Advances in Microbial Physiology Volume 15

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Advances in

MICROBIAL PHYS IOLO GY

This Page Intentionally Left Blank

Advances in

MICROBIAL PHYSIOLOGY edited by

A. H. ROSE School of Biological Sciences Bath University England

D. W. TEMPEST Laboratorium voor Microbiologie Universiteit van Amsterdam Amterdam-C Th-eNetherlands

Volume 15

1977

ACADEMIC PRESS London New York San Francisco A Subsidiary of Harcourt Brace Jouanouich, Publishers

ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road London NWI United States Edition published by ACADEMIC PRESS INC. 111 Fifth Avenue New York, New York 10003

Copyright 0 1 9 7 7 by ACADEMIC PRESS INC. (LONDON) LTD.

All Rights Reserved N o part of this book may be reproduced in any form by photostat, microfilm, or any

other means, without written permission from the publishers

Library ofCongress Catalog Card Number: 67-19850 ISBN: 0-12-027 7 15-8

Printed in Great Britain by William Clowes and Sons Limited London, Colchester and Beccles

Contributors to Volume 15 D. E. ATKINSON, Molecular Institute and Biochemistry Division, Department of Chemistty, University of California, Los Angeles, California 90024, U.S.A. A. T . BULL, Biological Laboratory, University ofKent, Canterbury C T 2 7NJ, England. (Present address :Department of Applied Biology, University of Wales Institute ofStience and Technology, C a r d g CFI 3 N U , Wales) A. G. CHAPMAN, Molecular Biology Institute and Biochemistry Division, Department of Chemistry, University of California, Los Angeles, California 90024, U.S.A. M. CRANDALL, School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506, U.S.A. I . E. D. DUNDAS, Institutt for Generell Mikrobiologi, Universitetet i Bergen, Bergen, Nonuay R. EGEL, Institut fur Biologie 111 der Universitat Freiburg, 0 - 7 8 0 0 Freiburg, Schanzlestrasse +I I ,Federal Republic of Germuny C. G. ELLIOTT, Botany Department, University of Glasgow, Glasgow, Scotland W. N. KONINGS, De artment ofMicrobiology, Biolo ‘cal Centre, University of Groningen, Kerk Lan 30, Haren, The Netherla s V. L. MACKAY, Waksman Institute of Microbiology, Rutgers University, New Brunswick, NewJersey 08903, U.S.A. A. P. J . TRINCI, Department of Microbiology, Queen Elizabeth College, University of London, London W8 7 A H England

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Contents The Physiology and Metabolic Control of Fungal Growth

. .

. . .

A T BULL and A P J TRlNCl

I . Introduction . . . . . . . . . . . . . . . . .‘ I1. Mathematical Modelling of Fungal Growth . . . . . . . . . A. WhyModel? . . . . . . . . . . . . . . . . B . Approaches to Modelling . . . . . . . . . . . . C. The Applicability of Classic Models to Fungal Growth . . . . . I11. Growth of Undifferentiated Mycelia . . . . . . . . . . . A. Regulation of Mycelial Form . . . . . . . . . . . . B . Polarization of Hyphal Growth . . . . . . . . . . . C . Regulation of Branch Initiation . . . . . . . . . . . D. Hyphal Growth Units of Different Strains and Species . . . . . E. Effect of Environment on Hyphal Growth Unit Length . . . . F. RegulationoftheSpacialDistributionofHyphae . . . . . . IV. Colony Growth . . . . . . . . . . . . . . . . . A. Colony Differentiation . . . . . . . . . . . . . B . Mould-Induced Changes in the Substrate . . . . . . . . C. Kinetics of Colony Expansion on Solid Media . . . . . . . D. Colony Expansion as a Parameter of Mould Growth . . . . . E. Comparison of the Colonization of Solid Substrates by Moulds and Unicellular Micro-organisms . . . . . . . . . . V . Fungal Growth in Submerged Liquid Culture.Technical Considerations VI . Kinetics of Fungal Growth in Submerged Liquid Culture . . . . . A. RatesofGrowth . . ,. . . . . . . . . . . . . B . Transient States and Oscillatory Phenomena . . . . . . . C. “Macroregulation” of Growth . . . . . . . . . . . D . Maintained and Starved States . . . . . . . . . . . VII . Transport Controlled Features of Growth . . . . . . . . . A . Transport-Limited Growth . . . . . . . . . . . . B . Transport Regulation . . . . . . . . . . . . . . C. Modulation of Fungal Transport Processes . . . . . . . . VIII . Metabolic Control in Fungi . . . . . . . . . . . . . A. Intermediary Metabolism . . . . . . . . . . . . B . Anaplerotic Metabolism . . . . . . . . . . . . . C. Aspects ofTermina1 Oxidation . . . . . . . . . . . IX. RNA Synthesis and Function: Rate-Limiting Parameters of Growth . . A . Efficiency of Protein Synthesis . . . . . . . . . . . B . Concerning Polyamines . . . . . . . . . . . . . X. Acknowledgements . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . vii

2 3 3 4 7 13 14 14 15 19 20 23 23 23 21 29 33

35 36 40 40 43 46 41 48 48 50 53 51 57 62 61 71 12 14 16 16

viii

CONTENTS

Physiology of Halobacteriaceae

. . .

1 E D DUNDAS

I . Introduction . . . . . . . . . . . . . . . . . I1. Classification of Extreme Halophiles . . . . . . . . . . I11. Intracellular and Extracellular Salt Concentrations . . . . . . A. Intracellular Salts . . . . . . . . . . . . . . . B . Extracellular Salts . . . . . . . . . . . . . . IV. Subcellular Structures . . . . . . . . . . . . . . A. Cell Envelopes . . . . . . . . . . . . . . . B . Ribosomes . . . . . . . . . . . . . . . . C. Vacuoles . . . . . . . . . . . . . . . . . D . Flagella . . . . . . . . . . . . . . . . . V. Halophilic Proteins . . . . . . . . . . . . . . . A. Metabolic Pathways . . . . . . . . . . . . . . B . Halophilic Enzymes . . . . . . . . . . . . . . VI . Lipids in Halobacteriaceae . . . . . . . . . . . . . VII . Electron-Transport Chain . . . . . . . . . . . . . . VIII . Transport Across Membranes . . . . . . . . . . . . IX. Effects of Light . . . . . . . . . . . . . . . . A . Photophosphorylation . . . . . . . . . . . . . B . Effect on Growth and Viability . . . . . . . . . . . X. Nucleic Acids and Their Enzymology . . . . . . . . . . XI . Phage-Host Relationships . . . . . . . . . . . . . XI1. Ecological Considerations on the Existence of Obligate Extreme Halophilism . . . . . . . . . . . . . . . . . XI11. Acknowledgements . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .

85 86 88 88 90 91 91 94 96 99 100 101 102 104 106 107 109 109 110 111 113 114 116 116

Sterols in Fungi: Their Functions in Growth and Reproduction CHARLES G . ELLIOT I. I1. I11. IV. V.

Introduction . . . . . . . . . . . . . . . . . Functions of Sterols: Possible Approaches to the Problems . . . . Sterols in Model Systems . . . . . . . . . . . . . . Subcellular Distribution of Sterols in Fungi. and States of Binding . . Effects of Sterols on Metabolism and Vegetative Growth . . . . . A. e t h i u m and Phytophthora . . . . . . . . . . . . . B . Saccharomyces and Other Fungi . . . . . . . . . . . VI . Effects of Sterols on Asexual Reproduction . . . . . . . . . VII . Sexual Hormones of Achlya . . . . . . . . . . . . . VIII . Effects of Sterols on Sexual Reproduction in Homothallic Species of Pythium and Phytophthora . . . . . . . . . . . . .

121 123 130 135 141 141 144 148 149 152

CONTENTS

ix

IX. Reproduction in Heterothallic Species of Pythium and Phytophthm

. . . . . .

X. Sterols and Sexual Reproduction in Ascomycetes and Basidiomycetes XI . Conclusion . . . . . . . . . . . . . . . . XI1. Acknowledgements . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

156 162 165 166 166

Active Transport of Solutes in Bacterial Membrane Vesicles

.

WIL N KONINGS

I . Introduction . . . . . . . . . . . . . . . . . I1. Membrane Vesicles . . . . . . . . . . . . . . . A. Isolation Procedures . . . . . . . . . . . . . . B . Physical Properties . . . . . . . . . . . . . . C. Purity of Membrane Preparations . . . . . . . . . D. Functional Properties . . . . . . . . . . . . . . E . Orientation of the Vesicle Membrane . . . . . . . . . F. Localization of D-Lactate Dehydrogenase in Membrane Vesicles from Escherichiu coli . . . . . . . . . . . . . . . . I11. Active Transport Coupled to Electron Transfer Systems . . . . . . A . Coupling to Respiratory Chain . . . . . . . . . . . B . Couplingto AnaerobicElectronTransfer Systems . . . . . . C . Coupling toCyclic ElectronTransferSystems . . . . . . . IV. Energy Coupling to Active Transport . . . . . . . . . . . A. Role ofAdenosine 5'-Triphosphate and the ATPase Complex . . . B . Mechanism of Energy Coupling . . . . . . . . . . . C. Energy-Dependent Binding of Solute to Carrier Proteins . . . . V. Conclusions . . . . . . . . . . . . . . . . . . VI . Acknowledgements . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

175 177 177 180 183 184 194 200 203 203 217 223 225 225 228 239 243 244 244

Adenine Nucleotide Concentrations and Turnover Rates. Their Correlation with Biological Activity in Bacteria and Yeast

.

.

ASTRID G CHAPMAN and DANIEL E ATKINSON

I . Introduction . . . . . . . . . . . . . . . . . I1. Concentrations and Fluxes ofAdenine Nucleotides in uiuo . . . . . A. Adenine Nucleotide Turnover . . . . . . . . . . . . B . Turnover ofATP . . . . . . . . . . . . . . . C. RegulationofATP Utilizationand Regeneration . . . . . . . D . Sampling of Microbial Cultures for Adenine Nucleotide Determinations E. Changes inAdenineNucleotide Concentrations . . . . . . .

254 256 256 261 268 269 272

X

CONTENTS

I11. Concentration of ATP. Total Adenine Nucleotide Concentration. and . . . . . . . Energy Chargein Relation to Cellular Activities A . Relation between ATP Concentration and Growth Rate . . . . . B . Variations in Adenine Nucleotide Levels during Growth . . . . C. Adenine Nucleotides in Mutant Strains Arrested in Growth . . . . D . Correlation between Kinetics in vitro and Observations in vivo . . . E. Relation between Energy Charge and Total Adenine Nucleotide Concentration . . . . . . . . . . . . . . . . F. Phage Infection . . . . . . . . . . . . . . . . G . Other Nucleotides . . . . . . . . . . . . . . . H . RNASynthesis . . . . . . . . . . . . . . . . I . Protein Synthesis . . . . . . . . . . . . . . . IV. General Discussion . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .

282 282 285 286 287 289 290 291 293 295 297 300

Physiology of Mating in Three Yeasts

.

.

MARJORIE CRANDALL RICHARD EGEL and VIVIAN L MACKAY

I . Introduction . . . . . . . . . . A. Ecology . . . . . . . . . . . . . . . . . B . General Characteristics C . Lifecycles . . . . . . . . . . I1 . Hamenula Winget . . . . . . . . . A . Mating Type Locus . . . . . . . . B . Haploid Functions . . . . . . . . C. DiploidFunctions . . . . . . . . 111. Schizaracchmomycespobe . . . . . . . A . Mating Type Locus . . . . . . . . B . HaploidFunctions . . . . . . . . C . DiploidFunctions . . . . . . . . IV . Saccharomyces cerevisiae . . . . . . . . A . Mating Type Locus . . . . . . . . B . Haploid Functions . . . . . . . C. DiploidFunctions . . . . . . . . V. Comparative Discussion . . . . . . . A. Steps in Yeast Conjugation Compared . . B . Evolutionary Aspects of Sexual Reproduction C. Comparison with Mammalian Systems . . VI . Acknowledgements . . . . . . . . References . . . . . . . . . . . Author Index . . . . . . . . . . . Subject Index . . . . . . . . . . .

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

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

307 309 310 310 313 313 314 327 331 331 336 347 350 350 354 377 384 384 390 391 392 392 399 419

The Physiology and Metabolic Control of Fungal Growth A. T. BULL* and A. P. J. TRlNCl Biological Laboratory, University of Kent, Canterbury CT2 7NJ, England; Department of Microbiology, Queen Elizabeth College, University of London, London W8 7AH. I. Introduction

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.

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.

11. Mathematical Modelling of Fungal Growth

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. . .

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. . .

.

. . .

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. .

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A. Why Model? . . . . . . B. Approaches to Modelling . . . . C. The Applicability of Classic Models to Fungal Growth . 111. Growth of Undifferentiated Mycelia . . . . . A. Regulation of Mycelial Form . . . . . . B. Polarization of Hyphal Growth . . . . . . . C. Regulation of Branch Initiation . . . . . . . D. Hyphal Growth Units of Different Strains and Species . . E. Effect of Environment on Hyphal Growth Unit Length . . F. Regulation of the Spacial Distribution of H p h a e . . . 1V. Colony Growth . . . . . . . . . . . A. Colony Differentiation . . . . . . . . . B. Mould-Induced Changes in the Substrate . . . . . C. Kinetics of Colony Expansion on Solid Media . . . D. Colony Expansion as a Parameter of Mould Growth . . E. Comparison of the Colonization of Solid Substrates by Moulds and Unicellular Micro-organisms . . . . . . . V. Fungal Growth in Submerged Liquid Culture. Technical Considerations VI. Kinetics o f Fungal Growth in Submerged Liquid Culture . . A. Rates of Growth . . . . . . . . . . B. Trmisient States and Oscillatory Phenomena . . . . C. "Macroregulatioti" of Growth . . . . . . . D. Maintained arid Starved States . . . . . . . VII. Trailsport Controlled Features of Growth . . . . . A. Traiisport-Liiiiited Growth . . . . . . . . B. Transport Regulation . . . . . . . . . C. Modulatioii of Fungal Transport Processes . . . .

* Present address: Department ol'Applied Biology. Uiiivcrsity ol'W i i h Technology, Cardill; CFI 3NU 1

Illbtittitc

2 3 3 4 7 13 14 14

15 19 20 23 23 23 21 29 33

35 36 40 40 43 46 47 4X *X 50

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A. T. BULL AND A. P. J. TRlNCl

VIII. Metabolic Control in Fungi . . . . . . . * A. Intermediary Metabolism . . . . . . . B. Anaplerotic Metabolism . . . . . C. Aspects of Terminal Oxidation . . . . . . . IX. RNA Synthesis and Function: Rate-Limiting Parameters of Growth A. Efficiency of Protein Synthesis . . . . . . B. Concerning Polyamines . . . . . . . . X. Acknowledgements . . . . . . . . . . References . . . . . . . . . . . .

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57 57 62 67 71 12 74 76 76

I. Introduction “The modern era of mould metabolism has only scarcely begun, but signs of an immense advance in the numerous phases of the field are unmistakable. Their importance is recognized, practically and academically’’.Thus commented Jackson Foster in the introduction of his unique contribution to the study of fungal physiology and biochemistry, “Chemical Activities of Fungi” (Foster, 1949). A vast mycological literature had accumulated by the time of Foster’s original persuasion, in 1936, to prepare a critical account of fungal metabolism and, it is worth recalling, several aspects of this subject had already been established during the previous century. Pre-Foster fungal physiology had relied extensively on a classical response-to-stimulus type of approach, an approach unfortunately that generated much conflicting and confusing data. It was Foster’s conspicuous talent and understanding that brought together much of this data in an intelligible form and was instrumental in orientating subsequent researches. I t is appropriate, therefore, by way of a preface to our main discussions, to examine the current status of fungal biochemistry. We would advocate, from an admittedly biased position, that fungi unquestionably are the organisms of choice for the study of numerous aspects of microbial biochemistry: one needs only to ponder their extreme morphological diversity and plasticity, their unrivalled biosynthetic capacities, especially with respect to secondary metabolites, and their utility as model systems in the biochemical analysis of mating behaviour, differentiation, ageing and so on, to find the validity of this assertion. And yet our knowledge of fungal biochemistry remains rather fragmentary, while specific reference to investigation of growth physiology shows that the majority of work has been done with bacteria. Two important reasons for this situation can be proffered that have their bases in: (a) long-standing technical difficulties of growing mycelial fungi; and (b) lack of adequate kinetic analyses of

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

3

their mode of growth. Accordingly, we have devoted a portion of this review to the practicalities of growing filamentous fungi, and have developed at greater length mathematical treatments of mould growth and discussed the applicability of some existent models. We have singled out the growth of surface colonies for particular discussion because, despite the fact that it is a technique used by mycologists ad libitum,it has received little critical analysis; moreover, it has been the subject recently of renewed interest. Strain instability also has plagued the efforts of the fungal physiologist, and this has been an especially acute problem in relation to the Fungi Imperfecti many of which are of major interest in the context of commercial fermentations. Problems of strain selection and strain degeneration can seriously hamper prolonged continuous-flow culture experiments with moulds and care has to be taken to check biological stability under these conditions. The scope of this article is such that we have, of necessity, been selective in our choice of topics to discuss. The selection has been resolved in two ways: by taking those areas of fungal physiology and biochemistry that have been researched in depth, and by attempting to indicate areas that are as yet largely unexplored but seem to us to warrant special attention. In addition we have tried to keep as a theme running throughout the discussion, the modulation of fungal metabolism in response to growth conditions. Palpable absences from this article are references to “secondary” or “shunt” metabolism, at which fungi are most adept, and to the metabolic control of differentiation: in both of these areas, notable advances have been made in recent years (see Smith and Anderson, 1973). But one of our prime objectives in the following pages is to demonstrate to the microbial physiologist and biochemist that the fungi arguably are among the most propitious and alluring of all micro-organisms that he can select for his studies and that von Haller’s eighteenth century portrayal of them as “a mutable and treacherous tribe” is no longer the most apposite of epithets. 11. Mathematical Modelling of Fungal Growth A.

WHY MODEL?

Any mathematical modcl of growth attempts to specify interrelationships between the many components of the system, physical, chemical and biological and, clearly, such components must be

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A. T. BULL AND A. P. J. TRlNCl

capable of being quantified. The system then is usually described for convenience by a series of differential equations which, depending on the complexity of the-model, may be solved manually or by the aid of computing techniques. A model is likely to be of practical value only if it is mathematically tractable and can be analysed to predict responses to defined environmental conditions, and if it provides a reasonable fit of the experimental data. Topiwala (1973) recently has cautioned that apparently successful models do not conclusively validate basic assumptions made in the model because alternative models may produce similar conclusions. Nevertheless, if a given theory of growth cannot be modelled satisfactorily it is unlikely to be a valid one. What then are the purposes of modelling? First, in formulating models, all descriptions and definitions need to be rigorous, free of ambiguity and be capable of being expressed in mathematical terms. Thus, the microbiologist is obliged to think precisely and systematically about the growth system under study, and for which experimental data can be obtained. Second, models have a conceptual function in helping to focus attention on, and in revealing, fundamental properties of the system. Consequently models enable predictions to be made of the behaviour of the biological system under a limitless range of conditions which may not have been investigated in the laboratory. Indeed computer simulation studies now provide important strategic approaches in both research and process microbiology. An instructive example in the fungal field is provided for the griseofulvin fermentation by Calam et al. ( 197 1). Finally, following on from the latter point, modelling can be a considerable guide in the design of experiments and in the interpretation of experimental data. B . APPROACHES TO MODELLING

Numerous types of mathematical models have been proposed to describe microbial growth and behaviour, and these reflect the particular approaches and objectives prescribed by the modeller. The majority of this research, reported in the chemical-engineering, biochemical and sanitary literature, is frequently overlooked by the microbial physiologist and we feel that it is pertinent to include a brief resume of the main criteria considered in model construction. Although the microbial physiologist most often is concerned with population dynamics, any population of micro-organisms comprises

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

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individuals whose size, composition and metabolism is distributed over a definable range that itself is dependent on the growth conditions. Thus, models that recognize the differing physiological states of individuals in a population are termed segregated. However, it is more usual to neglect the variations between individuals (thus simplifying the mathematics and avoiding statistical correlations) and instead to treat population dynamics in terms of variations in average properties; models of the latter type are called unsegregated (distributed or non-segregated). In passing we should note that, in models based on a distribution of individuals of different physiological states, the distribution per se may be dependent on the existent environment. These models are referred to as being structured. Again, however, most of the models with which a microbiologist is familiar disregard population structure and more simple unstructured modelling terms of reference are adopted. Unsegregated models also may be endowed with structure if composition changes of the population during the course of cultivation are admitted. For example, biochemical structure was incorporated into an unsegregated growth model by Ramkrishna el al. ( 1966) who divided microbial biomass into G mass (nucleic acids) and D mass (proteins) components. Because the growth rate of individual micro-organisms cannot be predicted with complete certainty, stochastic population, or probabilistic, models have been formulated that take into account the variability of generation times. Once more, due to the “formidable mathematical difficulties” that arise “when one attempts to model only very simple biological phenomena” (Frederickson et al., 19701, stochastic models are usually disregarded in favour of simpler deterministic models. In summary, therefore, most of the commonly used mathematical models of microbial growth are unsegregated, unstructured and deterministic, assumptions that are largely valid when population sizes are large, i.e. the kinetics of microbial growth can be developed on the lines of established chemical reaction kinetics. The classic example of a model of this type is that proposed by Monod ( 1942, 1949) and having its origins in the much neglected researches of M’Kendrick and Pai (191 1). The latter showed: (1) that exponential growth proceeded in batch cultures as long as the “nutriment” supply was unlimited; (2) that the rate of growth was proportional to the number of organisms present (i.e. that growth was autocatalytic); and (3) that they were clearly appreciative of such growth co-efficients as yield and

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A. T. BULL AND A.

P. J. TRlNCl

maintenance. Monod related the specific growth rate (p) to the concentration of growth limiting substrate (s) thus: p =pcmax SI(K,+ S), where pmax is the maximum value of p under a given set of growth conditions and K, is the substrate saturation constant ( K , = S at p =pmax/2). Although the Monod expression is formally analogous to the Michaelis-Menten equation relating enzyme reaction velocity to substrate concentration, there are important distinctions between the two models. Thus, the rate constant, p, in the Monod equation is a logarithmic function of S, whereas v in the Michaelis-Menten equation is related linearly to S. Further, whereas the Michaelis-Menten constant, K,, has a mechanistic basis, K , is an empirical constant. From its first proposition, the Monod model was recognized as an oversimplification but it should be remembered that its original objective was in the curve-fitting of experimental data. Two other unsegregated growth models, less widely used than that of Monod, will be mentioned briefly. The first of these, the logistic equation, includes a term to describe the decrease in growth rate as the limiting substrate becomes exhausted and predicts a maximum population or stationary growth phase in batch cultures. The logistic equacion can be variously expressed (cf. Tsuchiya et al. 1966; Hockenhull and MacKenzie, 1968; Maynard-Smith, 1968) one form of which is: x=

xOePt

l-px.(l - P )

where x and xo are microbial biomass concentrations at time t and o, and p is a constant which may be written: = Y(So+ YxJ where Y is the yield factor with respect to the growth-limiting substrate, concentration S,at zero time. Finally we wish to draw attention to a threeconstant growth model (Dabes et al., 1973) which embodies Blackman kinetics (i.e. the concept of a rate limiting step in a biological process). Dabes and his colleagues considered the situation in which two slow enzymic steps were separated by fast reactions, the overall equilibrium constant of which is not large, and their model, unlike the Monod and logistic equations, contain three constants :

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

7

B is the growth-limiting substrate saturation constant, and A is a constant incorporating overall and rate-limiting equilibrium constants. This model has the potential of indicating the proximity of ratelimiting reactions to each other from the magnitude of B relative to Ap,,,; as B approaches zero, the equilibrium constant between the reactions increases. In the following section we examine the applicability of existing models, particularly those referred to above, to describe growth of filamentous fungi. In short, how adequate for fungi are models developed to express the growth of unicellular micro-organisms? C . THE A P P L I C A B I L I T Y O F C L A S S I C MODELS T O FUNGAL GROWTH

At the outset, the extreme morphological plasticity of fungi must be recognized. In liquid culture, a particular species may grow as a diffuse mycelium, in the form of variously sized pellets or other aggregations or, quite frequently, may develop a yeast-like morphology. Diffuse mycelial and pellet growth will be considered here while the growth of surface colonies of fungi will be examined in detail in subsequent parts of this article (p. 29). 1. Distribution $Metabolic Activities in Mycelial Systems

I f we wish to approach fungal growth in terms of unsegregated, unstructural models, an immediate question arises : are metabolic activities distributed evenly among the hyphal cells of a multicellular mycelium? In other words, is the mycelium differentiated biochemically? We will assume for the present that significant morphological differentiation does not occur during unrestricted growth in batch cultures or in steady-state continuous-flow cultures. Unfortunately there have been very few studies of the distribution of activities along fungal hyphae. Fencl et al. (1969) used micro-autoradiography to analyse the distribution of RNA synthesis in Aspergillis niger mycelia growing in batch and multi-stage chemostat cultures. Synthesis of RNA was distributed evenly throughout the mycelium as long as batch cultures were growing exponentially, but differential synthesis occurred in stationary-phase mycelia. This fungus showed diauxic growth on a sucrose-nitrate medium, and unequal RNA and protein synthesis was evident in the mycelia of the second exponential phase (Machek and Fencl, 1973). However, when mycelia from either

8

A. T. BULL AND A. P. J. TRlNCl

the stationary or the second exponential phase were given a nutritional shift-up, the distribution of activity reverted to that typical of the first exponential phase. The results from multi-stage chemostat experiments substantiated these findings. A three-stage chemostat culture was established in which: (1) the dilution rate ( D )in the first stage was 0.04 h-’; (2) fresh medium was fed to the third stage to make D equal to 0.17 h-l; and (3) additions were not made to the second stage, i.e. conditions which were considered to resemble those at the onset of the stationary phase in a batch culture. In all three vessels the mycelium behaved as a homogenous entity, all hyphal cells synthesizing RNA even if at a very low rate as in the case of the stage 2 population. Data from Terui’s laboratory strongly support the findings of the Czech workers. Shinmyo and Terui ( 1970)observed uniform incorporation of 14C-adenineand I4C-guanosine into “pulpy” (i.e. diffuse) mycelia of A . niger growing as hanging drop microcultures, and also noticed that all hyphal cells had a uniform growth potential, i.e. longitudinal growth of apices or formation of branches in subapical cells. Similar observations to the latter have been made on chemostat cultures of A . nidulans (M. E. Bushell and A. T. Bull, unpublished experiments). On the basis of these analyses, therefore, it seems justifiable to treat rate relationships in moulds in terms of the average kinetics discussed above (Section 11, B; p. 4). I t must be stressed that our last conclusion applies on4 to liquid cultures of diffusely growing mycelia. Shinmyo and Terui (1970) pointed out that the distribution of growth activities in the hyphal cells of pellets was highly heterogeneous. Much earlier Camici et al. (1952) had reported that the centres of dense pellets of Penicillium chrysogenum contained mycelium that was frequently dead or autolysing, observations later confirmed in other species by Yanagita and Kogane ( 1963a). Indeed, the latter authors showed that extensive morphological differentiations occurred in mycelia adjacent to the central spaces in pellets while, at a metabolic level, RNA synthesis was strictly limited to the peripheral hyphae of the pellet. Thus, although pelleted growth may be desirable for the formation of certain fungal products, for example ergot alkaloids (Tonolo et al., 1961), citric acid (Clark, 1962) and certain enzymes (a-galactosidase; Kobayashi and Suzuki, 19721, their highly differentiated nature makes them totally unsuitable for most metabolic studies. In passing we should note that, like pellets, surface colonies of fungi

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

9

become differentiated metabolically, and usually morphologically, with increasing age and this process is clearly evident from changing enzyme patterns. Isaac (1964) described a clear differentiation of Rhizoctoniu soluni colonies with respect to the distribution of cellulase

activity; enzyme production was associated with short branched hyphae in old parts of the colony but not with young hyphae produced apically or by subapical branching. A study of phosphorus metabolizing enzymes in Aspergillus niger by Nagasaki ( 1968) illustrates a similar age-dependent differentiation in surface colonies. More recently Skowronski and Gottlieb ( 1970)analysed metabolic changes that occur in the peripheral (actively growing) hyphae from young and old colonies of R. soluni. They made the interesting conclusion that factors responsible for growth limitation in old colonies probably were located in the peripheral hyphae, such hyphae from old colonies having greatly diminished rates of respiration and protein synthesis. Moreover, the deficient protein synthesis appeared to be due to inhibitory factors in the soluble fraction of the hyphae; ribosomes from these hyphae retained the capacity to synthesize protein at close to maximum rates. The plugging of septa1 pores and the consequent prevention of translocation is a further manifestation of differentiation in a fungal colony (Trinci and Collinge, 1974b). We will return to this subject in Section 1V.A (p. 23).

2. Some Examples of Fungal Growth Modelling Only quite recently has it become widely appreciated that filamentous fungi possess the ability to grow exponentially (6.Mandels, 1965), and the perpetuation of the contrary view seems to rest on the fact that these organisms grow by linear apical extension of their hyphae. However, exponential increase in total mycelial length or mass does occur by the generation of new hyphal apices at a rate proportional to the total mycelial length; this condition is realized either by branch formation or by hyphal fragmentation by shearing forces in a stirred culture. In passing it may be noted that very little quantitative data have been published on hyphal fragmentation in stirred fermenters, and information on hyphal-length distribution at different shearing rates is lacking. Recently, however, Japanese workers have made a valuable contribution to the analysis of mycelial strength and have provided a comparative standard against which to assess the intensity of shearing

10

A. T. BULL AND A. P. J. TRlNCl

shock (Tanaka, Takahashi and Ueda, 1975; Tanaka, Mizuguchi and Ueda, 1975). In practice, the Monod model can produce good approximations of fungal gowth, and this is illustrated in Figure l a with reference to the batch cultivation of Geotrichum lactis in a defined glucose-nitrate medium. The departure of the observed data from that predicted is highly suggestive of oxygen limitation occurring when the biomass concentration exceeds about 2 g 1-I. The model also demonstrates the relative unimportance of K , as a determinant of growth under conditions of substrate excess. We noted earlier that the Monod model does not predict a decreasing growth rate as the substrate becomes limiting, and even when the value for K, is very small (Fig. la) a significant deceleration phase may be evident. Consequently, the logistic law, which embodies the principle of a maximum population, may be a more appropriate model to adopt. Constantinides et al. (1970) found that growth of Penicillium was very closely predicted by the logistic equation. Similarly, growth of wild-type Aspergzllus nidulans is fitted much more closely by the logistic than by the Monod equation (Fig. lb). The significant divergence of the Monod plot and the experimental data may be explicable in terms of an over-estimated rate of substrate utilization, or of a substantial channelling of carbon into extracellular product(s). The extensive synthesis of extracellular melanin that begins towards the end of exponential growth (Carter and Bull, 1969; Rowley and Pirt, 1972) is an argument in support of the latter hypothesis. Figure l a also demonstrates the utility of the logistic model; whereas the Monod equation accurately predicts exponential growth, the logistic equation in addition provides an acceptably accurate modelling of the decelerating growth rate phase. The pronounced morphological, as well as biochemical, differentiation which moulds may display necessitates models additional to those discussed so far. Thus, Emerson (1955) argued, on quite reasonable grounds, that the growth rate of a filamentous fungus should be somewhat less than exponential and he predicted that growth would follow a cube root law. Emerson’s own observations with Neurosporu cratsa sustained such a cube root growth model, and other workers have confirmed this fact subsequently. The ensuing confusion over exponential or cube-root expressions of fungal growth has persisted in the literature for many years and was only conclusively resolved by Pirt ( 1966). Pirt deduced that cube root growth was a characteristic of fungi growing as pellets, rather than as diffuse mycelia, and he argued that

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

I

I

I

5

10

I

15 20 Time ( h )

1

I

25

30

11

(a)

Time(h)

(b) FIG. 1. (a).Time-course of growth of a batch culture of Geotrkhum cundidum, from the data of Trinci (1971). Observed values for biomass production are indicated by . Computer simulations based on the Monod model for &value of 9 mg I-' are indicated by . . . ., and for a &value of 24 mg I-' by - - - -,and for the Logistic model by -. Computor simulations (Bushel1 et al., 1976) incorporate the growth constants of Fiddy and Trinci (1975). (b). Time-course of growth of a batch culture of Aspergillus nidulans 224 based on the data of Carter and Bull ( 1969). Observed values for biomass , and the concentration of glucose in the culture by A . production are indicated by . Predictions from the Monod model are indicated by - and from the Logistic model by - - - - (Bushel et ul., 1976).

12

A. T. BULL AND A. P. J. TRlNCl

once a pellet exceeded a critical size growth was restricted to a peripheral zone by constraints on substrate diffusion. Trinci ( 1970), and more recently Huang and Bungay ( 1973), have supplied convincing experimental proof of Pirt’s model and have provided quantitative data on the dimensions of the peripheral growth zone. To our knowledge, Blackman kinetics have not been used to model mould growth, but in their paper Dabes et al. (1973) have made some pertinent observations on yeast respiration and growth. Commenting on the data of Terui and Sugimoto (19691, Dabes and his colleagues argued that, while it was the availability of electrons that determined the maximum rate of respiration, the apparent &value for oxygen “is set by the cytochrome system close to the point of oxygen utilization”. The fit of respiration rates by the three-constant model is claimed to be good support for the idea of two widely separated rate-limiting steps in yeast respiration. These preliminary analyses recommend that more attention should be given to Blackman-type kinetics by the microbial physiologist. All of the models of fungal growth referred to above neglect mycelial differentiation or the effects of cell age. An attempt to model growth of Aspergillus awamori based on the existence of discrete states of mycelial differentiation has been made by Megee et al. (1970). Although they succeeded in modelling many features of mould fermentations on the basis of age-dependent parameters, the differentiation states incorporated into the model were defined with reference to surface colonies, and such an extrapolation may be unwarranted. The concept of a mean cumulative age” introduced by Aiba and Hara (1965) is also relevant in the context of age-related phenomena in filamentous fungi. The mean cumulative age, defined as the cumulative age of all mother and daughter cells of a particular mycelial system divided by the total number of cells in that system, provides a common time scale for comparing batch and continuous-flow cultures. Aiba and Hara (1965) illustrated their hypothesis by reference to the penicillin fermentation, and concluded that mean cumulative age analysis could offer scope for designing continuous processes from observations made on batch culture%. In coklusion, it is essential to recognize that mathematical description of microbial growth is a continuing quest and that refinement of models must be limited by our ability to define physiological changes in precise mathematical terms. Fungal physiologists, and mycologists 66

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

13

in general, usually find the rigorous demands of growth-modelling discouraging and also, perhaps, have been too disposed to seek simple mathematical expressions in the hope of deriving a universal quantitative description of batch growth. Dawson and Phillips (1974) have argued that most growth models have limited utility “if only because simple models have extremely restricted applications and complex models are self-defeating’’ in the sense that critical data needed to test their validity are lacking. At this stage, therefore, the mycologist might adopt mathematical modelling most advantageously as a conceptual aid in the problem of understanding fungal growth. 111. Growth

of Undifferentiated Mycelia

A distinction may be made between differentiated and undifferentiated mycelia (Steele and Trinci, 1975). A fungal spore germinates upon a solid medium to form an undifferentiated mycelium which increases in size and differentiates into a “mature” colony. The differences which distinguish the hyphae of undifferentiated mycelia from those at the margin of mature colonies are listed in Table 1. Most studies of the growth and cytology of moulds have been made upon hyphae at the margin of “mature” colonies. However, the basic features of fungal growth are more likely to be displayed by the hyphae of unTABLE 1. Comparison between the characteristics of hyphae of differentiated and undifferentiated rnycelia Undifferentiated mycelia Hyphae formed during exponential growth on solid media or submerged culture 1. The hyphae of a single mycelium have more or less the same diameter 2. Each hypha has the same maximum extension rate (Emax)

3. Hyphae have relatively short extension zones 4. Hyphae do not usually branch subapically

Differentiated mycelia Hyphae formed at the margin of colonies on solid media The hyphae are usually differentiated into wide “leading” hyphae and narrower branch hyphae “Leading” hyphae have faster rnaximum extension rates than primary and secondary branches “Leading” hyphae have relatively long extension zones “Leading” hyphae often branch subapically

14

A. T. BULL AND A. P. J. TRlNCl

differentiated mycelia since the mechanisms involved in regulating mycelial form may be obscured by the differentiation process involved in colony formation. A.

R E G U L A T I O N O F MYCELIAL FORM

The thallus of a mould, unlike that of a unicellular micro-organism, is well adapted to colonize solid substrates such as plant surfaces, soil

and solidified culture media. An advantage of the filamentous form is that the organism can increase in size indefinitely without altering the ratio between protoplasmic volume and surface area. Thus, exchange of substances between the mycelium and the medium involves transport over only short distances. The fact that hyphae branch at more or less regular intervals ensures that solid substrates are effectively and efficiently covered by the mycelium. At least three mechanisms must be involved in regulating the formation of undifferentiated mycelia: ( l ) Regulation ofhyphal polarity. Hyphal growth is polarized, i.e. extension is confined to the hyphal tip. (2) Regulation of branch initiation. Germ tubes increase in length to form “main” hyphae from which primary branches are produced. In their turn, the primary branches give rise to secondary branches and so on. The predictable form of a mycelium indicates that there is a mechanism which regulates the frequency of branch initiation. (3) Regulation ofthe spatial distribution of hyphae. Hyphae formed by an undifferentiated mycelium tend not to grow in contact with one another. Contact between adjacent hyphae is minimized by an “avoiding” reaction known as autotropism (Robinson, 1973). Thus there is a mechanism which regulates the spatial distribution of the hyphae within a mycelium. B.

POLARIZATION O F HYPHAL GROWTH

The mechanism which initiates and subsequently maintains the polarity of hyphal growth is not known. Growth initially becomes polarized during germ tube formation; fungal spores are often spherical and hence lack any obvious polarity. The onset of polarity may be prevented by manipulating the cultural conditions; for example, at temperatures of 37OC and below, conidia of Aspergillus niger germinate in the normal way and form a mycelium but, on incubation at 44OC, a supra-optimal temperature for this fungus, polarity is in-

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

15

hibited and the conidia increase in volume and dry weight to form giant, coenocytic, spherical “cells” which have very thick walls (Smith and Anderson, 1973). Growth of these structures becomes polarized (the cells germinate forming hyphal outgrowths) when the incubation temperature is lowered to 3 O O C . Mucor rouxii, which also forms a mycelium under normal conditions, grows in a non-polarized, yeastlike manner when it is cultured under anaerobic conditions in the presence of carbon dioxide (Bartnicki-Garcia, 1963). Thus nonpolarized (isotropic) growth may be associated with conditions which are unfavourable far growth. Hyphal polarity is clearly governed by some endogenous regulatory mechanism since the filamentous form is maintained in submerged culture. Further, entire filaments as well as the individual compartments of Geotrichum candidum hyphae grown in batch culture are polarized with respect to branch initiation (Fiddy and Trinci, 1975). Moulds grown in submerged culture are not of course subjected to the environmental gradients associated with colony growth on solid media (Park and Robinson, 1966). The tubular form of a hypha is presumably a consequence of the apical transport and deposition of the vesicles involved in hyphal extension. The vesicle may contain wall precursors and/or enzymes which synthesize and lyse cell-wall polymers (Bartnicki-Garcia, 1973). Hyphal extension (tip growth, branching, spore germination) always appears to be associated with the fusion of vesicles with the existing wall. The mechanism which regulates hyphal polarity presumably operates through its effect on the apical transport and/or deposition of these vesicles. Factors which disrupt normal vesicle transport and/or deposition may thus result in isotropic growth. C . REGULATION OF B R A N C H INITIATION

Growth of undifferentiated mycelia may be studied by following their formation from spores on solid media overlaid with cellophane (Trinci, 1974).The cellophane ensures that the mycelia are formed in a single plane and can thus be photographed in their entirety. Mycelia which have a total hyphal length of only a few millimetres may be regarded as undifferentiated. Certainly, such mycelia lack differentiation into leading hyphae of wide diameter and branch hyphae of narrower diameter (Trinci, 1973a). However, undifferentiated mycelia

16

A. T. BULL AND A. P. J. TRlNCl

may not form a distinct morphology and physiological state of development but simply represent a transient stage in colony formation (Steele and Trinci, 1975). Growth of an undifferentiated mycelium from a spore initially occurs under environmental conditions which remain relatively constant. The physical and chemical characteristics of the medium will only be significantly changed after it has supported a certain amount of biomass production. Therefore the cultural conditions which prevail during the initial stages of mycelial growth on a solid medium will be similar to those present during the early part of the exponential phase of' growth of a batch culture. Certainly the morphology of undifferentiated mycelia produced under these two cultural conditions is very similar (Steele and Trinci, 1975). 1. M aximurn Extension Rates o f Individual Hyphae

of Undgerentiated

Mycelia

The initial rate of extension of a branch of an undifferentiated mycelium is dependent upon its parent hypha. Branch hyphae of Aspergillus nidulans, Geotrichum candidum and Mucor hiemalis attain their maximum extension rates when they are, respectively, about 400, 400 TABLE 2. Mean and maximum extension rates of the hyphae of undifferentiated mycelia grown at 25OC. Mean values are quoted f standard errors of the mean. From Trinci (1974) Species

A.\pergillus nidulans Geolrichum candidum Mucor hiemalis Penicillium chrysogenum Neurospora crassa spco 1

Mean hyphal extension rate ( E , pm h-'1

** 8 * 0.3 21 *I

33 4 48 3 125 k 11

Maximum hyphal extension rate (Em.,, p n h-') 80 120 330

49

and850pm long (Trinci, 1974). These values thus represent the maximum lengths of the peripheral growth zones (Trinci, 1971) of these hyphae. The hyphae of an undifferentiated mycelium thus appear to have a maximum rate of extension which is strain specific (Table 2).

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

2. Mean Extension Rate o f the Hyphae

17

of Undgerentiated Mycelia.

The mean rate of hyphal extension ( E ) of a mycelium may be calculated from the equation:

where H o is the total hyphal length (pm) of the mycelium at zero time, H , the total hyphal length one hour later, Bo the number of hyphal tips at zero time, and B ; the number of hyphal tips one hour later. The mean hyphal extension rate of a mycelium may also be estimated (Steele and Trinci, 1975) from the equation: E=Gp

(2)

where G is the mean length of the hyphal growth unit (Trinci, 1973b) of the mycelium, and p the organism’s specific growth rate. The symbol a has been used to denote specific growth rate in several publications in this field. Since G and p are constants, E must also be a constant. The mean hyphal extension rate (calculated using equation 1) of Geotrichum candidum increased until the mycelium had formed three tips and thereafter remained constant (Trinci, 1974).The low standard deviations calculated for the mean hyphal extension rates of the mycelia of different moulds (Table 2) suggests that E is a specific feature of undifferentiated mycelia. 3. Growth of UndiJerentiated Mycelia from Spores

Undifferentiated mycelia initially increase in total length at an exponential rate (Trinci, 1974); in the case of Mucor hiemalis, exponential growth continued until the mycelium had a total hyphal length in excess of about 15 mm. The exponential phase is followed by a period during which there is a progressive deceleration in growth rate. The duration of the exponential phase is probably influenced by the length of the mould’s hyphal growth unit; that is, deceleration is likely to occur earlier for species which form dense mycelia (e.g. Penicillium chrysogenum) than for species which form sparse mycelia (e.g. Mucor hiemalid. The onset of the deceleration phase is probably correlated with certain adverse changes in the composition of the medium (e.g.

18

A. T. BULL AND A. P. J. TRlNCl

changes in pH value or secondary metabolites or nutrient concentration) and with differentiation of the mycelium (e.g. the formation of narrow branch hyphae and wide “leading” hyphae). During the early part of the stage during which the total hyphal length of the mycelium is increasing exponentially, branches are formed at relatively infrequent intervals. Eventually the number of branches increases exponentially at more or less the same specific growth rate as the total hyphal length of the mycelium (Fig. 2). f

5120

2560 I280 -

--

0 c -4Og 5

320 -

f

160-

80-

20 - 01

0

I 2

4

tip production I

I

I

6

8

10

12

Time ( h )

FIG. 2. Growth of mycelium of Geotrichum candidum on solid medium. The number of hyphal tips (O), total length (0)and length of the hyphal growth unit (0)are plotted as a function of time. The figure is reproduced by permission of Cambridge University Press.

The ratio between the total hyphal length of an undifferentiated mycelium and its number of branches has been called the hyphal growth unit(Caldwel1andTrinci, 1973;Trinci, 1973b).After spore germination, the hyphal growth unit of an undifferentiated mycelium increases in length until the germ tube produces its first branch. At this point the hyphal grown unit is halved. The amplitude of the oscillations in the length of the hyphal growth unit decreases progressively as the undif‘ferentiatedmycelium increases in size. Eventually the hyphal growth unit attains a more or less constant value (Fig. 2). Growth of an un-

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

19

differentiated mycelium may be thought of in terms of the duplication of a hypothetical “growth unit” which consists of a tip and specific length of hypha. The apparently contradictory observations that the biomass of a mould grown in batch culture increases at an exponential rate whilst individual hyphae extent at a linear rate are reconciled by the fact that there is an exponential increase in Up number (Caldwell and Trinci, 1973). Similarly, an individual bacterium may increase in mass at a linear rate whilst the population increases in number exponentially (Kubitschek, 1970).The “hyphal growth unit” of a mycelium is a physiological, but not a morphological, entity. It is simply the mean length of hypha per tip, and it clearly differs qualitatively from the “growth units” (i.e. the cells) of unicellular micro-organisms. The observation that the extension rate of the hyphae of an undifferentiated mycelium of Mucor hiemulis varied from 2 1 to 329 pmlh (Trinci, 1974)suggests that the length of hypha actually associated with each tip probably varies over wide limits ; the observed difference in extension rates presumably reflect differences in the length of hypha associated with each tip (Trinci, 1971). The relationship between hyphal length and tip number (i.e. the hyphal growth unit) may also be investigated by studying populations of undifferentiated mycelia (Caldwell and Trinci, 1973; Trinci, 197313; Trinci and Collinge, 1973; Morrison and Righelato, 1974).The hyphal growth unit of a population of undifferentiated mycelia of Neurospora crussu spco 1 was relatively constant (Trinci, 1973b)suggesting that there is a direct relationship between total hyphal length and tip number. D. H Y P H A L G R O W T H U N I T S O F D I F F E R E N T S T R A I N S A N D S P E C I E S

The hyphal growth unit is strain- (Trinci, 1973a, b ; Morrison and Righelato, 1974) and species-specific (Table 3). The observation that some spreading colonial mutants (spco) of Neurospora crassu have the same specific growth rate as the wild type (Trinci, 1973a) but different hyphal growth unit lengths indicates that these mutations affected the spatial distribution of the organism’s biomass but not its rate of production (Trinci, 1973a).The mean and maximum hyphal extension rates of these strains have presumably also been altered by the mutations. There is a considerable variation amongst the fungi in hyphal growth unit length (Table 3).

A. T. BULL AND A. P. J. TRlNCl

20

TABLE 3. Hyphal growth units ofundifferentiated mycelia of fungi grown at 25OC o n solid defined medium (Trinci, 197 1). The hyphae measured had 3-8 tips. The medium was supplemented with thiamine and biotin for the phycomycetes. Values are quoted k standard errors ot'the mean. ( I . J. Caldwell and A. P. J. Trinci, unpublished results) Hyphal growth unit (G,pm)

Species Cunninghamella sp Rhizopus slolonifer M ucor rammanianus MUCOThiemalis Aclinomucor repens" Aspergillus niger Aspergdlus wenlii Aspergillus gtganteus Penicillium clavtforme Penicillium chyogenum Geolrichum candiduma Cladosporium sp Verlicillium sp Fusarium vaucerium Fusarium avenaceum Trichoderma viride

35 f 9 124 f 31 31 f 10 95 f 22 352 k 97 I? f 14

6 6 k 15 7lf9 104 f 18 48 f 10 110 f 28 59f 1 1 8 2 2 17 682 f 26 620 f 164 160 f 31

"Grown at 3oOC.

E. EFFECT O F E N V I R O N M E N T A L C O N D I T I O N S O N H Y P H A L GROWTH U N I T LENGTH

1. Temperature

Hyphal growth unit length is riot affected by temperature (Trinci, 1973b). This suggests that specific growth rate ( p ) varies directly with mean hyphal extension rate ( E ) (i.e. the ratio Elp is a constant). This certainly appears to be so (Table 4).Presumably the maximum hyphal extension rate (E,,,,J of an undifferentiated mycelium also varies TABLE 4. Specific growth rate and mean hyphal extension rate of undifrerentiated mycelia of NeUrOSPOTa crassa spco I . p was determined in batch culture. From Trinci (1974) Temperature (OC)

25

Mean hyphal extension rate ( E , pn h-')

Specific growth rate (K, h-9

Elp

21 38

0.26 0.45

81 88

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

21

directly with specific growth rate. Thus temperature alters the rate at which the hyphal growth unit is duplicated but not its length. Similarly the mean cell mass of bacterial populations grown in batch culture is not altered by temperature (Schaechter et ul., 1958). 2. Inhibitors

Hyphal growth unit length may or may not be altered by inhibitors of mould growth (Caldwell and Trinci, 1973; Trinci, 1973a, b). The effect of an inhibitor depends upon the nature of’ the inhibition and the concentration employed. When hyphal growth unit length is not altered by an inhibitor, this suggests there is a direct relationship between the effect of the inhibitor on the mould’s specific growth rate and its effect on the mean extension rate of its hyphae (i.e. the ratio E / p is not altered by the inhibitor). Cycloheximide, unlike deoxycholate and triphenyl tin acetate, causes a decrease in hyphal growth unit length. 3. L-Sorbose L-sorbose inhibits the extension rate of Neurosporu crussu hyphae without apparently affecting the mould’s specific growth rate (Trinci and Collinge, 1973). Thus L-sorbose causes a dramatic decrease in hyphal growth unit length, inducing N . CTUSSU to branch profusely. Like the spco mutations of Neurosporu crussu (Trinci, 1973a), the maximum specific growth rate of L-sorbose treated mycelia remains unaltered but the spatial distribution of the mould’s biomass is changed. L-Sorbose, and substances which act like L-sorbose, would be expected to induce moulds to grow in a colonial or semi-colonial manner. 4. Medium Composition

Qualitative changes in medium composition may affect hyphal growth unit length as well as altering specific growth rate (Katz et al., 1972; Morrison and Righelato, 1974). The results of Katz et al. (1972) suggest that there is an inverse relationship between specific growth rate and hyphal growth unit length (Table 5 ) . It would seem that varying the composition of the medium altered the specific growth rate of Aspergillus niduluns without having a corresponding effect on the mean hyphal extension rate of its mycelia (i.e. the ratio, E / p did not remain

A. T. BULL AND A. P. J. TRlNCl

22

TABLE 5. Et'frct of medium composition on hyphal growth unit length Mediuni

Specific growth -rate (p, h-')

Hyphal growth unit length ( G ,pm)

Estimated mean rate of hyphal extension ( E , m h-l)''

(a) A\pergzl/u.\ uidu1an.s at 3OoC (calculated from the data of Katz el al., 1972) Malt extract Defined medium with acetate as the carbon source Defined medium with L-tryptophan as the nitrogen source

0.14

c. 33b c. 7Sb

11.9 10.2

0.11

c. 12Ob

13.2

0.36

(b) Periicillium chrysogenuin T 14 (Morrison and Righelato, 1974) Complex niediuni Defined medium

0.24 0.14

4 3 f 10 60k9

10.3 8.4

Estimated using Equation 2 (p. 1 7 ) . bEstimated from the data of Katz el al., (1972).

a

constant); medium composition appears to have had little effect on mean hyphal extension rate (Table 5 ) . 5 . Conclusions

Under a given set of environmental conditions the total hyphal length of a mycelium, and the number of its tips, increase exponentially at the same specific growth rate (pu).Thus the ratio between total hyphal length and tip number (i.e. the hyphal growth unit) is a constant as is the mean rate of hyphal extension ( E l . The relative constancy of the length of the hyphal growing unit during mycelial development suggests that branch initiation, like the division of unicellular microorganisms, may be regulated by the changes in cytoplasmic volume which accompany growth, i.e. when the mean volume of cytoplasm (length of hypha) per hyphal tip exceeds a critical volume (length) it induces the mycelium to initiate a new branch. Mutations or cultural conditions which cause a deceleration in the mean rate of extension of a mould's hyphae without altering ;he organism's specific growth rate (i.e. altering the ratio, E / p ) will result in

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

23

a decrease in the length of the hyphal growth unit. The spreading colonial mutants of Neurosporu crma are such mutants (Trinci, 1973a,b) and L-sorbose such a factor (Trinci and Collinge, 1973). Mutations or cultural conditions which cause an increase in the organism’s specific growth rate without causing a corresponding acceleration in the mean rate of extension of its hyphae (i.e. again altering the ratio EIp) will also result in a decrease in hyphal growth unit length. Presumably this is what happened when Kau et ul. ( 1972) altered the specific growth rate of Aspergzllus niduluns by changing the composition of the medium (Table 5 ) . Altering the specific growth rate of a mould by temperature changes causes a corresponding change in the mean rate of extension of its hyphae (i.e. temperature does not alter the ratio, Elp) and thus hyphal growth unit length is not affected by temperature. F. REGULATION O F T H E SPATIAL DISTRIBUTION O F HYPHAE

The spatial distribution of the hyphae of undifferentiated mycelia in part results from negative autotropism, i.e. hyphae tend to grow away from each other. Autotropism is thus a mechanism which helps to ensure that solid substrates are effectively and efficiently covered by mycelia. The hyphae at the periphery of a fungal colony grow radially outwards from the centre. This phenomenon has usually been explained in terms of a negative chemotropic response of the hyphae to some unknown factor(s) which accumulate in the environment. However, Robinson (1973) has recently suggested that the phenomenon can be explained in terms of a positive chemotropic response to oxygen. Negative autotropic responses of undifferentiated hyphae have been observed where the responding hypha was up to 30 jm away from the hypha to which it was reacting (A. P. J. Trinci, unpublished observation). Thus, negative autotropism may result from a response to some unknown substances which diffuse from hyphae and accumulate in the environment, or to a gradient in a nutritional factor (including oxygen) which is established in the immediate vicinity of hyphae.

