase-specifically shed into the medium (Udaka, 1976; Gruber et a f . , 1988). Approximately 105-106 S-layer protomers are necessary to cover completely an average-sized bacterial cell (Sleytr and Messner 1983, 1988a). So far, the most detailed biosynthetic studies have been performed on the Gram-positive eubacterium Bacillus brevis 47. This organism has a very complex cell-wall protein gene with tandemly arranged promoters and two translation-initiation sites (Yamagata et a f . ,1987; kdachi et a f . ,1989,1990). Protein-excretion studies were performed with a-amylase as a reporter molecule. The precursors synthesized on both translation sites were cleaved at the same position to give mature proteins with identical N-termini. The advantage of this complex organization of the regulatory region is not yet fully understood, but the region is obviously important in regulating strictly and growth-phase dependently the synthesis of S-layer proteins (Ebisu et a f . , 1990). Smit and his coworkers are currently investigating the minimal requirements for excretion of the S-layer protein of Caufobacter crescentus using fusion proteins of the S-layer with a reporter molecule (J. Smit, personal communication). The first investigations of the biosynthesis of prokaryotic glycoproteins were performed by Mescher and Strominger (1978) on Hafobacterium safinarium. Growing cells were treated with bacitracin, which revealed an involvement of lipid-linked intermediates in biosynthesis of the glycan chains of this glycoprotein. Wieland, Sumper and their coworkers have extended these studies in Hb. hafobium (for reviews, see Sumper, 1987; Lechner and Wieland, 1989). In addition to the structural elucidation of the glycan chains and characterization of the protein+arbohydrate linkage regions (Table 3), the biosynthetic pathway of this archaebacterial glycoprotein was determined. The repeating-unit saccharides of the glycosaminoglycan, which is linked to the Asn, residue of the polypeptide chain by a novel N-glycosidic linkage (Asn-GalNAc) (Paul et a f . , 1986), are assembled on the lipid carrier by polymerization of preformed repeating units (Wieland et a f . , 1981). The reaction is inhibited by bacitracin. This

250

P. MESSNER AND U. B . SLEYTR

implies involvement of lipid diphosphate on the biosynthetic pathway (Siewert and Strominger, 1967). Unexpectedly, the lipid component is a CM polyprenol of the eukaryotic dolichol type, rather than of the eubacterial undecaprenol type (Wright et al., 1967). Sulphated oligosaccharides are linked by another novel N-glycosidic linkage (Asn-Glc) to the polypeptide (Wieland et al., 1983). A dolichol monophosphate is used as the lipid carrier (Lechner et al., 1985a). In eukaryotes, many of the modification reactions involving glycoproteins take place in the Golgi complex. Since bacteria lack this organelle, biosynthesis of glycoconjugates has to follow different biosynthetic pathways. Transfer of oligosaccharides to proteins was studied by Lechner etal. (1985b) using a synthetic hexapeptide which cannot permeate the cytoplasmic membrane. Interestingly, this acceptor peptide became glycosylated with sulphated oligosaccharides in a suspension with intact halobacterial cells. This result demonstrates that transfer of sulphated oligosaccharides occurs on the halobacterial cell surface. Recently, the composition of the S-layer glycoprotein glycan of Methanothermus fervidus was discovered (Hartmann and Konig, 1989; Konig et al., 1989a). One of the constituent sugars was 3-O-methylglucose. Formation of 3-O-methylsugars, which also occur in other bacterial oligosaccharides (see Section III.A), was thought to be an obligatory step for translocation of lipid-linked oligosaccharides through the cytoplasmic membrane or to be a specific recognition marker for glycosylation (Lechner et al., 1985b). In Hb. halobium, only a transient 3-O-methylation was observed, which means that this specifically modified sugar was not detected in the mature glycoprotein. From cell extracts of Mt. fervidus, neither uridine diphosphate (UDP)- nor lipid pyrophosphate-activated glucose or 3-O-methylglucose could be detected, but these sugars were present in the mature S-layer glycoprotein (Hartmann and Konig, 1989). On the other hand, the corresponding sugar l-phosphates and nucleotide derivatives of mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine, together with UDP- and dolichol-activated oligosaccharides, were isolated. In contrast to Hb. halobium in which the lipid carrier is a Cmpolyprenol (Lechner et al., 1985a), in Mt. fervidus a C55 dolichol was discovered. Additionally, undecaprenol-activated (Wright et al., 1967) disaccharides were isolated (Hartmann and Konig, 1989). Thus, it can be assumed that dolichol is involved in the glycoprotein biosynthesis in both eukaryotes and prokaryotes, and that undecaprenol only plays a role in biosynthesis of prokaryotic peptidoglycan (Higashi et al., 1967) and pseudomurein (Konig et al., 1989b).

CRYSTAL.L.INE BACTERIAL CELL-SURFACE LAYERS

25 1

IV. Functional Aspects of S-Layers

Surface layers are an integral part of the cell envelope of a great variety of archaebacteria and eubacteria (Table 1). Because of their surface location, it is evident that functions have evolved as the result of specific interactions with particular environmental and ecological conditions (Sleytr, 1978; Sleytr and Messner, 1983, 1988a; Koval, 1988). Although knowledge about specific functions of S-layers is still limited to only a few examples, they generally have the potential to act as (a) protective coats, molecular sieves and molecule and ion traps, (b) promoters for cell adhesion and surface recognition, and (c) a cell shape-determiningmaintaining framework (Sleytr and Messner, 1983, 1988a; Smit, 1987; Konig, 1988; Koval, 1988; Hovmoller et al., 1988). During the last few years, deeper insights into specific functional aspects of S-layers were obtained. The following sections summarize the most relevant studies performed during the last few years. A . S-LAYERS RELATED TO PATHOGENICITY

Probably the best studied S-layer system as it pertains to pathogenesis is the crystalline array found on species in the genus Aeromonas. Aeromonas salmonicida is an important Gram-negative pathogen, causing furunculosis in fish (Kay and Trust, 1991). The S-layer (in previous studies termed the Alayer) is required for virulence since S layer-free mutants are avirulent (Kay et al., 1981). The A-layer physically protects the cell against proteolysis, complement and, in addition, is required for macrophage infiltration and resistance (Evenberg and Lugtenberg, 1982; Munn et al. , 1982; Trust et al., 1983; Evenberg etal., 1985). A lipopolysaccharide with a constant size-class distribution of the 0-polysaccharide chains is necessary for anchoring the A-layer to the cell surface (Chart et al., 1984; Evenberg et al., 1985; Griffiths and Lynch, 1990). There is ongoing work in the development of potent fish vaccines to overcome the problems of persistence of infection even in immunized fish (Evenberg et al., 1988; Kay and Trust, 1991). Aeromonas hydrophilia and A . sobria are involved in a variety of systemic infections in man. Among the investigated strains, some were found to be covered by an S-layer (Dooley and Trust, 1988; Paula et al., 1988). As was already known for A . salmonicida, these strains also possess a specific type of lipopolysaccharide which seems to be responsible for the structural integrity of the S-layer (Dooley and Trust, 1988; Paula et al., 1988). Interestingly, this subgroup of highly pathogenic strains shares as a common feature the presence of a certain somatic antigen (serogroup 0 : l l ) and additionally as a phenotypic feature the ability for auto-agglutination in

252

P. MESSNER AND U. 9. SLEYTR

liquid media (Janda et a f . , 1987; Kokka et al., 1990). These strains can therefore be recognized by two relatively simple tests, i.e. auto-agglutination and subsequently serologic reactivity against polyclonal 0: 11 antisera (Kokka and Janda, 1990). Another important pathogen is the Gram-negative bacterium Campylobacter fetus. Infections caused by this organism include abortion in sheep and cattle and various systemic infections or acute diarrhoea1 illness in humans (Pei et al., 1988). As with Aeromonas spp., the ability of C. fetus to cause these diseases appears to be associated with the presence of S-layers (Dubreuil et al., 1988; McCoy et al., 1975; Blaser et af., 1987). The S-layer makes the cells resistant to phagocytic uptake and to the bactericidal activity of serum (Blaser et al., 1988). Impaired ability of complement component C3b to bind to S-layers of C . fetus is the explanation for these resistances (Blaser et al., 1988). Interestingly, C. fetus can undergo antigenic changes at a relatively high frequency during the course of an infection. Pei et al. (1988) investigated S-layer proteins from variants of C . fetus and detected different molecular weights ( 9 0 , 0 ~ 1 5 0 , 0 0 0 )for these proteins. Similar results were reported by Dubreuil et af. (1990). Both cross-reactivity and inability to bind antisera indicated different antigenic specificities of these S-layer proteins. Because of spontaneous mutations of C . fetus strains a simple means for differentiation between S layer-carrying (S') and S layer-negative (S-) variants was sought. An assay was developed based on recognition by lectins of specific receptor structures in the bacterial lipopolysaccharide. Three lectins (wheat-germ agglutinin, Bandeiraea simplicifolia I1 agglutinin and Hefix pomatia agglutinin) were able to bind to the O-side-chain of type-A lipopolysaccharide from C . fetus but the presence of S-layers on intact cells blocked binding (Fogg et a f . , 1990). Since these three lectins obviously recognize only type-A lipopolysaccharide, this assay can also be used to differentiate lipopolysaccharides from C. fetus. To compare the virulence of S+ and S' strains of C. fetus and to determine the importance of S-layers in the virulence of this bacterium, a mouse model was developed that is also relevant for human infection (Pei and Blaser, 1990). Results obtained clearly demonstrated that the S-layer is an important virulence factor after parenteral or oral challenge of mice. Although the S-layer protein is not toxic per se, it enhances virulence considerably when present on the bacterial cell envelope. Wofinella recta, a Gram-negative putative oral periodontopathogen, was shown to be able to alter its S-layer and outer membrane-associated proteins as a function of its growth environment (Borinski and Holt, 1990). The surface characteristics of short-term subcultured strains of W . recta were significantly different from those of long-term subcultured strains.

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

253

Complete loss of the S-layer protein and loss of high molecular-weight proteins in the outer-envelope protein profile in long-term cultures may explain the profound effect of the interaction of W. recta with selected host cells, such as gingival fibroblasts. Kerosuo et al. (1990) investigated the phagocytic ingestion of clinical isolates of the Gram-negative bacteria Bacteroides buccae, B. oris and related strains in vitro by polymorphonuclear leucocytes. They found that, in many of these periodontopathogenic strains, surface hydrophobicity contributed significantly to adherence of the bacteria to leucocytes. Although the role of Slayers in the susceptibility of the bacteria to phagocytosis has not yet been conclusively confirmed there are indications that the layers play an important role in the phagocytic uptake of the bacteria. B. INTERACTION BETWEEN S-LAYERS AND OTHER BACTERIA OR BACTERIOPHAGES

Aquaspirillum serpens was reported to be resistant to predation by the Gram-negative bacterium Bdellovibrio bacteriovorus when the entire bacterium was covered by an S-layer (Buckmire, 1971). The effect of S-layers on predation was recently re-investigated in detail by Koval and Hynes (1991). They demonstrated that bacteria with a complete S-layer were fully protected. On the other hand, those strains with an incomplete S-layer, as might arise from instability of the layer under certain growth conditions, were parasitized when exposed to competent Bdellovibrio strains. S-Layers were also shown to act as specific sites for phage adsorption. Detailed studies have been performed on the S-layer of Bacillus sphaericus P-1. Analysis of this layer on 24 phage-resistant mutants (Howard and Tipper, 1973) showed that the crystalline arrays were present on all mutants although the molecular weight of the S-layer subunits had changed in many mutants. M. L. Callegari and her coworkers purified the S-layer of Lactobacillus helveticus strain CNRZ 892 and demonstrated that the crystalline array acts as a putative phage receptor (M. L. Callegari, L. Skchaud, M. Rousseau, V. Bottazzi and J.-P. Accolas, unpublished observation). C. SHAPE-MAINTAINING FUNCTION OF S-LAYERS

Since the establishment of the archaebacteria as a third kingdom of life (Woese, 198l), it became evident that most of these prokaryotes possess a very simple cell-envelope architecture (Fig. 2(a)) with Slayers as the only, sometimes quite rigid, wall component outside the cytoplasmic membrane

254

P. MESSNER AND U. R . SLEYTR

(e.g. in “square bacteria”, some methanogens, and almost all sulphate reducers, halophilic and extremely thermophilic So metabolizers; see Table 1) (Kandler and Konig, 1985; Konig, 1988). This “archaic” functional principle-like cell-shape determination and maintenance was investigated in detail in Sulfolobus acidocaldarius (Deatherage et al. , 1983), Thermoproteus tenax (Messner et al., 1986a; Wildhaber and Baumeister, 1987) and Methanocorpusculum sinense (Sleytr and Messner, 1989; Pum et al., 1991). The structural organization of the hexagonal S-layer of T. tenax is comparable to the organization found in capsids of eicosahedral viruses (Caspar and Klug, 1962). By ferritin labelling of intact cells of T. tenax (Messner et al., 1986a), it was demonstrated that six five-fold vertices (pentameric units) on each cell pole were incorporated in the S-layer lattice of this rod-shaped archaebacterium (Fig. 7(b); see also Section 1I.B). The highly lobed cell surface of Sulfolobus acidocaldarius (Deatherage et al., 1983) obviously contains not only five-fold vertices but also vertices with seven-fold symmetry. These lattice faults could be visualized by freeze etching of intact cells of M. sinense (Sleytr and Messner, 1989). We have analysed the lattice distortions in greater detail and have proposed a general principle for the generation of lobed cell structures and the cell-division process of those archaebacteria which lack a rigid envelope component (e.g. pseudomurein) (Pum et al., 1991). D. SLAYERS AS MOLECULAR SIEVES

As discussed in Section II.A, the limitations in resolution and structural fidelity, encountered in electron microscopy, do not provide enough detailed information on the mass distribution of S-layer lattices to predict the morphology of the pores to an accuracy required for determining the “functional pore size”. In addition to this, uncertainties regarding pore morphology, considering the molecular-sieve function of Slayers, the distribution and orientation of charged groups as well as the ability of protein domains and of the carbohydrate residues to interact specifically with ions or “permeable” molecules, has to be taken into consideration (S6ra and Sleytr, 1987a, 1988; Sleytr and Messner, 1988a). Information on the size and morphology of pores passing through S-layers is usually obtained from negatively stained or more recently from frozen-hydrated preparations of isolated S-layers. Data obtained by high-resolution electron microscopy indicate that, depending on the symmetry, the lattice constants and the molecular weight of the constituent protomers, S-layers of mesophilic eubacteria possess channels with a diameter of approximately 2-3.5 nm (Stewart et al., 1980; Smit et al., 1981; Burley and Murray, 1983;

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

255

Chalcroft et al., 1986; Dickson et al., 1986;Lepault et al., 1986; Baumeister et al., 1988). Pores of such size would enable free passage for nutrients and metabolic waste products but should prevent entry of molecules with molecular weights exceeding 10,00~15,000.Since most lytic enzymes present in natural habitats of S layer-carrying mesophilic eubacteria have molecular weights of 20,00WO,OOO, the crystalline surface lattices were thought to act as protective coats for whole cells by rejecting hostile macromolecules such as muramidases (Stewart and Beveridge, 1980; Beveridge, 1981; Burley and Murray, 1983; Sleytr and Messner, 1983). Most recently, detailed permeability studies on mesophilic bacilli could not confirm this assumption. It was clearly demonstrated that S-layers are permeable to muramidases and that strains resistant to these lytic enzymes have muramidase-resistant peptidoglycan layers (Sara et al., 1990b). Permeability studies on S-layers of thermophilic bacilli by use of structurally well-characterized solutes have provided information on exclusion limits of S-layers under well-defined environmental conditions (Sleytr and Sara, 1986; Sara and Sleytr, 1987~). For example, the crystalline arrays of strains of Bacillus stearothermophilus revealed sharp exclusion limits between molecular weights of 30,000 and 45,000, suggesting a limiting pore diameter of about 4.5 nm (Sara and Sleytr, 1987b; see also Section V.A). If S-layers act as molecular sieves, they may also function in preventing release of molecules from the cell (Wecke et al., 1974; Sleytr and Messner, 1983) and it was proposed that the protein meshwork could generate a functional equivalent to the periplasmic space of envelopes in Gram-negative eubacteria (Sleytr, 1981; Sleytr and Messner, 1983, 1988a; Baumeister et al., 1989). In conclusion, the limited data available do not yet allow us to draw general conclusions on the molecular-sieve and possible protective functions of S-layers. E. RELEVANCE OF CHARGED GROUPS ON S-LAYERS

With regard to surface recognition and permeability properties of S-layers, important aspects are the anisotropic distribution of charged groups on both surfaces of the crystalline array (Sara and Sleytr, 1987a; Sara et al., 1988; Pum et al., 1989a; Messner et al., 1990) and the evidence for location of charged groups in the pore area (Kupcu et al., 1991; S6ra et al., 1990a). The most detailed studies on the distribution and functional significance of charged groups on the outer and inner faces of S-layers were performed on the crystalline surface layer of Bacillus stearothermophilus NRS 1536f3c (Sara and Sleytr, 1987a). Chemical modifications of the exposed amino and

256

P. MESSNER A N D U. B. SLEYTR

carboxyl groups were performed on S-layers on whole cells, isolated S-layers and S-layer self-assembly products. Subsequent labelling experiments with polycationic ferritin revealed that only the inner face of the S-layer carries a net negative charge. On both surfaces, free amino and carboxyl groups were arranged in close proximity, leading to electrostatic interactions. It can be expected that net negatively charged S-layer lattices have the potential to function like the anionic exopolysaccharide glycocalyces that act as ion-exchange matrices to attract and bind inorganic and organic nutrients or toxic metals close to the cell surface (Beveridge, 1981, 1989). A crystalline array composed of identical subunits would have a topographically and physicochemically better-defined binding capacity than a fibrous matrix. It can also be assumed that a dense geometrically well-defined anisotropic charge distribution would maintain a Donnan equilibrium between the environment of the cell and the “periplasmic space” generated by the S-layer (Sleytr, 1981). This would promote scavenging of macromolecular nutrients from the environment (Sleytr and Messner, 1988a). In this context, it is also of interest that permeability studies on isolated S-layers demonstrated that the pore areas have a low tendency to foul, a property essential for maintaining an exchange of nutrients and metabolites between the cell and its environment (Sleytr and Sara, 1986; Sara and Sleytr, 1987b). Comparative studies on S-layers of numerous strains of bacilli have shown that the outer face of the S-layer lattice revealed free amino and carboxyl groups but did not show a net negative surface charge (Messner et a l . , 1986b; Sara and Sleytr, 1987a; Sara et al., 1988; Pum et al., 1989a). Labelling experiments with polycationic femtin, a positively charged marker molecule, revealed no labelling of the S-layer surface on intact cells. However, the inner S-layer face of in vitro self-assembly products, which on intact cells is in contact with the peptidoglycan layer, did bind the marker molecules. Even on Clostridiumsymbiosum HB25 which, under physiological conditions, has positive and negative charges in the glycan chains of the S-layer glycoprotein (Messner et al., 1990; see Table 3), no polycationic-ferritin labelling was observed. Obviously, charge interaction between free amino groups of amino sugars and phosphate groups, both as constituents of the glycan chain, are leading to a charge-neutral outer S-layer face on all other S-layers of members of the Bacillaceae family studied so far. F. RELEVANCE OF GLYCOSYLATION OF S-LAYER PROTEINS

Structurally diverse glycans were found on glycoproteins of several archaebacteria and eubacteria (Table 3 and Section 1II.A). A comparison of all known glycan-chain structures of S-layer glycoproteins reveals a similar

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

257

heterogeneity as was observd in the protein portions of these molecules (Messner et al. , 1984; Kandler and Konig, 1985; Sleytr et al., 1986). In this context, it is interesting to note that, upon prolonged continuous cultivation under optimal growth conditions, strains of Bacillus stearothermophilus lose the ability to glycosylate the S-layer protein (P. Messner, unpublished results). Thus, the functional significance of the carbohydrate moieties of these proteins has yet to be determined. In contrast to the very limited data on functional aspects of prokaryotic glycoproteins, a considerable amount of knowledge has accumulated on eukaryotic glycoproteins (for a review see Sharon, 1984; Berman and Lasky, 1985; Costerton et al. , 1985; Olden et al., 1985). For example, the glycan moieties can play an important role in (a) protection of the polypeptide against proteolytic degradation, (b) maintenance of protein conformation and stability and (c) surface or intercellular recognition and cell-adhesion phenomena. All of these functions could be relevant for prokaryotic organisms. The obvious diversity of both the constituent protein subunits and glycosylation of S-layers, even among closely related strains, coincides with the generally observed, non-conservative character of bacterial surface molecules, e.g. exopolysaccharides and lipopolysaccharides (Sutherland, 1977, 1985).

V. Application Potential of S-Layers

Due to the increased knowledge of the structure, assembly, chemistry, biosynthesis, pathogenicity and permeability properties of S-layers, a considerable potential for various biotechnological and non-biological applications for two-dimensional crystals have become evident in the last few years. A . S L A Y E R S AS ISOPOROUS ULTRAFILTRATION MEMBRANES

Both analysis of the mass distribution in S-layer lattices by high-resolution electron microscopy (see Section 1I.A) and information on the “functional” pore sizes derived from permeability studies (see Section 1V.D) revealed that the crystalline arrays can be characterized as ultrafiltration membranes with well-defined molecular weight cut-off values (Sleytr and Sara, 1986; Sara and Sleytr, 1987c, 1988). To produce S-layer ultrafiltration membranes, isolated S-layer fragments are deposited as coherent layers on microfiltration membranes which provide mechanical support. For enhancing the mechanical and chemical stability of the membranes, the S-layer protein is cross-linked (e.g. with glutaraldehyde). The most significant difference

258

P. MESSNER AND U. B. SLEYTR

between S-iayer ultrafiltration membranes and ultrafiltration membranes produced from synthetic polymers is the crystalline and isoporous active filtration layer in the former. In contrast, in ultrafiltration membranes produced from polymer solutions by a phase-inversion process, the active filtration layer shows an amorphous structure and a pore-size distribution. Up to now, S-layer ultrafiltration membranes have been prepared from S-layers of different mesophilic and thermophilic bacilli and selected archaebacteria (Sleytr and Sara, 1986; Sara and Sleytr, 1988; Sara et af., 1990a) with pore diameters ranging from 2 to 5 nm. The rejection curves of these membranes show a very steep increase as expected for membranes with a crystalline selective-filtration layer. Chemical and thermal resistances of these membranes were found to be comparable to those of polyamide membranes. Moreover, since S-layers, as two-dimensional crystals, are formed by periodic repetition of identical subunits, it is evident that functional groups on the polypeptide chain and/or the carbohydrate moities, such as carboxyl, amino or hydroxyl groups, must be present on each protomer in an identical position and orientation on the crystalline array. Thus, a broad spectrum of identical physicochemical properties on each molecular unit area can be obtained by application of chemical-modification procedures. The aim of such modification reactions was generation of neutral, negatively or positively charged surfaces as well as the introduction of more hydrophilic or hydrophobic residues. This broad-modification potential allowed, for the first time, tailoring of ultrafiltration membranes to exhibit minimized interactions with solutes and adsorption of molecules, generally leading to a significant decrease in flux in synthetic membranes (Sara and Sleytr, 1988; Kiipcii et af., 1991; Sara et af., 1990a). B. S-LAYERS AS SUPPORT FOR COVALENT ATTACHMENT OF MACROMOLECULES

Since the first description of immobilized enzymes in the early 1970s, biomolecules covalently linked to different matrices have gained importance in a large number of pharmaceutical and biotechnological processes; for reviews, see Mosbach (1987, 1988). The carriers normally used for binding enzymes, antibodies or other ligands (e.g. avidin, protein A) are amorphous polymers with a random structure and pore-size distribution. In such supports, macromolecules are bound on the surface as well as in the interior, frequently leading to high diffusion distances. With S-layer fragments, S-layer vesicles or S-layer ultrafiltration membranes (see above), it was demonstrated by high-resolution electron microscopy and by calculating the amount of bound protein in each S-layer area that molecules can be

259

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

immobilized as a dense monolayer, which frequently resembles the pattern of the crystalline support (Sira and Sleytr, 1989; Sara et al., 1989, 1990a; Kiipcii et al., 1991). The advantage that, depending on the shape and molecular weight, a defined number of molecules can be bound on the crystalline array was utilized for development of new types of affinity membranes, enzyme membranes and biosensors (Pum et al., 1989b, 1992; Sleytr et al., 1989). C. S-LAYERS AS SUPPORT FOR LANGMUIR-BLODGETT

FILMS

Since the most common type of archaebacterial envelope consists exclusively of an S-layer associated with a plasma membrane (Fig. 2(a)), it was recognized that the crystalline arrays may be used as support for rnonoor multilayers of surfactants o r for reconstituted biological membranes generated by the Langmuir-Blodgett technique (Sleytr and SAra, 1986; Pum et al., 1989b; Sleytr et al., 1989). Langmuir-Blodgett layers can have a well-defined structure and may function as hyperfiltration membranes like the lipid layer found as an integral part of biological (plasma) membranes. The combination of S-layers and Langmuir-Blodgett films, in which functional molecules such as carriers, pore-forming proteins, proton pumps, light-harvesting and receptor molecules or other biologically active molecules can be incorporated, opens a broad spectrum of new applications for development of biosensors and more complex molecular nanometer technologies (Pum et al., 1989b, 1992). D. S-LAYERS AS CARRIERS FOR ARTIFICIAL ANTIGENS; VACCINE DEVELOPMENT

As discussed in Section IV.A, S-layers can be demonstrated in a large number of pathogenic micro-organisms (see also Table 1). Identification of partial or complete sequence homologies of the S-layer subunits of different strains of individual pathogenic species could lead to the design of peptidesubunit vaccines (Evenberg et al., 1988; Dubreuil et al., 1990; Kay and Trust, 1990). More recently it has been demonstrated that S-layer fragments or selfassembly products can be used for a geometrically well-defined covalent attachment of haptens, immunogenic or immunostimulating substances. Such hapten-containing S-layer structures were shown to act as strong immunopotentiators (Sleytr et al., 1987). S-Layer glycoproteins have an additional advantage in that both the protein and/or the carbohydrate portion of the molecule can be used for binding of haptens (Messner et al. ,

260

P. MESSNER AND U. B. SLEYTR

1991). An alternative approach will involve inserting foreign proteins (antigenic determinants) into the S-layer sequence. Such S-layer products have the advantage that the immunogenic component is directly inserted into the high immune-stimulating S-layer protein (glycoprotein) (Szostak and Lubitz, 1991).

E. SECRETION OF S-LAYER PROTEINS, A MODEL FOR PRODUCING EXTRACELLULAR PROTEINS

Fundamental studies on the biosynthesis, regulation of synthesis and secretion of S-layer proteins appear most relevant for recombinant DNA technology involving transfer of recombinant DNA products out of bacterial cells. S-Layer overproducers have been isolated and could be of great industrial interest (Udaka, 1976). One appealing approach is to use the promotor of the S-layer protein of such an overproducer. Another possibility is to couple the signal sequences of S-layer proteins to the N-terminal end of proteins cloned in these bacteria and thus bring about an export of the gene product rather than its accumulation inside cells. Tsukagoshi and his collaborators (1984) have shown the feasibility of this approach with a strain of Bacillus brevis which secretes more than 10 g of Slayer protein per litre.

VI. Concluding Remarks

Up to now around 300 different species of prokaryotic organisms from most phylogenetic branches have been shown to possess a crystalline array of proteins or glycoproteins as the outermost cell-envelope layer (Table 1). It is likely that in many strains S-layers have not yet been detected because they are either masked by unstructured amorphous polymer material (e.g. capsules or slime) or simply damaged by conventional electronmicroscopical techniques. During the last two decades a considerable body of knowledge about the structure, chemistry and morphogenesis of S-layers has accumulated. S-Layers have been shown to be the simplest biological protein membranes developed during evolution (Sleytr, 1978). The information encoded in a single S-layer protein species guarantees maintenance of a closed, highly ordered porous protein meshwork on a growing cell surface. This minimum of genetic information makes it interesting to speculate that S-layer-like membranes could have fulfilled barrier and supporting

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

261

functions as required by self-reproducing systems (progenots) during the early periods of biological evolution (Sleytr and Plohberger, 1980; Sleytr, 1981). Nevertheless, relatively little is known about the functional significance of S-layers in different species or even strains. The observation that eubacteria and archaebacteria carrying crystalline arrays are ubiquitous in the biosphere, thus including organisms inhabiting the most extreme and diverse environments, leads to the conclusion that S-layers must have the potential to fulfill a broad spectrum of functions. It is most challenging to elucidate the specific adaptions and functional diversifications of S-layers within the archaebacterial and eubacterial phylogenetic lines. Depending on the type of prokaryotic envelope, S-layers can be associated with quite diverse supramolecular structures and may only function as a result of specific interactions with these structures. Future studies will primarily have to focus on the specific natural habitats of S layer-carrying organisms and the reasons why S-layers are frequently ‘‘lost’’ upon prolonged cultivation under optimal growth conditions. For such an ecological approach, it will be essential to mimic particular environments and potential hazards for the cells under strictly controlled laboratory conditions. The observation that structurally diverse S-layers can even be found among strains of the same species requires more detailed studies at the molecular level. It may turn out that physicochemical features of S-layer subunits responsible for functional similarities can be fulfilled by quite different protomers, o r that structurally diverse S-layers may have common conservative protein domains. With our advanced knowledge of the aminoacid sequences of S-layers, it will be possible to answer this question and to elucidate the taxonomical relationship of crystalline arrays. It has also to be stressed that still very little is known about the functional significance of the glycan chains of S-layers detected now in an increasing number of both eubacteria and archaebacteria (Messner et al., 1991a,b). Although the amplifying effect of microbial adsorption to various solids on metabolic activity has been reported for organisms from various ecological niches, most studies with strains possessing S-layers have been performed with cell suspensions. So far, general conclusions cannot be drawn, but adhesive properties due to the presence of an S-layer may enhance the chances of bacterial strains surviving under oligotrophic conditions due to accumulation of nutrients at solid-liquid interfaces (Gruber and Sleytr, 1991). Finally, most recently Slayers have shown a broad potential for biological and non-biological applications (Pum et al., 1989b; Sleytr et al., 1989). It is to be hoped that these studies will attract new investigators to the field.

262

P. MESSNER AND U. 6. SLEYTR

VII. Acknowledgements We are grateful to all the colleagues who have shared their ideas and unpublished work. We thank Eva Mikula and Elisabeth Finotti for their help with the manuscript. The work was supported in part by the “Osterreichischer Fonds zur Forderung der wissenschaftlichen Forschung” , projects S 50/02 and P 7757, and the “Osterreichisches Bundesministerium fur Wissenschaft und Forschung”.

REFERENCES

Abe, M. and Kimoto, N. (1984). Microbiology and Immunology 28, 841. Abe, M., Kimoto, M. and Yoshii, Z. (1983). FEMS Microbiology Letters 18, 263. Adachi, T., Yamagata, H., Tsukagoshi, N. and Udaka, S. (1989).Journal ofBacteriology 171, 1010. Adachi, T., Yamagata, H . , Tsukagoshi, N. and Udaka, S. (1990).Journal of Bacteriology 172, 511. Aebi, U., Smith, P. R., Dubochet, J., Henry, C. and Kellenberger, E. (1973). Journal of Supramolecular Structure 1, 498. Aldrich, H. C., Robinson, R. W. and Williams, D. S. (1986). Systematic and Applied Microbiology 7, 314. Al-Karadaghi, S., Wang, D. N. and Hovmoller, S. (1988). Journal of Ultrastructure and Molecular Structure Research 101, 92. Altman, E., Brisson, J.-R., Messner, P. and Sleytr, U. B. (1990). European Journal of Biochemistry 188, 73. Altman, E., Brisson, J.-R., Messner, P. and Sleytr, U. B. (1991). Biochemistry and Cell Biology 69, 72. Antranikian, G. , Herzberg, C., Mayer, F. and Gottschalk, G. (1987). FEMS Microbiology Letters 41, 193. Austin, J. W. and Murray, R. G. E. (1987). Canadian Journal of Microbiology 33, 1039. Austin, J. W. and Murray, R. G . E. (1990). Journal of Bacteriology 172, 3681. Austin, J. W., Engel, A., Murray, R. G . E. and Aebi, U. (1989). Journalof Ulfrastructureand Molecular Structure Research 102, 255. Austin, J. W., Stewart, M. and Murray, R. G . E. (1990). Journal of Bacteriology 172, 808. Barker, D. C. and Thorne, K. J. I. (1970). Journal of Cell Science 7 , 755. Baumeister, W. and Engelhardt, H. (1987). In “Electron Microscopy of Proteins, Membranous Structure” (J. R. Harris and R. W. Horne, eds), vol. 6, pp. 109-154. Academic Press, London. Baumeister, W. and Kiibler, 0. (1978). Proceedings of the National Academy of Sciences of the United States of America 75, 5525. Baumeister, W., Karrenberg, F., Rachel, R., Engel, A., ten Heggeler, B. and Saxton, W. 0. (1982). European Journal of Biochemistry 125, 535. Baumeister, W., Barth, M., Hegerl, R., Guckenberger, R., Hahn, M. and Saxton, W. 0. (1986). Journal of Molecular Biology 187, 241. Baumeister, W., Wildhaber, I. and Engelhardt, H. (1988). Biophysical Chemistry 29, 39. Baumeister, W., Wildhaber, I. and Phipps, B. M. (1989). Canadian Journal of Microbiology 35, 215. Baumeister, W., Santarius, U., Volker, S., Diirr, R., Lembcke, G . andEngelhardt, H. (1990). Systematic and Applied Microbiology 13, 105.

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

263

Belland, R. J. and Trust, T. J. (1985). Journal of Bacteriology 163, 877. Belland, R. J. and Trust, T. J. (1987). Journal of Bacteriology 169, 4086. Bergere, J.-L., Hayes, H. and Commissaire, J. (1986). Annales de l’lnstitut Pasfeurl Microbiologie 137B, 271. Berman, P. W. and Lasky, L. A. (1985). Trends in Biotechnology 3, 51. Betz, J. V. (1970). Journal of Bacteriology 103, 814. Beveridge, T. J. (1979). Journal of Bacteriology 139, 1039. Beveridge, T. J. (1981). International Review of Cytology 72, 229. Beveridge, T. J. (1989). In “Bacteria in Nature, Structure, Physiology, and Genetic Adaptability” (J. S. Poindexter and E. R. Leadbetter, eds), vol. 3, pp. 1-65. Plenum Press, New York. Beveridge, T. J. and Murray, R. G. E. (1974). Journal of Bacteriology 119, 1019. Beveridge, T. J. and Murray, R. G. E. (1975). Journal of Bacteriology 124, 1529. Beveridge, T. J. and Murray, R. G. E . (1976a). Journal of Ultrastructure Research 55, 105. Beveridge, T. J. and Murray, R. G. E. (1976b). Canadian Journal of Microbiology 22, 567. Beveridge, T. J. and Murray, R. G. E. (1976~).Canadian Journal of Microbiology 22, 1233. Beveridge, T. J., Stewart, M., Doyle, R. J. and Sprott, G. D. (1985). Journalof Bacteriology 162, 728. Beveridge, T. J., Harris, B. J., Patel, G. B. and Sprott, G. D. (1986a). Canadian Journal of Microbiology 32, 779. Beveridge, T. J., Patel, G. B., Harris, B. J. and Sprott, G. D. (1986b). Canadian Journal of Microbiology 32, 703. Beveridge, T. J., Sara, M., Pum, D., Sprott, G. D., Stewart, M. and Sleytr, U. B. (1988). In “Crystalline Bacterial Cell Surface Layers” (U. B. Sleytr, P. Messner, D. Pum and M. Sara, eds), pp. 26-30. Springer-Verlag, Berlin. Bingle, W. H., Doran, J. L. and Page, W. J. (1984). Journal of Bacteriology 159, 251. Bingle, W. H., Doran, J. L. and Page W. J. (1986). CanadianJournalofMicrobiology32,112. Bingle, W. H., Engelhardt, H., Page, W. J. and Baumeister, W. (1987a). Journal of Bacteriology 169, 5008. Bingle, W. H. and Smit, J. (1990). Plasmid 24, 143. Bingle, W. H., Whippey, P. W., Doran, J. L., Murray, R. G. E. and Page, W. J. (1987b). Journal of Bacteriology 169, 802. Blakemore, R. P. and Canale-Parola, E. (1973). Archiv fur Mikrobiologie 89, 273. Blaser, M. J. and Gotschlich, E. C. (1990). Journal of Biological Chemistry 265, 14529. Blaser, M. J., Smith, P. F., Hopkins, J. A,, Heinzer, I., Bryner, J. H. and Wang, W.-L. L. (1987). Journal of Infectious Diseases 155, 696. Blaser, M. J., Smith, P. F., Repine, J. E. and Joiner, K. A. (1988). Journal of Clinical Investigation 81, 1434. Blom, J. and Heltberg, 0. (1986). Acta Pathologica, Microbiologica et Immunologica Scandinavica, Section B 94, 301. Bonch-Osmolovskaya, E. A,, Miroshnichenko, M. L., Kostrikina, N. A., Chernych, N. A. and Zavarzin, G. A. (1990). Archives of Microbiology 154, 556. Borinski, R. and Holt, S. C. (1990). Infection and Immunity 58, 2770. Bowditch, R. D., Baumann, P. and Yousten, A. A. (1989). Journal of Bacteriology 171,4178. Braun, M., Mayer, F. and Gottschalk, G. (1981). Archives ofMicrobiology 128, 288. Breznak, J. A. and Canale-Parola, E. (1969). Journal of Bacteriology 97, 386. Brockl, G., Behr, M., Fabry, S., Hensel, R., Kaudewitz, H., Biendl, E. and Konig, H. (1991). European Journal of Biochemistry 199, 147. Buckmire, F. L. A. (1971). Bacteriological Proceedings, p. 43. Buckmire, F. L. A. and Murray, R. G. E. (1970). Canadian Journal of Microbiology 16,1011. Buckmire, F. L. A. and Murray, R. G. E. (1973). Canadian Journal ofMicrobiology 19,59. Buckmire, F. L. A. and Murray, R. G. E. (1976). Journal of Bacteriology 125, 290. Budel, B. and Rhiel, E. (1985). Archives of Microbiology 143, 117. Burchard, R. P. (1981). Annual Review of Microbiology 35, 497. Burggraf, S., Fricke, H., Neuner, A., Kristjansson, J., Rouvier, P., Mandelco, L., Woese, C. R. and Stetter, K. 0. (1990a). Systematic and Applied Microbiology 13, 263.

264

P. MESSNER AND U. B. SLEYTR

Burggraf, S., Jannasch, H. W., Nicolaus, B. and Stetter, K. 0.(1990b). Systematicand Applied Microbiology 13, 24. Burley, S. K. and Murray, R. G. E. (1983). Canadian Journal of Microbiology 29, 775. Caldwell, D. E . and Tiedje, J. M. (1975). Canadian Journal of Microbiology 21, 362. Carl, M. and Dasch, G. A. (1989). Journal of Autoimmunity 2 (Supplement), 81. Carl, M., Dobson, M. E., Ching, W. M. and Dasch, G. A. (1990). Proceedings of the National Academy of Sciences of the United States of America 87, 8237. Caspar, D. L. D. and Klug, A. (1962). ColdSpring Harbor Symposiumon Quantitative Biology 27, 1.

Caston, J. R., Carrascosa, J . L., de Pedro, M. A. and Berenguer, J. (1988). FEMS Microbiology Letters 51, 225. Cejka, Z. and Baumeister, W. (1987). FEMS Microbiology Letters 44, 13. Cejka, Z., Hegerl, R. and Baumeister, W. (1986). Journal of Ultrastructure and Molecular Structure Research 96, 1 . Chalcroft, J. P., Engelhardt, H. and Baumeister, W. (1986). Archives of Microbiology 144, 196. Chang, J.-J., Leonard, K., Arad, T., Pitt, T., Zhang, Y.-X. and Zhang, L.-H. (1982). Journal of Molecular Biology 161, 579. Chapman, J. A., Murray, R. G. E . and Salton, M. R. J. (1963). Proceedings of the Royal Society of London Series B, Biological Sciences 158, 498. Chart, H., Shaw, D. H., Ishiguro, E . E. andTrust, T. J. (1984). Journalof Bacteriology 158,16. Christian, R., Schulz, G., Unger, F. M., Messner, P., Kiipcii, Z. and Sleytr, U. B. (1986). Carbohydrate Research 150, 265. Christian, R., Messner, P., Weiner, C., Sleytr, U. B. and Schulz, G. (1988). Carbohydrate Research 176, 160. Chu, S., Cavaignac, S., Feutrier, J., Phipps, B. M., Kostrzynska, M., Kay, W. W. and Trust, T. J. (1991). Journal of Biological Chemistry 266, 15258. Cohen, S., Oren, A. and Shilo, M. (1983a). Archives of Microbiology 136, 184. Cohen, S., Shilo, M. and Kessel, M. (1983b). Ultramicroscopy 11, 330. Cohen-Bazire, G., Kunisawa, R. and Pfennig, N. (1969). Journal of Bacteriology 100, 1049. Costerton, J. W., Marrie, T. J. and Cheng, K. J. (1985). In “Bacterial Adhesion. Mechanism and Physiological Significance” (D. C. Savage and M. Fletcher, eds), pp. 3-43. Plenum Press, New York. Crowther, R. A. and Sleytr, U. B. (1977). Journal of Ultrastructure Research 58, 41. D’Aoust, J.-Y. and Kushner, D. J. (1972). Canadian Journal of Microbiology 18, 1767. Dasch, G. A. and Bourgeois, A. L. (1981). In “Rickettsiae and Rickettsia1 Diseases” (W. Burgdorfer and R . L. Anacker, eds), pp. 71-80. Academic Press, New York. Deatherage, J. F., Taylor, K. A. and Amos, L. A. (1983). Journal of Molecular Biology 167, 823. De Boer, W. E. and Spit, B. J. (1964). Antonie van Leeuwenhoek 30,239. Dickson, M. R., Downing, K. H., Wu, W. H. and Glaeser, R. M. (1986). Journal of Bacteriology 167, 1025. Dokland, T., Johansen, B. V., Farrants, G. W. and Olsen, I. (1988). Journal of Ultrastructure and Molecular Structure Research 100, 284. Dooley, J. S. G. and Trust, T. J. (1988). Journal of Bacteriology 170, 499. Dooley, J. S. G., McCubbin, W. D., Kay, C. M. and Trust, T. J. (1988). Journal of Bacteriology 170, 2631. Dooley, J . S. G., Engelhardt, H., Baumeister, W., Kay, W. W. and Trust, T. J. (1989). Journal of Bacteriology 171, 190. Doyle, R. J., Beveridge, T. J., Stewart, M. and Ezzell, E. (1986). Abstract, 86th Annual Meeting of the American Society for Microbiology, Washington D C , No. 518, 191. Dubourguier, H.-C., Samain, E., Prensier, G. and Albagnac, G. (1986). Archives of Microbiology 145, 248. Dubreuil, J. D., Logan, S. M., Cubbage, S., Ni Eidhin, D., McCubbin, W. D., Kay, C. M., Beveridge, T. J., Ferris, F. G. and Trust, T. J. (1988). Journal of Bacteriology 170, 4165.

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

265

Dubreuil, J. D., Kostrzynska, M., Austin, J. W. and Trust, T. J . (1990). Journal of Bacteriology 172,5035. Dupoy, G. (1973). Journal of Microscopy 97,3. Ebisu, S.,Tsuboi, A,, Takagi, H., Naruse, Y., Yamagata, H., Tsukagoshi, N. and Udaka, S. (1990). Journal of Bacteriology 172, 1312. Ellar, D. J. and Lundgren, D. G. (1967). Journal of Bacteriology 94, 1778. Engel, A., Massalski, A , , Schindler, H., Dorset, D . L. and Rosenbusch, J. P. (1985). Nature 317,643. Engelhardt, H.(1988). In “Methods in Microbiology” (F. Mayer, ed.), vol. 20, pp. 357413. Academic Press, London. Engelhardt, H., Saxton, W. 0. and Baumeister, W. (1986). Journalof Bacteriology 168,309. Evenberg, D. and Lugtenberg, B. (1982). Biochimica et Biophysica Acta 684, 249. Evenberg, D., van Boxtel, R., Lugtenberg, B., Schurer, F., Blommaert, J. and Bootsma, R. (1982). Biochimica et Biophysica Acta 684,241. Evenberg, D., Versluis, R. and Lugtenberg, B. (1985). BiochimicaetBiophysicaActa815,233. Evenberg, D., De Graaff, P., Lugtenberg, B. and Van Muiswinkel, W. B. (1988). Journalof Fish Diseases 11, 337. Evers, D., Weckesser, J. and Drews, G. (1984). Journal of Bacteriology 160, 107. Every, D. and Skerman, T. M. (1980). Journal of Bacteriology 141, 845. Faraldo, M. L. M., de Pedro, M. A. and Berenguer, J. (1988). FEBS Letters 235, 117. Fassel, T. A,, Schaller, M. J., Lidstrom, M. E. and Remsen, C. C. (1990). Journalof Electron Microscopy Technique 14,52. Fiala, G. and Stetter, K. 0. (1986). Archives of Microbiology 145, 56. Fiala, G., Stetter, K. O., Jannasch, H. W., Langworthy, T. A. and Madon, J. (1986). Systematic and Applied Microbiology 8, 106. Finch, J. T., Klug, A. and Nermut, M. V. (1967). Journal of Cell Science 2, 587. Fischer, F.,Zillig, W., Stetter, K. 0. and Schreiber, G. (1983). Nature 301,511. Fisher, J. A., Smit, J. and Agabian, N. (1988). Journal of Bacteriology 170,4706. Fogg, G. C., Yang, L., Wang, E. and Blaser, M. J. (1990). Infection and Immunity 58, 2738. Fujimoto, S., Umeda, A,, Takade, A,, Murata, K. and Amako, K. (1989). Infection and Immunity 57, 2563. Gerbl-Rieger, S., Schneider, S., Engelhardt, H., Peters, J. and Baumeister, W. (1988). In “Crystalline Bacterial Cell Surface Layers” (U. B. Sleytr, P. Messner, D . Pum and M. Sira, eds), pp. 65-69. Springer-Verlag, Berlin. Gerhardt, P. (1967). Federation Proceedings 26, 1504. Gerwig, G. J., deWaard, P., Kamerling, J. P., Vliegenthart, J. F. G., Morgenstern, E., Lamed, R. and Bayer, E. A. (1989). Journal of Biological Chemistry 264, 1027. Ghiorse, W. C. and Hirsch, P. (1979). Archives of Microbiology 123,213. Giesbrecht, P. (1969). In “Bildatlas pathogener Mikroorganismen” (G. Henneberg, ed.), vol. 111, pp. 1-141. Fischer, Stuttgart. Gilchrist, A. and Smit, J. (1991). Journal of Bacteriology 173,921. Glaeser, R. M., Chiu, W., Grano, D. and Taylor, K. (1980). In “Electron Microscopy at Molecular Dimensions” (W. Baumeister and W. Vogell, eds), pp. 22-26. Springer-Verlag, Berlin. Glauert, A. M. (1962). Proceedings of the 5th International Congress of Electron Microscopy, Philadelphia 2, RR-I. Glauert, A. M. and Thornley, M. J. (1971). Journal of Cell Science 8, 19. Goderdzishvili, M. G., Gostevji,V. V., Klitsunova,N. V.,Timakova, N. V.,Titova, I. V. and Ilyashenko, B. N. (1989). Zurnal Mikrobiologii, Epidemiologii i Imrnunbiologii 2 , 8. Goundry, J., Davison, A. L., Archibald, A. R. and Baddiley, J. (1967). Biochemical Journal 104, Ic. Griffiths, S. G. and Lynch, W. H. (1990). Archives of Microbiology 154,308. Gromov, B. V., Gavrilova, 0. V. and Konovalov, E. S. (1986). Mikrobiologija 55,821. Gruber, K. and Sleytr, U. B. (1988). Archives of Microbiology 149,485. Gruber, K. and Sleytr, U. B. (1991). Archives of Microbiology 156, 181.

266

P. MESSNER AND U. B. SLEYTR

Gruber, K., Tanahashi, H., Tsuboi, A,, Tsukagoshi, N. and Udaka, S. (1988). FEMS Microbiology Letters 56, 113. Haapasalo, M., Lounatmaa, K., Ranta, H., Shah, H. and Ranta, K. (1985). International Journal of Systematic Bacteriology 35, 65. Haapasalo, M., Lounatmaa, K. and Ranta, H. (1988). In “Crystalline Bacterial Cell Surface Layers” (U. B. Sleytr, P. Messner, D. Pum and M. SBra, eds), pp. 92-95. Springer-Verlag, Berlin. Hageage, G. J. and Gherna, R. L. (1970). Bacteriological Proceedings 57. Hageage, G. J. and Gherna, R. L. (1971). Journal of Bacteriology 106, 687. Haikara, A,, Lantto, R., Makinen, V. and Lounatmaa, K. (1985). Proceedings of the 20th Congress of the European Brewery Convention, Helsinki, 443. Hancock, R. E. W. (1987). In “Bacterial Outer Membranes as Model Systems” (M. Inouye, ed.), pp. 187-225. Wiley, New York. Harris, W. F. (1977). Scientific American 237, 130. Harris, W. F. (1978). South African Journal of Science 74, 332. Hartmann, E. and Konig, H. (1989). Archives of Microbiology 151, 274. Hastie, A. T. and Brinton, C. C. Jr, (1979). Journal of Bacteriology 138, 999. Haubold, R. (1978). Zeitschrift fur Allgemeine Mikrobiologie 18, 51 1. Hayes, H. J., Commissaire, J. and Bergere, J.-L. (1984). FEBS Letters 174, 284. Heinen, U. J. and Heinen, W. (1972). Archiv fur Mikrobiologie 82, 1. Hespell, R. B. and Canale-Parola, E. (1970). Archiv fur Mikrobiologie 74, 1. Higashi, Y., Strominger, J. L. and Sweeley, C. C. (1967). Proceedings of the National Academy of Sciences of the United States of America 57, 1878. Hollaus, F. and Sleytr, U. (1972). Archiv fur Mikrobiologie 86, 129. Holt, J. G . (ed.) (1984, 1986, 1989a,b). “Bergey’s Manual of Systematic Bacteriology”, vols 1 4 . Williams and Wilkins, Baltimore. Holt, S. C. and Beveridge, T . J. (1982). Canadian Journal of Microbiology 28, 1. Holt, S. C. and Canale-Parola, E. (1968). Journal of Bacteriology 96, 822. Holt, S. C. and Leadbetter, E. R. (1969). Bacteriological Reviews 33, 346. Houwink, A. L. (1953). Biochimica et Biophysica Acta 10, 360. Houwink, A. L. (1956). Journal of General Microbiology 15, 146. Houwink, A. L. (1963a). In “Structure and Ultrastructure of Microorganisms” (E. M. Brieger, ed.), p. 282. Academic Press, New York. Houwink, A. L. (1963b). In “Structure and Ultrastructure of Microorganisms” (E. M. Brieger, ed.), p. 294. Academic Press, New York. Hovind-Hougen, K. (1974). Acta Pathologica et Microbiologica Scandinavica, Section B 82,495. Hovind-Hougen, K. (1975). Acta Pathologica et Microbiologica Scandinavica, Section B 83,91. Hovind-Hougen, K. (1976). Acta Pathologica et Microbiologica Scandinavica, Section B, Supplement 255, 1 . Hovmoller, S. (1986). In “Techniques for the Analysis of Membrane Proteins” (C. I. Ragan and R. J. Cherry, eds), pp. 315-344. Chapman and Hall, London. Hovmoller, S., Sjogren, A. and Wang, D. N. (1988). Progress in Biophysics and Molecular Biology 51, 131. Howard, L. and Tipper, D. J. (1973). Journal of Bacteriology 113, 1491. Howard, L. V., Dalton, D. D. and McCoubrey, W. K. Jr, (1982). Journal of Bacteriology 149, 748. Howatson, A. F. and Russel, W. C. (1964). Journal of Applied Physics 35, 3086. Huber, H., Thomm, M., Konig, H., Thies, G. and Stetter, K. 0. (1982). Archives of Microbiology 132, 47. Huber, R., Langworthy, T. A,, Konig, H., Thomm, M., Woese, C. R., Sleytr, U. B. and Stetter, K. 0. (1986). Archives of Microbiology 144, 324. Imhoff, J. F. and Triiper, H. G. (1977). Archives of Microbiology 114, 115. Inatomi, K., Ohba, M. and Oshima, T. (1983). Chemistry Letters, 1191. Jackson, S . and Black, S. H. (1971). Archiv fur Mikrobiologie 76, 308. Jaenicke, R., Welsch, R., Sara, M. and Sleytr, U. B. (1985). Biological Chemistry HoppeSeyler 366, 663.

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

267

Janda, J. M., Oshiro, L. S., Abbott, S. L. and Duffey, P. S. (1987). InfectionandImmunity55, 3070. Jeffries, P. and Wilkinson, J . F. (1978). Archives of Microbiology 119, 227. Jensen, T . E. and Sicko, L. M. (1972). Cytobiologie 6, 439. Johnson, M. C., Ray, B. and Bhowmik, T. (1987). Antonie van Leeuwenhoek 53, 215. Jones, J. B., Bowers, B. and Stadtman, T. C. (1977). Journal of Bacteriology 130, 1357. Jones, W. J., Leigh, J. A , , Mayer, F., Woese, C. R. and Wolfe, R. S. (1983). Archives of Microbiology 136, 254. Kandler, 0. (1982). Zentralblatt fur Bakteriologie, Mikrobiologie und Hygiene, I . Abfeilung, Originale Reihe C 3, 149. Kandler, 0. and Konig, H . (1985). In “The Bacteria, Archaebacteria” (C. R. Woese and R. S. Wolfe, eds), vol. VIII, pp. 413-457. Academic Press, New York. Kari, M., Ronkko, R., Valtonen, A , , Piiparinen, H., Kukkonen, M., Poyry,T., Nohynek, L., Nurmiaho-Lassila, E.-L. and Lounatmaa, K. (1990). Journal of Basic Microbiology 30,99. Karlsson, B., Vaara, T., Lounatmaa, K. and Gyllenberg, H. (1983). Journal of Bacteriology 156, 1338. Kawata, T. (1981). Electron Microscopy 15, 115. Kawata, T. and Masuda, K. (1972). Japanese Journal of Microbiology 16, 515. Kawata, T., Masuda, K., Yoshino, K. and Fujimoto, M. (1974). Japanese Journal of Microbiology 18, 469. Kawata,T.,Takeoka, A.,Takumi, K. and Masuda, K. (1984). FEMS Microbiology Letters24, 323. Kay, W. W. and Trust, T. J. (1991). Experientia 47, 412. Kay, W. W., Buckley, J. T., Ishiguro, E. E., Phipps, B. M., Monette, J. P. L. and Trust, T. J. (1981). Journal of Bacteriology 147, 1077. Kay, W. W., Phipps, B. M., Ishiguro, E. E., Olafson, R. W. and Trust, T. J. (1984). Canadian Journal of Biochemistry and Cell Biology 62, 1064. Kellenberger, E. and Kistler, J. (1979). In “Unconventional Electron Microscopy for Molecular Structure Determination” (W. Hoppc and R. Mason, eds), pp. 49-79. Vieweg, Braunschweig. Kerosuo, E. (1988). Oral Microbiology and Immunology 3, 134. Kerosuo, E., Haapasalo, M., Ranta, H. and Lounatmaa, K. (1987). Journal of General Microbiology 133, 2217. Kerosuo, E., Haapasalo, M., Lounatmaa, K. and Ranta, H. (1988a). In “Crystalline Bacterial Cell Surface Layers” (U. B. Sleytr, P. Messner, D. Pum and M. Sara, eds), pp. 122-126. Springer-Verlag, Berlin. Kerosuo, E., Haapasalo, M., Lounatmaa, K., Ranta, H. and Ranta, K. (1988b). Scandinavian Journal of Dental Research 96, 50. Kerosuo, E., Haapasalo, M., Alli, K. and Lounatmaa, K. (1990). Oral Microbiology and Immunology 5, 202. Kessel, M. (1978). Journal of Ultrastructure Research 62, 203. Kessel, M. and Cohen, Y. (1982). Journal of Bacteriology 150, 851. Kessel, M., Radermacher, M. and Frank, J. (1985). Journal of Microscopy 139, 63. Kessel, M., Brennan, M. J., Trus, B. L., Bisher, M. E. and Steven, A. C. (1988a). Journal of Molecular Biology 203, 275. Kessel, M., Buhle, E . L. Jr, Cohen, S. and Aebi, U. (1988b). Journal of Ultrastructure and Molecular Structure Research 100, 94. Kessel, M., Cohen, S., Buhle, E. L. Jr, and Aebi, U. (1986). Proceedings of the 11th International Congress of Electron Microscopy, Kyoto 3 , 2471. Kessel, M., Volker, S., Santarius, U., Huber, R. and Baumeister, W. (1990). Systematicand Applied Microbiology 13, 207. Kirk, R. G . and Ginzburg, M. (1972). Journal of Ultrastructure Research 41, 80. Kist, M. L. and Murray, R . G . E. (1984). Journal of Bacteriology 157, 599. Kobata, A. (1984). In “Biology of Carbohydrates” (V. Ginsburg and P. W. Robbins, eds), vol, 2, pp. 87-161. Wiley, New York. Kokka, R. P. and Janda, J. M. (1990). Journal of Clinical Microbiology 28, 1297.