IV. Colony Growth A. COLONY DIFFERENTIATION

A mycelium increases in size and gradually differentiates into a “mature” colony which subsequently extends radially across the substrate at a linear rate. This differentiation probably occurs as a direct

24

A. T. BULL AND A. P. J. TRlNCl

response to the changes induced in the medium by growth of the mould. “Mature” colonies can be divided into at least four morphological zones (Yanagita and Kogane, 1962): (1)the extending zone (which is equal to the peripheral growth zone; Trinci, 197 1 ), made up of the peripheral, sparse network of vegetative hyphae not supporting aerial hyphae; (2) the productive zone, made up of a much denser network of vegetative hyphae supporting aerial hyphae; (3) the fruiting zone, where asexual and/or sexual reproductive structures are formed; and (4)and aged zone, made up of the “aged” and autolysing hyphae at the centre of the colony. Although it is convenient to recognize these zones, there is a continuous differentiation of the colony from its periphery to its centre. 1. The Peripheral Growth Zone The width of the peripheral growth zone of a “mature” colony remains approximately constant as it expands radially across the substrate (Yanagita and Kogane, 1962;Trinci, 1971).The hyphae in the peripheral growth zone, unlike those of young mycelia, are usually differentiated into wide “leading” hyphae and narrower branch hyphae (Butler, 1961; Trinci, 1973a). The “leading” hyphae are oriented radially outwards from the centre of the colony with their apices more or less at the same level, giving a smooth outline to the colony (Butler, 1966). Hyphae at the outer fringe of the peripheral growth zone are thin walled, full of protoplasm (Butler, 1966)and rich in RNA and DNA (Yanagita and Kogane, 1962). The cytoplasm in hyphae more distant from the margin of the colony is vacuolated and the degree of vacuolation increases distally (Park and Robinson, 1967). Intrahyphal hyphae (Lowry and Sussman, 1966 ; Trinci and Righelato, 1970)are rarely if ever formed in the peripheral growth zone, and the septa1 pores of septate hyphae usually remained unplugged (Trinci and Collinge, 1973). Anastomoses (Buller, 1933) are not usually formed between hyphae in the peripheral growth zone. In some moulds, e.g. Neurosporu crassu, formation of a mature colony involves a differentiation process which results in the formation of wide “leading” hyphae having a faster maximum extension rate than the hyphae of the organism’s undifferentiated mycelium (Trinci, 1973b; 1974). This type of differentiation may be lacking in some moulds or not so marked; for example, the maximum extension rate of the leading hyphae of Geotrichum candidum colonies is not very much

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

25

faster than that of hyphae of its undifferentiated mycelium (Trinci, 1974). In most fungi, the hyphae in the peripheral growth zone branch monopodially, i.e. wide, fast growing “leading” hyphae subtend narrower, slower growing branch hyphae. Butler (1961) found that, if the extension rate of the “leading” hyphae of Coprinus dimminutus colonies was taken as loo%, then the extension rates of the primary and secondary branches were 66% and la%, respectively. Primary branches formed by “leading” hyphae at the margin of colonies of Aspergillus niduluns and Geotrichum cundidum had extension rates which were 20% and 30% less than their parent hyphae (Trinci, 1970).Thus, the peripheral growth zone hyphae d o not all have the same maximum extension rate. Butler (1961)found a positive correlation between the extension rate and diameter of “leading” hyphae of colonies of Coprinus disseminutus. There was a similar correlation between the diameter of the leading hyphae of spreading colonial mutant colonies of Neurosporu crassu and their extension rates (Trinci, 1973b). Little consideration appears to have been given by mycologists to the reason why most moulds have a monopodial branching pattern. Leopold (1971) concluded that monopodial branching is the most economical in terms of branch length for the efficient exposure of the leaves of trees to light and drainage of river basins by streams. This suggests that a monopodial branching pattern is probably a very efficient and economical way for a mould to colonize solid substrates, i.e. it ensures efficient cover of the substrate by the mould at the expense of a minimum production of biomass. The leading hyphae of colonies of Allomyces sp. (Emerson, 1955)and Geotrichum cundidum (Trinci, 1970) branch dichotomously as well as laterally, whilst those of Aspergillus niduluns branch sup-apically producing two or more branches per tip (Trinci, 1970). After dichotomous branching in G. cundidum, the extension rate of the branches accelerates until each attains the extension rate of the parent hypha. Dichotomous or sub-apical branching is rarely observed in the undifferentiated mycelia of these same species. Sympodial branching patterns have been observed in Ascobolus immersus colonies where parent hyphal tips are successively overtaken by their branches (Chevaugeon, 1959). The density of the hyphae at the circumference of a mature colony remains more or less constant as it increases in radius. This observation suggests that there is a periodic generation of new leading hrphae

26

A. T. BULL AND A.

P. J. TRlNCl

as the colony increases in diameter. Presumably these new leading hyphae arise as the result of primary branches increasing in diameter and growth rate until they assume the position and characteristics of leading hyphae (Trinci, 1973b). The transformation of a primary branch into a leading hypha probably occurs as a chance event when such a branch happens to extend into a relatively uncolonized part of the substrate at the fringe of the colony. The density of hyphae (Plomley, 1959; Trinci, 197 1) and biomass per unit area (Gillie, 1968) increase from the margin of the colony inwards. In the case of hyphal density, the increase occurs exponentially, suggesting that growth within the peripheral growth zone is rapid. 2. The Productive Zone

Like the peripheral growth zone, the width of the productive zone remains more or less constant. This region consists of a dense mat of vegetative hyphae which, unlike the peripheral growth zone, supports aerial hyphae. Some or all of the aerial hyphae may eventually be associated with reproductive structures. In fungi such as Mucor mucedo, the vegetative aerial hyphae are morphologically distinct from the sporangiophores which will eventually support the sporangia. The differentiation from mycelial to aerial growth is presumably associated with the deceleration in growth rate observed in this region of the colony. The cytoplasm of hyphae in this zone is very vacuolated and well endowed with reserves such as glycogen and lipid (Butler, 1966). The walls of productive zone hyphae are generally thicker than those of hyphae in the peripheral growth zone. Branch hyphae formed within the productive zone become progressively narrower as branching proceeds, and their growth is more meandering and less radially directed (Plomley, 1959). The septa1 pores of septate hyphae are usually plugged (Trinci and Collinge, 1973) and additional septa may be formed (Butler, 1966). Intrahyphal hyphae may be produced in this zone, and anastomoses between hyphae may occur in fungi other than the phycomycetes (Buller, 1933). 3. The Fruiting Zone

The width of the fruiting zone is less constant than the two zones considered previously. The mycelium is very dense and consists of highly vacuolated hyphae, some of which may be autolysing. Intra-

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

27

hyphal hyphae may be present. The mycelium may support asexual and/or sexual reproductive structures, the former usually preceding the latter in their development. The induction of reproductive structures is probably associated with the establishment in the environment of conditions which are relatively unfavourable for growth; for example, exhaustion of a nutrient or accumulation in the medium of inhibitory products. The formation of reproductive structures in at least some species is associated with a turnover of certain cytoplasmic and wall polymers. Exhaustion of the carbon source in the medium induces formation of a-1,s glucanases in Aspergillus nidulans which degrade the a1,3 glucan component of the wall (Zonneveld, 1974). The breakdown products of such wall polymers may supply the carbon and energy required for sporulation. In the fruiting and aged zones of the colony, the nucleic acids which were present in the mycelium may become associated with the reproductive structures (Yanagita and Kogane, 19621, suggesting that there is a turnover of these polymers. 4. The Aged Zone

As a colony increases in radius a progressively larger proportion of it is made up of a central aged zone of indefinite diameter. It is composed largely of autolysing hyphae and reproductive structures. B. M O U L D - I N D U C E D C H A N G E S I N T H E SUBSTRATE

The spatially separated regions of the colony already described reflect temporal changes which occur as the substrate is progressively colonized by the mould. Differentiation of the mould almost certainly reflects, and is induced by, the changes in the substrate which result from mould growth. The physical changes include an increase in the relative humidity above the medium, greater temperature constancy and changes in medium viscosity (Park and Robinson, 1966). However, there is little doubt that the crucial changes in the medium which are correlated with differentiation of the mould are of the chemical type discussed below. 1. Nutrient Concentration

The concentration of nutrients in the environment decreases progressively as colonization proceeds, but whether or not this has a direct effect upon the growth rate of the mould depends upon the original nutrient concentration and the affinity of the mould (K,value)

28

A. T. BULL AND A. P. J. TRlNCl

for the particular nutrient which may eventually limit growth. Moulds, like bacteria, generally have a high affinity for essential nutrients h e . a low K,value); thus, the concentration of the limiting nutrient has to be decreased to a very low level before it causes a lowering in growth rate. For example, the K, (glucose) values of Fusarium aquaeductuum and Geotrichum candidum are 0.3 (Steensland, 1973)and 1.0 mg l-l, respectively (Fiddy and Trinci, 1975). In the case of these fungi, the concentration of glucose in the medium would have to fall below about 45 nig 1-I before growth rate became glucose-limited. Growth of course would not continue for long at such concentrations because the glucose would very quickly become exhausted (cf. Section 11, C,2; p. 9 ) . Nutrient concentrations will obviously have a very significant effect on the maximum biomass of mould per unit area of substrate (i.e. on the yield). I t is a common microbiological practice to compose media so that one particular nutrient, usually the carbon and energy source, becomes exhausted before the rest, and hence determines the final yield. However, it is often difficult to decide which is the limiting nutrient in many of the media conventionally used by mycologists; for example, Vogel’s medium for Neurospora crussa (Vogel, 1956) and Czapek Dox medium (Ainsworth and Bisby, 1961). With these media, it is unlikely that the concentration of the carbon source ultimately limits growth.

2. Oxygen Tension The oxygen tension at the base of a dense mycelial mat probably is decreased to a level at which it limits growth rate. The growth rate of such hyphae will then be limited by the rate of diffusion of oxygen from the air above the colony. A decrease in oxygen tension may lead to the accumulation of secondary metabolites in the medium (e.g. citric acid).

3 . Changes in PH value

The pH value of the substrate may change as a result of the utilization of nutrients during growth (e.g. when the nitrogen source is ammonium sulphate or sodium nitrate) and/or by accumulation of secondary metabolic products (e.g. citric acid). The pH value of the medium is particularly likely to change when it has little buffering capacity.

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Changes in the pH value of the medium are likely to affect the mould’s growth rate and may also encourage production of certain secondary metabolites. 4. Accumulation

of Secondary

Metabolites

Accumulation of products of secondary metabolism in the medium is likely to lower further the organism’s growth rate, and thus trigger a positive feedback mechanism. Sometimes crystals, or water-soluble pigments, may be deposited in the medium (Raper and Thom, 1949; Nobles, 1948). The changes in the substrate will increase progressively as the fungal biomass per unit area increases during colonization. The chemical changes which occur in one part of the medium will tend to spread throughout the medium by diffusion. However, the radial growth rate of the colonies of most fungi is likely to exceed the rate at which chemicals such as secondary metabolites diffuse through the medium. I t is unlikely, therefore, that secondary metabolites and products of autolysis formed at the centre of the colony will diffuse through the medium at a sufficiently fast rate to attain significant concentrations in the peripheral growth zone of the colony. Hyphae at the margin of a fungal colony continually extend into medium having a composition which is identical, or very similar, to the composition of the original uninoculated medium. C . KINETICS O F COLONY EXPANSION O N SOLID MEDIA

Pirt (1967) pointed out the need to define: (1) the factors which govern the radial growth rate of microbial colonies; (2) the relationship between radial growth rate and mass growth rate; and (3) the factors which cause differentiation within colonies. Some progress has now been made towards these ends. Gillie ( 1968) determined the dry weight of successive one-cm segments of linear colonies of Neurosporu crassu grown on solid medium in growth tubes. Such plots of mould dry weight against distance from the margin of the colony will of course reflect the temporal changes in biomass which occur as any given part of the medium is colonized by the mould. Gillie’s results showed that during the first two days of the growth of N. crussu on a given region of the medium the mould

30

A. T. BULL AND A.

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biomass increased with time. Subsequently as the mould started to autolyse there was a decrease in biomass per unit area of substrate. The radial expansion of fungal colonies may be divided into four phases (Trinci, 1969):(a)lag, the period between inoculation and germtube emergence; (b) exponential, during which the colony increases in radius at an exponential rate; (c) deceleration, the period between the termination of the exponential phase and the onset of the linear phase; and (d)linear, during which the colony increases in radius at a constant or linear rate. In some fungi there is a deceleration from the linear growth rate as the colony approaches the margin of the Petri dish (see the chapter by Carlile, in Hawker and Linton, 197 1). The exponential and deceleration phases are usually of comparatively short duration, and in Aspergillus nidulans are completed by the time the colony has grown one mm from the edge of the inoculum (Trinci, 1969). Thus for most of its growth a fungal colony expands at a linear rate. The rate of the linear phase is not influenced by the ploidy of the nuclei (Lhoas, 1968). 1. Influence

of Peripheral

Growth Zone Width on Radial Expansion

Pirt (1967) suggested that growth of a microbial colony was restricted to a peripheral annulus and that growth in the centre of the colony eventually stopped due to exhaustion, or near exhaustion, of a particular nutrient. In the case of fungal colonies it has been shown that the peripheral hyphae have a much higher metabolic activity than hyphae of other regions of the colony. The rates of uptake of [3Hlleucine,13H]uridine and [3H]N-acetylglucosamine,for example, are fastest in the peripheral 2 to 3 mm of Trichoderma uiride colonies indicating that the rate of synthesis of RNA, protein and chitin is fastest in this region of the colony (Galun, 1972). Similarly, hyphae of the peripheral 1.5 mm of Aspergillus niger colonies have a faster rate of 32Puptake than other parts of the colony (Table 6). The decrease in the rate of uptake of these various compounds with distance from the colony margin suggests that there is a decrease in growth rate from the periphery to the centre of the colony. Smith ( 1924) was the first to suggest that “although the actual.extension occurs at the tip, there are grounds for believing that the parts of the hypha behind the tip contribute to the activity of the latter”. This hypothesis was subsequently implicitly or explicitly supported by the

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TABLE 6. Uptake of 32P by colonies of Aspergillus n i p . Adapted from Yanagita and Kogane (1963b) Region o f the colony

Peripheral growth zone Productive zone Fruiting zone Aged zone

Width of 3zP uptake 32Puptake expressed as a the region (c.p.m./mgdry wt) per cent of the rate (mm) of uptake in the peripheral zone 1.5 2.0 4.5

3.5

4,800 1,500 300 140

100

31 6 3

~

work of Zalokar (19591, Clutterbuck and Roper (1966), Lhoas (19681, Trinci (1971) and Kau et al. (1972). Trinci ( 197 1) has shown for a number of fungi that the radial growth rate of their colonies (K,) is a function of the width of the peripheral growth zone ( w ) and the organism’s specific growth rate. Thus, K,= wp

(3)

The concept defined by Equation 3 may be considered in terms of unbranched leading hyphae traversing the peripheral growth zone of the colony. The protoplasm in such hyphae increases at, or close to, the mould’s maximum specific growth rate. This hypothetical hypha, and the colony, will only grow at a linear rate if the width of the peripheral growth zone remains constant, i.e. as the peripheral hyphae increase in length by a specific increment at the margin of the colony an equivalent increment of length is removed from the peripheral growth zone of the hypha at its inner margin. However, the hyphae in the peripheral growth zone branch, and thus some of the protoplasm produced by leading hyphae is almost certainly directed to support the initial growth of its branches (Trinci, 1970, 1974). Further, the specific rate of synthesis of protoplasm within the peripheral growth zone is likely to decrease with distance from the margin of the colony. Thus Equation 3 is unlikely to provide more than a first approximation of the rate of expansion of fungal colonies. To summarize, the peripheral growth zone has the following characteristics: (1) In colonies growing at a linear rate the width of the peripheral growth zone remains constant; (2) only growth within the peripheral zone contributes to radial expansion of the colony; regions of the colony distal to the peripheral growth zone increase in biomass (e.g. continue to branch and form a dense mycelium, and also

32

A. T. BULL AND A. P. J. TRlNCl

reproductive structures) but this growth rate does not contribute to radial expansion; and (3) growth within the peripheral growth zone is rapid and occurs at or close to the organism’s maximum specific growth rate for the prevailing conditions. The relationship defined by Equation 3 may be tested by calculating the theoretical radial growth rate of a colony from the width of its peripheral growth zone and the organism’s maximum specific growth rate and comparing this value with the observed colony radial growth rate (Trinci, 1971).

2 . Determination

of Peripheral

Growth Zone Width

The maximum width of the peripheral growth zone may be determined by calculating the minimum length of a hypha which extends at the same rate as the expansion rate of the colony. This may be done by severing peripheral growth zone hyphae and then determining their subsequent growth rate (Ryan et al., 1943; Clutterbuck and Roper, 1966; Lhoas, 1968; Trinci, 1971; Trinci, 1973; Trinci and Collinge, 1973). This method probably overestimates the length of the peripheral growth zone because of the damage which results from cutting. In the case of septate hyphae, the cutting damage appeared to be restricted to three intercalary compartments (Trinci, 197 1). The septa1 pores in the region of the cut may be plugged with Woronin bodies (Reichle and Alexander, 1965; Trinci and Collinge, 1974b)which limit loss of protoplasm. An alternative method of measuring peripheral growth zone length is to determine the length of a branch when it first attains its maximum extension rate (Trinci, 1974). For a number of fungi there is close agreement between the hypothetical colony radial growth rates, calculated from peripheral growth zone and specific growth rate measurements, and observed rates (Trinci, 197 1). The spreading colonial mutants of Neurospora crassa form colonies which are more compact and have a slower rate of expansion than colonies of wild type strains (Garnjobst and Tatum, 1967). Some of these mutants have the same maximum specific growth rate as the wild type but much slower colony radial growth rates. As predicted by Equation 3, there is a linear relationship between the rate of expansion of the colonies of these mutants and the width of their peripheral zones (Trinci, 1973b). In the case of fungi having septate hyphae it is possible that the peripheral growth zone only extends from the tip to the first septum and, in those fungi which have complete septa (i.e. lack pores), e.g.

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

33

Geotrichum candidum, Basidiobolus ranarum and Mucor hiemalis, there is little doubt that the peripheral growth zone is restricted to the apical compartment. The leading hyphae of G. candidum colonies had a mean apical compartment length of 290 k 99 pm compared with an experimentally determined peripheral growth zone width for the colonies of 423 k 129 p.Many fungi, such as Aspergillus nidulans and Neurospora crassa, form septa which initially have unoccluded central pores large enough to allow the translocation of vesicles and organelles such as nuclei (Trinci and Collinge, 1973). It is possible that the peripheral growth zone of these hyphae is limited by the plugging of the septal pores (Trinci and Collinge, 1973). The plugging of septal pores may be initiated by the establishment in the medium of conditions which inhibit growth (see Section IV, B; p. 27). Septa1 plugging may thus be considered as an ageing phenomenon. As mentioned earlier, the establishment in the substrate of conditions which are unfavourable for growth is probably related to the rate of increase in biomass per unit area of substrate. This in turn is probably a function of the organism’s specific growth rate and branching pattern, the rate of increase being fastest for strains with low hyphal growth unit values, i.e. strains which branch profusely. It is possible that there is a direct relationship between hyphal growth unit length and peripheral growth zone width (Trinci, 1973b). Certainly Morrison and Righelato (1974) have also come to this conclusion. D. C O L O N Y E X P A N S I O N A S A P A R A M E T E R O F M O U L D G R O W T H

Colony radial growth rate is only a reliable parameter of growth under conditions where it varies directly with the organism’s specific growth rate (i.e. the ratio KJp is a constant). It follows from Equation 3 that colony radial growth rate will only be directly related to the specific growth rate when the width of the peripheral growth zone of the colony remains constant. 1 . Eflect of Temperature

The width of the peripheral growth zone of a colony remains more or less constant when its rate of expansion is altered by temperature (Trinci, 197 1). Thus, colony radial growth rate may be used to determine the effect of temperature on mould growth (i.e. the ratio KJp is a canstant 1.

34

A. T. BULL AND A. P. J. TRlNCl

2. Effect o f Growth Inhibitors

Inhibitors such as qdoheximide apparently do not alter peripheral growth zone width (Trinci and Gull, 1970) at least at comparatively low concentrations. Under these circumstances, colony radial growth rate is a reliable parameter of growth (K,/c( is a constant). Several workers have shown that colony radial growth rate decreases with the logarithm of the inhibitor concentration in the medium (Trinci and Gull, 1970; Fevre, 1972; Bret, 1972). The basis for this relationship is not known. 3. Effect o f L-Sorbose It has been known for a long time that L-sorbose causes some moulds to grow in a “colonial” form, i.e. form dense colonies which have a lower rate of expansion (Tatum et al., 1959). L-Sorbose caused a

decrease in the hyphal growth unit of Neurosporu crassu (i.e. it branched more profusely) but did not affect the mould’s specific growth (Trinci and Collinge, 1973). The decrease in colony radial growth is correlated with a decrease in the width of the peripheral growth zone which in turn is presumably correlated with the observed increase in branching frequency. Cellobiose may also induce some fungi to grow in a “colonial” form in a similar way (Wilson and Niederpreum, 1967; Wilson, 1970). 4. Nutrient Concentration

The rate of expansion of glucose- and arginine- “limited” fungal colonies increased linearly with the logarithm of the nutrient concentration (Trinci, 1969; Gillie, 1968; Fiddy and Trinci, 1975). Again the basis of this relationship is not known. Most fungal colonies attain their maximum rate of expansion at very low nutrient concentrations (below about 150 mg I-’ for glucose and 80 mg I-’ for arginine). Fiddy and Trinci (1975) have shown that over the “glucose-limited” range there is a direct relationship between the radial growth rate of Geotrichum candidum colonies and the width of their peripheral growth zones. Thus, the deceleration in colony radial growth rate at glucose concentrations below about 100 mg 1-’ could be entirely accounted for by the observed decrease in peripheral growth zone width. Glucose concentration has little effect (Fiddy and Trinci, 1975) or no

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROVVTH

35

effect (Trinci, 1969) on internode length (the mean distance between adjacent primary branches produced by “leading” hyphae). Thus the variation in colony hyphal density with glucose concentration is not related to the frequency of branch initiation, but rather to their subsequent growth. At low glucose concentration branches are initiated, but most of them only grow for a short period of time before the substrate (glucose) in their vicinity becomes exhausted. At low glucose concentrations, therefore, very sparse colonies are formed but they expand at almost the maximum rate. This feature of the growth of fungal colonies clearly has some ecological significance.

5 . Growth of Diflerent Strains and Species Colony radial growth cannot be used as a parameter to compare the specific growth rates of different fungal species (Trinci, 197 1)or strains (Trinci, 1973a). The rapid rate of expansion of the colonies of many phycomycetes is largely due to their having wide peripheral growth zones rather than to their having particularly fast specific growth rates. Colonies of some phycomycetes, e.g. Rhizopus stolonijh, may have wide peripheral growth zones because their hyphae lack septa. In addition the density of hyphae at the margin of such colonies is much less than in the case of colonies of fungi like Penicillium chrysogenum which expand very slowly. E . C O M P A R I S O N O F T H E C O L O N I Z A T I O N OF S O L I D SUBSTRATES BY M O U L D S A N D UNICELLULAR M I C R O - O R G A N I S M S

The filamentous morphology of moulds enables them to colonize solid substrates more efficiently than non-motile, unicellular microorganisms. The polarization of growth within hyphae allows fungi to form colonies which have much wider peripheral growth zones than bacterial colonies (e.g. w equal to about 8.5 mm for Rhizopus stolonijer colonies (Trinci, 197 1) compared with about 90 pm for Escherichia colz colonies (Pirt, 1967)).Thus fungal colonies are able to expand across solid substrate at much faster rates than bacterial colonies although fungi usually have the slower specific growth rates. The wide peripheral growth zones of fungal colonies result in their having radial growth rates which usually exceed the rates of diffusion of chemicals in the medium. Thus secondary metabolites and other products formed at the centre of the colony diffuse through the medium at a slower rate

36

A. T.

BULL AND A. P. J. TRlNCl

than the rate of expansion of the colony, and hence do not affect the growth rate of the peripheral hyphae. Similarly the rate of diffusion of nutrients from uncolonized parts of the medium towards the margin colony is slow compared with the rate of expansion of most fungal colonies. The leading hyphae of fungal colonies are continually growing into uncolonized medium which has approximately the same composition and pH value as the uninoculated medium. However, in the case of bacterial colonies which expand at very slow rates (Pirt, 19671, there will be a tendency for secondary metabolites formed at the centre of the colony to diffuse through the medium and inhibit growth at the periphery of the colony. In addition the concentration of nutrients in the uncolonized region of the medium surrounding the bacterial colony may be significantly lowered or even exhausted because of diffusion towards the colony (Rieck et al., 1973). These effects probably explain the gradual deceleration in the rate of expansion of bacterial colonies with time (Pirt, 1967). The growth of fungal colonies is initiated at much lower nutrient concentrations than bacterial colonies, and fungal colonies attain their maximum rate of expansion at lower nutrient concentrations than bacterial colonies (4 g 1-I for Escherichia coli (Pirt, 1967)compared with 75 mg 1-' for Mucor hiemalis; Trinci, 1969). These latter differences probably resulted from the fact that fungi, unlike bacteria, have a mechanism which regulates biomass density per unit area of substrate according to the concentration of nutrients in the medium. Thus the filamentous habit enables moulds to distribute the biomass which a solid substrate will support to maximum advantage in spreading the colony. Finally the filamentous habit enables fungi, unlike bacteria, to penetrate a solid substrate such as agar-gelled media (Trinci, 1969; Trinci, 1973a); hyphae appear to grow down into the medium at a rate similar to their growth rate across its surface.

V. Fungal Growth in Submerged Liquid Culture. Technical Considerations A variety of operational difficulties accompany the submerged cultivation of fungi. These difficulties are compounded when continuous-flow cultures (the method of choice for analysing fungal growth and metabolism; Bull and Bushell, 1976)are selected, and they

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

37

have undoubtedly inhibited the widespread use of chemostats in fungal research. Basically, the problems are related to the rheological properties of fungal cultures, the propensity of fungi to accrete over fermenter walls and internal surfaces, and to an extremely variable growth morphology. In this section brief consideration is given to the practicalities of fungal fermentations and the solution of common technical problems. A critical assessment of methods for measuring mycelial growth has been made by Calam ( 1969) and, while this topic will not be developed further here, two points may be emphasized. First, that considerable care needs to be taken to obtain representative samples from fungal cultures, especially when small scale laboratory equipment is used, and the position of sampling points, velocity of sample take-off (Solomons, 1972) and culture heterogeneity all exert a pronounced effect on the quality of the sample. Second, it is worth noting that culture absorbance can be used as an accurate growth parameter provided that measurements are restricted to biomass concentrations of less than about 2 g I-' (Trinci, 1972; Solomons, 1975). The relevance of this observation lies in the possibility of continuously monitoring mycelial growth and, thereby, developing turbidostat systems for fungi. As far as we are aware turbidostat culture of moulds has yet to be exploited. The growth form of fungal cultures profoundly affects metabolism; the relationship may be direct and reflect growth of the organism in, say, a yeast-like, mycelial or pelleted form (see p. 14) or, indirect via changes in the rheological properties of the culture. Unlike bacteria, yeasts and fungal pellets, suspensions of diffuse mycelia are nonNewtonian in character; that is their apparent viscosities are a function of the shearing produced by agitation, and the suspension may be heterogeneous with respect to the mass transfer of substrates and products. The apparent viscosity is also dependent on the mould concentration, and Solomons and Weston (19611, among others, have shown conclusively that it may be impossible adequately to aerate high-viscosity cultures in laboratory-scale fermenters. Similarly, the critical dissolved oxygen tensions for fungal cultures become greater (respiration at a submaximum rate) as the culture viscosity increases (Phillips and Johnson, 1961 ; Steel and Maxon, 1966). Unfortunately, even though adequate aeration may be provided, the degree of mixing and distribution of nutrients in a fungal culture may have an adverse effect on growth (Donovick, 1960). These effects can be exacer-

38

A. T. BULL AND A. P. J. TRlNCl

bated in continuous-flow cultures of fungi that are commonly operated at low dilution rates, i.e. < 0.03 h-? At these low rates an increasing proportion- of the energy source is consumed for “maintenance” purposes, a situation that appears to be aggravated by poor mixing. Experimental support for this view was provided by Hansford and Humphrey (1966) who observed that higher fungal yields could be obtained at low dilution rates by use of multifeed distribution and improved mixing. Various other means have been proposed for circumventing growth limitations associated with the rheological characteristics of fungal cultures. Thus, increasing the agitation but not the air flow rate (Brierley and Steel, 19691, lowering the biomass concentration, or establishing pelleted growth (approximation to Newtonian fluid) enhance culture aeration. Some recent work in Moo-Young’s laboratory has revealed that addition of water-soluble polymers to fungal cultures increased both the mass transfer of oxygen to the liquid (Moo-Young et al., 19591, the specific growth rate and other growth parameters (Elmayergi and Moo-Young, 1973). Increased rates of potassium transport induced by higher concentration gradients across the mycelial surface were considered responsible for these effects. For further information on the rheology of mycelial cultures, the reader is directed to the review of Steel (1969) and the morphology model of Roels et af. (1974). Accreted fungal growth creates difficulties in any culture system, and particularly in continuous-flow types. It provides heterogeneous conditions where, for example, anaerobic metabolism may occur in a well aerated fermenter. Bungay et al. ( 1969), using an elegant microprobe technique, showed that respiration could be prevented in microbial accretions of only 150pm thickness. When the thickness of such accretions exceeds the penetration depth of nutrients, detachment from the support surface begins, and subsequent blockage of feed and emuent lines may quickly follow. Accreted growth in chemostats leads additionally to variable culture volumes, the possibility of inadvertent feedback conditions (Solomons, 1972)and an increase the value of D,,,, (Topiwala and Hamer, 197 1). Clearly, each of these effects may vitiate the maintenance of steady-state conditions. The prevention of accreted growth essentially is a biochemical engineering problem, and several fermenter designs and operating conditions have been proposed to alleviate the problem, especially in continuous flow systems. For

THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH

39

further information, the interested reader is directed to the papers of Righelato and Pirt (1967), Brunner and Rohr (19721, Bull and Bushel1 (19761, Means et al. (1962) and Dawson (1963). It is sufficient to say here that the conventional stirred-tank type of fermenter can now be adapted easily for continuous culture of fungi, and one such reliable design has been reported by Rowley and Bull (1973). It has been a common experience to find that the critical dilution rate of fungi in chemostat cultures is significantly less than the value of p,,,,,derived from batch cultures grown under similar conditions. Thus, studies with Aspergzllus nidulans, A. niger, Fusarium graminearum and Mucor hiemalis (Carter and Bull, 1969; Fencl and Novak, 1969; Ng et al., 1974; Solomons, 1972; Lynch and Harper, 1974) suggested that steady state dilution rates exceeding about 50%p,,,,, (batch) could not be established. Solomons has suggested (Solomons, 1972 ; Solomons and Scammell, 1974) that such premature washout from chemostats could be due to a growth rate dependency on vitamins, and that the higher growth rates in batch cultures were consistent with a sufficiency of growth factors being present in the spore inoculum. Thus, F. graminearium was found to have a requirement for both biotin and choline. An explanation of this phenomenon, based on the obligatory accumulation of a growth-limiting “intermetabolite”, was favoured by Novak and Fencl(1973).These authors obtained some evidence for the ammonium ion being the critical intracellular metabolite when A. niger was grown in a glucose-nitrate medium; depletion of the ammonium pool occurred when D was approximatrely 4O%pma,(batch),and subsequent nitrite accumulation was considered to cause culture intoxication and washout. However, in our experience, the growth form of the fungus can be the crucial factor in determining Dct.11. Experiments with the hyaline 13 me1 mutant of A. niduluns and the wild-type strain used by Carter and Bull (1969) revealed that D,,,, values within 1% of pm.xCOUld be obtained without any modification of the medium or incubation conditions (M. E. Bushell and A. T. Bull, unpublished results). Similarly the Dorltof both strains could be decreased to about 5 5 4 0 % ,urn.=when chemostat cultures were established from suboptimal spore inocula (
40

A. T. BULL AND A. P. J. TRlNCl

observed that the onset of pelleted growth in A . niger chemostat cultures predisposed them to washout. Finally, the effects of nonmetabolized polymer additions to fungal cultures add support to our conclusions. Elmayergi -and Scharrer ( 1973) found that carboxypolymethylene (0.3%w/w) significantly enhanced the specific growth rate of A niger in batch cultures (50 to 70% increases were recorded) and the only convincing explanation of this effect was the change in fungus morphology from a pelleted to a pulpy form. VI. Kinetics of Fungal Growth in Submerged Liquid Culture

The growth kinetics of mycelial fungi has been the subject of a recent excellent review by Righelato (1975) and in a parallel essay Bull and Bushel1 ( 1976) have examined the environmental control of fungal growth. These articles include the first comprehensive accounts of continuous-flow culture studies of fungi; the present discussion, therefore, is intended to orientate the reader and provide some terms of reference for discourses on the metabolic aspects that follow. A.

RATES OF G R O W T H

I t is interesting to recall that simple forms of continuous-flow cultivation of fungi were being attempted prior to the development of the chemostat theory. Wean and Young ( 1939), for example, developed what is now recognized as a fed-batch system for growth of Pythium debaryanum, and reported increased yields and pH stability compared to simple batch cultures. The mathematical treatment of such fedbatch cultures has appeared only recently (Pirt, 1974). Although prolonged linear rates of growth under quasi-steady state conditions are possible by this form of cultivation, the first genuine chemostat experiments with fungi are accreditable to Hofsten et al. (1953). Hofsten et al. ( 1953) grew Ophiostoma multiannulatum at a dilution rate of approximately 0.14 h-' and reported apparent yields from glucose of 0.43 to 0.70 g g-', depending on the concentration of glucose in the inflowing medium. The latter value indicates that carbon other than that from glucose was being assimilated. However, a definitive testing of the applicability of chemostat theory to filamentous fungi was not made until Pirt's extensive studies of the pencillin fermentation (Pirt and Callow, 1960; Pirt and Righelato, 1967 ; Righelato et al., 1968). Subse-

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quently the growth kinetics of several other fungi-mainly aspergilli-under continuous-flow conditions have been described. Typical steady state biomass and substrate concentrations as functions of dilution rate are shown in Fig. 3 for a glucose-limited chemostat culture of Aspergillus nzdulum 13 mel. The excellent agreements between experimental and theoretical values of X at dilution rates > 0.05 h-* demonstrates the validity of applying the general chemostat theory to filamentous fungi. The glucose saturation constant, K,, determined from a Lineweaver-Burk plot of S against D, was 0.1 1 g 1-' and is similar to values reported for other fungi. Although this value is small relative to SR,energy-substrate affinities several hundred-fold greater are known for Fusarium uquaeductuum(Steensland, 1973)and Neurosporu crmsa (Fiddy and Trinci, 1975).The small number of Ksdeterminations for other nutrients have been compiled by Bull and Bushell (1976) and Fiddy and Trinci (1975). Figure 3 also illustrates that, in accord with chemostat theory, the output of mycelium reaches

Dilution rote ( D ; h-' 1

FIG.3. Influence of dilution rate on the steady-state concentrations of biomass (0) and of residual glucose (0)in a glucose-limited chemostat culture of Aspergillus niduluns I3 me1 (ATCC 28270). The theoretical biomass production ( P ; 0)cuiyc was ca~cdatcd from the equation:

where Y = 0.43 g biomass g glucose-', S, = 15 g glucose I-', K,= 0.1 1 gglucosc I-I, ;und pa..= 0.205 11-! Biomass productivity was calculated as the product of D, and

x

e.,

determined by coniputor analysis. Unpublished data of M . E. Bushell and A. T. Bull.

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A. T. BULL AND A. P. J. TRlNCl

a maximum at a dilution rate of 0.165 h-l. Morphological differentiation of the culture occurred at the extremes of the growth rate range; thus, as D approached Dcrit,pelleted growth was induced, whereas at dilution tates below 0.05 h-' the mycelium became profusely branched and produced conidia. The decreased growth yields evident at low dilution rates can be accounted for by the existence of a maintenance energy requirement; that is, energy for macromolecular turnover, osmoregulation, and movement, for example. The glucose maintenance coefficient for A . nidulans 13 mel, calculated from qslucose measurements, is 0.019 glucose g biomass-' h-l, a value very similar to those for other moulds (0.022 g g-' h-l, Penicillium chrysogenum; Righelato et al., 1968; 0.018 g g-' h-*, wild type A . nidulans 224; Carter et al., 1971). In chemostat cultures the significance of the maintenance coefficient becomes marked as D is decreased to low values. Righelato ( 1975) has calculated that the glucose maintenance requirement was only 10% of the total glucose utilization rate when P. chrysogenum was grown at p,, but amounted to 70% of the consumed glucose when D was made 0.005 h-l; the latter growth rate is typical of penicillin fermentations. The proportion of glucose diverted for maintenance functions in A . nidulans 13 me1 growing at pmaX, 0.05 and 0.02 h-' was 4, 13 and 26%, respectively. These values are comparable to ones derived for bacteria where, at maximum growth rates, about 10% of the total energy output was used for maintenance purposes (Stebbing, 1973). Maintenance requirements for oxygen have been determined for several moulds, and the values of 0.55,0.7 1 and 0.75 mmol g biomass-' h-t respectively, for A . nidulans 224 (Carter et al., 19711, Trichoderma uiride (M. A. Zainudeen personal communication) and P. chrysogenum (Mason and Righelato, 1976) are consistent with complete oxidation of the glucose used to meet the maintenance requirement. Not surprisingly, maintenance energy requirements increase under conditions of environmental stress such as high salinity (Watson, 1970) and high temperature (MatschCand Andrews, 1973). Watson recorded a 10-fold rise in the glucose maintenance coefficient for Saccharomyces cerevisiae when sodium chloride was added to the growth medium by a concentration of 1 M . The number of careful measurements of pmaxforfilamentous fungi is depressingly small (see Solomons, 1975, for a compilation). Most values are in the range 0.2 to 0.3 h-' but rates as high as 0.6 h-'

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(Geotrichurncandidurn)and 0.8 h-' (Achlya bisexualis) have been reported by Trinci (1972) and Griffin et al. (1974). Recently the phenomenon of hypertrophic growth (Powell, 1972) has attracted attention; this is the condition in a two-stage chemostat where p in the second stage (p2)greatly exceeds p,,,.x. To our knowledge hypertrophic growth has not been reported for moulds but Vrana (1973) has provided an important insight into the phenomenon as it occurs in Candidu utilis. Under the conditions of Vrana's experiments, pm.x was 0.35 h-! When the dilution rate of the second stage (D2) was 1.10 h-', the rate of RNA synthesis was greater than that of DNA (i.e. the population departed from balanced growth, the organisms enlarged, produced filaments and multiple budding, and started to grow hypertrophically; p2 = 0.60 h-'). The lower differential rate of DNA synthesis produced a situation where a proportion of daughter cells lacked DNA. At the other end of the growth rate range there appears to be a finite limit below which part or all of the population differentiates and ceases to grow; this limit has been termed the minimum specific growth rate (p,,,I,J and has been determined experimentally with at least two species. Under glucose-limited conditions pmInfor P . chrysogenum was about 0.0 14 h-*. Below this growth rate the specific rate of penicillin synthesis (q,,J decayed (Pirt and Righelato, 19671, massive turnover of nucleic acids occurred and the culture conidiated (Righelato et ul., 1968). The Pu,l,of glucose-limited A. nidulans 13 me1 was approximately 10% ofpUm.x (M.E. Bushel1 and A. T. Bull, unpublished experiments); again it was identified with major morphogenetic change in the organism, and concomitantly the efficiency of ribosomes in protein synthesis reached a minimum (see Section IX, A; p. 72). Critical determinations of growth yields of fungi have been made with several species, and the reader is referred to the recent reviews by Righelato ( 1975), Solomons ( 1975) and Bull and Bushel1 ( 1976) for discussions of fungal growth efficiency. 8 . TRANSIENT A N D OSCILLATORY PHENOMENA

When a steady state is perturbed by a point or permanent change, a period of re-adjustment is required before a new steady state becomes established. Thls re-adjustment period is manifest as a transient state, the analysis of which may uncover control mechanisms for growth rate and provide practical information for the operation of steady-state

44

A. T. BULL AND A. P. J. TRlNCl

continuous-flow cultures. Hardly any studies of this sort have been made of filamentous fungi but the value of the approach has been amply demonstrated by work with yeasts. When the Monod model is applied to the analysis of perturbations resulting from stepwise changes in, say, the dilution rate or growthlimiting substrate concentration, a smooth transition towards the new steady-state biomass concentration is predicted. However, much more complex transient state kinetics usually are encountered, and the results of work by Gilley and Bungay (1967) with Saccharomyces cerevisiae clearly illustrate this point. Thus, an abrupt change in D caused the yeast population to oscillate in size, and the time required for the oscillations to damp out was related to nutrient concentration and to the size and range of the change in dilution rate. Comparable experiments with moulds have not been reported, but M. E. Bushell and A. T. Bull (unpublished data) have analysed the response of glucoselimited populations of Aspergillus nidulans to step-up and step-down changes in dilution rate. Under step-up conditions (see Fig. 41, whereas the increase in dilution rate was accommodated within three culture

UI 0

.-s m

Time ( h )

FlG. 4. Oscillations in biomass concentration, efficiency of protein synthesis and specific growth rate during a step up in dilution rate in a chemostat culture of AJpergillus niduluns 13 mel. At zero time, the dilution rate of the steady-state culture ( D = 0.125 ti') was increased to 0.175 h-'. Steady-state conditions at the higher dilution rate were established after about 12 h, that is after about three doubling times. Transient values ofp were calculated from the equation: dxldt = p X - DX.The efficiencyof protein synthesis ( E ) is given by the expression g protein g RNA-' multiplied by the specific growth rate.

'

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doublings, the protein synthesis requirements for the higher growth rate were met almost immediately (see Section IX, A; p. 72). Although the response to an altered growth environment mav be rapid, as in thc case of protein synthesis, other metabolic systems may take longer to stabilize, and this is seen in the flux of metabolites through the pentose phosphate pathways in A . nidulans (Carter and Bull, 1969). Following a stepwise decrease in the dissolved oxygen tension troni 150; to 2 1 111111 Hg, the respiratory quotient and activitv of the pentose phosphate pathway oscillated, and only attained new steady-state levels after seven culture doublings; the biomass concentration reached steadv state in about 60% of this time. I t is important to realize, therefbre. that even if’ biomass and growth-limiting substrate concentrations have reached steady states other culture parameters may still be exhibiting transient beha\ w u r . Pye (1969) made a very detailed investigation ofglycolvtic flux oscillations following the addition of glucose and trehalose to. yeast suspensions. Oscillations in NADH, concentration were thought to be induced by the allosteric activation of phosphofruktokinase. Such a control mechanism produced a predictable short-cycle oscillation and, due to the point stimulation (hexose addition), the oscillations eventually damped out. More recently Poole et al. (1973) have attempted to resolve oscillatory respiratory activity in synchronously dividing populations of Schizosaccharomyces pombe. Respiration was considered to comprise two components, only one of which oscillated and, although the control mechanism has not been identified, i t possibly relates to a Pasteur effect. Finally the phenomenon of rhythmic mycelial growth of fungi on solid and non-agitated liquid media has long been known and examined by mycologists. Nectria cinnabarina, for example, produces colonies consisting of concentric rings or spirals, about 1 mm apart, that define growth rate oscillations with a 16-hour period. Winfree (1973) has proposed a number of models that attempt to interpret rhythmic growth patterns of this type; he considers that the most acceptable model is based on a unique unstable state, established at spore germination, persisting in the peripheral growth zone of the colony and damping out rapidly behind this zone. Although the nature of this metabolic instability is imprecisely defined at present, it appears to be focused o n an oscillating flux of carbohydrate through catabolic and hyphal wall-synthesizing pathways (Lysek and Esser, 197 1); ’

46

A. T. BULL AND A. P. J. TRlNCl

Bornefeld and Lysek, 1972. The interested reader will find the stimulating papers of Winfree (1970,1973)a valuable source offurther information and ideas. A circadian rhythm’ of conidiation has been recognized for many years in Neurosporn cru.wz, and several classes of morphological mutants liave been described in which the periodicity has been disturbed. In the (01 niutaiits, for example, a partial block in pentose phosphate pathway has been implicated as a specific biochemical lesion (Brody, 1970; see also Section VIII, C; p. 70). Halaban (1975) recently has described a palch mutant that produced alternatively, o n a medium containing 5% glucose, sparse and dense aerial hyphae with a periodicity of about 50 hours but had retained the wild- type circadian rhythm of conidiation. She also showed that the period was a function of glucose concentration, and that it decreased with decreasing glucose concentration. In this niutant, the biochemical lesion was found to involve a loss of the low-affinity glucose transport system (see Section VII, C ; p. 5 3 ) . The causal relationship between this transport deficiency and the morphological growth rhythm has not been explored but its basis could reside in an altered plasma membrane. c . “MACROREGULATION”

O F GROWTH

A growth regulatory system recently has been described in Neurospora c r a m that may be an example of a distinct class of overall or macroregulatory” phenomena. Reissig and Glasgow ( 197 1) have proposed that a galactosaminoglycan found extracellularly, and in the walls, of N . c r a m is a mediator molecule in such a regulatory mechanism. The polysaccharide (MW about 1 x lo6 daltons) produced by colonial mutants and by wild-type strains causes a severe decrease in the growth rate when added to young cultures. The effects induced by galactosaminoglycan included vacuolation, agglutation, a lethal effect on conidia and an impairment of transport functions, from which data the plasma membrane appears to be a probable candidate for the site of interaction. Conidia exposed to galactosaminoglycan lose the capacity to transport substrates and to form colonies. Subsequently Glasgow and Reissig (1974)have investigated the killing kinetics of galactosaminoglycan and found that one to three “hits” were lethal. However, each “hit” required about lo5 molecules of the glycan. The low probability of a “hit” per molecule suggests that only a few of the total binding sites for galactosaminoglycan function as receptors for (6

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lethal events. The biochemical nature of the lethal event awaits elucidation. Similarly the occurrence of this phenomenon in fungi has not been surveyed but it is interesting to note that galactosaminoglycans are wall components of other fungi such as Aspergillus nidulans (Bull, 1970). D . MAINTAINED A N D STARVATION STATES

When a fungal population becomes depleted of nutrients its subsequent course of development depends on which nutrient is first exhausted. The classic study of differential nutrient exhaustion on fungal growth is that of Borrow et al. ( 1961) on Gibberellafujiikuroi. Their experiments, made in batch fermenters, revealed that, depending on the sequence of nutrient depletion: (1) growth could continue at a linear rate; (2) growth could cease but synthesis of storage and secondary metabolites continue, and (3) exhaustion of endogenous substrates could lead to autolysis. Lysis could be prevented by supplying carbonand energy-depleted populations with an appropriate substrate at the maintenance rate. The behaviour of non-growing, carbon-starved and maintained cultures has been intensively analysed with Penicillium chrysogenum and Aspergtllus nidulans 224. (Trinci and Righelato, 1970 ; Bainbridge et al., 197 1). Following the onset of glucose deprivation in glucose-limited chemostat cultures of these fungi, the biomass concentration declined with a half-life of about 50 to 60 hours. Massive degradation ofprotein, RNA and DNA had occurred after 120 hours and post-starvation levels were about 25% (P. chysogenum)and40% ( A . nidulans) of zero-time values. In contrast, changes in carbohydrate were much less significant and reflected the lack of carbon storage materials in the mycelium; little change in wall thickess was reported. Catabolic activities (q,,, TCA cycle flux) declined rapidly during the initial 10 hours of starvation. Attempts were made to monitor viability changes in starving A . nidulans cultures, and even after 96 hours the viability index was 0.6. However, viability determinations on filamentous organisms are difficult to make, and the value obtained is likely to have been an overestimate. When the glucose feed rate to chemostat cultures of the two species was lowered to the maintenance ration, fungal dry weight remained constant and, after fluctuating slightly, so did the protein and carbohydrate contents. Under these conditions the Penicillium mycelium differentiated, and conidia were produced in large numbers. This

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A. T. BULL AND A. P. J. TRlNCl

difyerentiation was reflected in the rapid and large turnover in RNA and DNA. On the other hand, the RNA content of maintained Aspergillus cultures varied little, and conidiation was very slight. Respiratory activity decayed following a lowering of the glucose supply rate to maintenance levels but the respiratory quotient remained close to unity. However, considerable changes in glucose catabolism were observed in A . nidulans (Bainbridge et al., 197 1). During the preceding steady state about 20% of the glucose was metabolized via the pentose phosphate pathway, but within 10 hours of switching to a maintained state the flux through the Embden-Meyerhof pathway was essentially 100%. This change is related to the minimal biosynthetic requirement for NADPH, in maintained populations. The capacity to synthesize penicillin was lost from maintained cultures of P. chysogenum at a rate related inversely to the previous steady-state dilution rate (Pirt and Righelato, 1967). Not all secondary biosyntheses decay in this fashion as demonstrated by the continued production of melanin by A . nidulans.

VII. Transport- Controlled Features of Growth A . TRANSPORT-LIMITED GROWTH

Detailed studies of transport phenomena in fungi have been made with relatively few species and most information is related to Saccharomyces cerevisae and other yeasts, Aspergillus nidulans and Neurospora crassa. Nevertheless a wide range of transport systems has been reported in fungi (Rothstein, 1966; Burnett, 1968; Kotyk and Janacek, 1970; Jennings, 1974). It is not out intention to re-iterate this body of information but, instead, we wish to explore the relationship between transport, and its control, and growth. In addition we will emphasize the significance of mechanisms which fungi possess for modulating their transport and assimilation processes in response to the growth environment. The kinetic treatment of transport-limited growth given by Van Uden ( 1967, 1969)is the most useful starting point for these discussions, but to date few other workers have followed his lead. Van Uden analysed transport-limited growth from the viewpoints of diffusion kinetics (passive diffusion mechanism) and Michaelis-Menten kinetics

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(facilitated diffusion and active transport mechanisms). For transport systems obeying Michaelis-Menten kinetics it can be shown that:

where kmaXis the maximum transport rate of the growth-limiting nutrient, Y is the yield factor, S, and S, are the concentrations of growth-limiting nutrient in the medium and the organism, respectively, and K,, and K,, are respectively the Michaelis constants of transport of the limiting nutrient into and out from the organism. Because the value of S2 is usually very low, the second term in parentheses can be omitted, while a maintenance term can be added to p in order to correct for the variation in yield with growth rate. Similarly Lax will be a function growth rate because of concomitant changes in organism size (and hence surface area). Tests of this model have been made with a non-growing yeast suspension and it was found that: (a) the rate of glucose assimilation by aerobic populations was limited by the rate of phosphorylation (by hexokinase) and was not transport-limited; and (b) assimilation by anaerobic populations was transport-limited and subject to competitive inhibition by L-sorbose (Van Uden, 1967). Moreover, transport-limited growth was observed in glucose-limited chemostats under anaerobic conditions and under aerobiosis with a respiratory-deficient strain. The K , (glucose)value was lower by a factor of ten than previously published values (e.g. Van Steveninck and Rothstein, 1965, reported a value of about 5 x M ) and it is tempting to speculate that the affinity difference is a consequence of selection under circumstances of substrate limitation. Van Uden ( 1969) has also examined diffusion transport of growthlimiting nutrients and found that in neither glycerol- nor oxygenlimited populations of Candida utilis was the diffusion process a significant control on the specific growth rate. However, it should be recalled that diffusion-limited situations are readily established in pelleted cultures of fungi. Virtually nothing is known about transport-limited growth of filamentous fungi, but the establishment of transport-limited cultures offers many interesting experimental possibilities, some which are clearly suggested from the work of Von Meyenburg ( 197 1) on bacterial growth. For example, mutants having increased K m values for various nutrients can be used to produce high densities of organisms in batch cultures under growth rate-limiting substrate concentrations.

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B . TRANSPORT REGULATION

Nutrient uptake is effectively controlled in fungi at the levels of transport activity and synthesis and clearly, if uptake is under metabolic control, nutrient assimilation (and hence growth) will be increasingly efficient. Fine control of transport in fungi frequently is achieved by feedback inhibition. Uptake of amino acids and pyrimidines, for example, is usually subject to very specific feedback inhibition. A detailed analysis has been made of the way by which acetate prevents utilization of a large number of sugars by Aspergillus nidulans (reviewed by Romano, 1973). Acetate exerts its effect on sugar transport, not on sugar metabolism, and acetyl-CoA appears to be the most likely regulator molecule. In this context it is interesting to speculate that the potentiating effects of acetate on secondary metabolite synthesis in fungi may be due to a sparing of sugar uptake rather than to its provision as a precursor. Sugar transport into filamentous fungi seems not to involve a phosphorylation step. Indeed, phosphorylation of sugar analogues such as Z-deoxy-D-glucose, which was taken as presumptive evidence of group translocation, has been shown conclusively to be a post- transport event mediated by intracellular kinases (Brown and Romano, 1969). Repression control of transport affects uptake, by fungi, of various nutrients including amino acids and vitamins (Wiley, 1970; Hiitter, 1973; Rogers and Lichstein, 1969). Amino-acid transport has been studied extensively in Neurospora crassa, a fungus that has three specific systems for neutral, basic and acidic amino acids, and a fourth nonspecific system. The operation of these transport systems is affected by the growth environment, and especially interesting is the fact that nonspecific transport was induced under conditions of nitrogen exhaustion (Benko et al., 1969). Ammonium ions probably repressed the latter system when there was a sufficiency of nitrogen, and the internal ammonium ion concentration fell four-fold following starvation. The results of the work of Wiley (1970)on tryptophan transport in N . c r a m has shown that, as in bacteria, repression of fungal transport systems caused a marked decrease in the concentration of specific binding proteins. Vitamin nutrition can exert a profound influence over amino-acid transport and the maintenance of pool sizes and, consequently, may determine whether or not growth is balanced. Studies of this kind have related mainly to yeasts (e.g. Moat et al., 1969) but biotin deficiency

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decreases the transport of glucose and ammonium ions into Aspergillus nidulans (Desi and Modi, 1975). The precise effect of biotin in the Aspergillus system is not known; the glucose-binding proteins from biotin-deficient and replete mycelia appeared to be identical, therefore the effect may be one of repression or may be indirect. As we have already noted, vitamin uptake itself may be under repression control. Thus the biotin uptake system of Saccharomyces cerevisiae is repressed by high exogenous concentrations of the vitamin (Cicmanec and Lichstein, 1974) and turnover of the system occurs under such conditions of repression. However, repression of synthesis enabled only a slow adaptation to a changing exogenous biotin status and rapid responses were accommodated by a biotin excretion mechanism. Cicmanec and Lichstein (1974) suggest that excretion prevented biotin accumulation during the period when repression and turnover of the transport system was occurring, and before it was fully repressed. Indeed, excretion may be a widespread control process in the regulation of sizes of fungal metabolite pools, even when uptake of the compound is controlled by a specific feedback inhibition. Excretion will be an especially important means of pool regulation under circumstances of endogenous overproduction. Regulation of amino acid and pyrimidine pools in yeast, via excretion, has been discussed by Grenson ( 1973) but data on emux mechanisms are scarce. The work of Jennings and his colleagues has revealed a particularly interesting control over the rate of sugar uptake by the marine fungus Dendvphiella salzna that simultaneously maintains osmotic homeostasis in the mycelium. 3-0-Methyl glucose was not metabolized by D . salzna but was accumulated by an active transport mechanism. However, the mycelial concentrations of arabitol and mannitol fell as the methyl glucoside was taken up, so that the total soluble carbohydrate pool size remained remarkably constant (Jennings and Austin, 1973). A channelling of polyols into polysaccharide synthesis appeared to occur in proportion to the amount of sugar transported. A stable internal osmotic pressure is of significance in fungi, where apical hyphal rupture is readily induced by osmotic shock. Sugar transport in Dendryphiella also is inhibited by acetate, but whether acetyl-CoA or a polyol is the regulating compound remains to be elucidated (Holligan and Jennings, 1973). Polyols are synthesized during acetate utilization, and if they do inhibit sugar transport their significance in the regulation of internal osmotic pressure is reinforced. Repression of nitrogen-metabolizing enzymes and nitrogen-

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A. T. BULL AND A. P. J. TRlNCl

transport systems by ammonium ions, or other nitrogen-containing metabolites, is a widespread phenomenon in fungi and illustrates the tightly integrated nature of regulatory mechanisms. Our understanding of ammonia regulation is especially detailed in Aspergillus niduluns, where two control systems have been revealed: (i) the extracellular ammonia concentration determines the rate of uptake of Lglutamate, urea and thiourea, and the activity of certain enzymes such as nitrate reductase; (ii) the intracellular ammonia concentration determines the rate of uptake of ammonia. Ammonia was preferentially utilized by A. niduluns and A. clauatus when growth occurred on a mixture of nitrogen sources (Robinson et al., 1973, 1974) and one repercussion of this situation relates to the practice of using ammonia gas for control of pH value in commercial fungal fermentation. Thus, the major nitrogen source and/or supplementary nitrogen compounds may be inefficiently utilized due to ammonia repression, or inhibition of the respective uptake systems. Similarly it has been argued that in the absence of extracellular ammonia it may be disadvantageous for a fungus to utilize any single nitrogen source exclusively (Pateman et ul., 1973) and advantageous simultaneously to utilize nitrate, glutamate or purines if they are available. The latter systems are not regulated by intracellular ammonia until its concentration exceeds about 4 mM at which point efflux commences. One class of mutants (gdhA) lack the normal NADP-linked glutamate dehydrogenase and are derepressed for both types of ammonia regulation, and Pateman et al. (1973) have presented a unifying model of theie ammonia effects in which NADP-linked glutamate dehydrogenase assumes a regulatory function. Plasma membrane-bound NADPlinked glutamate dehydrogenase is thought to complex with extracellular, but not intracellular, ammonia and thereby regulates the category (i) processes. A second glutamate dehydrogenase regulatory complex may be formed with intracellular ammonia when the enzyme is displaced from the membrane and affects ammonia uptake. When Lglutamate was the sole, or main, source of carbon, ammonia regulation of glutamate transport was alleviated and NADP-linked glutamate dehydrogenase was rapidly turned over (Kinghorn and Pateman, 1974). Although Pateman’s hypothesis requires rigorous experimental testing, it is consistent with many of the established observations, and notably that gdhA mutants are not defective in any known transport function. However, Arst and Cove ( 1973) have challenged the

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idea of a direct role for NADP-linked glutamate dehydrogenase in this context, and favour the view that a product of the areA locus (mutations which produce pleiotropic loss of ability to use sources of nitrogen other than ammonia) plays a more direct role. Until more data are forthcoming, resolution of this control system cannot be made. C. MODULATION O F FUNGAL TRANSPORT PROCESSES

Major imbalances in sizes of metabolite pools can have serious consequences for microbial growth and may be growth retarding, or even lethal. Therefore, ability to modulate substrate uptake and metabolism in the face of a changing nutrient status confers considerable selective advantage on an organism. As we have seen, lowering the polyol pool in Dendryphiella in response to non-metabolizable sugar accumulation provides osmotic homeostasis. Fungi can also respond to large fluctuations in nutrient availability in other ways. First, they can synthesize and maintain a large excess of transport components; this is the situation with phosphate uptake by Rhodotorulu mbru organisms (Button et ul., 1973) in which half of the components are operative at a growth rate of 0.5 ,urnaxand only 4% at prnam Clearly the maintenance-energy requirement for such a system will be high. Alternatively, fungi have evolved high- and low-affinity systems for uptake of individual nutrients, the operation of which is mediated by their extracellular concentrations. Two transport systems for glucose are known in Neurosporu crussu that are distinguishable on the basis of their affinities. Scarborough ( 1970a, b) described a low-affinity facilitated diffusion system (apparent K,,, = 8 mM) that was operative when the fungus was grown in glucosecontaining media (about 9 g1-3. However, when growth occurred in media containing a similar concentration of fructose, or on a low concentration of glucose (about 0.2 g P I , an alternative high-affinity active transport system was induced (apparent K, = 10 pM). A similar dual glucose transport system has been detected in ungerminated conidia of this fungus (Neville et ul., 1971). The differential operation of these alternative uptake systems has obvious implications in the efficiency of growth and germination. It can be argued that, when glucose is plentiful, active transport is uneconomical and the constitutive facilitated diffusion system can maintain an adequate intracellular concentration. On the other hand, the high-affinity system enables effective uptake under conditions of severe glucose limitation.

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I t is interesting to note that sugar assimilation as well as transport may be modulated according to prevailing growth conditions. For example, Roberts ( 1970) has described two systems for lactose and galactose metabolism in Aspirgillus niduluns that are regulated by pH value. The first, operative in galactose-grown mycelia at pH 4 to 6.5, involved p-galactosidase and transphosphorylation, while the nature of the second system has not been established fully but may involve direct oxidation of galactose (Fantes and Roberts, 1973). The second system was operative at a pH value of 7.5. Fantes and Roberts pointed out that pgalactosidase activity was essential for conidial germination, though not for mycelial growth, but a mechanistic interpretation of these observations remains to be made. Recognition of this alternative route helps to rationalize certain inconsistent data (Paszewski et ul., 1970)on the regulation of lactose utilization. Sucrose assimilation by the ergot fungus Cluviceps purpureu also provides an interesting example of metabolic modulation. Dickerson ( 1972) reported that when sucrose was the sole carbon source it was metabolized via p-fructofuranosidase; two molecules of sucrose were transfructosylated to form a trisaccharide (F2-6G1-2F) and assimilable free glucose. Further glucose release was provided from a second transfructosylation of the trisaccharide with sucrose to yield a tetrasaccharide (F2- 1F2-6G1-2F)plus glucose. With the exhaustion of glucose these oligosaccharides may be hydrolysed or transglycosylated with release of fructose; fructose assimilation occurred only in the absence of glucose, and was growth inhibitory at a concentration greater than 30% (w/v). I t has been proposed (Nisbet, 1975)that the transfer system regulates fructose concentrations so as to maintain non-inhibitory levels and to provide a carbohydrate reserve. Furthermore, the transferase system was operative under circumstances of low energy demand and low concentrations of free sugars; a hydrolytic system replaced transglycosylation under conditions of high catabolic activity (e.g. at the time of sphacelial to sclerotial differentiation in parasitic and axenic cultures), or when the availability of free sugars was high. Modulation of glycerol-assimilation pathways has been described for bacteria (Neijssel et uf., 1974) and similar high- and low-efficiency glycerol-uptake processes might exist in fungi. For example, glycerol kinase synthesis in Neurosporu crussu is induced by low temperature, whereas at normal growth temperatures (26OC) the enzyme is rapidly inactivated (North, 1973, 1974). The latter may represent a control

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system, albeit an uneconomic one, for regulating uptake of glycerol under circumstances where a simple diffusion process is adequate for the needs of the organism. Transport of anions, such as sulphate and phosphate, is known to occur via separate systems in a number of fungi. Sulphate transport in Neurospora crasa, for example, is mediated by two distinct permeases; permease I has low substrate affinity ( K , = 200 pM), and is associated with conidia, whereas permease I1 has a much greater affinity ( K , = 8 pM) and is mycelial in location (Marzluf, 1970). Synthesis of these transport systems is repressed by methionine. The specificity of fungal sulphate permeases has some interesting consequences for growth. In N . crmsa, chromate competes for sulphate permeases (Roberts and Marzluf, 197 1) while SO:-, S,Oi; SeOi-and MoOf enter Aspergtllusnidulansby a common permease. Tweedie and Segal( 1970) have argued that molybdate transport by the sulphate uptake system may be disadvantageous because the latter was controlled by sulphur nutrition; under conditions of sulphur sufficiency, the sulphate permease was repressed and molybdate assimilation via this route prevented. Uptake of Mo02,- by the sulphate- transport system also ceased under acidic conditions (below a pH value of 5 ) . Consequently molybdate uptake by A. nidulans may occur by some additional system. More recently Reinert and Marzluf (1974) have shown that N . c r a ~ ~possesses a a low-affinity transport system for glucose 6-sulphate that is distinct from both the glucose and the sulphate transport systems, and again is methionine repressible. Glucose 6-sulphate can serve as a sole source of sulphur but is not a significant source of carbon. An interesting example of modulation of transport systems is that for the uptake of phosphate by N . crassu. Two phosphate-transport systems have been described, one of which has high afinity, and is derepressed under phosphorus starvation conditions; the other lowaffinity system ( K m = 123pM) is sufficient to support exponential growth under conditions of phosphate sufficiency (Lowendorf et ul., 1974). These authors calculated that a phosphate flux of about 1.20 mmol. 1 cell water-' min-' would be necessary to support exponential growth of N . crassa at a rate of 0.27 h-l, and observed a flux (J)of 1.63 mmol . I cell water-: at pH 5.8, and an external phosphate concentration of 37 mM. A further finding of considerable interest was the dependence of K , on the pH value of the medium. Over the range of pH values from 4.0 to 7.3, theJ,,, oscillated around a mean value of

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1.42 mmol. 1 cell water-'min-', but the K , increased from 10 pM to 3.62 mM. Does the dependence reflect two separate transport systems? The data of Lowendorf et al. (1974) eliminated such an explanation, and, of the single-system models considered, OH- or H+modification of the transport mechanism was most consistent with the experimental evidence. The OH- ions could compete with PO:- at the binding sites, or PO:- may be cotransported with H+; in either case a high external pH value would affect transport. It should be emphasized that the effects of changes in external pH value on the efficiency of nutrient uptake may be explicable in terms other than those proposed by Lowendorf et al. (1974). Thus, a carrier that can exist in protonated and unprotonated forms will have different K , values for its substrate(s1,and the proportions of the two forms will be pH-dependent. An elegant illustration of such a modulation is found in the work of Tanner (19741, albeit with a microalga, on sugar transport. Over the range of pH values from 6.0 to 8.8, the amount of high-affinity (i.e. protonated) carrier ( K m = 2 10 pM) decreased commensurately with an increase in low-affinity (i.e. unprotonated) carrier ( K , = 50 mM). To our knowledge comparable systems have not yet been reported to occur in fungi. As a final example of mechanisms which fungi have evolved for modulating their assimilation of nutrients we will consider ammonia. Among the possibilities for ammonia assimilation in protists are routes involving aspartase, NADP-linked glutamate dehydrogenase (GDH) and the glutamine synthetase-glutamate synthase (GS/GOGAT) system. In bacteria, the significance of the GS/GOGAT system lies in its efficiency for scavenging ammonia in ammonia-limited environments; the apparent K , values for GDH and GS in Aerobacter aerogenes are 10 mM and 0.5 mM, respectively (Meers et al., 1970). In a recent review, Brown et al. (1974) stated that the only clearly demonstrable pathway of ammonia assimilation in moulds and yeasts is the synthesis of glutamic acid via glutamate dehydrogenase and that GOGAT had not been demonstrated in these organisms. Brown and Stanley (1972) had suggested a number of ways in which the relatively high K , of glutamate dehydrogenase for ammonia is counteracted in yeasts: (a) NH, rather than NH,+ is the native substrate, and the variation in K , with pH value ensures that the higher the intracellular pH value the more active is the glutamate dehydrogenase at low extracellular ammonia concentrations; (b) synthesis of a large excess of

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glutamate dehydrogenase also enables effective ammonia assimilation. Subsequently GOGAT has been demonstrated in several yeast genera (Roon et al., 1974; Johnson and Brown, 19741, but its presence in mycelial fungi has not been definitely established. Roon et al. (1974) found that the apparent K,,, of glutamate dehydrogenase and GOGAT were approximately equal (about 10 mM) in Saccharmyces cerevisiae and they suggested that the glutamate synthase (GOGAT) had a function that was auxiliary to glutamate dehydrogenase, or else had a different subcellular location. However, Johnson and Brown ( 1974) measured the contents of glutamine synthetase, as well as GOGAT and glutamate dehydrogenase, in several Schizosaccharomyces species, and concluded that assimilation of low or growth-limiting concentrations of ammonia was the likely function of the GUGOGAT pathway, and that the glutamate dehydrogenase route would operate in environments containing an excess of ammonia. VIII. Metabolic Control in Fungi A.

INTERMEDIARY METABOLISM

1. Fermentative versus Oxidative Metabolism

Most fungi are strictly aerobic organisms that can grow at wide extremes of dissolved oxygen tension but, additionally, a fav authenticated examples of facultative anaerobic species are known (Bull and Bushell, 1976). However, many fungi can ferment carbohydrates anaerobically with the formation of ethanol, lactic acid and other products, and the flux partitioning of substrates between the various glycolytic pathways is considered below. The rate of carbon dissimilation via fermentative and oxidative pathways in fungi is sensitive to oxygen availability, and especially to the concentration and nature of the carbon source. Until quite recently, the regulation of these pathways and its relationship to growth rate was known only for yeasts. Thus, early studies of batchgrown Saccharomyces cerevisiae organisms (Polakis and Bartley, 1965 ; Polakis et al., 1965; Gorts, 1967) demonstrated that, although glucose inhibited synthesis of enzymes of the TCA cycle, glyoxylate cycle and electron- transport chain, fermentative metabolism could provide a high rate of energy generation and support high specific growth rates

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(about 0.4 h-'1. Following glucose disappearance, the accumulated ethanol was assimilated by the now derepressed oxidative pathways. These workers also made the important discovery that carbon substrates that supported lower specific growth rates (compared with glucose) also caused much less repression of respiration. Van Wijk ( 1968) extended these investigations, and examined catabolite repression of respiratory enzymes and a-glucosidase in Sacch. ceverisiae and Sacch. carlsbergensis. He found that a strong positive correlation existed between the specific activities of a-glucosidase and enzymes of the TCA cycle during growth on various sugars, but oxygen availability did not change the correlation; i.e. the correlation was not due to a direct effect of respiration on a pool of repressing catabolite(s1.Subsequently glucose-limited chemostat experiments confirmed the relationship between the fermentation-oxidation state and specific growth rate (Fiechter and von Meyenberg, 1966; Beck and von Meyenberg, 1968). The degree of fermentative metabolism increased with dilution rate and, at dilution rate values greater than 0.2 h-l, the q 0 2was repressed and the respiratory quotient rose from unity to 7 (at D = 0.42 h-'). Of course, not all yeasts are sensitive to catabolite repression of respiration, and Kluyveromycesfragilis, for example, exhibits the Pasteur effect when grown on glucose (Chassang-Douillet et al., 1973). Detailed studies of fermentative-oxidative transitions in filamentous fungi have been made in continuous cultures of Aspergillus nidulans 224, and with two phycomycetes, namely Mucor genevensis and Cokeromyces poitrarsi. With glucose-limited cultures of A . nidulans, a qualitative change in carbon catabolism was observed at a dilution rate of 0.055 h-', corresponding to a stimulation of fermentation (Carter and Bull, 1969; Carter et al., 197 1); a decreased yield from glucose accompanied this change. Similarly, a shift to fermentative metabolism occurred at dissolved oxygen tensions below 30 mm Hg (Carter and Bull, 1969). An interesting feature of these shifts towards fermentation in A . nidulans was the differential enhancement of the pentose phosphate pathway compared to the Embden-Meyerhof pathway (see p. 59). Rogers et al. (1974) analysed the effects of oxygen on carbon metabolism in glucose-limited and glucose-excess continuous cultures of M . geneuensis. Under glucose-limitation (SR = 5 g l-'), a shift from fermentative to oxidative metabolism accompanied an increase in the dissolved oxygen tension (DOT) from < 0.1 to about 8 mm Hg, and concomitantly the yield increased five-fold. Respiration was severely

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repressed under conditions of glucose-excess (S, = 60 g 1-’ ; S = 20 to 40 g 1-l) even when the DOT was maintained at approximately 50 mmHg. An essentially similar change in glucose catabolism, with a change in DOT, was observed with cultures of C. poitrassi (Rogers and Gleason, 1974). Considerable evidence exists linking fermentative to oxidative transitions with yeast-mycelium dimorphism in fungi, particularly in members of the Mucorales. In other words, “filamentous morphology in fungi might be regarded in many instances as a morphogenetic expression of the Pasteur effect” (Terenzi and Storck, 1969). Terenzi and Storck induced a yeast morphology in M . rouxii by exposure of the organisms to phenethyl alcohol, an agent that did not affect the q 0 2 value or cytochrome synthesis but is believed to uncouple oxidative phosphorylation. Phenethyl alcohol progressively repressed conidium and mycelium development in Neurospora crassa (Turian et al., 19721, and increasingly restricted oxidative metabolism. In the absence of inhibitors, these morphogenetic events seem to be regulated specifically by glucose and not by the oxygen status of the medium (Rogers et al., 1974; Rogers and Gleason, 1974; Schulz et al., 1974; Friedenthal et al., 1974).

2. Flux Partitioning between the Embden-Meyerhof Pathway and the Pentose Phosphate Pathways A variety of glycolytic sequences are known to occur in fungi, including the Embden-Meyerhof (EM) pathway and the hexose monophosphate pathways (such as the pentose phosphate (PP) and Entner-Doudoroff pathways), and usually two such pathways (most often the EM and PP) operate simultaneously. When the EM and PP pathways are present together, the former usually predominates, and rather less than 30 to 40% of the carbon flux is via the PP route. The factors regulating partition of carbon between the alternative glycolytic pathways are not fully known; moreover little study has been made of their operation under varying growth conditions. In discussing this subject it is useful to reiterate the particular synthetic functions of the pentose phosphate pathway, viz. provision of pentose sugars, especially ribose 5-phosphate for nucleotide synthesis, and provision of NADPH, for synthesis of glutamate, fatty acids and sterols. Blumenthal ( 19651, in his review of fungal glycolysis, remarked that

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there had been no systematic studies made of the effects of physical and physiological variables on the relative functioning of glycolytic pathways. During the intervening decade, however, a few such analyses have been reported. Thus, Carter and Bull (1969) found that the proportion of glucose catabolized via the pentose-phosphate pathway by glucose-limited chemostat populations of Aspergzllus nidulans 224 (nitrate as nitrogen-source) varied directly with dilution rate up to a maximum of 37% at Furthermore, and in contrast to Escherichia coli (Bengali and Hempfling, 19721, enzymes of the pentose-phosphate pathway, such as glucose 6-phosphate dehydrogenase, were induced by glucose (Carter and Bull, 1969) and the flux through the pathway increased dramatically following transient rises in the glucose concentration (Carter and Bull, 1969; 1971). Subsequently Smith and his colleagues (Ng et al., 1974) extended this type of analysis to A. niger, and obtained analogous results with glucose-limited organisms (ammonia as nitrogen-source) but found citrate-limitation partially repressed both the EM and pentose-phosphate pathways. Earlier experiments by Smith and Ng (19721, using batch cultures, suggested that phosphorylating enzymes acting before fructose diphosphate cleavage (i.e. hexokinase and phosphofructokinase) appeared to regulate glycolysis, and application of the cross-over theorem (Chance et al., 1958) for identifying control sites in glycolysis revealed a distinct control point between fructose 6-phosphate and fructose 1,6-diphosphate. Phosphofructokinase is subject to multivalent allosteric control by a number of effector molecules, including ATP and citrate. Thus, excessive flux through the EM pathway is likely to be prevented at the level of phosphofructokinase by ATP and, because the equilibrium constant of the isomerase is close to unity, fructose 6-phosphate will not accumulate; instead glucose 6-phosphate is channeled into the pentose-phosphate pathway. The observed effects of citrate (Ng et al., 1974) also may be interpretable in terms of phosphofructokinase inhibition. Recently Mian et al. ( 1974) have reported similar growth rate-dependent fluxes of glucose through the different glycolytic pathways in yeasts. As the dilution rate of a glucose-limited chemostat culture was increased from 0.1 to 0.5 h-I, the flux through the pentose phosphate pathway increased linearly from about 25% to 60%. When steady-state cultures of A. nidulans were deprived of an exogenous carbon and energy source, or when .such cultures were

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supplied with a glucose feed only slightly in excess (1.5 times) of the maintenance ration, the flux through the pentose-phosphate pathway rapidly decayed and within, respectively, 4 and 10 hours the flux was almost undetectable (Bainbridge et al., 1971). These changes were entirely consistent with those of the macromolecular composition of starved and maintained populations, and reflect their negligible biosynthetic demand. Data on the effect of dissolved oxygen tension on flux partitioning is rather confused (see Blumenthal, 1965) and only one systematic examination has been described (Carter and Bull, 1969). When the dissolved oxygen tension of steady-state cultures ( D = 0.05 h-') of A . nidulans was decreased stepwise, relatively small flux changes occurred in the EM and pentose-phosphate pathways until values between 5 and 10 mm Hg were attained. At this point, however, the flux through the pentose-phosphate pathway rose from about 30%, and reached a maximum of 55% as the dissolved oxygen tension approached a critical value (1.75 mmHg; Carter and Bull, 1971); simultaneously, the specific activity of glucose 6-phosphate dehydrogenase increased three-fold. The sharp rise in pentose-phosphate metabolism at these low DOT values was paralleled by the appearance of NADPH,-linked dissimilating nitrate reductase activity (see below; p. 62). The pentose-phosphate pathway appears to play a vital role in differentiating fungal cultures. One of the first observations of this correlation was made by McDowell and De Hertogh ( 1968) who described a preferential stimulation of the enzymes of the pentose-phosphate pathway during sporulation of Endothia parasitica in submerged culture, while Carter and Bull (1969) revealed an identical situation in conidiating batch cultures of Aspergillus nidulans. Comparable studies of conidiation in A . niger (Smith et al., 197 1) initially did not corroborate this presumed association between a high rate of flux through the pentose phosphate pathway and morphogenesis. However, L-glutamate was a major component of the growth medium used by Smith et al., (197 1) and would likely relieve the increased demands for NADPH, occurring during sporulation. Subsequent experiments with nitratecontaining media produced results that were in full agreement with those of McDowell and De Hertogh ( 1968) and Carter and Bull ( 1969) (Ng et al., 1972). The last authors found that highest EM pathway activity occurred in vegetative cultures. Secondary metabolite formation in penicillia and aspergillus was favoured when glucose catabolism

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proceeded via the EM route (Bu’Lock et al., 1965; Carter and Bull, 1969; Shih and Marth, 1974). Finally, what is the function of nitrate in regulation of the pentose phosphate pathway? Apart from the work already described, nitrate has been shown to enhance the pentose-phosphate pathway in other fungi such as Dendryphiella salina (Holligan and Jennings, 1972). Recent data from Cove’s laboratory suggests that the availability of NADP is not a primary modulator of pentose-phosphate pathway activity, as proposed by Osmond and Ap Rees (1969). Hankinson and Cove (1974) and Hankinson (1974) have postulated that a product of the nirA gene in A . nidulans is necessary for synthesis of nitrate reductase, and of pentose-phosphate enzymes. The nir product is inactivated by nitrate reductase per se, but when nitrate is available it binds to nitrate reductase and hence the inactivation of the nir product is relieved. This effect of nitrate is augmented by its inhibiton of mannitol I-phosphate dehydrogenase (MDP)activity (Hankinson and Cove, 19751, an enzyme that, in vim, has fructose 6-phosphate reductase activity. Assuming that recycling of fructose 6-phosphate occurs via the pentose-phosphate pathway when A . nidulans is grown on nitrate, glucose-6-phosphate isomerase is essential for the recycling. However, mannitol phosphate dehydrogenase competes with the isomerase for substrate and nitrate-directed inhibition of the former enzyme may be important in alleviating such competition. B. ANAPLEROTIC METABOLISM

Anaplerotic sequences have evolved as a means of replenishing the supply of intermediates of the TCA cycle that are drawn off for biosynthetic purposes, thereby preserving the energy-generating function of the cycle. In fungi, anaplerotic metabolism is provided by : (i) the glyoxylate cycle; (ii) acetyl-CoA synthetase activity; and (iii) direct fixation of carbon dioxide. A variety of acceptor molecules for carbon dioxide have been reported to be present in fungi including pyruvate, phosphoenolpyruvate, acetyl-CoA and 3-methylcrotonyl CoA. The “malic” enzyme (NADP-linked t-malate enzyme) now is considered not to have an anaplerotic function, but to have catabolic activity and to be significant in the generation of NADPH,. 1. The Glyoxylate Cycle The conversion of isocitrate, by isocitrate lyase, to succinate and glyoxylate, and subsequent condensation of the latter with acetyl-CoA,

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enables the decarboxylating reactions of the TCA cycle to be bypassed and a net synthesis of succinate to occur. The glyoxylate cycle operates in fungi when growth occurs on acetate, and usually is inactive in fungi grown on a carbohydrate. For example, Aspergillus nidulans developed an active glyoxylate cycle when grown in acetate-containing media (Armitt et al., 19701, but when glucose was present (even the very small concentrations found at low dilution rates in glucose-limited chemostats; Carter, 19681, the cycle was inoperative. However, not all carbohydrates repress isocitrate lyase synthesis, and acetate was utilized preferentially when gresent in the medium along with sucrose (Armitt et al., 1970); nevertheless, it has to be pointed out that addition of sucrose to an acetate-containing medium prevented synthesis of glyoxylate-cycle enzymes in Neurospora crassa (Flavell and Woodward, 197 1). The question of whether the cycle is under induction or repression control has received some attention and, although the evidence has been somewhat conflicting, it is generally accepted that acetate does not act as an inducer. On the contrary, acetate metabolism probably effects removal of metabolite(s) that repress synthesis of glyoxylate-cycle enzymes and Kornberg ( 1966) concluded that phosphoenolpyruvate (or a related intermediate) was the repressor in bacteria. Data on the co-ordinacy of isocitrate lyase and malate synthase regulation in fungi has been equivocal (see Armitt et al., 1970; Flavell and Woodward, 197 11, and this suggests that effectors additional to phosphoenolpyruvate (PEP) may be involved in controlling their synthesis. The activity, as well as the synthesis, of the lyase in N . crassa may be regulated by phosphoenolpyruvate (Sjogren and Romano, 1967). 2. Carbon Dioxide Fixation The stimulatory effects of carbon dioxide on fungal growth have been reported on many occasions, and for a wide range of taxonomic and ecological groups (Rockwell and Highberger, 1927 ; Barinova, 1954; Cantino and Horenstein, 1956; Macauley and Griffin, 1969; Harvey and Hodgkiss, 1972; Hartman et al., 1972). For example, growth of Verticillium albo-atrum was very severely curtailed in a carbon dioxide- free atmosphere when glucose and glycerol were the carbon sources (94% and 97% decreases, respectively) whereas deprivation of carbon dioxide did not affect growth on TCA-cycle intermediates (Hartman et al., 1972). Under the latter conditions, the glyxoylate cycle is operative and fulfils an anaplerotic function. The essential role of carbon dioxide-fixing systems in fungal growth on carbohydrates also

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can be gauged from the properties of mutants. Skinner and Armitt ( 1972) produced pyruvate carboxylaseless mutants of Aspergillus nidulans that would not grow on sugars alone, but would do so when the medium was supplemented with amino acids that could furnish C, intermediates. A number of enzymes have been implicated in carbon dioxidefixation in fungi, and of these pyruvate carboxylase (E.C. 6.4.1.1.) has been reported most widely. Phosphoenolpyruvate carboxylating activity also has been observed, but the identity of such activity as PEP carboxylase (E.C. 4.1.1.3 1.) or PEP carboxykinase (E.C. 4.1.1.32.) is still subject to debate. Thus, Bushel1 and Bull (1974b) claimed that the high rate of PEP carboxylation by A. nidulans growing on glucose was due to PEP carboxylase and not to a carboxykinase working in reverse direction; PEP carboxykinase is usually ascribed a decarboxylating role (Scrutton, 197 1) and is important in gluconeogenesis. Hartman and Keen ( 1973) originally considered that carbon-dioxide tixation in V . albo-atrum was mediated by pyruvate carboxylase and PEP carboxykinase, but in the light of further research have concluded (Hartman and Keen, 1974b)that in uiuo the carboxykinase functions in gluconeogenesis. Moreover, the levels of PEP carboxykinase were low in mycelia grown on sugars or glycerol, and high on aspartate and TCA-cycle intermediates, while PEP carboxykinaseless mutants of Neurospora crassa could grow on sucrose but not on acetate as carbon sources (Flavell and Fincham, 1968). Recently Beever (1975) has produced evidence to suggest that the synthesis of PEP carboxykinase in N . crassa was under derepression and not induction control, and that a glycolytic intermediate caused the repression. PEP carboxykinase usually is assayed at pH 6.5 (Scrutton, 197 1)andthe pH optimumof the V. albo-atrum enzyme was found to be 6.2 (Hartman and Keen, 1974b). I t is significant, therefore, that the pH optimum of the A. nidulans PEPcarboxylating activity is 8.7 (M. E. Bushell and A. T. Bull, unpublished data), indicative of PEP carboxylase activity. Moreover, the A. nidulans activity also corresponds to PEP carboxylase by virtue of its lack of bound cofactors. However, comparatively little is known about the regulation of PEP carboxylase and PEP carboxykinase, most attention having been afforded to pyruvate carboxylase. All fungal pyruvate carboxylases appear to have biotin as a bound factor, and exhibit an optimum activity at a pH value of about 8. Additionally, Mg2+and K + or NH: are

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required for activity. Various metabolites act as modifiers of pyruvate carboxylase activity: L-aspartate is a negative modifier and causes feedback inhibition, while acetyl CoA has been shown to act as a positive modifier in Rhizopus nigricans (Overman and Romano, 1969), in Saccharomyces cereuisiae (Miller and Atkinson, 19721, and in Helminthosporium cynodontis (Clarke and Hartman, 19731, and to reverse aspartate inhibition in V . albo-atrum (Hartman and Keen, 1974). In contrast, acetyl CoA did not activate the pyruvate carboxylase from A. nzger (Bloom and Johnson, 1962; Feir and Suzuki, 19691, from A. nidulans (M. E. Bushell and A. T. Bull, unpublished data) or from N. crussa (Beever, 1973). Finally, studies of Penin'llium camemberti (Stan and Schormuller, 1968; Stan, 1972) and Sacch. cereuisiae (Miller and Atkinson, 1972) revealed that the activity of pyruvate carboxylase also is regulated by the adenylate energy charge of the cell. A model of regulatory interactions at the pyruvate branch point involving pyruvate carboxylase has been advanced by Miller and Atkinson, and is summarized in Fig. 5 .

FIG. 5. Diagram showing the action of pyruvate carboxylase and regulatory interactions at the pyruvate branchpoint. Positive and negative effects are shown. Anabolic and catabolic reactions by ----. Sites of modifier action reactions are shown by -, are indicated by-. When the adenylate energy charge is low, the ATP demand is high and oxidation of pyruvate to acetyl-CoA and its entry into the tricarboxylic-acid cycle are favoured. However, when the energy charge is high, ATP can be used for anabolic reactions. A high energy charge causes accumulation of acetyl-CoA, and oxidation of pyruvate is inhibited. In turn, the activity of pyruvate carboxylase is enhanced by mass action and allosterically activated by acetyl-CoA. The rate of carbon-dioxide fixation is subsequently regulated by feedback inhibition of pyruvate carboxylase by L-aspartate. After Miller and Atkinson (1972).

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Remarkably few data have been collected on the effects of culture conditions on carbon-dioxide fixation by moulds. Continued pyruvate carboxylase activity under conditions of nitrogen limitation has been revealed as the means of fumaric acid production in high glucosecontaining media by Rhizopus nigricans (Overman and Romano, 1969). The inability to remove respiratory carbon dioxide, either by fixation or culture ventilation (see Bull and Bushell, 19761, can produce growth inhibition. Thus the auto-inhibition of spore germination can frequently be attributed to carbon dioxide toxicity, and one interesting illustration of this phenomenon is provided by work on Agaricus bisphorus (Rast and Bachofen, 1967 ; Rast and Stauble, 1970).Accumulation of respiratory carbon dioxide prevents spore germination in this fungus, but the inhibition can be reversed by isovaleric acid. This volatile acid is converted to 3-methylcrotonyl-CoA via isovaleryl-CoA, the former intermediate compound being enzymically carboxylated with the eventual production of acetoacetate and acetyl-CoA. Bushell and Bull (1974b, and unpublished experiments) have investigated the fixation of carbon dioxide by glucose-limited chemostat populations of Aspergillus nidulans 13 mel, and found that the activities of pyruvate carboxylase and presumed PEP carboxylase were growth rate-dependent. The activity of pyruvate carboxylase increased linearly as the dilution rate was decreased from approximately 0.15 to 0.05 h-l, while that of PEP carboxylase passed through a maximum at D = 0.1 1 h-l. The sum of the two carboxylase activities agreed closely with in vim measurements of total H14C0, incorporation. The decreased rates of carbon dioxide fixation at high dilution rates were correlated with a decreasing total mycelial protein content; because the major incorporation of H14C0, was into protein, the decreasing carboxylase activities at D > 0.1 1 h-’ appear to reflect a lowered anaplerotic requirement in response to changing rates of protein synthesis. Although the affinity for HCO, of pyruvate carboxylase (about 11 mM) was twice that of PEP carboxylase, the V,,, values of the two enzymes was in a 1 :3 ratio, an observation suggesting modulation of carbon dioxide-fixing systems to meet increasing anabolic requirements. The effect of variations in steady state HC0,- concentration was also examined. As the HCO,- concentration was raised from 2.5 mM to 7.0 mM, the biomass yield was increased by 23%and, in keeping with the measured K , values, a differential synthesis of PEP carboxylase

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occurred. Such data could have important implications in the optimization of processes for production of fungal protein. Finally, some interesting reports have appeared recently on the activity of anaplerotic and gluconeogenic enzymes in yeasts growing diauxically on glucose and ethanol. First, Divjak and Mor (1973) claimed that PEP carboxykinase, as well as pyruvate carboxylase, was very active during glycolytic growth of Saccharomyces cerevisiae, an observation not in accord with the generally accepted regulation of this enzyme by glucose repression. Furthermore, PEP carboxylase was reported to be strongly repressed by glucose and was synthesized only after glucose depletion from the medium. A subsequent study of this system by Haarasilta and Oura (1975) has produced more readily interpretable data. Thus only pyruvate carboxylase was present throughout, and its activity was regulated by aspartate; glyoxylate enzymes and gluconeogenic enzymes (PEP carboxykinase and hexose diphosphatase), in contrast, were synthesized following the complete utilization of glucose. In addition to glucose repression of PEP carboxykinase, Haarasilta and Oura (1975) showed that the addition of glucose to cultures growing on ethanol caused a rapid decrease in PEP carboxykinase activity, and they suggested that this may be due to proteolytic inactivation of the type reported by Holzer et al. (1973)for yeast tryptophan synthase. At present, results from these two studies are difficult to reconcile but the suggestion of Haarasilta and Oura (1975) that the assay reaction mixture used by Divjak and Mor (1973) for PEP carboxykinase may have produced equivocal results, deserves serious consideration. C . ASPECTS O F TERMINAL O X I D A T I O N

Two features of respiratory metabolism common to most fungi are a functional TCA cycle and an electron- transport system closely similar to that found in animals and higher plants. Tricarboxylic-acid cycle activity has been reported for members of all of the main taxonomic groups, although it may be weakly functional or, occasionally, absent. The aquatic phycomycete Aqualinderella fermentans is an example of the latter type and it derives energy solely from a homolactic fermentation (Held et al., 1969). The TCA cycle appears to be fully operative in A . niger, even during the phase of growth in which citric acid is produced (Ahmed et al., 1972),and presumably sufficient citric acid is recycled to

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meet the cells' biosynthetic requirements. Fungal electron transport involves a flow of electrons from NADH, and substrate through a system including cytochromes 6, c and uug (see Fig. 6) and containing up to three energy conservation sites. Again in common with higher plants, fungal respiration may proceed via cytochrome pathways or alternate pathways (see p. 70). However, little quantitative information has been published on electron-transport chain components and how they are affected by, and related to, growth conditions and growth efficiency in fungi. Descriptions of respiratory components and oxidative SHAM

U I I

Succmmte-FP~Fe/S

I I I

\I

FIG. 6. Diagram showing the cytochrorne and alternative respiratory pathways that operate in lungi. Solid lines indicate the cytochrome pathway and dashed lines the alternative pathways; arrows indicate the sites of action of inhibitors. FP, and FP, indicate flavoprotein dehydrogenases, and Fe/S non-haem iron protein. SHAM indicates salicyl hydroxamate.

phosphorylation in fungi, reviewed by Lindenmayer ( 1965) and by Watson (19761, will not be restated here. Phenotypic modifications of the electron-transport chain and energy conservation sites have been clearly revealed in Torulu utilis growing under different conditions of nutrient limitation (Light, 1972). Mitochondria from carbon (glycerol)-limited chemostat cultures had three energy conservation sites associated with the electron-transport chain but, when iron- or sulphate-limitations were imposed, Conservation at site 1 (between NADH, and cytochrome b) was lost. Furthermore, NH;- and Mg*+-limitationexperiments prove that the loss of site 1 conservation reflected specific deprivations of iron and sulphur, and not repression caused by an excess of glycerol. Predictably the loss of an energy conservation site had a profound

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effect on growth efficiency, and the yield from glycerol was 33%lower under iron-limiting conditions compared with glycerol-limitation. Although inhibition of respiration, loss of conservation sites and uncoupling of oxidative phosphorylation all clearly affect growth by diminishing energy generation, the metabolic state of mitochondria may exert other regulatory effects. Synthesis of valine from pyruvate, and isoleucine from pyruvate and threonine, by Neurospora crassa, are dependent on the integrity of a five-component enzyme complex. Bergquist et ul. ( 1974b) showed that valine synthesis was severely depressed if respiration and oxidative phosphorylation were disturbed. This regulation of amino-acid synthesis, via the functional integrity of the enzymic machinery, is reversible and illustrates an additional rapidly responding mechanism of metabolic control. The manner in which energy metabolism regulates growth has been increasingly studied over the past decade; such studies in moulds have been few, and mainly concerned with Neurospora. Slayman ( 1973) found that the intracellular ATP content ((ATPI,)of N . crussu was not constant during exponential growth but, if the total adenylate nucleotides were examined, an average energy charge value of 0.72 obtained throughout the exponential phase. This value is within the range typical for metabolically active micro-organisms. When mycelia were transferred to buffer lacking a nitrogen source, the (ATP), stabilized at a level equivalent to 2.5 mM, the maximum observed during exponential growth. Using a non-growing system to study the effect of metabolic perturbations on the energy status of the fungus, Slayman (1973)derived a minimal ATP turnover rate of 0.44 mMmin-’ when respiration was inhibited with cyanide. However, under these conditions, continued adenylate kinase activity probably caused some resynthesis of ATP, a suggestion supported by the fact that the ATP turnover calculated from steady state respiration rates, was much higher ( 1.17 mM min-’1. Slayman has argued that this rapid turnover of ATP coupled with the facts that N . crassu lacks energy-storage compounds and cannot generate energy from its polyphosphate reserves, may make it very susceptible to “metabolic accidents” such as respiratory blockage. Short- and long-term compensatory mechanisms for circumventing such a block probably revolve around the feedback control of glycolysis by the adenine nucleotide ratio, and the induction of the alternate cyanide-resistant respiratory pathway (see below; p. 701, respectively. A pattern of fluctuation in the size of the ATP pool

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resembling that of N . C ~ U S S has U been reported for cultures of the woodrotting fungus Formes annosus (Johansson and Hagerby, 1974). A second peaking of the ATP pool in this species corresponds to the generation of energy from mobilization of storage materials. Similarly, a rapid turnover of ATP occurred in the presence of uncouplers such as pentachlorophenol. Oscillations in the sizes of adenosine nucleotide pools have recently been implicated in the circadian rhythm of conidiation (see Section VI, B ; p. 46) of N . crassa (Delmer and Brody, 1975). These authors observed no clear oscillation of (ATPL but a circadian oscillation of the energy charge from 0.65 to 0.93, correlating with the conidiation rhythm, was clearly demonstrated. Continuous illumination of N . crassa cultures obliterated the conidiation rhythm and produced an average energy charge in the peripheral zone hyphae of 0.73. Moreover, the oscillations in conidiation and energy charge could be phaseshifted by light. The underlying cause of the energy-charge oscillation may be a partial uncoupling of oxidative phosphorylation, but definite proof of this hypothesis has yet to be established. We have already suggested that respiration in Neurospora crassa and other fungi usually proceeds via the conventional electron- transport chain. However, growth of wild-type strains in the presence of antimycin or cyanide, or, of respiratory mutants, frequently induces an alternative, cyanide-resistant, route that is sensitive to salicyl hydroxamate (SHAM)(Fig. 6). Cyanide-resistant respiration was recognized in several fungi at the time Lindenmeyer ( 1965) reviewed the subject and, subsequently, has been studied in depth in N . crussa (Lambowitz and Slayman, 1971; Colvin et al., 1973; Edwards and Kwiecinski, 1973; Bergquist et al., 1974) and has also been reported in Mucor (Rogers, Clark-Walker and Stewart, 1974). The physiological significance of this alternative system has excited considerable attention, particularly since it is not coupled to phosphorylation. Edwards and Kwiecinski (1973) have proposed that the alternative oxidase in N . cras~a may be associated with the NADH, limb of the electron-transport chain; thus, derepression of the alternative oxidase might prevent the accumulation of reduced nicotinamide nucleotide, thereby stimulating ATP generation via substrate level phosphorylation. Work in Slayman’s laboratory (Slayman et al., 1975) has further investigated this possibility. Experiments with the mutant poky F revealed that the efficiency of the alternative pathway in ATP generation (via substrate level and

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site I phosphorylations) was about 13% that of the wild-type cytochrome pathway, and that sufficient ATP could be produced in the absence of the latter pathway to enable slow growth to occur. The percentage of ATP coming from substrate level and site-I phosphorylations, in the presence of cyanide, was dependent on the rate of glycolysis and hence on the nature of the carbon-energy substrate. Moreover, differences in growth rate of other respiratory mutants reflected the proportional flux of electrons along the cytochrome and alternative pathways. Because the latter system is inefficient in ATP production, regulatory mechanisms ensure that the cytochrome system operates preferential whenever possible. Thus, under normal growth conditions, electron flux is partitioned so that the cytochrome pathway is saturated and the alternative pathway only takes up excess electron flow; the means of flux partitioning is not known. Regulation at the level of synthesis also occurs, but again the mechanism has not been elucidated. However, the induction of cyanide-resistant respiration in M . genevensis occurred only at very low values of dissolved oxygen tension ( ( 5 mm Hg) (Rogers et d.,19741, a result that seems to implicate oxygen in the regulation of its synthesis. IX. RNA Synthesis and Function: Rate-Limiting Parameters of Growth

In this final section, we wish to consider some aspects of macromolecule synthesis, particularly the synthesis of RNA and protein, in terms of overall rate-limiting processes in fungal growth. Earlier in this article we referred to attempts at growth modelling based on the concept of a rate-limiting step (Dabes et al., 1973; see Section 11, B; p. 12) and it is in this context that the following discussion of RNA synthesis, and its functioning in protein synthesis, will be set. Inquiries of this nature have, until very recently, been restricted to prokaryotic micro-organisms, but sufficient data now are accumulating from experiments with yeasts and moulds to enable comparable analyses of fungal growth. A brief statement of macromolecular synthesis in bacteria will be a useful preliminary to the assessment of the fungal data. Koch (197 1) has provided a valuable analysis of the overheads for protein synthesis in enteric bacteria and, having shown that the ribosome is the most expensive item in the protein-synthesizing

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machinery, he argued that it should be used at maximum efficiency. The notion of ribosomes functioning with a constant efficiency was proposed some years earlier by Maaloe and his colleagues (see Maalse and Kjeldgaard, 19661.-They observed that the number of ribosomes per bacterial genome was proportional to the specific growth rate, and concluded that the organism was functioning with maximum economy. In other words, growth at twice a given rate would require twice as many ribosomes to accommodate the doubled rate of protein synthesis. The constant-efficiency hypothesis subsequently has gone into decline, and Koch’s own data have been instrumental in its reappraisal. For example, following a 17-fold step increase in dilution rate, the rate of protein synthesis in chemostat populations of E . coli increased seven-fold before any net increase in RNA had occurred. Koch concluded that slowly growing organisms possessed “extra” nonfunctioning but rapidly mobilized RNA. Finally, Koch has compared the step times for transcription and translation in micro-organisms, and remarks that the ribosome’s capacity for peptide-chain elongation is about three times less that of the RNA polymerase’s capacity to add nucleotides to the messenger RNA. With this background we will examine macromolecule synthesis in fungi. A.

EFFICIENCY OF PROTEIN SYNTHESIS

The value of continuous-flow cultures for the investigation of protein synthesis is forcibly demonstrated by the results of Koch’s experiments (see above). Nevertheless, the availability of such data for fungi is almost nil. A recent publication by Griffen et al. (1974)dealing with protein synthesis in Achlya bisexualis presents data purporting to be consistent with the hypothesis of a constant ribosome efficiency. The latter experiments were made in a variety of batch-culture systems, variously aerated and containing different nitrogen sources as a means of modifying the specific growth rate. Statistical analysis of the data of Griffen et al. ( 1974) by Bushel1 ( 1974) has revealed that the correlation between RNA concentration and p, and protein concentration and p, are unsatisfactory and this places some doubt on their interpretation in terms of the Maalse hypothesis. A critical discussion of relationships between different macromolecular species in Candzda utilis organisms was developed by Ahoy and Tannenbaum (1973).They expressed the efficiency ( E ) of RNA ( R )

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in protein synthesis ( P ) , in steady-state chemostat populations, as a function of dilution rate: E =(P/N x D

(g protein g RNA-lli’).