268

P . MESSNER AND U. B . SLEYTR

Kokka, R. P., Vedros, N. A. and Janda, J. M. (1990). Journal of Clinical Microbiology 28, 2240. Konig, H. (1988). Canadian Journal of Microbiology 34, 395. Konig, H. and Stetter, K. 0. (1986). Systematic and Applied Microbiology 7, 300. Konig, H. and Stetter, K. 0. (1982). Zentralblatt fur Bakteriologie, Mikrobiologie und Hygiene, I. Abteilung, Originale Reihe C 3, 478. Konig, H., Buckel, W. and Langworthy, T. A. (1985). FEMS Microbiology Letters 30,283. Konig, H., Hartmann, E . , Brockl, G., Biendl, E. and Behr, M. (1989a). Proceedings ofthe Xth International Symposium on Glycoconjugates, Jerusalem, 177. Konig, H., Kandler, 0. and Hammes, W. (1989b). Canadian Journalof Microbiology 35,176. Kornfeld, R. and Kornfeld, S. (1980). In “The Biochemistry of Glycoproteins and Proteoglycans” (W. J. Lennarz, ed.), pp. 1-34. Plenum Press, New York. Kornfeld, R. and Kornfeld, S. (1985). Annual Review of Biochemistry 54, 631. Koval, S. F. (1988). Canadian Journal of Microbiology 34, 407. Koval, S. F. and Hynes, S. H. (1991). Journal of Bacteriology 173, 2244. Koval, S. F. and Jarrell, K. F. (1987). Journal of Bacteriology 169, 1298. Koval, S. F. and Murray, R. G . E . (1981). Journal of Bacteriology 146, 1083. Koval, S. F. and Murray, R. G. E. (1984). Canadian Journal of Biochemistry and Cell Biology 62, 1181. Koval, S. F. and Murray, R. G. E. (1985). Canadian Journal of Microbiology 31, 261. Kiibler, O., Engel, A , , Zingsheim, H . P., Emde, B., Hahn, M., Heisse, W. and Baumeister, W. (1980). In “Electron Microscopy at Molecular Dimensions”, (W. Baumeister and W. Vogell, eds), pp. 11-21. Springer, Berlin. Kuen, B., Sleytr, U. B. and Lubitz, W. (1988). Biological Chemistry Hoppe-Seyler 369, 860. Kuhn, P. J. and Holt, S. C. (1972). Biochimica et Biophysica Acta 261, 267. Kupcu, S., Sara, M. and Sleytr, U. B. (1991). Journal of Membrane Science 61, 167. Kupcu, Z., Marz, L., Messner, P. and Sleytr, U. B. (1984). FEES Leners 173, 185. Kushner, D. J., Bayley, S. T., Boring, J., Kates, M. and Gibbons, N. E. (1964). Canadian Journal of Microbiology 10, 483. Lai, C.-H., Listgarten, M. A , , Tanner, A. C. R. and Socransky, S. S. (1981). International Journal of Systematic Bacteriology 31, 465. Lancy, P. Jr, and Murray, R. G . E . (1977). Canadian Journal of Microbiology 24, 162. Lapchine, L. (1976). Journal de Microscopie et de Biologie Cellulaire 25, 67. Lapchine, L. (1979). FEMS Microbiology Letters 5, 223. Lauerer, G., Kristjansson, J. K., Langworthy, T . A , , Konig, H. and Stetter, K. 0. (1986). Systematic and Applied Microbiology 8 , 100. Leadbetter, E. R. and Holt, S. C. (1968). Journal of General Microbiology 52, 299. Lechner, J. and Sumper, M. (1987). Journal of Biological Chemistry 262, 9724. Lechner, J. and Wieland, F. (1989). Annual Review of Biochemistry 58, 173. Lechner, J., Wieland, F. and Sumper, M. (1985a). Journal of Biological Chemistry 260,860. Lechner, J . , Wieland, F. and Sumper, M. (1985b). Journal of Biological Chemistry 260,8984. Leduc, M., Rousseau, M. and van Heijenoort, J. (1977). European JournalofBiochemistry80, 153. Leigh, J . A, , Mayer, F. and Wolfe, R . S. (1981). Archives of Microbiology 129, 275. Lepault, J . and Pitt, T . (1984). E M B O Journal 3, 101. Lepault, J., Martin, N. and Leonard, K. (1986). Journal of Bacteriology 168, 303. LeRuyet, P., Dubourguier, H.-C., Albagnac, G . and Premier, G. (1985). Systematic and Applied Microbiology 6, 196. Lewis, L. O., Yousten, A. A. and Murray, R. G . E. (1987). Journal of Bacteriology 169, 72. Listgarten, M. A. and Socransky, S. S. (1964). Journal of Bacteriology 88, 1087. Liu, Y., Boone, D . R. and Choy, C. (1990). International Journal of Systematic Bacteriology 40,111. Lounatmaa, K. and Brander, E. (1989). Journal of Bacteriology 171, 5756.

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

269

Lounatmaa, K., Vaara, T., Osterlund, K. and Vaara, M. (1980). Canadian Journal of Microbiology 26, 204. Lounatmaa, K., Jousimies-Somer, H., GrCnman, R. and Rintala, A. (1988). In “Crystalline Bacterial Cell Surface Layers” (U. B. Sleytr, P. Messner, D. Pum and M. Sara, eds), pp. 114-117. Springer-Verlag, Berlin. Luckevich, M. D. and Reveridge, T. J. (1989). Journal of Bacteriology 171, 6656. MacRae, J. D. and Smit, J . (1991). Applied and Environmental Microbiology 57, 751. McCoy, E. C., Doyle, D., Burda, K., Corbeil, L. B. and Winter, A. J. (1975). Infection and Immunity 11, 517. Maeba, P. Y . (1986). Journal of Bacteriology 166, 644. Manire, G. P. and Wyrick, P. B. (1979). In “Microbiology-1979” (D. Schlesinger, ed.), pp. 111-1 15. American Society for Microbiology, Washington, DC. Margaret, 8. S. and Krywolap, G. N . (1986). International Journal of Systematic Bacteriology 36, 145. Martikainen, P. J., Nurmiaho-Lassila, E.-L. and Lounatmaa, K. (1989). FEMS Microbiology Letters 59, 313. Masuda, K. and Kawata, T. (1978). Microbiology and Immunology 23, 941. Masuda, K. and Kawata, T. (1980). Microbiology and Immunology 24, 299. Masuda, K. and Kawata, T. (1981). Journal of General Microbiology 124, 81. Masuda, K. and Kawata, T. (1982). Journal of Bacteriology 150, 1405. Masuda, K. and Kawata, T. (1983). FEMS Microbiology Letters 20, 145. Masuda, K. and Kawata, T. (1986a). Microbiology and Immunology 30, 401. Masuda, K. and Kawata, T . (1986b). Proceedingsof the 11th International Congress of Electron Microscopy, Kyoto 4, 3365. Masuda, K., Itoh, M. and Kawata, T. (1989). Microbiology and Immunology 33, 287. Matthies, C., Mayer, F. and Schink, B. (1989). Archives of Microbiology 151, 498. Mayer, F., Lurz, R. and Schoberth, S. (1977). Archives of Microbiology 115, 207. Merkel, G . J., Durham, D. R. and Perry, J . J. (1980). Canadian Journalof Microbiology 26, 556. Mescher, M. F. (1981). Trends in Biochemical Sciences 6, 97. Mescher, M. F. and Strominger, J. L. (1976). Journal of Biological Chemistry 251, 2005. Mescher, M. F. and Strominger, J. L. (1978). FEBS Letters 89, 37. Mescher, M. F., Strominger, J. L. and Watson, S. W. (1974). Journal of Bacteriology 120,945. Messner, P. and Buckel, W. (1988). Systematic and Applied Microbiology 10, 226. Messner, P. and Sleytr, U. B. (1988a). In “Crystalline Bacterial Cell Surface Layers” (U. B. Sleytr, P. Messner, D. Pum and M. Sara, eds), pp. 11-16. Springer-Verlag, Berlin. Messner, P. and Sleytr, U. B. (1988b). FEBS Letters 228, 317. Messner, P. and Sleytr, U. B. (1988~).In “Bacterial Cell Surface Techniques” (1. C. Hancock and I. R. Poxton, eds), pp. 97-104. Wiley, Chichester. Messner, P. and Sleytr, U. B. (1991a). In “Microbial Cell Surface Analysis. Structural and Physicochemical Methods” (N. Mozes, P. S. Handley, H . J . Busscher and P. G. Rouxhet, eds), pp. 109-125. VCH Publishers, New York. Messner, P. and Sleytr, U. B. (1991b). Glycobiology 1, 545. Messner, P., Hollaus, F. and Sleytr, U. B. (1984). International Journal of Systematic Bacteriology 34, 202. Messner, P., Pum, D., Sara, M., S e t t e r , K. 0. and Sleytr, U. B. (1986a). Journal of Bacteriology 166, 1046. Messner, P., Pum, D . and Sleytr, U. B. (1986b). Journal of Ultrastructure and Molecular Structure Research 97, 73. Messner, P., Sleytr, U. B., Christian, R., Schulz, G . and Unger, F. M. (1987). Carbohydrate Research 168, 21 1. Messner, P., Bock, K., Christian, R., Schulz, G . and Sleytr, U. B. (1990). Journal of Bacteriology 172, 2576. Messner,P.,Kiipcii,S.,Sara,M., Pum,D. andSleytr,U. B. (1991). In “ProteinGlycosylation: Cellular, Biotechnological and Analytical Aspects” (GBF Monographs) (H. S. Conradt, ed.), vol. 15, pp. 111-116. VCH Publishers, Weinheim.

270

P. MESSNER AND U . B. SLEYTR

Michel, H., Neugebauer, D.-Ch. and Oesterhelt, D. (1980). In “Electron Microscopy at Molecular Dimensions” (W. Baumeister and W. Vogell, eds), pp. 27-35. Springer-Verlag, Berlin. Millonig, G., D e Rosa, M., Gambacorta, A. and Bu’lock, J. D . (1975). Journal of General Microbiology 86, 165. Mohr, V. and Larsen, H . (1963). Journal of General Microbiology 31, 267. Mosbach, K. (ed.) (1987). Methods in Enzymology 136. Mosbach, K. (ed.) (1988). Methods in Enzymology 137. Mullakhanbhai, M. F. and Larsen, H. (1975). Archives of Microbiology 104, 207. Munn, C. B., Ishiguro, E . E . , Kay, W. W. and Trust, T. J. (1982). Infection and Immunity 36, 1069. Murray, R. G . E . (1968). In “Microbial Protoplasts, Spheroplasts and L-forms” (L. B. Guze, ed.), pp. 1-18. Williams and Wilkins, Baltimore. Murray, R. G . E., Dooley, J. S. G., Whippey, P. W. and Trust, T. J. (1988). Journal of Bacteriology 170, 2625. Nermut, M. V. and Murray, R. G. E . (1967). Journal of Bacteriology 93, 1949. Nusser, E . and Konig, H. (1987). Canadian Journal of Microbiology 33, 256. Nusser, E., Hartmann, E., Allmeier, H., Konig, H., Paul, G . and Stetter, K. 0. (1988). In “Crystalline Bacterial Cell Surface Layers” (U. B. Sleytr, P. Messner, D . Pum and M. Sara, eds), pp. 21-25. Springer-Verlag, Berlin. Ohta, N., Chen, L.-S., Swanson, E . and Newton, A. (1985). Journalof Molecular Biology 187, 107. Olden, K., Bernard, B. A , , Humphries, M. J., Yeo, T.-K., Yeo, K.-T., White, S. L., Newton, S. A , , Bauer, H . C. and Parent, J. B. (1985). Trends in Biochemical Sciences 10, 78. Osborn, M. J. (1979). In “Bacterial Outer Membranes” (M. Inouye, ed.), pp. 15-34. Wiley, New York. Palmer, E. L., Mallavia, L. P., Tzianabos, T. and Obijeski, J. F. (1974a). Journal of Bacteriology 118, 1158. Palmer, E . L., Martin, M. L. and Mallavia, L. (1974b). Applied Microbiology 28, 713. Pangborn, J . and Starr, M. P. (1966). Journal of Bacteriology 91, 2025. Pate, J . L. and Chang, L.-Y. E . (1979). Current Microbiology 2, 59. Paul, G., Lottspeich, F. and Wieland, F. (1986). Journal of Biological Chemistry 261, 1020. Paula, S. J., Duffey, P. S., Abbott, S. L., Kokka, R. P., Oshiro, L. S., Janda, J . M., Shimada, T. and Sakazaki, R. (1988). Infection and Immunity 56, 2658. Paynter, M. B. and Hungate, R . E . (1968). Journal of Bacteriology 95, 1943. Pei, Z. and Blaser, M. J. (1990). Journal of Clinical Investigation 85, 1036. Pei, Z., Ellison 111, R. T., Lewis, R. V. and Blaser, M. J. (1988). Journal of Biological Chemistry 263, 6416. Pellerin, P., Fournet, B. and Debeire, P. (1990). Canadian Journal of Microbiology 36,631. Peters, J., Peters, M., Lottspeich, F., Schafer, W. and Baumeister, W. (1987). Journal of Bacteriology 169, 5216. Peters, J., Peters, M., Lottspeich, F. and Baumeister, W. (1989). Journalof Bacteriology 171, 6307. Phipps, B. M., Trust, T . J., Ishiguro, E. E. and Kay, W. W. (1983). Biochemistry 22,2934. Phipps, B. M., Engelhardt, H., Huber, R. and Baumeister, W. (1990). Journal of Structural Biology 103, 152. Pledger, R. J . and Baross, J. A. (1989). Systematic and Applied Microbiology 12, 249. Priischenk, R. and Baumeister, W. (1987). European Journal of Cell Biology 45, 185. Pum, D . , Sara, M. and Sleytr, U. B. (1989a). Journal of Bacteriology 171, 5296. Pum, D., Sara, M. and Sleytr, U. B. (1989b). Journalof Vacuum Scienceand Technology B7, 1391. Pum, D., Messner, P. and Sleytr, U. B. (1991). Journal of Bacteriology 173, 6865. Pum, D., Sara, M. and Sleytr, U. B. (1992). In “Immobilised Functional Biomolecules” (U. B. Sleytr, P. Messner, D . Pum and M. Sara, eds). Springer-Verlag, London. Rachel, R., Jakubowski, U., Tietz, H., Hegerl, R . and Baumeister, W. (1986). Ultramicroscopy 20, 305.

CRYSTALLINE BACTERIAL CELL-SURFACE LAYERS

27 1

Rachel, R., Engel, A. M., Huber, R., Stetter, K. 0. and Baumeister, W. (1990). FEBS Letters 262, 64. Ramaley, R. F., Turner, F. R., Malick, L. E. and Wilson, R. B. (1978). In “Biochemistry of Thermophily” (S. M. Friedman, ed.), pp. 89-102. Academic Press, New York. Ranta, H., Lounatmaa, K., Haapasalo, M. and Ranta, K. (1983). Scandinavian Journal of Dental Research 91, 458. Rasch, M., Saxton, W. 0. and Baumeister, W. (1984). FEMS Microbiology Letters 24,285. Ray, B. and Johnson, M. C. (1986). Cryo-Letters 7, 210. Remsen, C. C. and Triiper, H. G. (1973). Archives of Microbiology 90, 269. Remsen, C. C., Valois, F. W. and Watson, S. W. (1967). Journal of Bacteriology 94, 422. Remsen, C. C., Watson, S. W., Waterbury, J. B. and Triiper, H. G. (1968). Journal of Bacteriology 95, 2374. Remsen, C. C., Watson, S. W. and Triiper, H. G. (1970). Journal of Bacteriology 103,255. Reniero, R., Morelli, L., Callegari, M. L., Sommi, P. and Bottazzi, V. (1990). Annuli di Microbiologica 40, 83. Reyn, A., Birch-Anderson, A. and Murray, R. G. E. (1971). Acta Pathologica et Microbiologica Scandinavica Section B 79, 51. Reynolds, D . M., Laishley, E. J. and Costerton, J. W. (1981). Canadian Journal of Microbiology 27, 151. Ridgway, H. F . (1977). Canadian Journal of Microbiology 23, 1201. Ridgway, H. F. and Lewin, R. A. (1973). Journal of General Microbiology 79, 119. Rivard, C. J., Henson, J. M., Thomas, M. V. and Smith, P. H. (1984). International Journal of Systematic Bacteriology 34,91. Robards, A. W. and Sleytr, U. B. (1985). “Practical Methods in Electron Microscopy. Low Temperature Methods in Biological Electron Microscopy” (A. M. Glauert, ed.), vol. 10. Elsevier, Amsterdam. Robertson, J. D., Schreil, W. and Reedy, M. (1982). Journal of Ultrastructure Research 80, 148. Rogers, G. M. and Baecker, A. A. W. (1991). International Journalof Systematic Bacteriology 41, 140. Romesser, J. A,, Wolfe, R. S., Mayer, F., Spiess, E. and Walther-Mauruschat, A . (1979). Archives of Microbiology 121, 147. Salton, M. R. and Williams, R. C. (1954). Biochimica et Biophysica Acta 14, 455. Sara, M.and Sleytr, U. B. (1987a). Journal of Bacteriology 169, 2804. Sara, M.and Sleytr, U. B. (1987b). Journal of Bacteriology 169, 4092. Sara, M. and Sleytr, U. B. (1987~).Journal of Membrane Science 33, 27. Sara, M. andSleytr,U. B. (1988). In“Biotechnology”(H.-J. RehmandG. Reed,eds),vol.bb, pp. 615-636. VCH Publishers, Weinheim. Sara, M. and Sleytr, U. B. (1989). Applied Microbiology and Biotechnology 30, 184. Sara, M., Kalsner, I. and Sleytr, U. B. (1988). Archives of Microbiology 149, 527. Sara, M., Kiipcii, S. and Sleytr, U. B. (1989). Archives of Microbiology 151, 416. SBra, M., Kiipcii, S. and Sleytr, U. B. (1990a). The Genetic Engineer and Biotechnologist 10, 10. Sara, M., Moser-Thier, K., Kainz, U. and Sleytr, U. B. (1990b). Archives ofMicrobiology 153, 209. Sawatake, M. (1966). Proceedings of the 6th International Congress of Electron Microscopy, Kyoto 2, 279. Saxton, W. 0. and Baumeister W. (1986). Journal of Molecular Biology 187,251. Schallehn, G. and Wecke, J. (1974). Zentralblatt fur Bakteriologie, Mikrobiologie und Hygiene, I. Abteilung, Originale Reihe A 228, 63. Schenk, A. and Aragno, M. (1979). Journal of General Microbiology 115, 333. Schenk, S. F. and Earhart, C. F. (1981). Journal of Bacteriology 146, 398. Schiewer, U.and Jonas, L. (1977). Archiv fur Protistenkunde 119, 146. Schmidt, J. M. (1978). Current Microbiology 1, 65. Segerer, A,, Neuner, A., Kristjansson, J. K. and Stetter, K. 0. (1986). InternationalJournal of Systematic Bacteriology 36, 559.

272

P. MESSNER AND U. B. SLEYTR

Segerer, A. H., Trincone, A., Gahrtz, M. and Stetter, K. 0. (1991). International Journal of Systematic Bacteriology 41, 495. Sharon, N. (1984). Trends in Biochemical Sciences 9, 198. Shaw, P. J., Hills, G. J., Henwood, J. A,, Harris, J. E. and Archer, D. B. (1985). Journal of Bacteriology 161, 750. Siewert, G . and Strominger, J. L. (1967). Proceedings of the National Academy of Sciences of the United States of America 57, 767. Simon, R. D . (1981). Journal of Bacteriology 148, 315. Singer, S. J., Maher, P. A . and Yaffe, M. P. (1987). Proceedings of the National Academy of Sciences of the United States of America 84, 1015. Sjogren, A,, Hovmoller, S., Farrants, G., Ranta, H., Haapsalo, M., Ranta, K. and Lounatmaa, K. (1985). Journal of Bacteriology 164, 1278. Sjogren, A., Wang,D. N., Hovmoller, S.,Haapasalo,M.,Ranta,H., Kerosuo,E., JousimiesSomer, H. and Lounatmaa, K. (1988). Molecular Microbiology 2, 81. Sleytr, U. (1970). Archiv fur Mikrobiologie 72, 238. Sleytr, U. B. (1975). Nature 257, 400. Sleytr, U. B. (1976). Journal of Ultrastructure Research 55, 360. Sleytr, U. B. (1978). International Review of Cytology 53, I . Sleytr, U. B. (1981). In “Cell Biology Monographs. Cytomorphogenesis in Plants” (0.Kiermayer, ed.), vol. 8, pp. 3-26. Springer-Verlag, Vienna. Sleytr, U. B. and Glauert, A. M. (1975). Journal of Ultrastructure Research 50, 103. Sleytr, U. B. and Glauert, A. M. (1976). Journal of Bacteriology 126, 869. Sleytr, U. B . and Glauert, A. M. (1982). In “Electron Microscopy of Proteins” (J. R. Harris, ed.), vol. 3, pp. 41-76. Academic Press, London. Sleytr, U. B. and Messner, P. (1983). Annual Review of Microbiology 37, 311. Sleytr, U. B. and Messner, P. (1988a). Journal of Bacteriology 170, 2891. Sleytr, U. B. and Messner, P. (1988b). In “Crystalline Bacterial Cell Surface Layers” (U. B. Sleytr, P. Messner, D. Pum and M. Sara, eds), pp. 160-186. Springer-Verlag, Berlin. Sleytr, U. B. and Messner, P. (1989). In “Electron Microscopy of Subcellular Dynamics” (H. Plattner, ed.), pp. 13-31. CRC Press, Boca Raton. Sleytr, U. B. and Plohberger R. (1980). In “Electron Microscopy at Molecular Dimensions” (W. Baumeister and W. Vogell, eds), pp. 36-47, Springer-Verlag, Berlin. Sleytr, U. B. and Sara, M. (1986). Applied Microbiology and Biotechnology 25, 83. Sleytr, U. B. and Thorne, K. J . I. (1976). Journal of Bacteriology 126, 377. Sleytr, U. B. and Thornley, M. J. (1973). Journal of Bacteriology 116, 1383. Sleytr, U., Adam, H. and Klaushofer, H. (1967). Mikroskopie (Wien) 22, 233. Sleytr, U., Adam, H. and Klaushofer, H. (1968). Mikroskopie (Wien) 23, 1 . Sleytr, U., Adam, H. and Klaushofer, H. (1969). Archivfur Mikrobiologie 66, 40. Sleytr, U. B., Kocur, M., Glauert, A. M. andThornley, M. J. (1973). Archivfiir Mikrobiologie 94,77. Sleytr, U. B., Thornley, M. J. and Glauert, A. M. (1974). Journal of Bacteriology 118, 693. Sleytr, U. B . , Sara, M., Kupcu, Z. and Messner, P. (1986). Archives of Microbiology 146,19. Sleytr, U. B., Mundt, W. and Messner, P. (1987). European patent application EP 0 306 473 Al. Sleytr, U. B., Messner, P. and Pum D. (1988a). In “Methods in Microbiology” (F. Mayer, ed.), vol. 20, pp. 29-60. Academic Press, London. Sleytr, U. B., Messner, P., Pum, D. and Sara, M. (eds) (1988b). “Crystalline Bacterial Cell Surface Layers”. Springer-Verlag, Berlin. Sleytr, U. B., Sara, M. and Pum, D. (1989). Microelectronic Engineering 9, 13. Smarda, J . (1988). In “Crystalline Bacterial Cell Surface Layers” (U. B. Sleytr, P. Messner, D. Pum and M. Sara, eds), pp. 127-132. Springer-Verlag, Berlin. Smarda, J. (?991). Archiv fur Hydrobiologie, Supplement Algological Studies 64, 41. Smarda, J., Cislavski, J . and Komarek, J. (1979). Archiv fur Hydrobiologie, Supplement 56, 154. Smit, J. (1987). In “Bacterial Outer Membranes as Model Systems” (M. Inouye, ed.), pp. 343-376. Wiley, New York. Smit, J. and Agabian, N. (1982). Journal of Cell Biology 95, 41.

CRYSTALLINE BACTERIAL. CELLSURFACE LAYERS

273

Smit, J. and Todd, W. J. (1986). In “Ultrastructure Techniques for Microorganisms” (H. C . Aldrich and W. J. Todd, eds), pp. 469-516. Plenum Press, New York. Smit, J., Grano, D . A , , Glaeser, R. M. and Agabian, N. (1981). Journalof Bacteriology 146, 1135. Smith, S. H.and Murray, R. G . E. (1990). Canadian Journal of Microbiology 36,327. Sowers, K. R. and Ferry, J. G. (1983). Applied and Environmental Microbiology 45,684. Sowers, K. R., Baron, S. F. and Ferry, J. G. (1984). Appliedand Environmental Microbiology 47,971. Sprott, G . D., Sowden, L. C., Colvin, J. R., Jarrell, K. F. and Beveridge, T . J. (1984). Canadian Journal of Microbiology 30,594. Starr, M. P., Short, K. A. and Schmidt, J . M. (1984). International Journal of Systematic Bacteriology 34, 465. Steensland, H . and Larsen, H . (1969). Journal of General Microbiology 55, 325. Stetter, K. 0. (1982). Nature 300, 258. Stetter, K. O., Thomm, M., Winter, J., Wildgruber, G., Huber, H., Zillig, W., JanCkovic, D., Konig, H., Palm, P. and Wunderl, S. (1981). Zentralblatt fur Bakteriologie, Mikrobiologie und Hygiene, I. Abteilung, Originale Reihe C 2, 166. Stetter, K. O., Konig, H. andstackebrandt, E. (1983). SystematicandApplied Microbiology4, 535. Stetter, K. O., Lauerer, G., Thomm, M. and Neuner, A. (1987). Science 236, 822. Stewart, M.(1986). In “Ultrastructure Techniques for Microorganisms” (H. C. Aldrich and W. J . Todd, eds), pp. 333-364. Plenum Press, New York. . Stewart, M. and Beveridge, T. J . (1980). Journal of Bacteriology 142,302. Stewart, M., BeveridgeT. J. and Murray, R. G . E. (1980). Journalof Molecular Biology 173,l. Stewart, M. and Murray, R. G . E . (1982). Journal of Bacteriology 150, 348. Stewart, M., Beveridge, T. J. and Murray, R. G. E. (1980). Journal of Molecular Biology 137, 1. Stewart, M., Beveridge, T . J. and Sprott, G . D. (1985). Journal of Molecular Biology 183, 509. Stoeckenius, W. (1981). Journal of Bacteriology 148, 352. Stoeckenius, W. and Rowen, R. (1967). Journal of Cell Biology 34, 365. Sumper, M.(1987). Biochimicu et Biophysica Acta 906, 69. Sumper, M., Berg, E., Mengele, R. and Strobel, I. (1990). Journal of Bacteriology 172,7111. Sutherland, I. W. (1977). In “Surface Carbohydrates of the Prokaryotic Cell” (I. W. Sutherland, ed.), pp. 17-96. Academic Press, London. Sutherland, I. W. (1985). Annual Review of Microbiology 39, 243. Szostak, M. and Lubitz, W. (1991). In “Vaccines 91” (R. M. Chanock, H. S. Ginsberg, F. Brown and R. A. Lerner, eds), pp. 409414. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Takacs, B. J. and Holt, S. C. (1971). Biochimica et Biophysica Acta 233,258. Takagi, A , , Nakamura, K. and Ueda, M. (1965). Japanese Journal of Microbiology 9,131. Tang, M., Owens, K., Pietri, R., Zhu, X., McVeigh, R. and Gosh, B. K. (1989). Journal of Bacteriology 171,6637. Tanner, A. C. R., Listgarten, M. A,, Ebersole, J. L. and Strzempko, M. N. (1986). International Journal of Systematic Bacteriology 36, 213. Tauschel, H.-D. and Hoehniger, J. (1974). Canadian Journal of Microbiology 20, 13. Taylor, K. A., Deatherage, J. F. and Amos, L. A. (1982). Nature 299,840. Thompson, B. G., Murray, R. G . E . and Boyce, J. F. (1982). Canadian Journal of Microbiology 28, 1081. Thornley, M. J. (1975). CRC Critical Reviews in Microbiology 4, 65. Trust, T. J., Kay, W. W. and Ishiguro, E . E . (1983). Current Microbiology 9,315. Tsuboi, A,, Tsukagoshi, N. and Udaka, S. (1982). Journal of Bacteriology 151, 1485. Tsuboi, A., Uchihi, R., Tabata, R., Takahashi, Y., Hasiba, H., Sasaki, T., Yamagata, H., Tsukagoshi, N. and Udaka, S. (1986). Journal of Bacteriology 168, 365. Tsuboi, A., Uchihi, R., Adachi, T., Sasaki, T., Hayakawa, S., Yamagata, H., Tsukagoshi, N. and Udaka, S. (1988). Journal of Bacteriology 170,935.

274

P MESSNER AND U B SLEYTR

Tsuboi, A., Engelhardt, H., Santarius, U . , Tsukagoshi, N., Udaka, S. and Baumeister, W. (1989). Journal of Ultrastructure and Molecular Structure Research 102, 178. Tsukagoshi, N., Tabata, R., Takemura, T., Yamagata, H. and Udaka, S . (1984). Journalof Bacteriology 158, 1054. Udaka, S . (1976). Agricultural and Biological Chemistry 40, 523. Udey, L. R. and Fryer, J. L. (1978). Marine Fisheries Review 40, 12. Vaara, T. (1982). Canadian Journal of Microbiology 28, 929. Van Gylswyk, N. O., Morris, E . J. and Els, H. J. (1980). Journalof General Microbiology 121, 491. Van Iterson, W. (1956). Proceedings of the 3rd International Conference of Electron Microscopy, London, 602. Van Rijssel, M. and Hansen, T. A. (1989). FEMS Microbiology Letters 61, 41. Wahlberg, J., Tynkkynen, S., Taylor, N., Uotila, J., Kuusinen, K., Kuusinen, M., Haggblom, M., Viljanen, J., Villstedt, R., Nurmiaho-Lassila, E.-L. and Lounatmaa, K. (1987). FEMS Microbiology Letters 40, 75. Walsby, A. E. (1980). Nature 283, 69. Wang, D. N., Sjogren, A,, Hovmoller, S. and Lounatmaa, K. (1986). Proceedings of the 11th International Congress of Electron Microscopy, Kyoto 3, 2435. Watson, S. W. and Remsen, C . C . (1969). Science 163, 685. Watson, S. W. and Remsen, C. C. (1970). Journal of Ultrastructure Research 33, 148. Wecke, J., Reinicke, B. and Schallehn, G. (1974). Proceedings of the 8th International Congress of Electron Microscopy, Canberra 2, 644. Wehrli, E . and Egli, T . (1988). Systematic and Applied Microbiology 10, 306. Weiss, R. L. (1974). Journal of Bacteriology 118, 275. Wells, B., Horne, R. W., Lund, B. M. and King, N. R. (1983). Micron 14, 11. Wiegel, J. and Ljungdahl, L. G . (1981). Archives of Microbiology 128, 343. Wiegel, J . , Braun, M. and Gottschalk, G. (1981). Current Microbiology 5, 255. Wieland, F., Lechner, J., Bernhardt, G. and Sumper, M. (1981). FEBS Letters 132, 319. Wieland, F., Lechner, J. and Sumper, M. (1982). Zentralblattfiir Bakteriologie, Mikrobiologie und Hygiene, I. Abteilung, Originale Reihe C 3, 161. Wieland, F., Heitzer, R. and Schaefer, W. (1983). Proceedings o f t h e National Academy of Sciences of the United States of America 80, 5470. Wildgruber, G., Thomm, M., Konig, H., Ober, K., Ricchiuto, T. and Stetter, K. 0. (1982). Archives of Microbiology 132, 31. Wildhaber, I. and Baumeister, W. (1987). EMBO Journal 6 , 1475. Wildhaber, I., Santarius, U. and Baumeister, W. (1987). Journal of Bacteriology 169, 5563. Winter, A. J., McCoy, E. C., Fullmer, C . S . , Burda, K. and Bier, P. J. (1978). Infection and Immunity 22, 963. Woese, C . R. (1981). Scientific American 244, 98. Woese, C . R., Kandler, 0.and Wheelis, M. L. (1990). Proceedings ofthe NationalAcademy of Sciences of the United States of America 87, 4576. Woodcock, C . L. F., Engelhardt, H. and Baumeister, W. (1986). European Journal of Cell Biology 42, 21 1 . Word, N. S., Yousten, A. A. and Howard, L. (1983). FEMS Microbiology Letters 17, 277. Work, E. and Griffiths, H. (1968). Journal of Bacteriology 95, 641. Wright, A,, Dankert, M., Fennessy, P. and Robbins, P. W. (1967). Proceedings ofthe National Academy of Sciences of the United States of America 57, 1798. Yamada, H., Tsukagoshi, N. and Udaka, S . (1981). Journal of Bacteriology 148, 322. Yamagata, H., Adachi, T., Tsuboi, A , , Takao, M., Sasaki, T., Tsukagoshi, N. and Udaka, S. (1987). Journal of Bacteriology 169, 1239. Yoshii, Z . (1966). Proceedings of the 6th International Congress of Electron Microscopy, Kyoto 2, 277. Zabel, H.-P., Konig, H. and Winter, J. (1984). Archives of Microbiology 37, 308. Zabel, H.-P., Konig, H. and Winter, J. (1985). Systematic and Applied Microbiology 6, 72.

CRYSTALLINE BACTERIAL CELL-SURFACE I.AYERS

275

Zehnder, A. J. B., Huser, B. A., Brock, T. D. and Whurmann, K. (1980). Archives of Microbiology 124, 1. Zellner, G., Alten, C . , Stackebrandt, E., Conway de Macario, E. and Winter, J. (1987). Archives of Microbiology 147, 13. Zellner, G., Messner, P., Kneifel, H., Tindall, B. J., Winter, J. andstackebrandt, E. (1989a). Journal of General and Applied Microbiology 35, 185. Zellner, G., Stackebrandt, E., Kneifel, H., Messner, P., Sleytr, U. B., Conway de Macario, E., Zabel, H.-P., Stetter, K. 0. and Winter, J. (1989b). Systematic and Applied Microbiology 11, 151. Zellner, G., Stackebrandt, E., Messner, P., Tindall, B. J., Conway de Macario, E., Kneifel, H., Sleytr, U. B. and Winter, J. (1989~).Archives of Microbiology 151, 381. Zellner, G., Sleytr, U. B., Messner, P., Kneifel, H. and Winter, J. (1990). Archives of Microbiology 153, 287. Zillig, W., Stetter, K. O., Wunderl, S., Schulz, W., Priess, H. and Scholz, I. (1980). Archivesof Microbiology 125, 259. Zillig, W., Stetter, K. O., Schafer, W., Janekovic, D., Wunderl, S., Holz, I. and Palm, P. (1981). Zentralblatt fur Bakteriologie, Mikrobiologie und Hygiene, I. A bteilung, Originale Reihe C 2, 205. Zillig, W., Stetter, K. O., Prangishvilli, D., Schafer, W., Wunderl, S., Janekovic, D., Holz, I. and Palm, P. (1982). Zentralblatt fur Bakteriologie, Mikrobiologie und Hygiene, I. Abteilung, Originale Reihe C 3, 304. Zillig, W., Gierl, A , , Schreiber, G . , Wunderl, S., Janekovic, D., Stetter, K. 0. and Klenk, H. P. (1983a). Systematic and Applied Microbiology 4, 79. Zillig, W., Holz, I., Janekovic, D., Schafer, W. and Reiter, W. D. (1983b). Systematic and Applied Microbiology 4, 88. Zillig, W., Holz, I., Janekovic, D., Klenk, H.-P., Imsel, E., Trent, J., Wunderl, S., Forjaz, V. H., Coutinho, R. and Ferreira, T. (1990). Journal of Bacteriology 172, 3959.

This Page Intentionally Left Blank

Bacterial Motility and Chemotaxis MICHAEL D . MANSON Department of Biology. Texas A &M University. College Station. T X 77843-3258. USA

. . Cell-surface distribution of flagella .

. . . . A. . . B . Structureofthe flagellum: filament, hookand basal body . . C . Regulation of flagellar assembly and flagellar gene expression . Motility . . . . . . . . . . . . . . . A . Surface motility . . . . . . . . . . . B . Physicalconstraintsonbacterialswimming . . . . . C . Swimmingpatterns . . . . . . . . . . D . Flagellar rotation . . . . . . . . . . . E . Mechanicsof rotation . . . . . . . . . . F. Flagellarenergetics . . . . . . . . . . G . Protein interactions within the flagellar motor . . . . Chemoreception . . . . . . . . . . . . A . Classical phase of chemoreception . . . . . . . B . Identity of bacterial chemoreceptors . . . . . . C . Cellulardistributionofchemoreceptors. . . . . . D . Structure and ligand interactionsofchemoreceptors . . . E . Stimulation of transducers by attractants and binding proteins Chemotactic signal transduction . . . . . . . . . A . Transmembrane signalling . . . . . . . . . B . Intracellular signalling . . . . . . . . . . C . Events at the flagellar switch . . . . . . . . D . Adaptation of the chemotactic response . . . . . . E . Integratedmodelforchemotacticsignalling . . . . . Conclusion . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . .

I . Introduction 11. Flagella

111.

IV .

V.

VI . VII .

References

.

.

. .

.

. .

.

. .

.

. .

. .

.

.

. .

.

.

. . .

.

. . .

.

. . .

.

. . .

.

.

.

. . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . .

277 280 280 281 285 287 287 288 289 290 291 292 293 296 296 298 302 302 305 310 310 313 322 324 332 334 334 335

.

I Introduction The first experimental demonstration of bacterial chemotaxis was published by Wilhelm Pfeffer over 100 years ago (1884. 1888). A centennial ADVANCES IN MICROBIAL PHYSIOLOGY. VOL . 33 ISBN w2-OZ7733-6

Copyright0 1992. by AcademicPressLimited All rightsolreproduction in any form reserved

278

M. D. MANSON

symposium honouring his contribution was organized by the University of Tuebingen in June 1987. Other early contributions to the study of bacterial motility and chemotaxis have been reviewed (Weibull, 1960; Berg, 1975a). Although the capillary assay for chemotaxis in use today (Adler, 1973) still bears Pfeffer’s name, the modern era of research in bacterial motility and chemotaxis began with four major advances in the late 1960s and early 1970s. The first was the realization by Julius Adler and his coworkers at the University of Wisconsin at Madison that the Gram-negative enteric bacterium Escherichia coli senses chemical attractants with specific receptors. The second was the insight, developed by Robert Macnab and Daniel Koshland at the University of California at Berkeley, and Howard Berg at the University of Colorado at Boulder, that bacteria measure spatial gradients of chemicals by a temporal mechanism, sampling and comparing concentrations as they swim. The third was the discovery, made by Berg, and by Michael Silverman and Melvin Simon at the University of California at San Diego, that bacteria swim by rotating their flagellar filaments in response to a proton current through the cytoplasmic membrane. The fourth was the systematic and detailed genetic analysis of flagellar, motility and chemotaxis genes begun by Simon and Silverman, Sho Asakura at Nagoya University, Tetsuo Iino at Mishima University, Bruce Stocker at Stanford University, and J. S. Parkinson at the University of Utah. I dedicate this review to this second generation of pioneers. Bacterial chemotaxis represents one of the simplest behaviours that can be studied. Its popularity as an experimental system stems from several sources. The full arsenal of genetic and biochemical tools that has been developed to investigate the biology of enteric bacteria, in particular E. coli and Salmonella typhimurium, can be used with good effect to examine an entire behavioural repertoire. Admittedly, the repertoire is limited, but what it lacks in complexity is compensated by the depth and detail of the questions that can be posed and answered. Bacterial chemotaxis will probably be the first stimulus-response type of behaviour to be explained at the molecular level, from the reception of the signal from the environment to the change in motor behaviour that the stimulus evokes. The utility of chemotaxis as a model behavioural system, however, should not obscure the importance of bacterial motility and chemotaxis in a broader biological, clinical or ecological context. The significance of chemotaxis in survival strategies of bacteria may seem obvious at first glance but has been definitely established in relatively few instances. Even for the major players in this review, E. coli and S . typhimurium, the role of chemotaxis in the life cycle is far from completely understood. The fact that bacteria devote as much as 3-5% (about 50 genes) of their genome to motility and chemotaxis strongly suggests that chemotaxis imparts substantial selective advantage

BACTERIAL MOTILITY A N D CHEMOTAXIS

279

under some conditions, but the exact nature of these conditions is guesswork. As to clinical relevance, it is clear that motility and chemotaxis can be virulence factors for intestinal and urogenital-tract pathogens. Vibrio choferaeand Safmoneffastrains apparently utilize chemotaxis to direct their swimming as they penetrate the mucosal lining of the intestine to reach underlying cells, and the ability to carry out chemotaxis is highly correlated with the pathogenicity of the strains (Allweis et a f . , 1977; Freter and O’Brien, 1981a,b,c; Nevola et a f . , 1985). It seems likely that many other clinical ramifications of motility and chemotaxis await discovery. Moreover, flagella constitute one of the most effective bacterial antigens against which the immune system directs its counterattacks. Engaged in perpetual coevolutionary competition, some pathogens, including Safmoneffaspecies, can alter the antigenic type of their flagella (Lederberg and Iino, 1956; Silverman et a f . , 1979; Simon et a f . , 1980; Iino and Kutsukake, 1983). Chemotaxis also occurs in bacteria within the rhizosphere and in aquatic environments. Recent work has shown that Agrobacterium tumijuciens may be drawn to a plant wound through chemotaxis (Shaw et u f . , 1988), and that aggregation of cells of Rhizobium species around incipient root hairs is at least in part a chemotactic response (Gulash et a f . , 1984; Bergman et a f . , 1988). Many species from a wide variety of other eubacterial genera are chemotactic (Seymour and Doetsch, 1973; van der Drift et a f . , 1975). The best studied of these organisms with respect to chemotaxis are the Grampositive soil bacterium Bacillus subtifis (Ordal and Goldman, 1975; Ordal and Gibson, 1977; Ordal, 1985) and the freshwater stalked bacterium Caufobacter crescentus (Ely et a f . , 1986; Champer et a f . , 1987; Frederikse and Shapiro, 1989). Chemotaxis has also been observed in halophilic archaebacteria such as Hafobacterium hafobium (Schimz and Hildebrand, 1979; Spudich and Stoeckenius, 1979; Sundberg et a f . , 1990), although in that species it has received less attention than phototaxis (Spudich and Bogomolni, 1984, 1988). In the exceedingly complex biotic and chemical chains of marine environments, it seems certain that chemotaxis must play a major, if largely unexplored, role. Being deeply interested in behaviour and ecology, I am fascinated by the diverse manifestations of bacterial chemotaxis in nature. However, the natural history of bacteria is not the subject of this review, and the interested reader will have to turn elsewhere for information on these topics (Chet et a f . ,1971; Chet and Mitchell, 1976). My intent here is to summarize the basic features of bacterial motility and chemotaxis. In most instances, I will refer exclusively to E. cofi and S . typhimurium, which are so similar that I will distinguish between them only when there is a specific need to do so. This combined approach is made easier by the recent unification of the genetic

280

M. D. MANSON

nomenclature for the flagellar (Iino et al., 1988) and chemotaxis (DeFranco et al., 1979) genes of the two species. Information appearing since the last comprehensive reviews of the field (Ordal, 1985; Macnab, 1987a,b; Stewart and Dahlquist, 1987) will be emphasized. 11. Flagella

Until recently, it was safe to state that bacterial swimming is always propelled (a literal description, as I will soon describe) by a flagellum or flagella. Spirochaetes, without external flagella, generate thrust by rotating helical cell bodies counter to the rotation of the axial filaments, which are internalized flagella retained in the periplasmic space (Berg, 1976a; Berg et al., 1978; Canale-Parola, 1977). Like more conventional flagellar swimming, spirochaete motility is driven by a proton-motive force (Goulbourne and Greenberg, 1980). The vigorously swimming, unicellular marine cyanobacterium Synechococcus sp. is devoid of external or internal flagellar filaments (Waterbury et al., 1985). The mechanism of this flagellurnindependent locomotion is unknown, although it seems to be powered by a sodium-motive force (Willey et al., 1987), as is some flagellar motility (see later). Phage x (Meynell, 1961), which uses the flagellar filament as its primary receptor for adsorption to E. coli or S . typhirnuriurn, cannot infect nonflagellated bacteria (Shade et al., 1967; Icho and Iino, 1978; Kagawa et al., 1984). Therefore, resistance to this flagellatropic bacteriophage has been a useful method for selecting Fla- mutants. A. CELL-SURFACE DISTRIBUTION OF FLAGELLA

A good source of information about patterns of flagellation over the whole range of taxa is contained in general bacteriological reference texts. The monograph by Leifson (1960) is of special interest to the connoisseur of flagellar staining. What follows is a brief catalogue of The structure of flagellar filaments and the ways in which flagella may be distribukd over the cell surface. All extracellular flagellar filaments, at least when they are in their propulsive mode, assume a helical shape. The sense of the helix varies from species to species and may be either right handed or left handed. In E . coli and S . typhirnuriurn, the normal sense of the helix is left handed. The structure and assembly of the flagella of these two species is discussed by Macnab (1987a), Macnab and Parkinson (1991) and Jones and Aizawa (1991).

BACTERIAL MOTILITY AND CHEMOTAXIS

281

Polar flagella arise at the morphologically definable ends of rod-shaped or curved bacterial cells. The simplest case is a single polar flagellum, a condition known as monopolar. A number of flagella may arise at one pole of the cell to form a flagellar bundle. Cells with a single polar bundle are termed lophotrichous. Bacteria with bipolar flagellation can have either single or multiple flagella; if both poles have flagellar bundles the condition is termed amphitrichous. Some bacteria, especially spiral-bodied forms, show subpolar flagellation, in which the flagella arise near, but offset from, the apex of the pole. Some curved bacteria, such as Vibrio species, have a single medial flagellum arising on the concave side of the body. Lateral flagella occur in swarming bacteria, as discussed below, but they are also present in bacteria that swim with flagella arranged in a peritrichous fashion. These flagella are inserted at seemingly random spots on the lateral cell surface but coalesce to form a bundle when the cells are swimming. Peritrichous flagellation is very common in smaller rod-shaped bacteria, including E. coli and S. typhimurium. Those two species typically have five to 10flagella on each cell. In some bacteria, including Vibrio species, a single thick polar flagellum may be present along with thin peritrichous flagella (Allen and Baumann, 1971). B. STRUCTURE OF THE FLAGELLUM: FILAMENT, HOOK AND BASAL BODY

A schematic diagram of the basal body, hook and extreme proximal portion of the filament of a flagellum from S . typhimurium is shown in Fig. 1. The easily visible, helical, extracellular component of a bacterial flagellum is the filament. The centre of the filament is hollow. In many bacteria, including E. coli and S. typhimurium, the flagellar filament is a homopolymer consisting of the protein flagellin. Simple flageila have a uniform, smooth appearance to the filament surface. The subunits within the flagellar filament of S . typhimurium are arranged in an 11-start helical array (Macnab, 1978). The overall helical shape of the filament is determined by non-equivalent interactions of the flagellin subunits with their neighbours. The typical left-handed helix of filaments from E . coli and S . typhimurium is only one of 12 theoretically possible forms. Alterations in either the ionic strength or pH value of the medium, or mutations altering a single residue in flagellin, can give rise to alternative forms (Kamiya et al., 1982). Among mutant variants are straight and curly filaments (Iino and Mitani, 1966,1967; Martinez et al. , 1968). The latter are right-handed helices with half of the normal helical pitch. Mutants with such altered filaments are either poorly motile or non-motile. The C- and N-terminal regions of flagellin are conserved, whereas the mid-region of the protein, exposed on the surface of the filament, is highly

282

M. 0. MANSON

FliD (filament cap, flagellin assembly)

20 nrn

H FIG. 1. Schematic diagram of the filament, proximal hook and basal body of a flagellum from E . coli or S. typhirnuriurn. Structures which are isolated as part of the complex in cell-free preparations are shown in solid outline, whereas proteins that are left behind in the cell membrane or cytoplasm are outlined with pale stippling. The postulated export channel through the hollow core of the rod, hook and filament is outlined with heavier stippling. The protein designations follow the gene nomenclature advocated by Iino et al. (1988). The drawing is reproduced from Macnab and Parkinson (1991).

BACTERIAL MOTILITY AND CHEMOTAXIS

283

variable in composition (Iino, 1977; Lawn, 1977; Wei and Joys, 1985; Kuwajima et al. , 1989). The N-terminus is important for export of flagellin, and both the N- and C-termini are involved in the assembly of the subunits into the filament (Trachtenberg and DeRosier, 1988; Kuwajima et al., 1989). The variable region differs both within and among species, and it provides the diversity of the flagellar antigens among members of the Enterobacteriaceae (Edwards and Ewing, 1972). In S. typhimurium, phase variation creates an additional complication in that any given cell may have filaments made up of flagellin of one or the other of two antigenic types (Lederberg and Iino, 1956). In E. coli, flagellin is encoded by theJliC gene, formerly hag, which is one of the most highly expressed genes in a motile cell (Komeda and Iino, 1979). The designation hag was derived from the German word Hauchantigen (Hauch meaning “film”) , which refers to the fuzzy swarms produced by motile colonies growing in semisolid (0.3-0.4%, w/v) agar. The length of the filament is not constant and, over the population of cells, is a function of the rate of elongation of the filament and the rate of its breakage. The period of the flagellar helix is typically 2.0-2.5 pm (about the same as the length of the cell) and the helical diameter is 0.4-0.6 pm (slightly narrower than the cell body); these values vary from species to species and even strain to strain (Asakura, 1970). Complex flagella have a more complicated surface pattern than plain flagella, with a conspicuous helical pattern of ridges and grooves that may confer greater rigidity (Lowy and Hanson, 1965; Schmitt et al., 1974a,b; Trachtenberg et al., 1986, 1987). Some species, such as the monopolarly flagellated C. crescentus (Driks et al., 1989) and Rhizobium meliloti which has complex flagella (Pleier and Schmitt, 1989), have filaments composed of multiple types of flagellin. The three flagellins of C . crescentus are distributed in distinct regions along the axis of the filament, whereas the two nearly identical flagellins from R. meliloti may form heterodimers that contribute to the complexity of the filament structure in that species. Unstained flagellar filaments are too slender to be resolved by normal light microscopy, although unstained filaments are easily seen with transmission or scanning electron microscopy. Macnab (1976), however, has adapted high-intensity dark-field microscopy to visualize individual filaments on living, motile cells of S . typhimurium and other peritrichously flagellated bacteria. The thickened flagella that are visible using phasecontrast microscopy are sheathed, or covered with an outgrowth of membrane (Sjoblad et al. , 1983). The nature of the sheath membrane is still under investigation. Thomashow and Rittenberg (1985) suggest that the flagellar sheath in Bdellovibrio bacteriovorus constitutes a separate, stable domain, more fluid than a typical outer membrane. Immunogold electron

284

M. D. MANSON

microscopy of V . choferae has shown that the sheath and the outer membrane of the cell body have common lipopolysaccharide antigens (Fuerst and Perry, 1988), suggesting that the two membranes are chemically related, if not necessarily continuous. The filament is attached to the rotation-generating apparatus via the proximal hook, which provides a flexible coupling, or biological universal joint (Wagenknecht et a f . , 1984; Macnab, 1987a). In particular, cells with flagella embedded in their lateral surfaces must convert rotation generated in that plane into rotation of the filament around the long axis of the cell body. In E. cofi and S. typhirnuriurn, the flagellar hook is a helical polymer composed of one type of subunit, a product of theflgE (formerlyflaK) gene (Komeda et a f . , 1978). The hook polymer is elastic, not rigid like the filament. Unlike the length of the filament, which is determined statistically, the length of the hook is genetically determined. Normally, the hook comprises only a fraction of a helical turn. Mutations in thefliK (formerly flaE) gene lead to assembly of abnormally long hooks (called polyhooks) that encompass a number of helical turns (Silverman and Simon, 1972; Patterson-Delafield et al., 1973; Suzuki and Iino, 1981). Three hookassociated proteins, or HAPs, are required to form the junction of the hook and the filament (Homma et a f . , 1984b, 1985,1990). Mutants lacking HAPs secrete unassembled flagellin into the external medium (Homma et a f . , 1984a). The HAPs remain in the mature flagellum (Homma and Iino, 1985) and form structural elements as well as participate in assembly. They are the products of the flgK, flgL and PiD genes. The basal body, which can be isolated free from the cell (DePamphilis and Adler, 1971a), consists of four rings encircling a central rod in flagella from E. cofi and S. typhirnuriurn (DePamphilis and Adler, 1971b). Four is not an upper limit, however, because the basal body of the single polar flagellum of C. crescentus has five distinct rings (Stallmeyer et al., 1989). Electron micrographs indicate that the M-ring is within the cell membrane, the S-ring is located above the M-ring and just outside the cell membrane, the P-ring is at the level of the peptidoglycan layer of the cell wall, and the L-ring is within the outer lipopolysaccharide membrane (DePamphilis and Adler, 1971~). The M-, P- and L-rings are products of the fliF, flgZ and flgH genes, respectively. No gene product has been associated with the S-ring, and it seems likely that the M- and S-rings are composed of domains of the same polypeptide (Homma et a f . , 1987a). Since the basal bodies of flagella from Gram-positive bacteria have only the two inner rings (DePamphilis and Adler, 1971b), it is clear that the outer rings cannot play a general role in flagellar function. The rod is assembled from the products of at least three genes, flgB, flgF andJEgG, and theflgD gene product is required for modification of the rod

BACTERIAL MOTILITY AND CHEMOTAXIS

285

(Macnab, 1990). All axial components of the flagellar structure, namely the rod and hook proteins, the HAPs and flagellin, share certain structural features that are presumably important in their assembly, although in many instances there are no significant similarities in their amino-acid sequences (Homma et al. , 1990). The products of many of the 37 genes known to be involved in formation of intact flagella in E. coli or S . typhimurium are not found in the filamenthook-basal body structure that is isolated from cells (Macnab, 1987a, 1990). Some of these proteins may have a catalytic function during flagellar assembly. Others are almost certainly components of the intact, functional flagellum that are left behind in the cytoplasm or cell membrane when the basal body is extracted from the cell. Five of these proteins will be considered in discussions of motility and chemotactic control of flagellar motion. Elaborations on this basic flagellar structure occur in non-enteric species of bacteria. These embellishments are particularly evident in bacteria with flagellar bundles, or in those with complex or sheathed flagella. The fifth ring of the basal body from C. crescentus has already been mentioned (Stallmeyer et al. , 1989). In Wolinella succinogenes, large structures consisting of concentric rings have been observed surrounding the point at which the flagellum exists through the outer membrane (Kupper et al., 1989). In Aquaspirillum serpens, which has a polar flagellar bundle, regular arrays with a mean number of 15 particles surround the site of insertion of the basal body into the cell membrane (Coulton and Murray, 1978). It is unclear, however, whether these particles are needed for organizing the flagellar basal bodies to allow close packing or are involved in generating flagellar rotation (see later). C. REGULATION OF FLAGELLAR ASSEMBLY AND FLAGELLAR GENE EXPRESSION

The later steps in the pathway of flagellar assembly in S . typhimurium have been elucidated by Iino and his coworkers (reviewed in Iino, 1977; Macnab, 1987a) by examining structures formed by mutants blocked in formation of different flagellar gene products. Strains with mutations in the majority of the flagellar genes produce no structures detectable by electron microscopy. The simplest incomplete structure observed consists of a rod with the M- and S-rings attached. The P- and L-rings are then added, in that order. The proteins (FlgG and FlgH) that form these two rings are unique among flagellar gene products in that they are synthesized with typical cleavable signal sequences (Homma et al., 1987b,c; Jones et al., 1987, 1989). The hook and HAPs are added next, at the distal end of the rod. The filament grows out from the hook, with assembly of new flagellin subunits

286

M. D. MANSON

occurring only at the distal tip of the filament (Iino, 1969; Emerson et al., 1970). The hook and filament subunits diffuse to the distal ends of those structures through the hollow cores of the rod, hook and filament, and filament subunits are excreted in unassembled form if HAPS are lacking (Homma et al., 1984a). Indeed, the rate of elongation of the filament slows as the time required for flagellin subunits to diffuse through the core to the distal tip increases with increasing filament length (Iino, 1974). The diameter of the filament core is too small to permit passage of folded flagellin monomers. Flagellin is probably only partially folded and in an extended conformation during its transit through the growing filament, and there is a specific signal peptide-independent export pathway for flagellin and other axial components of the flagellum (Homma et al., 1990; Kuwajima et al., 1990). Flagellar filaments grown in vitro in the presence of high concentrations of already folded flagellin monomer also grow asymmetrically, extending at a linear rate at the end corresponding to the distal tip (Ishihara and Hotani, 1980). Early genetic studies on bacterial flagella were reviewed by Iino (1977) for S. typhirnurium, and Silverman and Simon (1977~)for E. coli. New flagellar genes were discovered at a furious rate for a number of years, but with the completion of the DNA sequence of the flagellar gene regions in those species it seems probable that the final tally of jig, Jlh and p i genes will remain at or close to the current number of 37 (Macnab, 1990). These genes are organized in 12 transcriptional units that are grouped in three chromosomal regions. These regions, designated Fla I, Fla I1 and Fla I11 (Silverman and Simon, 1973a) and containing theflg, JEhandpi genes, respectively, are located at 24 min, 4 1 4 2 min and 42-43 min on the chromosome of E. coli (Bachmann, 1990) and at equivalent positions on the chromosome of S. typhirnuriurn (Sanderson and Roth, 1988). Deletion analysis and phage p mutagenesis were used to order genes within these regions in E. coli (Silverman and Simon, 1973b, 1974a). Models that incorporate a hierarchy of control levels have been proposed for flagellar gene regulation in E. coli (Komeda, 1982,1986; Kutsukake et al., 1990). The molecular details of this regulation have been examined in the laboratories of Chamberlin (Helmann and Chamberlin, 1987; Helmann et al., 1988; Arnosti and Chamberlin, 1989) and Matsumura (Bartlett et al., 1988). RNA polymerase is directed to flagellar promoters by one or more flagellum-specific (T factors. Characteristic sequence motifs are associated with promoters of transcriptional units at different levels of the control hierarchy (Helmann and Chamberlin, 1987; Bartlett et al., 1988). Expression of all other flagellar genes depends on the products of thefEhC and PhD genes, which are called class 1 genes and which comprise the flagellar “master operon”. The FlhC a n d o r FlhD proteins may act as

BACTERIAL MOTILITY AND CHEMOTAXIS

287

o factors for expression of the second level (class 2) of genes in the flagellar hierarchy; class-2 gene products include most of the proteins of the hook-basal body complex. The product of another class-2 gene, the FliA protein, is a a-factor (Arnosti and Chamberlin, 1989; Ohnishi et al., 1990) required for expression of genes at the third level (class 3) of the hierarchy. It is noteworthy that expression of the master operon itself is under catabolite control (Adler and Templeton, 1967; Yokota and Gots, 1970; Silverman and Simon, 1974b; Komeda et a f . , 1975), and that its promoter has a sequence resembling the consensus binding site for CAMP-binding protein (Bartlett et al., 1988). Catabolite repression greatly decreases the expression of all flagellar, motility and chemotaxis genes in cells grown in the presence of glucose. Additional proteins may be involved in controlling gene expression. The &A (Komeda, 1982),JziS andflifliT (Kutsukake et al., 1990; Macnab, 1990) gene products have been proposed as negative regulators of class3 genes, and FlgJ (Komeda, 1982) has been described as a transcriptional activator specific to pic,the highly expressed gene that encodes flagellin. However, the roles of these gene products in regulation are not firmly established. 111. Motility

Bacteria can move in two or in three dimensions. Those that move on surfaces without flagella exhibit gliding motility, whereas those that rely on flagella to move on surfaces exhibit swarming motility. Any movement in three dimensions is called swimming. Phage x requires functional rotating flagella for infection (Shade et al. , 1967; Icho and Iino, 1978); it can be used to select mutants of E. coli and S. typhirnuriurn with immobile flagella as well as non-flagellated mutants. Motility in these two species has recently been reviewed (Jones and Aizawa, 1991). A. SURFACE MOTILITY

Bacteria can glide on a solid surface, or at gas-liquid or liquid-liquid interfaces. The mechanism by which gliding motility is generated is still under investigation. Indeed, it is not clear that the mechanism is the same in all bacteria, some of which glide relatively fast while others move glacially slow. For a review of gliding, see Burchard (1981) and Reichenbach (1981). Two forms of gliding are found in some species, like Myxococcus xanthus, which can move alone or in rafts containing hundreds or thousands of cells (Parish, 1979; Kaiser, 1986). These rafts act like “wolf packs”, surrounding and digesting other bacteria with proteases and nucleases excreted by the population of myxococci. Social gliding is also used to form cell aggregates

288

M. D. MANSON

that develop into fruiting bodies. Many other soil-dwelling bacteria, of which the best studied are Cytophaga sp. (Lapidus and Berg, 1982) and Flexibacter sp. (Dayrell-Hart and Burchard, 1979), are solitary gliders. Some mycoplasmas are also capable of gliding (Rosengarten and Kirchoff, 1987). Gliding, like swimming, is powered by a proton-motive force (Ridgway, 1977), at least in some species. Swarming also occurs in two dimensions but, like swimming, it is driven by flagella. In Proteus sp. (Palleroni et al., 1970; Williams and Schwarzhoff, 1978) and Vibrio parahaemolyticus (Ulitzur, 1974, 1975; Shinoda and Okamoto, 1977) swarming is associated with lateral flagella. Some species, like V . parahaernolyticus, swim with a single polar, sheathed flagellum in aqueous surroundings or swarm when they proliferate lateral flagella after contacting a surface (Belas and Colwell, 1982; Belas et al., 1986). The expression of genes encoding lateral flagella is induced by increased viscous drag on the polar flagellum (McCarter et al., 1988). The swimmer-swarmer transition in Serratia marcescens is similar, except that the flagellin found in flagellar filaments from the swimmer and swanner cells is the same in S . marcescens, suggesting that a single gene for flagellin is differentially regulated (Alberti and Harshey, 1990). In essence, swarming is swimming through a surface film of water, and it often involves large numbers of cells moving in rafts. B . PHYSICAL CONSTRAINTS ON BACTERIAL SWIMMING

For their size, bacteria are remarkably rapid swimmers. Cells of E. coli and S. typhirnuriurn swim at 20-40 pm s-l at physiological temperatures (Berg and Brown, 1972; Macnab and Koshland, 1972), with S . typhirnurium being the faster. Some small, polarly flagellated bacteria, like species of Bdellovibrio and Pseudomonas, are substantially faster. (The “zippy” little contaminants that appear unbidden in cultures of more lethargic species are often pseudomonads.) Since enteric rods average about 2 pm in length, cells of E. coli and S. typhimuriurn move at 1&20 body lengths in each second. That is about the same relative velocity as a 1 m long yellowfin tuna (Thunnus albacares, among the fastest of fish), which can swim in bursts from 5 up to 20 m s-’ (Walters and Fierstine, 1964). Bacteria live in a regime of low Reynolds number (a physical term that represents the ratio of inertial forces to viscous forces). The problems of “life at low Reynolds number” are entertainingly described in a delightful and insightful paper by Purcell (1977). For a man in water, the Reynolds number is of the order of lo4, for a guppy perhaps lo2. For a bacterial cell in dilute aqueous medium, the number is of the order of 10-4-10-5. In other words. inertia is irrelevant-a

BACTERIAL MOTILITY AND CHEMOTAXIS

289

bacterium does not glide. In fact, if a cell of E. coli stops actively swimming, of a body length). it will come to a halt in 0.6 ps and 0.01 nm (less than Purcell's analogy on the human scale is a person operating under the constraint that no body part can move faster than 1 cm min-' and condemned to swim in a pool filled with molasses. Strict reciprocal motion is fruitless at low Reynolds number; the net propulsion will be zero even if a paddle (or tail) is moved rapidly in one direction and slowly in the other. The clearest illustration of this principle is a scallop, whose strategy of slowly opening and then rapidly closing its shell is unavailing at low Reynolds number; a bacterium that swims like a scallop will never be discovered. Rotating propellors do not rely on reciprocal motion, and they work at low Reynolds number. Rotation of helical propellors is the mechanism that allows bacteria to swim, as will be discussed in the next section. But, before discussing the mechanism of propulsion, it is worthwhile to consider the motility patterns generated by swimming bacteria. C. SWIMMING PATTERNS

Swimming bacteria perform a three-dimensional random walk (Berg and Brown, 1972). This is true regardless of the pattern of flagellation, although the flagellar movements that lead to the random walk are various. For peritrichously flagellated bacteria, such as E. coli or S . typhimurium, a cell in the absence of any chemical gradient swims along a gently curved path for several seconds (it runs), goes through a brief (about 0.1 s) period of chaotic motion that reorients it, and then swims off in a new, random direction. Even during a run, rotational diffusion prevents the cell from swimming straight. Spirochaetes, and many lophotrichously or amphitrichously flagellated bacteria, undergo essentially 180" reversals in swimming direction. However, since they, like peritrichously flagellated cells, are unable to swim on straight paths, the net result is a three-dimensional random walk, with changes in direction that are on average larger than in run-tumble motility. Some of the monopolar bacteria go through brief periods of back up. Since they are less able to hold course while going in reverse, the direction they assume when they return to forward swimming is different from that in the previous run, and a random walk results. Finally, in Rhodobacter spheroides, runs alternate with pauses during which the right-handed helical filament coils up and the cell reorients passively due to Brownian motion before the next run (Armitage and Macnab, 1987). A similar alternation between runs and pauses occurs with R. meliloti, which has complex, righthanded helical filaments (Goetz and Schmitt, 1987). However, the complex flagellum of this species does not coil up during pauses.

290

M. D. MANSON

D. FLAGELLAR ROTATION

The demonstration that flagella rotate (Berg and Anderson, 1973; Berg, 1974; Silverman and Simon, 1974c) marked a major breakthrough in the study of bacterial motility. The helical filament is a propellor, and the flagellar basal body and proteins associated with it form a reversible, rotary motor. Individual filaments, or even bundles of filaments, are too thin to be visible on swimming cells except under high-intensity illumination with dark-field optics. Therefore, flagellar rotation was originally inferred from observations of counter-rotation of pairs of cells held together by antifilament antibody or from rotation of polystyrene beads attached with antibody to flagella of mutant cells that produce straight filaments (Berg, 1975b). Rotation of the cell body, driven by the flagellar motor, can be observed using tethered cells (Silverman and Simon, 1 9 7 4 ~ ).Before tethering, cells are mechanically sheared to reduce flagellar filaments to short stubs that can be attached to a glass surface with antibody. When the filament of a lateral flagellum in E. coli or S . typhimuriurn is held fast, the cell body rotates parallel to the surface at speeds of up to at least 10 Hz (10 rev s-’). Short cells, which produce less hydrodynamic drag, rotate more rapidly than longer cells (Manson et al., 1980; Berg, et al., 1982). Tethered cells alternate between periods of clockwise (CW) and counteret al., clockwise (CCW) rotation (Silverman and Simon, 1 9 7 4 ~ Larsen ; 1974b). (Convention decrees that the sense of flagellar rotation is defined by the direction in which the flagellum turns as it is viewed along the axis of the filament looking toward the cell.) The duration of the intervals in both directions follows a Poisson distribution, except that very short intervals are rare (Segall et al., 1982; Block etal. 1983). For E. coli, the mean intervals are of the order of one-half to several seconds. For most cells, the mean period of CCW flagellar rotation is longer than for CW rotation. However, both the mean interval length and the CCW:CW ratio vary with the metabolic state of the cells (Khan and Macnab, 1980a) and from cell to cell even within a population that is genetically and physiologically homogeneous (Spudich and Koshland, 1976). For peritrichously flagellated bacteria with left-handed helical filaments, CCW flagellar rotation corresponds to running and CW rotation corresponds to tumbling. During a run, the individual filaments coalesce into a bundle that pushes the cell. Clockwise flagellar rotation causes the bundle to fly apart, and tumbling ensues. If CW rotation is prolonged, hydrodynamic forces cause the filaments to be converted into a right-handed “curly” conformation. The right-handed helix first appears at the cell body and then propagates outward. If CW rotation is sufficiently prolonged, as it is in some strongly CW-biased mutants of S . typhimurium, then the cells can swim with a right-handed flagellar bundle (Rubik and Koshland, 1978; Khan et al.,

BACTERIAL MOTILITY AND CHEMOTAXIS

291

1978), although at decreased speed because of the suboptimal hydrodynamic properties of the right-handed filaments, which have a shorter period. In bacteria with monopolar flagella, reversal of the flagellar motors causes the cells to reverse, whether a bundle o r a single filament is present (Berg, 1975a,b). Backward swimming is less efficient, and its mean interval is shorter than the mean interval of forward swimming. In the spirochaetes and amphitrichously flagellated bacteria, the cell has no intrinsic polarity. In the large, spiral-bodied, bipolarly flagellated Spirillum volutuns, for example, the bundle at the leading end of the cell folds back over the body and helps pull the cell along, while the trailing bundle pushes (Berg, 1975a,b). Rotation of the helical cell body itself, in reaction to the rotation of the flagellar bundles, also contributes to the propulsive force. (A moment’s reflection will convince the reader that, for either spirochaetes or S . volutuns, the filaments at the two ends of the cell must turn in opposite directions for the cell to make any progress.) E. MECHANICS OF ROTATION

All recent models for flagellar rotation stipulate that rotation is generated within the cytoplasmic membrane. This rotation is transmitted through the rod to the hook and, via the hook, to the filament. The P- and L-rings may serve as bushings to allow the rod to pass through the outer layers of the cell envelope, whereas the M-ring may be more directly involved in the generation of rotation. One mechanical constraint on the flagellar motor is that it must have both a rotor and a stator. Since the flagellar filament is quite massive and encounters considerable viscous drag as it turns at speeds of up to several hundred hertz (Lowe et ul., 1987), the stator must be anchored to something even more massive (Berg, 1974). Otherwise, the stator would simply spin in the membrane, and the filament would hardly budge. The only obvious structure in the cell that has the necessary mass is the cell-wall peptidoglycan. Most models for the flagellar motor incorporate some element that is firmly attached to peptidoglycan. The products of five genes are specifically implicated in the generation of rotation. Three of these are proteins of the switch-motor complex (to be described later). The two remaining genes are motA and motB. Their products are not required for flagellar assembly, since null mutations in these two genes do not block synthesis of the basal body, hook and filament. The flagella made by motA- or motB- bacteria, however, are paralysed, that is, they do not turn. Electron-microscopic examination of freeze-fractured cell membranes at the site of insertion of the M-ring reveals a rather regular array of 10-12

292

M. D. MANSON

particles, or “studs”, in a motile strain of E. coli. These particles surround the doughnut-shaped depression left by the ring (Khan et al., 1988). The studs are absent or disorganized in either motA or motB mutants, suggesting that they may be made up of Mot proteins, or complexes containing Mot proteins. Block and Berg (1984) and Blair and Berg (1988) found, in agreement with earlier results of Silverman et al. (1976), that functional Mot proteins could be added to a pre-existing, paralysed flagellum. The time-course for restoration of motility was examined in tethered motA or motB mutants containing plasmid-borne motA+ or motB+ genes under lac regulatory control. Synthesis of MotA or MotB was induced by addition of isopropylthiogalactoside. After a lag of several minutes, previously non-rotating cells began to turn. The initial velocity was low, but increased in equal quantized increments. The final velocity was consistently eight times the velocity achieved in the first step (Blair and Berg, 1988). This response suggests that each flagellum has up to eight independent force generators that can be assembled after synthesis of the basal body is complete. The finding that quantized decreases in velocity were also observed suggests that the force generators can be released from the motile apparatus as well as added. Although the difference between the number of quantized steps (eight) and the number of studs (10-12) has not been resolved, it is attractive to think that the studs and force generators may be different manifestations of the same phenomenon. F. FLAGELLAR ENERGETICS

The energy for flagellar motility is provided by the proton-motive force in most bacteria, so that rotation normally is coupled to an inward current of protons. The motor is electrostatic rather than dependent on any particular facet of proton chemistry, since in some alkalophilic bacteria motility is driven by a sodium-ion current or sodium-motive force (Hirota and Imae, 1983). The most parsimonious interpretation of this diversity is that the basic mechanism of energy coupling in proton-driven and sodium ion-driven motors is similar. The experiments of Larsen et al. (1974a) showed that ATP is not directly involved in motility, although it is required for chemotaxis. This work was extended by the demonstration that artificially generated membrane potentials and/or p H gradients (the cell interior being negative or alkaline with respect to the medium) support flagellar rotation in starved, deenergized cells of a motile Streptococcus sp. (strain V4051; Manson er al., 1977, 1980). Similar evidence that the proton-motive force drives motility has been obtained with B. subtilis (Matsuura et al., 1977, 1979; Khan and Macnab, 1980b) and Rhodospirillum rubrum (Glagolev and Skulachev,

BACTERIAL MOTILITY AND CHEMOTAXIS

293

1978). Interesting features of energy coupling in Streptococcus are: (a) the speed of flagellar rotation is proportional to the proton-motive force when the motor runs at high load (as in a tethered cell); (b) the rotational speed is inversely proportional to the viscous load in the high-load regime; (c) the threshold proton-motive force for rotation is less than 10 mV (Khan and Berg, 1983); (d) membrane potentials or pH gradients of opposite polarity (inside positive or acidic with respect to the medium) also drive flagellar rotation (Manson et al., 1980; Berg et af., 1982). Technical problems have been encountered in generating controlled, artificial membrane potentials and pH gradients in Gram-negative bacteria such as E. cofi and S. typhimurium. These difficulties have thus far prevented a wedding of the genetic information available for these species with the type of physiological data obtained with Streptococcus sp. strain V4051 or B. subtifis. Estimates of the proton flux through the flagellar motor of Streptococcus V4051 yield a value of about 1200protons for each revolution (Meister et af., 1987), a value that is independent of rotational velocity or the load under which the motor operates. This number of protons provides roughly four times the minimum energy required when the motor operates under high load, assuming that the proton electrochemical potential is harnessed with 100% efficiency and the torque is applied at the outer edge of the M-ring (radius 12-13 nm). Up to a rotational velocity of 5 Hz, the stall torque and the running torque of the motor are nearly identical (Meister and Berg, 1987), indicating that proton flow through the motor or movement of motor parts cannot be rate-limiting in this speed regime. For motors operating under conditions of lower load, the efficiency of the motor decreases (Lowe et a f . , 1987), and the maximum velocity is set by physical constraints within the motor. Blair and Berg (1990) described motA mutants of E. cofi in which motor torque is normal at high load, but rotational speed at low load is decreased relative to the velocities of several hundred hertz obtained by the flagella of wild-type cells. The mutations in these strains may lower the transit velocity of protons traversing the cell membrane through MotA. Other ion-channel elements within the flagellar motor probably exist but remain to be identified. A number of models have been proposed to explain the function of the proton-driven motor, including ones based on the most recent data on the dynamics and energetics of motor function (Adam, 1977; Laeuger, 1977; Glagolev and Skulachev, 1978; Khan and Macnab, 1980b; Berg et af., 1982; Berg and Khan, 1983; Khan and Berg, 1983; Khan et a f . , 1985; Blair and Berg, 1990). G. PROTEIN INTERACTIONS WITHIN THE FLAGELLAR MOTOR

A heroic genetic study (Yamaguchi et a f . , 1986a,b) identified proteinprotein interactions within the switch-motor complex of the flagellum of S.

294

M. D. MANSON

typhimurium. Mutations in the three genes WiG, JEiM and JEiN) whose products comprise the complex can have any one of three consequences. Null (or knock-out) mutations lead to a non-flagellated phenotype. A few mis-sense mutations in each gene result in a non-motile (paralysed) phenotype, and a larger number of mis-sense mutations generate alterations in the CW-CCW rotational bias of the motor. Mutations causing non-motile phenotypes are clustered into particular regions in the DNA sequence of each of the three genes (Yamaguchi et al., 1986b). Furthermore, strains with Mot- mutations in each gene give rise to intergenic pseudorevertants, in which a second-site mutation in one or the other of the remaining two genes partially restores motility (Yamaguchi et al., 1986a). This suppression is highly allele-specific (a particular mutation in one gene is supressed by only one or a small subset of mutations in another gene). Because similar results are obtained with switch mutations in these three genes (see later), it appears that the protein products of these genes form a complex. Analysis of mutations affecting the switch-motor complex will be facilitated by the availability of the complete sequences of the wild-type genes from E. coli (Kuo and Koshland, 1986; Malakooti et al., 1989) and S. typhimurium (Kihara et al., 1989). The deduced amino acid-residue sequences themselves are not particularly revealing, except that FliG has clusters of positive and negative charge that could be involved in proton conduction. The FliM and FliN proteins have been found in the membrane fraction (Homma et al., 1988; Malakooti et al., 1989), although neither they nor FliG are predicted to be particularly hydrophobic. The best estimate is that all three proteins associate with the cytoplasmic face of the cell membrane, where they can interact with the M-ring, the Mot proteins and soluble chemotaxis proteins. The MotA protein is quite hydrophobic and is proposed to contain amphipathic helices that form a transmembrane channel (Dean et al., 1984). The probable role of MotA as a proton-channel element (or a sodium ionchannel element in alkalophiles) is bolstered by the finding that overproduction of MotA causes a two-fold decrease in the growth rate of E. coli when a MotB-TetA fusion protein containing the 60 N-terminal residues of MotB is also present in the cell (Blair and Berg, 1990). Overproduction of MotA in the total absence of MotB, or in the presence of wild-type MotB, does not inhibit growth (Wilson and Macnab, 1990; B. Stolz and H.C. Berg, personal communication). One interpretation of Blair and Berg’s result is that the N-terminal portion of MotB interacts with MotA to activate it as a proton-conducting channel that partially uncouples the cell membrane. Intact MotB, if it is localized at the basal body, may couple the MotA channel to the rest of the

BACTERIAL MOTILITY AND CHEMOTAXIS

295

motor, thereby slowing proton flux. Another possibility is that wild-type MotB is inserted into the membrane preferentially at particular sites in the vicinity of the basal bodies, and that these sites are not sufficiently numerous to activate enough MotA channels to generate a debilitating proton leakage. Interaction of MotB with MotA is not essential for their membrane association, since each protein inserts into the membrane when made in the absence of the other (Wilson and Macnab, 1990). However, when MotB is overproduced, it is far less stable in the absence of MotA than in its presence. Based on analysis of fusions to alkaline phosphatase (Manoil and Beckwith, 1985) and partial proteolysis of protoplasts, Chun and Parkinson (1988) concluded that only one N-terminal hydrophobic helix anchors MotB in the cell membrane, with the rest of the polypeptide chain located in the periplasm. This interpretation is consistent with theoretical considerations of the predicted MotB amino acid-residue sequence (Stader et al., 1986). Thus, MotB could provide the link to the cell wall essential for formation of the stator. The MotA protein may be held in position by MotB and either be located at the interface of the stator and rotor or serve to funnel protons to other stator components deeper within the motor. The MotA protein is produced in a 4:l excess relative to MotB in wild-type E. coli (about 600 copies in each cell compared with 150; Wilson and Macnab, 1990), so that each MotB polypeptide may be associated with up to four MotA molecules. The flagellar filaments of Mot- cells are not “locked in gear”; they can be passively rotated with a fluid flow that generates an external driving force (Ishihara et al., 1981). In contrast, non-rotating, de-energized flagella on Mot’ cells resist passive rotation imposed by “optical tweezers” (Block et al., 1989). The tethered cell body can be twisted through about 180” until stiff resistance is met, a compliance that is chiefly due to the flexibility of the hook. When excessive force is applied, the “in-gear” motor breaks, and the cell body rotates freely, like a Mot- cell. My research group is using intergenic suppression to investigate interactions of the Mot proteins with other components of the flagellum. Our preliminary results are as follows: (a) certain mis-sense mutations mapping to the 5’ terminus of motB are suppressed in an allele-specific fashion by mutations in JliM; (b) the suppressing fliM mutations give rise to a nonmotile phenotype in combination with motB+; (c) certain motA mutations are suppressed in a non-allele-specific manner by mutations in the Fla I region. These observations are augmented by the finding that one fliG is weakly suppressed by a mutation in motB (Yamaguchi et al., 1986a). The 5’ location of the suppressible motB mutations is consistent with the predicted cell-membrane association of the N-terminus of MotB. Perhaps MotB is positioned around the motor by an interaction with FliM. The

296

M. D. MANSON

lack of allele-specific suppressors of motA mutations suggests that the mechanism may be indirect. IV. Chemoreception Two major discoveries that charted the course of work on bacterial chemoreception for the ensuing two decades were made near the dawn of the 1970s. One was that bacteria have chemoreceptors in the true sense of the word,,namely proteins that bind to the effector ligands in a stereospecific way. The other was that bacteria sense spatial gradients by means of a temporal mechanism. It is instructive to consider these two breakthroughs and their immediate consequences before proceeding to a more detailed discussion of chemoreception. However, it should be noted that, in addition to being attracted to or repelled by specific chemicals, bacteria exhibit phototaxis (Spudich and Bogomolni, 1984; Haeder, 1987), magnetotaxis (Blakemore, 1982; Frankel and Blakemore, 1989), osmotaxis (Li et af., 1988; Qi and Adler, 1989), galvanotaxis (Adler and Shi, 1988) and thermotaxis (Maeda et al., 1976). A . CLASSICAL PHASE OF CHEMORECEPTION

Adler (1969) provided the first demonstration that E. coli possesses specific receptors that recognize the structure of an attractant chemical rather than the energy released during its metabolism. The data supporting this conclusion fell into six categories: (a) some chemicals that are extensively metabolized fail to attract bacteria; (b) mutant bacteria that have lost the ability to metabolize a chemical can still be attracted to it; (c) some essentially non-metabolizable analogues of metabolizable chemicals attract bacteria; (d) chemicals attract bacteria even in the presence of another metabolizable chemical; (e) attractants that are closely related in structure compete with each other but not with structurally unrelated compounds; (f) there are mutants which fail to carry out chemotaxis to certain attractants but are still able to metabolize them. Hazelbauer and Adler (1971) reported the first characterization of a bacterial chemoreceptor, namely the galactose-glucose-binding protein of E. coli. Later work by Adler’s group demonstrated that a number of sugars (Adler et al., 1973) and amino acids (Mesibov and Adler, 1972) act as attractants. A tabular summary of all known chemoreceptors in E. coli and S. typhimurium is provided by Macnab (1987b). Escherichia coli and S. typhimurium sense spatial chemical gradients by comparing the change in concentration of the chemical as they swim, and

BACTERIAL MOTILITY AND CHEMOTAXIS

297

they respond to those gradients by biasing their three-dimensional random walk. Berg and Brown (1972), using an automated tracking microscope that followed individual, free-swimming bacteria (Berg, 1971), showed that the mean length of a run for E. coli swimming up an attractant gradient or down a repellent gradient was longer than would be expected from random behaviour. This result is achieved by suppression of tumbles. Since CCW flagellar rotation corresponds to running and CW rotation to tumbling (Larsen, 1974b), runs up the gradient are extended because transition of flagellar rotation from CCW to CW is suppressed. The behaviour of an E. coli cell in the presence and absence of a chemotactic gradient is shown in Fig. 2.

FIG. 2. Idealized behaviour of a cell of E. coli or S. typhimurium swimming in the (A) absence or (B) presence of spatial gradients of chemotactically active compounds. The cell in (A) begins at the solid arrow and undergoes nine runs and eight tumbles (tumbles are the abrupt changes in direction). The runs are of variable length, and the net motion is a three-dimensional random walk. The cell in (B) is swimming in the presence of a gradient of attractant (open arrow pointing in the direction of a higher concentration) or repellent (closed arrow pointing in the direction of a higher concentration). The swimming path is identical with that in (A) except that the runs in the favourable direction (up the attractant or down the repellent gradient) have been lengthened by the amount cos 8, where 8 is the angle with the gradient vector. The result is a biased three-dimensional random walk, in which the step length in the favourable direction is increased. The drawing is based on a diagram from Macnab (1987b).

Macnab and Koshland (1972), using a rapid-mixing device discharging into a viewing chamber, showed that S. typhimurium responds to rapid addition of attractants with periods of uninterrupted smooth swimming (running) and to addition of repellents with briefer periods of uninterrupted tumbling. The length of the response to a particular compound depends on the magnitude of the change in concentration. The conclusion from these findings is that bacteria sense the change in concentration of chemicals with time, at least for large jumps in concentration.

298

M. D. MANSON

This conclusion was extended to very gradual temporal changes in concentration by Brown and Berg (1974), who showed that the mean run lengths of E. coli increase in a chamber in which an attractant is generated enzymically in a spatially uniform fashion. The ability to measure changes in concentration with time implies that the cells must be able not only to monitor the instantaneous concentration but also to store information about what the concentration was in the recent past. This chemical memory is provided by the process of adaptation, which is discussed in a later section. At least some gliding bacteria exhibit chemotaxis, such as the chemoautotroph Beggiatoa sp., which uses phobic taxis towards oxygen and hydrogen sulphide to migrate to the optimal depth in cyanobacterial mats (Nelson et af., 1986). Myxococcus xanthus contains developmentally regulated genes with significant homology to chemotaxis genes in E. coli (McBride et al. , 1989; Weinberg and Zusman, 1989; McCleary and Zusman, 1990; McCleary et al. 1990). These genes are called f r z , for “frizzy”, because of the ragged appearance of the aggregates of the mutant strains. Products of the frz genes are needed for normal aggregation and fruiting-body development, and frz mutants show altered motility patterns (Blackhart and Zusman, 1985). It seems likely that the relatively slow-moving gliding and swimming bacteria will employ different mechanisms for sensing chemical gradients. B . IDENTITY OF BACTERIAL CHEMORECEPTORS

The galactose-glucose-binding protein (GBP) is shared by the high-affinity galactose transport and galactose chemotaxis systems. However, mutant GBP from E. coli mutant strain AW551 is unaltered in galactose transport but non-functional in galactose chemotaxis (Hazelbauer and Adler, 1971). This discovery showed that the transport and chemoreceptor functions of GBP are separable phenomena. Subsequent work demonstrated that the periplasmic ribose-binding protein (RBP; Aksamit and Koshland, 1974) maltose-binding protein (MBP; Hazelbauer, 1975; Brass and Manson, 1984) and dipeptide-binding protein (DBP; Manson et al., 1986; Blank, 1987; Abouhamad et al. , 1991; Olson et al., 1991) are all chemoreceptors in E. coli, and all are also components of high-affinity transport systems for the attractants they bind. The majority of periplasmic binding proteins in E. coli function in transport but not in chemotaxis (Furlong, 1987; Macnab, 1987b). The three structurally similar proteins GBP, RBP and arabinose-binding protein (ABP; Vyas et al., 1991) are noteworthy in this regard; the first two are chemoreceptors while ABP is not. In GBP from strain AW551, residue G1y74 is replaced with an aspartate residue (Scholle et a f . , 1987). This

BACTERIAL MOTILITY AND CHEMOTAXIS

299

substitution perturbs a salt bridge between residues G ~ Uand , ~ Argsl of GBP and displaces five of six water molecules bound to the wild-type protein in this region (Vyas et al., 1988). The hydration energy associated with .these water molecules may be important in the interaction of wild-type GBP with other chemotaxis proteins. The Gly,, residue of R S P is in a structural region analogous to the region containing residue Gly, of GBP, whereas in this part of the primary sequence the ABP structure diverges from those of GBP and RBP. Two additional sites present at the periphery of GBP and RBP, but absent from ABP, may also be specifically involved in chemoreception. Transport as such is required to elicit the chemotactic response to sugars that are phosphorylated during uptake by the phosphotransferase system (PTS), although the response does not involve further metabolism of the sugar (Adler and Epstein, 1974; for reviews of the PTS and the chemotaxis it mediates, see Postma and Lengeler, 1985; Saier, 1989). The membranebound, sugar-specific enzyme I1 for any sugar transported by the PTS is a chemoreceptor. Enzyme I1 phosphorylates the sugar, an event that links transport into the chemosensory pathway (see later). However, extensive searches have failed to yield any chemotaxis-specific mutations affecting an enzyme 11, and it seems unlikely that there is any unique chemoreceptor (as distinct from transport) function associated with the PTS. Molecular oxygen is another attractant for which chemosensing is linked to metabolism. The attractant response to oxygen is processed through cytochrome oxidase (Shioi et al., 1988), which reduces oxygen to stimulate electron transport and to boost the proton-motive force. The response to oxygen seems to be a special case of proton-motive force chemotaxis (Taylor, 1983; Shioi andTaylor, 1984). In general, conditions that lead to an increase in the proton-motive force are responded to as attractants, and conditions that lead to a decrease in the proton-motive force are responded to as repellents (Miller and Koshland, 1977, 1980; Berg et al., 1982). Many repellents may act by diminishing the proton-motive force. The chemoreceptors that communicate most directly with the central processing machinery are the chemotactic signal transducers (transducers for short). Escherichia coli has four transducers (Boyd et al., 1981), whose genes have been sequenced (Boyd et al., 1983; Krikos et al., 1983; Bollinger etal., 1984). They include the serine transducer (Tsr), the aspartate-maltose transducer (Tar), the dipeptide transducer (Tap) and the ribose-galactoseglucose transducer (Trg). The tar gene of S. typhimurium has also been sequenced (Russo and Koshland, 1983); surprisingly, the Tar protein of this species does not mediate maltose chemotaxis (Dahl and Manson, 1985; Mizuno et al., 1986). Since S. typhimurium responds to serine, ribose, galactose and glucose, it is likely that it also has Tsr and Trg. In fact, the original data demonstrating that RBP and GBP compete for a single

300

M. D. MANSON

signalling site was obtained with S . typhimurium strains (Strange and Koshland, 1976). Two other transducers, one mediating a strong repellent response to phenol (Imae et a f . , 1987) and another of unknown function, named Tip (for taxis-involved protein; Russo and Koshland, 1986), have been reported in S. typhimurium. A related motile and chemotactic enteric species, Enterobacter aerogenes, has two transducers, named Tse and Tas, whose genes have been cloned and sequenced (Dahl et al., 1989). The tse gene of E. aerogenes is homologous to tsr and complements the serine-taxis defect of E. cofi tsr mutants; the Tsr and Tse proteins are 60% identical in their periplasmic receptor domains. The tas gene of E. aerogenes complements the aspartate-taxis defect (but not the maltose-taxis defect) of an E. cofi tar mutant. However, the predicted amino acid-residue sequence of the periplasmic receptor domain of Tas diverges greatly (only 20% identity) from that of E. cofi or S. typhimurium Tar, which are themselves 70% identical in this domain. Another related but non-motile species, Kfebsieflapneumoniae, contains at least two genes encoding transducers (Dahl et a f . , 1989). One of these genes can complement the serine-taxis defect of an E. cofi tsr mutant. The other does not complement any known specific taxis defect, although it restores run-tumble behaviour to an E. coli strain that swims smoothly because it lacks both Tsr and Tar (see later). It is unknown what, if any, function the transducers have in K . pneumoniae, or even if they are expressed in that species. The transducers are membrane-associated polypeptides (Ridgway et a f . , 1977) comprising 510-557 amino-acid residues. Their topology in the membrane is based both on theoretical considerations (Krikos et a f . , 1983) and analysis with TnPhoA fusions (Manoil and Beckwith, 1985, 1986; Gebert et a f . , 1988). The transducers have two helical hydrophobic regions that span the membrane. The N-terminus is in the cytoplasm and contains several positively charged residues that are followed by a stretch of hydrophobic residues that constitute the first transmembrane region (TM,). The positively charged residues and TMI resemble, in structure and function, the leader peptide (signal sequence) of an exported bacterial protein (Manoil and Beckwith, 1986; Gebert et a f . ,1988), but no cleavage of the leader occurs with the transducers. Next in sequence is the periplasmic receptor domain, which comprises about 160 amino-acid residues. The second hydrophobic transmembrane region (TM,) brings the polypeptide back through the membrane to the cytoplasmic signalling domain, which contains 300-350 residues. During membrane assembly of Tsr, TM2 acts as a stop transfer sequence (Manoil and Beckwith, 1986). Hazelbauer et af. (1990) have proposed a generalized model for the structure of a transducer monomer that invokes four-helix bundles in the periplasmic and cytoplasmic domains.

BACTERIAL MOTILITY AND CHEMOTAXlS

301

Transducers can be primary or secondary chemoreceptors, or both. The Tsr protein has been shown to be only a primary chemoreceptor. It binds L-serine with high affinity and many other amino acids with lower affinity (Mesibov and Adler, 1972; Springer et al., 1977b; Hedblom and Adler, 1980), and it is a receptor for the repellents L-leucine, indole and low external p H values (Tsang et al., 1973; Tso and Adler, 1974). It also functions as a thermoreceptor (Maeda et al., 1976; Lee et al., 1988), with temperatures up to 37°C acting as attractants. Serine at high concentrations blocks thermosensing (Maeda and Imae, 1979), presumably by inducing a conformation of Tsr in which the protein is no longer capable of responding to changes in temperature. Finally, Tsr mediates a repellent response to weak organic acids, which are detected because they lower the cytoplasmic pH value (Kihara and Macnab, 1981; Repaske and Adler, 1981). Recent evidence (Eisenbach et al., 1990a) implies that transducers themselves are low-affinity receptors for repellents rather than that transducers respond to changes in general properties of the membrane, such as its fluidity, induced by repellents. The Tar protein of S . typhimurium is also exclusively a primary chemoreceptor. It binds aspartate with high affinity (Clarke and Koshland, 1979) and some other amino acids with lower affinity. It also mediates the repellent response to cobalt and nickel ions (Tsang et al., 1973; Tso and Adler, 1974). The Tar protein of E. coli is more versatile, possessing all of the activities of that from S . typhimurium and interacting with the maltosebound MBP to initiate the response to that disaccharide attractant (Mesibov and Adler, 1972; Springer etal., 1977b). In the absence of Tsr, Tar mediates a positive response to increasing temperature, but the response is inverted by aspartate. An inverted response is also obtained with wild-type cells when serine and aspartate are both present (Mizuno and Imae, 1984). Weak organic acids and phenol act as attractants when sensed via Tar in E. coli (Kihara and Macnab, 1981; Repaske and Adler, 1981; Imae et al., 1987). The other two transducers are only secondary receptors for attractants. The Trg protein from E. coli or S. typhimurium can bind to ribose-bound RBP, or to galactose- or glucose-bound GBP, to mediate responses to those sugars (Hazelbauer and Adler, 1971; Kondoh et al., 1979; Hazelbauer and Harayama, 1981). The Tap protein, which is present only in E. coli, interacts with DBP to mediate taxis toward dipeptides (Manson et al., 1986; Abouhamad et al., 1991; Olson et al., 1991). Both Trg and Tap from E. coli can serve as repellent receptors for phenol, whereas Trg is an attractant receptor and Tap a repellent receptor for weak organic acids (Yamamoto et al., 1990). It was suggested that Tap duplicates the chemosensing functions of Tar (Wang et al., 1982), but later work found no evidence for this redundancy (Slocum and Parkinson, 1985).

302

M. D. MANSON

The transducers are not present in equal numbers in E. cofi (Koman e t a f. , 1979; Hazelbauer and Harayama, 1983) or in S. typhirnuriurn (Clarke and Koshland, 1979). The Tsr protein is the most abundant, with an estimated 2000-2500 molecules in each cell, while Tar has 1000-1500 copies in each cell, making these the two major transducers. Both Trg and Tap occur in several hundred copies in each cell (Koman et a f . , 1979; Slocum and Parkinson, 1983) and are collectively referred to as minor transducers.

C. CELLULAR DISTRIBUTION OF CHEMORECEPTORS

The binding proteins that serve as chemoreceptors are presumably distributed throughout the periplasmic space. The signal transducers could be randomly distributed in the cell membrane or localized to particular regions. The distribution must be compatible with the mechanism of signal transduction between the transducers and flagella, and should reflect the optimal strategy for sampling the chemical environment. A theoretical argument in favour of random distribution of receptors on the cell surface is made by Berg and Purcell(1977), an argument based on the properties of molecular diffusion in the vicinity of a surface, and receptors are treated as perfectly absorbing patches. If receptors are scattered randomly, only one-thousandth of the cell surface needs to be covered for a molecule to be captured with 50% efficiency compared with a situation in which the entire surface is absorbent. The reason is that a molecule tends to have a rather long dwell time on the surface, since Brownian motion causes it to collide many times with a circumscribed area of the surface. If receptors are patchily distributed, capture is much less efficient. In contrast, photoreceptors, suffer no disadvantage from a patchy. distribution, since photons do not diffuse. The finding that signal transducers are not localized near flagellar basal bodies (Engstroem and Hazelbauer, 1982) is consistent with their random dispersal.

D. STRUCTURE AND LIGAND INTERACTIONS OF CHEMORECEPTORS

By far the best understood ligand-chemoreceptor interactions are those involving periplasmic substrate-binding proteins. High resolution X-ray diffraction studies of GBP from E. coli (Vyas et a f . , 1988) and S. typhirnuriurn (Mowbray and Petsko, 1983) have revealed both the overall architecture of the protein and the configuration of the binding site. Like other binding proteins whose structures are known, GBP consists of large N-terminal and C-terminal domains, joined by a “hinge” made up of three peptide strands. The domains consist of alternating, antiparallel p-strands

BACTERIAL MOTILITY AND CHEMOTAXIS

303

and a-helices, with a P-sheet sandwiched between arrays of helices. Substrate binds in the cleft between the domains. Galactose and glucose can bind with equal, high affinity to GBP (Vyas et al., 1988). The 4-position hydroxyl group of the two epimers can form equivalent hydrogen bonds with the same aspartate residue in the binding site, although a different oxygen atom in the carboxyl group is involved in each case. The amino acid-residue sequence of RBP (Groarke et al. , 1983) maps very well onto the three-dimensional structure of GBP (Vyas et al., 1991), suggesting that the two proteins may interact in a similar fashion with Trg and with the membrane components of the transport systems to which they belong. The structure of MBP at 0.23 nm resolution has recently been published (Spurlino et al., 1991). The tertiary structure of MBP is quite dissimilar from that of GBP or RBP. Binding of substrate to GBP, RBP or MBP causes conformational changes in the proteins that can be detected by alterations in electrophoretic mobility (Boos et al., 1972) and isoelectric point of the protein (Boos et al., 1981; Mowbray and Petsko, 1982; Dahl and Manson, 1985). Substrate binding also changes intrinsic tryptophan fluorescence (Boos et al., 1972; McGowan et al., 1974; Zukin et al., 1977; Zukin, 1979) or the fluorescence of reporter groups (Zukin et al., 1977; Zukin, 1979). In the absence of substrate, the binding proteins are in an open configuration, in which the cleft stands agape. Bound substrate stabilizes a closed conformation of the protein in which the hinge has moved through an angle of 18”.The substrate is buried deeply within the cleft and is inaccessible to the medium, a circumstance that contributes to low dissociation rates of the binding proteins. Binding of the substrate may also cause “tweaking” of components of the secondary structure on the surface of the protein. Changes in the relative positions of the domains and relatively subtle reorientation of the secondary structure may both affect the interaction of binding proteins with other polypeptides. Specific point mutations affect the affinity of the histidinebinding protein (Ames and Spudich, 1976) and MBP (Treptow and Shuman, 1985, 1988; Duplay et al., 1987) with their respective membrane-bound partners in transport. Mutations affecting the interaction of binding proteins with signal transducers are discussed in the next section. It should be kept in mind that, because the induced levels of periplasmic binding proteins can be very high (1 mM for MBP (Dietzel et al., 1978), a 20- to 40-fold excess relative to Tar), the affinity of the binding proteins for transducers need not be high. The K D value for interaction of maltoseloaded MBP with Tar has been estimated to be about 250 PM and, with the membrane-associated transport components, to be about 90 p~ (Manson et al., 1985). This low affinity between MBP and Tar permits the cells to perceive changes in maltose concentration over a large range instead of

304

M. D. MANSON

having the response saturate when only a small fraction of MBP is ligand bound. No explicit structural data have been published for the periplasmic domain of a signal transducer. However, a model based on the predicted amino acid-residue sequence of the receptor domain of Tar from S. typhimurium has been proposed (Moe and Koshland, 1986). It is suggested that the polypeptide chain ascends from TMl through an a-helix that culminates in an apical loop (AL,) that connects to a descending a-helix. The descending helix is joined to another ascending a-helix that connects through a second apical loop (AL,) to a descending a-helix that leads to TM2. The two ascending and two descending a-helices comprise a four-helix bundle. It it thought that AL, and AL, are roughly antiparallel and may themselves contain a substantial amount of a-helical structure. Considerable evidence indicates that ligands interact with ALl and A b . Wolff and Parkinson (1988) found that three arginine residues (Arg@,Arg69 and Arg73) located in the N-terminal half of AL1 are critical for aspartate sensing by Tar from E. coli. These residues may come together to create a positively charged arginine pocket that could be important for aspartate binding and for maintaining and altering the conformation of this region of the protein. Mutant Tar proteins with substitutions at these residues show decreased affinities for aspartate (about 100 lower for changes at Arg73, 1000 times lower for changes at Arg6,, and more than 10,000 times lower for changes at Mowbray and Koshland, 1990). Lee and Imae (1988, 1990) have shown that Arg,, and Thr154residues (ThrlS6in Tsr) are crucial for aspartate sensing by Tar and for serine sensing by Tsr. The Thr154 residue in AL2 may be located opposite the Arg6, residue in AL1. These authors suggest that the a-carboxyl group and the a-amino groups of the ligand form ionic or hydrogen bonds with residues Arg,, and Thr154(ThrlS6),respectively. Analysis of their periplasmic domains reveals that all of the transducers from E. coli, S . typhimurium and E. aerogenes that respond to amino acids have the three arginine residues and the sequence Gln-Pro-ThrGln at residues 152-155 (E. coli Tar numbering) conserved (Dahl and Manson, 1989). These three arginine residues are not present in Trg and Tap, although arginine residues in different positions in the primary sequence of AL, from Trg may come together to form an arginine pocket (Hazelbauer et al., 1990). Many repellents with diverse chemical properties have been identified for E. coli and S. typhimurium. Most repellents are toxic to some degree, including indole, phenol, organic acids, alcohols and heavy-metal ions, although leucine, isoleucine and valine are also repellents (Tsang et al., 1973; Tso and Adler, 1974). Even though most repellents are sensed by the transducers, very little is known about the manner in which they are

PLATE 1. Two stereo pairs (3" offset) of a peptide backbone model of the maltose-binding protein from Eschen'chia coli. The protein is in the closed, ligand-bound conformation. (a) The protein viewed from the side of the cleft, with the N-terminal domain to the left and the regions that interact with Tar facing downward. (b) The protein viewed looking towards the opening of the cleft, with the regions that interact with Tar facing the viewer. The interacting regions are shown in red for continued overleaf

the N-terminal domain and in blue for the C-terminal domain. Residues 53, 55, 342, 345 and 367 (see text and Table 1) are shown in green with a diameter of one van der Waal's radius. Other residues are labelled to help orient the viewer, including the N-and C-termini of the protein and the beginning and end of the red and blue regions, as well as one residue in the middle of the blue region. The co-ordinates used in preparing this model were taken from Spurlino (1988). In regions important for chemotaxis, the model closely reflects the structure recently published by Spurlino et al. (1991).

BACTERIAL MOTILITY AND CHEMOTAXIS

305

recognized. Studies of chimeric transducers revealed that sensors for leucine (Tsr), nickel ions (Tar) and external pH values (Tsr and Tar) are in the periplasmic domain (Krikos et al. , 1985). In contrast, sensing of cytoplasmic pH values involves titration with hydrogen ions of a histidine residue in the cytoplasmic domain (Krikos et al. , 1985). A pK, value of 7.5 for histidine protonation makes this amino-acid residue a good candidate for a cytoplasmic p H sensor. This distribution of receptor functions is consistent with the observation that the periplasmic and cytoplasmic domains of Tar from S. typhimurium are preserved as stable, functional proteolytic fragments (Mowbray et al., 1985). Glycerol and ethylene glycol are repellents that are sensed at high concentration by any of the transducers (Oosawa and Imae, 1983). These molecules can dissolve into the membrane and change its thickness. One possible interpretation of this response is that alterations in the local environment of the transmembrane regions of the transducers may initiate signalling in some instances, and that these effects may provide general insight into the signalling mechanism (see later). . E. STIMULATION OF TRANSDUCERS BY ATTRACTANTS AND BINDING PROTEINS

My own laboratory has been studying how Tar from E. coli interacts with ligands as diverse as MBP and small molecules. The latter include glutamate, methionine and other amino acids, as well as dicarboxylic acids such as succinate, all of which bind with lower affinity than aspartate (Mesibov and Adler, 1972). The initial step was to identify mutations that disrupt only the chemotactic function of MBP (Manson and Kossmann, 1986). Three mutations of this type substitute residues Thr53 of MBP with isoleucine (T53I), Asp,, with asparagine (D55N) and Thr345 with isoleucine (T345I). Subsequent mutational analysis (Y. Zhang, C. Conway, K. Mossman, Y. Sub and M. D. Manson, unpublished observations) has identified regions of MBP that are specifically involved in chemotaxis (Table 1and Plate 1 (opposite)). With reference to the structure of MBP (Spurlino et al. , 1991), these include a-helix I1 and the two turns that flank it (residues 38-57) in the N-terminal domain and the penultimate and ultimate a-helices (XI11 and XIV) in the C-terminal domain (residues 337-370). Also, a deletion-substitution that replaces residues 364-370 of MBP with 11 other residues is partially defective in maltose transport and seriously defective for chemotaxis (Duplay and Szmelcman, 1987). Certain residues within these three helices are more critical for function than others, and the N-terminal domain is affected by mutations at more different sites than is the C-terminal domain. The AspSs residue apparently is a key element in the interaction of MBP

306

M. D. MANSON

TABLE 1. Amino acid-residue substitutions in the maltose-binding protein of Escherichia coli that cause specific defects in maltose taxis Amino acid-residue change

Residue number N-terminal domain

40 45 46 50 53 54 55

Pr-Glu GlujLys LywGlu ValhAla ValjPhe Thr-Ala ThrjIle Gly-Ile AspAla A s p Asn AspGly

Amino acid-residue change

Residue number N-terminal domain

56 62

Gly-tIle TWGlY TgLeu TgSer

C-terminal domain

342 345 361

Ala-Val Ala-Asp Thr-Ile ArgjCys

The amino acid-residue substitutions shown in this table were chosen on two criteria. First, they M maltose and lower the rate of cause loss of a chemotactic ring on swarm plates containing swarm expansion on such plates by at least 25%. Second, they permit cells to transport maltose at concentrations of 3 x lo-' M and 3 x M at rates at least 40% of the rate of cells containing the wild-type MBP.

with Tar. It is located at the apex of the turn that is C-terminal to a-helix 11,it is flanked by two glycine residues, and it is exposed and accessible. Since glutamate is the only other amino-acid residue we tested that functions reasonably well at this position, we believe the negative charge of the Asps5 residue is important. It is also likely to be important that helices 11, XI11 and XIV are on one surface of the protein and are distributed on both sides of the substrate-binding cleft. An attractive hypothesis (Spurlino et al., 1991) is that, when MBP is in the open conformation, helix I1 and helices XIII-XIV are the wrong distance apart to interact simultaneously with Tar. In the closed conformation, stabilized by maltose, these two regions may be brought into the correct spatial relationship to match complementary sites in Tar. Amino acid-residue substitutions in Tar can partially suppress defects in maltose taxis caused by the T53I and D55N substitutions in MBP (Manson and Kossmann, 1986). The R73W change gives the best suppression, and R73Q and R69C substitutions also suppress to some extent (Kossmann el al., 1988). In a parallel search, we did not find tar mutations that suppress the T345I substitution in MBP. These findings led to a mutational analysis of the periplasmic domain of Tar (P. Gardina, C. Conway, M. Kossmann and M. D. Manson, unpublished observations). The results are presented in Table 2 and Fig. 3 and can

307

BACTERIAL MOTILITY AND CHEMOTAXIS

TABLE 2. Amino acid-residue substitutions in the AL, and AL, segments of Tar from Escherichia coli that affect aspartate or maltose taxis. ALI Residue number

Amino acid-residue change

Aspartate chemotaxis-negative 64 Arg-tCys Arg+His Arg-Lys 69 Arg-tCys Arg+Gl y Arg+ His Arg+Lys Arg+Ser 70 Ser-tIle 73 Arg+Gln Arg+Trp Aspartate and maltose chemotaxis-negative 73 Arg+Lys Maltose chemotaxis-negative 75 Met+ Arg Met-Lys 76 Met+Arg Met+Lys Met-tThr 77 AspHis 83 Ser+ Arg

A L Residue number 154 152

Amino acid-residue change Thr-tlle Gln+Leu Gln+Lys

150 149

Phe+Ser Tyr+Ser

145 143

Asn+Lys Tyr-t Asn Tyr+Cys Tyr+Ser Leu+ Arg

141

A mutation is designated as having an aspartate chemotaxis-negative or maltose chemotaxisnegative phenotype if it decreases the rate of swarm expansion in soft agar containing the attractant at M to 70% or less of the rate for a Tar+ cell. All of the mutant Tar proteins support normal run-tumble behaviour when they are present as the sole major transducer. The residues are listed in reverse order for AL2 to reflect that it is antiparallel to AL,.

be summarized as follows: (a) mutations that primarily affect aspartate, as opposed to maltose, sensing are located in the portions of ALl and AL, that are closest in the primary sequence to TMI and TM2, respectively; (b) mutations that exclusively affect maltose sensing are located in the portions of AL, and AL, that are farthest from TMl and TM2; (c) in each loop one or more residues near the intersection of the aspartate- and maltose-sensing portions of ALl and AL, affect aspartate and maltose sensing about equally. Separation of the aspartate- and maltose-sensing portions of the receptor domain into two distinct but overlapping regions is, in our view, consistent

308

M. D. MANSON

FIG. 3. Model for the periplasmic domain of the Tar chemotactic signal transducer. The cylinders represent a-helices that are thought to be organized in a four-helix bundle. The pairs of helices leading to and from each of the apical loops have been drawn apart to enable easier visualization of the loop regions. The “beads” on the loops represent individual residues (see Table 2). Residues at which mutational changes interfere primarily with aspartate taxis are striped, residues at which mutational changes interfere primarily with maltose taxis are stippled, and residues at which mutational changes interfere with both aspartate and maltose taxis are solid. The numbers identify the beginning and ending residues of each loop. The structure is based on the model proposed by Moe and Koshland (1986).

with the partial independence and semi-additivity of the aspartate and maltose responses mediated by Tar (Mowbray and Koshland, 1987). Our model for the function of the periplasmic domain of Tar is based on the idea that, in the absence of ligand, ALl and AL2 interact strongly with one another, perhaps through hydrogen bonding (Fig. 4). Candidates for such bonding are the arginine residues at positions 64,69 and 73 in ALl and the Tyr149, Gln152,Thr154and GlnlS5residues in AL;?. These interactions may stabilize some conformation of the proposed four-helix bundle in the periplasmic domain of Tar (Moe and Koshland, 1986). Small ligands would

BACTERIAL MOTILITY AND CHEMOTAXIS

309

FIG. 4 Possible hydrogen-bonding interactions between AL1 and AL2 in the Tar chemotactic signal transducer. The region shown corresponds only to the side facing away from the viewer in Plate 1. The curvature was included to bring potentially interacting residues into proximity and has no known structural basis. Possible hydrogen bonds between the loops are shown as dashed lines. Arginine residues are considered to be hydrogen donors, while hydroxyl-containingamino-acid residues and amide-containing amino-acid residues can be either acceptors or donors.

interpose themselves between ALl and A b , prying the two loops apart. All amino-acid ligands probably interact with Arg,, and Thr15,, as postulated by Lee and Imae (1988, 1990). The interaction of the P-carboxyl group of aspartate with Arg,, and Arg73 would enhance binding (Mowbray and Koshland, 1990). Amino-acid residues that are not sterically hindered from binding between AL, and AL, may serve as low-affinity attractants. Dicarboxylic acids, such as succinate, could disrupt the A L l - A b interaction by binding to the arginine residues of ALl ,perhaps with involvement of residues of A b . Amino acid-residue substitutions that disrupt hydrogen bonding between ALI and AL2 may mimic events associated with attractant binding. We are examining whether such mutations may weaken the interaction of ALl with AL, sufficiently that the transducer will continually generate an attractant signal as though it were adapted to an attractant (see later). A similar mechanism may operate for interaction of MBP with Tar. Patterns of intergenic suppression (Manson and Kossmann, 1986; Kossmann el al., 1988) suggest that the and Asp,, residues of MBP interact with residues Arg6, and Arg73 of Tar. The a-helix I1 of MBP may associate with portions of ALI and AL2 to position residue near residues Arg,, and Arg,, of Tar. Residue Thr53 of MBP may strengthen the interaction by hydrogen bonding through its hydroxyl group. Hydrogen bonding between

310

M. D. MANSON

ALI and AL2would be disrupted in a manner similar to the one proposed for amino-acid or dicarboxylic-acid attractants. We do not know if Tar interacts with the C-terminal domain of MBP (helices XI11 and XIV), or whether an MBP molecule contacts only one or both subunits of the Tar dimer (see later). Investigations with Trg provide further information about the interaction between transducers and binding proteins. The R85H mutation in Trg impairs galactose taxis and almost completely eliminates ribose taxis, whereas the G151D mutation eliminates ribose taxis but leaves galactose taxis essentially unaffected (Park and Hazelbauer, 1986). The residues affected by these mutations are located in the two proposed apical loops of the receptor domain of Trg. Also, the first apical loop in Trg can be divided into two functionally distinct regions (Hazelbauer et al. , 1990). Mutational changes N-terminal to residue Gln79 lead to overmethylation of transducer in the absence of an attractant. Mutational changes C-terminal to residue Gln79 prevent Trg from responding to attractant, as though the mutant protein cannot interact effectively with GBP or RBP. These results are consistent with our findings for Tar, and suggest that Tar and Trg interact with the respective binding proteins in an analogous manner. .V. Chemotactic Signal Transduction

Until the last few years the central question in bacterial chemotaxis was: “How do the chemoreceptors communicate with the flagellar motor? ” This aspect of chemotaxis is now understood in considerable detail at the molecular level, while chemoeffector-chemoreceptor interactions, transmembrane signalling and switching events at the flagellar motor remain much less clear. The previous section discussed chemoreceptor structure and ligand binding. This section considers the subsequent steps in signal transduction. Much of the information presented in this section was garnered very recently; the only biochemical data published more than five years ago concern covalent modification of the signal transducers during adaptation. A. TRANSMEMBRANE SIGNALLING

Chemotactic signalling through the transducers is initiated by binding of the chemo-effector to the periplasmic receptor domain. Since the cytoplasmic domain of the transducers is the source of the intracellular signal (Krikos et al., 1985; Mutoh et al., 1986; Ames and Parkinson, 1988), conformational changes in the external receptor domain must be propagated through the

BACTERIAL MOTILITY AND CHEMOTAXIS

311

membrane to the cytoplasmic domain. To understand how this communication occurs, we must first consider the quaternary structure of the transducers. The first reports on subunit organization of the transducers suggested that they were homotetramers (Chelsky and Dahlquist, 1980a; Foster et al., 1985). More recent work exploiting site-directed cross-linking has favoured the idea that transducers exist as homodimers (Falke and Koshland, 1987; Milligan and Koshland, 1988). No available evidence discriminates between the possibilities that the transducers bind one chemo-effector molecule on each monomer or one on each dimer. With multimeric transducers, two basic strategies for transmembrane signalling are possible. The first relies on intramolecular events, the dimer having a primarily structural role rather than being essential to the signalling mechanism as such. In the second strategy, ligand-induced changes in the interaction of the subunits in their periplasmic domains modify the relative positions of the two cytoplasmic domains to create o r abolish a signalling site, as suggested by Krikos et af. (1983). Co-operativity has not been observed in the response to attractants (Manson et af., 1985; Mowbray and Koshland, 1990). However, there is recent evidence that intermolecular contacts may be involved in transducer-mediated signalling (Milligan and Koshland, 1991). The ligand-binding domain of Tar can be produced as a soluble protein of 18 kDa. This molecule forms a monqmer at low protein concentration and a dimer at higher concentration. The protein dimerizes even at low concentration when it is exposed to aspartate, suggesting that ligands may also be able to alter the interaction of the periplasmic domains of the transducer in an intact dimer as well. A major source of information on interactions of the transmembrane regions within the dimer has come from site-directed cysteine mutagenesis with the Trg (Hazelbauer et af., 1990) and Tar (Falke et af., 1988) transducers. Cysteine residues may be introduced into the transmembrane regions to determine which positions are protected from sulphydryl reagents such as N-ethylmaleimide by packing interactions with adjacent transmembrane helices. Another approach is to introduce a cysteine residue at a different position in TM, o r TM2 in two different copies of a transducer, and to mix the substituted transducers in a single cell. The residues that are in contact should form disulphide cross-bridges upon oxidation. The substitutions can be projected onto a helical wheel, and surfaces of interaction defined. Such analysis has led to the conclusion that the closest contact is between the two TMI regions of a dimer, and that TMI may also interact with TM2 in the same polypeptide and in the other subunit of the dimer. There is no evidence for contact between the TM2 regions of the two subunits.

312

M. D. MANSON

Theoretical considerations have led to the conclusion that the transmembrane a-helices of a transducer homodimer form a closely packed structure (Falke et al., 1988; Hazelbauer et al., 1990). In contrast, the transmembrane regions of at least some combinations of transducers in heterodimers are not predicted to form closely packed structures (G. Burrows and G. Hazelbauer, personal communication). The inability to form a stable transmembrane structure may be effective in preventing assembly of transducer heterodimers in the cell, and no evidence for their existence has been obtained. Substitutions in the transmembrane regions of transducers that introduce residues generally incompatible with membranes into the transmembrane regions of transducers often alter or disrupt their function (Oosawa and Simon, 1986; Ames and Parkinson, 1988). In some instances, such mutations can be supressed by intragenic second-site mutations. For example? a mutation that introduces a lysine residue at position 19 of Tar in the middle of TM, causes a dominant CW-biased phenotype. This mutation can be phenotypically suppressed by introduction of glutamate residues at position 17 in TMI and at positions 198 and 201 in an opposing region of TM2 (Oosawa and Simon, 1986). The notion is that a salt bridge between the lysine and glutamate residues may ameliorate the effects of placing charged residues into the transmembrane regions. The same lysine-residue substitution in TM, is also suppressed by several non-polar substitutions in TMI and TM2 and by a number of mutations that cause amino acid-residue replacements in the linker region (Oosawa and Simon, 1986; Ames and Parkinson, 1988), which is located in the cytoplasmic domain immediately C-terminal to TM2. This latter result suggests that TMI and the linker region interact, or that a conformational “quirk” generated by the substitution of a lysine residue in TMI is compensated for by the change in the linker region. Mutations in TMI can also be suppressed by mutations in a non-helical portion of the periplasmic domain immediately N-terminal to TM2 (J. S. Parkinson, personal communication). Viewed together, the findings already described point out that transmembrane signalling involves interactions among the transmembrane regions and between these regions and the adjacent periplasmic and cytoplasmic domains. A likely scenario is that binding of an attractant or a repellent generates relatively subtle shifts in the conformation of the postulated four-helix bundle in the periplasmic domain. These changes in turn lead to intra- and intermolecular distortion within the transmembrane region, which in turn alters the shape of the linker regions and, ultimately, the conformation and the activity of the signalling domains. A hypothetical model for transducer structure based on this scheme was presented in a recent review by Hazelbauer et al. (1990).

BACTERIAL MOTILITY AND CHEMOTAXIS

313

E. INTRACELLULAR SIGNALLING

Pathways for intracellular signalling during the chemotactic response in E. coli or S. typhimurium are shown in Fig. 6, which is placed near the end of this review to serve as a summary. This diagram results from 20 years’ effort in over a dozen laboratories and incorporates the results of hundreds of research papers. However, it was only within the past four years that the biochemical essence of the scheme, a cascade of protein phosphorylation, was elucidated. The characterization of the chemotactic signal followed two historical paths-ne genetic, the other physiological. Biochemical approaches contributed significantly only when the proteins that mediate the response were purified and added to in vitro or reconstituted in vivo systems. I will follow the genetic and physiological threads forward in time to show how they joined with biochemical data to culminate in our current understanding of the signalling process.