The ribosomal efficiency of the Candida increased linearly with dilution rate up to about 0.8 +ax, and thereafter remained constant. Analysis of our continuous-culture data for Aspergillus niduluns (Bushell and Bull, 19761, and recalculation of that published by Bu’Lock et al. (1974) for Gibberella JujiiRuroi, also revealed a linear increase of ribosomal efficiency with D which became constant at dilution rates greater than 0.65 p,,.x. In addition we (A. M. T. McGetrick and A. T. Bull, unpublished experiments) have investigated the influence of some culture variables on the ribosomal efficiency of A . niduluns, growing under steady-state conditions. The stability of the proteinsynthesizing system was remarkably high, the ribosomal efficiency changing hardly at all with the medium pH value (3.1 tb 6.9) or salinity (0 to 8% NaCI). However, the ribosomal efficiency doubled as the growth temperature was increased from 22°C to 3OoC, remained constant over the range 30°C to 42”C, and started to fall at still higher temperatures. These data are consistent with the view that lowering the temperature decreases ribosomal activity, and that more RNA is synthesized such as to maintain the rate of protein synthesis, and thus maintain p, at the fixed dilution rate. Bushell and Bull (1975) also calculated the ribosomal efficiency during the transient stage following a step-up in dilution rate of a chemostat culture of A . niduluns (see Fig. 4, p. 44). An almost immediate and marked increase (three-fold) in ribosomal efficiency occurred and overshot the ultimate steadystate value. This observation provides further confirmation of Koch’s view of excess protein-synthesizing capacity at low growth rates. The identity of the “extra” RNA is evident from the work of Varricchio and Monier (197 1) and Harvey (1973) with E . coli. Thus, the concentration of ribosome subunits and free ribosomes decreased with increasing growth rate, while the polysome concentration rose concomitantly. The subunit plus free ribosome pool functioned as a reserve so that the polysome population could increase quickly in response to increased growth rate. Harvey (1973) found that the percentage of polysomes increased from about 30 to 70% as p was increased from 0.3 to 1.2 h-’ and that the response to a nutritional stepup was very rapid (detectable after 30 seconds) and was completed

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within 2 to 5 minutes. The question is, does a similar system operate in fungi ? The data are less extensive, and relate to ribosome changes during spore germination, but results from work with species of Fusarium (Cochrane et al., 197 l),-Bhiropus (Roheim et al., 1974) and Neurospora (Mirkes, 1974) are entirely consistent with a rapid shift in free ribosomes to polysomes. The accumulated results signify that the constant ribosome efficiency hypothesis is valid in a limited sense, but must be related only to polysomes. The most recent confirmation of this view comes from the work of Plaut and Turnock (1975) on Physarum polycephalum. The proportion of ribosomes associated in polysomal complexes rose from about 50%to approximately 70% as the dilution rate of chemostat populations was increased from 0.02 to 0.07 h-'; concomitantly the efficiency of protein synthesis increased by 50%over the same range of dilution rates. However, one cautionary note needs to be added which is relevant to these studies; data on protein turnover rates are lacking, and if the latter occurs to a significant extent then ribosome efficiencies will have been underestimated. Extrapolation from the above observations suggests that, in C. utilis and A . nidulans, all ribosomal subunits and free ribosomes are associated in functional polysomes at growth rates significantly below ,umax(i.e. at 0.3 ,urnaxand 0.65 pmas, respectively). However, an alternative or supplementary rate limitation might be imposed by the peptidechain elongation rate which conceivably reaches a maximum at submaximum growth rates. Once again the quantity of data in this area is small, especially for fungi. Of interest is the observation that the peptide chain-elongation rate increased linearly with growth rate in E . coli, but at growth rates exceeding 0.4 pmax the elongation rate became constant (Forchhammer and Lindahl, 197 1 ) . The only comparable data for fungi relates to Saccharomyces cerevisiae, and Boehlke and Friesen ( 1975) have recently reported that the elongation rate varied directly with the specific growth rate of the yeast and ranged from 2.8 amino acids per second at 0.09 h-'to 10 amino acids per second at 0.43 h-'. With our present knowledge, it is impossible to ascert whether peptide-chain elongation per se is the primary rate limitation to growth rate in fungi. B . CONCERNING POLYAMINES

By way of concluding this treatment of growth regulation and macromolecule synthesis we would like to refer briefly to the role, or roles, of polyamines. These organic cations are thought to confer stability on nucleic acids and to stimulate their synthesis, as well as that

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of proteins. Fluctuations in endogenous polyamine concentrations have been shown to accompany changes in growth rate and to be correlated with differentiating systems (see Cohen, 197 1, for review). Thus, the view has steadily developed that polyamines may be important regulators of cell growth, and within the past few years work has been reported that supports such a claim in fungi. Studies with prokaryotic and eukaryotic organisms have suggested a degree of functional exchangeability between polyamines and magnesium ions, and that enhanced polyamine synthesis occurs under conditions of Mg2+(andK)' deprivation. The first convincing evidence for such a reciprocal interchange between Mgz+and polyamines in fungi was provided by the work of Viotti et al. ( 197 1) with Neurospora c r a m . This group observed a decrease in mycelial magnesium during the mid-exponential growth phase and a concomitant increase in spermidine and spermine. Moreover, under these conditions of magnesium insufficiency, the increased concentrations of polyamines appeared to prolong exponential growth at an unaltered rate for several hours; the rate of RNA synthesis was also sustained under these conditions. Viotti et al. ( 197 1) demonstrated that the Mg2tpolyamine reciprocity occurred at the ribosomal level. This type of analysis was further developed by Bushell (1974) and Bushell and Bull ( 1974a) using chemostat cultures of Aspergillus niduluns. Under carbon-limited growth conditions they found that the mycelial Mg2+concentration fell (nearly six-fold) with increasing dilution rate and, whereas the molar ratio MgZf:RNA appears to be growth-rate independent in carbon-limited bacteria (Tempest, 19691, it varied significantly in the fungal system. When the polyamines (spermidine and spermine) were considered in this context, however, the molar ratio polyamines plus Mg2+:RNA was constant at approximately two. Bushell found that the polyamine :Mg2+ratioincreased logarithmically as a function of ribosomal efficiency, a result that indicated an increasing differential requirement for polyamines at higher rates of protein synthesis. This finding is consistent with known functions of polyamines; as ribosomal efficiency approaches a maximum (see Section IX, A; p. 73) increased polyamine requirements for tRNA synthesis and aminoacylation, mRNA binding to 30s ribosomal subunits and the formation and stabilization of polysomal complexes are highly probable. Polyamine concentrations, and synthesis, also have had preliminary investigation in differentiating fungi. Thus, putrescine pools increased

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several fold in Physarum polycephalum during the emergence of plasmodia from the dormant spherule stage (Mitchell and Rusch, 1973). Spermidine concentration was found to correlate with protein synthesis activity during diiferentiation. I t is interesting to note that, although putrescine is a precursor of spermidine, the two polyamines are under independent control in Physarum, and both occur in high concentrations. The latter observation points to distinct but as yet undefined functions of the molecules,

X. Acknowledgements I t is our pleasure to thank all those students, research collaborators and colleagues whose contributions over the years have done so much to stimulate and sustain our personal interests in the fungi. During the preparation of this review we have enjoyed the helpful comments and criticisms of three colleagues in particular Drs. Michael Bushell, Howard Slater and Ben Zonneveld. Much of our own research has been funded by the Science Research Council to whom we wish to express our gratitude. REFERENCES

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Physiology of Halobacteriaceae I. E. D. DUNDAS lnstitutt for Generell Mikrobiologi. Universitetet i Bergen. Bergen. Norway. I . Introduction . . . . . I1 . Classification of Extreme Halophiles

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90 B . Extracellular Salts 91 IV . Subcellular Structures . . . . . . A . Cell Envelopes . . . . . . . . . . . . 91 B . Ribosomes . . . . . . . . . . . . . 94 C . Vacuoles . . . . . . . . . . . . . 96 D . Flagella . . . . . . . . . . . . . 99 V . Halophilic Proteins . . . . . . . . . . . 100 A . Metabolic Pathways . . . . . . . . . . . 101 B . Halophilic Enzymes . . . . . . . . . . . 102 . . . . . . . . . . 104 VI . Lipids in Halobacteriaceae VII . Electron-Transportchain . . . . . . . . . . 106 VIII . TransportAcrossMembranes . . . . . . . . . . 107 IX . Effects of Light . . . . . . . . . . . . . 109 A . Photophosphorylation . . . . . . . . . . 109 B . Effect o n Growth and Viability . . . . . . . . 110 X. Nucleic Acids and Their Enzymology . . . . . . . . 111 XI . Phage-HostRelationships . . . . . . . . . . 113 XI1. Ecological Considerations o n the Existence of Obligate Extreme Halophilism . . . . . . . . . . . . . 114 XI11. Acknowledgements . . . . . . . . . . . . 116 References . . . . . . . . . . . . . . 116

.

I Introduction

Since prehistorical time. salt has been used by Man to preserve otherwise highly perishable materials such as fish. meat and hides . Spoilage of such highly salted products under unfavourable storage conditions could nevertheless be a serious economic problem . 85

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Bacteriologists became interested in the halophilic bacteria responsible because of the economic interest involved and because of the intriguing problems inherent in the ability of living organisms to function and proliferate in saturated brines. In recent times, the practice of cool storage has largely obviated the economic problems due to halophilic spoilage, at least in industrialized areas. Interest in extremely halophilic organisms has not decreased however. Even with the recent advances in our understanding of the physiology of these micro-organisms the basis for extreme obligate halophilism is far from clearly understood and remains an exciting scientific challenge. In addition to this, many scientists not primarily interested in extreme halophilism find that halophilic bacteria, their enzymes and organelles, are uniquely well suited to a study of general biological phenomena such as electron transport or transport of molecules across membranes. This review will concern itself only with extremely halophilic organisms, mainly with those classified in the 8th edition of “Bergey’s Manual” ( 1974) in the family Halobacteriaceae. This arbitrary delimitation is a convenient one, but ignores the fact that these organisms only represent one extreme of a continuum in the relationship between organisms and the salts in their external and internal milieux. Limiting a review of halophilism to this group of organisms is thus an artificial and unsatisfactory restriction which nevertheless limits the review to a manageable size. Excellent reviews with similar scopes have previously been published (Larsen, 1967; Kushner, 1968) and, as the present review will concern itself only with more recent developments, readers are referred to these reviews for older material. Extremely halophilic bacteria contain characteristic lipids having ether-linked alkyl groups instead of normal ester-bound fatty acids. The chemistry, metabolism and function of these unusual lipids has been thoroughly reviewed recently by Kates (1972). 11. Classification of Extreme Halophiles

Halophilism is not an easy parameter to include rationally in a taxonomic scheme. Growth of all micro-organisms is influenced by the presence of salt in their milieu. The influence may be positive or negative, depending on concentration; the tolerance range may vary considerably and the relation may be of an obligate or facultative nature. Extreme halophiles have been defined as those organisms which show best growth with 2 6 3 0 % (w/v) sodium chloride in their growth

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medium (Larsen, 197 1). This definition would allow inclusion of organisms able to grow suboptimally in low concentrations of salt. In the 8th edition of “Bergey’s Manual” (1974), N. E. Gibbons defines as members of the family Halobacteriaceae rods and cocci requiring more than 12% (w/v) sodium chloride for growth. All members of the family are thus obligate halophiles and, to the extent that they show optimal growth with more than 20% (w/v)sodium chloride, they can be termed obligately extreme halophiles. The inclusion of coccoid forms among the Halobacteriaceae is amply justified by the growing awareness of the striking similarities between them and the rod-shaped Halobacteria spp. The two types of organisms may be isolated from the same natural sources and after the same enrichment procedures. Both are obligately extreme halophiles. Both are aerobic and have respiratory, never fermentative, metabolism. Both contain similar isoprenoid pigments, bacterioruberins and retinal (Kushwaha et al., 1973). Both rods (Larsen, 1967) and cocci (Brown and Cho, 1970; Steensland and Larsen, 197 1) lack muramic acid residues in their cell walls. Both have very high intracellular salt concentrations (Larsen, 1967). Both have unique cell-envelope lipids with di-ether linkages (Kates, 1972) and similar DNA base ratios (Kocur and Bohacek, 1972) and both have a major and a minor (satellite)DNA component (Moore and McCarthy, 1969a). I t is tempting to speculate on the possible correlation between some of the striking common characteristics of Halobacteria spp. and their obligate extremely halophilic nature. In spite of the notoriously pleomorphic nature of many members of the Halobacteriaceae family and of the many striking similarities between the hormally coccoid and other forms, one must accept the inclusion of coccoid forms in a separate genus, namely Halococcus, both because of their general shape, their thick polysaccharide-containing cell wall and because of their higher resistance to osmotic damage. The genus Halobacterium was divided into five species in the 7th edition of “Bergey’s Manual” (1957). The division was based on rather unsatisfactory criteria, nitrate reduction and chromogenesis among others. The 8th edition of “Bergey’s Manual” recognizes only two species, namely Halobacterium salinarium and Halobacterium halobium, mainly on the basis of some differentiation in DNA homology (Moore and McCarthy, 1969b). Three species are described as incerta sedis. Zwilling et al. ( 1969)attempted to elucidate the immunological relationships among some Halobacterium species. While showing a close immunological relationship among the Halobacterium strains tested, they

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were also able to demonstrate four immunologically distinct and mutually exclusive groups. Some groups included strains classified in separate species by “Bergey’s Manual”. Immunological studies of obligately extreme halophiles do present difficulties. The antigen will necessarily be used in a denatured state due to decreased salt concentration, and the formed antibody can in any case hardly be expected to be active towards the original antigen in its natural very salty environment. I t is entirely possible that these facts do not preclude a satisfactory immunological taxonomic approach. Our knowledge of the members of the genus Halobacterium seems at present to be insufficient for a subdivision into species on other than rather subjective criteria. It seems a distinct possibility that the great homogeneity among the Halobacteriaceae to some extent reflects a bias on the part of the investigators. The original interest in the organisms as spoilage agents of proteinaceous materials led to the development of specific enrichment and isolation methods which are still in general and nearly exclusive use (Gibbons, 1969; Eimhjellen, 1965). Enrichment, o r plating on rich media containing yeast and protein hydrolysates, can hardly be expected to yield a flora representative of the varied halophilic microbial flora adapted to survival and proliferation in a saltern or salt lake. Isolation of carbohydrate-metabolizing extremely halophilic bacteria (Tomlinson and Hochstein, 1972a) and of halophilic photosynthetic bacteria (Raymond and Sistrom, 1967, 1969) has been reported. A photosynthetic facultatively aerobic halophilic spirillum with a growth range of from 2.5% to 25% (w/v) sodium chloride and a Halobacterium sp. able to grow with only glutamine and arginine as carbon and energy sources have recently been isolated in the author’s laboratory from the mother liquor in a Portuguese saltern. These reports raise the speculation that extreme halophilism may commonly occur in bacteria with much more variegated general physiology than that represented in the family of Halobac teriacea. 111. Intracellular and Extracellular Salt Concentrations A . INTRACELLULAR SALTS

Since Christian and Waltho (1962) demonstrated the very high intracellular salt concentrations in species of Halobacterium and Halococ-

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cus, disappointingly little has been done to study the normal internal salt concentrations of extreme halophiles. Inferences as to the composition of the internal milieux have been drawn from the effects of salts on the metabolic machinery and internal structures, but little direct work has been done. It seems that the data of Christian and Waltho (1962) may be generally valid for organisms in the Halobacteriaceae family. In fact, serious doubts could be raised about including in the family any obligately extremely halophilic organisms with intracellular salt concentrations which varied markedly from that established by Christian and Waltho (1962). There are, in principle, at least two mechanisms by which an organism might cope with the adverse effects of an extremely high extracellular salt concentration. The Halobacteriaceae seem to have solved some of the problems by allowing an approximately isoosmotic internal salt concentration even if maintaining an extremely high molal concentration ratio of potassium to sodium. A high internal concentration of salt makes it essential that the whole metabolic and regulatory cell machinery be extremely halotolerant. Some organisms may counteract high extracellular salt concentrations by maintaining a lower intracellular salt concentration allowing for a more normal metabolic apparatus. Borowitzka and Brown ( 1974) argue that all unicellular organisms which can thrive in concentrated solutions of electrolytes or non-electrolytes must do so, at least partly, by accumulating a solute of compatible type. Working with a halophilic species of Dunaliella viridis, which apparently excludes sodium ions effectively allowing a moderate potassium uptake, they found intracellular glycerol contents of 4.4 molal after grown in 4.25 M sodium chloride. It seems possible that similar mechanisms might operate in other unicellular extremely halotolerant or halophilic organisms. Any mechanism for accumulating compatible electrolytes or non-electrolytes by production or uptake, whether glycerol or potassium ions, would require an energy expenditure. Van Uden ( 1968) suggested the use of chemostat cultures for studying maintenance requirements of growing cells. Watson ( 1970) reported that increased external salt concentrations forces Saccharomyces cerevisiae in chemostat cultures to expend extra maintenance energy resulting in lower ynTp values. Increases in external concentrations of sodium chloride also led to greatly increased glycerol production per unit of biomass formed. The intracellular concentration of potassium ions did

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not vary with increased external concentrations of sodium chloride. Some influx of Na' occurred, but a ratio of extracellular :intracellular Na' equal to 10: 1 was maintained. It would have been extremely interesting in view of the work of Borowitzka and Brown (1974) on Dunaliella viridis to know whether these yeast cells also accumulated glycerol internally when growing in the presence of high concentrations of sodium chloride. I t may well be that the ratio of surface to volume and the general biosynthetic capabilities of the organism determine what mechanisms can be evolved for coping with high external concentrations of salt, and that small extremely halophilic bacteria, with restricted metabolic capabilities, are forced to tolerate a high intracellular concentration of salt. It has been shown that Halobacteria spp. may maintain an extremely high intracellular :extracellular ratio of K' for several days in the absence of any added energy source (Gochnauer and Kushner, 197 1). Even artificial vesicles of cell envelopes devoid of cytoplasmic material were able to maintain high intracellular K'concentrations for several hours in the absence of added energy sources (Andersen, 1975). Lanyi and Silverman ( 1972) showed that Pretention in whole cells was not due to any binding to cytoplasmic material. The work by Andersen ( 1975) also indicated that the major factor in K'retention is the impermeability of the cell envelope. B. E X T R A C E L L U L A R SALTS

I t is to be expected that obligately extreme halophiles are particularly well adapted for growth in the saline environment where they are normally found. An interesting new Halobacterium strain, namely H . uolcanii, isolated from Dead Sea sediment shows optimal growth at the unusually low sodium chloride concentration of 12% and would thus not qualify as an extreme halophile but fulfills the criteria for inclusion in the genus Halobacterium. Optimal salt concentrations for this strain mimick strikingly the saline composition of Dead Sea water from which it was isolated (Mullakhanbhai and Larsen, 1975). The most commonly available source for isolation of Halobacteria spp. is salt produced by solar evaporation of sea water. This fact may have led to an overemphasis on the importance of sodium chloride in growth media. The requirements for potassium and calcium ions, and especially the requirement for high concentrations of magnesium ions,

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should not be surprising in view of the natural habitat where the organisms grow and proliferate. It is probably more correct to view the mother liquour from solar evaporation pans and not the harvested salt as a natural habitat for halobacteria. The composition of the mother liquor is characterized by its very high magnesium-ion content and by having a concentration of sodium chloride of about 4.5 M at the end of a salt harvest period (Lepierre, 1936). I t may be appropriate here to re-emphasize the point made by Weber (1949) that it might be fruitful to pay more attention to the original habitat in future work on extracellular salt relationships of halobacteria. The external salt concentration may have a great influence on the survival of halobacteria. Gochnauer and Kushner ( 197 1) showed that omission of K+ decreased the ability of H . halobium to survive in salt solutions. The lack of success in maintaining isolates of extreme halophiles by freeze-drying (Gibbons, 1969) could be due to difficultieswith the control of water activity in preparations where salts represent the bulk of the material. Eimhjellen ( 1965) reports that cells of Halobacten‘um spp. may survive in salt for several years. The survival rates are not likely to be identical for all members of the halophile flora. While the bacterial flora of newly harvested salt probably is not fully representative of that in the saltern, this original salt flora is subject to further changes on storage. K. Eimhjellen (personal communication) indicates that storage under liquid nitrogen is satisfactory for pure cultures of extreme halophiles.

IV. Subcellular Structures

Extreme halophiles possess a normal array of subcellular structures. Their composition and their relationship to salt concentration may be unique. Their functions in the cell are “normal”, but must be carried out in the abnormal environment of near-saturated salt solutions. A.

CELL ENVELOPES

I t is rather surprising that two so markedly morphologically different bacterial groups as the halococci and halobacteria should both lack the constituent peptidoglycan in their cell envelopes (Reistad, 1975 ; Steensland and Larsen, 1969). Cell envelopes of the two groups are

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very dissimilar in many other aspects. Halococcus cell walls have strong anatomical resemblances to the walls of the non-halophilic Micrococcaceae (Steensland and Larsen, 197 1). In contrast to halobacteria, halococcus celIs are very resistant to low ionic environments, and their walls are quite resistant to disrupture by mechanical treatment. I t is not clear what compounds might be responsible for the mechanical strength of halococcal cells. Residues of amino acids and hexosamines constitute only 7-15% of the dry weight of the cell walls. There is a marked similarity in the chemical composition of walls of difrerent Halococcus strains. Noteworthy is the presence of gulosaminuronic acid which previously only has been found in Vi’ibrio prirahaemolyticus (Reistad, 197 5). Cell envelopes of halobacteria on the other hand are most fragile structures. Lowering the ionic concentration. of the suspending medium to about 12% sodium chloride by dilution with water or various ionic or non-ionic solutions causes severe disarrangement of the envelope (Larsen, 1967, 1973). Slight mechanical stress, like picking up a colony with a dry inoculum loop, may cause massive disruption of cells. The spectacular chemi-osmotic and mechanical fragility of Halobaclerium envelopes stimulated work on their morphology and composition. The surface layer of a halobacterium presents a regular hexagonal pattern when replica preparations are viewed in the electron microscope. D’Aoust and Kushner (1972) indicate that this hexagonal pattern can best be described as a honeycomb network forming the outer layer of the envelope. After mechanical disruption of cells, the cell envelope fragments tend spontaneously to form closed vesicles (Steensland and Larsen, 1969; Stoeckenius and Rowen, 1967). The vesicles exhibit the same hexagonal pattern as the whole cells (Andersen, 1975), which is taken as an indication that they have the normal “outside out” orientation of whole cell envelopes. The orientation of the membrane is crucial in interpreting the results from experiments on transport across vesicle membranes. Andersen ( 1975) utilized vesicles from H . salinarium which apparently all had the normal outside-out topology. Kanner and Racker (1975) on the other hand found that a portion of their vesicles prepared from H . halobium seemed to have the inverted outside-in topology. The mechanism for vesicle formation is not well enough understood to predict the final topology of the vesicles, and extreme care must be taken in in-

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terpreting minor differences in experiments on transport across vesicle membranes. Andersen ( 1975) studied transport phenomena associated with Halobacterium vesicles. It is remarkable that even quite small vesicles are continuous structures and do not allow leakage of contained material. The ability to form such small closed vesicles seems to indicate a remarkable fluidity in the envelope material. Thin sections of halobacteria show that they are surrounded by a three-layered unit membrane which again for some strains may be surrounded by a second structure forming a complex envelope. The envelope proteins are acidic in nature with at least five mole percent excess of acidic amino-acid residues. Diaminopimelic acid has not been found (Steensland and Larsen, 1969). Mjelde (1971) found that most cell-envelope amino sugars of H . salinarium were present as glycoproteins. Detectable amounts of peptidoglycan were not found, strengthening the hypothesis that polysaccharides play a negligible role in cell-envelope structure. An unique feature of H . salinarium cell envelopes seems to be the presence of sulphated acidic polysaccharides as reported by Koncewicz ( 1972). Mescher and Strominger (1975) studied the effect of several antibiotics, supposedly active in interfering with peptidoglycan synthesis, on H . salinarium, H . halobium and H . cutirubrum. Insensitivity of these bacteria towards the antibiotics was to be expected as peptidoglycan is not a constituent of their cell walls. With the exception of bacitracin, the antibiotics were indeed quite ineffective. Bacitracin, however, induced sphere formation by the normal rod-shaped bacteria even at concentrations as low as a few micrograms per ml. Mescher and Strominger (1975) suggest that bacitracin interferes with synthesis of the cell-envelope glycoproteins and possibly also with synthesis of saturated isoprenoid chains of membrane lipids. The results support the suggestion that glycoproteins and lipids play a role in maintaining cell-envelope morphology. Hsia et al. (1971) concluded from work using electron spinresonance labels that dissolution of the cell membrane of H. salinarium in media of low ionic strength was accompanied by changes in protein conformation and in lipid-protein interactions. In the course of a thorough study of the electron-transport chain of halobacteria, Lanyi ( 197 1) also studied more closely the salt-dependent dissolution of cell envelopes. A sequential solubilization of envelope components on lowering the sodium chloride concentration was demonstrated, flavo-

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proteins and the outer layer of the envelope being removed first. The components solubilized by a slight decrease in sodium chloride concentration exhibited a marked specificity for sodium chloride for remaining in the envelope. Components which were solubilized only at low concentrations of sodium chloride showed little salt specificity, remaining attached when sodiurn chloride was substituted by sodium nitrate or perchlorate. Solubilization of the envelopes with hydrophobic bond-breaking agents, such as urea, and di- and tetramethyl urea, also indicates that components showing high specificity for sodium chloride and requiring high concentrations of salt for remaining in the membranes were bound by predominantly hydrophobic forces, while the binding of components with low salt specificity and requiring low concentrations of salt, were predominantly ionic in character. Brown and Stevenson (1971) have shown that the composition of membrane fractions from H . salinarium may vary with the phase of growth. I t was remarkable in this connection that the molar ratio of menaquinone-8 (vitamin K,-40) to carotenoid in the membrane remained constant regardless of growth phase. Lanyi ( 1972a, b) has shown that treatment of H . cutirubrum cells and cell-envelope vesicles with Triton X- 100 results in the appearance of menadione reductase, an enzyme normally bound to the inside of the envelope. Respiring cells were resistant to Triton X- 100 while respiration-inhibited cells lysed rapidly. Respiring cells did lose the integrity of their cell envelope, and resistance to Triton X-100 seems to be due to the inability of the detergent to penetrate the lipid phase of membranes of respiring cells (Lanyi, 1973). Studies on the effect of Triton X-100 in shifting the absorption spectrum of the membranebound carotenoid bacterioruberin also indicated that the lipidmembrane layer is protected against derangement by its respiratory activity. Lanyi (1972b) points out that such a protection against detergents by respiratory activity is analogous to the substrate stabilization effects on many enzyme systems. The effect seems to be a good example of a “normal function” operative under highly abnormal extremely halophilic conditions. 8. RIBOSOMES

Ever since Bayley and Kushner (1964) isolated ribosomes from H . cutirubrum and reported on their unique relations to high K+and Mg2+

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concentrations, interest in the function and structure of these organelles in halobacteria has remained high. A most important development occurred when Bayley and Griffiths ( 1968a, b) showed that halobacterial ribosomes function normally in protein synthesis when their specific ionic requirements were satisfied. There was no evidence for any abnormalities in the genetic code for the amino acids. Ionic requirements may be different for maintaining ribosome integrity and for achieving optimal amino-acid incorporation. Ribosome stability was achieved in 0.1 M Mg2+whileoptimal amino-acid incorporation occurred in the presence of 0.02-0.04 M Mg? The presence of 3.8 M K+ and 1.0 M Na+was required for stabi!ity and activity. The observation that high Na+ concentrations increased the fidelity of translation is noteworthy. Rauser and Bayley (1968) noted that the stability of polyribosomes from H . cutirubrum depended specifically on the concentrations of both Mg2+and NH: ions. Visentin et al. (1972) were able selectively to remove groups of proteins from the 30 S and 50 S subunits of 70 S ribosomal particles from H . cutirubrum. This work has been continued by Strcbm and Visentin (1973) who have determined the molecular weights of the selectively extracted ribosomal proteins and compared them with analogous proteins from Escherichia coli. The acidic nature of the proteins released form halobacterial ribosomes has been known since the work of Bayley and Kushner (1964). Strcbm et al. (1975) have further studied the selective removal of 5 S RNA and proteins from 50 S ribosomal subunits. An acidic alanine-rich 50 S ribosomal protein from H . cutirubrum shows an extensive amino-acid sequence homology with analogous ribosomal proteins from E . coli (Oda et al., 1974). Such amino-acid homologies re-emphasize the questions about the evolutionary relationships between extreme halophiles and other organisms. I t is suggested that the rather high degree of conservation in the amino-acid sequence of this protein indicates that it is important in the basic function of protein synthesis on ribosomes. Further work along these lines may clarify the interrelations between normal function and abnormal saline environments. Visentin et al. (1972) extracted rRNA particles from 70 S ribosomes of H . cutirubrum and found that the 16 S and 23 S rRNA from the halophilic organism had a higher percentage of G + C (56.1 and 58.8%) than the corresponding rRNA from E . coli (53.8 and 54.1%). I t may be that this higher G + C percentage reflects the higher ionic concentrations to which H . cutirubrum ribosomes are normally exposed.

96

I. E. D. DUNDAS C . VACUOLES

Some Halobacterium species are conspicuously vacuolated, a characteristic which they share with some cyanobacteria, green and purple sulphur-bacteria and a few non-photosynthetic bacteria. Falkenberg (1974) gave a good review of present knowledge about vacuoles especially in halobacteria. Cohen-Bazire et al. ( 1969) point out that gas vacuoles from halobacteria and photosynthetic bacteria are strikingly similar both in general morphology and in fine structure. Vacuoles from all of these prokaryotic organisms are cylindrical structures with conical ends; the bounding membrane is a single layer nonunit membrane. The controversy about whether vacuoles contain gas or not seems to have been definitively settled. The structures contain gas which is freely exchangeable with gas in the medium. Vacuoles will collapse on being subjected to sudden increases in pressure (Walsby, 197 1). It is generally held that the gas vacuoles serve to position the aquatic micro-organism at an appropriate depth in the water column. Halobacteria, being strictly aerobic and living in strong brines with limited oxygen solubility, would presumably profit from flotation. During evaporation, the brines in a saltern may contain appreciable concentrations of oxygen even at the greatest depth. This is presumably due to the turnover of relatively oxygenated surface water with the increasing density due to evaporation. In the absence of evaporation, saltern brines may however rapidly become quite anoxic and the Dead Sea is permanently anoxic at depths below 40 m. Vacuolation may be a rather unstable character, some Halobacterium strains losing their vacuoles on subculturing in the laboratory (Larsen el al., 1967). Some Halobacterium strains, notably those classified as belonging to the species H . halobium, seem to be vacuolated at all stages of the cell cycle. Some Halobacterium strains produce non-vacuolated cells which become vacuolated on ageing. Vacuolization in such strains seems to be dependent on the physiological state of the cells, older colonies exhibiting circular zones and sectors with different degrees of vacuo1iza t ion. Isolation of vacuoles by fractional centrifugation is hampered by the fact that these structures collapse on exposure to the pressure increases in the centrifuge tube. Falkenberg ( 1974) obtained highly purified

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preparations of vacuoles from halobacteria by graded filtration of a preparation of lysed cells followed by flotation centrifugation. Comparison of vacuole membranes from two species of Halobacterium and the cyanobacteriumAnabaenaflos-qua (Falkenberg, 1974 ; Falkenberg et al., 1972) demonstrated that the membranes contained only protein and that these proteins contained unusually high proportions of non-polar amino-acid residues. Sulphur-containing amino acids were not found. Vacuole proteins from the extreme halophiles were considerably more acidic than those from the cyanobacterial vacuoles. Krantz and Ballou ( 1973), investigating vacuoles from another strain of H . halobium, found small amounts of tightly bound Dgalactose and phosphate. It seems possible, however, that this material may represent tightly bound contaminants. All gas-vacuole membranes from different halobacteria seem to be composed mainly or totally of proteins, and their amino-acid composition is similar and quite characteristic. Vacuole membranes from Halobacterium species 5 and H . halobium Brown (Falkenberg, 1974) are very similar in the mole percentage of their amino-acid residues. Similarities also exist between vacuole proteins from H. halobium Brown and Halobacterium species 5 as compared with those of H . halobium Delft (Krantz and Ballou, 1973). These statements hold true even though Falkenberg (1974) has shown that the vacuole from Halobacterium species 5 contains two protein species of molecular weights 12,900 and 15,100 daltons, respectively. The amino-acid composition of the two protein species was quite similar. It is not so surprising that vacuole proteins are similar in the cyanobacteria Anabaena and Microcystis (Falkenberg et al., 1972). More surprising is perhaps the degree of similarity found between the vacuole proteins from Halobacterium and Cyanobacterium spp. (Table 1). While the results presented in Table 1 are based solely on statistical probabilities, they do seem to support the view that vacuoles from presumably distantly related organisms are markedly similar not only morphologically but also as regards their proteins. Vacuoles and their proteins seem to be very well suited for comparative studies to elucidate halophilic properties of structural proteins. The ability to assess small structural variations by measuring changes in resistance to pressure-induced collapse (Walsby, 197 1 ) opens up possibilities for studying otherwise undetectable salt effects on a halophilic structural protein (Falkenberg, 1974).

TABLE 1. Relationships between pairs of proteins as estimated by the sum of squared differences in the contents of 15 amino acid residues (SAQvalues) Protein species Ribosomal 70s Fractions Escherichia colilHalobacteriumculisubrum Escherichia colilHalobaclerium cutirubrum Halobacterium cutirubrumlHalobacterium cutirubrum Vacuole Proteins Halobacleriurn halobium (De1ft)lHalobacteriumhalobium (Brown) Halobacterium halobium ( Delft)/Halobacleriumsp. 5 Halobacleriurn halobium (Delft)/Microcystis sp. Halobacleriurn halobiurn (De1ft)lAnabaenasp. Microcysfis sp.lAnabaena sp. Halobaclerium sp. 5AIHalobacten'urnsp. 5B RNA Polymaases Halobaclehum cutirubrurn subunit atsubunit Halobacleriurn cutirubrum subunit alEscherichia coli

SAQValue 247

160 120

References Spahr ( 1952),Bayley (1966) Spahr (19621,Visentin el al. (1972) Bayley (1966),Visentin et al. (1972)

m

P 24

27 96 73 23

21 12 298

Krantz and Ballou (19731, Falkenberg, (1974) Krantz and Ballou (19731, Falkenberg, (1974) Krantzand Ballou (19731, Falkenberg, (1974) Krantz and Ballou (19731, Falkenberg, (1974) Krantz and Ballou (1973), Falkenberg, (1974) Krantz and Ballou (19731, Falkenberg, (1974) Lanyi (1974) Lanyi (1974)

0

C

z CI

L

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D. FLAGELLA

Members of the genus Halobacterium are motile, exhibiting polar flagellation. The flagella can be stained by silver impregnation methods (Blenden and Goldberg, 1965). Fully grown cultures contain, in addition to flagella lophotrichously attached to cells, large amounts of loose flagella. These loose flagella sometimes aggregate in thick spiral bundles visible under the light microscope. These bundles are analogous to those reported for Proteus and Bacillus species (Frank and Hoffman, 1968; Wilson and Combs, 1970). Attempts at harvesting halobacterial flagella by traditional procedures, that is by shearing flagella from cells by shaking, resulted in excessive contamination by cell debris. The fragile Halobacterium cells tend to break before flagella shear off. Loose flagella and the flagellar bundles can be harvested directly by fractional centrifugation and by isopycnic centrifugation in gradients of caesium chloride. While such preparations probably are not pure enough to warrant chemical studies, such as amino-acid analysis, they appear microscopically pure and can be used in morphological studies on the effect of salts and solvents on flagella aggregation and morphology. T. Torsvik in my department found that flagellar bundles dissagregate into individual flagella on exposure to M-NaCI. Even in the presence of 4 M-NaCI, the bundles would dissagregate if exposed to 6 M-urea. Exposure to 8 M-urea caused disintegration of individual flagella. Morphological changes on exposure to a salt-free environment were slow. Dialysis against water for several hours caused the flagella to loose their uniform, helical morphology, but they did not disintegrate completely. Conventional procedures for disintegration of flagella, such as lowering the pH value, did not cause disintegration of flagella from halophiles. Attempts at electrophoretic analysis of urea-disintegrated flagella were unsuccessful, possibly because of hydrophobic aggregation of the proteins. T. Torsvik has recently found that 0.5%MgCI, protects flagellar integrity even in the absence of other salts. This finding raises the hope of purifying flagellar protein by conventional physicochemical procedures. While the flagellar proteins from Halobacterium salinarium thus seem to be distinct from the flagellin of other bacteria, the energy requirements for motility resemble those of some

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aerobic non-halophilic bacteria. Arginine is able to sustain motility under anaerobic conditions, presumably as a result of ATP formation from the ornithine carbamoyl transferase-catalysed reaction.

V. Halophilic Proteins Lanyi (1974) has recently published an excellent review on the saltdependent properties of proteins from extremely halophilic bacteria. A traditional theory for explaining the properties of halophilic proteins holds that the role of Na' and K+ is to shield mutually repulsive negatively charged groups within the protein molecule. The well established facts that halophilic proteins generally are more acidic than their non-halophile analogues, and that many halophilic proteins become less compact on lowering the salt concentration in the media, agree with the theoretical model. Bulk protein from both Halobacterium and Halococcus strains have a much greater excess of acidic amino acids than their non-halophile counterparts (Reistad, 1970). Many individual protein species from extreme halophiles also contain unusually high proportions of acid amino-acid residues. Lanyi and Stevenson ( 1970) studied the enzyme menadione reductase in some detail with respect to its relationship to salts. They found that hydrophobic binding forces contribute largely to its stability at high salt concentrations. Lanyi ( 1974) makes the point that rather low salt concentrations would be suflicient to satisfy charge-shielding effects alone. At high salt concentrations, new hydrophobic interactions are formed within the molecule, such interactions not being stable enough at lower salt concentrations. The relative importance of hydrophobic interactions for many halophilic proteins is evident from a calculation of their hydrophobicity parameters (Lanyi, 1974) as compared with non-halophilic proteins. Lanyi also points out that the cation specificity exhibited by many halophilic structural and functional proteins need not be solely or even largely due to specifically steric effects. The preference of binding of specific ions could be due to a larger net free-energy difference obtained when the specific ion is removed from its hydration shell in the aqueous environment and placed near the site of binding. It is obviously important to differentiate between salt effects on the stability and the activity of proteins. From these considerations, one would naturally expect halophilic proteins as a general rule to be rather acidic

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and to contain a fairly high proportion of hydrophobic amino-acid residues. While this may be generally true, extreme hydrophobicity or electrostatic charge of proteins may obviously be due to other than halophilic considerations. Professor R. Dowben pointed out that protein-sequence relationships could be inferred from data on amino-acid composition of proteins (EidsP, 1972). Data on sulphur-containing amino acids and tryptophan are not useful in this connection due to their general unreliability. The sum of the squared differences of the molar concentrations of paired amino acids from two proteins (SAW is an index of their relatedness. Recently, Dayhoff et al. ( 1975) suggested a similar procedure for assessing protein relatedness. Table 1 (p. 98) gives SAQvalues based on 15 amino-acid residues from various halophilic and non-halophilic proteins. While the considerations are based solely on the statistical probability that proteins with low SAQ values have similar amino-acid sequences, one may assume that a value of SAQ smaller than 50 indicates a very high probability that large sequence homologies exist. It may be argued that the apparent relatedness of H . halobium and cyanobacterial vacuole proteins is due to factors other than sequence homology. It would be interesting to compare other enzymes and flagellar proteins by this method. A.

METABOLIC PATHWAYS

Larsen (1967) stated that there was no reason to believe that the general metabolic patterns of extreme halophiles differed basically from those of non-halophiles and that the uniqueness of the differences seemed to lie solely in the halophilic nature of the enzymes. There is no reason to modifjr this general view today. All enzymes of the citrate and glyoxylate cycles have been detected in H . salinarium (Aitken and Brown, 1969). I t was long an accepted general view that extremely halophilic bacteria utilize carbohydrates very poorly if at all, but it has been demonstrated that H . halobium, H . cutirubrum and H . salinarium do utilize glycerol, sodium succinate and sodium pyruvate especially in media with high levels of potassium (Gochnauer and Kushner, 1969). Tomlinson and Hochstein (1972a) isolated carbohydrate-metabolizing extremely halophilic bacteria which grew, albeit suboptimally, on various carbohydrate-containing media. One of the isolates (designated M 6) was studied more closely. It utilized both

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glucose and galactose with concomitant production of pyruvate and acetate (Tomlinson and Hochstein, 1972b). The same strain was able to metabolize glucose via a modified Entner-Doudoroff pathway in which oxidation of glucose preceeds phosphorylation (Tomlinson et al., 1974). That hexokinase activity also was detected in the crude extract would indicate that glucose might also be metabolized by other pathways. Lipid metabolism in halobacteria seems to be unusual in that it proceeds by the malonate instead of by the more usual mevalonic pathway (Bergey’s Manual, 1974). The end products, phosphate esters of 1,Z-di-O-(dihydrophytyll-glycerol, are certainly unique (JOO and Kates, 1968). Squalenes are also among the lipids found both in extreme (Tornabene et al., 1969) and in more moderately halophilic organisms (Mullakhanbhai and Francis, 1972). Several workers (Marshall and Brown, 1968; Brown and Pearce, 1969) have reported finding different concentrations of lipid constituents in H. halobium depending on the growth phase. Such results emphasize the need for well defined growth conditions for obtaining reproducible results. Halobacteria adapt well to chemostat cultivation which may be a method of choice for similar studies. It was early recognized that arginine might be an interesting amino acid in the metabolism of extreme halophiles (Weber, 1949). Ducharme et al. (1972) also found that arginine was rapidly removed from the medium by H. cutirubrum. Arginine metabolism in H. salinarium seems to be similar to that in many non-halophiles (Larsen, 1967). The amino acid is degraded via the arginine desimidase pathway, and the resulting citrulline converted to ornithine with production of carbamoylphosphate in a reaction catalysed by ornithine carbamoyltransferase. This enzyme shows many of the normal regulatory properties of the analogous non-halophilic enzymes (Dundas, 1972). Chemostat experiments indicate that arginine to a great extent functions as a readily available energy source in the medium. B.

HALOPHILIC ENZYMES

Lanyi and Stevenson (1969) assumed that the role of high concentrations of ions was not primarily to lower water activity in the medium, but to interact directly and specifically with the conformation of enzymes. They pointed out that the observed specificities of ion pairs might be due to the high ionic strengths in the media. Such pair specificities were not likely to be expressed at low concentrations of ions. Accepting the suggestion (Lanyi, 1974) that the typical halophilic

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enzyme is a protein with an excess of acidic and hydrophobic aminoacid residues, the possible specific and general effects of high concentrations of salts are so varied as to defy any simplistic unifjring model. Charge shielding may be accomplished by Father low concentrations of ions; salting out of hydrophobic groups would require higher salt concentrations. For these effects, ion specificity might be achieved by simple steric or thermodynamic considerations. In addition, ions may be directly involved as a substrate in the enzymic conversions as has been suggested by the work of Aitken and Brown (1972). Griffiths ( 1970a, b) purified amino-acyl-tRNA synthetase from H . cutirubrum and found that the transfer reaction required 0.4 M-NH,CI for maximum activity while the ammonium ion at the same time acted as a competitive inhibitor with the respect to the amino acid (Grifiths and Bayley, 1969). The different ionic interactions may specifically affect enzyme activity, stability, or both. The individual structure of an enzyme, such as the existence of sulphydryl groups in halophile isocitrate dehydrogenase (Hubbard and Miller, 1970)or protein subunits (Louis and Fittt, 1971a1, may further complicate the picture. It is not surprising that several enzymes from H . salinarium are rather thermophilic (Keradjopoulos and Wulff, 1974). The low-temperature lability of several enzymes, including threonine deaminase (Lieberman and Lanyi, 19721, aspartate carbamoyltransferase (Norberg et al., 1973) and alanine dehydrogenase (Keradjopoulos and Wulff, 1974), probably reflects the cold sensitivity of hydrophobic bonds important for enzyme structure. Many regulatory mechanisms operative for normal enzymes have been shown to be active for halophilic systems. Feedback and product inhibition have been found for several enzymes, including aspartate carbamoyltransferase (Liebl et al., 1969; Norberg et al., 1973) and ornithine carbamoyltransferase (Dundas, 1972). The regulatory properties reported for citrate synthase from H . cutirubrum are noteworthy as resembling those in Gram-positive organisms (Cazzulo, 1973).The low molecular weight of the halophilic citrate synthase might not correlate so much with its similarities with the enzymes from Gram-positive organisms as with the unusually low molecular weight found for DNAdependent RNA polymerase (Louis and Fitt, 197 lb), polynucleotide phosphorylase, and RNA-dependent RNA polymerase from the same organism (Louis et al., 1971). One is reminded of Ingram’s prediction (Ingram, 1947) that the solubility of halophilic enzymes in high concentrations of salts might

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involve their existence as smaller molecular aggregates. Sigmoidal kinetics suggestive of allosteric interactions have been found for several enzymes, namely threonine deaminase (Lieberman and Lanyi, 19721, ornithine carbamoyltfansferase (Dundas, 1972) and aspartate carbamoyltransferase (Liebl et d.,1970;Norberg et d.,1973).Aitken and Brown ( 1972) found that sigmoidal kinetics for activity of an isocitrate dehydrogenase from H . salinarium depended on high concentrations of sodium chloride allowing polymerization of enzyme subunits into trimers or tetramers. All work on halophilic enzymes thus far indicates that the mechanisms underlying activity and regulation are similar to those that operate with non-halophilic enzymes. Further insights into those features of halophilic enzymes that are of importance in permitting “normal” function in abnormally saline environments will probably necessitate isolation of pure enzyme preparations, both for studying stoicheiometrically the various enzyme-salt interactions and for determining hydrophobicity and acidity parameters based on amino-acid analysis. Purification of halophilic enzymes has always been a difficult task, due to their instability in media containing low concentrations of salt. Several approaches will facilitate future work. Chromatography on calcium apatite (Norberg and Hofsten, 1970; Hochstein and Dalton, 1973;Dundas, 1970)is especially appropriate as the procedure is not disturbed by high concentrations of salt. Many halophilic enzymes are protected by the presence of their substrates, and several halophilic enzymes can be re-activated from low-salt inactivation by addition of salt especially if they contain stabilizing disulphide bridges. The protective effects of polyamines (Hochstein and Dalton, 1968, 1973; Lanyi, 1969b1,glycerol and ethylene glycol (Lanyi and Stevenson, 1969) on various halophilic enzymes is especially interesting. In this connection, the point should be made that, working with highly concentrated solutions, molality and not molarity may be the correct concentration reference. In concentrated solutions of sucrose, ornithine carbamoyltransferase activity reflects the molal and not the molar concentration of salt in the assay solution. VI. Lipids in Halobaaeriaceae

The lipids in members of the Halobacteriaceae have a strikingly difrerent composition from those of more normal bacteria. Already in

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1962 it was reported that H. cutirubrum lip5ds consisted mainly of diphosphatidylglycerol assumed to contain only ether-linked alkyl groups (Sehgal et al., 1962). Subsequent work, mainly by Kates and his group, has confirmed this assumption and led to a closer understanding of many intriguing aspects of lipid structure and metabolism in members of the Halobacteriaceae (Kates, 1972). In an elegant paper, it was shown that the unique lipids with ether linkages were not restricted to organisms in the genus Halobacterium, but were also typical of the halococci (Kates et al., 1966). Most work on lipids from extreme halophiles has been. carried out on H. cutirubrum, but enough has been done on other extreme halophiles that organisms in the Halobacteriaceae, also as regards lipid structure and metabolism, emerge as an unusual but surprisingly homogeneous group. Most if not all of the lipids in these cells are found associated with the membranes of the cellular envelope. The lipids consist in all studied cases of polyisoprenoid chains bound by ether linkages to glycerol. Fatty-acid residues are not detectable by normal analytical procedures, but synthesis of trace amounts of fatty acids can be demonstrated using isotopically labelled acetate (Kates et al., 1968). This extremely low content of fatty-acyl residues is in striking contrast to the situation found for moderately halophilic and non-halophilic bacteria where fatty-acyl residues represent a major fraction of the lipid content. Relatively little has been done on the biosynthesis of fatty acids since Kates et al. (1968) demonstrated that a pathway for fatty-acid synthesis might exist. Working with cell-free extracts from H. cutirubrum, Pugh et al. (197 1) found a salt-tolerant fatty-acid synthetase. It thus seems that the malonyl-CoA pathway for fatty-acid synthesis is present in halobacteria. They found the fatty-acid synthesis to be severely inhibited by salt. The evolutionary implications of the existence of a severely saltinhibited malonyl-CoA pathway in halobacteria are far from clear. One might assume that the presence of only trace amounts of fatty acids is somehow important for the organisms, in which case the severe salt sensitivity of the biosynthetic pathway enzymes would be of minor importance. The major pathway for lipid synthesis in H. cutirubrum proceeds from acetate via mevalonate as an intermediate (Kates et al., 1968).