I . Genetics of Signalling Genes whose products are involved in chemotaxis to all attractants were first described by Armstrong et al. (1967), Armstrong and Adler (1969a) and Parkinson (1974,1976). There are six genes (Aswad and Koshland, 1975b; Collins and Stocker, 1976; Silverman and Simon, 1977a; Warrick et al., 1977; Parkinson, 1978; Parkinson and Houts, 1982) whose products are soluble proteins (Ridgway et al., 1977) that are required for normal chemotaxis (Macnab, 1987b). The genes can be divided into three groups based on the phenotypes of null mutations (mutations leading to a complete elimination of a functional gene product). Null mutations in the cheA, c h e w and cheY genes result in cells that are unable to tumble because their flagella turn only CCW; these mutations eliminate any response to repellents, which would normally cause CW rotation. Strains with null mutations in the cheR gene are smooth swimmers until they encounter a repellent, which causes them to tumble incessantly (Goy et al., 1978; Parkinson and Revello, 1978). Strains lacking CheR are defective in adaptation (see later) because they lack the ability to methylate transducers (Springer and Koshland, 1977; Silverman and Simon, 1977b; Springer et al., 1977a). Null mutations in the cheB and cheZ genes lead to a CW-biased, tumbly phenotype; cheB mutants are unable to demethylate transducers and are also defective in adaptation. Both cheB and cheZ mutants can respond with CCW flagellar rotation to large jumps in attractant concentration. It is noteworthy that mutations in each of the che genes produce abnormal patterns of swimming in the absence of chemotactic stimulation.

314

M. D. MANSON

The CheA protein is made with two different translation starts (Smith and Parkinson, 1980; Kofoid and Parkinson, 1991). This dual initiation leads to the synthesis of two polypeptides that differ in mass by 11,000 Da, namely CheAL (71 kDa) and CheAs (60 kDa). The CheAL protein is absolutely required for chemotaxis and CW rotation, but CheAs appears to have a more subtle, modulatory role (Matsumura et al., 1990; A. Wolfe, personal communication). Two of the che genes identified in early studies, namely cheC and cheD (Parkinson, 1974, 1976), turned out to represent specific alleles of genes whose null phenotypes are, respectively, non-flagellate and tsr-. The original cheC mutations caused smooth swimming. Later, tumbly cheC mutants were isolated; the CW-biased and CCW-biased mutants both contain mutations in fliM, one of the switch-motor complex genes (Parkinson et al., 1983). The cheD mutations, which also cause smooth swimming, are dominant over tsr+ and produce a locked signal that maintains a perpetual CCW flagellar rotation (Ames and Parkinson, 1988). These CCW-locked mutations map primarily in the region of tsr that codes for the cytoplasmic signalling domain, but some of them alter the transmembrane and linker regions. There are also dominant CW-locked (tumbly) mutations that map in the signalling or linker regions. Similar dominant mutations have been identified in the tar gene of E. coli (Mutoh et al., 1986). Finally, cells lacking the two major transducers, Tsr and Tar, are generally non-chemotactic because they are strongly CCW biased and have a much lower tumble frequency (Springer et al., 1977b; Hazelbauer and Engstroem, 1980). This result implies that these transducers are required for maintaining a normal basal level of CW signal. The six canonical che genes and the two mot genes are organized into tandem operons at 42 min on the chromosome of E. coli (Armstrong and Adler, 1969b; Silverman and Simon, 1977a) and at 40 min on the chromosome of S. typhimurium (Enomoto, 1966; Aswad and Koshland, 1975a; Collins and Stocker, 1976; Warrick et al., 1977). Beginning at their promoter-proximal ends, the upstream (mocha) operon contains the motA motB cheA and c h e w genes while the second (meche) operon contains the tar [tap]cheR cheB cheY and cheZ genes (Matsumura et al., 1977; Silverman and Simon, 1977a; DeFranco and Koshland, 1981). The tap gene is not present in S. typhimurium. Transcription of these operons requires the o-factor encoded by thefliA gene (Ohnishi et al., 1990; see also the section on control of flagellar gene expression). The relative stoicheiometry of gene products in the meche operon of S. typhimurium has been estimated to be 4:1:1:18:3 (DeFranco and Koshland, 1981).

BACTERIAL MOTIIJTY AND CHEMOTAXIS

315

2. Physical Properties of the Signal Bacteria can integrate responses from either one type of receptor (Berg and Tedesco, 1975; Spudich and Koshland, 1975) or from different receptors (Berg and Tedesco, 1975; Rubik and Koshland, 1978). For different attractants the additivity may not be complete, an eventuality termed desensitization by Rubik and Koshland (1978). The CCW responses to attractants and the CW responses to repellents tend to cancel each other out (Tsang et al., 1973; Berg and Tedesco, 1975), so that progressively higher concentrations of repellent decrease the CCW response to a given concentration of attractant, and vice versa. The sum of the response times (also called recovery times or transition times and equal to the time of CCW-only flagellar rotation observed after addition of attractant) for shifts of attractant concentration from zero to X and then from X to a higher concentration Y is the same as for a single shift from zero to Y . This response pattern indicates that cells monitor the percentage change in receptor occupancy rather than the rate of change of occupancy (Berg and Tedesco, 1975; Spudich and Koshland, 1975). For ribose and allose, which bind to RBP with 1000-fold different affinities, and for analogues of aspartate and serine that bind to Tar and Tsr, the receptorbinding curves and the dose-response curves for chemotaxis correlate very well (Aksamit and Koshland, 1974; Berg and Tedesco, 1975; Spudich and Koshland, 1975; Clarke and Koshland, 1979). Response latency (the time from delivery of a stimulus until a response commences) was determined by stimulating individual tethered cells with negatively charged attractants or repellents (a-DL-methylaspartate or benzoate) delivered from iontophoretic micropipettes (Segall et al., 1982). Cells turning CW (for attractant stimuli) or CCW (for repellent stimuli) were given pulses of chemo-effector, and the time until the first reversal measured. For attractants and repellents, latencies were about 200 ms in wild-type cells, reflecting the time required for the signal to be processed in the cell. Similar latencies were found in mutants deleted for the cheR and cheB genes, but latencies of several seconds following challenge with attractant stimuli were found for cheZ mutants, suggesting that the cheZ gene product plays a direct role in signalling. In an extension of this work, Segall et al. (1985) used iontophoretic stimulation of filamentous cells to show that an intracellular chemotactic signal can pass a distance of several micrometres through the cell to the responding flagellum. Dissipation of the signal amplitude with distance can be used to calculate a space constant. The calculation, which assumes certain properties of the bacterial cytoplasm, is consistent with the signal being a small protein diffusing through the cell.

316

M. D. MANSON

Two experimental approaches have been used to investigate the properties of the chemotactic response that allow E. coli to respond to shallow gradients. The first was to measure the impulse response (the complete behavioural response to a brief impulse of chemoeffector) of individually tethered cells (Block et al., 1982). The impulse response persists for about four seconds. For an attractant, the probability of CCW rotation increases for about one second followed by a period of about three seconds during which it decreases. For a repellent, the CCW probability decreases briefly followed by a longer period of increased CCW probability. For either stimulus, if the unstimulated CCW probability is set at zero, the areas under the probability curves sum to zero when integrated over the impulse response. The conclusion is that the cell integrates information over an interval of a few seconds; short- and long-term fluctuations average out. This time period is appropriate for detecting concentration changes during the three-dimensional random walk generated by swimming E. coli, which run for several seconds. The second approach involved exposure of tethered cells to shallow, exponentially increasing or decreasing gradients of attractant (Block et ul., 1983). When exposed to an increasing gradient, cells maintained a constant CCW bias relative to their unstimulated behaviour, whereas they showed a CW bias to decreasing gradients. The extent of the bias was proportional to the steepness of the gradient. The data are best interpreted by a two-state (CW and CCW) model for the flagellar motor in which values of the rate constants for interconversion between the two states are determined by the chemotactic signal. 3. Biochemical Nature of the Signal The relatively gargantuan, amphitrichous bacterium Spirillum volutuns exchanges the head and tail orientations of its two flagellar bundles in near perfect synchrony (Kreig et al., 1967), even though they are 50-100 pm apart. This degree of co-ordination, and the observation that membrane-active agents disrupt it, suggested that signals controlling flagellar movement must be rapidly propagated in this large bacterium. It was attractive to speculate that rapid signal propagation was a general property of bacterial behaviour, and that bacteria might behave like miniature nerve cells. The nature of the signal co-ordinating flagellar rotation in Sp. volutans is still unknown. Dissipation of the membrane potential while the protonmotive force is retained through the transmembrane p H gradient abolishes chemotaxis in Spirochaeta auruntiu (Goulbourne and Greenberg, 1981). However, early evidence for a role for changes in membrane potential in the

BACTERIAL MOTILITY AND CHEMOTAXIS

317

chemotactic response of E. coli (Szmelcman and Adler, 1976) was not reproducible (Snyder et al., 1981) and may have been the result of measuring changes in the proton-motive force of cells during the experiment (Eisenbach, 1982). Similarly, reports of the involvement of calcium ions in signalling (Ordal, 1977) could not be corroborated (Snyder et al., 1981), nor could the proposed central role of cyclic nucleotides and cyclases (Black et al., 1980, 1983) be verified (Eisenbach et al., 1985; Taylor et al., 1985; Tribhuwan et al., 1986; Vogler and Lengeler, 1987). Another small molecule that may play a role in signalling is acetyladenylate. It is reported to be required for generating CW flagellar rotation in E. coli (Wolfe etal., 1988). However, the effects of acetyladenylate could be indirect, such as by influencing some aspect of cellular metabolism that impinges on control of the flagellar switch. Motile Streptococcus sp. or B. subtilis that have their membrane potential held constant with the potassium ionophore valinomycin still respond chemotactically (Manson et al., 1977, 1980; Miller and Koshland, 1977, 1980). Many bacteria respond to increases and decreases in the magnitude of the proton-motive force as attractants and repellents, respectively (Miller and Koshland, 1977, 1980; Khan and Macnab, 1980a; Berg et al., 1982; Glagolev, 1984). The availability of the DNA sequences for, and purification of the protein products of, the che genes of E. coli (Matsumura et al., 1984; Mutoh and Simon, 1986; Hess et al., 1987; Kofoid and Parkinson, 1991) and S . typhimurium (Simms et al., 1985,1987; A. M. Stock et al., 1985,1987,1988; Stock and Stock, 1987) helped launch the final assault on the chemotactic signal. Analysis of che gene sequences led to the discovery of homologies with other two-component signalling systems in bacteria (A. M. Stock et al., 1985, 1988; J. B. Stock et al., 1989; Nixon et al., 1986; Ronson et al., 1987; Kofoid and Parkinson, 1988; Bourret et al., 1989). The two components consist of a sensor or modulator that receives input from environmental stimuli and a response regulator that generates an output activity in accordance with modulatory signals received from the sensor. Recent work has shown that all of these systems involve protein phosphorylation cascades. In vitro experiments with purified Che proteins have revealed the reactions involved in propagation of the chemotactic signal (Hess et al., 1987,1988a,b; J. B. Stock et al., 1989; Bourret et al., 1989). The critical role of CheY in determining the direction of flagellar rotation was shown in experiments with tethered cell envelopes of E. coli (Eisenbach and Adler, 1981; Ravid and Eisenbach, 1984). A flagellum on a cytoplasm-free envelope always spins CCW unless the envelopes are prepared in the presence of purified CheY protein (Ravid et al., 1986), which can be

318

M. D. MANSON

captured inside envelopes during their formation. This work complements studies in which overproduction of the CheY protein in vivo, even in CCWlocked cells that are genetically “gutted” of other che and transducer genes, causes exclusively CW rotation (Clegg and Koshland, 1984; Wolfe et al., 1987). The concurrent overexpression of CheZ counteracts this effect and restores CCW rotation (Wolfe et al., 1987), implying that CheZ antagonizes the effect of CheY. Phosphorylation of CheY, which presumably controls its activity in vivo, was first shown in vitro (Hess et af., 1987, 1988a,b). The phosphate group that is added to CheY comes from ATP, and ATP is required for bacterial chemotaxis (Larsen et af., 1974a; Aswad and Koshland, 1975a; Shioi et af., 1982). However, the initial event is an ATP-dependent autophosphorylation of CheA (Hess et af., 1987). The CheA protein can be divided into at least three functional regions (Oosawa et af., 1988); these are an N-terminal region required for interaction with CheY and CheB (see later), a region extending through the N-terminal and middle regions of the protein that is required for autophosphorylation activity, and a C-terminal region that seems to be the target for transducers or C h e w , which regulate autophosphorylation activity. The CheA protein phosphorylates itself at the N-3 position of the His48residue (Hess et al., 1988a) and therefore belongs to a family of histidine protein kinases (J. B. Stock et al., 1989). An 18 kDa N-terminal tryptic fragment prepared from phosphorylated CheA can transfer a phosphate group to CheY or CheB, but the same fragment from non-phosphorylated CheA is incapable of autophosphorylation (Hess et af., 1988a). The CheY protein is phosphorylated at the carboxyl group of its Asps7 residue (Sanders et al., 1989a), and the Asplz and Aspl3 residues are also needed for phosphorylation and dephosphorylation (Bourret et al., 1990). The phosphorylated form of CheY (CheY-P) is required for CW rotation of the flagella. The D13K protein is not phosphorylated in vitro but, in vivo, even in a cheA deletion strain, it confers a dominant CW phenotype. These properties imply that it is the conformation of CheY-P, rather than the presence of the phosphate, that promotes interaction with the motor. The Lyslo9residue is also intimately involved in the function of CheY. The K109R mutant protein phosphorylates almost normally, undergoes spontaneous dephosphorylation about five times more slowly than wild-type CheY, and is virtually unaffected by CheZ in its rate of dephosphorylation (Lukat et al., 1991). However, contrary to the naive expectation that accumulation of phosphorylated CheY would lead to a tumbly (CW) behaviour, strains producing K109R CheY are CCW locked. The best explanation of this finding is that the mutant protein cannot undergo the conformational change that normally accompanies phosphorylation.

BACTERIAL MOTILITY AND CHEMOTAXIS

319

The implication is that the Lys,, residue may interact directly with the phosphate group to stabilize the active conformation of CheY, perhaps in a manner analogous to activation of p21 Ras by salt-bridge formation between the &-aminogroup of the LysI6residue and the y-phosphate group of GTP. The requirement of CheY-P for CW flagellar rotation explains the CCWlocked behaviour of strains with null mutations in cheA and cheY. The CCW-locked behaviour of chew mutants can also be explained, since C h e w is required for communication between transducers and CheA (Conley et a f . , 1989; Liu and Parkinson, 1989; Sanders et a f . , 1989b). The C h e w protein may also modulate the specificity of CheA toward its two natural substrates, CheY and CheB (Gegner and Dahlquist, 1991). A rather precise stoicheiometry of C h e w relative to CheA and the transducers seems to be critical for appropriate signalling (Liu and Parkinson, 1989; Sanders et af., 1989b), indicating that some direct physical interaction of these components is taking place. In this regard, the location of cheA and chewadjacent to one another in the mocha operon allows for co-ordinate control of their expression. The C h e w protein apparently interacts directly with transducers (Liu and Parkinson, 1991). Certain chew mis-sense mutations can be suppressed in an allele-specific fashion by mis-sense mutations in tsr. The suppressible mutations form an overlapping set with a group of independently isolated, partially dominant, chew mutations, implying that dominance is conferred by an altered interaction of the mutant C h e w proteins with Tsr. It is noteworthy that the suppressing mutations in tsr occur in sequences encoding the extremely highly conserved region in the signalling domain that is shared by all transducers. The crystal structures of the CheY proteins from S. typhimurium (A. M. Stock et al., 1989) and E. coli (Volz et af.,1986; Matsumura et a f . ,1990) have been described. The protein is a pure receiver module (Kofoid and Parkinson, 1988; J. B. Stock et af., 1989). Most response regulators have such an N-terminal region that receives input from the transmitter module of the kinase, and a C-terminal effector domain that converts the signal into action. Different effector domains can bind to promoters to regulate gene expression (e.g. NtrC, OmpR, PhoB) or have enzymic activities (CheB). However, the phosphorylated receiver domain of CheY is apparently able to interact directly with the flagellar switch. Chemotactic signalling is controlled by transducers through modulation of the rate of CheA autophosphorylation. In vitro, accumulation of CheY-P in the presence of ATP is absolutely dependent on CheA and magnesium ions, and the rate of CheY phosphorylation is increased dramatically by C h e w (Borkovich et af., 1989). Membranes containing wild-type or CWlocked Tar transducers further stimulate accumulation of CheY-P, whereas

320

M. D. MANSON

membranes containing CCW-locked Tar do not. When CheZ is added to the reaction mixture, steady-state accumulation of CheY-P is considerably diminished. Finally, aspartate greatly inhibits stimulation of CheY-P production in the presence of membranes containing wild-type Tar, but it has little effect on stimulation of CheY-P production in the presence of membranes containing CW-locked Tar. Furthermore, the dependence of this inhibition on aspartate concentration is similar to the dose-response curve in the chemotactic response to aspartate. The most recent data (Borkovich and Simon, 1990) suggest that CheA exists in three states. The open state requires association of CheA and Che w with the cytoplasmic domain of the transducer, either in the absence of apartate for wild-type Tar or even in its presence for CW-locked Tar. In its open state, CheA reaches a steady-state level of phosphorylation within five seconds. Addition of non-radioactive ATP leads to rapid loss of phosphorus-32, so that activated CheA can catalyse y-phosphate exchange. In the absence of wild-type or CW-locked Tar, CheA assumes a closed state that autophosphorylates relatively slowly. In the closed state, CheA does not catalyse y-phosphate exchange with ATP, although both activated and inactivated CheA-P are rapidly dephosphorylated in the presence of ADP. The sequestered state of CheA is produced when aspartate is added to membranes containing wild-type Tar in the presence of C h e w and activated CheA. Under these conditions, CheA is converted into a form in which its covalently bound phosphorus-32 cannot be removed by additon of ADP or ATP. Weak Tar-mediated attractants, such as succinate, do not have this effect, nor can it be achieved with membranes containing CW-locked Tar. The sequestered form of CheA donates its phosphate to CheY only in stoicheiometric amounts, not in a catalytic fashion as do the open and closed forms of CheA. Autophosphorylation of CheA is inhibited by membranes containing wild-type Tar in the presence of aspartate, wild-type Tsr in the presence of serine, or CCW-locked Tar. Autophosphorylation of CheA stimulated by CW-locked Tar is also inhibited by CCW-locked or attractant-bound transducer under these conditions. On a mole for mole basis, Tsr is three-fold more active than Tar in stimulating formation of CheY-P, although ligand-bound Tar and Tsr are equally effective inhibitors of phosphorylation. In the model presented by Borkovich and Simon (1990), the signalling state of the cell is determined by distribution of CheA among the open, closed and sequestered forms. Repellents shift the equilibrium towards the open state, and attractants shift it towards the sequestered state. The closed state represents an unbound form of CheA that may be in equilibrium with the other two states. This model can account for integration from different

BACTERIAL MOTILITY AND CHEMOTAXIS

321

receptors. It also accounts for amplification of a repellent signal, since one transducer can activate CheA to catalyse phosphorylation of many CheY molecules. An additional layer of control may be imposed if the activity of CheZ is regulated by interaction with CheA, such as being activated to form a phosphatase by the sequestered form of CheA and/or inhibited by the open form of CheA. Since an increase of one attractant-bound transducer out of a population of thousands in a cell causes a significant, if brief, increase in CCW flagellar rotation (Segall et al., 1986), the sequestration model must also incorporate the concept that an attractant-bound transducer acts catalytically to inactivate a number of CheA molecules (Borkovich and Simon, 1990). The CheA and C h e w proteins form a complex in vitro, with two C h e w molecules associating with one stable CheA dimer (Gegner and Dahlquist, 1991). The dissociation constant for theCheA-Chew interaction is 17 p ~ , and co-operativity for C h e w binding was not observed. The rates of association-dissociation of the CheA-Chew complex are far too slow to generate a response with the measured latency of about 200 ms (Segall etal., 1982). Affinity columns have been used to identify physical complexes of Che proteins (Matsumura et al., 1990). An anti-CheA antibody column extracted a complex of CheAL-CheAs-Chew from the cytoplasm of cells having or lacking transducers. In contrast to the work reported in the previous paragraph, Matsumura et al. (1990) experienced difficulty in forming the CheA-Chew complex in vitro, suggesting that conditions in the cell that are difficult to duplicate in vitro favour complex formation. In complexes formed in vivo, the ratio of the components in the complex CheAL-CheAs-Chew is approximately 1:1:1, which is different from the 1:1:2 ratio expected from the results of Gegner and Dahlquist (1991), although these authors did not distinguish between CheAL and CheA,. It is possible that C h e w binds to only one form of CheA, but there are no data to support this conclusion. Indeed, C h e w can bind CheA, (Matsumura et al., 1990), and presumably it binds CheA,, since CheA, is dispensable for chemotaxis. Activity of the CheA,>-CheA,-CheW complex is affected by transducers. A complex from a strain with CW-locked transducers autophosphorylates more than the complex from a cell with only wild-type transducers, whereas the complex from a CCW-locked mutant has less autophosphorylation activity. The CheA,-CheA,-Chew complex from cells lacking transducers also has very low autophosphorylation activity. However, membranes isolated from a strain producing a CW-locked mutant transducer bestow a high level of autophosphorylation on a complex from a strain lacking transducers. The CheAL4heAsXheW complex has a much greater affinity

322

M. D. MANSON

for CheY than purified CheA or Chew, but the affinity is the same regardless of the history of exposure to different types of transducers. The CheZ protein offers a departure from this pattern, since it associates with CheY or with CheA,, regardless of their state of association (Matsumura et af., 1990). It also binds to non-phosphorylated CheY, indicating that these two proteins may exist in a complex in the cell. The stoicheiometry of the CheZ-CheA, complex is of the order of 10-30:1, suggesting that CheA, may help to control activity of CheZ. Relatively little is known about signalling in systems that do not involve transducers, although the early stages of signalling are clearly different (Niwano and Taylor, 1982; Postma and Lengeler, 1985; Shioi et al., 1988; Lengeler and Vogeler, 1989). The aerotactic, phosphotransferase (PTS) and methylation-dependent chemotactic pathways may converge at the level of CheA-Chew (Rowsell et al., 1988). Very recently, it has been shown that Hpr, the histidine-containing protein of PTS, is the critical link between the PTS-dependent and methylation-dependent signalling pathways (Gruebl et al., 1990). In strains lacking HPr, or in strains containing specific hpr mis-sense mutations, chemotaxis to all substrates taken up by the PTS is eliminated. It is still not certain which Che proteins interact with HPr. Cross-talk among sensors and response regulators (Ninfa et al., 1988; Igo et al., 1989) reflects the ability of the histidine kinase from one twocomponent regulatory system to phosphorylate, albeit with lower efficiency, a response regulator from a heterologous system. This finding is not surprising given the sequence homologies observed among kinases and response regulators. Cross-talk can lead to partial complementation of mutational defects in one system by overexpression of components of another system. Although the relevance of cross-talk within a cell under normal conditions is not known, PTS-mediated chemotaxis represents a physiologically significant example of cross-talk between a transport system and the chemotactic signalling machinery (Gruebl et al., 1990). C. EVENTS AT THE FLAGELLAR SWITCH

In a cell running and tumbling in the absence of a chemotactic gradient, the concentration of CheY-P must be maintained at a level very near the K D value for its interaction with the switch. Switching is an all-or-none event, and very abrupt (Berg, 1974,1976b). Thus, either binding of one molecule of CheY-P to a motor is sufficient to reverse its direction or, as appears more likely (Kuo and Koshland, 1989), a number of CheY-P molecules bind, perhaps in a highly co-operative fashion. An interesting feature of motor reversal is that the switching probabilities of individual flagella on one cell are independent if no mechanical coupling

BACTERIAL MOTILITY AND CHEMOTAXIS

323

among them exists (Ishihara et al., 1983; Macnab and Han 1983). Superimposed on this stochastic process at each flagellum is global chemotactic control that modifies the switching probabilities for all flagella. Since swimming cells have discrete transitions between running and tumbling, it is probable that interactions among filaments in the bundle co-ordinate switching events (Macnab, 1987b). Recently, a third state of the flagellar motor in E. coli has been described (Ravid and Eisenbach, 1983; Lapidus et al., 1988); this involves a brief cessation of rotation called a pause. These events are reminiscent of pauses observed as part of the swimming pattern of Rhodobacter sphaeroides and Rhizobium meliloti, described in an earlier section. Pauses are suppressed by attractant stimuli and may represent very brief reversals from CCW to CW rotation (Eisenbach et al., 1990b). Eisenbach (1990) considered the flagellar motor to be a three-state device rather than a two-state device, as previously proposed (Parkinson and Parker, 1979; Khan and Macnab, 1980a; Block et al., 1983). One role of pausing may be to produce brief tumbles (Eisenbach, 1990). Conditions producing a high frequency of pausing also decrease adsorption of phage x (Ravid and Eisenbach, 1983). Since cell envelopes depleted of cytoplasmic contents (Ravid and Eisenbach, 1984) and genetically “gutted” strains (Clegg and Koshland, 1984; Wolfe et al. , 1987) are CCW locked, a wild-type motor in the absence of signal input is in the CCW mode. The cheC, CCW-lockedfriM mutations, as well as similar mutations infriG andfriN, maintain the motor in this state even when CheY-P levels are normal. However, some mutations in these three fri genes lead to continuous CW rotation in the absence of any chemotactic input (Ravid and Eisenbach, 1984; Yamaguchi et al., 1986a; Wolfe et al., 1987). An unusual behaviour detected in some mutants having strongly CWbiased motors is reverse chemotaxis, in which compounds that usually repel now attract and vice versa (Rubik and Koshland, 1978; Khan et al., 1978). The explanation is that prolonged CW rotation forces flagellar filaments into right-handed helices that form a propulsive bundle. Normally repellent substances that increase CW rotation inhibit brief periods of CCW rotation that disrupt the right-handed bundle; therefore, runs in the direction of higher repellent concentration are extended. Conversely, normally attractive compounds that promote CCW rotation cause tumbling because they induce longer periods of CCW rotation, which unwind the right-handed bundle. Specific Che- mutations in JiG, friM and fliN give rise to intergenic suppressors in either of the other two genes of the complex (Yamaguchi et al. , 1986a). The patterns of suppression are allele-specific, supporting the concept that the three proteins are in direct contact. Mutations conferring a

324

M. D. MANSON

CCW phenotype are suppressed by mutations that themselves cause a CW phenotype and vice versa. Both cheY and cheZ mutations in E. coli can be phenotypically suppressed by mutations in t h e p i c ( c h e w andPiM (cheC)genes (Parkinson and Parker, 1979; Parkinson et al., 1983). The cheY mutants are CCW biased, whereas the cheZ mutants are CW biased; the double-mutant pseudorevertants exhibited a relatively normal run-tumble behaviour. The suppressing mutations alone also imparted a chemotaxis-defective phenotype; the cheY suppressors (scy) inPiC orPiM had CW-biased motors, while the cheZ suppressors (scz) had CCW-biased motors. The scy mutants have recently been used to select for suppressing mutations in cheY. This new set of cheY mutations is being used to map the surface of cheY that interacts with the FliG protein of the switch (Matsumura et al., 1990). A complementary study was carried out on S . typhimurium (Yamaguchi et al., 1986a), starting with Che- mutations i n p i c andPiM. Some of t h e p i c andJIiM mutations were suppressed by mutations in cheY and cheZ, and in one strain by a mutation in cheA. Again, suppression was allele-specific, and suppressing mutations conferred the complementary phenotype; a CCW switch mutation was suppressed by a CW mutation in the che genes, and vice versa. Unexpectedly, several CCW-biased cheZ mutations were isolated in this way. The products of those cheZ alleles may promote CCW rotation better than wild-type CheZ, either because they bind more tightly to the switch or because they destroy CheY-P more quickly. At present there are no biochemical or structural data available that demonstrate interaction of any soluble Che proteins with the switch. The CheY protein itself is capable of converting a cell devoid of other soluble che gene products from exclusively CCW to CW flagellar rotation, and the extragenic suppression data support interaction of CheY and CheZ with FliG and FliM. However, extragenic suppression can monitor indirect effects. For example, the CCW-biased mutation in cheA that suppresses a CW-biasedpic mutation almost certainly does so by lowering the level of CheY-P or by decreasing sequestration of CheZ by CheAs. D . ADAPTATION OF THE CHEMOTACTIC RESPONSE

The ability of bacteria to sense spatial chemical gradients by a temporal mechanism requires a form of short-term memory with a retention time of seconds. The biochemical properties of this memory are most easily studied during adaptation to large step increases or decreases in the concentration of attractants and repellents, although such large concentration changes are seldom encountered in natural microbial environments. Adaptation to such stimuli occurs on a time scale ranging from tens of seconds to minutes.

BACTERIAL MOTILITY AND CHEMOTAXIS

325

I . Biochemistry of Transducer Methy fation and Deamidation Methionine is required for normal chemotactic behaviour in E. coli (Adler and Dahl, 1967), because it is a precursor of S-adenosylmethionine (Armstrong, 1972; Larsen et a f . , 1974a; Aswad and Koshland, 1975a), a common donor in bacterial methylation reactions. Depletion of methionine from the environment of a methionine-requiring auxotroph leads to progressive but reversible loss of tumbling and loss of responsiveness to chemotactic stimuli (Aswad and Koshland, 1974; Springer et a f . , 1975). Kort et a f . (1975) showed that a cell-membrane protein involved in chemotaxis is methylated, and they dubbed it MCP for methyl-accepting chemotaxis protein. (Is it a sign of the times or of my age that none of my students identify MCP with “male chauvinist pig”?!) The methyl group of S-adenosylmethionine is used to form a y-carboxylmethyl glutamate ester (Kleene et a f . , 1977; Van der Werf and Koshland, 1977). The MCPs were shown by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis to form multiple bands in the 60,000-65,OOO range of molecular weights (Silverman and Simon, 1977b; Springer et a f . , 1977a, 1979) and to correspond to the products of the tar and tsr genes (Goy et a f . , 1977; Silverman and Simon, 1977b). It later became clear that the Trg and Tap transducers are also MCPs (Hazelbauer et a f . , 1980; Boyd et a f . , 1981; Slocum and Parkinson, 1983), which were probably overlooked initially because of their lower abundance. The early work on methylation is summarized in a review by Springer et af. (1979). The multiple bands formed by each transducer arise because each protein has multiple methylation sites (Boyd and Simon, 1980; Chelsky and Dahlquist, 1980b; DeFranco and Koshland, 1980; Engstroem and Hazelbauer, 1980). The methylated glutamate residues are located on two tryptic peptides (called K1 and R1, since they are arginine- and lysinecontaining peptides) in the cytoplasmic domain of the transducers (Kehry and Dahlquist, 1982a). Depending on the transducer, there are four or five sites of methylation (Kehry and Dahlquist, 1982a; Kehry et a f . , 1983; Terwilliger et a f . ,1986; Nowlin et a f . ,1987). Three of these are in K1 (which encompasses residues 295-317 in Tsr) and one or two are in R1 (residues 483-507 in Tsr). The regions in which KI and R1 are located are highly conserved among transducers, and they flank the even more highly conserved central core of the signalling domain (Krikos et a f . ,1983; Bollinger et a f . ,1984). Ames and Parkinson (1988) proposed that the methylation states of KI and R1 modulate the conformation, and thereby the activity, of the core signalling region, which probably interacts with CheA and Chew (Liu and Parkinson, 1991; J. S. Parkinson, personal communication). The methylated positions occur at intervals of seven residues so that, if the methylated regions are

326

M. D. MANSON

a-helical, the glutamate residues will be on the same face of the helix and available as substrates for CheR and CheB (Terwilliger and Koshland, 1984; Hazelbauer et al., 1990). Different sites have different intrinsic rates of methylation (Terwilliger et al., 1986), but there apparently is not, as originally proposed (Stock and Koshland, 1981; Springer et al., 1982), a particular order in which the sites are methylated. Mutations destroying the function of cheR (then called cheXin E. coli) are unable to methylate MCP (Silverman and Simon, 1977b; Springer and Koshland, 1977; Springer et al., 1977b). The cheR gene product is an S-adenosylmethionine-dependent methyltransferase. Methyl groups are removed by the gene product of cheB (formerly called cheX in S. typhirnuriurn) , which is a methylesterase (Stock and Koshland, 1978; Hayashi et al. , 1979), and are given off as methanol (Stock and Koshland, 1978; Toews and Adler, 1979). Activity of CheB is regulated by phosphorylation, and its control is a feature of adaptation. Methyltransferase and methylesterase have equivalent activities towards all transducers (Springer et af., 1979). They are active in vitro with MCP in membrane preparations (Terwilliger et al. ,1983; Weis and Koshland, 1988), and the Tar protein from S. typhirnuriurn can also be methylated in vitro after being solubilized by suitable detergents. This solubilized protein can also be methylated to a higher level in response to aspartate (Bogonez and Koshland, 1985). There is another activity associated with CheB that is related to its methylesterase function; it serves as a deamidase (Kehry et a f . , 1983). Two of the methylatable glutamate residues are derived by modification of glutamine residues that are part of the original translation product (Krikos et al., 1983; Bollinger et al., 1984). This modification makes new additional sites available for methylation (Kehry and Dahlquist, 1982b). These glutamine residues may balance the signalling state of transducers that are newly inserted into the membrane and have not yet equilibrated with the methylation4emethylation cycle. Alternatively, they may be required for proper assembly of the transducers into the membrane (Kehry et a f . , 1983; Bollinger et a f . , 1984).

2. Function of Covalent ModiJication in the Adaptation Process The first connection between methylation and adaptation was the observation that cells depleted of rnethionine lose the ability to tumble and recover more slowly, or not at all, from the smooth-swimming response elicited by attractants (Springer et af., 1975). One possible conclusion was that methionine was directly involved in production of a tumbly signal. Although

BACTERIAL MOTILITY AND CHEMOTAXIS

327

methyl-group transfer and turnover are involved in MCP-mediated chemotactic signalling in B. subtilis (Thoelke et al., 1987, 1988, 1989, 1990; Bedale et al., 1988), this is not so in either E. coli or S . typhimurium. Rather, methylation or demethylation restores the signalling state of the transducers to their baseline level after addition of an attractant or repellent (Goy et al., 1977). The time to reach a new level of methylation after addition of an attractant or repellent is very similar to the adaptation time or response time, i.e. the time elapsed after onset of the stimulus until cells return to normal run-tumble behaviour. The response time is related to the magnitude of the concentration change and reflects the percentage change in receptor occupancy. Changes in net methylation rates are determined by changes in the rate of demethylation (Toews et al., 1979). Addition of attractants transiently inhibits demethylation, while addition of repellents stimulates demethylation. Responses to step increases in attractant concentration or step decreases in repellent concentration are much longer than for equivalent step changes in the opposite directions (Goy et al., 1977). This same asymmetry explains why runs for cells swimming up a spatial gradient of attractant become longer whereas runs down the gradient do not become shorter (Fig. 2). When receptor occupancy and methylation are in balance, the ratio of CWCCW flagellar rotation produces the run-and-tumble behaviour that leads to the three-dimensional random walk. Thus, methylation-demethylation provides the slow, signal-cancelling enzyme 2 proposed by Macnab and Koshland (1972) and the slowly filling or emptying reservoir whose level is measured against receptor occupancy by the comparator in the model of Berg and Tedesco (1975). Mutants lacking the CheR methyltransferase are grossly defective in adaptation (Goy et al., 1978; Parkinson and Revello, 1978). Although cheR (cheX) mutants tumble following addition of repellents, the response, brief with wild-type cells, may continue for hours with the mutants. When these cells are subsequently exposed to a potent attractant, they switch over to incessant smooth swimming. Strains with cheB deletions are also unable to adapt, and they switch from tumbling in the absence of attractant to smooth swimming that can last for hours (Yonekawa et al., 1983). Earlier reports that cheB strains adapt fairly rapidly to attractant stimuli (Parkinson, 1976) are probably attributable to leakiness in the mutations. Deamidation of the transducers is also accelerated by repellent stimuli (Rollins and Dahlquist, 1981; Sherris and Parkinson, 1981), suggesting that the amide and methyl ester groups on the glutamate residues may be functionally equivalent at these positions in the transducers. Deamidation may allow one round of adaptation to repellent stimuli in cheR mutants (J. B. Stock et al., 1985b).

328

M. D. MANSON

Dunten and Koshland (1991) took advantage of the qualitative equivalence of amides and methyl esters of glutamate to test whether adaptation lowers the affinity of transducers for ligands or negates the signal produced by occupied ligands. Site-directed mutagenesis was used to generate Tar proteins with various combinations of glutamate and glutamine residues at the four methylation sites. In a cheRB deletion, the proteins with four glutamate or four glutamine residues had the same affinity (within a factor of two) for aspartate. However, the concentration of aspartate required to produce a given extent of smooth swimming progressively increased 200fold as the number of glutamine residues went from zero to four. They concluded that amidation and, by inference, methylation do not alter the affinity of Tar for its ligands very much but do profoundly influence the signal produced when the ligand is bound. This result is in contrast with the finding that the affinity of Tsr for serine is at least 100-fold lower, and that the affinity of Tar for aspartate about 10-fold lower, in a cheB (overmethylated) strain than in a cheR (undermethylated) strain (Yonekawa and Hayashi, 1986). The discrepancy may reflect a real difference between the effects of amidation and methylation on ligand affinities or may lie with other differences in the experimental procedures used in the two studies. The full ramifications of covalent modification of glutamate residues have been explored recently by analysis of the behaviour of site-directed mutant forms of the Trg transducer (Park et al., 1990). Although two of the five glutamate residues capable of methylation are synthesized as glutamine residues (3E, 2Q), this configuration is not essential for normal synthesis, membrane assembly o r stability of the transducer. Either Trg(5E) or Trg(5Q) can be methylated, although in Trg(5Q) there are fewer methylation sites, apparently because CheB is inefficient in deamidating two of the extra glutamine residues. Newly inserted Trg(5E) produces a strong CCW signal, and Trg(5Q) produces a strong CW signal. These signals persist until transducers equilibrate with the methylation-demethylation system, although some, such as Trg(SQ), never fully equilibrate. Strains containing multiple copies of the trg(5E) or trg(5Q) genes are impaired for chemotaxis, as judged by formation of smaller swarm rings in galactose- or ribose-containing semi-soft agar. With Trg(SE), this effect is due to the excess CCW signal produced by recently synthesized transducers. With Trg(SQ), however, the effect is exacerbated because the two residual glutamine residues are not as effective as methyl glutamate esters in balancing the CCW signal produced by attractants. When all five positions capable of methylation are occupied by alanine residues, the signalling state is intermediate between the one generated by Trg(5E) and the one generated by Trg(5Q). The effects of covalent modification on the signalling properties of transducers are summarized in Fig. 5 .

329

H3cfi H3C0

FIG. 5. A model of the effects of attractant binding and covalent modification on transducer function. (1) Ligand is not bound while the steady-state level of methylation averages about one methyl group on each transducer molecule. (2) Attractant binding triggers a CCW signal and activates methyl-accepting sites and deactivates the methylesterase to generate a net increase in methylation. (3) Adaptation is achieved by cstablishment of a new, higher steady-state level of methylation. (4) Loss of ligand removes the excitatory input and leaves an overmethylated transducer that produces a CW signal; adaptation is achieved through demethylation by the activated methylesterase. (4‘)Glutamine residues in Trg(5Q) generate a CW signalling state in the non-ligand-bound transducer. (2’) Glutamate residues in Trg(5E) generate a CCW signalling state in the nonligand-bound transducer. SAM indicates S-adenosylmethionine; SAH, S-adenosylhomocysteine. The drawing is reproduced from Park et al. (1990).

330

M. D. MANSON

Adaptation to oxygen and substrates transported by the PTS occurs normally in the absence of methylation (Niwano and Taylor, 1982). There has been considerable debate about whether adaptation as a result of methylation is essential for chemotaxis mediated by methyl-accepting transducers. Stock et al. (1981) reported that cheR mutants of S . typhimurium respond chemotactically on swarm plates and in temporal assays. Under some conditions, cheRB deletion mutants, which have a reasonably normal run-tumble frequency, also display significant chemotaxis on swarm plates and in capillary assays, and they respond and partially adapt to addition and removal of attractants (J. B. Stock et al., 1985a, b). Segall et al. (1986) countered with an extension of their analysis of the impulse response of tethered cells. They pointed out that, although a cheRB double mutant responds with the same latency to an attractant pulse as a wild-type cell, its recovery is not rapid enough to be of use to a bacterium swimming in a spatial gradient. This prediction is confirmed by the observation that an intact methylation-demethylation system is needed for migration of E. coli in non-saturating gradients of aspartate, although some residual taxis was observed in very steep gradients (Weis and Koshland, 1988). The final word seems to be that methylation-dependent adaptation is crucial for chemotaxis in the natural environments of bacteria, although some migration in artificial gradients is possible with methylation-independent adaptation.

3. Feedback Control of the Adaptation Response In principle, two different mechanisms could modulate the methylation level of transducers. Either the activities of the methyltransferase or methylesterase can be regulated, o r the availability of the glutamate residues as substrates for these enzymes can be controlled. A large body of data indicates that both mechanisms operate. Activation of CheB methylesterase activity by repellents depends on functional CheA (Springer and Zanolari, 1984; Stewart et al., 1990) and C h e w (Stewart et al., 1990) proteins. Methylesterase activity, best measured by the rate of methanol release in a continuous-flow filter assay (Kehry et al., 1984, 1985), can increase substantially. A 21 kDa C-terminal proteolytic fragment of the 37 kDa CheB protein of S.typhimurium has a 15-fold increased level of methylesterase activity (Simms et al., 1985). A similar result was observed in mutants with deletions removing the N-terminal45% of E. coli CheB (Stewart and Dahlquist, 1988), suggesting that the N-terminus of CheB can inhibit methylesterase activity. The CheA protein is not required for inhibition of methylesterase activity by attractants, although the C h e w protein apparently is (Stewart et al., 1990).

BACTERIAL MOTILITY AND CHEMOTAXIS

33 1

Furthermore, strains in which the N-terminal region of CheB is deleted are still subject to inhibition by attractant stimuli (Stewart and Dahlquist, 1988; Lupas and Stock, 1989). In contrast, there is no evidence that activity of CheR is regulated, and the methyltransferase is apparently constitutive. Activation of methylesterase depends on phosphorylation of the Nterminal domain of CheB (Lupas and Stock, 1989; Stewart et al., 1990). Phosphorylation may reverse inhibition of the C-terminal catalytic domain by the non-phosphorylated N-terminus. The CheB protein is a good substrate for phosphorylation by CheA-P in vitru (Hess et al., 1988b; Oosawa et al. , 1988; Lupas and Stock, 1989). Because the N-terminus of CheB is very similar to CheY and also has the aspartate residues that are conserved in the response regulators of two-component regulatory systems, it seems likely that the Asp,, residue of CheB is phosphorylated. Similarity to CheY is underscored by the observation that mutant CheB proteins with residues show drastic substitutions in the Asplo, Asp,, , Asp,, and decreases in their activity as CheA phosphatases, implying that they are phosphorylated poorly by CheA (Stewart et al. , 1990). There is no known phosphatase for CheB-P, but spontaneous dephosphorylation of CheB is rapid. The mutant CheB proteins described by Stewart et al. (1990) behave much like CheB proteins in which the N-terminal domain is absent. They have high levels of methylesterase activity in vivu and show normal inhibition of activity by attractants, although their activity is not stimulated by addition of repellents or removal of attractants. These properties and the requirement of C h e w for inhibition by attractants suggest that C h e w interacts directly with the C-terminus of CheB to lower its activity (Stewart et al., 1990). Changes in methylesterase activity in response to chemotactic stimuli can transiently affect methylation levels of all transducers (Sanders and Koshland, 1988). The effect on heterologous transducers probably does not occur via transducer heterodimers (e.g. Tar-Tsr), which apparently do not exist (Milligan and Koshland, 1988). Transient overmethylation of heterologous transducers, which should become CW signallers, is the probable explanation for overshoot (Berg and Tedesco, 1975), the term used for the period of enhanced CW flagellar rotation observed immediately after the period of CCW-only rotation induced by attractants. Once the adapted state has been reached, methylation of heterologous transducers returns to the baseline level of slightly less than one methyl group on each transducer molecule (Terwilliger et al., 1986), whereas the methylation of the attractant- or repellent-bound transducer remains at an increased or decreased level, respectively (Silverman and Simon, 1977b; Springer et al., 1977b, 1979; Kondoh et al., 1979). Ligands have been shown to affect the substrate properties of the methylglutamate residues in

332

M. D. MANSON

transducers (Stock and Koshland, 1981; Springer et al., 1982). Thus, the extent of transducer methylation and establishment and maintenance of the adapted signalling state in the cell depend on a combination of feedback regulation of CheB activity and ligand-induced conformational changes of the transducer substrates of methylesterase (Sanders and Koshland, 1988). E. INTEGRATED MODEL FOR CHEMOTACTIC SIGNALLING

A model that incorporates most of the known features of chemotactic signalling in E. coli and S . typhimurium is presented in Fig. 6. In the absence of attractants or repellents, the net conformation of the signalling domains of the transducers, averaged over their population in the cell, is such that CheA autophosphorylation maintains intermediate levels of CheA-P. Communication between transducers and CheA involves the C h e w protein, which forms a complex with CheA. Phosphate-group transfer to CheY maintains an intermediate level of the CW signaller CheY-P, which, through its interaction with the switch-motor complex, supports a level of CCW to CW transitions of the flagellar motor that generates normal run-tumble behaviour. Accumulation of CheY-P is limited by removal of the phosphate

FIG. 6. A schematic diagram showing intracellular signalling during bacterial chemotaxis. CheA, CheB, CheR, Chew, CheY, CheZ and a generalized transducer are indicated by the large capital letters A, B, R , W, Y, Z and T, respectively. Phosphate groups are indicated by P. The active, phosphorylated form of CheB is designated by a solid tail. Activities of CheB, Chew, CheY-P and CheZ that are thought to be involved in regulating the activity of other components of the chemotactic system are represented by large open arrows, and the constitutive methylating activity of CheR is shown as a large solid arrow. Phosphorylationdephosphorylation reactions are indicated by the thin, curving arrows, as is the possible regulatory interaction between CheA and CheZ. Consult the text for a full explanation of signalling pathways.

BACTERIAL. MOTILITY AND CHEMOTAXIS

333

group by CheZ. The CheZ protein may also antagonize CheY-P by binding to the switch-motor complex to promote CCW rotation. Phosphate-group transfer to CheB maintains a level of active CheB-P methylesterase that balances addition of methyl groups to transducers by the constitutive CheR methyltransferase. These two maintain the basal level of transducer methylation (one of four or five sites methylated). Accumulation of CheB-P is limited by autodephosphorylation. When an attractant is added, it converts its transducers into a conformation in which it blocks autophosphorylation of CheA. Levels of CheY-P fall within 200 ms, the flagella turn CCW, and the cells run. A t the same time the level of CheB-P falls, although somewhat more slowly than that of CheY-P. The resulting inhibition of methylesterase leads to an overall rise in the level of transducer methylation, which generates a conformation of the signalling domain in which the transducer again supports an intermediate level of CheA autophosphorylation. The consequence is that CheY-P and CheB-P also return to intermediate levels, and normal run-tumble behaviour is restored. The higher level of methylation of the attractant-bound transducer species persists, thereby cancelling the attractant-induced signal, while methylation of the other transducer species returns to the baseline level. Removal of attractant causes the overmethylated transducer to stimulate CheA autophosphorylation. Levels of CheY-P rise and induce CW rotation and tumbling, but overmethylated transducers are rapidly demethylated by activated CheB-P methylesterase to return the transducers to a signalling state that maintains an intermediate level of CheA autophosphorylation. Addition of a repellent has essentially the same effect as removal of an attractant, except that, during the adaptation process, the transducer species mediating the response is demethylated by activated CheB-P to a level below one methyl group on each transducer molecule. Similarly, removal of repellent evokes the same response as addition of an attractant. The activity of CheA is inhibited by the undermethylated transducer now freed from repellent until methylation, enhanced by inhibition of CheB activity, returns to the baseline level. The integrated activities of all of the transducers determine the behaviour of a cell swimming in a complex chemical environment. Gradual increases or decreases in concentration of attractants and repellents modulate the net signal that is produced. The cell compares the current state of receptor occupancy, sampled over about one second, with its memory of the prior state of receptdr occupancy, measured over the previous three seconds and stored in the form of methylation levels of transducers. The deceptively simple decision made by the cell, whether to keep running or to tumble, depends on the outcome of this multifactor biochemical analysis.

334

M. D. MANSON

VI. Conclusion

The study of bacterial chemotaxis, ushered in by the observations of Wilhelm Pfeffer over a century ago, has come a great distance. More than any other individual, it was Julius Adler who saw the potential of bacterial genetics to explore the molecular basis of the entire behavioural repertoire of an organism. Fundamental problems of receptor structure and function, intracellular signalling and second messenger action, and evolutionary selection in response to intense competition for resources in nutrientlimiting environments, have been addressed and in some measure solved. The unanticipated discovery of the proton-driven rotory motor of the bacterial flagellum has posed a bio-engineering puzzle that is still unresolved. Although the broad outline of most aspects of chemotaxis in E . cofi and S. typhimurium is now understood, many crucial details are still being worked out and will occupy researchers in the field for many years to come. The biological role of chemotaxis is still largely a mystery, even in the best-studied organisms, and certainly many fascinating aspects remain to be uncovered in the broad range of eubacterial and archaebacterial taxa. It is increasingly clear that aspects of bacterial sensory biology will apply to our understanding of sensory and regulatory phenomena in both prokaryotes and eukaryotes. The two-component regulatory systems (Nixon et a f . ,1986; J. B. Stock et a f . , 1989) that govern a wide range of bacterial signal transduction systems are one example. The homologies of these proteins are highlighted by the capability of making functional hybrid proteins. The chimeric Taz protein, consisting of the periplasmic domain of Tar and the cytoplasmic domain of the membrane-spanning osmoregulator EnvZ of E. cofi,modulates the histidine kinase activity of EnvZ in response to aspartate (Utsumi et a f . , 1989). The applicability of the motifs of transmembrane and intracellular signalling inferred from bacterial chemotaxis may be even broader. Chimeras between the Tar protein of S . typhimurium and mammalian insulin receptor, similar to Taz, also function in transmembrane signalling (Moe et a f ., 1989), and activation of CheY by phosphorylation and of H-ras by GTP binding may exhibit structural and functional similarities (Lukat et a f . , 1991). It seems certain that further parallels of this sort will emerge to enliven our investigations of bacterial motility and chemotaxis.

VII. Acknowledgements

I am grateful to all of my colleagues who provided advice and information. Jerry Hazelbauer and Bob Macnab kindly supplied illustrations, and Sandy

BACTERIAL MOTILITY AND CHEMOTAXIS

335

Parkinson and Ann Stock sent me manuscripts in advance of their publication. Special thanks go to Bob Macnab, Phil Matsumura and Sandy Parkinson, my co-conspirators in organizing the BLAST (Bacterial Locomotion and Sensory Transduction) Conference that took place in January, 1991, in Austin, Texas. Much of the new material in this article was first brought to my attention at that stimulating meeting. I also want to thank those who endured me during the writing of this review: my long-suffering graduate students and research associates; my bacterial physiology students whose exams took forever to be graded and whose lectures were not quite as well prepared as they might have been; and my son Josiah, who had an absentee father for several months. Tony Rose, editor of this series, was more than reasonable in extending submission deadlines and in arranging for the inclusion of a colour plate; his patience and composure are remarkable. Finally, my heartfelt appreciation goes to my wife, Lily Bartoszek, for her angelic forbearance and for her undertaking to proofread the entire manuscript with extreme diligence, even though, as a non-scientist, she probably was wondering what all the fuss was about. REFERENCES

Abouhamad, W. N., Manson, M. D., Gibson, M. M. and Higgins, C. F. (1991). Molecular Microbiology 5, 1035. Adam, G. (1977). Journal of Theoretical Biology 65, 713. Adler, J. (1969). Science, 166, 1588. Adler, J. (1973). Journal of General Microbiology 74, 77. Adler, J. and Dahl, M. M. (1967). Journal of General Microbiology 46, 161. Adler, J. and Epstein, W. (1974). Proceedings of the National Academy of Sciences of the United States of America 71, 2895. Adler, J. and Shi, W. (1988). Cold Spring Harbor Symposium on Quantitative Biology 53,23. Adler, J. and Templeton, B. (1967). Journal of General Microbiology 46, 175. Adler, J., Hazelbauer, G . L. and Dahl, M. M. (1973). Journalof Bacteriology 115, 824. Aksamit, R. R. and Koshland, D . E., Jr (1974). Biochemistry 13, 4473. Alberti, L. and Harshey, R. M. (1990). Journal of Bacteriology 172, 4322. Allen, R. D. and Baumann, P. (1971). Journal of Bacteriology 107, 295. Allweiss, B., Dostal, J., Carey, K. E., Edwards, T. F. and Freter, R. (1977). Nature (London) 266,448. Ames, P. and Parkinson, J. S. (1988). Cell 55, 817. Ames, G. F. L. and Spudich, E. N. (1976). Proceedings of the National Academy of Sciences of the United States of America 73, 1877. Armitage, J. P. and Macnab, R. M. (1987). Journal of Bacteriology 169, 514. Armstrong, J . B. (1972). Canadian Journal of Microbiology 18, 1695. Armstrong, J. B. and Adler, J. (1969a). Genetics 61, 61. Armstrong, J. B. and Adler, J . (1969b). Journal of Bacteriology 97, 156. Armstrong, J. B., Adler, J. and Dahl, M. M. (1967). Journal of Bacteriology 93, 390. Arnosti, D. N. and Chamberlin, M. J. (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 830. Asakura, S. (1970). Advances in Biophysics 1, 99, Aswad, D. and Koshland, D. E., Jr. (1974). Journal of Bacteriology 118, 640. Aswad, D. and Koshland, D. E., Jr (1975a). Journal of Molecular Biology 97, 207.

336

M. D. MANSON

Aswad, D. and Koshland, D. E., Jr (1975b). Journal of Molecular Biology 97, 225. Bachmann, B. J. (1990). Microbiological Reviews 54, 130. Bartlett, D. H., Frantz, B. B. and Matsumura, P. (1988). Journalof Bacteriology 170, 1575. Bedale, W. A., Nettleton, D. 0..Sopata, C. S., Thoelke, M. S. and Ordal, G. W. (1988). Journal of Biological Chemistry 27, 8453. Belas, M. R. and Colwell, R. R. (1982). Journal of Bacteriology 150, 956. Belas, R., Simon, M. and Silverman, M. (1986). Journal of Bacteriology 167, 210. Berg, H. C. (1971). Review of Scientific Instruments 42, 868. Berg, H. C. (1974). Nature (London) 249, 77. Berg, H. C. (1975a). Annual Review of Biophysics and Bioengineering 4, 119. Berg, H. C. (1975b). Nature (London) 254, 389. Berg, H. C. (1976a). Journal of Theoretical Biology 56, 269. Berg, H. C. (1976b). In “Cell Motility” (R. Goldman, T. Pollard and J. Rosenbaum, eds), pp. 47-56. Cold Spring Harbor Press, Cold Spring Harbor. Berg, H. C. and Anderson, R. A. (1973). Nature (London) 245, 380. Berg, H. C. and Brown, D. A. (1972). Nature (London) 239, 500. Berg, H. C. and Khan, S. (1983). In “Motility and Recognition in Cell Biology” (H. Sund and C. Veeger, eds), pp. 486-497. Walter de Gruyter, Berlin. Berg, H. C. and Purcell, E. M. (1977). Biophysical Journal 20, 193. Berg, H. C. and Tedesco, P. M. (1975). Proceedings of the National Academy of Sciences of the United States of America 72, 3235, Berg, H. C., Bromley, D. B. and Charon, N. W. (1978). In “Relations between Structure and Function in the Prokaryotic Cell” (Society for General Microbiology. Symposium No. 28) (R. Y. Stanier, H. J. Rogersand J. B. Ward, eds). Cambridge University Press, Cambridge. Berg, H. C., Manson, M. D. and Conley, M. P. (1982). In “Prokaryotic and Eukaryotic Flagella” (W. B. Amos and J. G. Duckett, eds), pp. 1-31. Cambridge University Press, Cambridge. Bergman, K., Gulash-Hoffe, M., Hovestadt, R. E., Larosiliere, R. C., Ronco, P. G., I1 and Su, L. (1988). Journal of Bacteriology 170, 3249. Black, R. A., Hobson, A. C. and Adler, J. (1980). Proceedings of the National Academy of Sciences of the United States of America 77, 3879. Black, R. A., Hobson, A. C. and Adler, J. (1983). Journal of Bacteriology 153, 1187. Blackhart, B. D. and Zusman, D. R. (1985). Proceedings of the National Academy of Sciences of the United States of America 82, 8767. Blair, D. F. and Berg, H. C. (1988). Science 242, 1678. Blair, D. F. and Berg, H. C. (1990). Cell 60,439. Blakemore, R. P. (1982). Annual Review of Microbiology 36, 217. Blank, V. (1987). Diploma Thesis: University of Constance, FRG (in German). Block, S. M. and Berg, H. C. (1984). Nature (London) 309, 470. Block, S. M., Segall, J. E. and Berg, H. C. (1982). Cell, 31, 215. Block, S. M., Segall, J. E. and Berg, H. C. (1983). Journal of Bacteriology 154, 312. Block, S. M., Blair, D. F. and Berg, H. C. (1989). Science 338, 514. Bogonez, E. and Koshland, D. E., Jr (1985). Proceedings of the National Academy of Sciences of the United States of America 82, 4891. Bollinger, J. C., Park, C., Harayama, S. and Hazelbauer, G. L. (1984). Proceedings of the National Academy of Sciences of the United States of America 81, 3287. Boos, W., Gordon, A. S . , Hall, R. E. and Price, H. D. (1972). Journalof BiologicalChemistry 247, 917. Boos, W., Steinacher, 1. and Engelhardt-Altendorf, D. (1981). Molecular and General Genetics 184, 508. Borkovich, K. A. and Simon, M. I. (1990). Cell 63, 1339. Borkovich, K. A,, Kaplan, N., Hess, J. F. and Simon, M. 1. (1989). Proceedingsofthe National Academy of Sciences of the United States of America 86, 1208. Bourret, R. B., Hess, J. F., Borkovich, K. A,, Pakula, A. A. and Simon, M. I. (1989). Journal of Biological Chemistry 264, 7085.

BACTERIAL MOTILITY AND CHEMOTAXIS

337

Bourret, R. B., Hess, J. F. and Simon, M. I. (1990). Proceedings of the National Academy of Sciences of the United States of America 87, 41. Boyd, A. and Simon, M. I. (1980). Journal of Bacteriology 143, 809. Boyd, A., Krikos, A. and Simon, M. (1981). Cell 26, 333. Boyd, A, , Kendall, K. and Simon, M. I. (1983). Nature (London) 301, 623. Brass, J. M. and Manson, M. D. (1984). Journal of Bacteriology 157, 881-890. Brown, D. A. and Berg, H. C. (1984). Proceedings of the National Academy of Sciences of the United States of America 71, 1388. Burchard, R. P. (1981). Annual Review of Microbiology 35, 497. Canale-Parola, E. (1977). Bacteriological Reviews 41, 181. Champer, R., Dingwall, A. and Shapiro, L. (1987). Journal of Molecular Biology 194, 71. Chelsky, D. 0. and Dahlquist, F. W. (1980a). Biochemistry 19, 4633. Chelsky, D. 0.and Dahlquist, F. W. (1980b). Proceedings of the National Academy of Sciences of the United States of America 77, 2334. Chet, I. and Mitchell, R. (1976). Annual Review of Microbiology 30, 221. Chet, I., Fogel, S. and Mitchell, R. (1971). Journal of Bacteriology 106, 863. Chun, S. Y. and Parkinson, J. S. (1988). Science 239, 276. Clarke, S. and Koshland, D. E., Jr (1979). Journal of Biological Chemistry 254, 9695. Clegg, D. 0. and Koshland, C. E., Jr (1984). Proceedings of the National Academy of Sciences of the United States of America 81, 5056. Collins, A. L. T. and Stocker, B. A. D. (1976). Journal of Bacteriology 128, 754. Conley, M. P., Wolfe, A. J., Blair, D. F. and Berg, H. C. (1989). Journalof Bacteriology 171, 5190. Coulton, J. W. and Murray, R. G . E. (1978). Journal of Bacteriology 136, 1037. Dahl, M. K. and Manson, M. D. (1985). Journal of Bacteriology 164, 1057. Dahl, M. K., Boos, W. and Manson, M. D. (1989). Journal of Bacteriology 171, 2361. Dayrell-Hart, B. and Burchard, R. P. (1979). Journal of Bacteriology 137, 1417. Dean, G. E., Macnab, R. M., Stader, J., Matsumura, P. and Burks, C. (1984). Journal of Bacteriology 159, 991. DeFranco, A. L. and Koshland, D. E., Jr (1980). Proceedings of the National Academy of Sciences of the United States of America 77, 2429. DeFranco, A. L. and Koshland, D. E., Jr (1981). Journal of Bacteriology 147, 390. DeFranco, A. L., Parkinson, J. S. and Koshland, D. E., Jr (1979). Journalof Bacteriology 139,107. DePamphilis, M. L. and Adler, J. (1971a). Journal of Bacteriology 105, 376. DePamphilis, M. L. and Adler, J. (1971b). Journal of Bacteriology 105, 384. DePamphilis, M. L. and Adler, J. (1971~).Journal of Bacteriology 105, 396. Dietzel, I., Kolb, V. and Boos, W. (1978). Archives of Microbiology 118, 207. Driks, A , , Bryan, R., Shapiro, L. and DeRosier, D. J. (1989). Journal of Molecular Biology 206, 627. Dunten, P. and Koshland, D. E., Jr (1991). Journal of Biological Chemistry 266, 1491. Duplay, P. and Szmelcman, S. (1987). Journal of Molecular Biology 194, 675. Duplay, P., Szmelcman, S., Bedouelle, H. and Hofnung, M. (1987). Journal of Molecular Biology 194, 663. Edwards, P. R. and Ewing, W. H. (1972). “Identification of Enterobacteriaceae” 3rd edn., pp. 146-258. Burgess Publishing Co. Minneapolis. Eisenbach, M. (1982). Biochemistry 21, 6818. Eisenbach, M. (1990). Molecular Microbiology 4, 161. Eisenbach, M. and Adler, J. (1981). Journal of Biological Chemistry 256, 8807. Eisenbach, M., Margolin, Y. and Ravid, S. (1985). In “Sensing and Response in Microorganisms” (M. Eisenbach and M. Balaban, eds), pp. 43-611. Elsevier, Amsterdam. Eisenbach, M., Constantinou, C., Aloni, H. and Shinitizky, M. (1990a). Journal of Bacteriology 172, 5218. Eisenbach, M., Wolf, A , , Welch, M., Caplan, S. R., Lapidus, 1. R., MacNab, R. M., Aloni, H. and Asher, 0. (1990b). Journal of Molecular Biology 211, 551. Ely, B., Geradot, C. J., Fleming, D. L., Gomes, S. L., Frederikse, P. and Shipiro, L. (1986). Genetics 114, 717.

338

M. D. MANSON

Emerson, S. U., Tokuyasu, K. and Simon, M. I. (1970). Science 169, 190. Engstroem, P. and Hazelbauer, G . L. (1980) Cell 20, 165. Engstroem, P. and Hazelbauer, G. L. (1982). Biochimica et Biophysica Acta 686, 19. Enomoto, M. (1966). Genetics 54, 715. Falke, J. J. and Koshland, D. E., Jr (1987). Science 237, 1596. Falke, J. J . , Dernburg, A. F., Sternberg, D. A., Zalkin, N., Milligan, D. L. and Koshland, D. E., Jr (1988). Journal of Biological Chemistry 263, 14850. Foster, D. L., Mowbray, S. L., Jap, B. K. and Koshland, D. E., Jr (1985). Journal of Biological Chemistry 260, 11706. Frankel, R. B . and Blakemore, R. P. (1989). Bioelectromagnetics 10, 223. Frederikse, P. H. and Shapiro, L. (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 4061. Freter, R. and O’Brien, P. C. M. (1981a). Infection and Immunity 39, 215. Freter, R. and O’Brien, P. C. M. (1981b). Infection and Immunity 39, 222. Freter, R. and O’Brien, P. C. M. (1981~).Infection and Immunity 39, 234. Fuerst, J. A. and Perry, J. W. (1988). Journal of Bacteriology 170, 1488. Furlong, C. E . (1987). In “Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology” (F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter and H. E. Umbarger, eds), vol. 1 , pp. 768-796. American Society for Microbiology, Washington DC. Gebert, J. F., Overhoff, B., Manson, M. D. and Boos, W. (1988). Journal of Biological Chemistry 263, 16652. Gegner, J. A. and Dahlquist, F. W. (1991). Proceedingsofthe National Academy of Sciencesof the United States of America 88, 750. Glagolev, A. N. (1984). Trends in Biochemical Sciences 9, 397. Glagolev, A. N. and Skulachev, V. P. (1978). Nature (London) 272, 280. Goetz, R. and Schmitt, R . (1987). Journal of Bacteriology 169, 3146. Goulbourne, E. A., Jr and Greenberg, E. P. (1980). Journal of Bacteriology 143, 1450. Goulbourne, E . A., Jr and Greenberg, E . P. (1981). Journal of Bacteriology 148, 837. Goy, M. F., Springer, M. S. and Adler, J. (1977). Proceedings of the National Academy of Sciences of the United States of America 74, 4964. Goy, M. F., Springer, M. S. and Adler, J. (1978). Cell 15, 1231. Groarke, J . M., Mahoney, W. C., Hope, J. N., Furlong, C. E., Robb, F. T., Zalkin, H. and Hermodson, M. A. (1983). Journal of Biological Chemistry 258, 12952. Gruebl, G., Vogler, A . P. and Lengeler, J. W. (1990). Journal of Bacteriology 172, 5871. Gulash, M., Ames, P., LaRosiliere, R. C. and Bergman, K. (1984). Applied and Environmental Microbiology 48, 149. Haeder, D. P. (1987). Microbiological Reviews 51, 1 . Hayashi, H., Koiwai, 0. and Kozuka, M. (1979). Journal of Biochemistry (Japan) 85, 1213. Hazelbauer, G. L. (1975). Journal of Bacteriology 122, 206. Hazelbauer, G. L. and Adler, J. (1971). Nature New Biology (London) 230, 101. Hazelbauer, G. L. and Engstroem, P. (1980). Nature (London) 283, 98. Hazelbauer, G. L. and Harayama, S. (1981). Cell 16, 617. Hazelbauer, G. L. and Harayama, S. (1983). International Review of Cytology 81, 33. Hazelbauer, G. L., Engstroem, P. and Harayama, S. (1980). Journal of Bacteriology 145,43. Hazelbauer, G. L . , Yaghmai, R., Burrows, G. G., Baumgartner, J. W., Dutton, D. P. and Morgan, D. G. (1990). In “Biology of the Chemotactic Response” (Society for General Microbiology Symposium, No. 46) (J. P. Armitage and J. M. Lackie, eds), pp. 107-134. Cambridge University Press, Cambridge. Hedblom, M. L. and Adler, J. (1980). Journal of Bacteriology 155, 1048. Helmann, J. D . and Chamberlin, M. J. (1987). Proceedings of the National Academy of Sciences of the United States of America 84, 6422. Helmann, J. D., Marquez, L. M. and Chamberlin, M. J. (1988).Journal of Bacteriology 170,1568. Hess, J. F., Oosawa, K., Matsumura, P. and Simon, M. I. (1987). Proceedingsof the National Academy of Sciences of the United States of America 84, 7609. Hess, J . F., Bourret, R . B. and Simon, M. I. (1988a). Nature (London) 336, 139.

BACTERIAL MOTILITY AND CHEMOTAXIS

339

Hess, J . F., Oosawa, K., Kaplan, N. and Simon, M. I. (1988b). Cell 53, 79. Hirota, N. and Imae, Y. (1983). Journal of Biological Chemistry 258, 10577. Homma, M. and Iino, T. (1985). Journal of Bacteriology 162, 183. Homma, M., Fujita, H., Yamaguchi, S. and Iino, T. (1984a). Journal of Bacteriology 158, 1056. Homma, M., Kutsukake, K., Iino, T. and Yamaguchi, S . (1984b). Journal of Bacteriology 157,

loo.

Homma, M., Kutsukake, K. and lino, T. (1985). Journal of Bacteriology 163, 464. Homma, M., Aizawa, S . , Dean, G. E . and Macnab, R. M. (1987a). Proceedings of the National Academy of Sciences of the United States of America 84, 7843. Homma, M., Komeda, Y., Iino, T. and Macnab, R. M. (1987b). Journalof Bacteriology 169, 1493. Homma, M., Ohnishi, K., Iino, T. and Macnab, R. M. (1987~).Journal of Bacteriology 169, 3617. Homma, M., Iino, T. and Macnab, R. M. (1988). Journal of Bacteriology 170, 2221. Homma, M., DeRosier, D. J. and Macnab, R. M. (1990). Journalof Molecular Biology 213, 819. Icho, T. and Iino, T . (1978). Journal of Bacteriology 134, 854. Igo, M. M., Ninfa, A . J., Stock, J. B. andSilhavy,T. J. (1989). GenesandDevelopment3,1725. Iino, T . (1969). Journal of General Microbiology 56, 227. Iino, T . (1974). Journal of Supramolecular Structure 2, 372. Iino, T. (1977). Annual Review of Genetics 11, 161. Iino, T. and Kutsukake, K. (1983). In “Nucleic Acid Research. Future Developments” (K. Mizobuchi, I . Watanabe and J. D. Watson, eds), pp. 395-406. Academic Press, New York. Iino, T. and Mitani, M. (1966). Journal of General Microbiology 44, 27. Iino, T. and Mitani, M. (1967). Journal of General Microbiology 49, 81. Iino, T., Komeda, Y., Kutsukake, K., Macnab, R. M., Matsumura, P., Parkinson, J. S., Simon, M. 1. and Yamaguchi, S. (1988). Microbiological Reviews 52, 533. Imae, Y., Oosawa, K. Mizuno, T., Kihara, M. and Macnab, R. M. (1987). Journal of Bacteriology 169, 371. Ishihara, A. and Hotani, H. (1980). Journal of Molecular Biology 139, 265. Ishihara, A., Yamaguchi, S. and Hotani, H. (1981). Journal of Bacteriology 145, 1082. Ishihara, A , , Segall, J. E . , Block, S. M. and Berg, H . C. (1983). Journalof Bacteriology 155, 228. Jones, C. J. and Aizawa, S.-I. (1991). Advances in Microbial Physiology 32, 109. Jones, C. J., Homma, M. and Macnab, R. M. (1987). Journal of Bacteriology 169, 1489. Jones, C . J., Homma, M. and Macnab, R. M. (1989). Journal of Bacteriology 171, 3890. Kagawa, H., Ono, N., Enomoto, M. and Komeda, Y. (1984). Journalof Bacteriology 157,649. Kaiser, A. D. (1986). Annual Review of Genetics 20, 539. Kamiya, R., Hotani, H . and Asakura, S. (1982). In “Prokaryotic and Eukaryotic Flagella” (W. B. Amos and J. G. Duckett, eds), pp. 5 5 7 6 . Cambridge University Press, Cambridge. Kehry, M. R. and Dahlquist, F. W. (1982a). Journal of Biological Chemistry 257, 10378. Kehry, M. R. and Dahlquist, F. W. (1982b). Cell 29, 761. Kehry, M. R . , Bond, M. W., Hunkapiller, M. W. and Dahlquist, F. W. (1983). Proceedings of the National Academy of Sciences of the United States of America 80, 3599. Kehry, M. R., Doak, T. G. and Dahlquist, F. W. (1984). Journalof Biological Chemistry 259, 11828. Kehry, M. R., Doak, T. G. and Dahlquist, F. W. (1985). Journal of Bacteriology 163, 983. Khan, S. and Berg, H. C . (1983). Cell 32, 913. Khan, S. and Macnab, R. M. (1980a). Journal of Molecular Biology 138, 563. Khan, S. and Macnab, R. M. (1980b). Journal of Molecular Biology 138, 599. Khan, S., Macnab, R. M., DeFranco, A . L. and Koshland, D. E., Jr (1978). Proceedings of the National Academy of Sciences of the United States of America 75, 4150. Khan, S . , Meister, M. and Berg, H. C . (1985). Journal of Molecular Biology 184, 645. Khan, S., Dapice, M. and Reese, T. S . (1988). Journal of Molecular Biology 202, 575.

340

M . D. MANSON

Kihara, M. and Macnab, R. M. (1981). Journal of Bacteriology 145, 1209. Kihara, M., Homma, M., Kutsukake, K. and Macnab, R. M. (1989). Journalof Bacteriology 171, 3247. Kleene, S. J., Toews, M. L. and Adler, J. (1977). Journalof Biological Chemistry 252,3214. Kofoid, E . C. and Parkinson, J. S. (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 4981. Kofoid, E. C. and Parkinson, J. S. (1991). Journal of Bacteriology 173, 2116. Koman, A,, Harayama, S. and Hazelbauer, G . L. (1979). Journal of Bacteriology 138,739. Komeda, Y. (1982). Journal of Bacteriology 150, 16. Komeda, Y. (1986). Journal of Bacteriology 168, 1315. Komeda, Y. and Iino, T. (1979). Journal of Bacteriology 139, 721. Komeda, Y., Suzuki, H., Ishidsu, J. and Iino, T. (1975). Molecular and General Genetics 142, 289. Komeda, Y., Silverman, M. and Simon, M. (1978). Journal of Bacteriology 133, 364. Kondoh, H., Ball, C. B. and Adler, J. (1979). Proceedings of the National AcademyofSciences of the United States of America 76, 260. Kort, E. N., Goy, M. F., Larsen, S. H. and Adler, J. (1975). Proceedings of the National Academy of Sciences of the United States of America 72, 3939. Kossmann, M., Wolff, C. and Manson, M. D. (1988). Journal of Bacteriology 170,4516. Krieg, N. K., Tomelty, J. P. and Wells, J . C. (1967). Journal of Bacteriology 94, 1431. Krikos, A., Mutoh, N., Boyd, A. and Simon, M. I. (1983). Cell 33, 615. Krikos, A,, Conley, M. P., Boyd, A., Berg, H. C. and Berg, H. C. (1985). Proceedings of the National Academy of Sciences of the United States of America 82, 1326. Kuo, S. C. and Koshland, D. E., Jr (1986). Journal of Bacteriology 166, 1007. Kuo, S. C. and Koshland, D. E., Jr (1989). Journal of Bacteriology 171, 6279. Kupper, J . , Wildhaber, I., Gao, Z. and Baeuerlein, E. (1989). Journal of Bacteriology 171, 2803. Kutsukake, K., Ohya, Y. and Iino, T. (1990). Journal of Bacteriology 172, 741. Kuwajima, G., Kawagishi, I., Homma, M., Asaka, J., Kondo, E. and Macnab, R. M. (1989). Proceedings of the National Academy of Sciences of the United States of America 86,4953. Laeuger, P. (1977). Nature (London) 268, 360. Lapidus, I. R. and Berg, H. C. (1982). Journal of Bacteriology 151, 384. Lapidus, 1. R., Welch, M. and Eisenbach, M. (1988). Journal of Bacteriology 170, 3627. Larsen, S. H., Adler, J., Gargus, J. J. and Hogg, R. W. (1974a). Proceedings of the National Academy of Sciences of the United States of America 71, 1239. Larsen, S. H., Reader, R. W., Kort, E. N., Tso, W.-W. and Adler, J. (1974b). Nature (London) 249, 74. Lawn, A. M. (1977). Journal of General Microbiology 101, 112. Lederburg, J. and Iino, T. (1956). Genetics 41, 744. Lee, L. and Imae, Y. (1990). Journal of Bacteriology 172, 377. Lee, L., Mizuno, T. and Imae, Y. (1988). Journal of Bacteriology 170, 4769. Leifson, E. (1960). “Atlas of Bacterial Flagellation” Academic Press, New York. Lengeler, J. W. and Vogler, A. P. (1989). FEMS Microbiology Reviews 5, 81. Li, C., Boileau, A. J., Kung, C. and Adler, J. (1988). Proceedings ofthe National Academyof Sciences of the United States of America 85, 9451. Liu, J. D. and Parkinson, J. S. (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 8703. Liu, J. D. and Parkinson, J. S. (1991). Journal of Bacteriology 173, 4941. Lowe, G., Meister, M. and Berg, H. C. (1987). Nature (London) 325, 637. Lowy, J. and Hanson, J. (1965). Journal of Molecular Biology 11, 293. Lukat, G. S . , Lee, B. H., Mottonen, J. M., Stock, A. M. and Stock, J. B. (1991). Journal of Biological Chemistry 266, 8348. Lupas, A. and Stock, J. (1989). Journal of Biological Chemistry 264, 17337. McBride, M. J . , Weinberg, R. A. and Zusman, D. R. (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 424. McCarter, L., Hilmen, M. and Simon, M. (1988). Cell 54, 345.

BACTERIAL MOTILITY AND CHEMOTAXIS

34 1

McCleary, W. R. and Zusman, D. R. (1990). Proceedings of the National Academy of Sciences of the United States of America 87,5898. McCleary, W. R., McBride, M. J. and Zusman, D. R. (1990). Journal of Bacteriology 172, 4877. McGowan, E. D., Silhavy, T. J. and Boos, W. (1974). Biochemistry 13,993. Macnab, R. M. (1976). Journal of Clinical Microbiology 4, 258. Macnab, R. M. (1978). CRC Critical Reviews in Biochemistry 5,291. Macnab, R. M. (1987a). In “Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology” (F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter and H. E. Umbarger, eds), vol. 1, pp. 70-83. American Society for Microbiology, Washington, DC. Macnab, R. M. (1987b). In “Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology” (F. C. Neidhardt, J . L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter and H. E. Umbarger, eds), vol. 1, pp. 732-759. American Society for Microbiology, Washington, DC. Macnab, R. M. (1990). In “Biology of the Chemotactic Response” (Society for General Microbiology Symposium, No. 46),(J. P. Armitage and J. M. Lackie, eds), pp. 77-106. Cambridge University Press, Cambridge. Macnab, R. M. and Han, D. P. (1983). Cell 32, 109. Macnab, R. M. and Koshland, D. E . , Jr (1972). Proceedings of the National Academy of Sciences of the United States of America 69,2509. Macnab, R. M. and Parkinson, J . S . (1991). Trends in Genetics 7, 196. Maeda, K. and Imae, Y. (1979). Proceedings of the National Academy of Sciences of the United States of America 76,91. Maeda, K., Imae, Y . , Shioi, J.-I. and Oosawa, F. (1976). Journal of Bacteriology 127, 1039. Malakooti, J., Komeda, Y. and Matsumura, P. (1989). Journal of Bacteriology 171,2728. Manoil, C.and Beckwith, J. (1985). Proceedings of the National Academy of Sciences of the United States of America 82,8129. Manoil, C. and Beckwith, J. (1986). Science 233, 1403. Manson, M. D. and Kossmann, M. (1986). Journal of Bacteriology 165,34. Manson, M. D., Tedesco, P. M., Berg, H. C., Harold, F. M. and van der Drift, C. (1977). Proceedings of the National Academy of Sciences of the United States of America 74,3060. Manson,M. D.,Tedesco, P. M. andBerg, H. C. (1980). JournalofMolecularBiology138,541. Manson, M. D., Boos, W., Bassford, P. J., Jr and Rasmussen, B. A. (1985). Journal of Biological Chemistry 260,9727. Manson, M. D., Blank, V., Brade, G . and Higgins, C . F. (1986) Nature (London) 321,253. Martinez, R. J., Ichiki, A. T., Lundh, N. P. and Tronick, S . R. (1968). Journal of Molecular Biology 34, 559-564. Matsumura, P., Silverman, M. and Simon, M. (1977). Journal of Bacteriology 132, 996. Matsumura, P., Rydel, J. J., Linzmeier, R. and Vacante, D. (1984). Journal of Bacteriology 160,36. Matsumura, P., Roman, S., Volz, K. and McNally, D. (1990). In “Biology of the Chemotactic Response” (Society for General Microbiology Symposium, No. 46) (J. P. Armitage and J. M. Lackie, eds), pp. 135-154. Cambridge University Press, Cambridge. Matsuura, S . , Shioi, J.-I. and Imae, Y. (1977). FEBS Letters 82, 187. Matsuura, S.,Shioi, J.-I., Imae, Y. and Iida, S . (1979). Journal of Bacteriology 140,28. Meister, M. and Berg, H. C. (1987). Biophysical Journal 52,413. Meister, M., Lowe, G . and Berg, H. C. (1987). Cell 49, 643. Mesibov, R. and Adler, J. (1972). Journal of Bacteriology 112,315. Meynell, E.W. (1961). Journal of General Microbiology 25, 253. Miller, J. B. and Koshland, D. E., Jr (1977). Proceedings of the National Academy of Sciences of the United States of America 74, 4752. Miller, J. B. and Koshland, D . E., Jr (1980). Journal of Bacteriology 141,26. Milligan, D . L. and Koshland, D. E . , Jr (1988). Journal of Biological Chemistry 263,6268. Milligan, D . L. and Koshland, D. E . , Jr (1991). FASEB Journal 5, A1434. Mizuno, T.and Imae, Y. (1984). Journal of Bacteriology 159,360.

342

M. D. MANSON

Mizuno, T., Mutoh, N., Panasenko, S. M. and Imae, Y. (1986). Journalof Bacteriology 165, 890. Moe, G. R. and Koshland, D. E., Jr (1986). In “Microbial Energy Transduction: Genetics, Structure, and Function of Membrane Proteins” (D. C. Youvan and F. Daldal, eds), pp. 163-168. Cold Spring Harbor Laboratory, Cold Spring Harbor. Moe, G. R., Bollag, G . E. and Koshland, D . E., Jr (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 5683. Mowbray, S. L. and Koshland, D. E., Jr (1987). Cell, 50, 171. Mowbray, S. L. and Koshland, D. E., Jr (1990). Journal of Biological Chemistry 265, 15638. Mowbray, S. L. and Petsko, G. A. (1982). Journal of Molecular Biology 160, 545. Mowbray, S. L. and Petsko, G. A.(1983). Journal of Biological Chemistry 258, 7991. Mowbray, S. L., Foster, D. L. and Koshland, D. E., Jr (1985). Journalof BiologicalChemistry 260, 11711. Mutoh, N. and Simon, M. I. (1986). Journal of Bacteriology 165, 161. Mutoh, N., Oosawa, K. and Simon, M. I. (1986). Journal of Bacteriology 164, 992. Nelson, D. C., Jorgenson, B. B. and Revsbach, N. P. (1986). Applied and Environmental Microbiology 52, 225. Nevola, J. J . , Stocker, B. A. D., Laux, D. C. and Cohen, P. S. (1985). Infection and Immunity 50, 152. Ninfa, A . J . , Ninfa, E. G., Lupas, A,, Stock, A,, Magasanik, B. and Stock, J . (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 5492. Niwano, M. and Taylor, B. L. (1982). Proceedings of the National Academy of Sciences of the United States of America 79, 11. Nixon, B. T., Ronson, C. W. and Ausubel, F. W. (1986). Proceedingsofthe NationalAcademy of Sciences of the United States of America 83, 7850. Nowlin, D. M., Bollinger, J. and Hazelbauer, G. L. (1987). Journal of Biological Chemistry 262, 6039. Ohnishi, K . , Kutsukake, K., Suzuki, H. and Iino, T. (1990). Molecular and General Genetics 221, 139. Olson, E. R., Dunyak, D. S . , Jurss, L. M. and Poorman, R. A. (1991). Journalof Bacteriology 173, 234. Oosawa, K. and Imae, Y . (1983). Journal of Bacreriology 154, 104. Oosawa, K. and Simon, M. I. (1986). Proceedings of the National Academy of Sciences of the United States of America 83, 6930. Ordal, G. W. (1977). Nature (London) 270, 66. Ordal, G. W. (1985). CRC Critical Reviews in Microbiology 12, 95. Ordal, G. W. and Gibson, K. J . (1977). Journalof Bacteriology 129, 151. Ordal, G. W. and Goldrnan, D. J . (1975). Science 189, 802. Palleroni, N. J . , Doudoroff, M., Stanier, R. Y ., Solanes, R. E. and Mandel, M. (1970). Journal of General Microbiology 60, 215. Parish, J. H. (1979). In “Developmental Biology of Prokaryotes” (J. H. Parish, ed.). University of California Press, Berkeley. Park, C. and Hazelbauer, G. L. (1986). Journal of Bacteriology 167, 101. Park, C., Dutton, D. P. and Hazelbauer, G. L. (1990). Journal of Bacteriology 172,7179. Parkinson, J. S. (1974). Nature (London) 252, 317. Parkinson, J . S. (1976). Journal of Bacteriology 126, 758. Parkinson, J . S. (1978). Journal of Bacteriology 135, 45. Parkinson, J. S. and Houts, S. E. (1982). Journal of Bacteriology 151, 106. Parkinson, J. S. and Parker, S. R. (1979). Proceedings of the National Academy of Sciences of the United States of America 76, 2390. Parkinson, J. S. and Revello, P. T . (1978). Cell 15, 1221. Parkinson, J . S . , Parker, S. R., Talbert, P. B. and Houts, S. E. (1983). Journalof Bacteriology 155, 265. Patterson-Delafield, J . , Martinez, R. J . , Stocker, B. A. D. and Yamaguchi, S. (1973). Archiv fur Mikrobiologie 90, 107. Pfeffer, W . (1884). Untersuchungen des Botanischen Instituts Tuebingen 1, 363.

BACTERIAL MOTILITY AND CHEMOTAXIS

343

Pfeffer, W. (1888). Untersuchungen des Botanischen Instituts Tuebingen 2, 582. Pleier, E. and Schmitt, R. (1989). Journal of Bacteriology 171, 1467. Postma, P. W. and Lengeler, J. W. (1985). Microbiological Reviews 49, 232. Purcell, E. M. (1977). American Journal of Physics 45, 3. Qi, Y. L. and Adler, J. (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 8358. Ravid, S. and Eisenbach, M. (1983). Journal of Bacteriology 154, 604. Ravid, S. and Eisenbach, M. (1984). Journal of Bacteriology 158, 222. Ravid, S., Matsumura, P. and Eisenbach, M. (1986). Proceedings of the National Academy of Sciences of the United Stares of America 83, 7157. Reichenbach, H. (1981). Annual Review of Microbiology 35, 339. Repaske, D. R. and Adler, J. (1981). Journal of Bacteriology 145, 1196. Ridgway, H. F. (1977). Journal of Bacteriology 131, 544. Ridgway, H. F., Silverman, M. and Simon, M. I. (1977). Journal of Bacteriology 132, 657. Rollins, C. and Dahlquist, F. W. (1981). Cell 25, 333. Ronson, C. W., Nixon, B. T . and Ausubel, F. M. (1987). Cell 49, 579. Rosengarten, R. and Kirchhoff, H. (1987). Journal of Bacteriology 160, 1891. Rowsell, E. H., Smith, J. M., Wolfe, A. J., Conley, M. P., Taylor, B. L. and Berg, H. C. (1988). FASEB Journal 2, A575. Rubik, B. A. and Koshland, D. E., Jr (1978). Proceedingsof the NationalAcademyofSciences of the United States of America 75, 2820. Russo, A. F. and Koshland, D. E., Jr (1983). Science 222, 1016. Russo, A. F. and Koshland, D. E., Jr (1986). Journal of Bacteriology 165, 276. Saier, M. H., Jr (1989). Microbiological Reviews 53, 109. Sanders, D. A. and Koshland, D. E., Jr (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 8425. Sanders, D. A,, Gillece-Castro, B. L., Stock, A. M., Burlingame, A. L. and Koshland, D. E., Jr (1989a). Journal of Biological Chemistry 264, 21770. Sanders, D. A, , Mendez, B. and Koshland, D. E., Jr (1989b). Journal of Bacteriology, 171, 6271. Sanderson, K. E. and Roth, J. R. (1988). Microbiological Reviews 52, 485. Schimz, A. and Hildebrand, E. (1979). Journal of Bacteriology 140, 749. Schmitt, R., Bamberger, I., Acker, G. and Meyer, F. (1974a). Archives of Microbiology 100, 145. Schmitt, R., Raska, 1. and Mayer, F. (1974b). Journal of Bacteriology 117, 844. Scholle, A , , Vreemann, J., Blank, V., Nold, A , , Boos, W. and Manson, M. D. (1987). Molecular and General Genetics 208, 247. Segall, J. E., Manson, M. D. and Berg, H. C. (1982). Nature (London) 296, 855. Segall, J. E., Ishihara, A. and Berg, H. C. (1985). Journal of Bacteriology 161, 51. Segall, J . E., Block, S. M. and Berg, H. C. (1986). Proceedings o f t h e National Academy of Sciences of the United States of America 83, 8987. Seymour, F. W. K. and Doetsch, R. N. (1973). Journal of General Microbiology 78,287. Shade, S . Z., Adler, J. and Ris, H. (1967). Journal of Virology I , 599. Shaw, C. H., Ashby, A. M., Brown, A , , Royal, C., Loake, G. J. and Shaw, C. H. (1988). Molecular Microbiology 2, 413. Sherris, D. and Parkinson, J. S. (1981). Proceedings of the National Academy of Sciencesof the United States of America 78, 6051. Shinoda, S. and Okamoto, K. (1977). Journal of Bacteriology 129, 1266. Shioi, J. and Taylor, B. L. (1984). Journal of Biological Chemistry 259, 10983. Shioi, J.-I., Galloway, R. J., Niwano, M., Chinnock, R. E. andTaylor, B. L. (1982). Journalof Biological Chemistry 257, 7969. Shioi, J., Tribhuwan, R. C., Berg, S. T. and Taylor, B. L. (1988). Journalof Bacteriology 170, 5507. Silverman, M. R. and Simon, M. I. (1972). Journal of Bacteriology 112, 986. Silverman, M. and Simon, M. (1973a). Journal of Bacteriology 113, 105. Silverman, M. and Simon, M. (1973b). Journal of Bacteriology 116, 114.

344

M . D. MANSON

Silverman, M. and Simon, M. (1974a). Journal of Bacteriology 117, 73. Silverman, M. and Simon, M. (1974b). Journal of Bacteriology 120, 1196. Silverman, M. and Simon, M. (1974~).Nature (London) 249, 73. Silverman, M. and Simon, M. (1977a). Journal of Bacteriology 130, 1317. Silverman, M. and Simon, M. (1977b). Proceedings of the National Academy of Sciences of the United States of America 74, 3317. Silverman, M. and Simon, M. I. (1977~).Annual Review of Microbiology 31, 397. Silverman, M., Matsumura, P. and Simon, M. (1976). Proceedingsof the National Academyof Sciences of the United States of America 13, 3126. Silverman, M., Zieg, J., Hilmen, M. and Simon, M. (1979). Proceedings of the National Academy of Sciences of the United States of America 76, 391. Simms, S., Keane, M. and Stock, J. B. (1985). Journal of Biological Chemistry 260, 10161. Simms, S. A., Stock, A. M. and Stock, J. B. (1987). Journal of Biological Chemistry 262,8537. Simon, M., Zieg, J., Silverman, M. and Doolittle, R. (1980). Science 209, 1370. Sjoblad, R. D., Emala, C. W. and Doetsch, R. N. (1983). Cell Motility 3, 93. Slocum, M. K. and Parkinson, J. S. (1983). Journal of Bacteriology 155, 565. Slocum, M. K. and Parkinson, J. S. (1985). Journal of Bacteriology 163, 586. Smith, R. A. and Parkinson, J. S. (1980). Proceedings of the National Academy of Sciences of the United States of America 77, 5370. Snyder, M. A., Stock, J. B. and Koshland, D. E., Jr (1981). Journalof Molecular Biology 149, 241. Springer, W . R. and Koshland, D. E., Jr (1977). Proceedings of the National Academy of Sciences of the United States of America 74, 533. Springer, M. S.and Zanolari, B. (1984). Proceedings of the National Academy of Sciences of the United States of America 81, 5061. Springer, M. S . , Kort, E. N., Larsen, S. H . , Ordal, G. W. and Adler, J. (1975). Proceedings of the National Academy of Sciences of the United States of America 72, 4640. Springer, M. S., Goy, M. F. and Adler, J. (1977a). Proceedings of the National Academy of Sciences of the United States of America 74, 183. Springer, M. S., Goy, M. F. and Adler, J. (1977b). Proceedings of the National Academy of Sciences of the United States of America 74, 3312. Springer, M. S . , Goy, M. F. and Adler, J. (1979). Nature (London) 280, 279. Springer,M. S.,Zanolari,B. andPierzchala,P. A. (1982). JournalofBiologicalChemistry257, 6861. Spudich, J. L. and Bogomolni, R. A. (1984). Nature (London) 312, 509. Spudich, J. L. and Bogomolni, R. A. (1988). Annual Review of Biophysics and Biophysical Chemistry 17, 193. Spudich, J . L. and Koshland, D. E., Jr (1975). Proceedings of the National Academy of Sciences of the United States of America 72, 710. Spudich, J. L. and Koshland, D. E., Jr (1976). Nature (London) 262, 467. Spudich, J. L. and Stoeckenius, W. (1979). Photochemistry and Photobiophysics 1, 43. Spurlino, J. C. (1988). Ph.D. Thesis: Rice University, USA. Spurlino, J . C., Lu, G.-Y. and Quiocho, F. A. (1991). Journal of Biological Chemistry 266, 5202. Stader, J., Matsumura, P., Vacante, D., Dean, G. E. and Macnab, R. M. (1986). Journal of Bacteriology 166, 244. S t a h e y e r , M. J . , Hahnenberger, K. M., Sosinsky, G. E., Shapiro, L. and DeRosier, D. J. (1989). Journal of Molecular Biology 205, 511. Stewart, R. C. and Dahlquist, F. W. (1987). Chemical Reviews 87, 997. Stewart, R. C. and Dahlquist, F. W. (1988). Journal of Bacteriology 170, 1728. Stewart, R. C., Roth, A. F. and Dahlquist, F. W. (1990). Journal of Bacteriology 172,3388. Stock, A. M. and Stock, J. B. (1987). Journal of Bacteriology 269, 3301. Stock, A., Koshland, D. E., Jr and Stock, J. (1985). Proceedings of the National Academy of Sciences of the United States of America 82, 7989. Stock, A., Mottonen, J., Chen, T. and Stock, J. (1987). Journal of Biological Chemistry 262, 535.

BACTERIAL MOTILITY AND CHEMOTAXIS

345

Stock, A. M., Chen, T., Welsh, D. and Stock, J. (1988). Proceedingsof the NationalAcademy of Sciences of the United States of America 85, 1403. Stock, A. M., Mottonen, J. M., Stock, J. B. and Schutt, C. E. (1989). Nature (London) 337, 745. Stock, J. B. andKoshland, D. E., Jr (1978). Proceedingsofthe NationalAcademyofSciencesof the United States of America 75, 3659. Stock, J. B. and Koshland, D. E., Jr (1981). Journal of Biological Chemistry 256, 10826. Stock, J. B., Maderis, A. and Koshland, D. E., Jr (1981). Cell 27, 37. Stock, J., Borczuk, A., Chiou, F. and Burchenal, J. (1985a). Proceedings of the National Academy of Sciences of the United States of America 82, 8364. Stock, J., Kersulis, G. and Koshland, D. E., Jr (1985b). Cell 42, 683. Stock, J. B., Ninfa, A. J. and Stock, A. M. (1989). Microbiological Reviews 53, 450. Strange, P. G. and Koshland, D. E., Jr(1976). Proceedingsofthe NationalAcademyofSciences of the United States of America 73, 762. Sundberg, S. A, , Alam, M., Lebert, M., Spudich, J. L., Oesterhelt, D. and Hazelbauer, G. L. (1990). Journal of Bacteriology 172, 2328. Suzuki, T. and Iino, T . (1981). Journal of Bacteriology 148, 973. Szmelcman, S. and Adler, J. (1976). Proceedings of the National Academy of Sciences of the United States of America 73, 4387. Taylor, B. L. (1983). Annual Review of Microbiology 37, 551. Taylor, B. L., Tribhuwan, R. C., Roswell, E. H., Smith, J. M. and Shioi, J. (1985). In “Sensing and Response in Microorganisms” (M. Eisenbach and M: Balaban, eds), pp. 63-71. Elsevier, Amsterdam. Terwilliger, T. C. and Koshland, D. E., Jr (1984). Journal of Biological Chemistry 259,7719. Terwilliger, T. C., Bogonez, E., Wang, E. A. and Koshland, D.E., Jr (1983). Journal of Biological Chemistry 258, 9608. Terwilliger, T. C., Wang, J. Y. and Koshland, D. E., Jr (1986). Journalof Biological Chemistry 261, 10814. Thoelke, M. S . , Bedale, W. A , , Nettleton, D. 0. and Ordal, G. W. (1987). Journal of Biological Chemistry 262, 281 1. Thoelke, M. S . , Parker, H. M., Ordal, E. A. and Ordal, G. W. (1988). Biochemistry 27,8453. Thoelke, M. S., Kirby, J. R. and Ordal, G. W. (1989). Biochemistry 28, 5585. Thoelke, M. S., Casper, J. M. and Ordal, G. W. (1990). Journalof Biological Chemistry 265, 1928. Thomashow, L. S. and Rittenberg, S. C. (1985). Journal of Bacteriology 163, 1047. Toews, M. L. and Adler, J. (1979). Journal of Biological Chemistry 254, 1761. Toews, M. L., Goy, M. F., Springer, M. S. and Adler, J. (1979). Proceedings of the National Academy of Sciences of the United States of America 16, 5544. Trachtenberg, S. and DeRosier, D. J. (1988). Journal of Molecular Biology 202, 787. Trachtenberg, S., DeRosier, D. J., Aizawa, S.-I., and Macnab, R. M. (1986). Journal of Molecular Biology 190, 569. Trachtenberg, S., DeRosier, D. J. and Macnab, R. M. (1987). Journalof Molecular Biology 195, 603. Treptow, N. A. and Shuman, H. A. (1985). Journal of Bacteriology 163, 654. Treptow, N. A. and Shuman, H. A. (1988). Journal of Molecular Biology 202, 809. Tribhuwan, R. C., Johnson, M. S. and Taylor, B. L. (1986). Journal of Bacteriology 168,624. Tsang, N . , Macnab, R. M. and Koshland, D. E., Jr (1973). Science 181, 60. Tso, W.-W. and Adler, J. (1974). Journal of Bacteriology 118, 560. Ulitzur, S. (1974). Archives of Microbiology 101, 357. Ulitzur, S. (1975). Archives of Microbiology 104, 67. Utsumi, R., Brissette, R. E., Rampersaud, A., Forst, S. A ,, Oosawa, K. and Inouye, M. (1989). Science 245, 1246. van der Drift, C., Duiverman, J., Bexkens, H. and Krijnen, A. (1975). Journalof Bacteriology 124, 1142. Van der Wed, P. and Koshland, D. E., Jr (1977). Journal of Biological Chemistry 252,2793. Vogler, A. P. and Lengeler, J. W. (1987). Journal of Bacteriology 169, 593.

346

M. D. MANSON

Volz, K., Beman, J. and Matsumura, P. (1986). Journal of Biological Chemistry 261, 4723. Vyas, N. K., Vyas, M. N. and Quiocho, F. A. (1988). Science 242, 1290. Vyas, N. K., Vyas, M. N. and Quiocho, F. A. (1991). Journal of Biological Chemistry 266, 5226. Wagenknecht, T., DeRosier, D . J., Aizawa, S . 4 . and Macnab, R . M. (1984). Journal of Molecular Biology 162, 69. Walters, V. and Fierstine, H . L. (1964). Nature (London) 202, 208. Wang, E. A., Mowry, K. L., Clegg, D. 0.and Koshland, D. E., Jr (1982). Journal of Biological Chemistry 257, 4673. Warrick, H. M.,Taylor, B. L. and Koshland, D. E., Jr (1977). JournalofBacteriology 130,223. Waterbury, J. B., Willey, J. W., Franks, D . G., Valois, F. W. and Watson, S. W. (1985). Science 230, 74. Wei, L. N. and Joys, T . M. (1985). Journal of Molecular Biology 186, 791. Weibull, C. (1960). I n “The Bacteria” (I. C. Gunsalus and R. Y. Stanier, eds), vol. 1 , pp. 153201, Academic Press, New York. Weinberg, R. A. and Zusman, D . R. (1989). Journal of Bacteriology 171, 6174. Weis, R. M. and Koshland, D. E., Jr (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 83. Willey, J. M., Waterbury, J. B. and Greenberg, E. P. (1987). Journalof Bacteriology 169,3429. Williams, F . D . and Schwarzhoff, R. H. (1978). Annual Review of Microbiology 32, 101. Wilson, M. L. and Macnab, R. M. (1990). Journal of Bacteriology 172, 3932. Wolfe, A. J . , Conley, M. P., Kramer, T. J. and Berg, H. C. (1987). Journal of Bacteriology 169, 1878. Wolfe, A. J., Conley, M. P. and Berg, H . C. (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 6711. Wolff, C. and Parkinson, J. S. (1988). Journal of Bacteriology 170, 4509. Yamaguchi, S., Aizawa, S., Kihara, M., Isomura, M., Jones, C. J. and Macnab, R. M. (1986a). Journal of Bacteriology 168, 1172. Yamaguchi, S., Fujita, H., Ishihara, A. and Macnab, R . M. (1986b). Journal of Bacteriology 166, 187. Yamamoto, K., Macnab, R. M. and Imae, Y. (1990). Journal of Bacteriology 172, 1634. Yokota, T. and Gots, J. (1970). Journal of Bacteriology 103, 513. Yonekawa, H. and Hayashi, H . (1986). FEBS Letters 198, 21. Yonekawa, H . , Hayashi, H . and Parkinson, J. S. (1983). Journal of Bacteriology 156, 1228. Zukin, R. S. (1979). Biochemistry 18, 2139. Zukin, R. S . , Hartig, P. R. and Koshland, D. E., Jr (1977). Proceedings of the National Academy of Sciences of the United States of America 74, 1932.

Corrigendum “Mechanical Behaviour of Bacterial Cell Walls” by John J . Thwaites and Neil H. Mendelson, vol. 32, p. 217, final paragraph, continued on p. 218. Further research, since the publication of the forementioned review, on the cell-wall growth model has shown that, although the equilibrium state referred to does exist, it would be, for autonomous wall growth, an unstable state. Thus, the wall thicknesshadius ratio h tends to the given equilibrium value only if there is a suitable control signal. In the same way some feedback mechanism is required in order to maintain constancy of cylinder radius. A full analysis will appear elsewhere.

This Page Intentionally Left Blank

Author Index

Numbers in bold refer to the pages on which references are listed at the end of each chapter A

Abbott, S. L., 216, 251, 252, 267, 270 Abe, M., 218, 262 Abouhamad, W. N., 298, 301, 335 Achstetter, T., 116, 140 Acker, G., 283, 343 Adachi, T., 214,244,246,249,262,273, 274 Adam, G., 293, 335 Adam, H., 218, 272 Adams, A. E. M., 129, 140, 142 Adebayo, A. A., 153, 154, 160, 206 Adler, J., 278, 280, 284, 287, 290, 292, 296,298,299,301,304,305,313,314, 317,325,326,334,335,336,337,338, 340,341, 342, 342, 344, 345 Adler, L., 145-205, 206, 209, 210, 211 Admassu, W., 7, 67 Aebi, U., 214, 218, 224, 262, 267 Agabian, N., 222,235,247,255,265,272 Ahmad, F., 7, 65 Aisaka, K., 178, 211 Aitken, J. R., 118, 119, 120, 121, 122, 125, 130, 132, 140, 141 Aitken, W. B., 181, 206 Aizawa, S.-I., 293, 294, 295, 339, 345, 346 Akita, H., 178, 211 Aksamit, R. R., 298, 315, 335 Alam, M., 279, 345 Albagnac, G., 219, 222, 264, 268 Alberti, L., 288, 335 Aldrich, H. C., 223, 262

Al-Hamdani, A. M., 173, 206 Al-Karadaghi, S., 216, 262 Allen, R. D., 281, 335 Alli, K., 253,.267 Allmeier, H., 242, 247, 270 Allweiss, B., 279, 335 Al-Mahmood, S., 46, 51, 63 Aloni, H., 301, 337 Alten, C., 223, 224, 274 Altherthum, F., 182, 206 Altman, E., 107,140,217,219,241,262 Amako, K., 215, 265 Ames, G. F. L., 303, 335 Ames, P., 279, 310,312, 314, 325,335, 338 Amherdt, M., 89, 143 Ammerer, G., 80, 81, 88, 143 Amory, D. E., 26, 28, 44, 46, 57, 63 Amos, L. A., 224, 236, 254, 264, 273 Amri, M. M., 16, 58, 63 Anand, J. C., 156, 158, 159, 160, 206 Anderson, R. A., 290, 336 Anderson, R. G. W., 127, 141 Andre, L., 169, 189, 196, 197,203,206, 21 1 Andrews, S., 158, 160, 206 Anraku, Y., 185, 210, 211, 212 Antalis, C., 114, 143 Antebi, A., 109, 110, 143 Antranikian, G., 219, 262 Arad, T., 217, 264 Aragno, M., 218, 271 Archer, D. B., 223, 271 Archibald, A. R., 218, 265

350

AUTHOR INDEX

Armitage, J. P., 289, 335 Armstrong, J. B., 313, 314, 325, 335 Armstrong, R. A., 160, 174, 211 Armstrong, W. M., 152, 184, 207 Arnosti, D. N., 286, 287, 335 Aronson, M., 53, 63 Arst, H. N., 178, 206 Asaka, J., 283, 286, 340 Asakura, S., 278, 283, 335, 339 Ashby, A. M., 279, 343 Asher, O., 301, 337 Assinder, S., 8,17,18,41,46,49,51,56, 60, 62, 70 Aswad, D., 313,314,318,325,335,336 Atkinson, B., 11, 37, 64 Attfield, P. V., 196, 206 Auk, R. G., 33, 63 Austin, A., 174, 209 Austin, J. W., 216, 233, 239, 259, 262, 265 Ausubel, F. M. 317, 334, 342, 343 Avron, M., 163, 181, 207 Axcell, B. C., 5, 63 Axcell, B. D., 59, 60, 66 Ay, H., 17, 66 Ayerst, G., 161, 206

B Bachmann, B. J., 152, 206, 286, 336 Back, J. F., 158, 197, 206 Bacon, R. A., 94, 102, 140, 142 Baddiley, J., 218, 265 Baeuerlein, E., 285, 340 Bajpai, P., 3, 51, 63 Baker, D. A., 54,55,57,58,63,91,92, 93, 102, 140 Baker, R. K., 85, 140 Balch, W. E., 89, 91, 140, 143 Baldassare, J. J., 131, 141 Ball, C. B., 301, 340 Ball, S. G., 198, 206 Ballou, C. E., 43,44, 49,51,63,64,65, 74, 114, 140, 142, 143 Bamberger, I., 283, 343 Banerjee, S., 19, 66 Bank, H., 163, 206 Bankaitis, V. A., 7S140, 141, 143 Barendrecht, H. P., 13, 64 Barford, J. P., 7, 56, 69

Barkai-Golan, R., 48, 64 Barker, D. C., 220, 262 Barnett, J. A., 159, 178, 179, 206 Baron, S. F., 223, 272 Barondes, S. H., 48, 64 Baross, J. A , , 225, 270 Barrow, G. M., 29, 39, 64 Barth, M., 225, 255, 262 Bartlett, D. H., 286, 287, 336 Bassford, P. J. Jr, 79, 141, 311, 341 Basu, J., 48, 64, 67 Bauer, H. C., 257, 270 Baumann, P., 238, 246, 263, 281, 335 Baumeister, W., 214,215,216,217,218, 219,222,223,224,225,228,229,230, 234,235,237,238,243,244,245,246, 248,254,255,267,268,270,271,273, 274, 262, 264, 265 Baumgartner, J. W., 300,304,311,312, 338 Bayer, E. A., 242, 265 Bayley, S. T., 224, 268 Beachey, E. H., 53, 68 Beavan, M. J., 12, 26, 30, 46, 57, 64 Beckers, C. J. M., 89, 91, 140 Beckwith, J., 79, 80, 142, 295, 300, 341 Bedale, W. A., 327, 336, 345 Bedouelle, H., 303, 337 Beever, R. E., 171, 172, 173, 174, 183, 184, 206 Behr, M. 237, 238, 244, 247, 263, 268 Beijerinck, M. W., 13, 20, 64 Belas, M. R., 288, 336 Belk, D. M., 13, 26, 30, 48, 57, 64 Belland, R. J., 233, 248, 263 Bellinger, Y., 169, 206 Beman, J., 319, 345 Bennett, S. N., 178, 212 Berenguer, J., 216, 264, 265 Berg, E., 240, 243, 273 Berg, H. C., 278,280,288,289,290,291, 292,293,294,295,297,298,299,302, 305,310,315,317,322,327,331,336, 337, 339, 340, 341, 343, 346 Berg, S. T., 299, 343 Bergh-e, J.-L., 219, 263, 266 Bergeron, J. J. M., 111, 141 Bergman, K., 279, 336, 338 Bergmann, J. E., 63, 64, 111, 140 Berman, P. W., 257, 263

AUTHOR INDEX

Bernard, B. A., 257, 270 Bernhard, G., 224, 250, 274 Bernstein, H. D., 75, 84, 140 Bernstein, M., 75, 99, 141 Berry, D. R., 18, 66 Betz, J. V., 219, 263 Beuchat, L. R., 197, 206 Beveridge,T. J.,214,215,217,218,219, 222,223,228,231,237,238,239,252, 255, 256, 263, 264, 266, 269, 273 Bexkens, H., 279, 345 Bhowmik, T., 219, 267 Biendl, E., 237,238, 244,247,263,268 Bier, P. J., 215, 243, 274 Biguet, J., 43, 71 Bingle, W. H., 216, 238, 246, 263 Birch-Anderson, A., 216, 271 Bisher, M. E., 237, 267, 267 Black, R. A., 317, 336 Black, S. H., 215, 266 Blackett, B., 53, 66 Blackhart, B. D., 298, 336 Blackwood, A. C., 179, 211 Blair, D. F., 292,293,294,295,336,337 Blake, C. C. F., 47, 64 Blakely, E. R., 179, 206 Blakemore, R. P., 215, 263, 296, 336, 338 Blank, R. P., 298, 336 Blank, V., 298, 341, 343 Blasco, F., 184, 211 Blaser, M. J., 214, 238, 245, 247, 252, 263, 265, 270 Blatt, M. R., 184, 206, 211 Blobel, G., 77, 79, 83, 85, 86, 87, 88, 140, 141, 144 Block, M. R., 89, 91, 99, 140, 142, 144 Block, S. M., 290, 292, 295, 316, 323, 339, 343 Blom, J., 220, 263 Blomberg, A , , 145-205, 206, 209, 211 Blommaert, J., 230, 265 Bock, K., 255, 256, 269 Boer, P., 43, 67 Bogomolni, R. A., 279, 296, 344 Bogonez, E., 326, 336, 345 Bohni, P. C., 85, 140, 141 Boileau, A. J., 296, 340 Bole, D. G., 104, 140 Bollag, G. E., 334, 342

351

Boller, T., 185, 195, 196, 207, 208 Bollinger, J. C., 299, 325, 326,336,342 Bonaly, R., 3, 8, 16, 18, 46, 51, 58, 63, 66, 68 Bonch-Osmolovskaya, E. A., 234, 263 Bond, M. W., 326, 339 Bonner, D. M., 152, 206 Boone, D. R., 225, 268 Boos, W., 298, 300, 303, 336, 337, 338, 341, 343 Booth, C., 109, 140 Booth, I. R., 189, 211 Bootsma, R., 230, 265 Boraschi-Gaia, C., 176, 210 Borczuk, A., 327, 345 Borer, R., 8, 58, 66 Boring, J., 224, 268 Borinski, R., 252, 263 Borkovich, K. A., 319, 32G1, 336 Borst-Pauwels, G. W. F. H., 183, 184, 206, 211 Botstein, D., 61, 64, 93, 101, 102, 103, 129, 130, 140, 142, 143 Bottazzi, V., 253, 271 Boudarel, M. J., 20, 69 Bourgeois, A. L., 217, 264 Bourne, H. R., 103, 132, 134, 140 Bourret, R. B., 317, 318, 336, 337, 339 Bowditch, R. D., 238, 244, 246, 263 Bowen, W. R., 26, 59, 64 Bowers, B., 43, 64, 71, 267 Bowlus, R. D., 146, 167, 168, 176, 203,212 Bowser, R., 138, 141 Boyce, J. F., 217, 273 Boyd, A., 299, 300, 305, 310, 311, 325,337, 340 Boyd, W. C., 48, 64 Brada, D., 97, 98, 142 Brade, G., 298, 341 Brake, A. J., 127, 141 Brander, E., 220, 268 Branton, D., 127, 142 Brass, J. M., 298, 337 Braun, M. L., 180, 206, 218, 219, 263, 274 Brennan, M. J., 216, 237, 267 Brennwald, P., 83, 84, 141 Breznak, J. A., 215, 263 Brinton, C. C. Jr, 218, 266

352

AUTHOR INDEX

Brissette, R. E., 334, 345 Brisson, J.-R., 219, 241, 262 Broach, J., 133, 142 Brock, T. D . , 223, 274 Brockl, G., 237,238,244, 247, 263,268 Brohult, S . , 25, 64 Bromley, D. B., 280, 336 Bronan, B., 12, 17, 39, 41, 64 Brown, A. D., 146, 151, 155, 156, 158,159, 160, 167, 169, 177, 181, 188, 189,191,192,194, 195,203,204,206, 207, 208, 209, 279, 343 Brown, C. M., 176, 207 Brown, D. A., 288, 289, 297, 298, 336, 337 Brown, F. F., 181, 207 Brown, J . H . , 53, 71 Brown, M. G . , 150, 151, 152, 212 Brown, M. S., 127, 141 Brownlee, C., 175, 199, 207 Brudzynski, A., 7, 64 Brundish, H . M . , 16, 70 Bryan, R., 283, 337 Bryner, J. H . , 252, 263 Buchanan, T. M . , 53, 69 Buckel, W., 217, 218, 219, 268, 269 Buckley, C. M . , 109, 110, 143 Buckley, J. T., 251, 267 Buckmire, F. L. A , , 215, 243, 253, 263 Biidel, B., 221, 263 Buhle, E. L. Jr, 224, 267 Bu’Lock, J . D., 7, 55, 64, 224, 270 Bunker, A., 5, 70 Burchard, R. P., 22, 263, 287, 288, 337 Burchenal, J., 327, 345 Burda, K . , 215, 243, 252, 269, 274 Burggraf, S., 223, 224, 263, 264 Burke, R. M . , 153, 171, 183, 207 Burkholder, P. R . , 60, 69 Burks, C., 294, 337 Burley, S. K . , 218, 255, 264 Burlingame, A. L., 318, 319, 332, 343 Burns, J . A., 10, 15, 16, 64 Burrows, G. G., 300,304,311,312,338 Busink, R., 178, 208 C Cabib, E., 43, 64, 71 Cairney, J . , 189, 211

Caldwell, D. E., 221, 264 Calibo, R. L., 16, 64 Callegari, M . L., 220, 253, 264, 271 Calleja, G . B., 3, 11, 12, 17, 20, 37, 39, 60, 62, 64, 66 Camp, T. R., 27, 64 Campbell, I . , 7, 64 Canale-Parola, E., 215, 263, 266, 280, 337 Canh, D. S., 180, 207 Caplan, S. R . , 301, 337 Carey, K. E., 279, 335 Carl, M., 214, 217, 238, 245, 247, 248, 264 Carlson, M., 61, 62, 64, 69 Carman, G . M., 122, 141 Carrascosa, J . L., 216, 264 Caslavska, J . , 221, 272 Caspar, D. L. D., 254, 264 Casper, J. M . , 327, 345 Castiau, C., 57, 68 Caston, J. R . , 216, 264 Cavaignac, S., 238, 245, 248, 264 Cejka, Z . , 219, 264 Ceriotti, A . , 106, 141 Chakrabarti, P., 48, 64, 67 Chalcroft, J . P., 215, 255, 264 Challinor, S. W . , 58, 64 Chalmers, K . , 195, 196, 208 Chamberlin, M. J . , 286, 287, 335, 338 Chambers, K . A., 131, 142 Champer, R., 279, 337 Champion, K. M., 118, 119, 120, 121, 122, 125, 130, 132, 141 Chang, J.-J., 217, 264 Chang, L.-Y. E . , 222, 270 Chapman, D., 195, 207 Chapman, J . A . , 216, 264 Chapman, S., 29, 64 Charalampous, Y., 8, 58, 66 Charon, N. W., 280, 336 Charpentier, C., 3, 18, 51, 66 Chart, H., 251, 264 Chasseboeuf, C., 8, 68 Cheeseman, J . M., 198, 207 Chelsky, D. O . , 311, 325, 337 Chen, E., 99, 144 Chen, L.-S., 57, 69, 246, 270 Chen, S.-M., 189, 207 Chen, T., 317, 319, 344

353

AUTHOR INDEX

Chen, Z.-Y., 41, 68 Chen-Wu, J. L.-P., 62, 69 Cheng, K. J., 257, 264 Chernych, N. A., 234, 263 Chester, V. E., 5, 61, 64 Chet, I., 279, 337 Chiang, A , , 81, 82, 143 Chiang, C., 190, 207 Chilvers, G. A., 152, 208 Ching, W. M., 238, 245, 247, 248, 264 Chinnock, R. E., 318, 322,343 Chiou, F., 327, 345 Chirico, W. J., 83, 88, 141 Chiu, W., 215, 265 Choy, C., 225, 268 Christian, R., 24&1,242,255,256,264, 269 Chu, S., 238, 245, 248, 264 Chudek, J. A., 164, 169, 173, 174, 176, 183, 208, 210 Chun, S. Y., 295, 337 Civan, M., 163, 211 Clark, M. E., 146,167,168,176,203,212 Clark, S., 136, 144 Clarke, K. J., 162, 163, 192, 209, 210 Clarke, S., 301, 302, 315, 337 Clary, D. O., 89, 100, 141 Clegg, D. O., 301, 318, 323, 337, 346 Clegg, J. S., 195, 207 Cleves, A. E., 43-139, 140, 141, 143 Clipson, N. J. W., 186, 207 Cohen, P. S., 279, 342 Cohen, R. E., 43, 44, 64 Cohen, S., 224, 264, 267 Cohen, Y . , 225, 267 Cohen-Bazire, G., 220, 264 Coleman, H. P., 31, 70 Collander, R., 181, 207 Collier, D. N., 79, 141 Collins, A. L. T., 313, 314, 337 Collins, J. C., 185, 208 Colman, A., 106, 141 Colvin, J. R., 222, 273 Colwell, R. R., 288, 336 Combermach, D. M., 7, 55, 64 Commerford, J. G., 187, 207 Commissaire, J., 219, 263, 266 Conde, J., 104, 143 Conley, M. P., 299, 305, 310, 319, 336, 337, 340, 343, 346