The ether-linked alkyl groups in halobacterial lipids have been shown to have a structure corresponding to dihydrophytyl (phytanyl) (Kates et al., 1965). Isotope-labelling experiments with whole cells show that glycerol is an effective precursor for both the glycerol and

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the phytanyl moieties of the lipid. The fact that the label in 1(3b3Hglycerol is totally retained in the glycerol moiety of the lipid indicates that aldo-keto isomerizations can not occur in this part of the pathway. Kates ( 1972) argues that dihydroxyacetone itself, or a separate pool of dihydroxyacetone phosphate, might serve as an intermediate, and points out that the unknown metabolic steps from dihydroxyacetone or dihydroxyacetone phosphate to the diphytanyl glycerol ether probably represent the really unique steps in the pathway for biosynthesis of ether linkages in a dialky glycerol ether. Biosynthesis of the phytanyl moiety probably proceeds by normal metabolic pathways as far as the production of dimethyl allyl pyrophosphate. The biosynthetic pathway for phytol from dimethyl allyl pyrophosphate which occurs in plants is largely unknown, but there is no specific reason to assume that this pathway is different in the halobacteria (Kates, 1972). Kates ( 1972) discusses the potential function of diphytanyl ether lipids in extremely halophilic bacteria. He suggests that the membrane lipids might contribute towards structural stability as the diether analogue of phosphatidylglycerophosphate has a strong affinity for Mg2+. This would also at least in part explain the importance of magnesium ions in maintaining the structural integrity of halobacteria. The saturated nature of the lipids would also protect the membranes against peroxidation under aerobic conditions. A most interesting point is made in suggesting that the lipid may play a role in selective ion transport by the membrane. Synthetic model membrands constructed with the diphytanyl ether analogue of phosphatidylglycerophosphate have properties which would render them highly selective for K+ transport (Kates,1972). If a fundamental common characteristic of extreme halophiles is their ability to counteract a high external concentration of salts by maintaining an internal compatible solute with high concentrations of potassium ions, then one would also expect membranes with lipids specially well suited for selective transport of potassium ions to be a common characteristic for this group of bacteria. In this connection, it is interesting to note that the lipids from the moderately halophilic bacterium H. volcanii were quite similar to those in previously investigated extreme halophiles. VII. Electron-Transport Chain

The presence in halobacteria of a fairly normal electron- transport chain, able to oxidize succinate, glycerol phosphate or NADH,, and

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containing cytochromes of the b, c and a types, was established early (Lanyi, 1968, 1969a). it may be remarked that some work on electrontransport using “particulate fractions”, obtained by homogenizing cells in media containing high concentrations of salt, may have given preparations containing closed envelope vesicles. As some components of the electron-transport chain are localized on the inner surface of the envelope (Lanyi, 1972a1, permeability phenomena may have influenced the results obtained. Cheah (1969) studied the properties of the electron-transport particles of H. cutirubrum and reported the presence of cytochromes o and a,, both reducible by ascorbate. The c-type cytochrome present could not be reduced by ascorbate alone, but was reduced by ascorbate-tetramethyl-p-phenylendiamine (TMPD). Electron-transport particles from H. iialobium contained cytochromes o and a, and a predominant complex of b-type cytochromes. Both the membrane-bound a- and b-type cytochromes could be reduced by ascorbate alone or ascorbate-TMPD (Cheah, 1970a). Thus, a feature peculiar to extreme halophiles seems to be the ability of their cytochromes a,, a, or o to be reduced by ascorbate alone. The ascorbate-induced respiration and the cytochrome oxidase activity in H. culirubrum proceed at higher rates in the presence of high concentrations of K+ than with equimolar Concentrations of Na’. This may reflect the higher intracellular concentrations of K+ in intact H. cutirubrum cells (Cheah, 1970b). On the basis of work on H . salinarium and H. cutirubrum, Cheah (1970a) proposes an electron-transport chain for these organisms in which ascorbic acid reduces some of the b-type cytochromes. A by-pass from the cytochrome-b complex to cytochrome oxidase was indicated by the insensitivity of ascorbate oxidation to low concentrations of 2-heptyl-4-hydroxyquinoline-Noxide which effectively inhibits reduction of cytochrome c by the cytochrome b complex. VIII. Transport Across Membranes Halobacterium salinarium is able to carry out energy-dependent transport of glutamate; this transport is specifically dependent on the Na+ outside the cells (Larsen, 1967). A thorough study of glutamate transport in H . salinarium vesicles has been made by Andersen ( 1975). The vesicles were composed of seemingly structurally complete cellenvelope material with a normal outside-out topology. Care was taken to ensure that the vesicles were empty of any cytoplasmic material. The

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contents of such vesicles could be changed by varying the composition of the media in which they were formed. With ascorbate as an oxidizable substrate, vesicles accumulated glutamine against a gradient of 1 :230,000. The transpoft mechanism had an absolute requirement for K+ inside the vesicles and Na'outside. Glutamate did not diffuse out of the vesicles even under conditions where the uptake mechanism was inhibited. As the amino acid exists in its free form inside the vesicles, it follows that the membranes must be impermeable to glutamate. The Na' gradient and the K+ gradient necessary for optimal glutamate uptake could not by themselves furnish the energy for uptake. Vesicles containing the same internal concentration of K+ or Na' as the suspending medium were able to effect glutamate uptake. Vesicles were able to accumulate glutamate even if the directions of the K'and Na+ gradients were reversed. In the absence of ascorbate, no glutamate uptake occurred, even in the presence of steep gradients of K + or Na'. Constructed pH value gradients across the vesicle membranes did not give rise to glutamate uptake. Ferrocyanide alone did not support glutamate uptake but, together with ascorbate, it was able to function as an electron donor to the electron-transport chain. Ascorbate was shown to reduce b- type cytochromes in the electron- transport chain. Adenosine triphosphate does not seem to be involved in glutamate uptake. The presence of ATP within or outside the vesicles did not give rise to any glutamate transport. Convincing evidence that phosphorylation reactions were not involved was obtained when it was shown that arsenate did not influence uptake, even if incorporated in the vesicles. Halobacterium vesicles were also shown to be capable of accumulating several other amino acids simultaneously with glutamate. The glutamate-uptake mechanism may be specific in that glutamate uptake was unaffected by the presence of other amino acids. Whole cells of H . salinarium were able to accumulate glutamate after starvation for eight hours, even in the absence of added respiratory electron donors. Very recently, MacDonald and Lanyi ( 1975) reported that cell-envelope preparations from H. halobium, which contains bacteriorhodopsin, were able on illumination to take up leucine against a 1000-fold gradient. In contrast to the impermeability of H. salinarium envelopes to glutamate diffusion, H. cutirubrkm envelopes seem to allow rapid loss of accumulated leucine. The leucine-uptake system in H . cutirubrum was also unaffected by arsenate, and required Na+ in the external medium. The work by Andersen (1975) and

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MacDonald and Lanyi (19 75) re-emphasizes the possibilities offered by extreme halophiles for studying fundamental biophysical phenomena. IX. Effects of Light

Natural aquatic milieux with very high concentrations of salt are also as a rule subjected to intense solar irradiation. It was natural to suppose that organisms able to benefit from this irradiation, or able to protect themselves against eventual deleterious effects of too intense light, would have some ecological advantage. Moore and McCarthy ( 1969b) suggested that halobacteria might have evolved from phototrophic bacteria. Photosynthesis, photophosphorylation, photooxidation of vital molecules and light re-activation after lethal ultraviolet irradiation have been demonstrated for members of the natural bacterial flora in’ salterns. Normal chlorophyll-mediated photosynthesis, however, has not been found in any organism which otherwise would qualify for inclusion in the family Halobacteriaceae. A . P H O T O P H O S P H O R Y LATI O N

An extremely exciting development occurred when Oesterhelt and Stoeckenius (1971) reported that the previously isolated “purple membrane” from H . halobium (Stoeckeniusand Kunau, 1968)contained bacteriorhodopsin as the single protein component. Photo-exitation of the “purple membrane” resulted in a net translocation of protons from the bacterial cells (Oesterhelt and Stoeckenius, 1973) and it was argued that the resultant electrochemical gradient might be used by the cell to satisfy, some aspects of its energy requirements. As was subsequently shown by Danon and Stoeckenius (19741, “purple membrane” is implicated in photophosphorylation in Halobacterium halobium. Under anaerobic conditions, the ATP level in H . halobium drops rapidly to a low level. Aeration or illumination will restore the level of ATP to its normal value. The fact that specific inhibitors of photosystem I1 in normal photosynthesis were ineffective against photophosphorylation in H . halobium was taken to indicate that the bacteriorhodopsin-mediated phosphorylation is different from normal non-cyclic chlorophyll-mediated photophosphorylation. Specific inhibitors of the ATP-synthesizing enzyme of nonhalophilic bacteria inhibited both oxygen- and light-mediated phosphorylation in H . halobium, indicating that one enzyme might be

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responsible for ADP phosphorylation in this organism. Uncouplers functioning as proton translocators also inhibited phosphorylation, implying that a proton gradient indeed might be active in the phosphorylation (Danon and Stoeckenius, 1974). The fact that supplying oxygen to the anaerobic cells caused the ATP levels to rise even in the absence of exogenous substrate indicates an endogenous electron donor as a n energy source. A possible, if unlikely, explanation for the light-mediated anaerobic phosphorylation might then be that illumination induced utilization of some endogenous anaerobic energy substrate. That such an explanation is fallacious is clearly shown by Racker and Stoeckenius ( 1974). Reconstituted phospholipid vesicles containing the purple-membrane fraction from H . halobium and mitochondria1 oligoniycin-sensitive ATPase were able to carry out light-dependent phosphorylation. Spin-label studies of the purple membranes from H . halobium indicate that they are extremely rigid structures and that their proton translocation occurs by some pore mechanism rather than being dependent on the mobility of the protein in the membranes (Chignell and Chignell, 1975). Oesterhelt et al. (1974) reported that the chromophore in bacteriorhodopsin, on illumination in the presence of hydroxylamine, yielded retinaloxime. The purple membrane became white due to loss of retinal after the treatment but could be reconstituted simply by adding retinal (Oesterhelt and Schuhmann, 1974). The reconstituted membrane functioned normally. The chromophore in the purple membrane seems to consist of a charge-transfer complex between retinyllysine and an appropriate side chain of the membrane protein. The ease in isolating the purple membrane, the possibility of incorporating it in model lipid membranes for studying basic aspects of proton translocation, light-mediated energy convertions and similar fundamental mechanisms, make its discovery one of the most exciting recent developments in biophysics. The intense interest in the field is evident from the not less than seven reports on research on bacterial rhodopsin published in the February 1975 issue of the Biophysical Journal. B . EFFECTS O N G R O W T H A N D VIABILITY

There seems to be no doubt that the carotenoid pigments of'Halobacterium spp. exert some protective effect against the deleterious effects

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of intense visible light (Larsen, 1967).While Dundas and Larsen (1962) measured the deleterious effect of visible light by the decrease in growth rate, Clausen (19721, using more intense light, was able to demonstrate a direct lethal effect on a colourless mutant of H . salinarium. Several potentially light-sensitive compounds are present in the cells. Catalase is rapidly destroyed by photo-oxidation in the colourless mutant but not in its pigmented progenitor. Flavins are also possible targets for photo-oxidative destruction. Menaquinon-8, a compound involved in the NADH, oxidative pathway in halophiles, is also quite sensitive to light derangement (Marque2 and Brodie, 1970). E. Clausen (unpublished observations) argues that the carotenoids of Halobacterium spp. may have a two-fold protective effect, viz. a direct shielding in the 380nm and 440-530nm range and as an indirect excitation energy scavenger” for illumination in the 400-430 nm range. These conclusions are based on the action spectrum for the carotenoid-protective effect and on measured inactivation rates for catalase activity. Flavins seem to be implicated as endogenous photosensitizers The cartotenoid pigmentation of extreme halophiles has also been assumed to be implicated in photoreactivation of ultraviolet-killed cells. Hescox and Carlberg ( 1972)reported that ultraviolet-treated populations of H . cutirubrum, with only 1% survivors, were nearly completely re-activated after 45-60 minutes illumination in the 300-400 nm range. The use of diphenylamine-treated cells is not ideal for studying the role of carotenoids in the photoreactivation mechanism, even if growth is reported not to be affected. The use of suitable nonpigmented mutants would obviate any undetected effects of diphenylamine. Hescox and Carlberg (1972) indicate that the high guanine + cytosine (G + C) content of halobacterial DNA may render it less susceptible to damage by thymine dimerization during ultraviolet irradiation. L6

X. Nudeic Acids and Their Enzymology

Moore and McCarthy ( 1969a)extended earlier work on nucleic acids from extreme halophiles to show that DNA satellite bands occurred in all species tested in the genera Halobacterium and Halococcus. They concluded that the satellite probably was not attributable to intracellular parasites or viruses, and that it could not be due to cultural contamination. The percentage of G + C in the main DNA component

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from five strains of Halobacterium and one of Halococcus varied from 66 to 69% while the corresponding satellite DNA varied from 57 to 60%. The amount of satellite DNA per cell was estimated to be about equal to 15 copies of a normal-bacteriophage genome. Renaturation studies wiih DNA indicated that the satellite probably does not represent multiple copies of an episomal fragment (Moore and McCarthy, 1969b). Base-sequence homology studies indicated a considerable degree of relatedness among Halobacterium strains. Both DNA from the satellite and the main component of a Halobacterium strain were equally able to form hybrid DNA duplexes with total DNA from other Halobacterium strains. The degree of homology between satellite and main component DNA, however, was not assessed. It would have been interesting to know the degree of homology between purified satellite DNA from different Halobacterium strains in view of speculations as to the possibility of satellite DNA representing specifically halophilic genetic information. Fitt and his coworkers have worked extensively on the nucleic-acid enzymology of H . cutirubrum. Peterkin and Fitt ( 197 1) purified polynucleotide phosphorylase 2 17 -fold and described it as a normal halophilic enzyme dependent on high concentrations of salt for activity and stability. As the enzyme could not be re-activated from the salt-free state, purification was carried out in the presence of salt. Deoxyribonucleic acid-dependent RNA polymerase, purified 150-fold from H. cutirubrum, seems to be a normal enzyme insomuch as it showed a specific requirement for native DNA as template (Louis and Fitt, 197 la) and also required the presence of all four trinucleotides for optimal activity. I t had an absolute requirement of Mg2+and Mn2+ for activity. Most striking was the fact that the enzyme, with calf thymus DNA as a template, behaved as a non-halophilic enzyme. The presence of 0.5-M NaCl or KCl in the assay medium resulted in a 94% decrease in enzyme activity compared with activity in the absence of K+and Na+. This salt inhibition, however, does not occur when native H . cutirubrum DNA is used as a template (Louis and Fitt, 197 lc). Under these conditions, optimum polymerase activity was observed in the presence of 2.5-M or higher concentrations of salts. Stability of the enzyme, as regards activity with both template types, required salt concentrations about 2-M, as is normal for many halophilic enzymes. The implications of an enzyme able to function normally with a “nonhalophilic” substrate and behaving as a typically halophilic enzyme with its normal substrate are quite interesting.

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Louis and Fitt (1971b) have also purified a RNA-dependent RNA polymerase from H . cutirubrum 2800-fold. This enzyme is relatively insensitive to changes in NaCl or KC1 concentrations both as regards activity and stability. They argue that the possibility of this enzyme being virus-induced is remote, as the H. cutirubrum strain used does not develop plaques on agar or grow more slowly than other strains of H . cutirubrum. In this connection, one is reminded of the fact that H . salinarium strain 1 was carried as a pure culture in several laboratories for about 20 years before it was shown that it harbours a phage (Torsvik and Dundas, 1974).Griffiths (1970a)has shown that at least two isoaccepting types of phenylalanyl-tRNA can be found in H . cutirubrum. Nucleic acids, their enzymology and their function in protein synthesis, representing fundamental functions common to all organisms, seem to be areas offering fruitful possibilities for comparative work on halophiles and non-halophiles. XI. Phage-Host Relationships

Until recently, halobacteria were among those few bacterial groups for which bacteriophages had not been found. During work on isolation of flagella from H . salinarium, phage particles were observed in the electron-microscope preparations (Torsvik and Dundas, 1974). Repeated subculturing of H . salinarium from late log-phase batch cultures led to eventual lysis of the cultures. Phages could be isolated from these lysates by isopycnic caesium chloride centrifugation (Torsvik, 1976).The phage particles contain double-stranded DNA in polyhedral heads and possess contractile tails. Lysis also occurs when H . salinarium is cultivated in a chemostat for prolonged periods of time. Aliquots of an inoculum, which gave rise to a lysing phage-producing culture, could be stored for several days at 4OC. O n transferring to fresh medium, these inocula invariably gave rise to lysing phageproducing cultures. Phage, purified by caesium chloride centrifugation, causes lysis when introduced into cultures at high levels of infection. I t has not been possible to induce phage-producing lysis by ultraviolet irradiation or by any other means. Repeated single-cell isolations gave clones which still produced phages. Colonies of H . salinarium on agar will, after several weeks growth, show central zones of lysis. Phages can be isolated from such colonies, and have been isolated from three different strains of halobacteria. No sensitive host has been found yet, and single phage particles do not form plaques.

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I t is suggested (Torsvik, 1976) that the phage is a temperate one that rarely mutates to virulence. The phage shows some halophilic qualities, and it is morphologically stable in 6%NaCl but begins to disintegrate on exposure t7, 4% NaC1. Using analytical centrifugation, it was shown that phage DNA is not identical with either the main component or the satellite of the host bacterium. The described bacteriophage for H. salinarium offers limited possibilities for studies on the phage-host genetic system in halobacteria. Among other factors, the absence of a sensitive host able to allow plaque formation renders experimentation dependent on the electron microscope for demonstration and enumeration of phage particles. Wais et al. ( 19751, on the other hand, report isolation of a phage able to infect H. cutirubrum with a plating efficiency of 1.0. This phage is dependent on rather high concentrations of salt in the suspending medium for survival, and is also morphologically quite similar to the phage for H. salinarium. The existence of bacteriophages for halobacteria opens up the exciting possibility of applying phage-host genetics to the problem of extreme halophilism. Also, phage-protein components offer a new array of halophile proteins for further comparative studies.

XII. Ecological Considerations on Existence of Obligate Extreme Halophilism I t is a common and understandable view point that concentrated brines represent an extreme environment. From the view point of obligate and extremely halophilic organisms, however, a low-ionic environment is an extremely lethal one and saturated brines the least stressfull environment possible. Some saline environments, like the Dead Sea, the Great Salt Lake in U.S.A. and similar bodies of water, may be large enough to maintain such high salinities at all times so that obligately extreme halophiles always have some region in the habitat where they may survive. Smaller bodies of water, pools and flats along. the seashore, where seawater has been concentrated by evaporation, are much more transiently highly saline environments. Nevertheless it has been reported that extremely halophilic bacteria can be isolated directly or by enrichment from such sources. Their existence in these environments seems to be generally assumed (Kushner, 1968). Reports have even been published on the isolation of extreme

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halophiles from enrichment cultures using inocula from salt-free or marine sources (Larsen, 1962, 1967). Work on adapting extreme halophiles to growth in the presence of low concentrations of salt, or marine bacteria to growth in media containing high concentrations of salt, have not given convincing results. I t is nevertheless too facile to assume that all reports on the isolation of extreme halophiles from sources which temporarily have low salt concentrations are the result of faulty sterile techniques. Salterns in temperate climates are inoperative in the winter months and become covered with a blanket of fresh water. Salt from these salterns contains a normal flora of halophilic organisms, and it is possible that some part of the saltern stored salt nearby, or implements used in the working of the saltern, carry over enough bacteria for re-inoculation of the brines when they again reach the necessary concentration. The same possibilities do not seem to exist for small flats and pools along the seashore which only contain concentrated brines in the summer months. Larsen ( 1967) argues convincingly that the number of mutations necessary for converting a marine bacterium into an extremely halophilic one is probably much too high for such a transformation to be successful in the laboratory, even assuming that the substitution of acidic for non-acidic amino-acid residues in the proteins is the only change necessary for obtaining halophilic character. It seems reasonable to assume that the amount of new genetic information necessary for changing a non-halophile into an extreme halophile is so great that it only can be obtained by normal evolution mechanisms in Nature or, possibly, by the acquisition by a non-halophile of a rather large amount of halophilic information by some sort of gene-transfer mechanism. The existence of extreme halophiles in transiently extremely saline bodies of water tempts one to speculate about these possibilities, and about possible roles for satellite DNA or for extremely halophilic phages in this connection. The collection of extremely halophilic bacteria as described in “Bergey’s Manual” ( 1974) represent a strikingly homogeneous group, and it is tempting to correlate the characteristics common to the group with their extremely halophilic character. Insomuch as this group may represent an artificially selected subgroup of extremely halophilic bacteria isolated from much the same sources and by very similar enrichment and isolation techniques, such correlations may be mis-

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leading. Gibbons ( 1960) and Weber ( 1949) report isolating colourless halophilic bacteria from saline lakes. I found that the majority of organisms of the bacterial population, even in red brines of a saltern, may be colourless or white. Efforts should be made to broaden our concepts of how to isolate extremely halophilic bacteria with regard to enrichment techniques. The view that marine and freshwater environments are “normal” and brines abnormal leads to the assumption that the halophilic way of life has evolved from life forms in normal environments. Assuming that the current concept of prebiological evolution and the origin of life is plausible, then concentration of preformed prebiological organic molecules to a level where generation of “organisms” could occur would cause a concomitant increase in the concentration of any salts present in the “primordial soup”. An anaerobic extreme halophile might thus represent a rather primitive life form. The fact that some extreme halophiles are able to carry out anaerobic photophosphorylation (Danon and Stoeckenius, 19741, by a seemingly more primitive mechanism than normal photosynthesis, might be relevant in this connection. With the current and increasing interest in halophilism and extremely halophilic bacteria, it is important that we do not become hampered by unnecessarily narrow preconceptions of the possible range within extremely halophilic bacteria. To the extent that this article has broadened the view in this respect, my goal has been achieved. XIII. Acknowledgements I wish to thank the Gulbenkian Institute of Science and the staff at

the Laboratory of Microbiology at Oeiras, Portugal, for making possible a one-month visit to study the halophilic bacterial flora of Portuguese salterns. REFERENCES

Aitken, D. M. and Brown, A. D. (1969). Biochimica et Biophysics Acta 177, 351. Aitken, D. M. and Brown, A. D. (1972). Biochemical Journal 150, 645. Andersen, K. (1975). Doktor Ingenisr. Thesis: The Technical University of Norway, Trondheim. Bayley, S. T. (1966).Journal of Molecular Biology 15, 420. Bayley, S. T. and Grifiths, E. (1968a). Biochemistry, New Y o d 7, 2249. Bayley, S. T. arfd Griffiths, E. (1968b). Canadian Journal ofBiochemistry 46, 937.

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Sterols in Fungi: Their Functions in Growth and Reproduction CHARLES G. E L L I O T Botany Department, University of Glasgow, Scotland. I. Introduction

.

.

.

.

.

.

.

.

.

11. Functions of Sterols: Possible Approaches to the Problems . . . . . . . 111. Sterols in Model Systems

.

.

.

.

.I21

.

. 130 . 135 . 14 I

.

IV. Subcellular Distribution of Sterols in Fungi, and States of Binding . . V. Effects of Sterols on Metabolism and Vegetative Growth A. Pythium and Phytophthora . . . . . . . .

VI. VII. VIII. IX. X. XI. XII.

. 125

.I41

B. Saccharomyces and Other Fungi . . . . . . . . 144 . . . . . . 148 Effects of Sterols on Asexual Reproduction . . . . . . . . .I49 Sexual Hormones ofkhlya Effects of Sterols on Sexual Reproduction in Homothallic SpeciesofPythium and Phytophthora . . . . . . . . . . . .I52 Reproduction in Heterothallic Species of Pythium and Phytophthora . 156 162 Sterols and Sexual Reproduction in Ascomycetes and Basidiomycetes Conclusion . . . . . . . . . . . . . 165 . . . . . . . . . . .lG6 Acknowledgements References . . . . . . . . . . . . . 166

I. Introduction

My interest in the subject of this review began with the observation that Phytophthoru cuctorum would grow on a simple medium containing sucrose, asparagine, mineral salts and thiamin, but that on this medium it remained purely vegetative. On the other hand, when grown on oatmeal agar, oospores were produced in abundance. The factors in oats responsible for the difference were found to be sterols (Elliott et ul., 1964). Addition of sterols to the simple basal medium changed the mode of growth from vegetative to reproductive. Similar findings were reported about the same time for various species of Phytophthoru and Pythium (Hendrix, 1964, 1965; Haskins et al., 1964; Harnish et ul., 1964; Leal et ul., 1964). It is worth noting that, in 121

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1937, Leonian and Lilly had found that some substance extracted by alcohol from peas was required for reproduction of Phytophthoru. However they considered the active compound in the extract not to be a sterol but some unknown compound which they could not separate from sterols. Since that time, advances in our knowledge of the chemistry and biosynthesis of sterols in other organisms, the development of analytical techniques (particularly gas-liquid chromatography), and the availability of a wide range of pure sterols, have clarified the problem, and it is now clear that it is indeed the sterols which are the required growth factor in Phytophthoru spp. Species of Pythium and Phytophthoru are unable to synthesize sterols. Thus sterols are not detectable in mycelium grown in the absence of sterols (Elliott et ul., 1964; Schlosser and Gottlieb, 1966; McCorkindale et ul., 1969) and incorporation of labelled sterol precursors (acetate and mevalonate) into sterol-like material does not occur (Hendrix, 1966, 1975b; Schlosser et ul., 1969). Richards and Hemming (1972) reported that mevalonate was incorporated into farnesol, geranylgeraniol, dolichols and ubiquinones in Phytophthoru cuctorum. Squalene was not labelled; but D. Gottlieb (personal communication) finds that squalene is synthesized, but is not cyclized to sterol. Langcake (1975) reports that Phytophthoru infeestuns can convert lanosterol to a compound which he thought was possibly cholesterol, but cholesterol, cholestanol, sitosterol and stigmasterol are not converted to other sterols. Knights and Elliott ( 1976) find that A7 and A5*’-sterolsare converted to the corresponding A5-sterolsby Phytophthoru cuctorum. Species of Pythium and Phytophthoru are all plant pathogens, and it is evident that they obtain the sterols necessary for their reproduction from their hosts. It is perhaps surprising that more fungi cannot synthesize sterols, as they mostly inhabit environments where sterols would be expected to occur. The sterol content of spores of Plusmodiophoru brassicue varies significantly according to the sterol composition of the host, suggesting at least partial heterotrophy (Knights, 1970). The rusts however synthesize sterols. The predominant sterols in their uredospores are A’-stigmastenol, A7*24(28’-stigmastadienol and A’-ergostenol (Jackson and Frear, 1968; Nowak et ul., 1972). A7-Sterolswere found in rust-infected leaves and not (or only exceptionally) in healthy leaves (Nowak et al., 1972; Lin and Knoche, 1974). This indication that the fungus synthesizes sterols was confirmed by the finding that these par-

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

123

ticular sterols became labelled when germinating uredospores were supplied with [14Cl-mevalonateor acetate (Lin et al., 1972). I t is notable that, unlike other basidiomycetes, the rusts do not contain ergosterol. Those oomycetes which synthesize sterols (e.g. species of Achlya, Saprolegnia) also do not contain ergosterol, but rather fucosterol and its relatives (McCorkindale et al., 1969). Ergosterol was not detected either in two chytridiomycetes and two hypochytridiomycetes (Bean et al., 1972). The occurrence and biosynthesis of sterols in fungi have been reviewed by Weete (1973, 1974) and also by Goodwin (1973). A list of fungi and the sterols found in them is given by Bean (1973). The lipids of yeast were reviewed by Rattray et al. (1975). Biological aspects of sterols in fungi were reviewed by Hendrix ( 1970). 11. Functions of Sterols: Possible Approaches to the Problems

The object of this review is to discuss the function of sterols in fungi, with special reference to sexual reproduction ; the effect on sexual reproduction is the most striking aspect of the sterol requirement in Phytophthora. A general approach to this problem in fungi might be to consider what happens when sterol synthesis is inhibited. This inhibition might be the result of gene mutation, so that some step or steps in the biosynthesis of sterol cannot be carried out. As already indicated it appears that pythiaceous fungi cannot convert squalene to sterols. Species of Pythium and Phytophthora are very favourable organisms for studying the function of sterols. One can simply compare the mycelium grown with and without sterol. Evidently, in these fungi, sterols are not essential for vegetative growth. If sterols are essential for

FIG. 1. Structure of stigmastane, indicating the numbering of carbon atoms in sterols.

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CHARLES G. E L L I O l l

I

IV

&

H b:H

11

111

V

VI

HO@H,C kH,

&OH VIII

VI I

IX

FIG. 2. Ring systems of sterols. See Table 1 for sterols in which these occur. TABLE 1. List of principal sterols mentioned in the text, with systematic names and index to their structures shown in Figs. 2 and 3. Common name

Systematic name C,

B-nor-5-cholesten-3~-ol

Coprostanol Cholestanol Cho lest ero I A’-Cholestenol 7 - Dehvdrocholesterol Epicholcsterol Zymosterol

5/hholestan-S/J-ol 5a-cholestan-3/?-ol 5-cholesten-3~-ol 5a-cholest- 7-en-3p-01 5,7-cholestadien-3~-ol 5-cholesten-3a-ol 5a-cholesta-8,24-dien-3/3-ol

Campesterol A5-Ergostenol A7-Ergostenol

(24R)-24-methyl-5-cholesten-3~-ol (24S)-24-methyl-5-cholesten-3/3-ol (24S)-24-methyl-5a-cholest-7-en-3g-ol

C,

Side chain

sterol

B- Norcholesterol

C,

Ring system I

A

I1 I11 IV

A A A

V VI VI I VIII

A A B

sterols

A

Jterols

IV

C

IV V

D D

125

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

+A+* A

C

G

B

D

E

F

H J FIG. 3. Side chains of sterols.

K

TABLE 1 . 4 co n t i n u ed) Common name

Systematic name

Ring system

(24R)-24-methyl-5,2ZE-cholestadien- IV Ergosterol Episterol Ergostadienol AS.l.24 (28)-

3/3-01 (24R)- 24- methyl-5,7,22E-cholestatrien3/3-01 24-methylene-5a-cholest-7-en-3P-01

VI V

Side chain

E E F

(24R)-24-inethyl-5a-cholesta-8(9),22E-VIII

E

dien-3/3-ol. 24-inethylene-5,7-cholestadien-3/3-ol

VI

F

C,, sterols IV (24R)-24-ethyl-5-cholesten-3/3-ol V (24R)-24-ethyl-5a-cholest7-en-3/3-01 (24R)-24-ethyl-5,7-cholestadien-3~-olVI IV (24S)-24-ethyl-5,22E-cholestadien-

G G G H

Ergostatrieiiol

3/3-01

(24S)-24-ethyl-5,7,22E-cholestatrien8/3-01 24E-ethylidene-5-rholesten-3/3-ol

(24R)-24-ethyl-5,22E-cholestadien-

VI

H

IV IV

J

IX

B

K

3/3-01 Lanosicrol

C,, sterol 4a,4/3, 14a-trimethyl-5a-cholesta-8,24: dirn-3~-ol

126

CHARLES G . ELLIOlT

vegetative growth in other organisms, then pythiums and phytophthoras must make some substance which can do the same job. But it is apparent that this hypothetical substance cannot fulfil the role in reproduction which sterols have. In species of Saccharomyces, mutants in which the later stages of ergosterol synthesis are blocked, are known. These mutations were selected as conferring resistance to polyene antibiotics (see p. 145). Other ways in which sterol synthesis can be inhibited are to subject the organism to anaerobic conditions (see p. 1461, or to use drugs which inhibit particular steps in biosynthesis (pp. 147 and 162). An alternative and complementary approach is to consider the effect of molecular shape on activity. The approach is complementary because, when the terminal stages of synthesis are blocked, a precursor of different structure may accumulate and it may have some but different activity. Both the configuration of the ring system (Fig. 2) and of the side chain (Fig. 3) are important. The configurations of the ring systems are illustrated in photographs of models in Fig. 4(a-f). There is a fundamental difference in shape between molecules with the 5a and 58 configuration. In the former, there is a regular alternation of the carbon atoms above and below the plane through all the rings. In the latter, the molecule is bent at the A ring end (Figs. 4(aHb)).The position of the hydroxyl group at C-3 also is important. Ifit is in thepposition, the oxygen atom lies in the plane of the rings of the 5a or A5 sterols (i.e. an equatorial configuration; Figs. 4(b) and (c)).A double bond between C-5 and C-6 flattens the B ring somewhat, and alters the position of the C-4 hydrogens relative to those at other positions in ring A (see Figs. 4(b) and (c)),but a more extreme flattening of the B ring occurs with a double bond between C-7 and C-8 (with or without As).I t will be noticed that A7 brings the “axial” methyl groups (C-18 and C- 19) closer together (compare Figs. 4(d) and (el with (a), (b) and (c)). A double bond between C-8 and C-9 results in greater joint flattening of the B and C rings (Fig. 4(f)). An indication of the overall degree of flatness of the molecule is given by the number of the axial ahydrogen atoms which are coplanar. For Sa-cholestanol, those attached to carbons 1, 3, 5 , 7, 9, 12 and 14 are coplanar; for A5cholestenol, 1, 3, 9, 12 and 14; for A8-cholestenol,1,3, 12 and 14, and for A7-cholestenol,1, 3 and 14. In the last instance, it is notable how far the a-hydrogen attached to C- 12 is from this plane. The numbering of the carbon atoms is indicated in Fig. 1.

STEROLS IN FUNGI: THEIR FUNCTIONS IN GROWTH AND REPRODUCTION

127

The shape of the side chain is modified by substituents at C-24and double bonds at various positions. An ethyl or methyl group which replaces one of the hydrogen atoms at C-24can be either in the a or p position (Figs. 3, C and G;compare with D and K).The ethyl group of sitosterol (Figs. 2 and 3, IVG) and stigmasterol (IVH) is in the a position; that of poriferasterol (IV K),and the methyl group of ergosterol (VI D)are orientated. In the R and S terminology, sitosterol is (24R)-24-ethyl-5-cholesten-3p-o1. But, under the rules of terminology, the double bond at C-22 causes the ethyl group of stigmasterol to be designated S, and stigmasterol is (24S)-24-ethyl-5,22E-cholestadien3p-01. These variations in the shape of the sterol molecule can result in great variation in biological activity. A very high degree of specificity in their activity is one of the principal characteristics of steroids (Grant, 1969). A third method of approach is to study changes in sterol content or state of binding during the growth cycle, and to corrblate these with metabolic changes in the mycelium. In higher organisms, sterols have three principal functions; as precursors of other steroids, as hormones, and as membrane components (Heftmann, 1971). Heftmann (1970, 1971) points out that plants, as well as animals, produce sterols and from them CZ1, C,, and C,, steroids, and he argues that the fundamental biochemical similarity between all living organisms implies that these compounds have similar roles in plants and animals. In recent years, there have been great advances in our understanding of the action of steroid hormones at molecular level (Jensen and DeSombre, 1972;O’Malley and Means, 1974).A great deal is known about how these hormones bind to cytoplasmic receptors which are characteristic of the target tissues, and how these hormone-receptor complexes then bind to chromosomes leading to synthesis of RNA and specific proteins. At the physiological level, one is struck by the remarkable diversity of effects that hormones induce. We are still far from understanding how events at the molecular level account for this diversity, except that it is evident that a hormone can switch on a sequence of genetic events. Thus, administration of ecdysone to Drosophilu larvae brings about puffing (the sign of gene activity) first in one band of the giant chromosomes, followed by puffing in other bands in a definite order (Ashburner, 1970),so that a complex pattern of metabolic activity can be built up. One of the physiological effects of hormones is on transport across

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CHARLES G. E L L I O l l

FIGS. 4(a-1). Photographs of models of sterols to illustrate the configuration of the ring systmi. (a) Coprostanol; (b) Cholestanol; (c) Cholesterol; (d) A’-Cholestenol; (e) 7-Dehydrocholesterol; (D A8-Cholestenol.

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CHARLES G. ELLlOlT

membranes. Indeed, this is the principal effect of the mineralocorticoids and the vitamin D complex, but oestrogens too, for example, modify transport into the tissues that show the more dramatic responses to the hormone. Formerly, the possibilities that hormone action resulted from direct effects on permeability or on enzyme activity were considered (see Tepperman and Tepperman, 1960; Riggs, 1970). In work on fungal sterols, we are faced with a particular difficulty. As will be discussed later in this review, sterols as components of membranes have a pronounced effect on permeability, in model systems at least. Therefore we have to try to distinguish whether the effects of sterols on, say, species of Phytophthora, result from direct consequences of the presence of sterols in the plasma or mitochondria1 membranes, or whether there are effects which can only be attributed to a hormone-type action. On the whole, it seems that sterols in model membranes affect simple diffusion, but hormones in cells control active energy-requiring transport of specific ions or molecules. 111. Sterols in Model Systems

Nes ( 19741, in an admirable article, has reviewed the occurrence of sterols and sterol-like substances, and drawn attention to the small range of structures found in the dominant sterols of eukaryotes. Evidently only a few structures can fulfil the particular role which fiee sterols perform in the membranes where they occur. Few organisms are known which do not use sterols for this role. One is Tetruhymena pynyormis, which produces instead tetrahymenol, a compound which can adopt a shape similar to that of a sterol. When supplied with exogenous cholesterol, Tetrahymenu pyntormis incorporates it into its membranes (converting it into A5~7~22-cholestatrienol) and the amount of tetrahymenol synthesized is greatly decreased (Connor et al., 1969). Evidently the sterol is a more satisfactory molecule for performing the role in membranes. However, Nozawa et al. (1975) found that, when T. pyriiormis is grown with ergosterol, the replacement of tetrahymenol by ergosterol is accompanied by extensive changes in phospholipid and fatty-acid composition, and also by ultrastructural changes visible in the electron microscope. Some mycoplasmas require an exogenous source of sterol for growth (Edward and Fitzgerald, 1951; Smith and Lynn, 1958); these are now placed in the genus Mycoplasma. The genus Acholeplasma comprises organisms which do not require sterols. Achole-

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plasma laidlawii and A. granularum synthesize cartotenoids which Nes (1974) supposes play a similar role to sterols, but A. axanthum does not make pigmented carotenoids (Tully and Razin, 1969, 1970). Sterols are adsorbed into the membranes of both the sterol requirers and nonrequirers (Smith and Rothblat, 1960; Tully and Razin, 1969). The effect of exogenous sterol supplied in serum to A. laidlawii is to decrease the amount of carotenoid in the membrane (Smith, 1963) bht, when the sterol is added without serum, it is incorporated without significantly affecting the carotenoid content (Razin and Cleverdon, 1965).

FIG. 5. Diagram showing possible mode of interaction between cholesterol and lecithin with stearoyl and linolenyl chains (Vandenheuvel, 1963). CCC indicates the carbon atoms of glycerol; P, phosphorus; N, nitrogen ofcholine; and 0, the hydroxyl oxygen of' cholesterol.

The way in which cholesterol can fit into a phospholipid layer has been discussed by Finean ( 19531, Vandenheuvel (1963) and Brockerhoff ( 1974). The dimensions of the cholesterol molecule are such that, if the 3-hydroxyl of the sterol is associated with the nitrogen of the choline of the lecithin, the end of the cholesterol side chain fits neatly under the fatty-acyl chain where it bends at the cis double bonds (as in linoleic acid; Fig. 5; Vandenheuvel, 1963). Brockerhoff (1974) proposed a model based on hydrogen bonding between the 3hydroxyl group of the sterol and the carboxyl oxygen of the fatty-acyl chain. Edwards and Green (1972) showed that extra carbon atoms in the side chain decrease the ability of sterols to pack into phospholipid layers, campesterol being less readily incorporated than cholesterol, and sitosterol than campesterol. This could be related to the well known fact that ergosterol and sitosterol are less readily absorbed in the human intestine than cholesterol (Glover and Morton, 1958). Our understanding of the role of sterols in membranes has been es-

132

CHARLES G. ELLIOlT

pecially advanced by the use of physicochemical techniques applied to model systems-the lipid monolayer spread on water, and the bilayer “liposome” produced by mixing lipid with salt solution-as well as on membranes themselves. The membranes of mycoplasmas in particular are admirable objects for such experiments (Razin, 1973). The physicochemical studies were reviewed by Oldfield and Chapman (1972). Demel el al. (1967) and Chapman et al. (1969) showed that incorporation of cholesterol into a lecithin monolayer decreased the area occupied by each molecule. The magnitude of the affect increased with increasing proportion of cholesterol (De Kruyff et al., 1972). Darke et al. (19721, in a study using nuclear magnetic resonance, showed that cholesterol and lecithin formed a complex in which their molar ratio was 1 : 1 ; when the proportion of cholesterol in a bilayer was less than half, its distribution was clustered. However, Verkleij et al. (1974) found, using freeze-fractured liposomes, that lecithin bilayers containing only 1&15 mole per cent cholesterol were homogeneous. Rand et al. (1975) described a 1 : 1 molar complex between cholesterol and lysolethicin, and emphasized that their interaction was quite different from the interaction of cholesterol with the diacyllecithins. Addition of cholesterol to a lecithin decreases the amount of movement which the fatty-acyl chains of the lecithin can undergo. Thus the temperature at which the transition between the more rigid gel phase and the less rigid crystalline phase occurs is lowered, and the amount of heat adsorbed at the transition point is decreased (Ladbrooke et al., 1968). More recently, the use of electron spin resonance (ESR also called electron paramagnetic resonance, EPR), in which the freedom of movement of the electron of an introduced free radicle can be studied, has provided much information. For example, Hsia and Boggs (1972) and Mailer et al. ( 1974) showed that cholesterol restricts the freedom of movement of the hydrocarbon chains of fatty acyl groups, and Butler et al. (1970) compared the effects of a number of steroids. Kroes et al. ( 1972) similarly showed increased viscosity in erythrocyte membranes from guinea pigs fed a cholesterol-supplemented diet; their erythrocytes contained about twice the control amount of cholesterol. Feinstein et al. (1975) used a fluorescent probe to show that cholesterol decreased membrane fluidity. Rothman and Engelman (1972) showed with models how the presence of cholesterol in the membrane restricts the movement of the carboxyl half of the fatty-acyl chain, while some movement is still possible at the free end.

STEROLS I N

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Work with artificial membranes has shown that sterols affect their permeability. When they contain cholesterol, their permeability to water is decreased (Finkelstein and Cass, 19671, the rate at which liposomes swell in hypotonic solutions is diminished (De Gier et al., 1968) and the rate of loss of sequestered glucose is also decreased (Demel et al., 1968). Both cells of Acholeplasma laidlawii, and liposomes prepared from their lipids, showed lower permeability to glycerol and erythritol when the cells has been grown in cholesterol-containing medium (De Kruyff et al., 1972; McElhaney et al., 1973). The latter authors, and also De Kruyff et al. (1973), emphasized the effect of fatty-acid composition on membrane permeability, but incorporation of cholesterol had no effect on the fatty-acid composition, and thus the diminished permeability with cholesterol could be attributed to cholesterol (De Kruyff et al., 1972). Rottem et al. (1973) adapted the sterol-requiring species Mycoplasma mycodes var. Capri to grow without sterol, and showed that the adapted cells were more fragile osmotically, and were more permeable to erythritol. The effects of other sterols besides cholesterol on molecular spacing in monolayers and on permeability of liposomes have been studied by Demel et al. (1972a, b). Their papers give no indication of the variability of the results, but it appears that the effects of sterol structure on both phenomena are very similar and are comparable to the effects on cell permeability (De Kruyff et al., 1972, 1973). The ESR studies by Butler et al. ( 1970) also give essentially similar ordering of the compounds. In general, cholesterol (Figs. 2 and 3, IVA) has the greatest effect in all of these systems, with cholestanol (IIIA), A’cholestenol (VA), 7 -dehydrocholesterol (VIA) and B-norcholesterol (IA) similar. Molecules without a side chain (5a-androstan-3p-01 and 5-androst-en-Sp-01) have little effect, as is also the case with 3ahydroxysterols (e.g. epicholesterol; VIIA) and coprostanol (IIA) with its 58 configuration. Of the sterols with extra carbon atoms in the side chain, sitosterol (with a saturated side chain; IVG) has a similar effect to cholesterol, and stigmasterol (IVH) and ergosterol WID) (with a double bond at C-22) are less effective. These conclusions are in agreement with the effects of steroids on growth of mycoplasmas (Smith, 1964; Smith and Lynn, 1958; Rottem et al., 1971). I t is interesting to note that, in agreement with Nes’s (1974) hypothesis, carotenoids have similar effects to sterols on permeability. Huang and Haug (1974) grew A . laidlawii on acetate-containing

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CHARLES G. ELLIOlT

medium (and thus increased the mount of carotenoids) and on propionate (thereby lowering the content of carotenoids). The carotenoid-enriched cells had less fluid membranes (as indicated by the ESR spectrum) and the cells were tougher to lyse and less permeable to glycerol. The effect of sterol composition on permeability and fragility of membranes has also been studied with erythrocytes and their ghosts by Bruckdorfer et al. (1968a, b). Erythrocytes readily lost cholesterol to sterol-depleted plasma or pure phospholipid dispersions. When these treated cells were re-incubated with dispersions of lecithin and sterol, the sterol was absorbed into the membranes, different sterols being taken up to different extents. Ergosterol was taken up to a remarkably poor extent. I t was found (Bruckdorfer et al., 1969) that the erythrocytes incub'ated in lecithin alone (which lost 3348% of their cholesterol) were very fragile and permeable to glycerol. As compared with cells re-incubated with cholesterol, those in which there was partial replacement of cholesterol (IVA) by A'-cholestenol (VA), 7 -dehydrocholesterol (VIA) or B-norcholesterol (IA) showed considerably decreased fragility, while incorporation of stigmasterol (IVH)or ergosterol (VIE) effected slight increases and decreases, respectively, although they replaced cholesterol to a very limited extent. The S-oxosteroids increased fragility and also permeability to glycerol. Coprostanol (IIA) and 7 -dehydrocholesterol (VIA) caused some decrease in permeability, but A'-cholestenol (VA) and Bnorcholesterol (IA) had little effect. Edwards and Green (1972) found that, in a replacement system, sitosterol (IVG) and campesterol (IVC) were incorporated into erythrocyte membranes to a lesser extent than cholesterol, the enlarged side chain being inimical to adsorption. In higher plant tissues, the effects of sterols on permeability have been studied by Grunwald (1968, 1971). He found (1968) that cholesterol was highly effective in reversing the methanol-induced leakage of betacyanin from beetroot cells, but sitosterol (IVG) and stigmasterol (IVH) were less effective; ergosterol (VIE) increased the leakage. With barley roots (Grunwald, 197 11, cholesterol was most effective at 10 pM at decreasing ethanol-induced permeability (but it increased leakage at 100 pM);campesterol (IVC)was less effective and stigmasterol (IVH) and sitosterol (IVG) had little effect. The evideuce already discussed shows that the occurrence in

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membranes of 3/3-hydroxysterolswith 5a- or A5-configurationsand a side chain decreases their permeability, and cholesterol is one of the most effective in this respect. But it must be remembered that not all membranes are alike in their lipid composition. Besides species differences, the membranes of different organelles are different, in sterol as well as phospholipid composition (Dod and Gray, 1968; Siekeviu, 1970). The differences are no doubt related to the structural requirements associated with the different enzymic proteins which are bound to the membranes; the lipid composition would require to be adapted to the physiological activity of the membrane. Also, Smith ( 1969) has argued that sterols in mycoplasmas are actively associated with transport across the membranes; especially, he believed that the sterol glucosides found in those species which use glucose as an energy source are sterols caught in the act of moving glucose into the cell. More recently other transport mechanisms have been found (see Razin, 1973, p. 66). Wiley and Cooper (1975) studied the effect of cholesterol enrichment on transport of Na' and K+across erythrocyte membranes, and suggested that the excess cholesterol could influence the position of the protein carrier within the membrane, or decrease its ability to move within a more viscous micro-environment. IV. Subcellular Distribution of Sterols in Fungi, and States of Binding

A number of fungi absorb sterols from their medium, even those which synthesize them (Hartman and Holmlund, 1962). Study of the uptake of sterols from solutions of various concentrations provides evidence as to the mechanism of uptake. With simple physical adsorption, we expect, from the Freundlich adsorption isotherm, a linear relation between the logarithms of the concentration supplied and the conceytration in the adsorbing surface. The evidence in Phytophthora cactorum (Elliott and Knights, 1974) is in agreement with this expectation, as are the much more extensive data on mycoplasmas (Smith and Rothblat, 1960) and mammalian tissue-culture cells (Rothblat et al., 1967). The uptake of cholesterol by Acholeplcwmu laidlawii was also shown to follow first-order kinetics by Gershfeld et al. (1974). Uptake of various sterols by Phytophthera infestans was investigated by Langcake (1975) who found that cholesterol (IVA) and cholestanol (IIIA) were taken up much more than sitosterol (IVG) and stigmasterol (IVH),

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CHARLES G. ELLIOlT

while ergosterol (VIE) was very poorly taken up. Hendrix (1975a) found that sitosterol and cholesterol were taken up less readily by Achlya spp. than by Pythium spp. The sterol taken up by Phytophthora and Pythium spp. is recoverable partly as free sterol and partly as ester (Hendrix et al., 1970; Gain, 1972; Elliott and Knights, 1974; Hendrix, 1975b). Hendrix et al. (1970) considered that all of the sterol could be readily extracted from the mycelium, but Elliott and Knights (1974) found that about 10%of the cholesterol taken up from the medium was not recovered with a 7 h Soxhlet extraction with acetone, but it was recovered if the acetoneextracted mycelium was hydrolysed with 6-N HCl or by pyrogallol in methanolic potassium hydroxide. We have also shown (B. A. Knights and C. G. Elliott, unpublished observations) that the residue remaining after prolonged extraction with acetone contains sterol by combustion of the residue and analysis of the carbon dioxide produced for radioactivity. Cholesteryl oleate added to cultures growing without sterol is taken up by P. cactorum much more slowly than free cholesterol (Elliott and Knights, 1974). We believe that the ester must be converted to free sterol before it can be absorbed. The free sterol content of the mycelium reaches a constant value per unit weight about 12 h after addition of free sterol to the culture, and despite the slower rate of uptake of oleate the free sterol content per unit weight reaches the same constant value within 24 h of adding the ester (Fig. 6). The amount of sterol ester in the cells follows a different pattern according to whether the culture is supplied with free sterol or with ester. With free sterol supplied, much sterol is taken up, and much is found as ester, in harvests made soon after addition of the sterol to the cultures; later the amount of ester declines. Presumably the ester formed in the early stages is de-esterified, thus keeping the free sterol content constant as the weight of mycelium increases. With oleate supplied, less sterol is taken up at first, and little of it is found as ester (Fig. 6; Elliott and Knights, 1974). These results are consistent with the view that free sterols are found in membranes, while the sterol present in the cell in excess of that required free in the membranes is esterified and located elsewhere (Nes, 1974).We have not investigated the distribution of free sterol and ester in P. cactorum, but Sietsma and Haskins (1968) fractionated cells of Pythium acanthium grown with cholesterol, and reported that 79%of the

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-0-0

;---fp---.---:--

Time ( h ) from addition of cholesterol to medium

FIG. 6. Changes with time in sterol and sterol ester content of mycelium of Phytop h h m cactorum, following addition of cholesterol or cholesteryl oleate to six-day-old cultures. U indicates changes in the content of free sterol in ecetone extracts of mycelium (free cholesterol supplied in medium); o----O of sterol ester in extracts of mycelium (free cholesterol in medium);. --- Aof free sterol in acetone extracts of mycelium (cholesteryloleate supplied in medium);A---Aof sterol ester in extracts of mycelium (cholesteryl oleate in medium). Data of Elliott and Knights (1974).

sterol was in the protoplast membrane, 18%in mitochondria and 3%in endoplasmic reticulum. Their assay was by the Liebermann-Burchard assay, and esters were not investigated. Langcake (1975)reported that 71% of the cholesterol taken up by Phytophthora infeestuns was in the supernatant after centrifugation of blended mycelium for 15 min at 2 O O O g and 86% of the sterol of this was sedimented after 30min at 160,000g. The subcellular distribution of ergosterol in the ascomycete Aspergdlus niger was studied by Barr and Hemming (1972).They found that about half of the free sterol was in the mitochondria1 fraction (identified by the presence of ubiquinone), but the largest proportion of ester was in the heaviest fraction (sedimentingat 500 g after 10 mid. More critical information comes from the work on yeast (Saccharomyces cereviseae) by Hossack et al. (19731, who found that the membranous fraction (identified by phosphatidylinositol kinase) contained primarily free sterol, while the fraction consisting of low-density vesicles contained sterol esters and very little free sterol. Such vesicles are present in the growing hyphal tip (Bartnicki-Garcia, 1973kin yeast in the young bud (Moor, 1967)-and they contribute directly to the extending plasmalemma. I t seems possible that the sterol esters of the

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CHARLES

G. ELLIOlT

vesicles serve as a ready source of the sterol required for the new membrane. Hallermeyer and Neupert ( 1974) found that ergosterol was present in the outer mitochondria1 membranes of Neurospora crassa, but there was no sterol in the inner membrane. Assay of the amounts of free sterol and ester in mycelium is however dependent on the methods of chemical extraction used. Adams and Parks ( 1967) showed that different extraction procedures removed different amounts of sterol from yeast. Saccharomyces cerevisiae contains a mannan in the cell wall which binds sterol (Thompson et al., 1973), making it water soluble and resistant to extraction with the usual lipid solvents; this bound sterol can be extracted after hydrolysis with alkaline pyrogallol (Adams and Parks, 1968). Sterol-binding polysaccharides were also described from Rhizopus arrhizus, Penicillium roquefortii and Saccharomyces carlsbergensis by Pillai and Weete ( 1975). Hence, studies on variation in the sterol content of mycelium during the growth cycle, using a single method of extraction only (as was done by Van Etten and Gottlieb (1965) with Penicillium atrouentum) are of limited value. In our analysis of sterols in Neurospora crassa (Elliott et al., 19741, we extracted the dried mycelium first with acetone, and then subjected it to alkaline pyrogallol hydrolysis. The acetone extract contained “readily extractable” free sterol and sterol esters; the alkaline pyrogallol extract was of “tightly bound” sterol. The occurrence of a large tightly bound fraction was characteristic of conidia and of senescent mycelium. It appeared that there was transfer of sterol from a readily extractable to a tightly bound state with the onset of senescence. Sterols were synthesized during active growth of the mycelium, with the amount of ester more or less parallel to free sterol. When accumulation of dry matter ceased, the free-sterol fraction continued to increase in size for a couple of days, apparently at the expense of the esters. Increase of free sterol at the expense of esters was noted in yeast by Madyastha and Parks (1969). In Phycomyces blakesleeanus, the composition of the free-sterol fraction is relatively constant during growth of the culture, but there are marked changes in the ester fraction. Thus, the proportion of episterol varies from 31 to 37% in the free-sterol fraction but from 45 to 76% in the ester fraction at different stages of growth; ergosterol forms from 49 to 66% of the free sterols but from 18 to 48% of the esters. The proportion of ergosterol esterified increases during the first 30 h of incubation, then declines somewhat and increases again after 45 h (Bartlett and Mercer, 1974). The composition of ester and free-sterol

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fractions differ also in Saccharomyces cereuisiae (Barks et af., 19741, and in Leptosphaeria typhae (Alais et ul., 1974).Atallah and Nicholas (1974)have suggested that esters may be important during biosynthesis; hence, perhaps, the greater diversity in the esterified sterols. DupCron et a f . (1972) found a small proportion of the sterol of potato tuber tissue in the supernatant after centrifugation of the material at 100,OOOg. This had a very large amount of sterol ester. They considered the occurrence of ester in the cytosol reflected a function in transport of sterol round the cell. The amount of sterol ester did not decline during starvation in Phycomyces blahleeanus (Bartlett and Mercer, 1974); thus sterol ester can act as a reserve for sterol but not for energy production. In potato tuber during storage, the sterol esters increase while the glucosides decrease; the free-sterol content remains more or less constant (DupPron et ul., 1971). Synthesis of sterols and sterol esters was followed during growth of Saccharomyces cereuisiae by Bailey and Parks (1975).They found that synthesis of C,, sterols continued throughout the logarithmic phase of growth, but the amount of sterol ester remained at a constant low value until the onset of the resting phase when it rapidly increased. While ergosterol formed the major sterol at an early stage of growth (92%of all sterol), it decreased to 40%at a later stage, concomitant with the appearance in substantial quantities of ergosterol precursors, especially A5.7-ergostadienoland zymosterol, and these were found mainly in the ester fraction. While zymosterol (Figs. 2 and 3, VIII B) is readily methylated at C-24, its esters are not, and, in general, esters of the precursors are not metabolised to ergosterol. Thus Bailey and Parks (1975)suggest that sterol esters may have an important role to play in the regulation of sterol synthesis. Alais et al. (1974)compared the sterols of Leptosphaeria typhae grown in light and in the dark. In the light-grown cultures, ergosterol was the only sterol found in the free state but, in the dark-grown cultures, there were small amounts of episterol, A7-ergostenol and ( ?) 24-ethyl-5cholestenol. The ester fractions also differed, the dark-grown cultures containing a considerable proportion of C,, and C,, sterols. It is to be noted that light induces formation of sexual structures in this fungus. Safe (1973)investigated the sterols of Mucor rouxii in the filamentous and yeast-like phases induced by growth under aerobic and anaerobic conditions, respectively, and found differences in sterol composition and binding. Under aerobic conditions, 90% of the sterol readily extractable with chloroform-methanol was ergosterol, but the tightly bound fraction (obtained by alkaline pyrogallol hydrolysis of the

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chloroform-methanol extracted mycelium) contained only 33% ergosterol together with A7J4(28)-ergostadienol (25%). Under anaerobic conditions, several sterols appeared in the readily extractable fraction as well as in the tightly bound fraction, but their proportions in the two fractions were different. Differences in the relative proportions of ergosterol and di-unsaturated sterols in the two fractions were also found in Neurospora crassa (Elliott et al., 1974). Safe and Caldwell(1975) reported further studies on the distribution of sterols between the cell wall and cytoplasm of Mucor rouxii in relation to the mode of binding, and they studied a yeast-like phase induced by phenethyl alcohol as well as that induced by anaerobiosis. Changes in the binding state of sterols in yeast were described by Adams and Parks (1967). Their technique was to grow cells anaerobically and then aerate them in a medium containing KH,PO,, 1% glucose and ['4C-methyllmethionine, so that the ergosterol synthesized was labelled at c-28. Cyclic changes in the amount of sterol extractable after saponification with 40% potassium hydroxide and after hydrolysis with hot 0.1-N HCI, were observed. The amounts of sterol recovered by these two methods, when added together, gave a constant value from the fourth hour after the beginning of aeration; up to this time, sterol was being synthesized. It was suggested that the sterol extracted with the base and acid treatments represented different pools, and that there was a shift of some sterol from one pool to the other according to the metabolic state of the cells. Starr and Parks ( 1962) observed that the time of occurrence of the drop in the amount of base-extractable sterol of cells aerated in glucose was determined by the glucose concentration, and was associated with exhaustion of sugar from the medium and a switch to respiratory utilization of the ethanol which had accumulated during fermentative metabolism. When the cells were aerated with acetate as energy source, the amount of baseextractable sterol increased to a maximum value which then remained constant (Adams and Parks, 1967). Also, when cells were aerated with glucose in the presence of acriflavine, which prevents adaptation to respiratory competence, the drop in base-extractable sterol did not occur (Adams and Parks, 1969). Synthesis of sterols during ascus formation in Saccharomyces cerevisiae has been investigated by Esposito et al. ( 19691, Henry and Halvorson (1973) and Illingworth et al. (1973). Incorporation of labelled acetate into lipid occurs in two stages (Esposito et al., 19691, the second of

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which co-incides with the appearance of the ascospores (Henry and Halvorson, 1973). Illingworth et al. (1973) noted an increase in free sterol proportional to the amount of new membrane formed, but there was a much greater increase in the amount of sterol ester, which was associated with the development of vesicles. Crystals observed in hyphae of Neurospora crassu (Tsuda and Tatum, 196 1) and Phymatotrichum omnivorum (Baniecki and Bloss, 1969) and considered to be ergosterol, have recently been shown to be proteinaceous (Hoch and Maxwell, 1974).