Constantinou, C., 301, 337 Conway de Macario, E., 224, 274, 275 Conway, E. J., 152, 184, 207 Cooke, R. C., 173, 206 Cooke, R. J., 26, 59, 64 Corbeil, L. B., 215, 252, 269 Corry, J. E. L., 146, 155, 161, 207 Cosgrove, D. J., 155, 207 Costa, M., 180, 187, 209 Costerton, J. W., 222, 257, 264, 271 Cottarel, G., 198, 206 Cottrell, S., 19, 66 Coulson, G. E., 162, 163, 192,209,210 Coulton, J. W., 285, 337 Courtwright, J. B., 178, 207 Coutinho, R., 225, 275 Cowan, W. D., 19,64 Cowling, T. G., 29, 64 Craig, E. A., 82, 83, 104, 141, 144 Cram, W. J., 167, 207 Critchley, I. A., 52, 64 Cronan, J. E., 178, 211 Crooke, E., 79, 141 Crosby, J., 117, 141 Crowe, J. H., 195, 207 Crowe, L. M., 195, 207 Crowther, R. A., 219, 264 Csonca, L. N., 182, 205, 207 Cubbage, S.,215,237,238,239,252,264 Culbertson, M. R., 185, 208 Cullis, P. R., 181, 207 Curtis, A. S. G., 12, 64 Curtis, N. S., 5, 6, 7, 23, 32, 64 Cusack, S., 53, 71 Cybulska, E. B., 48, 71 D DaCosta, M. S., 171,173,174,187,207, 210 Dahl, M. K., 299,300,303,304,313,337 Dahl, M. M., 325, 335 Dahlquist, F. W., 280, 311, 319, 321, 325,326,327,330,331,337,338,339, 343, 344 Dainty, J., 164, 207 Dalbey, R., 79, 141 Dalton, D. D., 235, 266 Dalton, T. P., 126, 141 Danders, D. A., 318, 319, 332, 343

354

AUTHOR INDEX

Diala, E. S., 19, 51, 65 Diaz, R., 89, 141 Dickeson, S. K., 126, 141 Dickson, M. R., 215, 255, 264 Dietzel, I., 303, 337 Dijkema, C., 180, 212 Dijkhuizen, L., 178, 207 Dingwall, A., 279, 337 Dittrich, H. H., 8, 65 Dixon, F. J., 63, 67 Doak, T. G., 330, 339 Dobberstein, B., 77, 84, 140, 143 Dobson, M. E., 238,245,247,248,264 Docherty, J., 23, 61, 65 Doetsch, R. N., 279, 283, 343, 344 Dokland, T., 217, 264 Dominguez, A., 8, 58, 65 Donath, C., 101, 141 Dooley, J. S. G., 216,237,238,239,248, 251, 264, 270 Doolittle, R., 279, 344 Dooms, L., 177, 212 Doran, J. L., 238, 263 Dorman, T. E., 109, 110, 143 Dorset, D. L., 231, 265 Dostal, J., 279, 335 Doudoroff, M., 288, 342 Douglas, H. C., 60, 69 Douglas, L. J., 52, 64, 65 Dovie, R. J., 49, 65 Dowhan, W., 118, 119, 120, 121, 122, 125, 130, 132, 140, 141, 142 Downing, K. H., 215, 255, 264 Doyle, D., 215, 252, 269 Doyle, R. J., 217 223, 263, 264 Drews, G., 221, 265 Drickamer, K., 47, 65 Driks, A., 283, 337 Dubochet, J., 214, 218, 262 Dubourguier, H.-C., 219,222,264,268 Dubreuil, J. D., 215,237,238,239,243, 252, 259, 264, 265 Duffey, P. S., 216, 251, 252, 267, 270 344,345,346 Deshaies, R. J.,80,81,82,83,85,86,88, Duffus, J. H., 8, 43, 58, 65 Dufour, P. J., 26, 28, 44, 46, 57, 63 140, 141, 143 Duiverman, J., 279, 345 Destruhaut, A., 7, 68 Duncan, M. J., 109, 143 Deutch, J. M., 41, 68 Devreux, A., 5,15,20,43,57,58,65,68 Dunphy, W. G., 89, 112, 113, 141 Dunten, P., 328, 337 deWaard, P., 242, 265 Dunwell, J. L., 8, 65 de-Zoysa, P., 23, 61, 65

Dankert, M., 250, 274 D’Aoust, J.-Y., 224, 264 Dapice, M., 292, 339 Dasch, G. A., 214, 217, 238, 245, 247, 248, 264 Date, T., 79, 141 Daum, G., 80, 81, 88, 120, 141, 143 Davidow, L. S., 109, 110, 143 Davidson, H. W., 91, 140 Davies, J. M., 199, 207 Davies, P. J., 192, 193, 210 Davies, T. M. C., 7, 56, 66 Davis, R. H., 5, 11, 39, 41, 64, 65 Davison, A. L., 218, 265 Day, A. W., 53, 65 Dayrell-Hart, B., 288, 337 De Boer, W. E., 222, 264 De Bruijne, A. W., 164, 207 De Clerke, J., 5, 17, 65 De Gier, J., 181, 207 De Graaff, P., 259, 265 De Greef, W. J., 181, 207 De Koning, W., 178, 207 de Kruijff, B., 181, 207 De Kruyff, B., 181, 207 de Pedro, M. A., 216, 264, 265 De Rosa, M., 224, 270 Dean, G. E., 294, 295, 337, 339, 344 Dean, N., 106, 107, 108, 141, 142, 143 Deatherage, J. F., 224, 236, 254, 264, 273 Debeire, P., 242, 270 DeFranco, A. L., 280,314,325,337,339 Degani, H., 163, 181, 207, 209 Dehoux, P., 83, 143 Del Villano, B.C., 63, 67 Delmotte, F., 51, 63 Demburg, A. F., 312, 338 Demel, R. A., 181, 207 Denor, P. F., 178, 207 DePamphilis, M. L., 284, 337 DeRosier, D. J., 283,284,285,337,339,

355

AUTHOR INDEX

Dunyak, D. S., 301, 342 Duplay, P., 303, 305, 337 Dupoy, G., 215, 265 Durham, D. R., 216, 269 Durr, M., 185, 207 Durr, R., 225, 262 Duteurtre, B., 16, 58, 63 Dutton, D. P., 300, 304, 311, 312,338, 342 E Eakle, K. A,, 99, 141 Earhart, C. F., 216, 271 Earl, A., 19, 51, 65 Ebersole, J. L., 217, 273 Ebisu, S., 238, 245, 247, 248, 249, 265 Eddy,A. A.,9,12,14,15,16,17,18,21, 23, 26, 43,44,46, 51, 55, 57, 65, 151, 184, 185, 207, 208 Edgley, M. A., 177, 188, 194,204,207, 208 Edwards, P. R., 283, 337 Edwards, T. F., 279, 335 Egilsson, V., 19, 65 Egli, T., 224, 274 Ehrenreich, J. H., 111, 141 Einspahr, H., 47, 65 Einspahr, K. J., 162, 208 Einstein, A., 24, 65 Eisenbach, M., 301, 317, 323, 340, 343 Elbers, P. F., 43, 67 Ellar, D. J., 218, 265 Ellis, R. J., 54, 65 Ellison 111, R. T., 252, 270 Elmore, M. J., 189, 211 Elorza, M. V., 43, 54, 57, 65, 68, 69 Els, H. J., 219, 273 Ely, B., 279, 337 Emala, C. W., 283, 344 Emde, B., 214, 268 Emerson, S. U., 286, 338 Emr, S. D., 74,85,99,140,141,142,143 Engel, A. M., 215, 231, 237, 262, 265, 268, 270 Engelhardt, H.,215,216,218,219,225, 227,228,229,230,237,248,255,262, 263, 264, 265, 270, 273, 274 Englehardt-Altendorf, D., 303, 336 Engstroem, P., 302, 314, 323, 338

Enomoto, M., 280, 314, 338, 339 Epstein, W., 299, 335 Eshdat, Y., 13, 53, 65, 69 Esmon, B. E., 43, 54, 65, 77, 114, 115, 118, 141, 143 Esser, K., 19, 65, 66,69 Evans, E., 79, 85, 141 Evans, I. H., 19, 51, 65 Evans, W. E., 192, 193, 210 Evenberg, D., 216, 230, 251, 259, 265 Evers, D., 221, 265 Everson, J. S . , 53, 66 Every, D., 216, 265 Ewing, W. H., 283, 337 Eylar, E. H., 43, 65 Ezzell, E., 217, 264 F Fabry, S., 237, 238, 244, 247, 263 Falke, J. J., 311, 312, 338 Fangman, W. L., 62, 70 Faraldo, M. L. M., 216, 265 Farkas, V., 43, 44,65 Farquhar, M. G., 111, 141 Farrants, G. W., 216,217,228,264,272 Fasano, O., 133, 142 Fassel, T. A., 216, 265 Feldman, R. I., 75, 141 Fennessy, P., 250, 274 Fernandez, M. P., 43, 71 Ferreira, T., 225, 275 Ferris, F. G.,215,237,238,239,252,264 Ferro, S., 17, 18, 54, 68, 142 Ferro-Novick, S., 75,91,93,94,96,102, 140, 141, 142, 143 Ferry, J. G., 223, 272 Feutrier, J., 238, 245, 248, 264 Fiala, G., 224, 265 Field, C., 60, 68, 74, 76, 100, 113, 117, 140, 141, 142 Fierstine, H. L., 288, 346 Finch, J. T., 218, 265 Fink, G. R., 109,110,143,184,202,208, 211 Finkelstein, D. B., 196, 209 Firon, N., 20, 53, 60,65, 69 Fischer, F., 224, 265 Fisher, D. J., 26, 65 Fisher, J. A., 246, 247, 265

356

AUTHOR INDEX

Fleeson, E. H., 14, 71 Fleet, G. H., 43, 44, 65, 67 Fleming, D. L., 279, 337 Flores-Carrkon, A , , 114, 141 Flowers, T. J., 185, 208 Flynn, G. C., 99, 144 Fogel, S . , 279, 337 Fogg, G. C., 252, 265 Forjaz, V. H., 225, 275 Forst, S. A., 334, 345 Foster, D. L., 305, 311, 338, 342 Foster, R., 164, 169, 172,173,174, 176, 183, 208, 210 Fournet, B., 242, 270 Frank, J., 222, 267 Frank, R., 84, 143 Frankel, R. B., 279, 338 Franks, D. G., 280, 346 Frantz, B. B., 286, 287, 336 Franzusoff, A., 113, 115, 116, 117, 118, 140, 141 Frederikse, P. H., 279, 337, 338 Freter, R., 279, 335, 338 Frevert, J., 43, 65 Fricke, H., 224, 263 Friis, J., 158, 208 Fryer, J. L., 214, 216, 273 Fuerst, J. A , , 284, 338 Fugit, D. R., 62, 67 Fujii, T., 59, 68 Fujirnoto, M., 214, 267 Fujimoto, S., 215, 265 Fujino, S., 5, 17, 59, 65 Fujita, A., 52, 65, 66 Fujita, €I., 286, 339, 346 Fukui, S., 15, 16, 20, 57, 61, 68, 71 Fuller, R. W., 117, 127, 141 Fullrner, C. S., 215, 243, 274 Furlong, C. E., 298, 303, 338

Garnbacorta, A., 224, 270 Gancedo, C., 177, 178, 179, 189, 208 Gancedo, J. M., 178, 189, 208, 209 Gao, Z., 285, 340 Garcia, P. D., 86, 87, 142 Gardner, W. R., 153, 154, 206 Gargus, J. J., 290, 292, 340 Garrison, I. F., 19,21,46,48,49,57,58, 60, 70 Garrod, D. R., 11, 37, 64 Gatenbeck, S., 179, 208 Gavrilova, 0. V., 221, 265 Gebert, J. F., 300, 338 Gegner, J. A., 319, 321, 338 Geilenkotten, I., 12, 54, 66 Gekko, K., 168, 208 Geradot, C. J., 279, 337 Gerbl-Rieger, S., 215, 265 Gerhardt, P., 217, 265 Gerwig, G. J., 242, 265 Gething, M. J., 104, 141, 142 Getz, G. S., 19, 66 Geys, K., 14, 66 Gezelius, K., 169, 208 Gherna, R. L., 220, 266 Ghiorse, W. C., 222, 265 Ghosh, A., 8, 48, 58, 66, 67 Gibbons, N. E., 224, 268 Gibson, K. J., 279, 342 Gibson, M. M., 298, 301, 335 Gierl, A., 224, 275 Giesbrecht, P., 221, 265 Gilchrist, A., 246, 265 Gillece-Castro, B. L., 318,319,332,343 Gilliland, R. B., 7, 8, 9, 12, 14, 15, 18, 54, 58, 60, 66 Gilman, A. G., 132, 141 Gilmore, R., 79, 85, 141 Giner, A., 83, 84, 143 Ginzburg, M., 84, 224, 267 Giummelly, P., 46, 51, 63, 66 G Glaeser, R. M., 215,222,255,264,265, Gaber, R. F., 184, 202, 208 272 Gadd, G. M., 163, 164, 165, 169, 172, Glagolev, A. N., 292, 293, 317, 338 173,174,176,183,190,195, 196,208, Glare, T. R., 152, 208 209, 210 Glauert, A . M., 216,218,219,228,234, Gallili, G., 54, 66 235, 265, 272 Galloway, R. J., 318, 322, 343 Glaver, H. M., 175, 208 Gallwitz,D., 101,110,134,141,142,143 Gleason, M. L., 103, 123, 125, 144 Gama, F. M., 51, 70 Glick, B. S., 89, 91, 140, 141, 142

357

AUTHOR INDEX

Glover, C. V. C., 62, 69 Goderdzishvili, M. G., 218, 265 Goebl, M., 118,119,120,121,122,125, 132, 141 Goetz, R., 289, 338 Gokhale, D. V., 7, 69 Goldman, D. J., 279, 342 Goldschmidt-Clermont, P. J., 131, 141 Goldstein, I. J., 48, 60, 66, 69 Goldstein, J. L., 127, 141 Goma, G., 7, 68 Gomes, S. L., 279, 337 Gbmez, A., 114, 141 Goncalves, M. H., 51, 70 Goodman, J. M., 85, 144 Gordon A. S., 303, 336 Gorham, J., 148, 151, 164, 212 Goring, T. E., 3, 14, 15, 16, 18, 19,48, 49, 58, 60, 70 Gosh, B. K., 218, 246, 273 Gosteva, V. V., 218, 265 Gots, J., 287, 346 Gotschlich, E. C., 214,238,245,247,263 Gottschalk, G., 218, 219, 262, 263, 274 Goud, B., 132, 133, 134, 135, 136, 137, 142, 144 Goulbourne, E. A. Jr, 280, 316, 338 Goundry, J., 218, 265 Goy, M. F., 301,313,325,327,338,340, 344, 345 Graff, G., 19, 67 Grano, D. A., 215, 222, 255, 265, 272 Greenberg, E. P., 280, 316, 338, 346 Greene, R., 118, 119, 140 Greenshields, R. N., 7, 56, 66 Greenway, H., 185, 210 Greig, R. G., 27, 66 GrCnman, R., 220, 269 Griff, I. C., 100, 141 Griffin, D. M., 154, 158, 209 Griffin, S. R., 43, 44, 46, 66 Griffiths, G., 127, 142 Griffiths, H., 111, 217, 274 Griffiths, S. G., 216, 251, 265 Groarke, J. M., 303, 338 Groesch, M., 94, 142 Gromov, B. V., 221, 265 Gross, C., 85, 143 Gruber, K.,218,235,247,249,261,265, 266

Gruebl, G., 322, 338 Guckenberger, R., 225, 255, 262 Gulash-Hoffe, M., 279, 336 Gustafsson, L., 153, 154, 169, 170, 173, 176,183,187,193,200,204,206,208, 209 Gustin, M. C., 154, 185, 208 Guthrie, B., 79, 141 Gyllang, H., 5, 6, 19, 20, 66 Gyllenberg, H., 221, 267 H Haapasalo, M., 216, 217, 218,220,225, 230, 237, 253, 266, 267, 271, 272 Haber, J. E., 202, 209, 210 Haeder, D. P., 296, 338 Hageage, G. J., 220, 266 Haggblom, M., 218, 274 Haguenauer-Tsapis, R., 85, 142 Hagues, G., 10, 68 Hahn, M., 214, 225, 255, 262, 268 Hahnenberger, K. M., 284, 285, 344 Haikara, A., 218, 266 Hajibagheri, M. A,, 185, 207, 208 Hall, A., 132, 142 Hall, R. E., 303, 336 Ham, R. K., 32, 70 Hammes, W., 242, 268 Hammond, J. B. W., 175, 208 Han, D. P., 323, 341 Hancock, R. E. W., 231, 266 Hand, S. C., 146,167,168,176,203,212 H a m , B. C., 84, 85, 142 Hanna, D. E., 62, 69 Hansche, P. E., 62, 66 Hansen, T. A., 219, 274 Hansen, W., 83, 84, 86, 87, 142, 143 Hanson, J., 283, 340 Harayama, S., 301, 302, 336, 338, 340 Harder, W., 178, 207 Hardwick, K. G., 107, 108, 142, 143 Harned, H. S., 151, 208 Harold, F. M., 292, 317, 341 Harris, B. J., 223, 263 Harris, J. E., 223, 271 Harris, J. O., 15, 44, 66 Harris, R. F., 149, 150, 153, 154, 159, 160.. 165., 174. 193, 206, 208 Harris, W. F., 236, 266

358

AUTHOR INDEX

Harrison, S. C., 127, 142 Harshey, R. M., 288, 335 Hartig, P. R., 303, 346 Hartmann, E., 242, 247, 250, 266, 268, 270 Hartong, B. D., 5, 66 Hartwell, L. H., 154, 210 Hartwig, J. H., 131, 142 Haselbeck, A., 43, 66 Hasiba, H., 238, 273 Hastie, A. T., 218, 266 Haubold, R., 216, 266 Hawthorne, D. C., 60, 69, 158, 208 Hayakawa, S., 246, 273 Hayashi, H., 326, 328, 338, 346 Hayduck, E., 14, 66 Hayes, H. J., 219, 263, 266 Hazelbauer, G. L., 279, 296, 298, 300, 301,302,304,310,311,312,314,323, 325,326,335,336,338,340,342,345 Heckels, J. E., 53, 66 Hedblom, M. L., 301, 338 Hedman, K., 111, 143 Hegerl, R., 219, 228, 262, 264, 270 Heimsch, R. C., 7, 67 Heinen, U. J., 216, 266 Heinen, W., 216, 266 Heinzer, I., 252, 263 Heisse, W., 214, 268 Heitzer, R., 250, 274 Hellebust, J. A., 182, 208 Heller, K. B., 182, 208 Helm, E., 10, 66 Helmann, J. D., 286, 338 Helmkamp, G. M., 120, 126, 141, 142 Heltberg, O., 220, 263 Hemmingsen, S. M., 54, 65 Hendershot, L. M., 104, 140 Hennig, K., 17, 66 Henry, C., 214, 218, 262 Henry, S. A., 122, 141 Hensel, R., 237, 238, 244, 247, 263 Henson, J. M., 223, 271 Henwood, J. A., 223, 271 Henzel, W. J., 99, 144 Herbaut, J., 43, 71 Herero, E., 43, 54, 60, 69 Herman, P. K., 74, 142 Hermodson, M. A., 303, 338 Herrera, V. E., 59, 60, 66

Herz, J., 84, 143 Herzberg, C., 219, 262 Hespell, R. B., 215, 266 Hess, J. F., 317,318,319,331,336,337, 338, 339 Heugebauer, D.-Ch., 224, 269 Hicke, L., 91, 92, 93, 98, 140, 142 Higashi, Y., 250, 266 Higgins, C. F., 189, 211, 298, 301, 335, 341 Hildebrand, E., 279, 343 Hills, G. J., 223, 271 Hilmen, M., 288, 340 Hinnen, A., 85, 142 Hinrichs, J., 19, 65, 66 Hirota, N., 292, 339 Hirsch, P., 11, 37, 64, 222, 265 Hixson, S. H., 114, 141 Hobot, J. A., 183, 208 Hobson, A. C., 317, 336 Hocking, A. D., 157,158,159,172,173, 181, 208, 210, 212 Hodgson, J. A., 18, 66 Hoehniger, J., 221, 273 Hoekstra, F. A., 155, 208 Hoesch, L., 176, 210 Hofer, M., 180, 208 Hofnung, M., 303, 337 Hogg, R. W., 290, 292, 340 Hoggan, J., 19, 64 Hollaus, F., 219, 243, 257, 266, 269 Holligan, P. M., 175, 179, 208 Holm, K., 83, 84, 141 Holmberg, S., 18, 19, 48, 61, 66 Holt, S. C., 215,217,218,219,220,221, 225, 252, 263, 266, 268, 273 Holz, I., 224, 275 Homma, M., 283, 284, 285, 286, 294, 339, 340 Hondmann, D. H. A., 178, 206, 208 Hope, J. N., 303, 338 Hope, M. J., 181, 207 Hopkins, J. A., 252, 263 Horak, J., 180, 207 Horie, Y., 59, 68 Horne, R. W., 44, 68, 216, 274 Hossack, J. A., 182, 208 Hotani, H., 286, 295, 339 Hottiger, T., 195, 196, 208 Hough, J. S., 7,15,21,44,46,64,66,68

359

AUTHOR INDEX

Houts, S. E., 313, 342 Houwink, A. L.,214,215,216,219,224, 266 Hovestadt, R. E., 279, 336 Hovind-Hougen, K., 215, 266 Hovmoller, S., 216, 220, 228, 229, 237, 248, 251, 262, 266, 272, 274 Howard, L. V., 218,235,243,253,266, 274 Howatson, A. F., 217, 266 Howe, H. B. Jr, 178, 211, 212 Hozumi, H., 12, 39, 70 Huang, J. S., 41, 71 Huber, H., 222, 223, 237, 266, 273 Huber, R., 222, 225, 266, 267, 270 Hudson, P. B., 177, 211 Hughes, R. C., 48, 66 Hull-Pillsbury, C., 17, 62, 67 Hulsmans, E., 190, 211 Hult, K., 179, 208 Hurnphries, M. J., 257, 270 Hungate, R. E., 223, 270 Hunkapiller, M. W., 326, 339 Hunt, T. P., 11, 39, 41, 64 Huser, B. A., 223, 274 Hussain, T., 3, 18, 51, 66 Hyashenko, B. N., 218, 265 Hynes, S. H., 253, 268 I Ichiki, A. T., 281, 341 Icho, T., 280, 287, 339 Igo, M. M., 322, 339 Iida, K., 131, 144 Iida, S., 292, 341 Iino, T., 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 339, 340,342,345 Irnae, Y., 292, 296, 300, 301, 304, 305, 309, 339, 340, 341, 342, 346 Imhoff, J. F., 221, 266 Imsel, E., 225, 275 Inatomi, K., 224, 266 Ince, E., 160, 174, 211 Ingledew, W. M., 15, 69 Ingraham, J. L., 182, 208 Ingram, J. M., 179, 208 Inouye, M., 334, 345 Ishido, T., 8, 67 Ishidsu, J., 287, 340

Ishiguro, E. E., 216,230,251,264,267, 270, 273 Ishihara, A., 286,295,323,339,343,346 Isomura, M., 293, 294, 295, 346 Ito, K., 178, 211 Itoh, M., 219, 269 Iwano, K., 13, 68

J Jackson, P., 62, 68 Jackson, S., 215, 266 Jacobson, L., 184, 211 Jaenicke, R., 232, 266 Jakovcic, S., 19, 66 Jakubowski, U., 228, 270 James,A. P., 8,15,17,18,20,47,51,68 Janda, J. M., 216,251,252,267,268,270 JanCkovic, D., 222, 273, 275 Janmey, P. A., 132, 144 J a m , K., 13, 65 Jannasch, H. W., 223, 224, 264, 265 Jansen, H. E., 8, 14, 15, 25, 66 Jap, B. K., 311, 338 Jarrell, K. F., 222, 223, 268, 273 Javekovic, D., 224, 275 Jayatissa, P. M., 15, 26, 30, 46, 66 Jeffries, P., 216, 267 Jenkins, D., 11, 32, 37, 64, 69 Jennings,D. H., 148,153,171,172,173, 174,175,177,179,183,184,186,190, 199, 207, 208, 209, 212 Jensen, T. E., 221, 267 Jermini, M. F. G., 157, 209 Jeunehornme-Ramos;C., 57, 68 Johansen, B. V., 217, 264 Johnson, B. F., 3,11, 12,20,37,62,64, 66 Johnson, L. M., 118, 119, 140 Johnson, M. C., 219, 267, 271 Johnson, M. S., 317, 345 Johnston, J. R., 18,23,6&1,65,66,67, 69 Joiner, K. A., 252, 263 Jonas, L., 221, 271 Jones, C. J., 285,293,294,295,339,346 Jones, E. W., 127, 128, 142 Jones, J. B., 223, 267 Jones, M. N., 27, 66 Jones, S. T., 7, 67

360

AUTHOR INDEX

Jones, W. B. G., 183, 209 Jones, W. J., 223, 267 Jorgenson, B. B., 298, 342 Jousimies-Somer, H . , 216,220,269,272 Jovall, P.-A., 176, 177, 179, 187, 209 Joys, T. M., 283, 346 Julius, D., 113, 115, 142 Jurss, L. M., 301, 342 K Kaasen, I., 175, 208 Kabat, E. A . , 49, 67 Kabcenell, A . K., 91, 93, 94, 132, 133, 134, 135, 136, 137, 142, 143, 144 Kagawa, H . , 280, 339 Kahn, R. A . , 102, 103, 143 Kainz, U., 217, 218, 255, 271 Kaiser, A . D., 287, 339 Kaiser, C., 95, 96, 100, 142 Kalsner, I., 254, 255, 256, 259, 271 Kamada, K., 17, 18, 48, 67, 71 Kamerling, J. P., 242, 265 Kamiya, R., 281, 339 Kandler, O., 214, 228, 239, 254, 257, 267, 268, 274 Kaplan, N., 317, 318, 319, 336, 339 Kari, M., 218, 267 Karlsson, B., 221, 267 Karrenberg, F., 219,222,225,229,230, 234, 237, 255, 262 Kasahara, H., 8, 67 Kasai, M., 185, 212 Kataoka, H . , 16, 64 Kataoka, T., 133, 142 Kates, M., 224,'268 Kato, S., 12, 67 Kaudewitz, H., 237, 238, 244, 247, 263 Kaufman, W. J., 32, 69 Kawagishi, I., 283, 286, 340 Kawata,T.,214,215,219,220,231,234, 267, 269 Kay, C. M., 215, 237, 238, 239, 252, 264 Kay, W. W., 216, 230, 238, 245, 248, 251, 259, 264, 267, 270, 273 Kaziro, Y., 134, 142 Keane, M., 330, 344 Kearney, J. R., 104, 140 Keen, J. H., 127, 142

Keenan, M. H. J., 9, 11, 12, 18,25,26, 28, 29, 30, 31, 36, 39, 70 Kehry, M. R., 325,326, 330, 339 Kellenberger, E., 214,218,228,262,267 Kendall, K., 299, 325, 337 Kepes, F., 75, 141, 142 Kern, K. A . , 114, 143 Kerosuo, E., 216, 217, 220, 225, 230, 237, 253, 267, 272 Kersulis, G., 327, 345 Kessel, M., 216,221,222,223,224,225, 237, 264, 267 Khan, S., 290, 292, 293, 317, 323, 336, 339 Kidney, E., 20, 71 Kielland-Brandt, M. C., 18,61,66,178, 202, 208, 211 Kihara, M., 293,294,295,300,301,339, 340,346 Kihn, J. C., 17, 28, 31, 46, 67 Kijima, M., 5, 12, 19, 59, 67 Kikuchi, Y., 62, 65 Kilmartin, J. V., 129, 142 Kimoto, N., 218, 262 King, L., 19, 66 King, N. R., 216, 274 Kirby, J. R., 327, 345 Kirchhausen, T., 127, 142 Kirchoff, H . , 288, 343 Kirk, R. G., 224, 267 Kirsop, B. H., 54, 55, 57, 58, 63 Kirst, G. O., 185, 210 Kist, M. L., 215, 234, 267 Kistler, J., 228, 267 Klaushofer, H., 218, 272 Kleene, S. J., 325, 340 Klenk, H.-P., 224, 225, 275 Klionsky, D. J., 74, 142 Klitsunova, N. V., 218, 265 Kloppel, I., 180, 208 Klug, A . , 254, 264, 265 Kneifel, H . , 224, 274, 275 Knight, S. G., 190, 207 Kobata, A . , 243, 267 Kobayashi, H., 7, 56, 62, 65,66, 67 Koch, A . L., 155, 163, 209, 210 Koch, B. D., 82, 83, 141 Koch, G. L. E., 109, 140 Koch, R., 14, 17, 67 Kocur, M., 217, 272

36 1

AUTHOR INDEX

Kofoid, E. C., 314, 317, 319, 340 Koh, T. Y., 158, 209 Kohno, K., 104, 142 Koiwai, O., 326, 338 Kokka, R. P., 216, 251, 252, 267, 268, 270 Kolb, V., 303, 337 Koman, A., 302, 340 Komarek, J., 221, 272 Komeda, Y., 280, 282, 283, 284, 286, 287, 294, 339, 340, 341 Kondo, E., 283, 286, 340 Kondoh, H., 301, 331, 340 Kong, Y.-C., 179, 209 Konig, H., 219,222,223,225,228,232, 237,238,239,242,244,247,250,251, 254,257,263,266,267,268,270,273, 274 Konovalov, E. S., 221, 265 Kopecka, M., 43, 67 Koppensteiner, G., 156, 209 Kornfeld, R., 243, 268 Kornfeld, S., 114, 142, 243, 268 Kort, E. N., 325, 340, 344 Korus, R. A., 7, 67 Koshland, D. E. Jr, 278,288,290,294, 297-302,303,304,305,308,309,311, 312,313,314,315,317,318,322,323, 325,326,327,328,330,331,332,334, 335-46 Kosic-Smithers, J., 104, 141 Kossmann, M., 305, 306, 309, 340, 341 Kostrikina, N. A., 234, 238, 245, 248, 259, 263 Kostrzynska, M., 238, 245, 248, 259, 264, 265 Kotyk, A. 180, 207 Koval, S. F., 215, 223, 225, 231, 251, 253, 268 Kozuka, M., 326, 338 Kozutsumi, Y., 104, 142 Kramer, T. J., 104, 141, 346 Krieg, N. K., 316, 340 Krikos, A., 299,300,305,310,311,325, 326, 337, 340 Krinjen, A., 279, 345 Kristjansson, J. K., 223, 224, 225, 263, 268, 271 Krudy, G., 114, 141 Krumhaar, H., 13, 15, 69

Kruyt, H. R., 14, 23-4, 67 Krywolap, G. N., 220, 269 Kuang, W., 99, 144 Kubler, O., 214, 217, 262, 268 Kudo, S., 59, 67 Kuen, B., 245, 248, 268 Kues, U., 19, 65, 69 Kuhara, S., 62, 65, 66 Kuhn, P. J., 221, 268 Kuhn, W., 25, 67 Kukkonen, M., 218, 267 Kundu, M., 48, 64, 67 Kung, C., 154, 185, 208, 296, 340 Kunisawa, R., 220, 264 Kuo, S. C., 294, 322, 340 Kupcu, S., 259, 261, 268, 269, 271 Kupcu, Z., 240,241,242,255,258,259, 264,268,272 Kupper, J., 285, 340 Kurane, R. ,.20, 67 Kurata, K., 8, 67 Kurihara, T., 81, 82, 143 Kuriyama, H., 7, 56, 67 Kushner, D. J., 224, 264, 268 Kusserow, R., 13, 67 Kutsukake, K., 279,280,282,284,286, 287, 339, 340, 342 Kuusinen, K., 218, 274 Kuusinen, M., 218, 274 Kuwajima, G., 286, 340 Kylin, A., 165, 169, 200, 202, 210 L Laeuger, P., 293, 340 Lagunas, R., 178, 209 Lahrer, F., 169, 206 Lai, C.-H., 217, 268 Laishley, E. J., 222, 271 Lamed, R., 242, 265 Lampen, J. O., 43, 48, 54, 57, 67, 71 Lancy, P. Jr, 217, 268 Lands, W. E. M., 19, 67 Landsteiner, K., 49, 67 Lange, H., 13, 62, 67 Langworthy, T. A., 219, 222, 223, 265, 268 Lantto, R., 218, 266 Lapchine, L., 215, 216, 268 Lapidus, I. R., 288, 301, 323, 337, 340

362

AUTHOR INDEX

Laracy, E. P., 171, 172, 173, 174, 183, 184, 206 LaRosiliere, R. C., 278, 279, 336, 338 Larsen, S. H., 224, 270, 273, 290, 292, 297, 318, 325, 340, 344 Larsson,C., 153,154,170,183,187,192, 194, 200, 204, 206, 208, 209 Lasky, L. A., 257, 263 Lauchli, A., 185, 208 Lauerer, G . , 222, 223, 268, 273 Laux, D. C., 279, 342 Lawn, A. M., 283, 340 Le Tourneau, D., 179, 212 Leadbetter, E. R., 217, 218, 219, 266, 268 Lebert, M., 279, 345 Lecar, H., 202, 210 Lechner, J., 214,224,237,240,242,243, 244, 245, 247, 249, 250, 268, 274 Lecker, S., 79, 141 Ledgerburg, J., 279, 283, 340 Leduc, M., 218, 268 Lee, B. H., 334, 340 Lee, L., 301, 304, 309, 340 Leeson, E. A , , 161, 162, 163, 192, 209 Legisa, M., 178, 209 Lehinger, A. L., 16, 67 Lehle, L., 114, 142 Leifson, E., 280, 340 Leigh, J. A., 222, 223, 267, 268 Lematre, J., 3, 18, 51, 66 Lembcke, G . , 225, 262 Lemmon, S. K., 127, 128, 142 Lemmon, V. P., 127, 142 Lemontt, J. F., 61, 67 Lengeler, J. W., 299,317,322,338,343, 345 Leonard, K., 217, 255, 268 Leonard, R., 217, 264 Lepault, J., 218, 255, 268 Lerner, R. A., 63, 67 LeRuyet, P., 219, 268 Levi, C., 8, 43, 58, 65 Levin, B., 19, 66 Levin, R. L., 164, 209 Levinson, A., 136, 144 LeVitre, J., 109, 110, 143 Levy-Rick, S., 12, 64 Lewin, L. M., 8, 58, 67 Lewin, R. A., 222, 271

Lewis, C. W., 60, 61, 66, 67 Lewis, D. H., 168, 175, 209 Lewis, L. O., 218, 230, 243, 268 Lewis, M. J., 107, 108, 109, 142, 143 Lewis, R. V., 252, 270 Li, C., 296, 340 Li, E., 114, 142 Liao, X., 83, 84, 141 Lidstrom, M. E., 216, 265 Liljenberg, C., 181, 206 Lill, R., 79, 141, 142 Lim, C. N., 126, 141 Lin, E. C. C., 182, 208 Lin, M. Y . , 41, 71 Lindman, B., 198, 199, 209 Lindner, P., 16, 67 Lindquist, W., 15, 17, 18, 44, 67 Lingappa, B. T., 195, 211 Lingappa, V. R., 78, 87, 136, 144 Linnemans, W. A. M., 43, 67 Linzmeier, R., 317, 341 Lipke, P. N., 17, 62, 67 Lipschitz-Farber, C., 163, 209 Lis, H., 47, 69 Listgarten, M. A., 215, 217, 268, 273 Liu, J. D., 319, 325, 340 Liu, Y., 225, 268 Lively, M. O., 85, 140 Ljungdahl, L. G . , 219, 220, 274 Llobell, A., 179, 208 Lloyd-Hind, H., 7, 68 Loake, G. J., 279, 343 Lockhart, J. A , , 153, 209 Lodish, H. F., 74, 144 Logan, S. M., 215, 237, 238, 239, 252, 264 Lostau, C. M., 57, 65 Lottspeich, F., 238, 243, 244, 245,246, 248, 270 Lounatmaa, K., 216,217,218,220,221, 225,230,237,253,266,267,268,269, 271,272,274 Low, P. S., 168, 209 Lowe, D. A., 179, 209 Lowe, G . , 291, 293, 340, 341 Lowy, J., 283, 340 Lu, G.-Y., 303, 305, 306, 344 Luard, E. J., 153, 154, 158, 171, 172, 174, 176, 184, 209 Lubitz, W., 248, 260, 268, 273

AUTHOR INDEX

Lucas, C., 180, 187, 209 Luchi, F., 179, 208 Luckevich, M. D., 218, 269 Lugtenberg, B., 216, 230, 251, 259, 265 Lukat, G. S., 318, 334, 340 Lund, A., 5, 68 Lund, B. M., 216, 274 Lundgren, D. G., 218, 265 Lundh, N. P., 281, 341 Lupas, A., 322, 331, 340, 342 Lurz, R., 220, 269 Lusena, C. V., 12, 64 Liittge, U., 164, 211 Lynch, W. H., 216, 251, 265 Lyons, T. P., 15, 44, 46, 68 M Maaloe, O., 182, 208 McAlister, L., 196, 209 Macauley, R. J., 7, 15, 16, 69 McBride, M. J., 298, 341 McCammon, K., 104, 141 McCandlish, D., 10, 68 McCarter, L., 288, 340 McCleary, W. R., 298, 341 McConahey, P. J., 63, 67 McCoubrey, W. K. Jr., 235, 266 McCoy, E. C., 215, 243, 252, 269, 274 McCubbin, W. D., 215, 237, 238, 239, 252, 264 McCullough, W., 175, 179, 209 McCusker, J. H., 202, 209, 210 McGee, T. P., 118, 119, 120, 121, 122, 125, 130, 132, 141 McGill, C., 133, 142 McGowan, E. D., 303, 341 Machesky, L. M., 131, 141 MacKay, V. L., 61, 67 MacKenzie, K. F., 191, 192, 195, 209 McLean-Bowen, C. A., 182, 209 McLoughlin, A. J., 12, 17, 39, 41, 64 McMurrough, I., 43, 68 Macnab, R. M., 278, 28&90, 292,293, 294,295,296,297,300,301,304,313, 317,323,327,335,337,339,340,341, 344, 345, 346 McNally, D., 314, 319, 321, 322, 324, 341 MacRae, J. E., 222, 269

363

McVeigh, R., 218, 246, 273 MacWilliam, I. C., 43, 44, 46, 66 Maderis, A., 330, 334, 345 Madon, J., 224, 265 Maeba, P. Y., 222, 269 Maed, M., 162, 208 Maeda, K., 296, 301, 341 Magasanik, B., 322, 342 Maher, P. A , , 231, 272 Mahoney, W. C., 303, 338 Makinen, V., 218, 266 Malakooti, J., 294, 341 Malaney, G. W., 176, 209 Malehorn, D. E., 118, 119, 125, 127, 140, 143 Malhotra, V., 89, 142, 143 Malick, L. E., 216, 270 Malkow, A., 13, 17, 68 Mallavia, L. P., 217, 270 Mandel, M., 288, 342 Mandelbrot, B. B., 41, 68 Mandelco, L., 224, 263 Mandersloot, J. G., 181, 207 Manire, G . P., 217, 269 Manner, D. J., 8, 43, 58, 65 Manney, T. R., 62, 68 Manoil, C., 295, 300, 341 Manson, M. D., 277-334,335,336,337, 338, 340, 341, 343 Mao, J., 109, 110, 143 Margaret, B. S., 220, 269 Margaritis, A., 3, 51, 63 Margolin, Y., 317, 337 Margui, A. 43, 47, 68 Markwell, M. A. K., 53, 68 Marquez, L. M., 286, 338 Marrie, T. J., 257, 264 Marshall, J. H., 179, 209 Martikainen, P. J., 221, 269 Martin, M. L., 217, 270 Martin, N., 255, 268 Martin, P. A., 61, 67, 69 Martinac, B., 154, 185, 208 Martinez, R. J., 281, 341, 342 Martinson, E., 5, 6, 19, 20, 66 Marz, L., 242, 268 Massalski, A , , 231, 265 Masschelein, C. A., 15, 20, 43, 57, 68 Masuda, K., 215, 219, 220, 231, 234, 267, 269

364

AUTHOR INDEX

Masy, C. L., 17, 28, 31, 46, 67 Matsumoto, S., 62, 65, 66 Matsurnura, M., 16, 64 Matsumura, P., 279,280,282,286,287, 292,294,295,298,314,317,319,321, 322,324,337,338,339,341,343,344, 345 Matsuura, S., 292, 341 Mattey, M., 178, 209 Matthies, C., 222, 269 Maurer, R., 130, 140 Maxwell, W. A., 182, 209 May, J. W., 179, 209 Mayer, F., 218,219,220,222,223,262, 263, 267, 268, 269, 271 Mayorga, L. S., 89, 141 Mazur, P., 163, 206 Meade, J., 62, 68 Meakin, P., 41, 68 Medalia, O., 53, 63 Meikle,A., 163,164,165,173,174,176, 190, 209 Meister, M., 291, 293, 339, 340, 341 Meleg, M., 19, 48, 60, 70 Mendez, B., 331, 343 Mendik, F., 8, 14, 15, 25, 66 Mengele, R., 240, 243, 273 Merkel, G. J., 216, 269 Merkel, J. R., 189, 207 Mescher, M. F., 224,239, 242, 249, 269 Mesibov, R. 296, 301, 305, 341 Messner, P., 213-261, 262, 264, 268, 269, 272, 274, 275 Mestdagh, M. M., 17, 28, 31, 46,67 Metcalf, E. C., 184, 212 Meyer, D. I., 86, 87, 143 Meyer, F., 283, 343 Meynell, E. W., 280, 341 Michel, H., 224, 269 Mihara, A., 178, 211 Miki, B. L. A., 8, 15, 17, 18,20,23,47, 48, 49, 51, 68 Mill, P. J., 15, 16, 17, 18,43,44,45,46, 55, 57, 58, 68 Miller, J. B., 317, 341 Miller, R. J., 20, 61, 69 Milligan, D. L., 299,311,312,317,331, 338, 341 Millonig, G., 224, 270 Milner, R. J., 152, 208

Mirelman, D., 53, 62, 63, 68 Miroshnichenko, M. L., 234, 263 Misra, L. M., 104, 105, 143, 144 Misumi, Y., 62, 66 Mitani, M., 281, 339 Mitchell, R., 279, 337 Mizuno, T., 299,300,301,339,340,341, 342 Moe, G. R., 304, 308, 334, 342 Mohr, V., 224, 270 Moir, D. T., 109, 110, 143 Molenaar, C., 134, 142 Moll, M., 16, 58, 63 Mornose, H., 13, 68 Monette, J. P. L., 251, 267 Monsigny, M., 48, 51, 63,66 Moor, H., 162, 210 Moradas-ferreira, P., 51, 70 Morales, C., 153,154,170,183,187,209 Moran, J. W., 171, 173, 188, 209 Morelli, L., 220, 271 Morgan, D. G., 300,304,311, 312,338 Morgenstern, E., 242, 265 Morimoto, K., 59, 68 Morris, E. J., 219, 273 Morris, G. J., 161, 162, 163, 192, 209, 210 Mortimer, R. K., 202, 210 Mosbach, K., 258, 270 Moser-Thier, K., 217, 218, 255, 271 Mostek, J., 19, 69 Mota, M., 51, 70 Mottonen, J. M., 334, 340, 344, 345 Moustacchi, E., 192, 211 Mowbray, S. L., 302,303,304,305,308, 309, 311, 338, 342 Mowry, K. L., 301, 346 Mowshowitz, D. B., 62, 70 Mueller, S. C., 127, 142 Mukherjee, K., 48, 64 Mulder, C. J., 5, 63 Mulholland, J., 101, 143 Mullakhanbhai, M. F., 224, 270 Muller, H., 138, 139, 142 Mundt, W., 254, 259, 272 Munn, C. B., 251, 270 Munns, R., 182, 185, 210 Munro, P. A., 20, 71 Munro, S., 104, 106, 142 Murakami, T., 7, 56, 67

365

AUTHOR INDEX

Muramatsu, M., 101, 142 Murata, K . , 215, 265 Murata, M . , 17, 19, 48, 67 Murray, R. G. E., 214, 215, 216, 217, 218,230,231,232,234,239,243,255, 262,263,264,267,268,270,271,272, 273, 285, 337 Mutoh,N.,299,300,310,311,314,317, 340, 342

N Nagarajan, L., 47, 68 Naji, B . , 8, 68 Nakajima, T., 138-9, 142 Nakamura, K . , 219, 273 Nakano, A., 97, 98, 101, 142 Naruse, Y . , 238, 245, 247, 248, 265 Nasim, A., 12, 64 Neidhardt, F. C., 182, 208 Neigeborn, L., 61, 64 Neish, A. C., 179, 211 Nelson, D. C., 298, 342 Nermut, M. V . , 218, 265, 270 Nettleton, D. O., 327, 336, 345 Netto, C. B., 7, 68 Neuner, A . , 224, 225, 263, 271, 273 Nevola, J . J . , 279, 342 Newell, P. C., 2, 68 Newman, A. P . , 96, 142 Newton, A., 246, 270 Newton, R . , 33, 63 Newton, S. A., 257, 270 Ni Eidhin, D . , 215, 237, 238, 239, 252, 264 Nichols, R . , 175, 208 Nicolaus, B . , 223, 264 Niedermeyer, W., 162, 210 Niederpruem, D. J., 173, 180,181,206, 207 Nilsson, A., 169, 186, 203, 206, 210 Nilsson, L., 177, 210 Ninfa, A . J . , 322, 334, 339, 342, 345 Ninfa, E. G . , 322, 342 Nishihara, H . , 15,16,17,18,20,23,46, 48, 49, 51, 57, 58, 68 Nishikawa, N., 12, 18, 67, 71 Niwano, M., 318, 322, 330, 342, 343 Nixon, B. T., 317, 334, 342, 343 Nobel, P. S., 149, 210

Nobre, M. F., 171, 174, 187, 210 Nohr, B., 10, 66 Nohynek, L., 218, 267 Nold, A,, 298, 343 Nordstrom, K . , 178, 210 Norkrans, B . , 156, 165, 169, 198, 200, 201, 202, 208, 210 Normington, K., 104, 142 Norrish, R. S., 191, 210 Northcote, D. H . , 44, 68 Northup, J. K . , 132, 137, 142 Norton, R. S . , 169, 172, 208, 210 Novick, P. J . , 17, 18,43, 54, 60, 65,68, 74,75,76,77, 100,102,113, 114,129, 130,132,133,134,135,136,137,138, 140, 141, 142, 144 Nowlin, D. M., 325, 342 Nudd, R. C., 20, 71 Nunokawa, Y . , 8, 67 Nurmiaho-Lassila, E.-L., 218,221,267, 269, 274 Nurse, P., 127, 143 Nusser, E., 223, 242, 247, 270 Nyns, E. J . , 12, 54, 66

0 Oakenfull, D., 158, 197, 206 Ober, K . , 223, 274 Obijeski, J. F., 217, 270 O’Brien, P. C. M . , 279, 338 Oesterhelt, D., 224, 269, 279, 345 Ofek, I . , 20, 53, 60, 63, 65, 68, 69 Ohba, M . , 224, 266 Ohnishi, K . , 287, 314, 339, 342 Ohsumi, Y . , 185, 211, 212 Ohta, N . , 246, 270 Ohya, Y., 286, 287, 340 Okamoto, K . , 288, 343 Olafson, R. W . , 216, 230, 267 Olden, K . , 257, 270 Oldfield, A. I . , 58, 68 Oliver, D. B . , 79, 80, 142 Olsen, I., 217, 264 Olson, E. R . , 298, 301, 342 Onishi, H., 155,157,160,161,168,180, 193, 210, 211 Ono, N., 280, 339 Oosawa, F., 296, 301, 341

366

AUTHOR INDEX

Oosawa, K., 300, 305, 310, 312, 317, 318, 331, 334, 338, 339, 342, 345 Orci, L . , 89, 91, 99, 142, 143 Ordal, E. A., 327, 345 Ordal, G. W., 280, 317, 327, 336, 342, 344,345 Oren, A., 224, 264 Orpin, C. G . , 176, 210 Ortega, J . K . E., 155, 207 Ortega, M. D., 185, 202, 211 Orton, W. L., 12, 15, 16, 17, 18,47,48, 49, 70 Osawa, T., 48, 66 Osborn, M. J . , 230, 243, 270 Oshima, T., 224, 266 Oshiro, L. S., 216, 251, 252, 267, 270 Oshumi, Y . , 185, 210 Osinchak, J. E., 189, 207 Osmani, S. A., 175, 179, 209 Osmond, B.C., 61, 64, 130, 142 Osterlund, K . , 221, 269 Ouchi, K . , 8, 67 Overbeek, J . Th. G., 14, 24, 27, 69 Overhoff, B., 300, 338 Owen, B. B . , 151, 208 Owen, W. G . , 202, 210 Owens, K . , 218, 246, 273 P Padmanabha, R., 62, 69 Page, W. J . , 238, 263 Pakula, A. A., 317, 318, 336 Palade, G. E., 74, 1 1 1 , 141, 143 Palaina, J., 104, 143 Palleroni, N. J . , 288, 342 Palm, P., 222, 224, 273, 275 Palmer, E. L . , 217, 270 Paltauf, F., 120, 141 Panasenko, S. M . , 299, 301, 342 Pangborn, J., 216, 270 Parent, J . B . , 257, 270 Parish, G. R . , 162, 210 Parish, J . H . , 287, 342 Park, C., 310, 328, 336, 342 Parker, D. S., 32, 69 Parker, H . M., 327, 345 Parker, K. R . , 210 Parker, S. R . , 323, 324, 342 Parkinson, J . S., 278, 280, 282, 295,

301-2, 304, 310, 312, 314, 317, 319, 323,324-5,327,335,337,339-43,346 Parks, E. H., 47, 65 Parks, L. W . , 182, 209 Parnham, C. S., 5, 65 Parry, J. M . , 192, 193, 210 Passioura, J . B . , 149, 210 Pastan, I. H . , 127, 142 Pasteur, L., 13, 69 Pastor, F. I. J., 43, 54, 60, 69 Pate, J. L., 222, 270 Patel, G. B., 15, 69, 223, 263 Patterson-Delafield, J . , 284, 342 Paul, G., 242, 247, 249, 270 Paula, S. J . , 216, 251, 270 Paulson, J. C., 51, 53, 68, 71 Pawiroharsono, S . , 8, 68 Payen, R., 195, 210 Payne, G. S . , 128, 143 Payne, R. W., 159, 178, 206 Paynter, M. B . , 223, 270 Pearce, W. A., 53, 69 Pearse, B. M. F., 127, 143 Pederson, A., 171, 206 Pei, Z., 239, 252, 270 Pelham, H. R . B., 104, 106, 107, 108, 109, 141, 142, 143 Pellerin, P . , 242, 270 Pena, A., 184, 210 Perlin, D. S., 202, 209 Perry, J . J . , 216, 269 Perry, J. W . , 284, 338 Peters, J . , 215, 238, 243, 244, 245, 246, 248, 265, 270 Peters, M., 238,243,244,245,246,248, 270 Peterson, M., 138, 139, 142 Petina, A , , 17, 68 Petsko, G. A., 302, 303, 342 Pfaff, E., 101, 143 Pfeffer, S. R . , 89, 141 Pfeffer, W., 277, 278, 334, 342 Pfennig, N., 220, 264 Pfyffer, B. U . , 176, 210 Pfyffer, G. E., 168, 175, 176, 210 Phaff, H. J . , 43, 67 Phillips, S. R . , 47, 65 Phipps, B. M . , 216, 225, 230, 234,238, 245, 248, 262, 264, 267, 270 Piendl, A., 20, 69

367

AUTHOR INDEX

Pierzchala, P. A,, 330, 344 Pietri, R., 218, 246, 273 Piiparinen, H., 218, 267 Pind, S., 91, 143 Pinette, M. F. S., 155, 163, 209, 210 Pipasts, P., 19, 48, 60,70 Pitt, J. E., 146, 155, 210 Pitt, J. I., 157, 158, 159, 160, 173, 206, 210, 212 Pitt, T., 217, 218, 264, 268 Pledger, R. J., 225, 270 Pleier, E., 283, 343 Plohberger, R., 232, 233, 261, 272 Pliitner, H., 91, 140, 143 Pollard, A., 168, 210, 212 Pollard, T. D., 131, 141 Pomper, S., 60, 69 Poon, N. H., 8,15,17,18,20,23,47,48, 49, 51, 53, 65, 68 Poorman, R. A., 301, 342 Poritz, M. A., 83, 84, 142, 143 Porter, A. M., 7, 15, 16, 69 Porter, G., 83, 84, 141 Postma, E., 200, 212 Postma, P. W., 299, 322, 343 Poulain, D., 43, 71 Power, D. M., 58, 64 Powers, S., 133, 142 Poyry, T., 218, 267 Pragishvilli, D., 224, 275 Pranger, R., 134, 142 Prehn, S., 84, 143 Premier, G., 219, 222, 264, 268 Prentiss, P. G., 150, 151, 152, 212 Price, H. D., 303, 336 Priess, H., 224, 225, 275 Prince, I. G., 7, 56, 69 Pringle, J. R., 129, 140, 154, 210 Prior, B. A., 171, 188, 212 Priischenk, R., 225, 270 Pum, D., 218, 223, 232, 233, 236, 254, 255,256, 259, 261, 263, 269, 270, 272 Purcell, E. M., 288-9, 302, 336, 343 Puzicha, M., 110, 143

Q Qi, Y. L., 296, 343 Quiocho, F. A., 49, 69, 298, 299, 302, 303, 305, 306, 344,345, 346

R Rabinowitz, M . , 19, 66 Rachel, R., 222, 228, 237, 262, 270 Radermacher, M., 222, 267 Raghav, R., 7, 69 Rainbow, C., 5, 21, 47, 51, 69 Ramaley, R. F., 216, 270 Ramirez, A., 20, 69 Ramirez, J. A., 202, 210 Ramos, J., 184, 210, 211 Rampersaud, A., 334, 345 Randall, L. L., 79, 143 Ranta, H., 216,217,218,220,225,230, 237, 266, 267, 271, 272 Ranta, K., 216, 217,218,220,266,267, 271, 272 Rapport, T. A., 75, 84, 140 Rasch, M., 222, 271 Raschke, W. C., 43, 44, 63, 114, 143 Raska, I., 285, 343 Rasmussen, B. A., 311, 341 Rast, D. M., 168, 175, 176, 210 Rauch, B., 162, 211 Ravid, S., 317, 323, 337, 343 Ray, B., 219, 267, 271 Read, R., 53, 69 Reader, H. P., 60-1, 66, 67, 69 Reader, R. W., 290,292,297,318,325, 340 Redding, K., 117, 141 Reed, R. H., 153, 154, 163, 164, 165, 167,169, 172,173, 174,176, 183,190, 195, 196, 208, 209, 210 Reedy, M., 224, 271 Reese, T. S., 292, 339 Reichenbach, H., 11, 37, 64, 287, 343 Reinicke, B., 255, 274 Reiter, W. D., 224, 275 Remsen, C. C., 216,220,221,222,234, 265, 271, 274 Reniero, R., 220, 271 Repaske, D. R., 301, 343 Repine, J. E., 252, 263 Revello, P. T., 313, 327, 342 Revsbach, N. P., 298, 342 Rexach, M., 91, 92, 140 Reyn, A., 216, 271 Reynolds, D. M., 222, 271 Rhiel, E., 221, 263

368

AUTHOR INDEX

Ribas, J.-C., 179, 208 Ribes, V., 83, 84, 143 Ricchiuto, T., 223, 274 Richter, J. P., 8, 68 Ridgway, H. F., 222,271,288,300,313, 343 Riezman, H., 100, 143 Rihova, L., 180, 207 Rintala, A., 220, 269 Ris, H., 287, 343 Rittenberg, S. C., 283, 345 Rivard, C. J., 223, 271 Robards, A. W., 228, 271 Robb, F. T., 303, 338 Robbins, P. W., 250, 274 Roberts, C. F., 175, 179, 209 Robertson, J. D., 224, 271 Robinson, R. A., 150, 210 Robinson, R. W., 223, 262 Robson, F. O., 7, 64 Rodriguez, L., 54, 69 Rodriguez-Navarro, A., 184, 185, 202, 210, 211 Rollins, C., 327, 343 Roman, H., 60, 69 Roman, S., 314,319,321,322,324,341 Romesser, J. A., 223, 271 Romisch, K., 84, 143 Ronco, P. G., I1 279, 336 Ronkko, R., 218, 267 Ronson, C. W., 317, 334, 342, 343 Roomans, G. M., 184, 211 Rose, A. H.,8,11,12,15,26,30,37,43, 44, 46, 48, 57,64,65, 66, 68,69, 182, 206, 208 Rose, D., 160, 165, 211 Rose, M. D., 104, 105, 143, 144 Roseman, S., 162, 211 Rosenbusch, J. P., 231, 265 Rosengarten, R., 288, 343 Rosevear, P. R., 114, 141 Rossi, G., 94, 142 Roswell, E. H., 317, 345 Roth, A. F., 330, 331, 344 Roth, J. R., 286, 343 Roth, T. F., 127, 144 Rothblatt, J. A., 80, 81, 86, 87, 88, 143 Rothman, J. E., 74,89,91,99,100,103, 104,112,113,123,125,140,141,142, 143, 144

Rothstein, A., 183, 209, 211 Rothstein, R. J., 62, 69 Rousseau, M., 281, 253, 268 Rouvier, P., 224, 263 Rouxhet, P. G., 17, 26, 28, 44,46, 57, 63, 67 Rowen, R., 224, 273 Rowsell, E. H., 322, 343 Royal, C., 279, 343 Rubik, B. A., 290, 315, 323, 343 Rudin, A. D., 14, 16, 18,26,43,44,46, 65 Rudolph, H. K., 109, 110, 143 Ruiz, T., 54, 69 Ruohola, H., 91, 92, 93, 94, 102, 140, 142, 143 Russel, D. W., 127, 141 Russel, W. C., 217, 266 Russell,I., 3,7,13,18,19,20,21,36,46, 48, 49, 55, 57, 58, 60,62, 69, 70 Russell, P., 127, 143 Russo, A. F., 299, 300, 343 Rydel, J. J., 317, 341 S Sadler, I., 81, 82, 143 Saier, M. H. Jr, 299, 343 Saimi, Y., 185, 208 St Johnston, J. H., 5, 9, 18, 58, 68, 69 Sakazaki, R., 216, 251, 270 Salama, S. R., 118, 119, 125, 127, 143 Salhi, O., 3, 18, 51, 66 Sallans, H. R., 179, 211 Salminen, A., 102, 132, 133, 134, 137, 138, 140, 142, 143 Salton, M. R. J., 216, 221, 264, 271 Samain, E., 222, 264 Sambrook, J. F., 104,109,110,141,142, 143 Sancho, E. D., 185, 211 Sander, C., 101, 136, 141 Sanders, D. A . , 331, 343 Sanders, S. L., 80, 81, 88, 143 Sanderson, K. E., 286, 343 Sands, S. M., 60, 69 Santarius, U., 223, 262, 267, 273, 274 Sira, M., 217, 218, 220, 223, 232, 233, 254,255,256,257,258,259,261,263, 266, 268, 269, 270, 271, 272

AUTHOR INDEX

Saraste, J., 111, 143 Sasaki, T., 214, 238, 244, 246, 273, 274 Sato, T., 185, 211 Savel, J., 19, 69 Sawatake, M., 218, 271 Saxton, W. O., 219, 222, 228, 262, 265, 271 Schaefer, W., 250, 274 Schafer, W. 224,238,243,244,245,246, 270, 275 Schallehn, G., 219, 255, 271, 274 Schaller, M. J., 216, 265 Schauer, I., 85, 143 Schaus, M., 198, 206 Scheffers, W. A., 200, 212 Schekman, R., 17,18,43,54,60,65,68, 74,75,76,77,80,81,85,86,88,91,92, 93, 94, 95, 96, 97, 98, 100, 102, 113, 114,115,116,117,118,128,139,140, 141, 142, 143 Schenberg-Frascino, A., 192, 211 Schenk, A., 218, 271 Schenk, S. F., 216, 271 Schiewer, U., 221, 271 Schimz, A , , 279, 343 Schindler, H., 231, 265 Schink, B., 11, 37, 64,222, 269 Schlesinger, M. J., 60, 71 Schleyer, M., 91, 92, 140 Schmidt, J. M., 222, 271, 273 Schmidt-Lorentz, W . , 157, 209 Schmitt, H. D., 101, 110, 143 Schmitt, R., 283, 289, 338, 343 Schmutz, P., 195, 196, 208 Schneider, S., 215, 265 Schneider, W. J., 127, 141 Schoberth, S., 220, 269 Scholle, A., 298, 343 Scholz, I., 225, 275 Schonfeld, F., 10, 13, 14, 69 Schori, L., 53, 63 Schreiber, G., 224, 265, 275 Schreil, W., 224, 271 Schiickling, K., 14, 66 Schultz, J., 62, 69 Schulz, G., 240,241,242,255,256,264, 269 Schulz, W., 224, 225, 275 Schurer, F., 230, 265 Schutt, C. E., 319, 345

369

Schuyler, G. T., 126, 141 Schwaninger, R.,91, 143 Schwarzhoff, R. H., 288, 346 Scott, W. J., 146, 156, 158, 159, 211 Scrutton, M. C., 175, 179, 209, 211 Scrutton, N. S., 179, 211 Sealy-Lewis, H. M., 180, 212 SeetararnaRao, B., 7, 69 Segall, J. E., 290, 315, 321, 330, 336, 339,343 Segerer, A., 225, 271 Segev, N., 93, 101, 102, 140, 143 Seiko, Y., 7, 56, 67 Seligy, V. L.,3,8,15,17,18,20,23,47, 48, 49, 51, 62, 66,68 Semenza, J. C., 107, 108, 142, 143 Sentandreu, R., 8,43,54,57,60,65,68, 69 Separi, F., 51, 66 Serafini, T. A., 89, 103, 123, 125, 143, 144 Serrano, R.,184, 202, 211 Sewell, J. L., 102, 143 Seyffert, H., 13, 14, 69 Seymour, F. W. K., 279, 343 Shade, S. Z., 280, 287, 343 Shah, H., 216, 217, 218, 266 Shao, M.-C., 114, 141 Shapiro, L., 279,283,284,285,337,338, 344 Shapleigh, E., 48, 64 Sharon, N., 20,47,48,53,60,63,64,65, 66,69, 257, 271 Sharp, P., 7, 56, 66 Sharpe, V. J., 182, 208 Shaw, C. H., 279, 343 Shaw, D. H., 251, 264 Shaw, P. J., 223, 271 Shere, S. M., 184, 211 Sherman, F., 62, 69, 183, 198, 209, 211 Sherris, D., 327, 343 Shi, W., 296, 335 Shieh, W., 57, 69 Shilo, M., 224, 264 Shim, J., 96, 142 Shimada, T., 216, 251, 270 Shimazu, T., 59, 68 Shinitizky, M., 301, 337 Shinoda, S., 288, 343

370

AUTHOR INDEX

Shioi, J.-I., 292,296,299,301,318,322, 341, 343, 345 Shipp, E. A., 176, 209 Short, K. A., 222, 273 Shporer, M., 163, 211 Shropshire, W. Jr, 155, 207 Shuman, H. A., 303, 345 Sicko, L. M., 221, 267 Siegel, V., 78, 83, 84, 143 Siekevitz, P., 111, 141 Siewert, G., 250, 271 Silbereisen, K., 14, 70 Silhankova, L., 19, 69 Silhavy, T. J., 303, 322, 339, 341 Silver, P., 81, 82, 143 Silverblatt, F. J., 20, 53, 69 Silverman, M., 278, 279, 284, 286, 287, 290,292,300,313,314,325,326,331, 336, 340, 341, 343, 344 Simms, S. A., 317, 330, 344 Simon, M. I., 278, 279, 280, 282, 284, 286,287,288,290,292,299,300,310, 311,312,313,314,317,319,325,326, 331,336,337,338,339,340,341,342, 343,344 Simon, R. D., 221, 271 Simons, K., 111, 127, 136, 141, 142 Simpson, J. R., 146, 167, 169, 207 Singer, S. J., 63, 64, 111, 140, 231, 272 Singh, A., 179, 198, 211 Singh, K. K., 195, 209 Singh, M., 179, 211 Sison, Y., 8, 58, 66 SivaRaman, H., 7, 69 Sjoblad, R. D., 283, 344 Sjogren, A., 216, 217, 220, 228, 229, 237, 248, 251, 266, 272, 274 Skehel, J. J., 53, 71 Skerman, T. M., 216, 265 Skulachev, V. P., 292, 293, 338 Slayman, C. L., 184, 206, 211 Sleytr, U. B., 213-261, 262, 263, 264, 265,266,268,269,270,271,272,274, 275 Slifkin, M., 49, 65 Sloan, J., 179, 209 Slocum, M. K., 301, 302, 325, 344 Slonim, A. E., 176, 209 Smarda, J., 221, 272 Smit, J., 222, 227, 230, 232, 233, 235,

246,247,249,251,255,263,265,269, 272 Smith, D. C., 168, 175, 209 Smith, J. E., 19, 64 Smith, J. L., 186, 207 Smith, J. M., 317, 322, 343, 345 Smith, M. B., 158, 197, 206 Smith, P. F., 252, 263 Smith, P. H., 223, 271 Smith, P. R., 214, 218, 262 Smith, R. A., 109, 143, 314, 344 Smith, R. H., 8, 58, 69 Smith, S. H., 215, 234, 272 Smith, S. N., 160, 174, 211 Snyder, M. A., 317, 344 So, L. L., 60, 69 Socransky, S. S., 215, 217, 268 Solanes, R. E., 288, 342 Solinova, H., 19, 69 Sols, A., 178, 189, 208 Somero, G. N., 146, 167, 168, 176,203, 212 Sommi, P., 220, 271 Sonoda, Y., 7, 56, 67 Sopata, C. S., 327, 336 Sosinsky, G. E., 284, 285, 344 Sowden, L. C., 222, 273 Sowers, K. R., 223, 272 Spedding, P. L., 20, 71 Spencer, D. M., 20,61,69,179,180,211 Spencer, J. F. T., 19, 20, 61, 69, 179, 180, 187, 206, 211 Spencer Phillips, P. T. N., 187, 207 Speth, V., 13, 65 Spiess, E., 223, 271 Spit, B. J., 222, 264 Spoerl, E., 182, 209 Sprague, G. F., 178, 211 Springer, M. S., 301,313,325,326,327, 330, 331, 332, 338, 345 Springer, W. R., 313, 314, 344 Sprott, G. D., 222, 223, 263, 273 Spudich, E. N., 303, 335 Spudich, J. L., 279, 290, 315, 344, 345 Spurlino, J. C., 303, 305, 306, 344 Stackebrandt, E., 222, 223, 224, 273, 274, 275 Stader, J., 294, 295, 337, 344 Stadtman, T. C., 223, 267 Stahl, P. D., 89, 141

371

AUTHOR INDEX

Stahl, U., 12, 19, 66, 69 Stallmeyer, M. J., 284, 285, 344 Stanier, R. Y., 288, 342 Stanley, S. O., 176, 207 Stannard, J. N., 183, 209 Stark, H. C., 62, 70 Starr, M. P., 216, 222, 270, 273 Steams, T., 103, 143 Steensland, H., 224, 273 Stein, P. C., 27, 64 Stein, W. D., 163, 181, 211 Steinacher, I., 303, 336 Sternberg, D. A., 312, 338 Stetter, K. O., 222, 223, 224, 225, 232, 237,242,247,263,264,265,266,268, 270, 271, 273, 274, 275 Steudle, E., 164, 211 Steven, A. C., 216, 237, 267 Stevens, T., 115, 118, 143 Stevenson, D. G., 36, 70 Stewart, G. G.,3,5,7,13,13,14,15,16, 18,19,20,21,23,26,30,36,46,48,49, 53,56,55,57,58,60,61,62,64,65,69, 70 Stewart, M., 215, 216, 217, 219, 223, 228, 233, 234,254,262,263,264,273 Stewart, R. C., 280, 330, 331, 344 Stock, A. M., 317, 318, 319, 322, 326, 332, 334, 340, 342, 343, 344, 345 Stock, J. B., 162,211,317,318,319,322, 327,330,331,334,339,340,342,344, 345 Stocker, B. A. D., 278, 279, 313, 314, 337, 342 Stockhausen, F., 13, 14, 70 Stoeckenius, W., 224,225,273,279,344 Stokes, R. H., 150, 210 Stone, D. E., 82, 144 Stossel, T. P., 131, 142 Strange, P. G., 300, 345 Stratford, M., 2-63, 70 Strathern, J., 133, 142 Strobel, I., 240, 243, 273 Strom, A. R., 175, 208 Strominger, J. L., 224, 242, 249, 250, 266,269,271 Strzempko, M. N., 217, 273 Styles, C. A., 184, 202, 208 Styrvold, 0. B., 175, 208 Su, L., 279, 336

Suddath, F. L., 47, 65 Sumper, M., 214, 224, 237, 240, 242, 243,244,245,247,249,250,268,273, 274 Sundberg, S. A., 279, 345 Sung, J . , 41, 71 Suptijah, P., 51, 66 Sussman, A. S., 195, 211 Sussman, I., 181, 207 Sutherland, I. W., 230, 257, 273 Sutherland, L., 189, 211 Suzuki, H., 287, 314, 340, 342 Suzuki, T., 20, 67, 180, 211, 284, 345 Svennerholm, L., 53, 68 Swanson, E., 246, 270 Swart, K., 180, 212 Sweeley, C. C., 250, 266 Sweet, D. J., 108, 109, 142 Szmelcman, S., 303, 305, 317, 337, 345 Szostak, M.,.260, 273 T Tabas, I., 114, 142 Tabata, R., 233, 238, 244, 246, 273 Tada, K., 203, 212 Takacs, B. J., 220, 273 Takade, A., 215, 265 Takagi, A., 219, 245, 273 Takagi, H., 238, 247, 248, 265 Takakuwa, M., 181, 212 Takao, M., 214, 274 Takashashi, Y., 238, 244, 273 Takata, Y., 18, 71 Takeda, K., 20, 67 Takemura, T., 233, 246, 273 Takeoka, A , , 214, 267 Takumi, K., 214, 267 Talbert, P. B., 313, 327, 342 Tambo, N., 12, 39, 70 Tanahashi, H., 218, 235, 247, 249, 261, 266 Tanfuji, M. 185, 212 Tang, M., 218, 246, 273 Tanner, A. C. R., 217, 268, 273 Tanner, R. D., 176, 209 Tanner, W., 43, 66 Tartakoff, A., 111, 144 Tauschel, H.-D., 221, 273 Taylor, A. E., 58, 68

372

AUTHOR INDEX

Taylor, B. L., 299, 313, 317, 322, 330, 342, 343, 345, 346 Taylor, K. A., 215, 224, 236, 254, 264, 265, 273 Taylor, N. W., 12,15,16,17,18,47,48, 49, 70, 218, 274 Tedesco, P. M., 292,315,317,327,331, 336, 341 Teixeira, J., 46, 51, 70 TeKippe, R. J., 32, 70 Temkin, M., 119, 140 Templeton, B., 287, 335 ten Heggeler, B., 219, 222, 225, 229, 230, 234, 237, 255, 262 Terada, O., 178, 211 Terwilliger, T. C., 325, 326, 331, 345 Thain, J. F., 151, 211 Thevelein, J. M., 175, 211 Thies, G., 223, 266 Thoelke, M. S., 327, 336, 345 Thomas, M. V., 223, 271 Thomashow, L. S., 283, 345 Thomm, M., 222, 223, 266, 273, 274 Thompson, B. G., 217, 273 Thompson, G. A. Jr., 162, 208 Thorne, K. J. I., 219,220,242,262,272 Thorne, R. S. W., 10,14,60,61,66,70 Thorner, J., 113, 115, 127, 141, 142 Thornley, M. J., 216,217,265,272,273 Thornton, R. J., 5, 8, 70 Thrash-Bingham, C., 62, 70 Tiedje, J. M., 221, 264 Tietz, H., 228, 270 Tilbury, R. H., 155, 211 Tilcock, C. P. S., 181, 207 Timakova, N. V . , 218, 265 Timasheff, S. N., 168, 208 Tindall, B. J., 224, 274, 275 Tipper, D. J., 218, 253, 266 Tirtiaux, C., 198, 206 Titova, I. V., 218, 265 Tkacz, J. S., 48, 71 Todd, W. J., 235, 272 Toews, M. L., 326, 327, 340, 345 Tokuyasa, K. T., 63, 64, 286, 338 Tollervey, D., 83, 84, 143 Tom, G. D., 178, 211 Tomelty, J. P., 316, 340 Tonetti, F., 8, 68 Tonge, R. J., 58, 64

Tonoike, R., 13, 68 Toraya, T., 15,16,20,23,48,49,51,57, 68 Trachtenberg, S., 283, 345 Trent, J., 225, 275 Treptow, N. A., 303, 345 Tribhuwan, R. C., 299, 317, 343, 345 Trollmo, C., 196, 197, 211 Tronchin, G., 43, 71 Tronick, S. R., 281, 341 Trumbly, R. J., 62, 71 Trumbore, M. W., 189, 207 Triiper, H. G., 220, 221, 266, 271 Trus, B. L., 216, 237, 267 Trust,T. J., 215,216,230,233,237,238, 239,245,248,251,252,259,263,264, 265,267, 270,273 Tsang, N. 301, 304, 315, 345 TSO,W.-W.,31,290,292,297,304,318, 325, 340,345 Tsuboi, A., 214,217,238,244,245,246, 247, 248, 265, 266, 273, 274 Tsuboi, K. K., 177, 211 Tsukagoshi, N., 217,233,238,244,245, 246,247,248,260,262,265,266,273, 274 Tulej, R., 5, 63 Tunblad-Johansson, I., 171, 177, 179, 181, 187, 190, 206, 209, 211 Tung, B., 19, 66 Tuorila, P., 24, 27, 71 Turner, F. R., 216, 270 Tynkkynen, S., 218, 274 Tzianabos, T., 217, 270

U Uchihi, R., 238, 244, 246, 273 Udaka, S., 217,233,238,244,245,246, 247,248,249,260,262,265,266,273, 274 Udey, L. R., 214, 216, 273 Ueda, M., 219, 273 Ulitzur, S., 288, 345 Ullrich, A., 99, 144 Umeda, A., 215, 265 Umesh-Kumar, S., 47, 68 Unger, E. R., 19, 66 Unger, F. M., 240, 241, 242, 264, 269 Unz, R. F., 26, 71

373

AUTHOR INDEX

Uotila, J., 218, 274 Utsumi, R., 334, 345 Uwajima, T., 178, 211

V Vaara, M., 221, 269 Vaara, T., 217, 221, 267, 269, 273 Vacante, D., 295, 317, 341, 344 Vacata, V., 202, 210 Vallejo, C. G., 202, 211 Valois, F. W., 222, 271, 280, 346 Valtonen, A., 218, 267 van Boxtel, R., 230, 265 Van Deenen, L. L. M., 181, 207 van der Drift, C., 292, 317, 341 van der Werf, P., 325, 345 Van Dijken, J. P., 200, 212 Van Eyk, R. V. W., 181, 207 Van Gylswyk, N. O., 219, 273 van Heijenoort, J., 218, 268 van Heusden, G. P. H., 119, 140 Van Iterson, W., 217, 274 Van Laere, A. J., 176, 190, 211, 212 Van Muiswinkel, W. B., 259, 265 Van Rijssel, M., 219, 274 Van Roekel, T., 155, 208 van Roey, G., 5, 65 Van Rooijen, R., 180, 212 Van Schaftingen, E., 190, 212 Van Steveninck, J., 164, 207 Van Uden, N., 180, 187, 197, 209, 212 Van Zyl, P. J., 171, 188, 212 Vedros, N. A., 252, 268 Veide, A., 179, 208 Verachtert, H., 177, 212 Verduyn, C., 200, 212 Verkleij, A. J., 181, 207 Versluis, R., 230, 259, 265 Viljanen, J., 218, 274 Villanueva, J. R., 8, 54, 58, 65, 69 Villstedt, R., 218, 274 Vincent, B., 11, 37, 64 Vingron, M., 84, 136, 141, 143 Visser, J., 178, 180, 206, 208, 212 Viswanath-Reddy, M., 178, 211, 212 Vliegenthart, J. F. G., 242, 265 Vogel, J. P., 104, 105, 143, 144 Vogler, A. P., 317, 322, 338, 340,345 Volker, S., 223, 225, 262, 267

Volz, K., 314, 319, 321, 322, 324, 341, 345 von Borstel, R. C., 210 von Smoluchowski, M., 24, 27, 71 Vreeman, J., 298, 343 Vyas, M. N., 298,299,302,303,345,346 Vyas, N. K., 49, 69,298,299,302,303, 346

W Wada, Y., 185, 212 Wagenknecht, T., 284, 346 Wagner, P., 101, 143 Wahlberg, J., 218, 274 Walker, T., 3, 20, 62, 66 Walsby, A. E., 154, 155, 212 Walter, P., 78, 83, 84, 85, 86, 87, 136, 142, 143, 144 Walters, V., 288, 346 Walther-Mauruschat, A . , 223, 271 Walworth, N. C., 132, 133, 134, 135, 136, 142, 144 Wang, D. N., 216, 220, 228,229,237, 248, 251, 262, 266, 272, 274 Wang, E. A ,, 252, 265, 301, 345, 346 Wang, J. Y., 326, 345 Wang, S.-Y. C., 179, 212 Wang, W.-L. L., 252, 263 Ward, M. E., 53, 66 Warrick, H. M., 313, 314, 346 Watanabe, Y., 39, 70, 181, 212 Watari, J., 18, 71 Waterbury, J. B., 220, 271, 280, 346 Waters, G., 83, 89, 141 Waters, M. G., 86, 87, 144 Watson, D. H., 8, 71 Watson, S. W., 220,221,222,234,269, 271, 274, 280, 346 Watson, T. G., 183, 199, 200, 212 Wattenberg, B. W., 89, 141 Way, J., 81, 82, 143 Webb, J. R., 84, 143 Weber, B., 176, 210 Wecke, J., 219, 255, 271, 274 Weckesser, J . , 221, 265 Weeks, M. G., 20, 71 Wehrli, E., 224, 274 Wei, L. N., 283, 346 Weibull, C., 278, 346

374

AUTHOR INDEX

Weidman, P. J . , 89, 141 Weinberg, R. A , , 298, 340, 346 Weiner, C., 24&1, 242, 264 Weis, R. M., 326, 330, 346 Weis, W., 53, 71 Weiss, J . B . , 79, 141 Weiss, R. L., 224, 274 Weitz, D. A . , 41, 71 Welch, M., 301, 323, 337, 340 Wells, B . , 216, 274 Wells, J . C., 316, 340 Welsch, R., 232, 266 Welsh, D., 317, 319, 344 Wen, D., 60, 71 Wenham, S., 6, 23, 64 Wenzel, K . , 8, 65 Werner-Washburne, M . , 82, 83, 141, 144 Wethered, J . M., 173, 174, 176, 183, 184, 212 Wheeler, K . A . , 159, 212 Wheelis, M. L., 214, 274 Whippey, P. W . , 216, 263, 270 White, F. H . , 20, 71 White, S. L . , 257, 270 Whitters,E. A , , 118,119,125, 127,141, 143

Whurmann, K . , 223, 274 Wickner, R. B . , 198, 206 Wickner, W . , 74, 79, 85, 141, 142, 144 Wiegel, J . , 219, 220, 274 Wieland, F. T., 103, 123, 125,144,224, 240,242,243,249,250, 268,270,274 Wiemken, A., 176, 185, 195, 196, 207, 208, 212

Wigler, M., 133; 142 Wilcox, C. A . , 99, 144 Wilderer, P. A . , 11, 37, 64 Wildgruber, G., 222, 223, 273, 274 Wildhaber, I., 224, 227, 229, 230, 234, 235, 237, 254, 262, 274, 285, 340 Wiles, A. E., 14, 25, 71 Wiley, D. C., 53, 71 Wilkie, D., 19, 20, 51, 61, 65, 69, 71 Wilkinson, J. F., 216, 267 Willey, J. M . , 280, 346 Williams, D. S., 223, 262 Williams, F. D., 288, 346 Williams, N. J., 12, 15, 18, 19, 48, 71 Williams, R. C., 221, 271

Williamson, D. H . , 151, 208 Willingham, M. C., 103, 127, 142, 143 Wilson, C. B., 63, 67 Wilson, D. W . , 99, 144 Wilson, M. L., 294, 295, 346 Wilson, P. D. G., 41, 53, 70 Wilson, R. B . , 216, 270 Wilson, T. H . , 182, 208 Windisch, S., 5, 36, 71, 156, 209 Winslow, C. E. A . , 14, 71 Winter, A. J., 215, 222, 224, 243, 252, 269, 273, 274, 275

Winters, L., 162, 163, 192, 210 Wirtz, K. W. A . , 125, 144 Wise, J. A . , 83, 84, 141 Wiseman, A . , 12, 15, 18, 19, 48, 71 Witter, L. D., 171, 173, 188, 209 Witteveen, C. F. B., 178, 208 Woese, C. R . , 214, 222, 223, 224, 253, 263, 266, 267, 274

Wold, F., 114, 141 Wolf, A . V . , 150, 151, 152, 212, 301, 337

Wolfe, A . J . , 314, 317, 318, 323, 337, 343, 346

Wolfe, P. B . , 85, 144 Wolfe, R. S., 222, 223, 267, 268, 271 Wolff, C., 304, 305, 309, 340, 346 Woodcock, C. L. F., 218, 274 Woodward, M. P . , 127, 144 Woof, J. B . , 12, 71 Word, N. S., 218, 243, 274 Work, E., 217, 274 Wright, A , , 250, 274 Wu, W. H., 215, 255, 264 Wuestehube, L., 93, 102, 140 Wunderl, S., 222, 225, 273, 275 Wyn Jones, R. G., 148, 151, 164, 168, 210, 212

Wyrick, P. B . , 217, 269

Y Yaffe, M. P . , 231, 272 Yaghmai, R., 300, 304, 311, 312, 338 Yagi, T., 203, 212 Yamada, H . , 214, 217, 274 Yamagata, H . , 233, 238, 244, 245,246, 247, 248, 249, 262, 265, 273, 274

375

AUTHOR INDEX

Yamaguchi, S., 280, 282,284, 286, 295,

2

339, 342

Yamamoto, K., 301, 346 Yamashita, I . , 61, 71 Yancey, P. H . , 146, 167, 168, 176,203, 212

Yang, L., 252, 265 Yarbrough, L. R . , 126, 141 Yarrow, D. Y . , 159, 178, 206 Yates, J., 7, 56, 66 Yeo, K-T., 257, 270 Yeo, T.-K., 257, 270 Yin, H . L., 131, 144 Yokota, T., 287, 346 Yonekawa, H . , 327, 328, 346 Yoshida, S., 5, 71 Yoshida, T., 17, 59, 65 Yoshii, Z . , 217, 218, 262, 274

Yoshino, K., 214, 267 Young, T. W., 63, 71 Yousten, A . A., 218,230,238,243,246, 263, 268, 274

Yusa, M., 27, 36, 71

Zabel, H.-P., 223, 274 Zalkin, H . , 303, 338 Zalkin, N., 312, 338 Zanolari, B . , 330, 344 Zavarzin, G. A . , 234, 263 Zehnder, A. J. B . , 223, 274 Zellner, G., 223, 224, 274, 275 Zerial, M . , 136, 141 Zhang, L.-H., 217, 264 Zhang, W., 43, 44, 64 Zhang, Y.-X., 217, 264 Zhou, X.-L., 154, 185, 208 Zhu, X., 218, 246, 273 Zieg, J., 279, 344 Zillig, W., 222, 224, 225, 265, 273, 274 Zimmerman, U . , 155, 165, 211, 212 Zingsheim, H. P., 214, 268 Zlotnik, H . , 43; 71 Zukin, R. S., 303, 346 Zusman, D. R . , 298, 336, 340, 341, 346

Zwetkowa, N., 17, 68

This Page Intentionally Left Blank

Subject Index

Abbreviations: CCW, Counter-clockwise; CW, Clockwise; NSF, NEthylmaleimide-sensitive factor; PC, Phosphatidylcholine; PI, Phosphatidylinositol. A

Acetate production, 189, 194 Acetogenium kivui, S-layer glycoprotein, gene, 246 Acetyladenylate, in intracellular signalling, 317 ACT1 gene, 129 actP mutants, 129 suppressor mutations, 129, 130 Actin, 129 secretion polarized to bud, regulation, 132 Actin-binding proteins, 130, 131 Actin-cytoskeleton, functions, Golgi-complex functions coupled to, 129-132 orientation correlated with cell growth polarity, 129, 131 structurekomponents, 129 see also SAClp Activation energy, 29 in flocculation, 29-30, 31, 39 Adaptation of chemotactic response, see Chemotactic signal transduction Adenosine triphosphate, see ATP S-Adenosylmethionine, 325 Adhesins, flocculation, see Flocculation ADP-ribosylation factor (ARF), see ARF Aeration, flocculation stimulation, 20 Aeromonas hydrophila, S-layer, role in pathogenicity, 251-252

Aeromonas salmonicida, S-layer, in pathogenicity, 251 S-layer protein, gene encoding, 248, secondary structure, 239 structural domains, 248 Aeromonas sobria, S-layer in pathogenicity, 251-252 Aggregation of micro-organisms, 2, 39 chain-forming, see Chain-forming strains of yeast definition, 3 mating, 2, 3 significance of, 2, 6 2 4 3 see also Flocculation Agitation, effects on flocculation, see Flocculation Agrobacterium tumifaciens, chemotaxis, 279 A-layer, 251 see also S-layer Alcian blue, 46 a-factor, precursor, accumulation in secP mutant, 115 secreted in chcl mutants, 128 see also Prepro-a-factor Amino acids, as compatible solutes, 175-176 in Slayer hydrolysates, 237 Amphitrichous cells, 281 Anaerobic bacteria, crystalline surface layers, 216-217

378

SUBJECT INDEX

Ancylobacter aquaticus, turgor pressure,

155 Anhydrobiotic organisms, 195 Anions, uptake with changes in media salinity, 184 Anoxigenic phototrophic bacteria, 220 Antifoaming agents, 8 Antigens, artificial, S-layers as carriers, 259-260 Aquaspirillum serpens, double S-layer, 234 flagella, basal body, 285 S-layer role in predation, 253 Arabinitol, 168 accumulation, 169, 171, 173-174 Debaryomyces hansenii, 170, 171, 187, 193 non-growing phases, 170, 173 regulation, 187, 188 solute-specific, 171 Zygosaccharomyces rouxii, 171, 179, 188 biosynthesis, pathway, 177, 179 catabolism, pathway, 179 in Debaryomyces hansenii, dynamics of increases, 170, 171 regulation of accumulation, 187, 193 osmoregulatory role, 171, 173-174 evidence for, 169 uptake mechanism, 180, 181 Arabinose-binding protein (ABP), 298 Archaebacteria, 214, 222-225 chemotaxis, 279 Slayer, cytoplasmic membrane interactions, 230, 231 double, 234 gene sequences, 244-247 growth, 235-236 proteins, glycosylation of, 239 role, 253-254 see also S-layer ARFgene, 101, 102-103 ARFl gene, 102 arfl' mutant, 103 ARF2 gene, 103 ARF (ADP-ribosylation factor), 102, 103 localization in Golgi complex, 103

Arginine residues, in periplasmic domains of transducers, 304 A R 0 7 gene, 198 Arrhenius plots, 29 Aspartate, CheY phosphorylation, effect on, 320 Aspartate-maltose transducer, see Tar protein Aspergillus, optimum water potential of species, 158 Aspergillus chevalieri, minimum water potential, 161 Aspergillus japonicus, glycerol utilization, pathway, 178 Aspergillus nidulans, compatible solutes in, 172, 174 glycerol utilization pathway, 178 intracellular sodium/potassium ion levels, 184 potassium ions as predominant cation, 183 Aspergillus niger, glycerol formation, 178 polyol content, 171, 172 regulation, 190 Aspergillus wentii, osmotic potential, 153 turgor relationship to water potential, 154 ATP, CheA phosphorylation, 320 in chemotaxis, 292, 318, 320 CheY phosphorylation, 318 generation, at low water potentials, 198-199 per unit of glucose, 199 in protein transport, to endoplasmic reticulum, 87 to Golgi complex, 92-93 utilization, increase at low water potentials, 199-200 in vesicle budding, 89 ATPase, Ca2+,in endoplasmic reticulum retention, 109 in Deb. hansenii, glycerol accumulation regulation and, 187 gene, 202 inorganic ion transport and, 184, 202 ATP-binding proteins, SEC12p and

SUBJECT INDEX

N-thylmaleimide-sensitivefactor as, 99 Attenuation, 5 Attractants (chemotactic), 298, 299 chemoreceptor interactions, 302-305 chemoreceptors for, 298, 299-300 in chemotactic signalling model, 332-333 transducer stimulation, 305-310 see also Chemotactic signal transducers; Chemotactic signal transduction Autoflocculation, 20 B

Bacillus alvei, , S-layer glycoprotein, 241 Bacillus brevis, S-layer overproducer strain, 260 Bacillus brevis HPD31 strain, S-layer protein, gene, 247 Bacillus brevis 47 strain, S-layer genes, 244, 247 sequence, 244 S-layer proteins, biosynthesis, 249 structure, 244 Bacillus licheniformis 7491C mutant NM105, S-layer glycoprotein, 246 Bacillus sphaericus, S-layer assembly, 234 S-layer subunit incorporation site, 235 Bacillus sphaericus 2362, S-layer glycoprotein, gene, 246 Bacillus sphaericus P-1, S-layer role in phage adsorption, 253 Bacillus stearothermophilus, S-layer , assemblylstructure, 232, 234 charged groups on, relevance, 255-256 glycoproteins, 240, 242, 243 glycosylation ability loss with cultivation, 257 permeability studies, 255 subunit incorporation site, 235 Bacillus stearothermophilus PV72, S-layer protein, gene, 248 Bacillus subtilis, chemotaxis, 279 Bacitracin, 249

379

Bacteria, adhesion to host cells via lectins, 53, 63 cell-surface layers, 213-275 nomenclature, 214, see also S-layer classification and crystalline surface layers, 214-225 flocculent yeasts binding to, 13, 20 in heterologous flocculation, 20, 21 motility and chemotaxis, see Chemotaxis; Motility permeability to glycerol, 182 tethered cells, rotation, 290, 315-316 see also individual bacterial species Bacterial swimming, 280, 287, 288-289 in chemotactic signalling model, 333 flagellar rotation, see Flagellar rotation mechanism, 289, 29&291 patterns, 289 physical constraints, 28,8-289 rate, 288 three-dimensional random walk, 289 biasing by chemical gradient, 296297 see also Flagellar rotation; Motility, bacterial Bacteriophage, adsorption via flagella, 280 S-layer role in adsorption, 253 Bacteriophage x , adsorption to flagellar filament, 280 Bacteroides buccae, pathogenicity and S-layer, 253 Barium ions, 15 Basipetospora halophila, optimum water potential, 158 Bdellovibrio bacteriovorus, flagellar sheath, 283 S-layer role in predation by, 253 bet mutants, 96 BET1 gene, 96 BETlp, 96, bet1 sec22 double mutants, 96 Binding proteins, see Periplasmic binding proteins; specific binding proteins BiP (binding protein), 103-104 Bipolar flagellation, 281

380

SUBJECT INDEX

Bordetella pertussis, S-layer, 237 BOSl gene, 96 Boyle-van’t Hoff relation, 151, 163-164 Brewing, 4-9 attenuation in, 5 fermenter design, 6-7 flocculation, 4-6 importance, 4 measurement, 10 see also FLOl phenotype; Flocculation flocculent strains, see Flocculent strains top-hottom- fermenting strains, 6, 7 see also Wort Brownian motion, 14, 2>25 collision frequency and, 26-27 particle mass and size, 14, 24-25, 27 yeast cell movements not due to, 25 BSDZ gene, 121 BSD2 gene, 121 BSR4 gene, 122 bsr mutants, 122, 123 Budding bacteria, crystalline surface layers, 222 Burns test, 10 C CAAX box, 134 Caesium chloride, 33 Calcium-ATPase, in endoplasmic reticulum retention, 109 Calcium-bridging hypothesis, see Flocculation Calcium ionophore A23187, 109 Calcium ions, as dipositive not divalent, 46 in endoplasmic reticulum retention of proteins, 109-1 11 in flocculation, 14 activation of, 15-16 criticism of calcium-bridging hypothesis, 46 in transport from endoplasmic reticulum to Golgi complex, 93 Calcium phosphate precipitation, 13 Campylobacter fetus, S+ and S-, type-A lipopolysaccharide assay, 252

virulence comparison, 252 S-layer, antigenic changes, 252 gene, 247 in pathogenicity of, 252 structure, 243 Campylobacter fetus 84-32 strain, S-layer protein, gene, 247 Candida spp., surface lectins in adhesion to host cells, 52 Candida krusei, osmotic hypersensitivity, 192 Carbohydrate, assembly, yeast Golgi complex and, 113-114 structure in yeast, 114 see also Oligosaccharides; Sugars Carboxyl groups, in flocculation, 45,46, 48 Carboxypeptidase Y (CPY), 83 biogenesis in secl4-I” mutant, 118 precursor, accumulation in sec7’” mutant, 115 translocation into endoplasmic reticulum, 83, 87 Carcinogens, flocculation loss, 19 Cascade theory, of flocculation, 38-41 Catabolite repression, flagellar assembly decreased, 287 Cations, intracellular, changes with medium changes, 183-184 see also individual cations; Inorganic ions Caulobacter crescentus, chemotaxis, 279 complex flagella and flagellins, 283 flagella, basal body rings, 285 S-layer extension, 235 S-layer glycoprotein, biosynthesis, 249 surface-layer protein (rsaA) gene, 246 CDP-choline pathway, 122 mutations in, SEC14p requirement bypass, 122, 123, 125-126 Cell, envelope, major classes, 227 outer component, see S-layer shrinkage with low water potential, 161

SUBJECT INDEX

381

size, changes with water potential CheB protein, 318, 330, 331 changes, 161-162 activities associated, 326, 330 Cell-cell, in chemotactic signalling model, 333 interactions, in flocculation, see Chew interaction, 331 Flocculation phosphorylation, methylesterase repulsion, see Repulsion activation, 331, 333 Cell-surface layers, see Bacteria, cellcheC gene, mutations, 314 surface layers cheD gene, mutations, 314 Cell-wall, ‘Chemical potential of water’, 148 elasticity, water loss and, 166165,166 Chemical reactions, flocculation yeast, structure, 4 3 4 4 analogy, 29-30, 31-32, 39 Chain-forming strains of yeast, 2 Chemolithotrophic bacteria, 221-222 in classification system, 8 Chemoreception, 296310 flocculation by, 8, 41 chemical gradients sensed, 296297 flocculation comparison, 3 shallow gradients, 298, 316 Chaperones, 54 chemical memory, 298, 324 HSP70 as, 82 see also Chemotactic signal Chaperonins, 79 transduction, adaptation CHCl gene, 128 classical phase, 296298 chcl mutants, 128 recognition of attractant not energy che (genes), 313-314 release, evidence, 296 in chemotactic signal analysis, 317 three-dimensional random walk in chemotactic signalling model, biasing, 297-298, 316 332-333 transducers, see also, Chemotactic sequence homologies with other signal transducers signalling systems, 317 transport and metabolism required in, see also individual genes 299 Che (proteins), Chemoreceptors, 296, 298-302 complexes identification, 321 for attractants, see Attractants see also individual proteins binding proteins, see Periplasmic cheA gene, null mutation, 313 binding proteins cheA mutants, 319 cellular distribution, 302 CheA protein, 318 random, 302 Chew complex/interaction, 320, 321, enzyme I1 for sugars, 299 332 for oxygen, 299 dual initiation sites, 314 primary and secondary, 301 in model of chemotactic signalling, for repellents, see Chemotactic signal 332-333 transducers; Repellents phosphorylation, 319, 320, 332 structure and ligand-interactions, inhibition/stimulation, 320 302-305 phosphorylation of CheB, 331 transducers as, see Chemotactic signal states, 320, 332 transducers determining signalling state, Chemosensory pathway, 299 320-321 Chemotactic signal, openlclosedtsequestered, 320 biochemical nature, 316322 repellents/attractants effects, see also CheA protein; CheY 320-321 protein; Intracellular signalling CheA-CheA-Chew complex, 321 cross-talk, 322 cheB gene, 326 genetics and, 313-314 cheB mutants, 327, 328 physical properties, 315-316

382

SUBJECT INDEX

Chemotactic signal transducers, 299-300 binding proteins affinity, 303-304 cellular distribution, 302 Chew protein interaction, 319 copy number/cell, 302 cytoplasmic domain, 305, 310-311 deamidation, 326, 327 as homodimers, 311, 312 methylation, 325-326, 327 action, 327 in adaptation, 327-330 feedback control of adaptation, 33G332 overmethylation, 331, 333 sites, 325 periplasmic domain, see Tar protein as primary or secondary chemoreceptors, 301 repellent sensing, 301, 304-305 low-affinity receptors, 301 required for clockwise (CW) signal, 314, 333 stimulation by attractants, 305-310, 312 see also Tar protein stimulation by binding proteins, 305-3 10 structure and topology, 300 transmembrane regions (TM, and TMJ, 300, 307 cysteine mutagenesis, 311 of homodimers and heterodimers, 312 mutations and suppression of, 312 in signal transduction, 312 transmembrane signalling, 31G312, 334 conformational changes, 312 tryptic peptides (K1 and Rl), 325 see also individual transducers; Tar protein Chemotactic signal transduction, 310-333 adaptation of response, 324-332 covalent modification, 326330 covalent modification model, 329-330 feedback control, 330-332 methylation affecting signal produced, 328

methylation and deamidation, 325-326 to oxygen and phosphotransferase substrates, 330 short-term memory, 298, 324 flagellar switch events, 322-324 integrated model, 332-333 intracellular, see Intracellular signalling transmembrane, see under Chemotactic signal transducers Chemotaxis, bacterial, 277-346 adaptation of response, see Chemotactic signal transduction ATP requirement, 292, 318 gliding bacteria, 298 historical aspects, 278 importance, 278-279 biological, 334 clinical, 279 survival strategies and selective advantages, 27&279 as model behavioural system, 278 proton-motive force, 299 reverse, 323 in rhizosphere and aquatic environments, 279 signal transduction, see Chemotactic signal transduction transport required in, 299 see also Chemoreception; entries beginning Chemotactic cheRB deletion mutants, 328, 330 cheR mutants, 313, 315, 326, 328, 330 adaptation defective, 327 c h e w gene, expression control, 319 mutations, 319 null mutation, 313 Chew protein, CheA protein cornpledinteraction, 320, 321, 332, CheB interaction, 331 function, 319 in model of chemotactic signalling, 332-333 transducer interaction, 319 cheY gene, mutations, 319, 324 null mutation, 313, 319

SUBJECX INDEX

CheY-P, 318, 320, 321 accumulation, 319 factors affecting, 319, 320 limited by CheZ, 320, 332 as clockwise (CW) signaller, 318,319, 332-333 levels in absence of chemotactic gradient, 322 in model of chemotactic signalling, 332-333 CheY protein, crystal structures, 319 events at flagellar switch, 322,324,332 in flagellar rotation direction, 317-318, 319 clockwise, 318, 319, 332-333 K109R mutant, 318 Lys residue involvement, 318-319 in model of chemotactic signalling, 332-333 phosphorylation, 318, 319, 332 aspartate effect, 320 CheA protein in, 320, 321 CheZ effect, 319, 320, 332 see also CheY-P cheY suppressors, 324 cheZ gene, mutations, 315, 324 null mutation, 313 CheZ protein, in chemotactic signalling model, 333 CheY, effect on, 319, 320, 332 flagellar rotation direction, 318, 333 function, 3,22, 333 regulation, by CheA, 321, 322 cheZ suppressors, 324 Chitin, abnormal deposition in sadc9 mutants, 130 in yeast cell wall, 43 Chlamydiae, crystalline surface layers, 217 Chloride ions, accumulation in vacuoles, 185 uptake, by fungi, 184 Choline kinase gene (CKZ), 122 Chrysosporium fastidium , glycerol increase with increasing salinity, 172 intracellular sodium/potassium ion levels, 184

383

osmophilic response and water potential, 157 osmotic potential, 153 CKAZ gene, 62 CKI ,gene, 122 ckI null mutations, 122 Clathrin, antibodies, 128 coats, structure, 127 gene for heavy-chain, 128 localization, 128 protein transport to cell surface, role disputed, 128 retrieval function, 128 role in Golgi-complex protein retention, 127-128 Clockwise rotation, see Flagellar rotation Clostridium symbiosum, S-layer glycoprotein, 241 Clostridium symbiosum HB25, S-layer charges, 256 Clostridium thermohydrosulfuricum, S-layer, 226 S-layer glycoproteins, 241, 242 Clostridium thermosaccharolyticum, S-layer glycoproteins, 241, 242 Coflocculation, 21, 22, 23 different non-flocculent yeasts, 51, 52 see also Flocculation Colloidal particles, 14, 23-24 forces attractinghepulsing, 14 Colloidal suspension, 23-24 particle size restrictions, 24 Colloidal theory, 17, 27 of flocculation, see Flocculation ‘Compatible solute’ concept, 146 see also under Osmoregulation Complementation groups, flocculation mutants, 61 sec mutants, 75, 76 Cotranslational translocation, 79, 87 Counter-clockwise (CCW) rotation, see Flagellar rotation CPTI gene, 122 Crystalline surface layers on bacteria, see S-layer cwp operon, 244 CYC8 gene, 61-62

384

SUBJECT INDEX

Cycloheximide, flocculation inhibited, 54, 57 polyol accumulation and, 188, 189, 194 Cysteine mutagenesis, Tar and Trg proteins, 311 Cysteine residues, in S-layer proteins, species with, 237, 247 Cytochemical methods, Golgi complex identification, 112, 113 Cytochrome oxidase, 19 Cytoskeletal proteins, SEC2p homology, 138 see also Actin-cytoskeleton Cytosolic transport factors, in protein transport, to endoplasmic reticulum, 82-85, 88, 89 see also RNA, 7SL; SSA gene products to Golgi complex, 89, 91 D Dahlem conference, 11 DDEL sequence, 108 Deamidase, 326 Deamidation, of transducers, 325-326, 327 Debaryomyces hansenii, adjustment to water potential changes, 171, 186 compatible solutes, amino acids, 176 polyols, see Debaryomyces hansenii,. intracellular polyols (below) glucose transport, 199 glycerol, content, 169, 171, 186 transport system, 180, 187 increased maintenance costs at low PH, 200 intracellular polyols, arabinitol, 170, 171, 187, 193 dynamics during growth cycle, 170-171, 187 erythritol, 174, 187 glucose-limitedchemostat cultures, 170-171

glycerol increase, 169, 171, 186 growth medium influence, 174, 187 metabolism of, 177, 178 regulation of, 171, 186-187 requirement for growth, 203 solute-specific, 171, 187 total polyol pool, 171 uptake and accumulation, 174, 187 at low water potentials, 171, 186 polyol accumulation required, 186, 203 respiration/fermentation affected, 198 mutants, impaired glycerol production, 203 osmotic dehydration, resistance to, 165 osmotic hypersensitivity, 192 osmotolerance, 165, 171, 186 adaptation to reduce cost of osmoregulation, 201 sodiumlpotassium ion changes with salinity, 171, 183, 187, 202 turgor pressure, 154 Deflocculation, thermal, 18 see also Floc(s), dispersal Dehydration, cellular, 165, 167, 195 resistance, see Osmotolerance structural changes with, 161, 162 see also Osmotic response; Water potential Deinococcus radiodurans, S-layer gene, 245 S-layer glycoprotein, 243 Demethylation, in adaptation of chemotactic response, 327 Dendryphiella salina, arabinitol production, pathway, 179 glucose transport system, 199 inorganic ions not accumulated in vacuoles, 186 intracellular sodium/potassium ion levels, 184 polyol concentration increase, 173, 174 nitrogen-compound in media, 175 2-Deoxy-D-glucose, 17 Desiccation, 195 trehalose, accumulation of, 195 protective function, 195-196

SUBJECT INDEX

Desulfotomaculum nigrificans, S-layer, 226 assemblies, 232 glycoproteins, 243 Dipeptide-binding protein (DBP), 298, 325 Dipeptide transducer (Tap), 299 as repellent receptor, 301 DnaJ protein, 82 Dolichol, in S-layer glycoprotein biosynthesis, 250 Domains, in taxonomy, 214 Drosophila melanogaster, phosphatidylinositoU phosphatidylcholine transfer protein, 126 Dunaliella salina, 162, 163 half-life of glycerol leakage, 181 ion accumulation in vacuoles, 185

E EDTA, floc dispersal, 3, 12, 15, 47 EGTA, 93 Elasticity, of cell walls, 164 Electrostatic charges, 14, 24 collision frequency and, 27 see also Flocculation; Repulsion Endoglycosidase H, 114 Endoplasmic reticulum (ER), 74 phosphatidylcholine synthesis, 123, 125 protein flux from (levels), 103 protein transport to, from cytoplasm, see Protein transport protein transport from, to Golgi complex, see Protein transport quality-control function, 103 retention of proteins, 103-111 BiP and KAR2 gene, 104-105 calcium-ion possible mechanism, 110 calcium-ion role, 109-11 1 disruption of calcium-ion-protein matrix effect, 110 HDEL sequence in, 106107, 108 incompletely assembled polypeptides, 103-1 06 mechanisms, 106, 110 mutants defective, see erdl mutants

385

of resident proteins, 106109 saturability of system, 106 summary of model, 110 in secretory pathway, 74 signal recognition particle (SRP) receptor, 78 Energetics, flagellar, 288, 292-293 Energy, expenditures, at low water potentials, 200 supplies, minimum water potential determined by, 201 Enterobacter aerogenes, transducers, 300 Epistasis analysis, sec mutants, 76 1,2-Epoxypropane, 46 ERDl gene, 108 sequence, 107-108 erdl mutants, failure to retain invertase, 107 Golgi-complex defect in, 108 KAP2p secretion, 107 erdl null mutants, 108 ERDlp, integral membrane protein, 108 location in Golgi complex, 108 erd2 mutants, Golgi-complex dysfunction, 109 ERD2p, as HDEL receptor, 108, 109 multiple functions, 109 erd mutants, 107-109 Ergosterol, 182 Erythritol, as compatible solute, 173 in Debaryomyces hansenii, 174,187 increase with increasing salinity, 171, 173 formation and utilization, pathways, 179-180 osmoregulatory role, fungi species, 173 glycerol accumulation regulation, 187 Escherichia coli, chemoreception, 296298 motility response, 297-298 response to shallow gradients, 298, 316 chemoreceptors, 296

386

SUBJECT INDEX

Escherichia coli (cont) chemotactic signal transducers, 299, 300 chemotaxis, 278 membrane potential in, 316317 nature of intracellular signal, 317 che and mot genes, location, 314 DnaJ protein, 82 FFH protein, 84 flagella, filaments, helix, 280, 281 genes, 286 numberkell, 281 rotation, intervals between, 290 structure, 283 flagellin, genes, 283 flocculent yeasts binding to, 13 glycerol transport and permeability to, 182 motA mutants, 292, 293 protein translocation, 79 co- and post-translational, 79 signal hypothesis for protein transport analogy, 79 signal-peptidase, 79, 85 swimming, rate, 288 Tar protein, 301 tsr mutants, 300 Escherichia coli strain AW551, mutant galactose-glucose-binding protein (GBP), 298 Ethylene glycol, as repellent, 305 N-Ethylmaleimide, microsome treatment and translocation failure, 87, 88 N-Ethylmaleimide-resistantfactor, SSAlp and SSA2p, 88 N-Ethylmaleimide-sensitive factor (NSF), 88, 89 attachment proteins, see SNAP functions, fusion of Golgi complex-derived vesicles, 89 fusion of transport vesicles to Golgi complex, 89, 91 SEC18p homologyhelationship, 99-100 Eurotium amstelodami, 158

F Facultative anaerobic bacteria, crystalline surface layers, 216 Fatty-acyl-CoA esters, long-chain, in vesicle budding, 89 FEHDEL sequence, 106 Fermentation, changes, polyol production, 169 continuous, 7 hunglstuck, 58 low water potentials affecting, 198 prolonged, 19 Fermenter, design, 6 7 tower, 7, 56 Ferritin, polycationic, labelling of S-layer, 256 Filamentous fungi, polyol concentration, 172-173, 174 regulation, 190 polyol metabolism, 178 Fimbriae, 53 Fission of cells, S-layer, 236 Flagella, 28C287 antigenic types, 279 assembly, flagellin folding, 286 genes in, 284-285 incomplete ring structures, 285 rate of elongation, 286 regulation, 285-287 sigma factors, 286, 287 basal body, rings, 284, 291 in rotation, 291 bipolar, 281 cell-surface distribution, 28CL281 complex, 283 energetics, 288, 292-293 filaments, 280, 281 helical shape, 281, 283 left-handed helix, 280, 281 length, 283 as propellor, 290 right-handed helix, 290 rotation, 284 genes, 284 catabolite control, 287

SUBJECT INDEX

chromosomal regions (Fla I, Fla 11, Fla III), 286 mutations, 291, 294, 295 number of, 285, 286 regulation, 286-287 sequences, 294 transcriptional units, 286 hook, in flagellar assembly, 285-286 hook-associated proteins (HAPS),284 in assembly, 285-286 lateral, 281 motor, 290, 291, 293 protein interactions within, 293-296 reversal, 322-323 as three-state device, 323 motor-switch complex, FliG, FliM and FliN proteins, 294 MotA and MotB roles, 294-295 mutations affecting, 291, 294, 295, 314 protein interactions in, 291,293-296 see also Flagella, switch; Flagellar rotation M-ring, 284, 291 insertion, ‘studs’ at, 291-292 numberkell, 281 polar, 281 polyhooks, 284 structure, 281-285 basal body, 284-285 cytoplasmic components, 285 hook, 284 see also Flagella, filament switch, events at, 322-324 see also Flagella, motor-switch complex; Flagellar rotation see also individual bacterial species Flagellar bundle, 281 counter-clockwise (CCW), response times, 315 Flagellar rotation, 290-291 biasing of CW/CCW by gradients, 297-298 proportional to gradient, 316 cessation (pause) state, 289, 323 in chemotactic signalling model, 333 CheY role in determining direction, 317-318, 319, 332-333 see also CheY protein

387

clockwise (CW), 290, 333 cheC mutants, 314 CheY protein role, 318, 319, 332 mutations affecting, 313, 323 response to repellents, 297,313,315 reverse chemotaxis and, 323 suppression of transition to, 297 tumbling, 290, 297 co-ordination, signal in, 316 counter-clockwise (CCW), 290 cheC and cheD mutants, 314 CheY-P levels reduced and, 333 CheZ promoting, 320, 333 in model, 332-333 mutations affecting, 313, 319, 323 response to attractant, 290,313,315 running, 290, 297 wild-type motor in absence of signal, 323 detection, 290 force generators, 292 genes involved, 291, 294 intervals between, 290 mechanics, 291-292 motor for, see Flagella, motor MOT proteins, 291-292, 294-295 overshoot, 331 passive, 295 peptidoglycan attachment in, 291 proton-motive force proportionality, 293 restoration by MOT proteins, 292 rotor and stator, 291, 295 tethered cells, 290, 315-316 velocity, proton flux and, 293 see also Flagella, motor-switch complex Flagellar sheath, 283-284 Flagellation, bipolar, 281 monopolar, 281, 289 flagellar rotation, 291 patterns, 280-281 peritrichous, 281, 289 subpolar, 281 Flagellatropic bacteriophage, 280 Flagellin, 281 C- and N- terminal regions, 281, 283 in flagellar assembly, 286

388

SUBJECT INDEX

Flagellin (cont) genes, 283 types in Salmonella typhimurium, 283 flg, (genes) 286 flgA gene, 287 fIgB gene, 284 flgD gene, 284 flgE (PaK) gene, 284 flgF gene, 284 flgG gene, 284 flgH gene, 284 flgl gene, 284 flgK gene, 284 FlgL gene, 284 flh, (genes) 286 flhC gene, 286 flhD gene, 286 Pi, (genes) 286 fliA gene, 314 FliA protein, 287,314 fliC gene, 283,287 fliD gene, 284 fliF gene, 284 fliC mutations, 323 suppression, 324 FliG protein, sequence/structure, 294 FliK (PuE) gene, 284 fliM mutations, 295,314,323 suppression, 324 FliM protein, 294 fliN mutations, 323 FliN protein, 294 fliS gene, 287 fliT gene, 287 FLOI gene, 60,62 FLOl phenotype, 49 killer L virus and, 63 mannose inhibition of flocculation,

17,49,56 sugar specificity of lectins, 4,9 FLO (genes), 6 M l regulatory nature, 6142 Floc(s), compaction, 36,42 compression, 36-38 energy from agitation/gravity,

thermal, 18 washing effects, 15 dissociation temperature, 12,18,

4546 formation, gravity effect, 33,36 fractal structure, 4142 gravity effect, compression by, 37-38 on floc formation, 33,36 on floc morphology, 33 liquid exudation from, 36 loose fluffy, 36 ‘melting’ temperature, 12,18,4546 morphology, 12 agitation and gravity affecting,

33-35 size, 33-35 agitation effect, 12,32,33 bimodal, 11, 39,41 single-cell fraction relationship, 12 size-density relationship, 39 spherical, 33,34 Flocculation, activation, aeration effects, 20 by calcium ions, direct effects, 15,

15-16 glucose in, 17 indirect effect of calcium ions, 16 by inorganic ions, 15-16 low salt concentrations, 16 temperature-sensitive, 18 activation energy, 29-30,31,39 adhesins, 23,47 lectins role, 47,48 as surface proteins, 47 agitation absent, 25,36 agitation effects, 9, 10,25-26,42 collision frequency curves, 28,

29 collision frequency increase, evidence against, 28-39 on dynamic equilibrium, 32 energy of collision, 29-30,42 energy for floc compression, 37-38,

42

37-38 cubical, 33,34

on morphology and size, 12,32,

dispersal, EDTA, 3,12,15, 47

rapid flocculation by, 25-26,32 summary of, 42

33-35

SUBJECT INDEX

agitation, minimum threshold, 11, 28 pH effect, 29, 30 bimodal distribution of cells, 11, 39, 41 explanation by, cascade theory, 41 bond strength, 11-12 high and diffusive flocs, 37 low and compaction, 37 weak, agitation effect on, 32 bond structure, 44-45 calcium-ion role, 44-47 carboxyl groups in, 45, 46, 48 hydrogen bonding, 46, 47 lectin role, 45, 47-48 phosphate groups, evidence against, 45, 46 protein-carbohydrate, 47 calcium-bridging hypothesis, 44-47 criticisms, 4 6 4 7 evidence supporting, 44-46 calcium ions role, 14, 44 in calcium-bridging hypothesis, 44-45, 46 in lectin hypothesis, 47 in promotion of flocculation, 14, 15-1 6 carboxyl groups in, 45, 46, 48 cascade theory, 38-41 cell suspension, evidence against, 25 by chain-formers, 8, 41 chain-forming aggregates comparison, 3 characteristics, classification, 7-9 chemical reactions analogy, 29-30, 31-32 second-order, 39 by clustering of clusters, 40, 41 coflocculation, see Coflocculation colloidal theory, 14, 18, 27 evidence against, 14, 25 surface protein effects combined with, 14 suspensions and Brownian motion, 23-25 definition, 3-4 derepression, 57 dynamic equilibrium (steady state as), 11, 31, 32, 42 early, in nitrogen-deficient worts, 58 electric double layer, 27

389

energy of collision, 26, 28-30, 39, 42 extent, 11, 30-33 fimbriae association, 53 foaming and, %9 genes, 60, 62 genetics, 14, 6 M 3 FLO gene discovery, -1 FLO gene regulatory nature, 61-62 mitochondria role, 19 suppression and instability, 61 gravity effect, see Floc(s) heterologous, 2&23 historical use of term, 13 homologous, 20 industrial, 4-9 inhibition, 16, 55-57 directhndirect effects of sugars, 17 1,2%epoxypropane, 46 high salt concentrations, 16 low pH and ethanol, 56-57 mannose, 49, 51 non-specific chaotropic effects of salts, 16 overcoming in premature flocculation, 58-59 by sugars, 3, 16-17, 55-56, 58 instability, 5, 61 lectin hypothesis, 45, 4 7 4 8 binding to mannan side branches, 51 evidence for, 4 7 4 8 mechanisms and sugar specificity, 48-49 sugar-binding sites, 48, 49 lectins, in control of onset, 53-55 processing/secretion/activation stages, 53-55 see also Lectins loss, 61 carcinogens causing, 19 petite strains, 19, 20 by proteases, 46 measurement, 9-12 equation, 11 methods, 11, 12 temperature for floc dissociation, 18 mechanism, 43-53 calcium-bridging, see Flocculation, calcium-bridging hypothesis (above)

390

SUBJECT INDEX

Flocculation mechanism (cont) lectin hypothesis, see Flocculation, lectin hypothesis (above) phosphate groups in, 45, 46 receptors, 49-51 yeast cell-wall composition, 4344 minimum collision energy, 29-30 morphology of flocs, 12, 33-35 mutual, 21, 47 mannan side-branch arrangement and receptors, 51 mechanism and compact floc formation, 38 as ongoing process, 11, 31, 32 onset control, 5340, 55 activation or exposure, 57 inhibition, 55-57 inhibition relief, 55-56 inositol-deficiency, 58 nitrogen-source depletion, 57-58 nutrient depletion, 57 ratio of signal nutrient to sugar, 58 synthesis or secretion, 57-58 tunicamycidcycloheximide blocking, 54, 57 pH effect, 15, 17-18 carboxyl groups involved, 45 inhibitory, 56 on minimum agitation threshold, 29, 30 physics of, 2343 agitation absent, effects, 36 agitation and flocculation rate, 25-26 cascade theory, 38-41 collision frequency, 2627, 29 energy of collision, 28-30, 39 extent and equilibrium, 3G33 floc compression by agitation/ gravity, 3638 fractal structures, 4142 morphology, 33-35 suspensions and Brownian, motion, 23-25 physiology, 13-23 historical perspective, 13-14 inorganic-ion effects, 14-16 mitochondria1 involvement, 19-20 pH effect, 15, 17-18

protein denaturation effect, 1&19 temperature effect, 18 premature, 5, 58-60 high molecular-weight polysaccharide, 60 mechanisms, 5 M O process, controlling steps, 53-54 protein precipitation theory, 13-14 rate, agitation effects, 25-26 cell concentration relationship, 39 increase due to pH changes, 18 initial, 11 sedimentation rate versus, 12 rate-limiting step, 39 doublet formation, 39 receptors, 23, 47, 4748, 49-51 a-mannan, 48, 49, 50, 51 formation, 54 mutual flocculation, 51 repulsion of cells, activation energy to overcome, 29-30 cascade theory and, 3 9 4 1 causes despite neutralization, 27 in determining rate, 11, 39 steric hindrance, 27, 30 water displacement resistance, 27 sedimentation, bow-wave effect, 39, 40 sedimentation rate, cell numbers and, 24, 25 selective advantages, 62 single-cell fraction, 11, 39 agitation effect, 30 in bimodal distribution, 11, 39, 41 as constant proportion, 31 doublet formation as rate-limiting step, 39 flocculent nature, 31 stationary phase, 8 strains associated, see Flocculent strains suppression and suppressor genes, 61 surface charge role, 14, 26 due to phosphate groups, 26 neutralization effect, 14, 17, 24, 27 repulsions due to, 26 surface proteins, 18-19 activation, 54