V. Effects of Sterols on Metabolism and Vegetative Growth A. P Y T H I U M

A N D PHYTOPHTHORA

An immediate effect of the uptake of sterol by Pythium sp. is to make the cells less permeable. Cholesterol-grown cells “leak” nucleosides and proteins less than ones grown without sterol (Sietsma and Haskins, 1968). Child et al. ( 1969) compared leakage of material from mycelium grown with and without cholesterol; after an initial loss attendant on transferring cholesterol-grown cells to fresh medium or water, there was no further leakage of material absorbing at 260 nm, of nitrogen or of protein, and no change in conductivity of the suspending fluid, whereas loss continued with cells grown without added cholesterol. Loss of carbohydrate however was greater with cholesterol-grown cells. Species of Phytophthora and Pythium grow faster in the presence of sterols than on medium without added sterols. This is true both for dry weight and hyphal extension. The amount of increase in growth with sterols is not as great as with lecithin, but the growth rate with lecithin and sterol is greater than on medium supplemented with lecithin only (Hohl, 1975). With Pythium acanthium, addition of cholesterol to the medium shortens the lag phase of growth or accelerates growth in the early exponential phase; the total mycelial mass grown with or without cholesterol is the same (Child et al., 1969), or eventually the mycelial mass without cholesterol may exceed that grown with cholesterol (Brushaber et al., 1972). In several other Pythium species and in Phytophthora cactorum, cholesterol-grown cultures attained a greater maximum weight (Schlosser and Gottlieb, 1968; Elliott, 1972a). Sterols require to be added to the medium for asexual reproduction

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CHARLES G. ELLIOlT

by sporangia (Hendrix, 1965, 1967; Chee and Turner, 1965; Elliott, 1972a; Langcake, 1974). Their incorporation also increases the ability to survive at high and low temperatures (Haskins, 1965; Sietsma and Haskins, 1967). Sterols -increased the number of propagules produced by Pythium sylvaticum, but those grown without sterols survived in soil as well as those grown with sterols (Kaosiri and Hendrix, 1972). Various sterols have different effects on vegetative growth and they affect aspects of growth differently (Langcake, 1974). Langcake’s results with P. inzstans are summarized in Table 2. The difference between cholesterol and stigmasterol is particularly intriguing. The effect of sitosterol is intermediate but generally much closer to that of TABLE 2. Ellects of several sterols on growth of Phytophthoru infeestuns (Langcake, 1974, 1975)

Stcrol

;Idclrd None

Cfiolrstrrol Cholrst;inol Si Iostrrol Stigniastrrol Ergosrcrol L;iii~~tc-r~l

Stcrol content ofmycelium (% ofdrv wt)

Dry weight of eight-day cultures (mg)

Colony diam. at 14 days (cm)

Sporangium production’

-

15.8 28.3 27.9 27.8 24.6

12.0 39.9 54.0 46.1 58.9 35.1 15.5

0.6 28.6 20.5 33.3 25.1 22.1 15.2

0.19 0.19 0.09 0.11

Trace -

-

6.0

~

A plug I cm in diameter was cut from the culture and shaken in 2 ml water. The number of sporangia liberated was counted in a 10 pl sample.

cholesterol. Cholesterol gives heavier cultures than stigmasterol (although the difference in weight is not actually significant) but, with stigmasterol, hyphal elongation is much faster (the difference being highly significant). Could a difference in their effect on the cell membrane account for these phenomena? The more permeable membrane might facilitate deposition of material at the hyphal tip, and so promote faster elongation; the less permeable might retain material within the cell and thereby increase its weight. Sterols appear to promote more active cellular metabolism. Schlosser and Gottlieb ( 1968) investigated glucose metabolism in Pythium ultimum grown with and without cholesterol. They found that glucose uptake was faster in cultures with cholesterol than without, but the difference was less than one would expect from the rate of dry

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matter production. However, the cultures containing cholesterol produced 65%more carbon dioxide per unit of glucose consumed, indicating a much greater energy production in the sterol-containing mycelium. Pythium acanthium grown with cholesterol produced a greater quantity of lipid than when grown without it (Brushaber et al., 1972). Sietsma (1971) showed that, in Pythium spp. grown in the presence of cholesterol, most of the enzymes of the tricarboxylic-acid cycle, some aminotransferases and glutamate dehydrogenase showed higher activity than when the
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weight of tissue). The authors suggested that sterols occurring in high localized concentration in older roots changed the fungus from its aggressive pectolytic enzyme-producing phase to a less aggressive reproductive phase. Defago et al. (1975) showed that the relationship between the amount of cholesterol supplied to Pythium paroecandrum and its pathogenicity depended on other constituents of the host plant. Thus, with sugar beet (Beta vulgaris) and tomato (Lycopersiconesculentum), the severity of disease symptoms decreased with increasing sterol concentration, as the plants contained saponins to which sterol-containing mycelium is sensitive, whereas mycelium without sterol is not sensitive to saponins. Pea (Pisum sutivum) and soy bean (Glycine max) contain no saponins, and here disease severity increased with increasing sterol concentration. With lucerne (Medicago sativa), the severity of symptoms increased with sterol concentration despite the presence of saponins, as the plant contains tannic acid, and cholesterol in the mycelium imparts some resistance to tannic acid. Langcake (1974) tested the suggestion of Elliott and Knights (1969) that, in plant tissues containing a high ratio of sterol precursors (e.g. cycloartenol) to sterols, the precursors would interfere with growth and sporulation of Phytophthora and Pythium spp. and confer resistance on the host tissues. Langcake (1974) found, contrary to expectation, that leaves of the potato varieties Majestic and Ulster Chieftain, which were susceptible to blight (caused by Phytophthora infestand, contained more cycloartenol relative to sitosterol than the resistant Pentland Dell. He also found that extracts of Majestic and Pentland Dell leaves, added to medium at concentrations adjusted to give the same amounts of sitosterol, had a similar ability to promote growth of P . infeestans, despite the difference in their cycloartenol content. B. S A C C H A R O M Y C E S A N D O T H E R F U N G I

The study of sterols in Saccharomyces cerevisiae has recently been considerably advanced by the use of mutants with altered sterol composition, and by a physiological technique which similarly allows replacement of ergosterol by other sterols. Sterol-requiring yeast mutants were described by Karst and Lacroute ( 1973) and by Sprinson and his coworkers (Gollub et al., 1974; Trocha et al., 1974). These mutants are “petite”, and the sterol deficiency is apparently due to a defect in biosynthesis of haem (Bard et al., 1974). Haem is required in sterol syn-

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thesis for oxygenation reactions concerned with removal of the C- 14 methyl group. The other kind of sterol mutants investigated are the polyeneresistant strains. Polyene antibiotics act by complexing with sterols in membranes (Lampen, 1966; Kinsky, 1967). They inhibit growth of fungal cells, but not in species of Phytophthora or Pythium grown without sterols-the antibiotics may even increase growth of Pythium (Defago et al., 1969). However, when these fungi are grown with sterol, they become sensitive to polyenes (Schlosser and Gottlieb, 1966; Van Etten and Gottlieb, 1967; Schlosser, 1972; Sietsma and Haskins, 1968; Child et al., 1969) and soluble material leaks out of the cells. Mutants of Neurospora crassa ( Grindle, 197 31 and Saccharomyces cereuisiue (Woods, 1971; Molzahn and Woods, 1972; Bard, 1972; Parks et al., 1972) resistant to polyene antibiotics contain different sterols from the wild-type sensitive strain, and it appears that each of four genetically different mutants of Sacch. cereuisiae has a different lesion in the biosynthetic pathway from zymosterol to ergosterol (Barton et al., 1974). The sterols of double mutants were as expected from the lesions observed in single mutants (Barton et al., 1975). Another mutant selected for nystatin resistance in the presence of cholesterol by Karst and Lacroute (1974) was found to be unable to convert squalene to lanosterol. Bard (1972) remarked that the sterols in his mutant strains must be effective in meeting the structural and functional requirements of the cell, but that the antibiotic has a greater chemical affinity for the sterols terminal in the biosynthetic pathways, i.e. ergosterol and cholesterol. Fryberg et al. (1974) adapted yeast to grow in concentrations of 30, 100, 200 and 300 units of nystatirdml, and they found that the strains which were resistant to the highest concentrations were blocked earliest in the synthetic pathway (and contained A8*24(28)ergostadienol (Figs. 2 and 3 VIIIF)) while those resistant to the lowest concentrations were blocked at the latest stage (and produced A’***ergostadienol WE), instead of ergosterol). Karst and Lacroute (1973) noted that a sterol-requiring mutant of yeast growing in ergosterol-containing medium was sensitive to nystatin at a concentration of 8 pg/ml but, when grown with cholesterol, sitosterol or stigmasterol, its sensitivity was less, 15 pg/ml being required for inhibition. Several people have investigated the ability of various sterols, when added to the medium together with the antibiotic, to reverse the anti-

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fungal activity of the polyenes (Gottlieb et al., 1960; Perritt et al., 1960; Zygmunt and Tavormina, 1966; Lampen et al., 1960; Gale, 1974) and in some cases the complexing of sterol and polyene in the absence of fungus was studied.-But these investigations have not included a sufficient range of sterols to provide any clue as to why absence of ergosterol confers resistance to nystatin on the yeast mutants. The physiological technique of replacement of ergosterol by other sterols depends on the fact that, during anaerobic growth, yeast cells require an exogenous source of sterol and unsaturated fatty acid (Andreasen and Stier, 1953, 1954).This sterol requirement arises from the need for molecular oxygen in the formation of 3-oxidosqualene, the first step in the cyclization of squalene to sterol. Proudlock et al. ( 1968) found a relatively “all or none” effect in the ability of sterols of various structures to support anaerobic growth. A number Of 5 a or A5 sterols (including, remarkably, 5a-cholestan-3a-ol) supported growth equally well. Hossack and Rose (1976) supplied anaerobic cells with a sterol other than ergosterol, and achieved considerable enrichment of the cells with the foreign sterol. The effects of this modification on the ability of protoplasts to stretch was investigated. The main finding was that protoplasts containing sterols with a double bond in the side chain (ergosterol, stigmasterol) are considerably more stable and burst less readily than those with a saturated side chain (cholesterol, 7-dehydrocholesterol, A’-ergostenol, campesterol or sitosterol). The presence of Azz sterols in the membranes thus endows them with a limited capacity to stretch (Hossack and Rose, 1976). As already noted, ergosterol and stigmasterol decrease the packing of molecules in a film less than sterols with a saturated side chain (Demel et al., 1972a). I t is obvious that species of Phytophthora and Pythium could provide much precise information on the effect of sterols on membrane structure and permeability, since the sterol content can be so directly controlled. I am not aware of such experiments having been reported, although protoplasts of Pythium sp. were prepared by Sietsma et al. (1968, 1969). The amount of sterol supplied to yeast cultures during anaerobic growth affects the performance of the cells when they are subsequently aerated. Cultures grown anaerobically with high concentrations of ergosterol in the medium adapt to aerobic growth more rapidly than,those with only low concentrations-the activities of succinate dehydrogenase and cytochrome oxidase increase linearly with

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time (and not by an S-shaped curve) in the presence of high concentrations of ergosterol (Hebb and Slebodnik, 1958). The effect of ergosterol on the ultrastructure of Saccharoqces cerevisiue was described by Wallace et al. (1968). Adding ergosterol to aerobically-grown cultures produced no visible effect. Cultures incubated anaerobically without sterol or unsaturated fatty acid showed only vague outlines of mitochondria. With oleic or palmitoleic acids or Tween 80, but no sterol, extensive membranous structures appeared in the cells, but not normal mitochondria. However, with Tween 80 and ergosterol, mitochondria profiles were much more clearly defined, and extra membranes were not found. It seems that such mitochondria would be ready to assemble and use the respiratory enzymes immediately they were formed, With the obligate aerobe, Candida parapsilosis, addition of ergosterol and unsaturated fatty acid to the medium during growth under low aeration greatly enhanced the clarity of the mitochondrial profiles without increasing growth rate (Kellermanet al., 1969). The conclusion here seems to be that ergosterol is fulfilling a structural role in mitochondrial membranes. This was also the conclusion of Thompson and Parks (1972) from their investigation of cytochrome oxidase. Associated with an 80-fold purified preparation of the enzyme, and necessary for its activity, was a lipid component which included ergosterol. However, ergosterol itself, when added to the enzyme, did not promote its activity, but unsaturated fatty acids did. Notable use of nystatin-resistant mutants has been made by Thompson and Parks ( 1974) to study physiological activity in relation to sterol structure. Wild-type cells contained ergosterol ; one mutant contained A5*7~22J4(28)-erg~~tatetren~l; two others contained As*nnergostadienol. When grown fermentatively in glucose-containing media, all strains had similar temperature optima but, when required to respire ethanol, the strains with A8J2-ergostadienol had lower temperature optima, and lower upper limits for growth, than those containing the A5*’-sterols.Differences were also found in the activity of sterol methyl transferase in mitochondria, attendant on the alteration in sterol structure from A5n7to As. The As-sterol appears to be less effective as a repressor of the enzyme than ergosterol. The effects of inhibitors of sterol synthesis are discussed in Section X,p. 162 in connection with sexual reproduction; but here we will note that certain fungicides are inhibitors of sterol synthesis (triarimol,

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Ragsdale and Sisler, 1972; S-1358, Kato et al., 1974, 1975). Ragsdale (1975) showed that, in Ustilago maydis, triarimol inhibits removal of the 14-methyl group, inserkion of the double bond at C-22 and the reduction of the double bond at C-24(28). The morphological effects were that sporidia enlarged and became multicellular and multinucleate; no more sporidia were budded off from these cells. The controls without triarimol continued sporidial production, the cells remaining unbranched, unicellular and uninucleate (Ragsdale and Sisler, 1972, 1973). The related compound triforine also inhibits ergosterol synthesis in Neurospora c r a m (Sherald et al., 1973). These authors also found that certain mutants of Cladosporium cucumerinum and Ustilago maydis, selected for resistance to triforine, were resistant to triarimol, and its other analogues, ancymidol and EL-241. VI. Effects of Sterols on Asexual Reproduction A well nourished mycelium is a sine qua non for asexual reproduction, and it is perhaps because of this that sterols are required for sporangium formation in species of Phytophthora (see page 14 1).

However, induction of asexual reproduction in fungi presumably involves the switching on of genes which are repressed during earlier stages of growth. We know nothing of the means whereby this is brought about at the chromosomal level. Could a steroid hormone be involved ? In the imperfect ascomycete Stemphylium solani, ergosterol can replace the requirement for light for induction of conidiation (Sproston and Setlow, 1968). The ergosterol was dissolved in 5% dimethyl sulphoxide in 0.1-M KH,PO,; the solvent itself induced some conidium formation. Ergosterol at 0.5 pg/ml further enhanced conidium production, although concentrations above 0.8 ,ug/ml caused inhibition of conidium formation. Ethanol, propylene glycol and ethylene glycol also increased conidiation. Sproston and Setlow ( 1968) suggested that the similar inducing effect of ergosterol and of ultraviolet radiation and solvents could be due to the freeing of internally bound sterol by the latter agents, and this release of free sterol is the triggering mechanism for conidium formation. Baniecki and Bloss ( 1969) reported that ergosterol stimulated conidium production by Phymatotrichum omnivorum but, in this case, it did not substitute for the light requirement; both light and sterol were re-

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quired. I t seems that ergosterol itself is not required but that its vitamin D product is, since vitamins D, and D, and dihydrotachysterol were stimulatory to conidiation. VII. Sexual Hormones of Achlya I begin the discussion of the effect of sterols on sexual reproduction in fungi with the sex hormones of Achlya spp., for here the involvement of sterols, through steroid hormones, is clearly understood, thanks to the elegant work of Dr. Alma Barksdale and her associates. The control of reproduction in Achlya spp. by a series of hormones was demonstrated in the classic experiments of Raper ( 1939, 1940). He showed that a hormone, A, produced by female plants of A . bisexualis and A . ambisexualis induced formation of antheridial branches in the male plant. The male plant was then induced to produce a second hormone, B, which had the effect of stimulating production of oogonia by the female plant. Raper supposed there was a third hormone, C, produced by the oogonial initials, which attracted the antheridial branches; when the antheridial branches made contact with the oogonial initials, the antheridia were delimited, and a fourth hormone, D, then led to delimitation of the oogonia from the oogonial initials. Following the work of Raper and Haagen-Smit (1942), hormone A was isolated from culture filtrates and partly characterized by McMorris and Barksdale (1967) who named it antheridiol. Its structure was elucidated by Arsenault et al. (1968), and confirmed by synthesis (Edwards et al., 1969, 1972; Fig. 7, I and 11). 23-Deoxyantheridiol (Fig. 7, I and VI) was also found in culture filtrates of female plants (Green et al., 1971). Antheridiol is active towards species of Achlya and Thraustotheca, but it is not active towards Allomyces and Dictyuchus (A. W. Barksdale, personal communication). Antheridiol is taken up by both male and female plants of Achlya spp., but only male plants react to it (Barksdale and Lasure, 1973). The primary morphological effect of antheridiol on the male plant is to induce branching within the space of two hours or so. Other compounds can also induce branching, but it is only antheridiol-induced branches which can develop into antheridia. In media containing a high concentration of nitrogenous nutrients, however, the antheridiol-induced branches may become vegetative (Barksdale, 1970). The branches are attracted to polystyrene balls soaked in antheridiol, and antheridia are delimited by the hyphae in

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* +$

G. ELLIOlT

I

* yl-$

0

0

I1

111

V 0

0 IV

VI

CH,OH

(CHJ,CHCOO VI I FIG. 7 . Formulae of antheridiol and its isomers. I, ring system; 11, the 22s 2SR side chain of antheridiol; 111, the 22s 23s side chain; IV, the 22R 23s side chain; V, the 22R 23R side chain; VI, the side chain of 23-deoxyantheridiol. VII, structure of oogoniol- 1 .

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contact with the balls; thus it appears that the functions of hormone C, postulated by Raper, are fulfilled by antheridiol (Barksdale, 1963). While branching is the first visible result of antheridiol treatment of a male plant, this is of course the consequence of a number of processes at a molecular level. Kane et al. (1973) and Horowitz and Russell ( 1974) showed that the antheridiol-induced branching is inhibited by actinomycin D, and thus depends on RNA synthesis. Silver and Horgen ( 1974) reported that antheridiol treatment induced accumulation over an 8-h period of a species of RNA rich in adenylic acid, presumably messenger-RNA; addition of actinomycin D or cordycepin inhibited its accumulation. Cordycepin (3’-deoxyadenosine) inhibits the addition of polyadenylic acid to RNA and conversion of heterogenous nuclear-RNA to messenger-RNA (Darnel et al., 197 1). Thomas and Mullins (1967, 1969) found that the level of cellulase in cells rose following antheridiol treatment, reaching a maximum in two hours. Cellulase production, as well as branching, was inhibited by puromycin and p-fluorophenylalanine. Mullins and Ellis ( 1974) reported that antheridiol treatment brought about accumulation of vesicles at the points where branches were formed; such vesicles contain cellulase (Nolan and Bal, 1974). Thus antheridiol has similar properties to the more extensively studied steroid hormones of mammals and insects, in that it stimulates production of RNA and protein and induces a series of specific morphogenetic events. The principal sterols of Achlya bisexualis are fucosterol, 24methylenecholesterol, 7 -dehydrofucosterol and cholesterol ( Popplestone and Unrau, 1973). 7-Dehydrofucosterol and fucosterol can be converted to antheridiol by the fungus (Popplestone and Unrau, 1975). Antheridiol and its C-22 and C-23 isomers have been synthesized and it was shown that the configuration of the side chain profoundly affects the activity of the compound (Barksdale et al., 1974). The 22s 23R compound (antheridiol; Fig. 7, I and 11) is active at a concentration of 6 pg/ml, but the 22R 23s ( I and IV) and 22s 23s ( I and 111) compounds have less than one thousandth of the activity of antheridiol (active at 20 ng/ml) and the 22R 23R (I and V) compound has even less activity (active at 400 ng/ml). 7 -Deoxy- 7 -dihydro-antheridiol is active at 0.11 ng/mg. Replacement of the 22-hydroxyl group of 7-deoxy-7dihydro-antheridiol acetate by an 0x0 group decreases its activity to about one-fiftieth (Barksdale et al., 1974). 23-Dehydro-antheriodiol

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(Fig. 7, I and VI) (found in culture solutions) has no activity at 28 ng/ml (Green et al., 197 1). Various male and hermaphroditic strains of Achlya spp. (and one weak female strain) have been found to convert antheridiol to a more polar compound, metabolite A, and in some strains to a second metabolite B. Production of metabolite A is inhibited by actinomycin D and cycloheximide. Evidently an enzyme is produced which destroys the hormone. Neither metabolite is identical with hormone B (Musgrave and Nieuwenhuis, 1975). Hormone B is produced by male strains of A . ambisexualis when stimulated by antheridiol, and it induces formation of oogonia in the female plant. Male plants respond to antheridiol, female plants to hormone B. Hermaphroditic strains of A . heterosexualis produce hormone B without the necessity for stimulation by antheridiol. Such strains also react to both antheridiol and hormone B, which is the basis of their hermaphroditic behaviour (Barksdale and Lasure, 1973). However, it was found that treatment of strain 8-6 of A . heterosexualis with antheridiol greatly stimulates its production of hormone B. 7-Deoxy7 -dihydro-antheridiol also stimulates production of hormone B, but the 22R 23s isomer of antheridiol, and fucosterol, are inactive (Barksdale and Lasure, 1974). The structure of three compounds having hormone B activity, named oogoniol-1, -2 and -3, has recently been described by McMorris et al. (1975). They are C,, steroids, but differ from antheridiol in not having a lactone ring in the side chain, and in being esterified at C-3 (Fig. 7, VII). While hormones A and B control the formation of the male and female organs-antheridia and oogonia-respectively, their conjugation is controlled by a third hormone, the delimiting hormone. A. W. Barksdale (personal communication) believes this hormone D is produced by antheridial branches and acts on both antheridia and oogonia. VIII. Effects of Sterols on Sexual Reproduction in Homothallic Species of Pythiwn and Phytophthoru

The most striking effects of adding sterols to cultures of Pythium and Phytophthora are the ensuing morphogenetic changes. In Phytophthoru cactorum, the outer parts of the colony first become much more extensively branched. Gametangia (oogonia and antheridia) are then produced on these branches and subsequently the oospores develop inside the oogonia.

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The structure of sterols in relation to their activity in effecting reproduction has been extensively studied (Haskins et al., 1964; Elliott et al., 1966; Elliott, 1972b).The results with P. cactorum (Elliott, 1972b, and unpublished work) are as follows. The most active are the C,, plant sterols, which have an ethyl group with the a-configuration at C-24 of the side chain (sitosterol; Figs. 2 and 3, IV G, p. 124) and stigmasterol (IV HI) or a 24-ethylidene group (fucosterol (IV J) and avenasterol). The C,, sterols have much lower activity, i.e. fewer oospores are produced with the same sterol concentration. The C,, sterols (24methyl) are intermediate in their effect. The configuration of the 24methyl group is important, the a configuration conferring greater activity than the p ; thus campesterol (IV C) is more active than A’-ergosterol (IV D), and stigmasterol (IV H) than poriferasterol (IV K)at all concentrations. A double bond in the B ring is essential; cholestanol (111A) is much less active than cholesterol (IV A), and ergostanol (I11D) than A5-ergostenol (IV D). However, A’-cholestenol (V A) and 7dehydrocholesterol (VI A) gave more oospores at high concentrations and fewer at low concentrations than cholesterol (IV A), and the same holds for A’-stigmastenol (V G) and 7-dehydrositosterol (VI G) as compared with sitosterol (IV G). The double bond at C-22 does not appear to have any significant effect as indicated by equal activity of A5-ergostenol (IV D) and brassicasterol (IV E), and of sitosterol (IV G ) and stigmasterol (IV H). Defago et al. (1969) reported that polyene antibiotics could induce oospore formation in Pythium acanthium in the absence of sterols. Hendrix and Gutman (1968, 1969) found oestradiol inhibited production of oospores by cholesterol or sitosterol in six strains of Pythium; there was no significant effect with two other pythia or with Phytophthora cactorum. In most, but not all, cases, oestradiol decreased vegetative growth (colony diameter), but reproduction was inhibited at lower concentrations of oestradiol than was growth rate. Inhibition of oospore formation by oestradiol could be reversed by increasing the concentration of cholesterol or sitosterol, but to a different extent in various fungi. The 17p-hydroxyl group is important in this interference; 17a-oestradiol and 17p-oestradiol-17-acetate are not inhibitory, although 17p-oestradiol-3-acetate is (Hendrix and Guttman, 1972). The sterol side chain occupies the 17s position. Experiments on the effect of C,,, C,, and C,, steroids on the permeability of liposomes were reported by Bangham et al. ( 1965), Sessa and Weissmann (1968) and Heap et al. (1970). The synthetic oestrogen,

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diethylstilbestrol, also inhibits oospore production in P. cactorum ( G .A. Bean and C. G. Elliott, unpublished results). The most notable point about the relative activities of various sterols in promoting oospore production in P. cactorum is that they bear little relationship to their effects on structural stabilization and permeability of lipid films and cell membranes. Thus, in the latter systems, AZ2sterols generally have different effects from those with saturated side chains (e.g. Demel et a/., 1972a, b; Rottem et a/., 1971; Hossack and Rose, 19761, and cholestanol (and sometimes sitosterol also) is generally equivalent to cholesterol. Thus it would seem that the effect on reproduction cannot be simply one on permeability of membranes. It may be, of course, that the composition of higher-plant membranes is such that the rules for the effects of sterols on simple lecithin films and animal membranes do not apply, but Grunwald’s (1968, 197 1) results suggest this is not so.Perhaps the degree of leakiness obtained with the C,, sterols is required for the deposition of the cellulose walls of higher plants and of the oomycetes. Phytophthora species too, being higher-plant pathogens, have access to the predominantly C,, sterols of higher plants. But the evident importance of the C-24 ethyl or methyl group and of its a configuration suggest that this structure has in itself a particular metabolic significance. Thus, it could be that C-29 is involved in the formation of a structure similar to the lactone ring in the side chain of antheridiol. Antheridiol biosynthesis involves oxidation of C-29 of fucosterol to form a carboxyl group. I t is therefore perhaps highly relevant to sexual reproduction that species of Pythium and Phytophthora metabolize cholesterol and sitosterol to more polar compounds (Hendrix et al., 1970; Elliott and Knights, 1974; Hendrix, 1975a, b) although Hendrix (1975b) could find no relationship between sterol metabolism and reproduction; the polar metabolite was formed both by species which produced oospores under the experimental conditions and by those which did not (i.e. heterothallic species in single culture). Polar metabolites are produced from cholesterol and sitosterol supplied to species of Pythium and Phytophthora but not to Achlya bisexualis or A. ambisexualis (Hendrix, 1975a). The metabolite described by Hendrix is slightly more polar than kryptogenin (Fig. 8; Hendrix, 1975a). Heftmann (1971) goes so far as to speak of all A5-sterols being “metabolized to the same hormone”.

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The low activity of cholestanol is particularly interesting as, although few oospores are formed, the number of oogonia produced is as high as with cholesterol, but most of them abort (Elliott, 1968, 1972b). Abortive oogonia are also produced with cholestanol by Pythium periplocum and P. prolatum (Hendrix and Guttrnann, 1972)and by Phytophthora dreschleri (G. A. Bean and C. G. Elliott, unpublished observations). It was found (Elliott, 1968)that cholestanol added with cholesterol lowered the number of oospores compared with that produced with cholesterol alone, with “kinetics” resembling competitive inhibition. But this was so only at high concentrations of sterol; at low concentrations, the substances acted synergistically. I considered that these results show that sterols have effects on sexual reproduction during at least two separate stages of development (gametangialformation and conjugation), and that the steric requirements for activity at these stages are different. Such an interpretation is compatible with a hormone-type action, but not with a simple permeability model.

HO FIG. 8. Structure of kryptogenin.

It will not be easy to prove that an endoge.noushormone is involved in regulation of reproduction in these homothallic species. In heterothallic species, there is a much better chance of identifying a hormone produced by one of the interacting strains. Indeed Kouyeas (1953) observed oospore formation in the heterothallic Phytophthora paracitica in one or both members of a mated pair when they were separated by a porous filter. Also, he reported oospore formation by one strain when it was grown in the culture filtrate of the other. However, similar experiments reported by Stamps (19551, Apple (1959)and Brasier (1972) gave negative results. In the Zygomycetes, the hormonal role of trisporic acid was apparent from work with heterothallic species, in which the two strains make characteristic contributions to synthesis of the compound ( Gooday, 197 4). Werkman and Van den Ende ( 197 4) investigated the effects of homothallic species on the precursors of tri-

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sporic acid, and concluded that the control of sexual development in homothallic species was not essentially different from that in heterothallic forms. IX. Reproduction in Heterothallic Species of Pythium and Phytophtlroru

Before describing reproduction in the Pythiaceae and the little that is known of its control, it would seem desirable to give some account of the system of “relative sexuality” as it exists in Achlya spp. An understanding of the morphological events at mating is essential for an investigation of their chemical control. The various isolates of Achlya ambisexualis and A . bisexualis can be placed in a single series as regards their interactions one with another, ranging from strong male on the one hand to strong female on the other. In an intermediate position are strains which can act as male or female depending upon whether they are paired with an isolate which lies further to the female or male side (Barksdale, 1960). The strains differ in their production and response to antheridiol and hormone B (Barksdale and Lasure, 1973). In pairings between a homothallic and a female heterothallic strain, the homothallic strain produces antheridial branches which interact with oogonia produced on the heterothallic strain. When male strains were paired with homothallic strains, it was not generally possible to say whether any of the oogonia were produced as a result of interstrain interaction, or whether they were all due to self stimulation by the homothallic strain; but in one particular combination, both mature and abortive oogonial initials were induced by the male (Barksdale, 1960). Mating in the heterothallic species Pythium sylvaticum was described by Papa et al. (1967). Isolate 1063-7 was always the male (antheridial) parent, and isolate 1063-8 the female (oogonial)parent. At the boundary between the two strains, the appearance of the male side was “smooth”, and the female “rough” due to penetration of male hyphae some distance into the female culture. Pratt and Green ( 197 1) showed that there were differences in the strength of the sexual response among a large number of isolates; they could be arranged in an approximate gradient. In any one mating, one strain generally behaved as male, one as female. Thus, the situation in Pythium sylvaticum resembles that in Achlya bisexualis; but sexuality in Phytophlhora is more complicated. Heterothallic species of Phytophthora can normally be assigned to one

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or other of two mating types or compatibility groups, namely A1 and A2 (Savage et al., 1968). Mating occurs between an A1 strain and an A2 strain, and not between two A l s or two A2s. Strains reacting with neither tester were isolated by Shepherd and Pratt ( 1973). Within each

of the mating types, differences in sexual potency occur. Galindo and Gallegly ( 1960) grew pairs of strains of P. infestans into opposite sides of a plain agar block and traced the origin of the antheridial and oogonial branches. They found that any one isolate behaved chiefly or entirely as male in some matings, as female in others, and in others the same isolate produced both antheridia and oogonia. In each mating type (A1 and A2), the strains could be arranged in a series, strong male (acting as male to most other strains) to strong female (female to other strains). I t is to be noted that Galindo and Gallegly’s results indicate that all oospores are the result of crossing between the two mating types. Savage et al. (1968) reported that either mating type could produce the antheridia and oogonia in the interspecific crosses P. infestans A1 x P. capsici A2 and P . capsici A1 x P. infestans A2 ; they implied that the phenomenon was a general one. Kouyeas (1953) arranged strains of P . parasitica in a series depending on the strength of their sexual reaction. Oogonium size was characteristic of some strains. Kouyeas (1953) used the distribution of oogonial sizes in crosses to determine the frequency with which each of the interacting strains functioned as female parent. Huguenin (1973) labelled one of the interacting strains of P. palmivora with the fluorescent dye, calcofluor white. According as the oogonium, the antheridium, both or neither were fluorescent, the origin of the antheridia and oogonia could be determined. Three A1 strains (26, K and 570) and two A2 strains (L and 36) were studied. When paired with strain L, 570 formed the oogonial parent in 7 1.5%of pairings, K formed the oogonia in 36.8% of pairings and 26 in 3.1%. When paired with strain 36, K formed the oogonia in 100%of pairings, 570 in 73.0% and 26 in 62.0% of pairings. These relationships are shown diagramatically in Fig. 9. It will be noted that the relative sexual expression of strains K and 570 differs according to the strain they are paired with. In addition, a substantial proportion of self fertilization was observed, particularly of the A2 strain when it carried the fluorochrome. Stamps ( 1953) reported that, in some matings between strains of P. cryptogea and P . cinnamoni, all oospores were hybrids, but in other matings selfing of the qptogea or the cinnamoni partner could occur as well.

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Brasier (1972)studied the interaction between a number of strains of P . palmivora. He scored the numbers of oospores formed on each side of the line where the two isolates met, and found significant variation between strains in their-mean score, both among the A1 mating-type strains and among the A2s. This indicated a gradient in sexual potency in both mating types, with, one might suppose, decreasing ability of strains to act as female parent, and increasing maleness, along the gradient. Brasier, however, thought that the result of the interstrain A1

9

A2

1

L

6

FIG. 9. Relative sexuality of three A1 strains of Phytophthorapalmiuora when paired with two A2 strains. From Huguenin (1975).

interaction was to make each strain produce selfed oospores. He noted that some strains produced oospores in single culture much more readily than others I$ Stamps, 1953; Apple, 1959)and that the strains which produced the most oospores in paired matings were those which produced the most oospores in single culture. He also found that some strains of each mating type (again those which tended to produce oospores in single culture) produced oospores when grown in combination with the homothallic P. heveae; no interspecific hyphal pairing was detected. I t seems hardly possible to draw a general conclusion from these morphological observations on interstrain pairings. Is the effect of the strains on one another to stimulate one to act as male parent, the other as female in a strict interstrain relationship? Can one strain act as both male and female parent in a cross? To what extent can one strain induce the other to undergo self reproduction? It seems that different species (and different strains of one species) do different things. But genetical evidence should provide more definite evidence on the occurrence and extent of selfing. Sansome (1970)discussed the possibility of selfing to account for the peculiar segregation ratios observed for mating type and other

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characters in P. drechsleri by Galindo and Zentmyer (1967).However, one must ensure that the characters used to evaluate the extent of selfing are suitable. Mating type in P. drechsleri is unsuitable because its inheritance has unusual features, whereas drug resistance showed simple Mendelian inheritance in the same crosses (Khaki and Shaw, 1974). Khaki and Shaw (1974)crossed strains resistant to p-fluorophenylalanine or chloramphenicol with wild type; the resistant F, progeny were crossed with both parents, and F, progeny of different mating type were intercrossed. Segregation in crosses of the F, to the sensitive parent [Rr+(A2)x r+~+(Al), r+being the wild-type drug-sensitive allele and R the resistant mutant] would be sensitive to selfing, which would give rise to deviation from the expected 1 : 1 ratio or to heterogeneity TABLE 3. Segregation for drug resistance in Phytophlhora drechsleri, showing the results of backcrosses of F, (and for mutant C1 of two heterozygous F, cultures) to a sensitive parent. Data of Khaki and Shaw (1974).

Mutant FA 1

c1

C 1(F2) c 2

Total

Resistant

Semitive

68 60 25 18 43 214

51 48 19 25 32 181

between crosses. Khaki and Shaw's (1974)data (Table 3) are homogenous (& = 3.14,P 0.7-0.5)and the totals give ax;,, value of 2.75 (P 0.1-0.05)against the expected 1 : 1. However, there is a fairly consistent excess of resistant offspring. If a proportion p of the resistant parents undergo selfing, the expected proportions, 4 and 4, become 4 + @ and 4- &, and the data give a value for p of 16.7%. Such a hypothesis however is unnecessary. There is no indication of selfing of the heterozygous resistant F, (of the other mating type) in the backcrosses to the resistant parents, as these crosses gave no sensitive offspring. The conclusion then is that all oospores are of hybrid origin, but it would be much more certain if doubly marked strains were available. Khaki and Shaw (1974)collected oospores from the region where the two cultures met (D.S. Shaw, personal communication). It should be noted however that, when the wild-type strains of P. drechsleri (6500A1

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and 6503 A l l are mated by inoculating them on opposite sides of a petri dish, oospores are found not only where the two cultures meet but throughout much of the A1 culture. Oospores are not formed in either strain grown by -itself under otherwise similar conditions. The effect of sterols on reproduction in heterothallic species have received far too little attention. Child and Haskins (1971) found that addition of cholesterol and a number of other sterols to paired cultures of Pythium sylvaticum and P. catenulatum greatly stimulated oospore production; in the absence of sterols, only a weak mating reaction occurred. They noted that, in these matings, oospores were produced not only where the strains met but also within the area occupied by one of the strains. In a mating between P. sylvaticum 1063-7 and 1063-8, oospores were formed within 1063-7 which Papa et al. ( 1967 had shown functioned as male (antheridium producing) in this mating. Child and Haskins (1971)also found that, when P. sylvaticum 1063-7 was grown by itself with cholesterol in the medium, oospores were produced; this did not occur in the absence of cholesterol, nor did it occur when strain 1063-8 was grown with cholesterol. Similar results were found with single cultures of the P. catenulatum strains, the responsive strain again being that which had the morphology of the male strain. They supposed that sterol regulated promotion of oogonial structures. Thus, a tendency to femaleness was induced in male strains, but maleness was not induced in female strains. One might speculate that the increased sterol supply stimulated the female strain to produce more of a hormone (analogous to antheridiol) which induced the male strain to produce another hormone (like hormone B) to which both it and the female strain responded by producing oogonia. Cholesterol could also directly stimulate production of hormone by the male strain, leading to selfing. Pratt and Mitchell (1973)investigated the effect of cholesterol on matings of Pythium sylvaticum and Phytophthora capsici. The strains were inoculated singly into medium with or without cholesterol ( 10 mg/l), and allowed to grow into opposite sides of plain agar blocks between the two nutrient media. With the Pythium isolates, addition of cholesterol to the male culture only had little effect, but a great increase in the number of oospores maturing in the plan agar block was observed when cholesterol was added to the female cultures, and a still greater stimulation occurred when it was added to both. This result is compatible with Child and Haskins’s (1971)results if the effect of

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cholesterol is to stimulate the female strains to produce an antheridiollike hormone. With Phytophthora capsici, Pratt and Mitchell ( 1973)found that the number of oospores formed was greatly increased by adding cholesterol to the A1 strain, more still by adding it to the A2 strain, and most of all when added to both. They suggested that addition of cholesterol to only one strain led to increased oospore production because either strain might function as both male and female. Their interpretation was that the effect of cholesterol on oospore development relates to the amount present in the gametangial initials, supposing that a large amount occurred in the larger oogonium, a small amount only in the smaller antheridium. In conclusion, the results with Pythium species seem to be fairly easily interpreted in terms of a hormonal system similar to that in Achlya spp. In the heterothallic phytophthoras, there is a separation of strains into two mating types, within each of which there is a gradation from strong males to strong females. The mating-type system, which seems to be superimposed on the relative sexuality system, is in some ways similar to the mating-type system in the heterothallic ascomycetes such as Neurospora crmsa, but it is not such an effective barrier to self fertilization as obtains in N . crassa. Although much more work needs to be done, one can make the general hypothesis that the interaction of strains of different mating type results in crossing along the line where the two cultures meet, and the interstrain reaction may stimulate selfing in one or both of the partners further back from the junction. One can hardly doubt that such a system is hormonally controlled. An interesting point is that A2, but not Al, cultures of Phytophthoru spp. can be induced to form oospores by volatile metabolites of Trichoderma spp. (Brasier, 197 1, 1975; Pratt et al., 1972). More recently, it has been reported that oospores can be induced to form in A2 cultures (but not A l ) by treatment with the fungicide chloroneb (Noon and Hickman, 1974) and even simply by cutting the hyphae with a scalpel (Reeves and Jackson, 1974). It thus seems that a variety of agents, perhaps relatively non-specific, can overcome the barrier to self compatibility in A2 strains, whereas with A1 strains this is not so. Damage to A2 hyphae of a kind to induce branching is, maybe, the stimulus to oogonium formation. But stimulation of A1 strains is only possible by A2 strains. The natural interaction of strains must be specific. Further genetical evidence is needed to clarify the extent of crossing and selfing.

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X. Sterols and Sexual Reproduction in Ascomycetes and Basidiomycetes The clearest evidence for involvement of sterols in reproduction in ascomycetes is in the work of Nelson et ul. (1967) on Cochliobolus curbonum. Perithecia of this fungus are produced in culture by inoculating two compatible mating types on opposite sides of a strip of sterilized senescent maize leaf lying on the surface of Sach’s agar (Nelson, 197 1). When SKF 3301-A, a compound known to inhibit sterol synthesis in other organisms, was added four days after mating, perithecial development was inhibited by appropriate concentrations of the drug. Its addition seven days after mating impaired ascus development, and at 11 days ascospore formation was affected. At the same time, vegetative growth and conidium formation (normally sparse in this kind of experiment) were greatly increased. Chromatographic differences in the non-saponifiable extract of matings with and without SKF 3301-A were noted. The inhibition of development was prevented by adding squalene, sitosterol, ergosterol, cholesterol or cholestanol. According to Holmes and Di Tullio (19621, SKF 3301-A inhibits sterol synthesis at a stage between mevalonic acid and squalene. Mating is not successful if the two strains of Cochliobolus are paired on filter paper lying on Sach’s agar, but Fries and Nelson (1972) found that a chloroform-methanol extract of maize leaves would stimulate perithecial development if applied to the filter paper at any time prior to perithecial initiation; addition of a water extract of the leaves also further stimulated perithecial production. Sterols from the chloroform-methanol extract were effective, but not as effective as the crude extract; fatty acids from the saponified extract had no effect. When sterols were added to matings on filter paper, perithecia were produced, provided that zinc was also added (Nelson, 1971). The effects of inhibitors of sterol synthesis were investigated in Sorduriujmicolu (Elliott, 1969). In medium containing SKF 3301-A, there was an abrupt change in hyphal growth rate when the colony had covered about half the petri dish, and where this change occurred a ring of perithecia developed ; subsequently perithecia were produced in concentric rings. Except at the highest concentrations tested, perithecial production exceeded that in the drug-fiee controls. O n the other hand, a different compound, AY9944, inhibited perithecium formation at concentrations which had little effect on hyphal growth rate.

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This compound inhibits saturation of the double bond at C-7 in animal material (Chappel et al., 1964; Horlick, 1966) and in high concentration it also inhibits reduction of A'* (Lutsky et al., 1975). In the alga Chlorellu ellipsoidea it inhibits conversion of A* to A7 in the sterol molecule as well as reduction of A14 and insertion of Azz (Dickson and Patterson, 1972). However, it is not possible to say whether the effect on reproduction in Sordariufimicola is related to any effect on sterol synthesis; there was no evidence of annulment of the inhibition by cholesterol (Elliott, 19691, but it seems unlikely that the cholesterol added was sufficiently taken up by the fungus. Zearalenone (Fig. 101, a metabolite of Gibberella zeae (Fusariumroseum) which has oestrogenic properties in animals (see Mirocha and Christensen, 1974), was found to affect sexual reproduction in some 39 ascomycetes and also several species of Pythium and Phytophthoru (Nelson, 1971). Nelson (1971) studied particularly the effect on Cochliobolus curbonum, and found it to stimulate perithecial production at concentrations of 0.01 to 1.Opg per culture. It was most effective when applied just prior to the time the sexual process was initiated, and its stimulatory abilities were quantitatively affected by the time of application. Higher concentrations (10 pg or more per mating) decreased the number of perithecia formed. Concentrations of the

Go I

HO

\

HO

0& O H

0 I1

I11

FIG. 10. Structural formulae of oestrone (I) and alternative ways of presenting the structural formula of zearalenone (11 and 111).

164

CHARLES G . ELLIOlT

compound which stimulated sexual development had no effect on vegetative growth. These points suggest that zearalenone is serving as a hormone-type regulato-r. Wolf and Mirocha ( 1973) showed that zearalenone affects reproduction in Gibberella zeae, the fungus which produces it, the exogenous compound augmenting the effect of that produced endogenously. Synthesis of zearalenone by G. zeae was inhibited by “Dichlorvos”, which also inhibits perithecial production; this effect was annulled by zearalenone (Wolf et al., 1972; Wolf and Mirocha, 1973). It seems highly significant that an oestrogen should have a hormone-like effect on reproduction. The sterols which affect reproduction in Cochliobolus carbonum could be metabolized to smaller molecules with hormonal effects, just as in animals. Various ascomycetes and Fungi Imperfecti have remarkable abilities to transform steroids (Charney and Hertzog, 1967). Such faculties would be meaningless if they were not required for the fungus’s own metabolism. My colleagues and I are approaching the question of involvement of sterols in reproduction by examining sterile mutants of Neurospora crassa (Elliott et al., 1974). We suppose that, if it were possible that sterility resulted from altered sterol metabolism, qualitative or quantitative differences in sterol content as compared with the fertile wild type might be detectable. Yanagishima ( 1969) reported that Saccharomyces cerevisiae secretes hormones which cause swelling of cells of opposite mating type. It was also reported that testosterone causes swelling of cells of the a but not the a mating type and that oestradiol causes swelling of a but not a (Yanagishima et ul., 1970). The yeast hormones were considered to be steroids (Takao et al., 1970), but the a hormone was subsequently identified as n-octanoic acid (Sakurari et al., 1974). On the other hand, a factor produced by a cells which causes elongation of a cells (Duntze et al., 1970) and which inhibits DNA synthesis in them (Throm and Duntze, 1970; Bucking-Throm et al., 1973) is a peptide of molecular weight of between one and two thousand daltons (Duntze et al., 1973). The sterols of Coprinus lagopus were studied by Defago et al. (1971). They found that monokaryotic and vegetative dikaryotic mycelium, whether grown in shaken or static culture, contained mostly A59’ergostadienol (50 to 70 times as much as of ergosterol), but that the fruiting bodies contained about equal quantities of these two

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sterols. In shake cultures, sterol synthesis continued for a longer period, and the sterol content reached a higher value per culture, than in static cultures. However, in shake culture, the amount of sterol per unit weight decreased between the third and ninth days and then rose; the primordia of the fruiting bodies appeared at about 15 days. In static culture, the sterol content per unit weight increased between two and six days, and fruiting bodies were then initiated; the sterol content of the mycelium then declined. In this study, the sterol was recovered only by one method, that is by extraction of the non-saponifiable material. Holtz and Schisler (197 1, 1972) did not detect any sterol in the vegetative mycelium of Agaricus bisporus, but free sterols (ergosterol, A5n7ergostadienol and A7-ergostenol) were found in fruit bodies. Sterols were, however, found in both vegetative mycelium and fruit bodies by Byrne and Brennan (1975). XI. Conclusions

Work on model membranes, and on the homone system of Achlya spp., have together provided a framework with which we can interpret the effects of sterols on growth and development in species of Pythium and Phytophthora. We have reached the stage where we can formulate hypotheses and test them. More work needs to be done with model systems on the C,, arid C,, sterols but, as far as the information goes, it is clear that stigmasterol lowers permeability less than cholesterol in such membranes. These sterols appear to have different effects on vegetative growth of Phytophthora in25tans (Langcake, 1974, 1975).With more information of this kind, we could see how their effects are correlated with the results with the models. In particular, we need information on the permeability of fungal membranes; work with yeast protoplasts shows the way in which this might be carried out. As regards sexual reproduction, the comparison of cholestanol and cholesterol in P. cactorum indicates that sterols have separate effects on reproduction at oogonium formation and at conjugation, which is readily comprehensible on the basis of a hormonal system like that in Achlya spp., but not if reproduction depended primarily on permeability of membranes. There is obviously a great deal to be clarified about reproduction in the heterothallic Pythium and Phytophthora; what we do know enables us to formulate a hypothesis on lines similar to those for Achlya.