391

SUBJECT INDEX

characterization needed, 48 denaturation effect, 18-19 exposuretunmasking, 57 formation and foaming, 8 genes and regulatory genes for, 53 loss from cell surface, 19, 48 role, as adhesins, 47, 48 ‘salting in and salting out’, 16 symbiotic theory of, 13 termination, 5 theories of, 13-14 as unstable property, 5, 61 viral gene transfer or viral transfer by, 63 Flocculent strains, 3 calcium-binding sites, 15 cells, rolling movements, 37 cell walls, isolated, flocculent character, 15 structure, 43-44 classification, 7-9 Gilliland, 7, 8, 9 FLOl and NewFLO phenotypes, 49 ideal characteristics, 54 sedimentation without agitation, 36 selection, 5 superflocculent, 41 surface charge, pH affecting, 17-18 top-/bottom-fermenting, 6, 7 for tower fermenters, 7 types, 22, 23 variability, 48, 49 ‘weak’, strong flocculation bonds, 37 ‘Floc points’, 12 Foaming, 8 flocculation and, 8-9 FOR2 gene, foaming property, 8 Fractal structures, 41 flocs as, 4142 Freeze-etched preparations, S-layer in, 226, 228 Freezing-point depression,, 15CL151 intracellular osmolality, 152 FRO1 gene, foaming property, 8 Fructose-containing media, minimum water potential values, 160 frz genes, 298 Fungi, initial osmotic response, see Osmotic response

non-osmotolerant, 156, 159 osmotolerance, see Osrnotolerance; Osmotolerant strains water-osmosis and, articles on, 146, 147 see also specific speciedtypes G Galactose, binding to galactose-glucosebinding protein (GBP), 303 Galactose-glucose-binding protein (GBP), 296, 298-299 conformational changes after substrate binding, 303 galactose/glucosebinding affinity, 303 interactions with other proteins, 303 mutation and effects of, 298-299 structure, 302-303 transport and chemoreceptor functions, 298 Gas-vesicle protein, gene for, 155 Gas vesicles, in turgor pressure measurement, 155 GDP-mannose, in protein transport, 93 Gelsolin, 131 Gene-disruption experiments, 83 Genes, flagella, see Flagella in protein translocation to endoplasmic reticulum, 80-82 in protein transport to Golgi complex, 94-1 03 see also individual genes Genetics, of flocculation, see Flocculation fungal water relations, 147 of intracellular signalling, 313-314 protein transport, see Protein transport; specific mutants Geotrichum candidum, arabinitol osmoregulatory role, 173 Gilliland classification, 7, 8, 9 Gliding bacteria, chemotaxis, 298 Gliding motility, 287-288, 298 P-Glucan, in yeast cell wall, 43 D-Glucosamine, 198, 199 Glucose, binding to galactose-glucose-binding protein (GBP), 303

392

SUBJECT INDEX

Glucose (cont) cost of maintenance and, 199 in flocculation, 17 osmotic hypersensitivity and, 192 transport, 198-199 low water potential effect, 198 Glutamate, 186187 covalent modification in chemotaxis, 327, 328 methylated, in chemotaxis, 325, 326 Glycans, in S-layer glycoproteins, 24&1, 242, 243 Glycerol 3-phosphate, 177, 190 Glycerol-3-phosphate dehydrogenase, 178, 186, 188 DNA clones, 189 synthesis in Saccharomyces cerevisiae, 188-189,193 Glycerol, accumulation, see Glycerol as compatible solute catabolism by oxidation, 178-179 growth on, 178 membrane permeability, 181 in bacteria, 182 production, constitutive, in Zygosaccharomyces rouxii, 187, 203, 204 cost of maintenance and, 199-200 minimum water potentials and, 20>204 NADH oxidation, 175 pathways and species, 177-178,189 Phycomyces blakesleeanus spores, 190 potassium-ion independence, 190 regulation, 186-187, 188-189, 193 in Saccharomyces cerevisiae, 188-189, 193, 204 protective effect against heat, 197 release from Saccharomyces cerevisiae, in absence of osmotic stress, 189 as repellent (chemotactic), 305 retention, regulation in Zygosaccharomyces rouxii, 187-1 88 transport, 180 in bacteria, 182 membrane-stretchingchannels, 189

in regulation of accumulation, 187, 189 Saccharomyces cerevisiae, 189, 203 uptake, 180, 187, 188 Debaryomyces hansenii, 187 Saccharomyces cerevisiae, 189 utilization, oxidative pathway, 178-179 pathway and genes, 178 phosphorylative pathway, 178 Glycerol as compatible solute, 168 evidence for osmoregulatory role, 169 major role evidence, 169, 171, 172 NMR evidence, 169, 172 exclusion from vicinity of proteins, 168 increase with increasing salinity, 169, 171 Chrysosporium fastidium, 172 Debaryomyces hansenii, 169, 170, 171, 173, 186 Debaryomyces hansenii mutants, 203 dynamics in growth cycle, 170, 173 external stress solute effect, 174 glucose-limited chemostat cultures, 171 Penicillium chrysogenum, 171-1 72 Saccharomyces cerevisiae, 169, 188-190 Zygosaccharomyces rouxii, 169, 171, 188 reasons for glycerol as preferred osmolyte, 173 regulation of accumulation, 186 Debaryomyces hansenii, 186187 Saccharomyces cerevisiae, 188-190, 193 time-course of accumulation, 173 Glycerol-containing media, optimum water potentials, 159 Glycerol kinase, 178, 186 Glycerol oxidase, 178 Glycerol-sodium symporter, 180 Glycoproteins, eukaryotic, relevancelfunction, 257 secretory, N-linked oligosaccharides on, 77, 114 in Slayer, see S-layer

SUBJECT INDEX

Glycosylation, 43 in assays, in vitro transport from endoplasmic reticulum, 77, 92 compartmentalization of Golgi complex, evidence, 114-115 in flocculation, 54 protein secretion in yeasts, 43 SEC12p, 97 S-layer proteins, 239-243 mechanisms, 250 sites, 245 Golgi complex, actin-cytoskeletal functions coupled to, 129-132 model, 131-132 cis-face, 111 transport vesicle fusion and uncoating, 89, 90, 91 cisternae, 111 yeast, 116, 117 clathrin role, 127-128 compartmental organization, 111-1 12, 112-1 17 mammalian, 111-1 12 compartmental organization in yeast, 113-1 17 evidence, 114-116 mnn mutants, 114 model, 116-117 oligosaccharide modifications, 113-117 organelle identification methods, 112-113 set? mutants, 115-116, 117 secl4" mutant, 117 defect in erdl mutants, 108 detection methods, 112-113 evidence for, in yeast, 112-113 functions, 111 actin-cytoskeletol functions coupling, 129-132 see also SAC/ gene; saclts mutants marker (KEX2p), 113 medial aspect, 111 membranes, SEC14p in, 119, 120 morphology, 111 phospholipid-transfer protein involvement, 117-127 conservation of SEC14p function/ structure, 125-127

393

SEC14p as transport factor, evidence, 118 see also SEC14p protein transport through, 76, 112, 115-1 17 genes involved, see SEC7 gene; SECII gene in vitro analysis method, 89, 112 model, 116117 protein transport to, see Protein transport regulatory role in protein transport, 111,120-125 resident proteins, 127 retention problem, 127-128 in SECl2p biogenesis, role, 97 SEC14p-positive structures, 119 as secretory organelle, 74, 111-132 trans-face, 111 Golgi complex-derived secretory vesicles, 74, 76 'docking' with plasma membrane, 135 fusion, SEC2p and SEC4p upstream of SECl5p action point, 138 fusion with plasma membrane, 76, 132-1 39 GTP-binding proteins in, 132-136 SEC gene products, 76, 132 see also GTP-binding proteins; SEC4p patching, SEClSp function and, 138 SEC4p.GTP binding of, 135 Gram-negative bacteria, anaerobic, crystalline surface layers, 216217 crystalline surface layers, 215-216 S-layer, membrane interactions and assembly, 230, 233 structure, 231 Gram-positive bacteria, crystalline surface layers, 217-220 S-layer, assembly, 230, 233 peptidoglycan layer associated, 228, 234 Gravitational term, in water potential, 148-149 Gravity, effect on flocs, see Floc( s)

394

SUBJECT INDEX

Growth, cardinal temperature, 157, 159 cardinal water potentials, 156-161 inhibition, solute-specific, 160 Growth cycle, osmotic hypersensitivity and, 192 polyol level changes, 170, 171 arabinitol, 170, 173 glycerol, 170, 173 sodium and potassium ion changes, 183 Growth rate, maximal, in nonosmotolerant strains, 159 GTP , binding to SEC4p, 135 affinities, 137 SEC4p function requiring, 133 GTP analogue (GTPTS), 91, 93 GTP-binding domains, in protein translocation into endoplasmic reticulum, 84 of signal recognition protein SRP54, 84 GTP-binding proteins, 132 mammalian, 136 in regulation of vesicular transport, 103, 132 SEC4p as, 133 see also SEC4o SEC gene proddcts potentiating, 137-1 39 in transport from endoplasmic reticulum to Golgi complex, 93, 101-103 see also ARFlp; SARlp; YPTlp in uncoating of transport vesicles, 89, 90 Guanosine triphosphate, see GTP GUT1 and GUE? genes, 178 H

hag gene (fliC gene), 283, 287 Halobacterium halobium, chemotaxis, 279 S-layer glycoprotein, biosynthesis, 249 gene sequence, 245 structure, 240, 242, 243, 245

Halobacterium salinarium, cell-envelope glycoprotein, 242 S-layer glycoprotein, biosynthesis, 249 Haloferax volcanii, S-layer gene sequence, 245 S-layer glycoprotein, 240, 242, 243 Hansenula anomala, compatible solutes in, 169 HDEL sequence, 104, 106, 108 receptor, 107, 108, 109 retention or retrieval of proteins?, 106107, 109 retrieval, 107, 109 Heat conditioning, 196 osmotolerance and, 196, 196-197 Heat-induced trehalose accumulation, 196 Heat shock, induction of KARZ expression, 104-1 05 Heat-shock proteins, 197 HSP70, 82, 88 KARZ gene in, 104 SSA subgroup, 82, 88, 104 synthesis during osmotic conditioning, 197 Histidine, 80 Histidine-binding protein, 303 Histidine kinase, 322 Histidinol, 80 HOL' mutants, 80 selection method, 80, 82 holl mutants, 184 hol-, phenotype, 80 Hook, see Flagella Hook-associated proteins (HAPS), see Flagella Hpr, in signalling pathways, 322 HRBP (human retinaldehyde binding protein), 119, 127 HSP70, see Heat-shock proteins, HSP70 Human retinaldehyde binding protein (HRBP), 119, 127 Hydrofluoric acid, 46 Hydrogen bonding, in flocculation, 46, 47 Hydroxymethylgutaryl-CoA reductase, 94

395

SUBJECT INDEX

I Immobilized enzymes, 258 Immunofluorescence microscopy, Golgi complex identification, 113 Inorganic ions, accumulation in vacuoles, 185 flocculation affected by, 14-16 intracellular levels, changes with media, 183-184 role in osmoregulation, 182-185 transport, 184-185 minimum water potentials and, 202 see also Potassium ions; Sodium ions Inositol deficiency, flocculation onset, 58

Intracellular signalling, 313-322, 334 bias proportional to gradient, 316 biochemical nature of signal, 316-322 acetyl-adenylate, 317 CheY, see CheY protein membrane potential, 316317 other pathways, 322 transducer systems, 317-322 see also CheA protein; Chew protein; CheY protein genetics of, 313-314 genes, 313 impulse response, 316 pathways, 313, 332-333 physical properties of signal, 315-316 response latency, 315 response times, 315, 327 Invertase, secretory, accumulation, class A sec mutants, 75 cotranslational translocation, 87 HDEL conferring endoplasmic reticulum retention of, 106, 107 intracellular pools, in a c P mutants, 129 oligosaccharide modifications, in sec mutants, 114 precursor accumulation, in secF mutant, 115 secretory pathway, 54 In vitro protein translocation systems, to endoplasmic reticulum, 8688 from endoplasmic reticulum, 77, 91-94

through Golgi complex, 89, 112 see also Protein transport Ion channels, mechano-sensitive, 154, 185 in vacuolar membrane, 185 K

kar2-159 mutant, translocation defect, 105 KAR2 gene, 104 as essential gene, 104 yeast BiP encoded, 104 see also KAR2p KAR2p, BiP homology, 104 expression, heat shock inducing, 104-1 05 role, 104-105 models to explain, 105-106 secretion, slow in erdl mutants, 107 signal peptide and HDEL sequence, 104 transcriptional regulation, 104 KDEL sequence, 106 KEX2p, 113 antiserum, 117 failure to retain in chcl mutants, 128 function in late Golgi complexcompartment, 113 as Golgi-complex resident protein, 127 SEC7p localization relationship, 117 SEC14p colocalization, 119 Klebsiella bulgaricus, 46, 51 Klebsiella rnarxianus, 51 Klebsiella pneurnoniae, transducers, 300 Kluyverornyces lactis, endoplasmic reticulum retention signals, 108 SECl4 gene, 126 SEC14p homologues, 126 1

Lactic acid bacteria, yeast flocculation, 13 Lactobacillus acidophilus, Slayer, 226 assembly, 234 Lactobacillus helveticus, S-layer role, 253

396

SUBJECT INDEX

Lampropedia hyalina, S-layer structure, 239 Langmuir-Blodgett films, supports, 259 Lectin hypothesis, of flocculation, 45, 47-48 Lectins, 48, 62-63 barley, in wort, 59 calcium and manganese in, 47 carboxyl-rich, 48 in Campylobacter fetus lipopolysaccharide detection, 252 definition, 48 in flocculation, see Flocculation mannose-specific, 53 polysaccharide affinity, premature flocculation due to, 60 sugar-binding sites, 48, 49 sugar-specificity, 49, 53 use by infecting organisms, 52-53, 63 viral, 53 of yeasts and bacteria, 51-53 of yeast virus, 63 Lipid, bilayer, S-layer interactions, 230, 231 carrier, in S-layer biosynthesis, 249-250 membrane composition, glycerol permeability and, 181, 182 osmotic stability of membrane, 182 Lipopolysaccharide, in Aeromonas spp., S-layer anchoring, 251 Carnpylobacter fetus, detection, 252 Lithium chloride, growth inhibition, 160 Lophotrichous cells, 281 L-ring, flagella, 284, 291 M Magnesium ions, in flocculation, 15, 16 Maintenance, costs of, at low water potentials, 199-201 Maltose, flocculation inhibition, 17, 55 Maltose-binding protein (MBP), 298, 303 affinity for Tar protein, 303 mutations, 303, 305, 306 amino-acid substitutions, 306 suppression by Tar protein changes, 306

Tar protein interaction, see Tar protein Maltotriose, 19, 20 Mammalian systems, BiP (binding protein), 103 Golgi complex, 111, 113 KDEL sequence, 106 protein transport from endoplasmic reticulum to Golgi complex, 89-91 protein transport into endoplasmic reticulum, 78-79, 79 signal recognition protein (SRP54), 84 a-Mannan, as flocculation receptor, 48,49,50,51 side branches, 50, 51 unmasking and flocculation, 47 in yeast cell wall, 43 Mannitol, as carbon reserve, 175 as compatible solute, 174 medium influence on, 174 minor role, 172, 173 formationhtilization, pathways, 179 fungi producing, 168 increase with non-growing phase, 173 uptake mechanism, 180 Mannoproteins, alterations in mnn mutants, 114 in yeast cell wall, 43 Mannose, compartmentalization of Golgi complex and, 114-117 flocculation inhibition, 17 in yeast glycoproteins, 114 a-Mannosidase, 57 a-( 1-3)-Mannosyltransferase, absence from mnnl mutants, 114 compartmentalization in Golgi complex, 114 model for, 116117 meche operon, 314 Membrane, lipid composition, 181-182 permeability, polyols, 181-182 see also Plasma membrane; specific membranes Membrane potential, in chemotaxis, 316 Methanocorpusculum sinense, S-layer structural organization, 254

397

SUBJECT INDEX

Methanospirillum spp., 228 Methanothermus fervidus, S-layer glycoprotein, gene, 247 glycan structure, 250 structure, 237, 242 Methanothermus sociabilis, S-layer glycoprotein, gene, 247 Methanotrix spp., 228 Methionine, 325 in adaptation of chemotactic response, 326, Methyl-accepting chemotaxis proteins (MCP), 325, 326 Methylation, of transducers, see Chemotactic signal transducers Methylesterase, 326, 330 activation, 33Ck331 see also cheB gene 3-O-Methylglucose, 250 Methyltransferase, 326, 327 see also cheR gene Micro-organisms, aggregation, see Aggregation of micro-organisms infecting, lectin use, 52-53, 63 Microsomal membrane proteins, in protein transport reaction, 87 Microsomal washing, translocation after, 87 Middle-wall protein (MWP), in Bacillus brevis S-layer, 244 Mitochondria, in flocculation, 19-20 secretion process affected, 20 Mitochondria1 mutants, yeasts, 19, 20 mnn mutants, 114 mnnl mutants, 114 mnn9 mutant, 115 mocha operon, 314 Monopolar celldflagellation, 281,289,291 Mot, proteins 292 motA gene, 291, 314 motA mutations, suppression, 295 MotA protein, overproduction, 294 as proton-conducting channel, 294 role, 294 motB gene, 291, 314 mutations, suppression by fliM mutations, 295

MotB protein, cell-membrane association, 295 overproduction, 295 role, 294, 295 Motility, bacterial, 277-346, 287-296 energetics, 288, 292-293 see also Proton-motive force gliding, 287-288, 298 importance, 278-279 patterns, 289 run-tumble, 289 chemotactic gradients and, 297 in chemotactic signalling model, 333 flagellar rotational direction, 290 see also Bacterial swimming; Flagellar rotation surface, 287-288 swimming, see Bacterial swimming swimming-swarming, 288 see also Chemotaxis; Flagella; Flagellar rotation M-ring, 284, 291 Mucor hiemalis, osmotic potential, 153 turgor relationship to water potential, 154 Muramidases, S-layer permeability to, 255 Myxobacteria, crystalline surface layers, 222 Myxococcus xanthus, gliding motility, 287-288

N NADH, in glycerol formation, 189 NADPH-cytochrome-c reductase, 93 Neurospora crassa, glycerol formationhtilization, 178 inorganic ion transport, 184 osmotic potential, 152 NewFLO phenotype, 17, 49 sugar specificity of lectins, 49 Nitrogen-source, depletion, in flocculation onset, 57-58 Non-flocculent strains, 5, 23 chain forming induced in, 8 in classification system, 8 coflocculation of, 51, 52 mutual flocculation, 22, 23

398

SUBJECT INDEX

Non-osmotic volume of cells (V,,), 163-164 Non-osmotolerant fungi, 156, 159 N P L l gene, protein transport into nucleus, 81 NSF, see N-Ethylmaleimide-sensitive factor (NSF) N U C l gene, 98 Nuclear magnetic resonance (NMR) spectroscopy, glycerol as compatible solute, 169, 172 Nucleus, protein transport, 81, 82 Null mutations, genes involved in chemotaxis, 313 see also individual null mutations

0 Oleic acid, 181 Oligosaccharides, core, in Saccharomyces cerevisiae, 114 modifications in sec mutants, 114-1 15 N-linked, in S-layer, 242, 243 on yeast glycoproteins, structure, 113-114 0-linked, in S-layer, 242, 243 in S-layer glycoproteins, 24&1, 242 transfer and mechanism, 250 olil gene, 19 Organelles, membrane, barrier function of, 77 see also Endoplasmic reticulum (ER); Golgi complex, O S M l and O S M 2 genes, 198 Osmolality, 149 Osmolytes, see Osmoregulation, compatible solutes ‘Osmophilic’ organisms, 155, 156, 157 Osmophilic response, Zygosaccharomyces rouxii mutant, 158 Osmoprotectants, 168 see also Osmoregulation, compatible solutes Osmoregulation, 167-190 adaptive, 167 cellular functions involved, summary, 204-205

compatible solutes, 146, 167-182 biophysicaVbiologica1 properties, 168 definition, 167 minimum water potential influenced by, 202-204 polyols, see Glycerol; Polyols trehalose and amino acids, 175-176 definition and use of term, 167 inorganic ions role, 182-185 intracellular levels, 183-184 transport, 184-1 85, 202 osmotic hypersensitivity and, 193-194 regulation of polyol accumulation, see Polyols as compatible solutes solute compartmentation, 185-1 86 steady-state, 167 ‘Osmotic’, conditions for use of term, 161 Osmotically active volume (V,,,), 163-1 64 Osmotic hypersensitivity, 19@197 determinants, 193-1 96 compatible solutes in, 193-194 osmoregulation and, 193-194 POIYOIS,193-194 proteins, 194 trehalose, 194-196 genes, 198 glucose in media, 192 growth cycle affecting, 192 heat conditioning not affecting, 196 sodium chloride in media, 192 thermotolerance and, 196-197 viability decrease, 192-193 Osmotic potential, 149, 151-153 adjustment, see Osmoregulation calculation, 149, 153 determination methods, 151-152,153 increased turgor and, water loss reduction, 165 Osmotic pressure, 149 Osmotic response, 161-166, 167, 186 Boyle-van’t Hoff relation and nonosmotic volumes, 163-164 microscopic observations, 161-163 water loss, cell-wall elasticity and, 164-165, 166 initial turgor pressure and, 165

399

SUBJECT INDEX

see also Osmoregulation Osmotic shock, 19 sensitivity, genes involved, 198 see also Osmotic hypersensitivity tolerance, see Osmotolerance Osmotolerance, 155-161, 193 alternative terms for, 155 cardinal water potentials of growth, 156-161 solute-specific properties, 158, 160 w,, 156-158 wmin, 157, 159-161 Vopt, 158-159 cellular factors determining, 197-204 compatible solute accumulation, 193-194 see also Osmoregulation; Polyols determination, growth-related and survival studies, 156 genes determining, 198 glycerol->phosphate dehydrogenase and, 193 heat tolerance and, 196197 high sterol level contributing, 181 initial osmotic response, see Osmotic response optimum water potential independent of predominant solute, 158, 159 potassium-ion transport and, 184,202 protein synthesis and, 194 temperature and pH value affecting, 161 terminology and choice of terms, 155, 156, 160 trehalose levels and, 176, 195-196 see also Water potential Osmotolerant species, 156 growth at low water potentials, 159-161, 165 maximal growth rate, 159 resistance to water loss on sudden exposure, 165 Outer-membrane, S-layer interactions, 230, 231 Outer-membrane proteins (OMP), crystalline (cOMP), 230, 231, 237 S-layer protein differentiation, 237 regular (rOMP), 230, 231, 237

Outer-wall protein (OWP), in Bacillus brevis S-layer, 244 oxi2 gene, 19 Oxygen, attractant response, 299 Oxygenic photosynthetic bacteria, 221 P Pathogenicity, S-layer in pathogenicity, 25 1 Penicillium chrysogenum, intracellular sodium/potassium ion levels, 183 osmotic potential, 153 polyols content, 171 regulation, 190 Penicillium ochro-chloron , potassium ions as predominant cation, 183 trehalose in, 176 Peptidoglycan, in flagellar rotation, 291 S-layer associated, 228, 234 Periplasmic binding proteins, 298, 299 affinity for transducers, 30>304 interactionswith transducers, 305-3 10 Trg protein, 310 see also Tar protein see also Chemoreceptors; Galactoseglucose-binding protein (GBP); other binding proteins Peritrichous cells/flagellation, 281, 289 Permeability coefficients, biological membranes, 163 see also Plasma membranes petite mutants, 6, 19, 20 cost of maintenance at low water potentials, 199-200 PH, effect on flocculation,see Flocculation effect on low water potential tolerance, 161, 200 increased maintenance costs at, 200 Phagocytosis, S-layers in, 252, 253 Phase variation, 283 Phosphate, in flocculation, 16 yeast surface charge, 26, 44

400

SUBJECT INDEX

Phosphatidylcholine (PC), synthesis, CDP-choline pathway, 122, 123, 125-1 26 in endoplasmic reticulum, 123, 125 methylation pathway, 123, 125 Phosphatidylethanolamine, in sphaeroplasts, lysis resistance, 182 Phosphatidylinositol 4,5-bisphosphate (PIP2), 131-132 Phosphatidylinositol/ phosphatidylcholine transfer protein, SEC14p as, 119-120 ubiquity, 125 see also Phospholipid-transfer proteins (PL-TPs); SEC14p Phosphodiester groups, in flocculation, 46 Phospholipids, bulk mobilization model, rejected, 123, 125 retrieval to endoplasmic reticulum, 123, 125 role in protein transport, 118 Phospholipid-transfer proteins (PL-TPs) , 120 defect, see s e ~ l 4 - 1 mutant '~ experimental system for in vivo study, 120 in vitro properties, 120 phospholipid retrieval to endoplasmic reticulum, 123, 125 protein transport through Golgi complex, 117-127 see also Golgi complex; SEC14p substrate specificity, 120 of SEC14p, 120 see also, Phosphatidylinositol/ phosphatidylcholine transfer protein; SEC14p Phosphomannomutase, 75 Phosphotransferase system (PTS), 299, 322 Photoreceptors, 302 Phycomyces blakesleeanus, 155, 190 Phytophthora cinnamomi, proline accumulation, 176 Pichia querquum, 180 - Pili, 53

PIT1 gene, 119 Plasma membrane, depressions in, water potential changes causing, 162, 163 H+-ATPase in, inorganic ion transport, 184, 202 osmotic stability, lipid composition affecting, 182 permeability to polyols, 181-182 lipid composition changes, 181, 182 SEC4p association, 134 S-layer interactions, 230, 231 vesicle fusion, see Golgi complexderived secretory vesicles Plasmolysis, 162-1 63 water potential at, 165 Plating media, osmotic hypersensitivity, 191-192 PMRI gene, 109 pmrl mutants, 110 pmrl null mutations, 110 Polyethylene glycol in media, water potentials, 156, 160 Polyhooks, 284 Polyhydroxy alcohols, see Polyols Polyol dehydrogenases, 180 Polyols, aldose/ketose reduction to, 175 as carbon reserves, 175 membrane permeability, 181-182 metabolism, 177-180 roles in fungi, 175 translocation in fungi, 175 transport and uptake, 180-181 Polyols, as compatible solutes, 168-174 accumulation strategies, 186 distribution in fungi, 168, 171 dynamics during growth cycle, 170, 173 arabinitol/mannitol during nongrowing stages, 170, 173 glycerol during growth phases, 170, 173 evidence for water relations role, 169, 171-1 72 factors influencing type of, 172 culture age, 173 fungi producing, 168, 171 properties, 168

SUBJECT INDEX

regulation of accumulation, 186-190 Debaryomyces hansenii, 186-187 Phycomyces blakesleeanus, 190 Saccharomyces cerevisiae, 188-190 Zygosaccharomyces rouxii, 187-1 88 uptake and accumulation, 174 see also Arabinitol; Glycerol; Mannitol Polypeptides, incompletely assembled, endoplasmic reticulum retention of, 103-106 Post-translational translocation, 79, 86, 87, 88 Potassium ions, glycerol production independent in Saccharomyces cerevisiae, 190 glycerol transport and, 180 intracellular level changes, with external salinity, 183, 202 non-ionic solute in medium, 183-1 84 predominant cation in fungi, species, 183 transport, 184, 202 voltage-gated channels, 185, 202 Prepro-a-factor, glycosylation, Golgi complex compartmentalization and, 117 HIS4p chimera, 80 in vifro transport from endoplasmic reticulum to Golgi complex, 92 post-translation translocation, 86, 88 translocation, SSA proteins in, 88 see also a-factor, precursor Prepro-carboxypeptidase Y (CPY), post-translation translocation, 87 P-ring, flagellum, 284, 291 Pro-carboxypeptidase Y (ProCPY), pl conversion to p2 form, 1 15 Profilin, 131 Proline, as compatible solute, 176 Promoters, Bacillus brevis S-layer gene, 244 Protein precipitation theory, flocculation, 13 Proteins, glycosylation, see Glycosylation interactions within flagellar motor, 293-296

401

as osmotic hypersensitivity determinant, 194 osmotolerance and, 194 secreted by yeast, see Secretory pathways, yeast secretion, S-layer role in bacteria, 260 S-layer, see S-layer sorting, in Golgi complex, 111 surface, in flocculation, see Flocculation Protein transport, 73-144 blocked in class A sec mutants, 75-76 GTP-binding protein role, see GTPbinding proteins intercompartmental and intracompartmental, 74 through Golgi complex, see Golgi complex see also Secretory pathways, yeast Protein transport, from cytoplasm to endoplasmic reticulum, cotranslational, 79, 87 criteria for signifying, 86 eventsktages, 77-78 genetic analyses, 79-86 7SL RNA in yeast, 83-84 cytosolic factors in, 82-85 HOL' mutants, 80 protein translocation genes (SEC61, SEC62, SEC63), 8&82, 88 Saccharomyces cerevisiae role in studies, 79 signal-peptide processing, 85-86 SSA gene products, 82-83, 88 genetic analysis, mutants, 8&82 in vitro systems, 86-88 mutants characterized, 87-88 mammalian/prokaryotic models, 77-79, 87 mammalian, 78-79, 79 post-translational, 79, 86, 87, 88 Protein transport, from endoplasmic reticulum to Golgi complex, 88-1 11 eventdstages, 88-89, 91, 94 in vifro analysis in yeast, 91-94 assay characterization, 91-94 as evidence of in vivo system, 93 requirements, 92-93

402

SUBJECT INDEX

Protein transport, from endoplasmic reticulum to Golgi complex in vitro analysis in yeast (cont) 'semi-intact' cells, 92-93 transitional vesicle isolation, 94 mammalian paradigm, 89-91 molecular analysis of genes stimulating, 94-103 early SEC gene products, 95-100 GTP-binding proteins, 101-103 see also individual SEC genes; sec mutants requirements, 89, 92-93 retention of proteins, see Endoplasmic reticulum (ER) vesicle budding, requirements for, 89 yeast system advantages, 74, 91, 139 see also Transport vesicles Proton flux, in flagellar rotation, 293 Proton-motive force, in bacterial motility, 288, 292-293 threshold, 293 in bacterial swimming, 280 in chemotaxis, 299 in gliding motility, 288 Proton pump, 184 Protoplasts, glycerol production increase, in osmotic stress, 190 osmotic potential determinations, 151, 152 shrinkage, plasmolysis and, 162, 163

neutralization effect, 14, 17, 24 27 steric, 27 Respiration, low water potentials affecting, 198 Reynolds number, low, 288 Rhizobium spp., chemotaxis, 279 Rhizobium meliloti, complex flagella and flagellins, 283 motility pattern, 289 Rhodobacter spheroides, swimming pattern, 289 Rhodotorula gracilis, polyol uptake mechanisms, 180 Ribose-binding protein (RBP), 298, 299 structure, 303 Ribose-galactose-glucose transducer (Trg), see Trg protein Rickettsiae, crystalline surface layers, 217 Rickettsia prowazekii, surface-protein antigen (SPA), gene, 247 RNA, 7SL component of signal recognition protein, 78 absent from Saccharomyces cerevisiae, 84 Schizosaccharomyces pombe, 8S84, 85 Yarrowia lipolytica, 83-84 rsdl mutants, 130 Run-tumble motion of bacteria, see Flagellar rotation; Motility, bacterial

R 5

rab proteins, 91, 136 Redox sink, polyols role, 175 Repellents (chemotactic), 304-305 CheB methylesterase activation, 327, 330 in chemotactic signalling model, 333 demethylation stimulated, 327, 333 low-affinity response by transducers, 301, 305 motility response, 297, 313, 315 Repulsion, interparticle, 11, 14 causes after charge neutralization, 27 collision frequency and, 27 in flocculation, see Flocculation

SAC1 gene, 122, 129

sequence and features, 131 SAClp, antibodies, 131 orientation and localization, 131 SEC14p influenced by, 132 secretory and cytoskeletal effects, model, 131-132 structure, 131 saclCSmutants, 130 suppression of actl-I& and secl&l" mutants, 130 suppression of secretorykytoskeletal defects, 130, 131

403

SUBJECT INDEX

sacl" mutants, genetic interactions with secls mutations, 13&131 Saccharomyces cerevisiae, 6 7SL RNA absent from, 84 ATPase mutants, 202 BiP homologue, 104 bud growth, 129 cell shrinkage as osmotic response, 161, 162 rapidity, 163 cell wall, structure, 4 3 4 4 clathrin function studies, 128 desiccation tolerance, trehalose role, 195-196 energy expenditures at low water potentials, 199, 200 glucose transport system, low waterpotential effect, 198-199 glycerol content correlated to salinity, 169, 188-190, 193 NMR studies, 169 regulation, 188-190 glycerol production, 177 high costs, reasons, 203 maximum, 204 NADH oxidation, 175, 204 potassium-ion independence, 190 protoplasts, 190 regulation, 188-189 glycerol uptake and transport, regulation, 189 glycerol utilization, 178 Golgi-complex identification, 112-1 13 HDEL and DDEL recognition, 108 heat conditioning, 196 inorganic ion transport, 184, 202 maintenance costs, 199-200 at low pH, 200 membrane-lipid composition, 181 minimum water potential, Zygosaccharomyces rouxii comparison, 203 non-osmotic volume, 164 oligosaccharides on glycoproteins, 113-1 14 osmoregulation in, 169, 188-190, 193 osmotic hypersensitivity, 191, 192 viability decrease, 192-193 osmotic potential determination, 151-1 52

osmotolerance, 188-190 protein synthesis and, 194 trehalose levels and, 195 plasmolysis not observed, 163, 164 polarized mode of secretion in, 129 polyol content, 169 glycerol as major solute, 169 regulation, 188-190 polyol uptake, 180, 189 resistance to environmental factors, growth cycle and, 192 respiration/fermentation at low water potentials, 198 SEC14p in, 126 comparison with other yeasts, 126127 secretory pathway function, see Secretory pathway, yeast signal recognition protein (SRP54), 84 sodium/potassium ion changes with salinity, 183 sphaeroplasts, lipid composition affecting stability, 182 trehalose, 195-196 as compatible solute, 176, 195 content, 175-176 vacuole size, decrease with dehydration, 162 water loss, on sudden osmotic dehydration, 165 Saccharomyces cerevisiae NCYC 1195 strain, flocculation, 28, 29, 30 floc morphology, 34-35 Saccharomyces cerevisiae Y41 strain, 194 Saccharomyces pastorianus, 6 Salinity, compatible solute increase, amino acids, 176 polyols, 169, 170-171 intracellular levels of inorganic ions and, 183-184 osmotic hypersensitivity, 191 see also Osmoregulation; Osmotolerance; Sodium chloride Salmonella s p p . , chemotaxis and motility, clinical relevance, 279

__

404

SUBJECT INDEX

Salmonella typhimurium, chemotaxis, 278 che and mot genes location, 314 flagella, filaments, helix, 280, 281 genes, 286 number on each cell, 281 protein-protein interactions, 293-294 straight-curly in mutants, 281 structure, 281-283 flagellar gene mutations, 290, 294 clockwise (CW) rotation, 290, 294 non-motile, 294 suppression, 294 flagellin, types, 283 plasmolysis, 162 swimming, rate, 288 Tar protein, 301 transducers, 299-300 Salt tolerance, 160 see also Osmotolerance SARI gene, 101 SECl2 genetic interaction, 101 SARlp, structure and possible function, 101 Schizophyllum commune, 180 Schizosaccharomyces pombe, 7SL RNA in, 83-84, 85 glycerol catabolism by oxidation, 178-179 mitochondria1 role in surface protein formation, 20 SECI4 gene, 126 SEC14p homologues, 126 signal recognition protein (SRP54), 84, 85 Schwanniomyces alluvius, 20 Sclerotinia sclerotinorum, osmoregulation, 173 scy mutants, 324 scz mutants, 324 Sea, water potential and costs of maintenance in, 20C201 SEC genes, 75-76 BET1 gene interactions, 96 see also individual geneslmutants sec mutants, 75 class-I, 95 vesicle formation block, 95

class-1 and class-11, 95-96 double mutants, 95-96 class-I and class-11, epistatic relationship, 95 class-1 and c,lass-11, genetic interactions, 96 class-11, 95 transport-vesicle consumption block, 95 class A, 75 complementation groups, 75-76 evidence of linear pathway, 76 stages of pathway affected, 75-76 class B, 75 complementation groups, 75, 76 early, see sec mutants, class-I/-I1 epistasis analyses, 76, 95 isolation, 75 oligosaccharide modifications of invertase, 114 see also individual mutations; SEC proteins SEC proteins, 75-76 early, 75, 95 seealso SEC12p; SEC18p; SEC23p; sec mutants, class 1/11 late-acting, 76, 132 see also SEC2p; SEC4p; SECl5p participation at multiple steps in protein transport, 98, 100, 112 for passage through Golgi complex, 76 see also SEC7p; SEC14p SEC2 gene, 138 SEC2p, 138 coiled-coil domain in function of, 139 localization, 139 structure and cytoskeletal protein homology, 138 truncation and thermosensitivity, 139 sec2" mutants, 139 secM'' mutant, 133, 134 SEC4 gene, 132 duplication, suppression of sec mutants, 137 SEC2 and SECIS gene interactions, 137-1 39 sequence, 133 SEC4p, 132, 133 action upstream of SECl5p action point, 138

SUBJECT INDEX

activation and GTP binding, 135 function, auxiliary factors in, 137 cycling model, 135-136 evidence for cycling model, 136 mammalian system similarity, 136 membrane-binding requirement, evidence, 134 as GTP-binding protein, 133 interaction with other late sec mutants, 137 kinetics of association with plasma membrane, 134 localization, 133 post-translational modification, 134 purification and GTP-GDP affinities, 137 recycling from membrane to secretory vesicles, 13S134 release after secretory-vesicle fusion to membrane, 135 sequence and structure, 133, 134 C-terminus, 134 S E C ~ P " ~136 '~~, sec4-Ile133 allele, 136 Sec6" mutant, 133 saclts mutants interactions, 130 SEC7 gene, 117 SEC7p, 117 antisera to, 117 functions, 117 sec7" mutant, 115 secq' mutant, sucl'" mutants interactions, 130 SECIl gene, sequence, 8.5-86 secll'" mutants, 76 as signal-peptidase mutants, 8.5 SEC12 gene, SARl genetic interaction, 101 sequence, 97 secl2 mutant, 93 SEC12p, biogenesis, Golgi-complex role, 97 glycosyl modification and slow glycosylation, 97 localization and possible recycling, 97, 98 SECl4 gene, 118 clones, Schizosaccharomyces pombe, 126

405

in Kluyveromyces lactis, 126 PI-PC-transfer protein gene, 120 PIT1 gene identity, 119 sequence, 118 SECl4p, antibodies and Golgi-complex structures, 119 colocalization with KEX2p, 119 conservation of structure and function, 125-127 as cytosolic species, 118, 119 function in vivo, 119 evidence, 121-122 models, 121 models rejected, 123, 125 in Golgi-complex membranes, 119,120 homologues in other yeast species, 126127 human retinaldehyde-binding protein (HRBP) homology, 119, 127 as phosphatidylinositolphosphatidylcholine transfer protein, 119-120 evidence, 119, 121 mammalian comparison, 119, 127 significance of discovery, 120 phospholipid mobilization model, 123-1 25 phospholipid retrieval role disputed, 125 PI:PC ratio in Golgi-complex membranes control, 12&125 evidence, 121-122 model (in vitro activity as artefact), 121 model (in vitro reflecting in vivo function), 121 model (phospholipid mobilization), 123, 125-126 protein transport in late Golgicomplex compartment, evidence, 118 requirement bypassed by phosphatidylcholine synthetic defects, 122, 123, 126 model to reconcile, 123, 125-126 requirement bypassed by sucIcs mutants, 130 SAClp influence on, model, 132 structure, 118

406

SUBJECT INDEX

sec14" mutants, 117, 119 Golgi complex-like cisternae accumulation, 117 secl61" mutants, 118, 119 block at late Golgi-complex compartment, 119, 130 phospholipid transfer defect, 12G121 SAC1 gene and, 130 suppressors, 121-122, 130 SEClS gene, 137-138 SEClSp, 137-138 functions, 138 increased, secretory vesicle fusion impaired, 138 SEC17p, role, 100 SEC18 gene, 99 secl8 mutant, 93 SEClSp, as peripheral membrane protein, 99 SEC17p role in delivery of, 100 as yeast N-ethylmaleimide-sensitive factor (NSF), 99-100 secl9 mutants, 76 SEC23 gene, sequence, 98 sec23 mutant, 93 SEC23p, 93-94 cytoplasmic location, 98 function, transport-vesicle formation stimulation, 99 required for cell growth, 98 unglycosylated, 98 sec23" mutant, 93 SEC53 complementation group, 75 SECS3 gene, 75 SEC59 complementation group, 75 SECS9 gene, 75 SEC61 gene, 80 sec61 mutants, 82 in vitro translocation system, 87-88 SEC62 gene, 80 nucleotide sequence, 81, 88 sec62 mutants, 8G81, 82 in vitro analysis, 88 SEC62p, 81 role in protein translocation to endoplasmic reticulum, 81 SEC63 gene, 80, 81, 88 sec63 mutants, 81, 88 SEC63p, membrane-spanning regions, 81

role in endoplasmic reticulum and nuclear transport, 82 secA gene product (SecAp), in E. coli, 79 secA gene product (SecAp) ATPase, 79 Secretory granules, 74 Secretory pathway, eukaryotic, 74 Secretory pathway, yeast, 43, 75-144 components involved in multiple steps, 98, 100, 112 elucidation, 75-77 order of organelle involvement, evidence, 76-77 sec mutants, 75-77 see also sec mutants in flocculation control, 53-54 Golgi complex as secretory organelle, see Golgi complex GTP-binding protein role, see GTPbinding proteins late stages, actin involvement, 129 mammalian systems used in resolving, 87 protein transport to-from endoplasmic reticulum, see Protein transport regulatory role of Golgi complex, see Golgi complex yeast system advantages-significance, 74, 91, 139-140 Secretory vesicles, see Golgi complexderived secretory vesicles SecYp, 79 Self-flocculation, 20 see also Flocculation Serine transducer (Tsr), see Tsr protein Serratia rnarcescens, motility, 288 Shear forces, floc size and agitation effect, 32, 33 Sigma factors, flagellar gene regulation, 286, 287, 314 Signal hypothesis, in protein transport, 78 Signalling systems, 317 see also Chemotactic signal transducers; Intracellular signalling Signal-peptidase, Escherichia coli, 79, 85 mammalian, 79, 85

SUBJECT INDEX

mutants, decreased protein transit rate, 85 subunit homology between species, 86 Signal peptide, processing, 79, 85-86 S-layer protein, 248-249 Signal recognition protein (SRP), 78, 83 7SL RNA component, see RNA activities in yeast, 83-84 canine, 83 mechanism of action, 78 polypeptide subunits, 78, 84 cDNA clones and sequencing, 84 Escherichia coli FFH protein homology, 84 polypeptide subunit SRP54, 7SL RNA as,sociation, 85 GTP-binding domains, 84 homologies, 84 yeast-mammalian comparison, 84-85 receptor, 78 Signal sequences, 78 removal, 79, 85-86 Signal transduction, see Chemotactic signal transduction S-layer, 213-275 alternative terminology, 214 application potential, 257-260 as carriers of artificial antigens, 259-260 as isoporous ultrafiltration membranes, 257-258 as model for extracellular protein production, 260 as support for Langmuir-Blodgett films, 259 as supports for macromolecule attachment, 258-259 in vaccine development, 259-260 bacteria with, and characterization of, 215-225 biological significance, 225, 26G261 biosynthesis, 248-250 lipid carriers in, 249-250 3-O-methylglucose, 250 pathways, 250 signal peptide, 248-249 charges on surface, 255-256

407

chemical analyses, 237-244, 261 amino acids, 237, 238 glycosylation, 239-243 molecular weights of subunits, 237 punctate-perforate layers, 239 purification techniques, 239 SDS-PAGE, 237, 245, 247 secondary structure, 238, 239 see also S-layer, (g1yco)proteins crystalline outer-membrane proteins differentiation, 237 detachment and disintegration methods, 231 discovery, 214, 260 double, 234 evolution and, 260-261 extension patterns, 235 fission of cells and, 236 functional aspects, 225, 225-226, 251-257, 261 adhesive properties, 261 bacteria-bacteriophage interactions, 253 charged groups on, relevance, 255-256 glycosylation relevance, 256257 as molecular sieves,, 254-255 pathogenicity, 251-253 in predation, 253 scavenging of nutrients, 256, 261 shape-maintaining function, 253-254 genes, 244-248, 260 little homology between strains, 248, 261 nucleotide sequences, 238,244-248 genetic studies, 230, 244-248 (glyco)proteins, 237, 239, 242 biosynthesis, 248-250 cysteine residues, 237, 247 eubacteria, 239 genes, 244-248 glycans, 24CL241, 242, 243 glycan structure, 243, 245, 257 glycosylation sites, 245 hapten binding, 259 linkages, 242, 243 signal peptide, 248-249 structures, 240-241, 242-243

408

SUBJECT INDEX

S-layer (cont) glycosylation, 239-243 loss with cultivation, 257 relevance, 256257 lattice subunit bonding, 231, 232 location, 227-228, 230 loss with cultivation, 214 monomer number, 233 morphogenesis and self-assembly, 231-236 in archaebacteria, 235-236 double layers, 232 dynamics, 233 incorporation sites of new subunits, 235 multilamellar planar sheets, 232-233 phases, 232 sites of lattice assembly, 233 subunit synthesis rate, 233 as only cell-wall component, 228,235, 253, 260 orientation and order, 235 overproducers, 260 peptidoglycan layer-associated, 228, 234 permeability studies, 255, 256 properties, 214 protomers number in each cell, 249 secretion, relevance, 260 structure, 227-236, 254 common lattice types, 228-229 diversity between strains of species, 22%230,261 of exposed surface, 229 hexagonal symmetry, 228,229,254 plasma membrane and outermembrane associations, 23G231 pore morphology,, 229, 254 pore size, 254-255 regularity of, 214 ultrastructure, 228-231 taxonomical significance, 214, 230 tubular sheath external to, 227, 228 ultrafiltration membranes, 257-258 modification, 258 SNAP (soluble NSF attachment proteins), 89 a-SNAP, SEC17p as, 100

in vitro assay, 100

responsiveness t o SEC18p, 100 Sodium chloride, external levels, intracellular polyol levels correlating, 169, 170, 173 glucose-transport system affected by, 198, 199 growth inhibition, 160 increased costs of maintenance, 199-200 ecological implications, 2W201 osmotic hypersensitivity, 191 see also Osmoregulation; Salinity Sodium dodecyl sulphatepolyacrylamide-gel electrophoresis (SDS-PAGE), 237, 245, 247 Sodium ion-hydrogen ion (NA+/H+) antiporter, 184 Sodium ions, accumulation in vacuoles, 185 flocculation induced by, 15 intracellular level changes, external salinity increases, 183, 202 non-ionic solute in medium, 183-1 84 transport, 184-185, 202 in vacuole, 185 Sodium-motive force, 280 Solutes, accumulation, in vacuoles, 185 compartmentation in fungi, 185-186 compatible, see under Osmoregulation direct-indirect effects on water availability, 146, 148 intracellular concentration changes, see Osmoregulation ionic, effects on intracellular potassium-sodium ions, 183 mechanism t o stabilize proteins, 168 minimum water potential affected by, 160 molality, water potential relation, 150, 151, 152 non-ionic, effects on intracellular potassium-sodium ions, 183-184 optimum water potential independent of, 158, 159

409

SUBJECr INDEX

stress, nature of and polyol accumulation, 174 spaP gene, 247 Sphaeroplasts, lipid composition affecting stability, 182 potassium-ion channels, 185 Spirillum volutans, motility, 291, 316 Spirochneta aurantia, chemotaxis, 316 Spirochaetes, crystalline surface layers, 215 motility, 280, 291 S-ring, flagellum, 284 SSA genes, 82, 83 SSA proteins, 82, 88 ssal mutant, 82 SSAlp, 88 depletion, precursor accumulation, 83 ssa2 mutant, 82 SSA,2p, 88 SSCl gene, 104 secretion of proteins engineered for expression in yeast, 109 Steric hindrance-repulsion, 27 Sterols, decreased permeability to glycerol, 181 Stigmasterol, 182 Streptococcus spp., flagellar energetics, 293 Strontium ions, 15 Sugars, binding sites on lectins, 48, 49 chemotactic response, 299 flocculation inhibited by, 3, 16-17 as direct or indirect effect, 17 see also Flocculation; specific sugars specificity of lectins, 49, 53 Sugar tolerance, 160 Sulfolobus acidocaldarius, S-layer structural organization, 254 Supports for macromolecules, S-layers as, 258-259 Suppression of mutations, actlls mutants, 129, 130 causing counter-clockwise and clockwise phenotypes, 323-324 flagella genes, 294 maltose-binding protein (MBP), 306 motA and motB, 295 secl4-1‘” mutants, 121-122, 130

transmembrane regions of transducers, 312 Suppressor genes, flocculation suppression, 61 Surface charge, yeasts, see Flocculation; Yeast Swarming motility, 287, 288 Synechococcus spp., motility, 280 Syneresis, 36 T Tap protein, see Dipeptide transducer (Tap) tar gene, 299, 300, 314, 325 Tar protein, 299 amino-acid substitutions, MBP mutations suppressed, 306 attractants, 305 interactions with, 305-310 CheY phosphorylation, 319 chimeras, 334 copies in each cell, 302 cysteine mutagenesis, 311 cytoplasmic domain, 305 ligand interactions, 304, 305-310 maltose-binding protein (MBP) interaction, 305 affinity, 303 possible mechanism, 309-310 residues, 305-306, 309 as methyl-accepting chemotaxis protein (MCP), 325 methylation, 325, 326 signal produced by ligand influenced by, 328 mutant, reduced affinity for aspartate, 304, 306, 307 periplasmic domain, 304, 305 AL, and AL, loops, 304, 307, 308-3 10 hydrogen-bonding interactions, 308-309 model, 308-309 monomer and dimer forms, 311 mutational analysis, 306307 mutations affecting aspartate, 304, 306, 307 mutations affecting aspartate and maltose, 307-308

410

SUBJECT INDEX

Tar protein, periplasmic domain (cont) mutations affecting maltose, 307 proposed structure, 304, 308 as primary chemoreceptor, 301 site-directed mutagenesis, 328 tas gene, 300 Taz protein, 334 Temperature, cardinal, for growth, 157 collision frequency and, 29 effect on flocculation, 18 floc melting, 12, 18, 4 5 4 6 low, transport to Golgi complex inhibited, 92 optimum water potential and, 159 osmophilic and halophilic response affected by, 157 water potential relationship, 157 Temperature-conditional mutants, 158 Temperature-sensitive mutants, bet mutants, 96 flocculent yeasts, 18 sec61, sec62, sec63, 80, 81 in secretory pathway elucidation, 75-77 see also individual sec mutants Tethered cells, 290, 315-316 Thermocouple psychrometry, 154 Thermodynamic activity of water (aw), 149 Thermodynamic state, of water, see Water Thermodynamic water equilibrium, 146, 151 Thermoproteus tenax, S-layer growth, 235, 236 S-layer structural organization, 254 Thermoreceptor, serine transducer (Tsr), 301 Thermotolerance, 196, 197 Thraustochytrium aureum, amino acids as compatible solutes, 176 Thraustochytrium roseum, amino acids as compatible solutes, 176 Tip (taxis-involved protein), 300 Tonoplast response, to dehydration, 162 Torulopsis halonitratophila, halophilic response and temperature, 157

Transducers, see Chemotactic signal transducers Transitional vesicles, 74 Transition metal ions, in flocculation, 15 Transmembrane signalling, 31G312, 334 Transport, periplasmic binding proteins in, 298, 299 Transport vesicles, 8 M 9 budding, from endoplasmic reticulum, 89, 91 early SEC gene products in, 95-96 diameter, 95 N-ethylmaleimide-sensitivefactor (NSF) functions, 89 formation, calcium ion fluxes and, 110 fusion and uncoating, 89, 90, 91 early SEC gene products in, 95-96 homogeneous population and true transport intermediates, 94 isolation, 94 see also Protein transport, from endoplasmic reticulum to Golgi complex Trehalose, accumulation, stress treatments inducing, 196 as compatible solute, 175-176 desiccation protection, 195-196 osmotic hypersensitivity and, 194-196 osmotic shock tolerance, 176,194-196 translocation in fungi, 175 Trehalose-6-phosphate synthase, 196 trg gene, strains with multiple copies, 328 Trg protein, 299 cysteine mutagenesis, 311 G151D mutation, 310 interaction with binding proteins, 310 as methyl-accepting chemotaxis protein (MCP), 325 R85H mutation, 310 as secondary chemoreceptor, 301 site-directed mutagenesis, 328 TRKl gene, 184 Trypsin, flocculent cell digestion, 18-19 tse, gene, 300, 314 tsr gene, 300, 325 mutations, 300

41 1

SUBJECT INDEX

Tsr protein, 299 assembly, 300 in CheY-P formation, 320 copies in each cell, 302 periplasmic domain for serine sensing, 304 as primary transducer, 301 as thermoreceptor, 301 b r mutants, 300 Tunicamycin, 54, 57 TUPl gene, 6 1 4 2 Turgor pressure, 153-155 measurement methods, 153 gas vesicles, 155 probe, 1 5 4 155 negative, plasmolysis and, 163 at plasmolysis point, 162 as prerequisite for growth, evidence against, 153-154 regulation, 154 water potential relationship, 154 Turgor-regulating cells, 154 Two-dimensional crystals, application potential, 257-260 see also S-layer

U Ultrafiltration membranes, isoporous, S-layers as, 257-258 Undecaprenol, 250 V Vaccine, development, S-layers in, 259-260 Vacuolar membrane, transport systems in, 185 Vacuoles, 185 decrease in size with cellular dehydration, 162 inorganic ions and solute accumulation, 185 van der Waals’ forces, 14, 24 van’t Hoff relation, see Boyle-van’t Hoff relation Vectors, in S-layer gene cloning, 246,247 Vesicles, see Golgi complex-derived secretory vesicles; Transport vesicles

Vibrio cholerae, chemotaxis and motility, clinical relevance, 279 flagellar sheath, 284 Vibrio parahaemolyticus, motility, 288 Viral lectins, 53 Viral proteins, secretion, 63 Volume-regulating cells, 154 Volumetric elastic modulus ( E ) , 164

W Water, chemical potential of, 148 concentration, 148 pure, concentration, 148 thermodynamic state, 148-155 parameters, 148 units of parameters, 148 water potential, see Water potential Water activity, 146, 149 Water equilibrium, thermodynamic, 146, 151 Water loss, cell-wall elasticity and, 164-165, 166 see also Osmotic response Water permeability coefficient, biological membranes, 163 Water potential, 148, 148-151, 149 cardinal, of growth, 156-161 see also Osmotolerance of cell, 151-155 cell-size changes with changes in, 161-162 as colligative property of solution, 150 components, 151-155 matrix potential term, 149 osmotic potential, 151-153 turgor pressure, 153-155 during heat treatment, 197 of environment, 151 gravitational term, 148-149 growth affected by, 156 high, species unable to grow at, 157, 158 at incipient plasmolysis ( y ~ , , , ~ ~ , , , )165 , low, cell shrinkage, 161 compatible-solute accumulation influencing, 202-204

412

SUBJECT INDEX

Water potential, low (cont) cost of maintenance at, 199-201 energy (ATP) generation at, 198-1 99 energy supplies influencing, 200, 201 glucose-transport systems, 19&199 increased ATP utilization, 200 inorganic ion responses, 182-185 ion transport-accumulation determining, 202 osmotic response, see Osmoregulation; Osmotic response respiration-fermentation affected by, 198 water loss and cell-wall elasticity, 165, 166 in osmotic hypersensitivity, 191, 192 vmax,156158 ymin,159-161 factors determining, 196204 see also Osmotolerance; Water potential, low Wopt, 158-159 of pure water, 149 sensing mechanisms in fungi, 204-205 solute particle rnolality relation, 150, 151, 152 temperature relationship, 157 vacuole size decrease with changes in, 162 Water stress plating hypersensitivity, see Osmotic hypersensitivity Water-osmosis, 146 articles published, 146147 Wine fermentations. 4 Wolinella recta, S-layer in pathogenicity, 252-253 Wolinella succinogenes, flagella, basalbody rings, 285 Wort, high molecular-weight factors, 59 nitrogen content, 58 pH value, 18 proteins, in flocculation, 13, 14 see also Brewing; Flocculation Wreck and check approach, 82, 86, 87

X Xeromyces bisporus, 157, 159 Xerotolerance, see Osmotolerance Xylulose, 179

Y Yarrowia lipolytica, 7SL RNA in, 84-85 YCSS, yeast cell agglutination, 18 Yeast, cell-wall elasticity, 164 cell-wall structure, 43-44 degeneration (flocculation decline), 5 4 flocculation, see Flocculation; Flocculent strains gathering of cells, 3, 39 see also Flocculation non-flocculent strains, see Nonflocculent strains osmoregulation, see Osmoregulation; specific yeasts secreted proteins, 43 secretory pathway, see Secretory pathway, yeast semi-intact cells, 92 surface charge, 14, 26, 44 see also Flocculation viability decrease, low water potential, 193 virus, 63 Yeast gum, 14 YPTl gene, 101 yptl mutants, 102, 110 Yfllp, calcium-ion function and, 102, 110 function, 101, 102, 110 stimulation of secretory pathway, 102 SEC4p homology, 133 sequence and structure, 134 2

Zoophthora radicans, osmotic potential, 152 Zwitterionic water molecules, 27 Zygosaccharomyces rouxii, arabinitol production, pathway, 179

SUBJECf' INDEX

glycerol production, 187, 203, 204 minimum water potential, pH affecting, 161 Saccharomyces cerevisiae comparison, 203 osmophilic mutant, 158 osmosensitive mutants, 203 osmotolerance and water potential, 158, 203 polyol content, 169, 203

41 3

glycerol as major solute, 169, 171, 187-1 88 regulation of, 187-188 solute-specific increase in arabinitol, 171, 179, 188 polyol metabolism, 177 reduced water loss on sudden osmotic dehydration, 165 viability decrease with low water potential, 193

This Page Intentionally Left Blank