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The role which sterols play in controlling reproduction in ascomycetes is not yet clear. The work with zearalenone suggests that any steroid hormone which might be involved is more likely to be a small molecule (CI9or C,,),not a true sterol. The importance of sterols as essential structural components of membranes, and their consequent importance in efficient metabolism, are well established in yeast, but their precise functions are still hardly understood. In the zygomycetes, control of reproduction by trisporic acid is well understood (Gooday, 1974). The precise function of sterols here is unknown. Bu'Lock and Osagie ( 1973) observed an increased ergosterol content in mated, as compared with single, cultures of Blukesleu tn'sporu, but this could be attributed merely to the autocatalytic stimulation of isopentenoid synthesis by trisporic acid. In the basidiomycetes, consideration of the problem of sterol function has hardly begun. XII. Acknowledgements It is a pleasure to acknowledge my indebtedness to my colleague Dr. Brian A. Knights for advice on many points of chemistry mentioned in this review, and also because it is his collaboration which has made possible the work on sterols with Phytophthoru and Neurosporu done in this Department. Much of this work has been supported by grants from the Science Research Council. I am also grateful to Professor A. H. Rose for his comments on the draft of this review. REFERENCES

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Active Transport of Solutes in Bacterial Membrane Vesicles WIL N. KONINGS Department of Microbiology, Biological Centre, University of Groningen, Kerklaan 30, Haren, The Netherlands I . Introduction

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Isolation Procedures . . . . Physical Properties . . . . Purity of Membrane Preparations . . . . Functional Properties . . . . . . . Orientation of the Vesicle Membrane . . . . Localization of D-Lactate Dehydrogenase in Membrane Vesicles . . . . . . . . . from Escherichia coli . Active Transport Coupled to Electron Transfer Systems . . . A. Coupling to Respiratory Chain. . . . . . . . B. Coupling to Anaerobic Electron Transfer Systems . . . C. Coupling to Cyclic Electron Transfer Systems . . . . Energy Coupling to Active Transport . . . . . . . A. Role of Adenosine 5’-Triphosphate and the ATPase Complex . B. Mechanism of Energy Coupling . . . . . . . C. Energy-Dependent Binding of Solute to Carrier Proteins . . Conclusions . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . References . . . . , . . , . . . . . A. B. C. D. E. F.

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180 183 184 194 200 203 203 217 223 225 225 228 239 243 244 244

I. Introduction Growth and survival of bacteria can only occur if the organisms are able to transfer solutes from the external milieu into the cytoplasm. For most solutes, the cytoplasmic membrane forms the only osmotic barrier within the bacterial envelope; thus an understanding of the mechanism by which solutes can pass the cytoplasmic membrane is an essential prerequisite for a better understanding of the physiological and ecological features of bacteria. 175

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Solute translocation through the cytoplasmic membrane might occur by processes which do not require metabolic energy, like passive diffusion and facilitated diffusion, or at the expense of metabolic energy by the mechanisms of group translocation and active transport (for definitions see Kaback, 1972). The metabolic energy-dependent processes are thought to be the major bacterial mechanisms involved in the accumulation of solute in the cytoplasm. The two processes differ in an important aspect: in group translocation, the transported molecule is changed covalently during passage through the membrane, while in active transport the solute is accumulated in an unmodified form in the interior of the cell. The initial studies on energy-dependent accumulation of solutes were performed with intact bacterial cells, and the pioneering work performed in the Institut Pasteur in Paris led to a widespread interest in bacterial transport phenomena. These studies supplied essential infomation about the nature, specificity and kinetic properties of the bacterial transport systems. In order to explain the results of these studies, several models were devised. It became increasingly apparent, however, that the results of studies with whole cells were subject to many interpretations and could supply only limited information about the molecular mechanisms of transport. The main problems arose trom the diffculty of separating reactions occurring in the cytoplasm (and periplasmic space) from those occurring in the cytoplasmic membrane; consequently, it was not possible to obtain much insight into the energy requirements of transport processes. This led to a search for adequately defined experimental systems, which in essence retained only the structural and functional properties of the cytoplasmic membrane. A major step in that direction was the isolation of bacterial cytoplasmic membrane vesicles by Kaback ( 197 1). Transport studies in these membrane vesicles contributed considerably to our understanding of the molecular mechanism of transport, and in the present review I shall focus attention mainly on the developments derived from these studies. A number of aspects of bacterial transport, such as studies in whole cells, genetics of bacterial transport systems and the role of the periplasmic binding proteins, will only be mentioned where relevant to this discussion. The reader interested in a summary of bacterial transport in general, or in specific aspects of bacterial transport systems,’ is referred to several excellent reviews (Cirillo, 1961 ;

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 177

Holden, 1962; Pardee, 1968; Kaback, 1970a, b; Lin, 1971; Oxender, 1972; Halpern, 1974; Boos, 1974, 1975; Hamilton, 1975; Simoni and Postma, 1975). 11. Membrane Vesicles A.

ISOLATION PROCEDURES

The isolation of the cytoplasmic membrane from bacterial cells, in such a way that they form closed membrane vesicles which are physiologically active with regard to integrated membrane functions, has been described by Kaback ( 197 1). The procedure consists in essence of two steps: (1) conversion of the organism into an osmotically sensitive form, and (2) controlled lysis of that form in the presence of nucleases and a chelating agent (Fig. 1). The osmotically sensitive form, termed protoplasts for Gram-positive organisms and sphaeroplasts for Gram-negative organisms, can be obtained by two distinctive methods, viz. the penicillin method and the lysozyme-EDTA method. SPHAEROPLAST

n

GRAM-NEGATIVE CELL

CM

GRAM-POSITIVE CELL ~treotment ~ in ~ o hypotonic medium

~

o~noio/ ,\ ,

hypotonic medium

a00 VESICLES

FIG. 1. Scheme for the isolation of bacterial membrane vesicles. LPS indicates lipopolysaccharide layer, CW cell wall, CM cytoplasmic membrane.

The former method involves exposure to penicillin of cells growing rapidly in the presence of a suitable osmotic stabilizer, like hypertonic sucrose. This results in unbalanced growth in which the cell outgrows its peptidoglycan shell. With actively growing cells this method leads to

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the formation of sphaeroplasts, or protoplasts, which lyse rapidly in a hypotonic environment. The penicillin method is a rather tedious procedure, requiring several dilution steps, and therefore has been used only occasionally.The more generally employed procedure is the lysozyme-EDTA method. In Gram-negative bacteria, the peptidoglycan layer of the cell wall is located between an external lipopolysaccharide layer and the cytoplasmic membrane. In order to expose the peptidoglycan layer to the hydrolytic action of lysozyme (muramidasel, treatment of the cells with EDTA at alkaline pH values is required. Such a treatment of Gram-negative cells, suspended in a hypertonic medium, results in the rapid formation of sphaeroplasts (Fig. 1). It should be mentioned here that both the penicillin method and the lysozyme-EDTA method result in sphaeroplasts which still contain the lipopolysaccharide layer, and fractions of this layer will be present in the final membrane vesicle preparation. In order to obtain optimal results, each organism requires specific conditions of incubation and treatment; thus, variations have to be applied to the nature and/or molarity of the incubation buffer, temperature, and duration of incubation with lysozyme and EDTA. For some organisms, more extensive modifications of the lysozyme-EDTA method have been developed. For Pseudomonas aeruginosa, the use of chelating agents such as Tris-HC1 and EDTA has been avoided by performing the lysozyme treatment in a sucrose solution in the presence of 2.5% (w/v)lithium chloride (Stinnett et al., 1973). For Gram-positive organisms, which lack the lipopolysaccharide layer, the peptidoglycan layer is directly accessible for lysozyme, and therefore protoplasts can be obtained rapidly from Bacillus subtilis without the use of EDTA (Konings et al., 1973). For Staphylococcus aureus, rapid formation of protoplasts was obtained by degradation of the cell wall with lysostaphin in a hypertonic medium (Short et al., 197 2a, b). The second step in the formation of membrane vesicles is the controlled lysis of the protoplasts or sphaeroplasts. Transfer of these osmotically sensitive forms to a hypertonic medium results in swelling, followed by lysis and release of the intracellular contents. The cytoplasmic membrane re-anneals by an unknown mechanism, yielding closed and empty membrane vesicles which can be easily sedimented. During lysis, the intramembranal milieu equilibrates with the external medium; therefore, the greater the lysis ratio (i.e. the ratio of the

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 179

volume of protoplasts or sphaeroplasts to the volume of the lysis solution) the more dilute are the intramembranal contents. During lysis, intracellular DNA is released which adheres to the membranes and makes the preparation difficult to handle. The addition of deoxyribonuclease (DNase) to the lysate results in a rapid decrease of the viscosity of the solution. In order to remove RNA as well as DNA, the lysis fluid is usually supplemented with EDTA and ribonuclease (RNase). The EDTA facilitates release of RNA from the membrane which is subsequently degraded by the RNase. Since Mg2+isrequired for DNase activity, it is necessary to add an excess of magnesium salt to the lysates after they have been exposed to EDTA. After lysis, the membrane vesicles are extensively washed in an EDTA-containing buffer and isolated by differential centrifugation. The whole isolation procedure requires several homogenization steps. In order to obtain membrane vesicles with the highest possible transport activity, it appears to be essential to perform this homogenization in the most gentle way so as to avoid mechanical damage of the membranes (Altendorf and Staehelin, 1974; Futai, 1974a). Usually this goal is reached by performing homogenization of the membranous pellets with a hypodermic syringe fitted with an 18 gauge needle. Further, drastic changes of the incubation temperature should be avoided. For some organisms, good results have been obtained when all steps were performed at room temperature, except for the lysozyme treatment which usually was performed at 37OC (A. Bisschop and W. N. Konings, unpublished results). A considerably shorter procedure is available for the isolation of membrane vesicles from Gram-positive organisms (Konings et al., 1973). Due to the absence of the lipopolysaccharide layer, it is possible to combine the conversion of cells into an osmotically sensitive form and the lysis step. Treatment of the cells with lysozyme in a hypotonic medium leads to partial hydrolysis of the cell wall, and this results in extrusion of the cytoplasmic membrane followed by lysis. After further degradation of the remaining pieces of the cell wall, the cytoplasmic membranes are isolated by differential centrifugation, as already described. This procedure circumvents protoplast formation and has the further advantage that it is less time-consuming and gentler since it requires fewer homogenization steps. The procedure has special advantages for organisms like B. subtilis that produce exoproteases. During protoplast formation, these enzymes are excreted into the incubation medium and partially degrade membrane proteins. As a conse-

180

WIL N. KONINGS

quence the membrane vesicles obtained are labile and lose their activity within a few hours (Konings and Freese, 1972). The “one step” isolation procedure drastically diminishes this effect, and the membrane vesicles may be kept at room temperature for a prolonged period of time without significant loss of activity. A modification of the lysis procedure is also employed for the preparation of membrane vesicles from anaerobically grown Escherichia coli (Konings and Kaback, 1973). Lysis of the sphaeroplasts is performed in small volumes of hypotonic medium (usually 2 g wet-weight of sphaeroplasts in 10 ml of 10 mM potassium phosphate buffer, pH 6.6, containing 2 mM magnesium sulphate), avoiding extensive washing of the vesicles. This procedure, which has also been employed for other anaerobic organisms (Konings et al., 19751, yields membrane vesicles which retain components involved in anaerobic electrontransfer systems, in contrast to procedures which require large lysis volumes. For the isolation from the phototrophic organism Rhodopseudomonas sphaeroides of membrane vesicles which perform active transport processes, it appeared essential to perform the lysozyme-EDTAand the lysis step in media with a controlled redox potential between zero and 100 mV (Hellinperf et al., 1975). The membrane vesicles are usually suspended in 0.1 M potassium phosphate buffer (pH 6.6) to a concentration of 10 mg membrane protein per ml. Small aliquots are rapidly frozen in liquid nitrogen. When the membrane vesicles are kept at temperatures below -8OOC (usually in liquid nitrogen) the activity can be retained for several months. Prior to the experiments, the membrane vesicles are rapidly thawed by incubation at 46OC, and during the experiment they are usually kept at room temperature. B.

PHYSICAL PROPERTIES

The structures observed in electron micrographs (see Fig. 2) of ultrathin sections of membrane vesicle preparations reveal structures which are almost exclusively intact membranous sacs (Kaback, 197 1; Konings et al., 19731, surrounded by a single trilaminar layer, which is 6.5-7.0 nm thick. The diameter of the vesicles varies from 0.1 to 1.5pm for E . coli (Kaback, 1972) and from 0.1-0.5 p for B. subtilis (Konings et al., 1973). These diameters are smaller than those from the corresponding sphaeroplasts and protoplasts. The surface to volume ratio, which for a

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 181

FIG. 2. Electron micrographs of thin sections through Bacillus subtilis cells, protoplasts and membrane vesicles. A. Whole cells; B. Details of cell layers ofwhole cells; C. Details of the cytoplasmic membrane of protoplasts; D. Detail of a vesicle membrane; E. Survey of a typical membrane preparation. Taken from Konings et al. (1973).

182

WIL N. KONINGS

sphere varies as the reciprocal of its radius, is therefore much smaller than andN. protoplasts, or the corresponding 182 it is for sphaeroplastsWIL KONINGS whole cell. The inner volume of the vesicles is 2-4 pl per mg membrane sphere (Kaback, varies as the reciprocal ofand its radius, much protein 19 7 Oa; Konings Freese, is19therefore 7 2). During lysissmaller of the than it is for sphaeroplasts and protoplasts, or the corresponding sphaeroplasts, or protoplasts, the original cytoplasmic membrane obwhole cell. Theinto inner vesicles is 2-4 pl membrane viously breaks a volume numberof ofthe pieces resulting in per the mg formation of protein (Kaback, 1970a; Konings and Freese, 1972). During lysis of the several vesicles per cell. sphaeroplasts, protoplasts, the original cytoplasmic membraneand obThe vesicles or appear to be devoid of cytoplasmic constituents viously breaks into a number of pieces resulting in the formation of cell-wall fragments, as is clearly shown by electron micrographs of several vesicles per cell. serial thin sections of vesicle preparations (Fig. 2) (Konings et al., 1973). The vesicles to beoften devoid of cytoplasmic constituents and Frequently, oneappear or more concentrically arranged “internal cell-wall fragments, as is clearly shown by electron micrographs of serial thin sections of vesicle preparations (Fig. 2) (Konings et al., 1973). Frequently, one or more often concentrically arranged “internal

-

0 8 - 0

t Osmolority ( m M )

FIG.3. Effect of the osmolarity of the incubation medium on the initial rate (3 min; 0) and steady state level (0)of t-glutamate uptake by membrane vesicles from Bacillus subtilh W23. Uptake experiments were performed in K- hosphate buffer pH 6.6 with ascorbate ( 10 mMkphenazine methosulphate (10 p&f as energy source. The osmolarity was adjusted by variation of the buffer concentration. (Taken from W. N. Konings, unpublished results).

P

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 183

vesicles” are observed, which most probably are the result of enclosures of small vesicles within bigger ones. Only a small fraction of these internal vesicles (15%, see p. 196) result from invagination. An essential feature of any system that it is to be used as a model for the study of solute transport is that it must have a continuous surface (i.e. it must be able to retain transported solutes). Electron micrographs of serial thin sections of membrane vesicles indicate that the vesicles are closed structures (Konings et al., 1973). This contention is supported by studies of the surface of membrane vesicles by electron micrographs of negatively stained vesicles (Kaback, 1972; Konings et al., 1973; Altendorf and Staehelin, 1974). More convincing evidence of membrane continuity has been provided by experiments demonstrating that the vesicles are osmotically sensitive (Kaback and Deuel, 1969; Kaback, 197 1) so that they shrink and swell appropriately when the osmolarity of the medium is increased or decreased. The diffusion barrier properties of the membranes of the vesicles are also demonstrated by the observation that the initial rates, and the steadystate levels, of transport strongly depend on the outside osmolarity. The highest initial rates, and steady-state levels, are obtained at an osmolarity which is slightly higher outside than within the membrane vesicles (Fig. 3). C.

PURITY OF M E M B R A N E VESICLE P R E P A R A T I O N S

The purity and homogeneity of the membrane vesicles have been established by a number of criteria. Membrane vesicles from E. coli contain less than 5% of the cell’s DNA and RNA, but 1 6 1 5 % of the protein and at least 70% of the phospholipids initially present in the whole cells (Kaback, 197 1, 1972). Moreover, almost all of the cytoplasmic enzymes are lost, as is demonstrated by polyacrylamide disc gel electrophoresis (Kaback, 19711, and less than 1% of the activities of cytoplasmic enzymes such as glutamine synthetase, p-galactosidase, fatty acid synthetase and leucine-activating enzyme are found in the vesicle preparation. Of the “periplasmic enzymes” (Heppel, 1967), only 2% or less are found in membrane vesicle preparations from Gram-negative organisms. The membrane vesicles contain very low concentrations of endogenous energy sources such as NADH, D-lactate and succinate, as is indicated by the low endogeneous rates of oxygen consumption and active transport of solutes (Barnes and Kaback, 1971; Konings et al.,

184

WIL N. KONINGS

1973) (see p. 203). Expressed as a function of dry weight, the membrane vesicles are approximately 60-70% protein, 3040% phospholipid and 1% carbohydrate (Kaback, 1971). Vesicles prepared from the ML strains of E. coli have less than 3% (w/w) lipopolysaccharide whereas vesicles prepared from a number of other strains of E coli and Salmonella typhimurium have 7 to 17% (Kaback, 1972).

D . FUNCTIONAL PROPERTIES

In contrast to the cytoplasmic enzymes, membrane vesicles retain a number of membrane-associated enzymes and perform several integrated membrane functions. 1. Phospholipid Synthesis

Membrane vesicles from E. coli catalyse, in the presence of cytidine 5’-triphosphate, synthesis of phosphatidylethanolamine and phosphatidylglycerol from serine and a-glycerol phosphate, respectively; they also produce endogenously phosphatidic acid or diglyceride (Weissbach et al., 197 1; Thomas et al., 1972, 1973). Furthermore, these membrane vesicles can synthesize cyclopropane fatty acids by transferring the methyl group from S-adenosylmethionine to an unsaturated fatty acid moiety esterified to endogenous phosphatidylethanolamine (Cox et al., 1973). In addition, the vesicles catalyse exchange of the y phosphate of ATP, or other nucleotide triphosphates, with the phosphate group of phosphatidic acid, and phosphorylation of ADP by a process independent of oxidative phosphorylation can be observed (Thomas et al., 1972, 1973). 2. Nucleotide Metabolism

Membrane vesicles contain enzymes involved in the degradation of nucleotides, and those from B. subtilis contain both exo- and endonuclease activity. Components involved in the process of genetic transformation also appear to be present in membrane vesicles from competent cells of B. subtilis, and these vesicles demonstrate a higher binding of deoxyribonucleicacid than do vesicles from non-competent cells (Joenje t t al., 1974, 1975).

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 185

3. Electron- TranJfer Systems

An important feature of membrane vesicles from bacteria is the presence of functional electron-transfer systems such as the respiratory chain, anaerobic electron-transfer systems and cyclic electron-transfer system. A wide diversity exists in the electron carriers which participate in these systems, but they usually include dehydrogenases, quinones, non-haem iron proteins, flavines, several types of cytochromes and terminal oxidases. In membrane vesicles from most of the bacteria so far investigated, these electron carriers are at least partially retained. Special precautions, however, have to be taken in order to retain sufficient levels of all electron carriers in the membrane vesicles kom some organisms (Dutton et al., 1975; P . A. M. Michels and W. N. Konings, unpublished results). a. The Respiratory Chain. Membrane vesicles from aerobically grown aerobic or facultatively aerobic bacteria contain a respiratory chain to which several dehydrogenases can be coupled. The nature of these dehydrogenases varies in the different organisms, and in many depends strongly on the growth conditions (Konings and Freese, 1972; Dietz, 1972; Short and Kaback, 1974; kczorowski et al., 1975). Membrane vesicles from aerobically grown E. coli contain high activities of TABLE 1. Oxidation of substrates by membrane vesicles from Escherichia coli, Bacillus

subtilis and Staphylococcus aureus. Rate of oxygen uptake (ng-atoms/min/mg membrane protein)

Substrate (20 m M )

Escherichia coli ML 308-225

None

<1

l lactate

330

L-Lactate Succinate NADH a-Glycerol phosphate

91

540 270

-

Bnn'llus subtilis 60015

Staphylococcus aurew U-7 1 glucose

gluconate

-=1 -=1

<4

-=4

14

<1 34

82 20 174 14

12 70 14

630 56

234 190

Escherichia coli ML 308-225 was grown in a medium containing 1% Na-succinate (Barnes and Kaback, 197 I), Bacil1u.1subtilis 60015 in nutrient sporulation medium (Konings and Freese, 1972). and Staphylococcur auras U-7 1 in a synthetic medium containing glucose or a complex medium containinggluconate as the primary energy source (Short and Kaback, 1974).

186

WIL N. KONINGS

D-Lactate dehydrogenase

NADH +f p Dehydrogenase Succinate dehydrogenase

/ 1

__+

Fe-Q

cytb,

cy' Fe-Q

--f

a2

c y t o -02

FIG. 4. Respiratory chain of Escherichia coli. f, indicates flavoprotein, Fe-Q non-haem iron-ubiquinone, cyt cytochrome. After Cox et al. ( 1970).

D-lactate dehydrogenase, succinate dehydrogenase or NADH dehydrogenase. Membrane vesicles from B. subtifis contain high activities of NADH dehydrogenase and succinate dehydrogenase; growth of B . subfilis on glycerol results in the induction of L-a-glycerol phosphate dehydrogenase and growth on L-lactate, of L-lactate dehydrogenase (Konings and Freese, 197 1, 1972). In vesicles from other organisms yet other dehydrogenases are found, such as L-malate dehydrogenase in Azotobacter vinelandii (Barnes, 19 7 2) and D-glucose dehydrogenase in Pseudomonas aeruginosa (Stinnett et af., 1973). Most dehydrogenases are coupled very effectively to the respiratory chain, as is evident from the observations that the corresponding substrates are oxidized by the membrane vesicles at a high rate (Table 1; Barnes and Kaback, 197 1; Konings and Freese, 1972; Short et al., 1972a). In membrane vesicles from E. coli, oxidation of D-lactate, L-lactate, succinate, or a-glycerol phosphate results in a stoicheiometric conversion to pyruvate, fumarate or dihydroxyacetone phosphate, respectively (Kaback and Milner, 1970; Barnes and Kaback, 1970). Upon addition of the substrates, an extensive reduction of respiratory chain intermediates (including flavins and cytochromes) is observed (Fig. 4). In membrane vesicles from E . coli, addition of the substrates D-lactate, succinate, or NADH results in reduction of flavoprotein, and cytochromes b, a, and a2 (Barnes and Kaback, 197 1). Together with cytochrome o these cytochromes belong to all the classes of cytochromes known to be present in E . coli (Smith, 1961). Similar observations have been made with membrane vesicles from B. subtifis. With NADH as substrate, essentially complete reduction of the flavoproteins, and the cytochromes b, c,, c, and a has been observed (Konings and Freese, 1972). Further evidence for the involvement of the respiratory chain in oxidation of

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 187

the substrates is obtained from inhibition experiments with respiratory-chain inhibitors. The sites of inhibition by amytal, 2heptyl-4-hydroxyquinoline-N-oxide (H0QNO 1 and cyanide have been well established in E . coli (Cox et ul., 1970) and B . subtilis (Miki et al., 1967). The location of the amytal-sensitive site in the respiratory chain of E . coli is the flavine group between D-lactate dehydrogenase and cytochrome b , ; HOQNO acts between cytochrome b, and cytochrome u2, perhaps at a quinone-containing component, and cyanide blocks cytochrome u2. These inhibitors severely block oxidation of Dlactate, succinate and NADH (Table 4 ; p. 208). b. Anaerobic Electron-Transfer Systems. Growth of E. coli under anaerobic conditions in the presence of nitrate results in induction of the anaerobic electron-transfer system with nitrate as a terminal electron acceptor (Koningsand Boonstra, 1976). The terminal oxidase in this electron-transfer system is nitrate reductase, which catalyses reduction of nitrate to nitrite. In E . coli, formate serves as the most effective electron donor for nitrate respiration (Taniguchi and Itagaki, 1960; Wimpenny and Cole, 1967; Cole and Wimpenny, 1968; RuizHerrera and DeMoss, 1969; Lester and DeMoss, 1971). Membrane vesicles isolated from E coli, grown anaerobically in the presence of Nitrate Respiration Formate

NADH Deh ydrogenase

/

I

Fumarate Reduction L-a-Glycerolphosphate

dehydrogenase

-+

fp

-

(FebMQ

- (cyt b , )

fumarate reductase

tiumarate

FIG. 5 . Anaerobic electron-transfer systems of Escherichia coli. f indicates flavoprotein, Fe-Q non-haem iron-ubiquinone, Fe- MQ non-haem iron-menaquinone, and cyt cytochrorne. The involvement of the electron-transfer components which are placed in brackets is not unambiguously established.

188

WIL N. KONINGS

nitrate, retain the nitrate respiration system. These membrane vesicles contain a high formate dehydrogenase and nitrate reductase activity, and electrons are transferred effectively from formate dehydrogenase to nitrate reductase (Konings and Kaback, 1973; Boonstra et al., 1975a). This electron transfer occurs most likely via ubiquinone (Enoch and Lester, 1974), non-haem iron protein (Taniguchi and Itagaki, 1960) and cytochrome b, (Fig. 5 ) (see for review: Konings and Boonstra, 1976). Nitrate respiration has been demonstrated also in membrane vesicles fiom the obligately anaerobic Veillonelfaalcalescens organisms that had been grown in the presence of nitrate (Konings et al., 1975). In these membrane vesicles, several dehydrogenases are coupled to nitrate respiration, viz. L-lactate dehydrogenase, formate dehydrogenase, L-malate dehydrogenase, L-a-glycerol phosphate dehydrogenase and NAD H - dehydrogenase. In another anaerobic electron-transfer system, fumarate functions as terminal electron acceptor (see for review: Konings and Boonstra, 1976). Growth of E. coli anaerobically on glycerol as a carbon source and fumarate as electron acceptor results in the induction of anaerobic L-a-glycerol phosphate dehydrogenase and fumarate reductase. These two enzymes constitute a hnctional complex which is membranebound and which catalyses dehydrogenation of L-a-glycerol phosphate and reduction of hmarate without involving any cofactor (Fig. 5). The terminal oxidase, fumarate reductase, converts fumarate to succinate. Membrane vesicles from these cells retain high activities of L-a-glycerol phosphate dehydrogenase and fumarate reductase, and electron transfer occurs via flavins (Miki and Lin, 1973) menaquinones and, most likely, non-haem iron proteins (Singh and Bragg, 1975). It is of interest that Singh and Bragg (1975) recently demonstrated, with a cytochrome deficient (haem A-) mutant of E. coli, that the participation of cytochromes in this electron-transfer system is not essential. c. Cyclic Electron Transfer Systems. Phototrophic bacteria contain cyclic electron-transfer systems in addition to the respiratory chain. Electrons are derived from reduced bacteriochlorophyll, in a lightdependent process, and transferred to acceptors (the nature of which is a point of discussion) and thereupon via quinones and cytochromes back to oxidized bacteriochlorophyll (Fig. 6). These systems may be operative under aerobic as well as anaerobic conditions. Most of this photosynthetic apparatus is localized in invaginations of the cytoplasmic membrane, the so-called chromatophores (Tuttle and Gest,

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 189

FIG.6. Cyclic electron-transfersystem of Rhodopseudomom sphaeroides. P,,, indicates bacteriochlorophyll with absorption band at 870 nm, Pa photorhedoxin, Q ubiquinone, and cyt cytochrome.

1959; Oelze and Drews, 19721, and several procedures have been described for their isolation (Tuttle and Gest, 1959; Cohen-Bazire, 1963; Oelze and Drews, 1972; Holt and Marr, 1965). Recently a procedure was developed for the isolation of cytoplasmic membrane vesicles from the facultative phototrophic bacterium Rhodopseudomonas sphaeroides (Hellingwerf et al., 1975). These membrane vesicles are distinct from chromatophores in that they are oriented as the cytoplasmic membrane (see p. 194), in contrast to chromatophores, and perform active transport processes. Furthermore, the average diameter of the vesicles is several times the diameter of the chromatophores (Oelze and Drews, 1972; Gibson, 1965). Membrane vesicles isolated from cells grown anaerobically in the light contain a functional cyclic electrontransfer system, as is demonstrated by the observations that light can supply the energy for active accumulation of amino acids and lipophilic cations, and by the presence of bacteriochlorophyll (Hellingwerfet al., 1975).

4. Ca2+-Mg2+-Activated ATPase

Membrane vesicles from several strains of E. coli, prepared by the lysozyme-EDTA method, hydrolyse ATP at a high rate. The rate of hydrolysis is increased by destroying the permeability barrier of the vesicles with detergents like Triton X-100 or toluene, and also by sonication (VanThienen and Postma, 1973; Futai, 1974a; Hare et d.,1974; Short and Kaback, 1974).The significance of these observations will be

190

WIL N. KONINGS

discussed below in the section on Orientation of vesicle membrane

(p. 194).Only a fraction (20-60%) of the CaP+-MgP+-dependent ATPase activity present in intact cells is retained in the membrane vesicles (Short and Kaback, 1975; Futai, 1974a). Washing of the vesicles with, for instance, medium containing low concentrations of salt (van Thienen and Postma, 1973: Short and Kaback, 1975)results in further losses of the ATPase activity, indicating that a considerable fraction of ATPase is not firmly bound to the membrane vesicles. Although the components involved in oxidative phosphorylation (the electron-transfer systems and CaP+-Mg4+-ATPase) are present in the membrane vesicles, oxidative phosphorylation does not occur in the presence of oxidizable substrates (Konings and Freeze, 1972; Klein and Boyer, 1972). This lack of oxidative phosphorylation capacity is not due to an ineffective coupling of ATPase to the energy-generating site(s)of the respiratory chain, but to the inability of ADP to reach the properly located ATPase at the inner side of the membrane (Van Thienen and Postma, 1973). Membrane vesicles, prepared from sphaeroplasts of E. coli lysed in the presence of ADP and inorganic phosphate (Pi),produced ATP with D-lactate and succinate as electron donor, and also when an artificial pH-gradient was formed across the membrane (Tsuchiya and Rosen, 1976). Synthesis of ATP required Mg2+and ADP and was inhibited by dicyclohexylcarbodiimide and carbony1 cyanidep- trifluoromethoxyphenylhydrazone.Such a synthesisof ATP was not observed in membrane vesicles prepared from a mutant lacking the ATPase. In this respect, it is of interest that membrane vesicles from E. coli, prepared by breakage of the cells by passage through a French-press cell, are capable of catalysing oxidative phosphorylation with several physiological oxidizable substrates (Hertzberg and Hinkle, 1974). 5 . Group- Translocation Transport Systems

In a group- translocation transport system, the transported solute is covalently changed during transport through the cytoplasmic membrane. Two group translocation systems, the phosphoenolpyruvate phosphotransferase system (PTS) and the adenine phosphoribosyltransferase system, have been investigated in detail in membrane vesicles. In addition, evidence has been presented for a group translocation function of the enzyme system acetyl coenzyme A:

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 191

butyryl coenzyme A transferase for uptake of butyrate in membrane vesicles from E. cob (Frerman, 1973) and of a system in which I7phydroxysteroid dehydrogenase is an essential component for the translocation of testosterone in membrane vesicles from Pseudomonas testeronii (Watanabe and Po, 1974). a. The Phosphoenolpyruvate Phosphotransferase System (PTS). The PTS has been described by Kundig et al. (1964).This system was shown to be responsible for the translocation, and concomitant phosphorylation, of several carbohydrates in a number of facultative aerobes (Hengstenberg, 1973; Cirillo, 1973; Harris and Kornberg, 19721, but apparently not in obligate aerobes (Romano et al., 1970). Besides its primary role as a translocating system for sugars, the PTS has been implicated in playing an important role in a number of regulatory functions (Roseman, 1972). Translocation and phosphorylation of sugars occur by the PTS via a series of reactions, as is schematically shown in Fig. 7 for E. coli. The protein fractions that take part in this phosphoryl transfer are both cytoplasmic and membrane constituents and can be classified into two groups : the general (non-sugar specific)proteins and the sugar-specific proteins.

\

f

I

CYTOPLASMIC MEMBRANE

r

PEP

xnz;

Pyruvate

I

Sugar- phosphate

)Sugar

\

FIG. 7 . The phosphoenol pyruvate phosphotransferase system in Escherichza coli. PEP indicates phosphoenol pyruvate, H Pr histidine-containing phosphocarrier protein, IIA Enzyme 11-A, 11-B Enzyme 11-B, and 111 Factor 111. Taken from Kundig (1976).

192

WIL N. KONINGS

The general PTS proteins, Enzyme I and the phosphocarrier HPr, are both cytoplasmic proteins. The main function of these proteins is the formation of phosphorylated HPr, which serves as the central phosphoryl donor for all membrane-associated PTS reactions. Both Enzyme I and HPr have been purified to homogeneity for several organisms (Anderson et al., 1971; Kundig, 1976). Enzyme I has a molecular weight of approximately 80,000 daltons and HPr is a histidine-containing protein with a molecular weight of 9,600 daltons. Both proteins seem to be constitutively synthesized. The sugar-specific proteins comprise a family of pairs of sugar-specific proteins, each pair being necessary for the phosphorylation of one sugar. Of each pair, at least one protein is a firmly-bound membrane component. A sugarspecific protein, which is soluble, has been called Factor 111. The sugar-specific proteins are either constitutively synthesized or inducible. In any given bacterial cell many different sugar-specific pairs of PTS proteins (11-A/II-Bor III/II-B) may function simultaneously in the transfer of the phosphoryl moiety to a given sugar, all utilizing the same central phosphoryl donor P HPr. The sequence of phosphoryl transfer proceeds from P HPr to one of the sugar-specific PTS proteins and then to the sugar. The last step, the formation of sugar phosphate, requires the second sugar-specific PTS protein and this protein is always membrane-bound. The number of sugars that are transported via the PTS varies for different organisms. In Staphylococcus aureus the PTS has been reported to be involved in the phosphorylation of hexoses (glucose, N-acetylmannosamine, fructose), glycosides (a-methylglycoside, salicin, thio/?-D galactosides), alditols (glycerol, mannitol, sorbitol) and disaccharides (maltose, melibiose, lactose) (Hengstenberg, 1973; Cirillo, 1973). In E. coli, however, the PTS has been reported to be involved in the transport of relatively few sugars (glucose, fructose, mannose, mannitol and probably maltose; Tanaka et al., 1967). The information available about the PTS has been reviewed extensively (Roseman, 1972; Kaback, 1970a, b; Kundig, 1976; Kornberg, 1972).Evidence has been presented that the components of the PTS are present in the membrane vesicles from E. coli, B. subtilis and S. lyphimurium at sufficiently high concentrations to allow vectorial phosphorylation of glucose and related monosaccharides (Kaback, 1969a).This means that the sugars are phosphorylated during the transport process through the vesicle membrane, which results in the ac-

- -

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 193

cumulation at the inside of the sugar-phosphate. The most direct evidence of this mechanism is given by double isotope experiments in which the intravesicular pool is preloaded with 14C-glucoseunder conditions in which there is no phosphorylation of the sugar. After removal of the external isotope, the preloaded vesicles are exposed to 3H-glucose in the presence of phosphoenolpyruvate. The phosphorylated sugar found inside is almost exclusively 3H-glucose 1phosphate. Experiments with mutants of E. coli and S . typhimurium defective in Enzyme I or HPr demonstrated that uptake and phosphorylation of the sugar by the membrane preparations were obligatorily dependent on phosphoenolpyruvate and that the effect of phosphoenolpyruvate had to be mediated by the phosphoenolpyruvate phosphotransferase system (Kaback, 1968; 1969a). In addition, it was demonstrated that a stoicheiometric relationship exists between the disappearance of 3*Pphosphoenolpyruvate and the appearance of 32P in the sugar phosphate, suggesting that phosphoenolpyruvate provided the energy for the simultaneous uptake and phosphorylation of sugar. The uptake of sugar via the PTS is subject to rigorous control (Kaback, 1969b). Glucose or a-methylglucoside transport and phosphorylation are non-competitively inhibited by glucose 6phosphate, glucose 1-phosphate and by a variety of related hexose phosphates. The inhibitory sites for the 6-phosphate and 1-phosphate esters are accessible from either side of the membrane and are distinct and separate; moreover inhibition of glucose transport by glucose 1phosphate was antagonized by glucose 6-phosphate and vice versa. These experiments, especially when considered in conjunction with the independent experim2nts of Lowry et al. (1971)and Kornberg (19721, strongly suggest that sugar phosphates (glucose 1-phosphate in particular) may be central metabolites in the regulation of carbohydrate transport and utilization in general. b. The Adenine Phosphoribosyl Transferme System. Another group translocation system is the adenine phosphoribosyltransferase system which is involved in the uptake of purines (Hochstadt-Ozerand Stadtman, 1971a, b, c). In the transport process, adenine is converted to AMP according to the following reaction : adenine + PRPP

adeninephosphoribosyltransferase

t

AMP+PPi

(1)

194

WIL N. KONINGS

Studies in membrane vesicles demonstrated that AMP is accumulated inside the membrane vesicles during adenine transport. The enzyme adenine phosphoribosyl- transferase is located at the outside of the membranes and variations in enzyme activity are reflected by changes in adenine transport. The transport of purine nucleosides in E . coli requires an additional membrane-bound enzyme (Hochstadt-Ozer, 1972) adenosine phosphorylase, which catalyses the reaction : adenosine + Pi

-

adenine + ribose 1-phosphate

(2)

The free adenine is then transported by reaction 1. Transport of pyrimidines in Salmonella typhimurium involves a transport system coupled to the respiratory chain (Hochstadt-Ozer and Rader, 1973); this seems to be analogous to the transport systems that will be discussed later (p. 203). 6 . Active Transport Solute transport by an active transport mechanism occurs via a specific carrier protein present in the cytoplasmic membrane; it is generally believed that the solute combines with this carrier. The carrier-solute complex formed at the outside of the membrane crosses the membrane and is modified at the inner surface in such a way that the carrier has a lowered affinity for the solute. This results in release of the solute at the inside of the membrane. The carrier can move back to the outside surface of the membrane and again combine with solute. This process requires metabolic energy and results in the accumulation of solutes against an electrochemical or osmotic gradient. Membrane vesicles from many organisms perform active transport of a wide variety of solutes in the presence of a suitable energy source. The available information about active transport in membrane vesicles will be discussed on p. 203. E. ORIENTATION OF VESICLE MEMBRANE

For interpretation of the experimental data on integrated membrane functions, it is essential to have knowledge regarding the orientation of the vesicle membrane with respect to the orientation of the cytoplasmic membrane of intact cells. There is a considerable amount of evidence that membrane vesicles isolated by the lysozyme-EDTA procedure, with gentle homogenization steps, have the same orienta-

ACTIVE TRANSPORT OF SOLUTES I N BACTERIAL MEMBRANE VESICLES 195

FIG. 8. Electron microscopy of freeze-etched cells, protoplasts and membrane vesicles from Bacillus subtilis W23. A. Replica of an intact cell showing two fracture faces of the cytoplasmic membrane. B. Replica of an intact cell showing the inner fracture face (left and the outer fracture face (right) of the cytoplasmic membrane. C. Replica of a protoplast showing the inner fracture face (left) and the outer fracture face (right) of the toplasmic membrane. D. Replica of a membrane vesicle showing the inner fracture ace (left)and the outer fracture face (right) of the membrane. The arrows indicate the direction of the shadow. Taken from Konings et ul. (1973).

7

196

WIL N. KONINGS

tion as the cytoplasmic membrane in whole cells. The major lines of evidence are: (i) Transport of solutes from the external medium into the vesicles is supposed to occur only by “right-side out” membrane vesicles. The initial rates of transport and the steady-state levels of accumulation of solutes in membrane vesicles from E. cofi (Lombardi and Kaback, 1972),Staph. aureus (Short et af., 1972b)and B . subtifis (Konings et af., 1973)are similar to those observed in intact cells. (ii) Freeze-etch electron microscopy studies demonstrate that the inner and outer fracture faces of the cytoplasmic membrane from intact cells are morphologically different. The inner (convex)fracture face has a high particle density whereas the outer (concave) fracture face has a low particle density (Fig. 8). The texture observed in the fracture faces of membrane vesicles is very similar to those observed in intact cells (Kaback, 197 1 ; Konings et af., 1973; Altendorf and Staehelin, 1974); an orientation with a higher particle density in the outer fracture face than the inner fracture face was never observed in membrane vesicles. However, such an orientation has been observed in about 15% of the vesicles which are enclosed in other vesicles (Konings et al., 1973). Probably these “internal” vesicles result from invaginations of the outen membrane layer. These observations demonstrate that the particle distribution is a good indication of the orientation of the membrane. (iii) The localization in the rpembrane of the membrane-bound enzymes, succinate dehydrogenase and NADH dehydrogenase, was studied with the membrane-impermeable electron carrier 5-N-methylphenazonium-3-sulphonate (MPS) in membrane vesicles and intact cells from B. subtilis (Konings, 1975). In both preparations, succinate dehydrogenase appears to be exposed to the outside while NADH dehydrogenase is localized at the inside. Furthermore, studies with antibody against D-lactate dehydrogenase demonstrated that this membrane-bound enzyme is exclusively present on the inner surface of the vesicle membrane (see below; p. 200). (iv) Rosen and McClees ( 1974) have demonstrated that inverted membrane vesicles, prepared by passing cells through a French-press cell, do not transport proline in the presence of the electron donor Dlactate, but do catalyse calcium accumulation. In contrast, vesicles prepared by osmotic lysis do not exhibit calcium transport but accumulate proline effectively in the presence of D-lactate. (v) Recently, Short et af., (1974a) demonstrated that essentially all vesicles in a preparation catalyse active transport. Vinylglycolate (2-

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 197

hydroxy 3-butenoate) is transported by membrane vesicles from E. coli ML 308-225 by the lactate transport system. Subsequently, vinylglycolate is oxidized by membrane-bound D- and L-lactate dehydrogenases to yield a reactive electrophile (presumably 2-0x0 3butenoate) which then reacts with sulphhydryl-containing proteins on the membrane. Essentially all of the vinylglycolate taken up is covalently bound to the vesicles. The limiting step for this labelling of the membrane protein is the transport of vinylglycolate. With SHlabelled vinylglycolate, it was possible to estimate the number of vesicles which transport this compound in the presence of the artificial electron donor ascorbate-PMS (Konings and Freese, 197 1; Konings et al., 197 1) by examining the vesicles by radioautography in the electron microscope. Each vesicle that takes up vinylglycolate is overlaid with exposed silver grains. Essentially all of the large vesicles in a preparation could be labelled in this way, while the size of the smaller vesicles is such that their proximity to individual silver grains in the emulsion may be limited. The same radio-autographic results were obtained with [SH]aceticanhydride, a reagent which reacts non-specifically with the vesicles. The major reason why the evidence presented above for a right-side out orientation of the membrane vesicles is not generally accepted lies in the difficulty of accounting for the relative effects of different physiological electron donors on transport in E. coli membrane vesicles. Membrane vesicles from E. coli catalyse active transport of amino acids and several other metabolites in the presence of an appropriate electron donor (see below; p. 203). In the absence of an electron donor, hardly any uptake is observed. As will be discussed below there is no relationship between the ability of the vesicles to oxidize a particular electron donor and the ability of that electron donor to catalyse active transport. For instance D-lactate, as well as a number of other compounds, is oxidized by E. coli membrane vesicles at a high rate, yet D-lactate is by far the best physiological energy source for transport (Table 2). When the membrane vesicles are oriented as the cytoplasmic membranes of intact cells, these and other observations can be explained by a specific localization of the coupling site for transport in a segment of the respiratory chain between D-lactate dehydrogenase and cytochrome 6 (see Fig. 4; Kaback and Barnes, 1971). An alternative explanation, however, can be offered if some of the

198

WIL N. KONINGS

TABLE 2. Respiration and transport by membrane vesicles from Eschrichia coli ML 308-225. Proline transport and the rate of oxygen consumption were measured with membrane vesicles from cells grown in a glucose-medium. (Taken from Kaback and Hong, 1973). ~~~

Electron donor None D-Lactate D,L-a-Hydroxybutyrate Succinate L-Lactate NADH

Transport of proline (nmoles/min/mg protein)

Rate of oxygen uptake (ng-atoms/min/mg protein)

0.02 1.26

<1 300 60 125 50 620

0.09 0.07 0.20

0.38

vesicles in a preparation are inverted. This explanation is based on the following assumptions : (i) electron donors which are ineffective with regard to transport are oxidized only by enzymes located at the inner side of the cytoplasmic membrane; (ii) the permeability of the vesicle membrane to these electron donors is low; and (iii) the observed oxidation of these electron donors by a vesicle preparation is due primarily to inverted vesicles. Several lines of evidence argue against the first assumption. It has been demonstrated that all of the electron donors which are oxidized by vesicles reduce the same cytochromes both qualitatively and quantitatively (Barnes and Kaback, 1971; Konings and Freese, 1972). If a percentage of the vesicles was inverted, and only these inverted vesicles would oxidize an ineffective electron donor such as NADH, it is dificult to understand how NADH could reduce all of the cytochromes in the preparations. Furthermore, although NADH is generally a poor electron donor for transport in E. coli membrane vesicles, it is the best electron donor for transport in B. subtilis membrane vesicles (Konings and Freese, 1971, 1972). Moreover, it has been demonstrated that intact cells ofB. subtilis, which are depleted for endogenous energy sources, oxidize NADH at a high rate and that NADH effectively drives transport of amino acids (Konings, 1975). Hampton and Freese (1974) observed that the oxidation of NADH in membrane vesicles from B. subtilis show biphasic kinetics, while oxidation of this substrate by whole cells has only one affinity constant; thus they concluded that some of their vesicles were open or inverted. These authors also observed that treatment of the membrane vesicles with proteolytic enzymes caused a 50%

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 199

decrease in NADH oxidation rate. This effect on NADH oxidation was not observed in membrane vesicles prepared according to Konings et al. ( 1973). Furthermore these membrane vesicles have a several-fold higher transport activity than the preparation of Hampton and Freese ( 1974).These apparently contradictory results can be best explained by the differences in isolation procedure, the main one being that the procedure applied by Konings et al. ( 1973) is more rapid and requires fewer homogenization steps. Observations made with mutants of E. coli deficient in certain dehydrogenases also argue against the assumption that electron donors which have a low efficiency in the energization of transport are oxidized mainly by inverted vesicles. Vesicles prepared from a mutant deficient in D-lactate dehydrogenase exhibit normal transport in the presence of the electron donor succinate, but not with D-lactate (Hong and Kaback, 1972). Since succinate oxidation by both wild-type and mutant vesicles is similar, it seems apparent that the coupling between succinate dehydrogenase and transport is increased in the mutant vesicles. In vesicles prepared from double mutants defective in both Dlactate dehydrogenase and succinate dehydrogenase, the coupling between L-lactate dehydrogenase and transport is increased and Llactate is the best physiological electron donor for transport. In vesicles prepared from a triple mutant defective in D-lactate dehydrogenase, L-lactate dehydrogenase and succinate dehydrogenase, the coupling between NADH dehydrogenase and transport is markedly increased, and NADH drives transport as well as D-lactate in wild-type vesicles (F. Grau, J. S. Hong and H. R. Kaback, unpublished results). The following observations have been presented for a partial inversion of the membrane vesicles. Calcium and Mg2+-stimulatedATPase is localized at the inner side of the cytoplasmic membrane and this enzyme has been used as a marker for the orientation of the membrane vesicles (Van Thienen and Postma, 1973; Hare et al., 1974). Theenzyme is detectable at the outside of the membrane vesicle preparation (Van Thienen and Postma, 1973) and with antibody to the purified enzyme about 50% of the total vesicle population can be agglutinated (Hare et al., 1974; Futai, 1974a).This led to the conclusion that a significant number of the vesicles are inverted, or that ATPase becomes translocated to the outer surface of the vesicle membrane during lysis. However, it has been demonstrated that ATPase is not firmly bound to

200

WIL N. KONINGS

the membrane and becomes easily dissociated during vesicle preparation so that 6040% of the ATPase activity of the cell is lost during preparation of membrane vesicles (Short and Kaback, 1975).These findings indicate that ATPase may be translocated from the inside to the outside surface during isolation of the vesicles, and that its inhibition by antibody is not dependable as a tool for determination of the orientation of the membrane vesicles. Another line of evidence for the inversion of membrane vesicles comes from studies of the localization of membrane-bound enzymes with the membrane-impermeable electron acceptor ferricyanide (Futai, 1974a, b ; Weiner, 1974). With ferricyanide, no activity of L-aglycerol phosphate dehydrogenase or NADH dehydrogenase was found in whole cells or sphaeroplasts unless the permeability barriers were destroyed by toluene. However, in lysozyme-EDTA vesicles, approximately 50% of the total enzyme activity is accessible to ferricyanide. From these observations it was concluded that either 50% of the enzymes have moved to the outer side of the membrane during vesicle preparation, or that 50% of the vesicles were inverted. Recent experiments, however, indicate that such a conclusion is not justified. Membrane vesicles from B. subtilis (Bisschop et al., 1975b)and from anaerobically grown E coli (Boonstra et al., 1976b)perform active transport of amino acids under anaerobic conditions in the presence of NADH or formate, and ferricyanide as electron acceptor. Therefore, these membrane vesicles must be right-side out. Evidence was presented that ferricyanide accepts electrons from a terminal part of the respiratory chain, or from the nitrate respiration system, and therefore enzyme activity measurements with ferricyanide do not supply information about the localization of these dehydrogenases. The only conclusion that may be drawn from ferricyanide reduction experiments is that a component(s) of the respiratory chain becomes accessible to ferricyanide in membrane vesicles. F.

LOCALIZATION O F D-LACTATE DEHYDROGENASE IN M E M B R A N E V E S I C L E S F R O M E S C H E R I C H I A COLI

Membrane vesicles from E. coli contain a high activity of D-lactate dehydrogenase which is coupled to the respiratory chain. In one of the initial publications on active transport processes in membrane vesicles (Kaback and Milner, 19701, it was demonstrated that oxidation of Dlactate via the respiratory chain supplies the energy for active transport

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 201

of amino acids. This, and many other studies made subsequently (see p. 203), strongly indicated that D-lactate dehydrogenase has a specific function in the energization of active transport processes. It was, therefore, of interest to obtain more information about the enzyme Dlactate dehydrogenase and about the binding of this enzyme to the membrane. The membrane-bound D-lactate dehydrogenase has been solubilized and purified to homogeneity (Kohn and Kaback, 1973; Futai, 1973). The enzyme has a molecular weight of about 75,000 daltons and contains approximately one mole of flavin adenine dinucleotide per mole of enzyme. D-Lactate dehydrogenase does not require nicotinamide adenine dinucleotide (NAW) because NAD+ has no effect on the catalytic conversion of D-lactate to pyruvate. By applying the specific activity of the purified enzyme to the D-lactate dehydrogenase activity in membrane vesicles from wild-type E. coli, it was estimated that these membrane vesicles contain 0.07 nmoles D-lactate dehydrogenase per mg membrane protein. Mutants of E. coli and Salmonella tyPhimurium have been isolated which are defective in D-lactate dehydrogenase, and membrane vesicles from these mutants do not catalyse D-lactate oxidation or D-lactate-dependentactive transport (Hong and Kaback, 1972). Reeves et al. (1973) demonstrated that a guanidine-HC1 extract from wild-type membrane vesicles, containing D-lactate dehydrogenase activity, is able to reconstitute D-lactate oxidation and D-lactatedependent transport in membrane vesicles from these mutants deficient in lactate dehydrogenase activity. Similar results were obtained with purified D-lactate dehydrogenase (Futai, 1974b; Short et al., 1974a). The reconstituted D-lactate dehydrogenase membranes carry out Dlactate oxidation and catalyse transport of a number of substances when supplied with D-lactate. Binding of the enzyme to membrane vesicles of the wild type has no effect on the rate of D-lactate oxidation, nor on the ability of the membranes to catalyse active transport. Reconstitution of D-lactate dehydrogenase deficient membranes with increasing amounts of D-lactate dehydrogenase results in a corresponding increase in the rate of D-lactate oxidation, and D-lactatedriven transport approaches an upper limit which is similar to the specific transport activity of wild-type membrane vesicles. However, the quantity of enzyme required to achieve maximum initial rates of transport varies somewhat for different transport systems.

202

WIL N. KONINGS

The flavin moiety of the holoenzyme appears to be critically involved in the binding of D-lactate dehydrogenase to the membrane (Short et al., 1974b). 2-Hydroxy-3-butynoate (Walsh et al., 1972a) irreversibly inactivates D-lactate dehydrogenase and L-lactate dehydrogenase, as well as D-lactate-dependent transport in membrane vesicles from E. coli (Walsh et al., 1972b; Walsh and Kaback, 1974). The compound is a substrate for the membrane-bound, flavin-linked, D-lactate dehydrogenase which undergoes turnover some 15 to 30 times prior to inactivation. Inactivation is due to covalent attachment of a reactive intermediate to flavin adenine dinucleotide at the active site of the enzyme. Treatment of the purified enzyme with hydroxybutynoate also results in inactivation by modification of the flavin adenine dinucleotide coenzyme bound to the enzyme. Enzyme labelled in this manner does not bind to membrane vesicles from D-lactate dehydrogenase-deficient mutants, which indicates that the flavine coenzyme itself may mediate binding, or that covalent inactivation of the flavin results in a conformational change that does not favour binding. D-Lactate dehydrogenase in reconstituted D-lactate dehydrogenase membrane vesicles appears to be localized on the outer surface of the membranes, as opposed to the inner surface of wild-type membrane vesicles. I t has been discussed above that membrane vesicles from E. coli ML 308-225 transport 2-hydroxy- %butenoate(vinylglycolate)via the lactate transport system and that this compound is oxidized by Dand L-lactate dehydrogenase on the inner surface to yield a reactive electrophile which subsequently reacts with sulphhydryl-containing proteins on the membrane. In reconstituted D-lactate dehydrogenase membranes such a labelling with vinylglycolate does not occur, which suggests that D-lactate dehydrogenase is present at the outer surface of these vesicles. This suggestion is supported by studies using antibody against D-lactate dehydrogenase (Short and Kaback, 1975). Incubation of E. coli ML 308-225 membrane vesicles with anti-D-lactate dehydrogenase does not inhibit D-lactate dehydrogenase activity unless the vesicles are disrupted physically, or sphaeroplasts are lysed in the presence of antibody. Treatment of reconstituted D-lactate dehydrogenase vesicles with anti-D-lactate dehydrogenase, however, results in a drastic inhibition of D-lactate dehydrogenase activity. The titration curves obtained with reconstituted D-lactate dehydrogenasemembrane vesicles are almost identical with those obtained for the homogeneous preparation of D-lactate dehydrogenase. These results

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 203

provide further support for the conclusion reached in the section “Orientation of the vesicle membrane” (p. 194) that essentially all of the vesicles are oriented in the same way as the cytoplasmic membrane in whole cells. 111. Active Transport Coupled to Electron-Transfer Systems A.

C O U P L I N G TO R E S P I R A T O R Y CHAIN

The isolation of membrane vesicles from bacteria has opened up the possibility of examining the energetics of active transport of solutes, independent of the general metabolism of the cell (Kaback, 1971). Membrane vesicles from E. coli oxidize the electron donors D-lactate, succinate and NADH at a high rate. Kaback and Milner (1970) observed that, especially, the oxidation of D-lactate stimulated markedly the transport of amino acids by membrane vesicles from E. coli (Fig. 9). Other electron donors in E. coli, such a$ succinate, Llactate, D,L-hydroxybutyrate and NADH, also could energize transport, but these electron donors were less effective energy sources for active transport than D-lactate (Barnes and Kaback, 197 1). Furthermore, in membrane vesicles from cells that had been induced to syn-

Time(min)

FIG. 9. Effect of o-lactate (0)on proline uptake by membrane from Escherichia coli M L 308-225. The response in the absence of lactate is shown by open circles. The lithium D-lactate concentration was 20 mM. Taken from Kaback and Hong ( 1973).

TABLE 3. Active transport systems coupled

to

electron-transfer systems in vesicles from bacteria

~~

Electron transfer system

soul.cr~0"

vcsiclrs ~~

I.:.\clicric.liia coli

Bacillus 1icheniJonnis

~~

Transport systems

~

Electron donors ~~~~~

Acceptors

References

~

Respiratory chain

Nine amino-acid D-Lactate; transport systems; ASC-PMS; P-galactosides; succinate; galactose; arabinose; NADH ; L-a- glycerolglucuronate; gluconate ;hexosephosphosphate; phate; deoxycytidine; formate'; D-alanine' valinomycin induced Rb' or K+ uptake; dipeptides ;succinate; D-hCtate ; L-lactate ; pyruvate; Cat+

Nitrate respiration system Fumarate reductase system

Amino acids; P-galactosides

Respiratory chain

Respiratory chain

Oxygen

Barnes and Kaback, 1970, 197 1 ; Kaback and Hong, 1973; Konings el a/., 197 1; Dietz, 1972; Kaback and Milner, 1970; Gordon el al., 1972; Komatsu andTanaka, 1973; Matin and Konings, 1973; Murakawa el al.. 197 1, 1973; Rayman el al., 1972; Lo et al., 1974; Bhattacharyya et al., 197 1;Kaworowski el al., 1975; Rosen and McClees, 1974; Tsuchiya and Rosen, 1975a; 1975b Konings and Kaback, 1973; Boonstra et al., 1975 ; Boonstra e t a l . , 1976 Konings and Kaback, 1973; Boonstra el al., 1975a

L-a-Glycerol phosphate'; formate' L-a- Glycaolphosphate'; formate.

Nitrate, chlorate, ferricyanide Fumarate

Nine amino-acid transport systems; succinate; fumarate; L-malate;citrate ; D-hCtate; t-lactate ; manganese

NADH; ASC-PMS; NADPH ; L-a-glycaolphosphate'; L-lactate'

Oxygen, ferricyanide

Konings and Freese, 197 1, 1972; Konings et al., 1972; Bhattacharyya, 1975; Konings and Bisschop, 1973; Matin and Konings, 1973; Bisschop et al., 1975a, 1975b

Amino acids

NADH; ASC-PMS

Oxygen

MacLeod et al., 1973

Amino acids, P-galactosides

Sacillus

megaterium SlaFhvlococcu.5

aureu.\

Respiratory chain Respiratory chain

L-Proline; CaP+

ASC-PMS

Oxygen

Konings el a/., 197 1 ; Bronner el al.,

Twelve amino acid transport systems ; valinomycin-induced Rb+ uptake

L-a-

Glycerolphosphate'; L-lactate'; Asc-PMS

Oxygen

Short el al., 1972a, 1972b; Short and Kaback, 1974; Lombardi et al., 1973

1975

Psrudomonns putida

Respiratory chain

Amino acids

ASC-PMS; D-lactate

Oxygen

Konings el al., 197 1 Sprott and MacLeod, 1972

Psrudomonm

Respiratory chain

Amino acids; oxalate

ASC-PMS

Oxygen

Croen el al., 1976

Pseridomonas spp.

Respiratory chain

L-Glutamate; succinate; D-hctate; L-lactate

ASC-PMS;NADH; succinate; L-lactate

Matin and Konings 1973

Marine P s e u d m n a s B- 16

Respiratory chain

Neutral amino acids; a-aminoisobutyrate

NADH ;ASC-TMPD; ethanol

Sprott and MacLeod, 1974; Fein and McLeod, 1975

P.~rudomonas aerugino.w

Respiratory chain

Gluconate

ASC-PMS

AzolobacI~rninelandii

Respiratory chain

D-Glucose; Caz+

L-Malate (+FAD); Oxygen NADH ; NADPH; ASC-PMS; ASC-TMPD

Barnes, 1972, 1973, 1974

Respiratory chain

Amino acids

ASC-PMS; Oxygen valinomycin induced Rb+ or K+ uptake

Konings el a / . , 197 1 ; Kaback, 1974; Lombardi e t a / . , 1973

Respiratory chain

Amino acids; citrate; cytidine; uridine

D-Lactate; L-a-glycerolphosphate

Konings el a / . , 197 1 ; Hong and Kaback. 1972; Hochstadt-Ozcr and Rader; 1973; Kaback, 1974

Respiratory chain

Citrate

D-Lactate; Asc-PMS Oxygen

oxalalicus

Oxygen

Oxygen

Guymon and Eagon, 1974

Johnson el a/.. 1975

TABLE 3.4continued) Sourcr of vcsiclcs

Electron transfer system

Transport systems

Electron donors

Acceptors

References

Respiratory chain

D-Fructose ; L-Malate L-rhamnose; glucose; amino acids

Oxygen

Wolfson and Krulwich; 1974; Wolfson el a/., 1974; Levinson and Krulwich, 1974

Pro/rio mirnbilic

Respiratory chain

L-Proline

Asc-PMS

Oxygen

Konings el al., 197 1

M icrobncleriicm

Respiratory chain

L-Proline

Succinate; NADH; ASC-PMS; ASC-TMPD

Oxygen

Hirata el al., 197 1

7'hiobncillus neopv1itnnu.r

Respiratory chain

Amino acids

NADH ; ASC-TMPD

Oxygen

Matin el a/., 1974

Veillonrlln alcalescens

Nitrate respiration system

Amino acids

L-Lactate"; NADH; Nitrate a-glycerolphosphate ; formate ; L-malate

Konings el a/., 1975

Htrodo~.\rudomonn.c sphaeroides

Amino acids Respiratory chain; Amino acids Cyclic electrontransfer system

NADH ;Asc-TMPD

Hellingwerf el a/., 1975

A rltrrohncler

/yridinoli.\

ptrlri

Oxygen

Light - induced electron flow

'These substances are only effective electron donors in vesicles horn cclls induced for the corresponding deliydi-ogcnascs.

Hellingwerfel a / . , 1975

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 207

thesize L-a-glycerol phosphate dehydrogenase, formate dehydrogenase or D-alanine dehydrogenase, L-a-glycerol phosphate, formate or D-alanine, respectively, also stimulated amino-acid transport (Dietz, 1972; Kaczorowski et al., 1975). In later publications it was demonstrated that oxidation of these electron donors could energize active transport of a wide variety of solutes; but the highest initial rates of transport were always observed with D-lactate as energy source (Kaback, 1972; Barnes and Kaback, 1971; Kaback and Barnes, 1971; Lombardi and Kaback, 1972; Lombardi et al., 1973). Similar effects of electron donors on transport of solutes were observed in membrane vesicles from many other Gram-negative as well as many Gram-positive bacteria (Table 3). Vesicles from the Gram-positive B . subtilis accumulate amino acids in the presence of NADH and, to a lesser extent, with NADPH (Konings and Freese, 20 -

-

Time (min)

FIG. 10. Effect of ascorbate-phenazinemethosulphate and NADH on L-glutamate uptake by membrane vesicles from Bacillus subtilis W23. Uptake of L-glutamate was determined in the presence of potassium ascorbate (10mMbphenazine methosulphate (10 pM) (01, NADH (10 mM) ( A ) , potassium ascorbate (10 mM) (m), phenazine methosulphate (10 mM) (01, or without further additions (0).

208

WIL N. KONINGS

1971, 1972). L-Lactate and L-a-glycerol phosphate can energize transport in vesicles from cells in which the respective dehydrogenases have been induced. In vesicles from Staph. aureus, amino-acid transport is energized by L-a-glycerol phosphate or L-lactate (Short et al., 1972a, 1972b; Short and Kaback, 1974). In a number of membrane vesicles, transport of several solutes was also energized by a non-physiological electron-donor system, namely ascorbate plus phenazine methosulphate (PMS)(Konings and Freese, 197 1, 1972; Konings et al., 197 1). Ascorbate alone caused only a small stimulation of transport, while PMS had no effect at all (Fig. 10). Accumulation of solutes in membrane vesicles is only observed in the presence of electron donors. N o other intermediate metabolites or cofactors, like ATP, phosphoenolpyuvate, glucose, hexose phosphates and many others, energized transport in membrane vesicles to any extent whatsoever (Kaback and Milner, 1970; Konings and Freese, 1972).These observations strongly point to a coupling of active transport to the respiratory chain in membrane vesicles from aerobically grown bacteria. This contention is supported by the observations described in a previous section (p. 198) that all substrates that are oxidized by membrane vesicles from E. coli, B. subtilis and Staph. aureus reduce the same cytochromes as dithionite. The same observation is made for the non-physiological electron donor ascorbate-PMS. Conclusive evidence of the involvement of the respiratory chain in the transport processes in membrane vesicles fiom aerobically-grown micro-organisms has been obtained from studies with respiratory chain inhibitors. As shown in Table 4, D-lactate-dependent lactose TABLE 4. Effect of respiratory-chain inhibitors o n lactose transport and D-lactate oxidation by membrane vesicles from Escherichia coli ML 308-225 (Taken from Kaback and Hong, 1973). In ti i bi tor

Concentration

-

Percentage inhibition of Lactose uptake

D-Lactate oxidation

94 76 70

98 84 52 60

Anacrobiosis Sodiuni cyanide HOQNO Sodiuni aniytal

zx 1u5 5 ,X 10-3

-

Sodium oxaniate

10-2 10-3

87 63

10-3

70

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 209

transport by E. coli membrane vesicles is strongly inhibited by anaerobiosis, and by the respiratory chain inhibitors cyanide, 2-heptyl-4hydroxy-quinoline-N-oxide(HOQNO) and amytal, and all of these inhibitors effectively block oxidation of D-lactate. Also, the specific Dlactate dehydrogenase inhibitors, oxamate and oxalate, effectively block transport energized by D-lactate. Further evidence for the involvement of the respiratory chain has been obtained from studies with mutants deficient in components of the respiratory chain. A mutant of E. coli with a defect in 5-aminolaevulinic acid synthesis does not form functional cytochromes when grown in the absence of this haem precursor (Devor et al., 1974), and membrane vesicles prepared from these cells do not catalyse active transport of lactose in the presence of D-lactate. However, membrane vesicles prepared from cells grown in the presence of 5-aminolaevulinic acid, accumulate lactose in the presence of D-lactate to the same extent as membrane vesicles from wild type E. coli (Devor et al., 1974). Furthermore, membrane vesicles from a mutant of B. subtilis defective in menaquinone synthesis do not perform active transport of amino acids with NADH as energy source, but a large stimulation of transport is observed with this electron donor when the respiratory chain is restored by addition of the menaquinone analogue, menadione (Bisschop et al., 1975a). 1. Amino-Acid Transport Systems

In membrane vesicles from E. coli, D-lactate and ascorbate-PMS markedly stimulate both the initial rates and the steady-state levels of accumulation (at which there is a balance between influx and emux rates) of the L-isomers of proline, glutamic acid, aspartic acid, tryptophan, serine, glycine, alanine, phenylalanine, tyrosine, cysteine, leucine, isoleucine, valine, and histidine (Lombardi and Kaback, 1972). Transport of glutamine, arginine, cystine, methionine and ornithine is stimulated only marginally by these electron donors. Evidence has been presented for the essential role of periplasmic binding proteins in the transport of leucine, isoleucine, valine, glutamine, lysine and arginine (see, for review, Boos, 1975). During the isolation of the membrane vesicles, these binding proteins are removed (Kaback, 1972).The transport systems which are retained in membrane vesicles therefore appear to be unrelated to transport systems mediated by periplasmic binding-proteins. Amino acids for which binding proteins

210

WIL N. KONINGS

have been demonstrated, and which are transported by the membrane vesicles (leucine, isoleucine, valine, histidine, glutamate, lysine), are transported by more _than one transport system in whole cells (Ames and Lever, 1970; Rosen, 1971; Halpern and Even-Shoshan, 1967). Amino acids which are not accumulated by the vesicles (glutamine, asparagine and arginine) appear to be transported by only one transport system which is mediated by a binding protein in whole cells (Weiner and Heppel, 197 1; Berger and Heppel, 1972; Rosen, 1973a). Binding proteins have not been demonstrated in Gram-positive organisms, and membrane vesicles from B. subtilis (Konings and Freese, 1972)and Staph. aureu5 (Short et al., 1972b)perform active transport of the amino acids glutamine, asparagine and arginine in contrast to membrane vesicles from E. coli. Accumulation of amino acids by the membrane vesicles occurs by an TABLE 5. Michaelis constants for amino-acid transport in membrane vesicles from Escherichia coli, Bacillus subtilis, Staphylococcus aureus. (Data from Lombardi and Kaback, 1972; Konings and Freese, 1972; Short et al., 1972b) ~

~~

Amino acid

~~

~

Escherichia coli ML 308-225

Bacillus subtilis 60015

Staphylococcus aureus U-7 1

K,value (pM) Glycine Alanine Valine Leucine lsoleucine Serine Threonine Asparagine Glutamine Aspartare Glutamate Lysine Histidine Argininr Phenylalanine Tyrosine Trypt o phiin Cvstcine . Metliionine Proline

1.6 8.4 2.0-29 1.1-18 1.7-21 2.6 5.4

-

11.0 2.9 1 0.24

-

0.4 0.7 0.3 38

1 .o

9 9 80 5 9 40 1 (30) (6) 20 50 17

-

17 ( 10) 2

20 16.7 16.7 14.3 14.3 15.2 14.7 14.3 12.5 43.5 38.5 10.1

-

-

25.0 28.6 26.2

(3) (1-10) 3.8

3.5

-

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 21 1

active transport process since virtually all radio-activity inside the vesicles can be recovered as the unchanged amino acid, and the steadystate concentrations reached inside the vesicles are many times (for some amino acids up to 100-fold) the concentration in the external medium, assuming that all of the amino acids are in free solution in the intravesicular pool. Transport of amino acids by the membrane vesicles from B . subtilis, E. coli and Staph. aureus is mediated by 9-12 distinct systems, each of which is specific for a group of structurally related amino acids (Table 5 ) (Konings and Freese, 1972; Lombardi and Kaback, 1972; Short et al., 1972b). These observations indicate that structurally related amino acids are transported by the same membrane carrier protein. Each system appears to have affinity for a limited number of substrates. For instance, in membrance vesicles from B . subtilis, the dicarboxylic amino acids glutamic acid and aspartic acid inhibit each other’s transport competitively, and the Michaelis constants for transport are equal to the affinity constants found during the inhibitory action (KJ. Structurally related compounds, namely the dicarboxylic amino acids, inhibit transport of the dicarboxylic acids noncompetitively; other amino acids have hardly any effect on the transport of these dicarboxylic amino acids (Konings et al., 1972; Bisschop et al., 1975a). There is also genetic evidence which corroborates the assignment of a group of amino acids to one transport system (Halpern, 1974). Membrane vesicles prepared from some transport-deficient mutants demonstrate the same transport defect as the whole cells. For instance, membrane vesicles from D-serine-resistant mutants of E . coli show a specific defect for glycine and alanine transport (Kaback and Kostellow, 1968); membrane vesicles from E. coli W 157 d o not transport proline (Kaback and Stadtman, 1966; Kaback and Deuel, 1969) and membrane vesicles from B . subtilis 60346 are defective in the transport of glutamate and aspartate (Bisschop et al., 1975a). The maximal transport rates of the different amino acids in the membrane vesicles vary from organism to organism, and may also vary between different vesicle preparations from one organism. For some amino acids, maximal transport rates in membrane vesicles of up to 25 nmoles/mg membrane proteidmin have been observed (Short et al., 1972b). Quantitative comparisons between vesicles and whole cells are difficult to make, especially if the activity manifested by intact cells towards a particular solute is a composite of more than one uptake

212

WIL N. KONINGS

system. However, if the initial rates of uptake (expressed in nmoles transported solutehmole cytochrome b+o/min) are compared for vesicles and whole cells of E. coli that had been given the same treatment as was applied during the preparation of the vesicles (except for the lysozyme treatment) it is observed that approximately 70%or more o f the transport activity towards lysine, serine and glutamate is retained in the membrane vesicles (Lombardi and Kaback, 1972). The apparent Michaelis constants for transport of amino acids in membrane vesicles from B. subtilis (Konings and Freese, 19721, E. coli (Lombardi and Kaback, 1972)and Staph. aureus (Short et al., 1972b)are in the micromolar range. These values are in agreement with results obtained with whole cells. Transport of most amino acids appears to be mediated by only one transport system, and the initial rate of transport, as a function of the external amino-acid concentration, shows monophasic kinetics. Biphasic kinetics are only observed for the transport of leucine, isoleucine, valine and histidine in E. coli membrane vesicles (Lombardi and Kaback, 1972). 2. Sugar Transport Systems

Transport of p-galactosides, such as lactose, has been investigated in detail in E. coli (see, for review, Kepes, 1970). Since all attempts to implicate the phosphoenolpyruvate phosphotransferase system in the transport of galactosides were uniformly negative (Kaback, 1970a), the ef’f’ectof D-lactate on the uptake of/%galactosideswas investigated in E. coli membrane vesicles. In membrane vesicles from cells containing the M protein (the product of the y-gene of the lac-operon; Fox et al., 1967), D-lactate markedly stimulated the initial rate of transport of lactose and other /3-galactosides (Barnes and Kaback, 1970, 197 1 ; Kaback and Barnes, 197 1). At steady-state levels of accumulation, internal concentrations were reached which were 100-fold or more the concentration in the external medium. The galactosides are not metabolized by the membrane preparations and can be recovered from the vesicles in an unmodified form after transport (Barnes and Kaback, 1970).The effects of the different electron donors which are oxidized by E. coli membrane vesicles were analogous to the effects on amino-acid transport; the highest initial rates are obtained with D-lactate followed by D,L-hydroxybutyrate, succinate, L-lactate and NADH (Barnes and Kaback, 197 1).

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 213

Transport of arabinose, glucuronate, gluconate and glucose 6phosphate is, in E. coli membrane vesicles, coupled to oxidation of Dlactate in a similar manner to that described for amino acids and /?galactosides (Kaback, 1972; Dietz, 1972; Lagarde and Stoeber, 1974) (Table 3). Transport of these sugars is inhibited by the same conditions that affect amino acid transport and /?-galactosidetransport. Evidence has been presented that transport of these sugars does not involve the phosphoenolpyruvate phosphotransferase system, and that induction of the parent cells for these transport systems is required; the kinetic constants for transport of these sugars are given in Table 6 (p. 214). A gluconate-transport system is also present in membrane vesicles from Pseudomonus aemginosa which concentrates free gluconate with high affinity (K-: 20 pM) in the presence of ascorbate-PMS (Guymon and Eagon, 1974; Stinnett et al., 1973). D-Galactose is transported in the presence of D-lactate by lac ymembrane vesicles from galactose-induced E. coli strains ML 3 and ML 35, and by strains ML 32400 (Horecker et al., 1960) and W 3092 cy(Wu, 196 7 ) which transport galactose constitutively. The galactosetransport system in the membrane vesicles does not require the galactose-binding protein, and this protein is absent from the vesicles (Kenvar et al., 1972). Accumulation of galactose by the membrane vesicles is mediated by a low-affinity transport system (Table 6). These findings, together with the observation that the membrane vesicles fail to transport /?-methylgalactoside,indicate that the galactose-transport system retained by the vesicles is the so-called “galpermease” system (Ganesan and Rotman, 1966). Studies on whole cells and membrane vesicles from Arthrobacter pyridinoh demonstrated that this organism accumulates D-fructose and L-rhamnose via a phosphoenolpyruvate phosphotransferase system and a respiratory chain-coupled transport system. The respiratory chain-coupled system is stimulated by addition of L-malate. Information obtained with mutants deficient in the D-fructose-specific component of the respiratory chain-coupled system suggested that transport of D-fructose via this constitutive system is needed for induction of synthesis of the PTS components (Wolfson and Krulwich, 1974; Wolfson et al., 1974; Levinson and Krulwich, 1974). Membrane vesicles from Azotobacter uinelandii catalyse the active transport of D-glucose via an inducible glucose-transport system (Barnes, 1973, 1972). The best electron donors for energizing glucose transport

214

WIL N. KONINGS

TABLE 6. Michaelis constants for sugar transport in membrane vesicles lion1 E.rchuichin coli. (Taken from Kaback and Hong, 1973)

sugaI

Vesicles from strain

KaPpvalue

(W) L;ICIOSC. Ar;ibinosr Ga lactosc GI IIclll'ollil t r Gliicosc 6-ohomhate

ML 308-225 M L 30 ML 35 M L 30 GN-2

200 140 50 30 250

are L-malate and tetramethylphenylenediamine (TMPD) reduced by ascorbate. Other electron donors such as NADH, NADPH and Dlactate are oxidized at a high rate by these membrane vesicles, but are far less efficient in energizing glucose transport. Evidence based on the inhibitory effect of respiratory-chain inhibitors on the oxidation rate of the electron donors, and their effect on transport, has been presented which indicates that glucose transport is linked to two distinct sites of the respiratory chain. 3 . Carboxylic Acid- Transport System

Membrane vesicles from Bacillus subtilis catalyse, in the presence of NADH or ascorbate- PMS, active transport of the monocarboxylic acids L- and D-lactate (Matin and Konings, 1973); the dicarboxylic acids L-malate, fumarate and succinate (Konings et al., 1972; Bisschop et al., 1975a) and the tricarboxylic acid citrate (W. N . Konings, unpublished observations). Transport of L- and D-lactate occurs via a common transport system (L. de Jong and W. N . Konings, unpublished observations) as is indicated by the mutal competitive inhibition by these monocarboxylic acids of each other's transport. Furthermore, the affinity constants of the transport system for the monocarboxylic acids, as determined during inhibition (apparent inhibition constants KJ, are the same as the affinity constants determined during the transport process (apparent K,,,). In addition, the transport of D- and L-lactate are similarly affected by other compounds; only carboxylic acids, such as L-aspartate, L-glutamate, glycolate, glyoxalate, glycerate and pyruvate, inhibit transport of the monocarboxylic acids significantly. The inducible membrane-bound enzymes L- and D-lactate

ACTIVE TRANSPORT

OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 21 5

dehydrogenases are not directly involved in the transport of their substrates. Vesicles, prepared from cells which are not induced for these enzymes, transport these substrates equally well and lactate can be recovered in an unmodified form. Escherichia coli membrane vesicles also accumulate L- and D-lactate in the presence of asc-PMS (Matin and Konings, 1973). In these membrane vesicles, a high activity of Land D-lactate dehydrogenases is present and consequently L- and Dlactate are found internally as pyruvate. However, in this organism one must rule out a direct role of the dehydrogenases since 2-hydroxy-3butynoate (Walsh et al., 1972), an inhibitor of D-lactate dehydrogenase, has no effect on the transport of D-lactate (Short et al., 1974b). Furthermore, membrane vesicles from a D-lactate dehydrogenase-less mutant of E. coli (Hong and Kaback, 1972) accumulate both monocarboxylic acids at a high rate (L. de Jong and W. N. Konings, unpublished observations). Membrane vesicles from E . coli also perform active transport of pyruvate, but such a transport system has not been demonstrated in membrane vesicles from B . subtilis (Matin and Konings, 1973). The C,-dicarboxylic acids, L-malate, fumarate and succinate, are actively transported by membrane vesicles from B . subtilis (Bisschop et al., 1975a). At steady-state levels of accumulation, internal concentrations are reached of up to 45 times the external concentrations. Transport of these dicarboxylic acids occurs via a single specific transport system with Michaelis constants of the same order of magnitude as those observed for the amino-acid transport systems (Table 7). They are in sharp contrast, however, to the affinity constants determined in whole TABLE 7 . Michaelis constants for transport of carboxylic acids in membrane vesicles from Bacillus subtilis W23. (Data taken from Matin and Konings, 1973; Bisschop el al., 1975b) Transported solute

K,,,value (pM)

D-Lactate L-Lactate L-Malate Fumarat e Succinate

22 60 13.5 7.5 4.3

210

WIL N. KONINGS

cells of B . subtilis (Fournier et al., 1972; Ghei and Kay, 1972; Willecke and Lange, 1974) for which Michaelis constants of between 100 and 700 ,uM have been reported. The reason for this discrepancy between whole cells and vesicle studies is unknown. The C,-dicarboxylic acidtransport systems have also been reported for membrane vesicles from E. coli (Rayman et al., 1972; Murakawa et al., 1973; Matin and Konings, 1973) and Pseudomom spp. (Matin and Konings, 1973). A transport system for the tricarboxylic acid citrate has been described in whole cells of B. subtilis (Willecke and Pardee, 197 1) and evidence was presented that citrate is transported as a complex with Mg2+(Willeckeet al., 1973). These observations have been confirmed in studies with membrane vesicles (W. N. Konings, unpublished observations).Vesicles from non-induced cells do not transport citrate in the presence of ascorbate PMS as electron donor, while vesicles from induced cells accumulate citrate with ascorbate-PMS at a high rate in the presence of Na+and Mg? In the absence of Na', both the initial rate and the steady-state level of accumulation are decreased by a factor of two, and in the absence of MgP+hardlyany accumulation occurs. 4, Inorganic Cation- Transport Systems

Available information about transport of cations by membrane vesicles is limited. At this moment only evidence for the existence of calcium- and manganese-transport systems has been presented. Bronner et al. (1975) made the initial observation that membrane vesicles from Bacillus megaterium accumulate calcium ions in the presence of ascorbate-PMS. Similar observations were made for membrane vesicles from Azotobacter vinelandii (Barnes, 1974). Accumulation of calcium ions was investigated in detail in membrane vesicles from E. coli (Rosen and McClees, 1974; Tsuchiya and Rosen, 1975a, b). Transport of calcium ions appears to be directed from the inside to the outside, and therefore is responsible for the active extrusion of calcium from the E. coli cells. Membrane vesicles prepared by the lysozyme-EDTA method accumulate amino acids at a high rate in the presence of D-lactate or other electron donors but exhibit little energy-dependent calcium uptake. On the other hand, membrane vesicles prepared by lysis with the French pressure cell accumulate calcium at a high rate in the presence of electron donors, but not amino acids. Membrane vesicles prepared by this method appear to

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 217

be inverted with respect to the orientation of the cytoplasmic membrane of whole cells. It is of interest that these membrane vesicles also demonstrate active transport of calcium in the presence of ATP. Membrane vesicles from B. subtilis perfom active transport of manganese in the presence of NADH or ascorbate-PMS. This uptake occurs via a high affinity system (K, = 13 pM) and is not inhibited by other divalent cations like CaP+or MgP+(Bhattacharyya, 1975). In the presence of the antibiotic valinomycin, the membrane permeability greatly increases, specifically towards potassium, rubidium and caesium (Shemyakin et al., 1963, 1969; Harold, 1970). Valinomycin forms a one-to-one complex with potassium ions (Tosteson et al., 1968) which then diffuses across the membrane and thus facilitates the net movement of potassium through the membrane (Pressman et al., 1967). This ionophore might be considered as a model for natural potassium carriers. Bhattacharyya et ul. (1971) have reported that addition of valinomycin to E. coli membrane vesicles results in the accumulation of K'or Rb' by a temperature- and energy-dependent process. These studies have been extended with Rb'as a potassium analogue (Lombardi et al., 1973). In nearly all respects, the valinomycin-induced Rb+ uptake is analogous to the respiration-linked transport of sugars and amino acids. D-Lactate and ascorbate-PMS are the most effective electron donors in E. coli and M. denitntcans vesicles, while a-glycerolphosphate and ascorbate-PMS are most effective in membranes from Staph. aureus. In E. coli vesicles, two moles of Rb' are transported per mole of D-lactate oxidized, and both lactate-dependent Rb'uptake and D-lactate oxidation are blocked by anoxia, oxamate, amytal, HOQNO and cyanide. B. C O U P L I N G T O A N A E R O B I C E L E C T R O N - T R A N S F E R S Y S T E M S

The demonstration that transport of a wide variety of compounds in membrane vesicles from aerobically grown cells can be energized by electron transfer in the respiratory chain, with oxygen as a terminal electron acceptor, raises the question of how the energy for transport is supplied under anaerobic conditions. Several lines of evidence obtained in whole cells indicated that at least ATP is able to drive active transport under anaerobic conditions (Or et al., 1973; Klein and Boyer, 1972; Schairer and Haddock, 1972; Berger, 1973; Parnes and Boos,

218

WIL N. KONINGS

I

I

I

I

2 3 Time( min 1

I

4

I 5

FIG. 11. Effect of formate and nitrate on the anaerobic uptake of L-proline by membrane vesicles from Escherichia coli M L 308-225, grown anaerobically in a glucosenitrate medium. Uptake of L-proline was determined in the presence of potassium formate (10 mM) and potassium nitrate (10 m M ) (01, potassium formite (10 mM) (V) potassium nitrate (10 mM) (A),or without electron donor or acceptor added (0). Taken from Boonstra et al. (1976a).

1973; Van Thienen and Postma, 1973; Berger and Heppel, 1974). A possible role for anaerobic electron- transfer systems in active transport was not considered, mainly because these systems have not been studied extensively in many organisms (Konings and Boonstra, 1976). Anaerobic active transport of lactose has been studied in whole cells of E. coli ML 308-225, a strain which is constitutive for the M-protein of the lactose-permease. Cells grown on glucose in the presence of nitrate (i.e. under conditions which induce the anaerobic nitrate respiration system) exhibit a marked increase in lactose transport in the presence of formate and nitrate (Fig. 11) (Konings and Kaback, 1973). In contrast, cells grown anaerobically on glucose in the absence

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 219

of an electron acceptor fail to show an increase in lactose transport upon the addition of formate and nitrate. These data indicate a coupling of lactose transport to the electron-transfer system, formate dehydrogenase-nitrate reductase. More evidence for such a coupling has been obtained from studies with membrane vesicles from E. coli grown anaerobically on glucose in the presence of nitrate. In order to demonstrate anaerobic transport coupled to electron transfer in membrane vesicles, a modified isolation procedure was required (see isolation procedures; p. 177 ). Components of anaerobic electron-transfer systems are apparently loosely bound to membranes and are removed during more drastic isolation procedures. Membrane vesicles from E. coli grown anaerobically on glucose and nitrate retain the nitrate respiration system. In this electron-transfer system the electron donor, formate, is oxidized by membrane-bound formate dehydrogenase and electrons are transferred via cytochromes of the btype (see, for review, Konings and Boonstra, 1976) to the terminal oxidase, nitrate reductase. This results in the reduction of nitrate to nitrite. In the absence of electron donors and acceptors, these membrane vesicles accumulate lactose and amino acids at a relatively high rate, indicating that these vesicles are not as depleted of endogenous energy sources as those prepared by the original procedure (Konings and Kaback, 1973).The formate dehydrogenase-nitrate reductase electrontransfer system is coupled to anaerobic transport of lactose and amino acids as was demonstrated by the marked stimulation of uptake in the presence of both the electron donor, formate, and the electron acceptor, nitrate (Fig. 11).Moreover, a strong stimulation of amino acid uptake is observed with chlorate, an analogue of nitrate, as electron acceptor. Ferricyanide which most likely accepts electrons from the electron-transfer system at a level after cytochrome b, can also replace nitrate (Boonstra et al., 1976b). Further evidence for the involvement of electron transfer in anaerobic transport has been obtained from studies with electrontransfer inhibitors. The formate-plus-nitrate-dependenttransport of amino acids and lactose is inhibited almost completely by 2-n-heptyl4-hydroxyquinoline-N-oxide(HOQNO), an inhibitor at the level of cytochrome 6 , and by cyanide, an inhibitor of nitrate reductase itself (Konings and Kaback, 1973; J. Boonstra, H. J. Sips and W. N. Konings, unpublished results).

220

WIL N. KONINGS

The cytochrome of the b-type in the nitrate respiration system is auto-oxidizable (Ruiz-Herrera and DeMoss, 1969) and transport of amino acids and lactose can also be energized by ascorbate-PMS with oxygen as terminal electron acceptor. Formate also, can effectively energize transport under aerobic conditions ; under these conditions the addition of nitrate has no significant effect on the rate of uptake. The electron donors NADH and ascorbate-PMS, however, fail to stimulate transport under anaerobic conditions in the presence of nitrate, indicating that in these membrane vesicles only formate dehydrogenase is coupled effectively to nitrate reductase (Koningsand Kaback, 1973; Boonstra et ul., 1975a). Under other growth conditions, however, different electron donors also donate electrons to this electron-transfer system. In membrane vesicles from E coli, grown anaerobically on glycerol in the presence of nitrate, L-a-glycerol phosphate plus nitrate stimulate amino acid trans-

Time (rnin)

FIG. 12. Effect of L-lactate and nitrate on the anaerobic uptake of L-glutamate by membrane vesicles from the obligately anaerobic Veillonella alcalescenr, grown in a medium containing L-lactate and nitrate. L-glutamate uptake was determined in the presence of lithium L-lactate (10 mM) and potassium nitrate (10 mM) (01, lithium Llactate (10 mMf (01,potassium nitrate (10 mM) ( A ) or without electron donor or acceptor added (W.

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 221

port, but the extent of stimulation is lower than with formate plus nitrate (Boonstra et al., 1975a). A similar coupling between anaerobic transport and the electrontransfer system with nitrate as terminal acceptor has been demonstrated in strictly anaerobic organisms. Membrane vesicles from the strict anaerobe Veillonella alculescens, grown on lactate in the presence of nitrate, catalyse active transport of L-glutamate and other amino acids under anaerobic conditions in the presence of the electron donor Llactate and the electron acceptor nitrate (Fig. 12). L-Lactate alone, or nitrate alone, have hardly any effect on L-glutamate uptake. L-Lactate could be replaced by NADH, L-a-glycerol-phosphate, formate or Lmalate, indicating that in these membrane vesicles several dehydrogenases are coupled effectively to nitrate respiration. None of these electron donors could energize transport under aerobic conditions, as was to be expected since Veillonella alcalescem does not contain a functional respiratory chain (Konings et al., 1975). Another anaerobic electron-transfer system in E. coli utilizes fumarate as electron acceptor. In this electron transfer system, fumarate is reduced by the terminal oxidase fumarate reductase at the expense of an electron donor. A coupling of anaerobic transport to this electron-transfer system has been suggested by uptake experiments in whole cells. Butlin ( 1973) and Rosenberg et ul. ( 1975) demonstrated that mutants of E. coli which are deficient in Ca2+-and Mg2+-stimulatedATPase (unc A) are able to catalyse active transport of serine and phosphate under anaerobic conditions in the presence of fumarate as electron acceptor. In whole cells of E. coli ML 508-225, grown anaerobically on glycerol in the presence of fumarate, a marked stimulation of lactose uptake is observed upon the addition of L-a-glycerol phosphate plus fumarate. Under these conditions L-a-glycerol phosphate dehydrogenase and fumarate reductase are induced. Such a stimulatory effect of L-a-glycerol phosphate plus fumarate is not observed in cells grown anaerobically on glucose alone, on glucose in the presence of nitrate, or in cells grown aerobically on glycerol. More evidence for a coupling between active transport and anaerobic electron transfer to fumarate has been obtained with membrane vesicles from cells grown on glycerol in the presence of fumarate. These membrane vesicles, isolated with the same procedure as used for vesicles from glucose-nitrate grown cells, have a high en-

222

WIL N. KONINGS

FIG. 13. Effect of L-a-glycerol phosphate and fumarate on the anaerobic uptake of lactose by membrane vesicles from Escherichia coli ML 308-225, grown anaerobically in a glycerol-fumarate medium. Lactose uptake was determined in the presence ofsodium sodium L - a L-a-glycerol-phosphate (10 mM) and potassium fumarate (10 mM) (O), glycerol-phosphate (10 mM) (v),potassium fumarate (10 mM) (01,or without electron donor or electron acceptor added @I. Taken from Boonstra et al. (1976a).

dogenous rate of lactose uptake and addition of the electron donor La-glycerol phosphate alone, or of fumarate alone, stimulates lactose uptake to some extent (Fig. 13). In the presence of both L-aglycerol phosphate and fumarate, however, a stimulation of amino acid and lactose uptake is observed which is significantly higher than the sum of the stimulations exerted by the electron donor or acceptor alone (Konings and Kaback, 1973; Boonstra et al., 1975a). In agreement with these observations, the membrane vesicles contain high activities of anaerobic L-a-glycerol phosphate dehydrogenase and fumarate reductase, and fumarate reduction occurs at a high rate in the presence of L-a-glycerol phosphate (Boonstra et al.,

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 223

1975a). Further evidence for the involvement of electron transfer to fumarate is presented by the observation that HOQNO inhibits by more than 70% transport energized by L-a-glycerol phosphate plus fumarate (W. N. Konings and H. R. Kaback, unpublished results). The electron donor L-a-glycerol phosphate is, in these vesicles, also coupled to nitrate reductase, and the stimulation observed with this electron donor in the presence of nitrate is higher than with fumarate (Boonstra et al., 1975a). I t is of interest that these membrane vesicles also reduce nitrate at a high rate in the presence of formate, and that formate plus nitrate catalyse transport of lactose even better than L-a-glycerol phosphate plus fumarate; formate plus fumarate, however, did not stimulate transport to a significant extent (Boonstra et al., 1975a). These observations indicate that, in membrane vesicles from cells grown anaerobically on glycerol in the presence of fumarate, two anaerobic electron- transfer systems are present both of which are coupled to anaerobic active transport. The data obtained from the uptake experiments suggest that these electron-transfer systems have some common electron-transfer intermediates. Moreover, these membrane vesicles contain a functional respiratory chain, and active transport can be obtained with the electron donors ascorbate-PMS, succinate, NADH and D-lactate, with oxygen as terminal electron acceptor (Konings and Kaback, 1973; Boonstra et al., 1975a). C . C O U P L I N G TO CYCLIC E L E C T R O N - T R A N S F E R

SYSTEMS

Cytoplasmic membrane vesicles from the phototrophic bacterium Rhodopseudomonas sphaeroides, grown anaerobically in the light, retain a functional cyclic electron-transfer system (see Section IIA; p. 177). In these membrane vesicles, illumination results in the oxidation of bacteriochlorophyll (P-870) and the excitated electrons are transferred via an electron acceptor, ubiquinone, cytochromes 6 and t back to bacteriochlorophyll. This electron flow in the cyclic electrontransfer system generates the energy for active transport of amino acids, as is demonstrated by the high rate of amino-acid accumulation upon illumination of the membrane vesicles (Hellingwerf et a/., 1975) (Fig. 14). In the dark, these accumulated amino acids are rapidly lost from the vesicles. Inhibitors of the cyclic electron-transfer system such as HOQNO and antimycin A strongly inhibit amino-acid transport,

224

WIL N. KONINGS

Light

oc1

ob

F

Ilo

:1

2b

Light

i5

40

5:

do

Time (min 1

FIG. 14. Effect of light on the anaerobic uptake of L-alanine by membrane vesicles from Rhodopseudomonas sphaeroides grown anaerobically in the light. The reaction mixture was incubated for five minutes in the light before alanine was added. Light was turned on and off as indicated. Taken from Hellingwerf et al. (1975).

while inhibitors which affect the respiratory chain, such as amytal or anaerobiosis, are essentially without effect on light-stimulated aminoacid transport. The initial rates of transport are strongly dependent on the light intensity and increase seven- to eight-fold from 0.002 Js-' cm2 up to saturation levels at 0.2 Js-I cm+. It is of interest in this respect that light can also supply energy for active transport via a completely different system in Halobacterium halobium. Membrane vesicles from this halophilic bacterium have been isolated by sonication of whole cells (MacDonald and Lanyi, 1975). The membrane vesicles contain a single protein (bacteriorhodopsin) and, upon illumination, the chromophoreretinal of this protein undergoes reversible bleaching (Oesterhelt and Stoeckenius, 1973 ; Oesterhelt and Hess, 1973; Stoeckenius and Lozier, 1974). These photochemical events have been correlated with the vectorial release and uptake of protons (see below; p. 228). The membrane vesicles accumulate leucine during illumination against a large concentration gradient. This leucine transport requires sodium ions in the external medium and is stimulated by the presence of potassium ions in the internal medium.

ACTIVE TRANSPORT

OF SOLUTES

I N BACTERIAL MEMBRANE VESICLES 225

IV. Mechanism of Energy Coupling to Active Transport A.

ROLE O F ADENOSINE S’-TRIPHOSPHATE

A N D THE

ATPaSe

COMPLEX

The demonstration that electron flow in the bacterial electrontransfer systems generates energy for active transport of a wide variety of solutes poses the question of whether ATP plays an obligatory role as an energy intermediate between electron flow and active transport. For a long time, energization of active transport has been considered to occur via either ATP or a high-energy intermediate A-B, especially by proponents of the permease model for active transport (Cohen and Monod, 1957). It has been demonstrated extensively that electron flow in the respiratory chain, the anaerobic electron-transfer systems and the cyclic electron-transfer system, results in synthesis of ATP by a Ca2+and Mg2+-activated ATPase. All attempts to demonstrate synthesis of ATP, or of other nucleoside triphosphates, in membrane vesicles under the conditions employed for the active transport experiments were negative (Hirata et al., 1971; Short et al., 1972a; Konings and Freese, 1972). Synthesis of ATP could be observed only in membrane vesicles which were prepared in the presence of ADP and Mg2+ (Tsuchiya and Rosen, 19761, indicating that the oxidative phosphorylation system is retained in membrane vesicles. Furthermore, inhibition of ATP synthesis by arsenate, in the absence of inorganic phosphate, or by dicyclohexylcarbodiimide (DCCD) or oligomycin, has no significant effect on transport (Kaback and Milner, 1970; Konings and Freese, 1972). Moreover, mutants of E. coli with uncoupled oxidative phosphorylation exhibit normal transport activities under aerobic conditions in both whole cells and membrane vesicles (Prezioso et al., 1973). Although these observations demonstrate that active transport in bacterial membrane vesicles does not depend on the synthesis of either ATP o r an energy-rich phosphate intermediate, the possibility still exists that ATP might serve as a source of energy for active transport under certain conditions. Several attempts to energize active transport by exogenous ATP gave negative results (Kaback and Milner, 1970; Konings and Freese, 1972). These failures have been attributed to the low permeability of vesicle membrane for ATP and/or to rapid hydrolysis of ATP by ATPase (Simoni and Postma, 1975). However, ac-

226

WIL N. KONINGS

tive transport activity was also not observed in membrane vesicles which were prepared by lysis of sphaeroplasts in a medium containing high concentrations of ATP, or an ATP-generating system consisting of ADP, creatine kinase and creatine phosphate (Koningsand Kaback, 1973). At this moment, only one report (Van Thienen and Postma, 1973) claims a stimulation by ATP of serine transport in membrane vesicles from E. coli under conditions in which ATP has been shocked into the vesicles at high concentrations. However, the transport rates obtained under these conditions were small as compared with the rates obtained with the electron donors ascorbate-phenazine methosulphate or D-lactate. Experiments performed with inverted membrane vesicles from E. coli, prepared by lysis with a French pressure cell, demonstrated that ATP could supply energy for active transport of Ca2+ at a rate which is half that obtained with NADH (Rosen and McClees, 1974; Tsuchiya and Rosen, 1975a, b). It should be of interest to investigate whether ATP, generated in membrane vesicles prepared in the presence of ADP and MgZ+cansupply the energy for active transport. Evidence for a role for ATP in the energization of active transport has also been obtained from transport studies with whole cells incubated under anaerobic conditions (Pavlasova and Harold, 1969; Klein and Boyer, 1972),and under aerobic conditions (Berger and Heppel, 1974; Berger, 19731, for transport systems in which a periplasmic binding protein is involved (Boos, 1975). The role of the ATPase complex in active transport has been studied extensively in mutants of E. coli defective in the membrane bound Ca2+, Mg2+-activatedATPase (Schairer and Haddock, 1972; Prezioso et al., 1973; Rosen, 1973b; Or et a/., 1973; van Thienen and Postma, 1973; Yamamoto et al., 1973; Berger, 1973; Altendorf et al., 1974; Berger and Heppel, 1974; Boonstra et al., 1975b).These mutants are defective in both oxidative phosphorylation and ATP-driven transhydrogenase activity. The precise role of the ATPase complex in active transport is not completely clear since hydrolysis of ATP is not an absolute requirement for active transport; moreover, mutants defective in the ATPase complex reveal different active transport activities. One class of these mutants has normal transport activities under aerobic conditions, but diminished activities under anaerobic conditions (Schairer and Haddock, 1972; Or et al., 1973; Parnes and Boos, 1973; Rosenberg et al., 1975) and membrane vesicles isolated from these

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 227

mutants exhibit normal respiratory chain-linked transport activities (Prezioso et al., 1973). A second class of mutants, when grown aerobically, do not perform active transport under aerobic conditions in both whole cells and membrane vesicles (Rosen, 1973b). Finally, aerobically grown cells of a third class of mutants (Simoni and Shallenberger, 1972) have normal aerobic transport activity (Berger and Heppel, 1974) but these activities are defective in membrane vesicles (Altendorf et al., 1974). However, normal transport activities under aerobic and anaerobic conditions are observed in membrane vesicles isolated from mutants of the latter two classes grown anaerobically in the presence of nitrate (Boonstra et al., 1975b). Evidence has been presented that the lesion in the ATPase complex in these mutants is associated with a marked increase in the proton permeability of the membrane, and that this defect, as well as the defect in active transport, can be cured by treatment with dicyclohexylcarbodiimide (DCCD) (Rosen, 1973a, 1973b; Rosen and Adler, 1975; Van Thienen and Postma, 1973). Addition of DCCD has been shown to inhibit the E. coli ATPase secondarily by binding to a membrane site and thereby decreasing the proton permeability of the membrane. These observations have led to the suggestion that, in addition to its catalytic activity, ATPase plays a structural role in the membrane, and that the complex masks a proton channel through the membrane. In the mutants, this complex is either missing or readily solubilized, which leads to an enhanced proton permeability (Altendorf et al., 1974; Rosen and Adler, 1975). This hypothesis, however, does not explain the normal aerobic and anaerobic transport activities in membrane vesicles from mutant cells grown anaerobically in the presence of nitrate, because these vesicles also lack ATP activity (Boonstra et al., 197513). Growth of these mutants under conditions that suppress the defect in active transport also affects the sensitivity of their vesicles to extraction with chaotropic agents. Pate1 et al. (1975) demonstrated that strong chaotropic agents cause the vesicles to become specifically permeable to protons in a manner that is completely reversed by treatment with a variety of carbodiimides. Vesicles from aerobically grown mutant cells are affected by the chaotropic agents in a similar way as vesicles from the wild-type E. coli, but vesicles from mutant cells, grown anaerobically in the presence of nitrate, are resistant to the effects of these agents.

228

WIL N. KONINGS 8 . MECHANISM OF ENERGY C O U P L I N G

Several theories have been presented which attempt to answer the question how energy released by electron flow in the electron-transfer systems is coupled to active transport. Most of the available evidence to date is in line with the prediction made according to a chemi-osmotic coupling model proposed by Mitchell (1966) or a direct coupling model as presented by Kaback and Hong (1973). The chemi-osmotic coupling hypothesis rests upon the following postulates : (i) the cytoplasmic membrane is essentially impermeable to most ions and in particular to OH-and H+; (ii)the respiratory chain is an alternating sequence of hydrogen and electron carriers, arranged across the membrane in loops. Oxidation of a substrate results in the translocation of protons from one side of the membrane to the other; in any loop, two protons pass across. Translocation of protons is equivalent to the movement of OH-in the opposite direction, so that oxidation of a substrate results in the distribution of H+and OH-at opposite sides of the membrane. Both a pH gradient and an electrical potential are therefore established across the membrane, and the sum of these forces constitutes the proton motive force:

Aq-ZA PH ApH+is the proton motive force, Aq the electrical potential, and ApH the pH value difference between interior and exterior; Z = 2.3 RRT/F, in which R is the gas constant, T the absolute temperature and F the Faraday constant. Z has a numerical value of about 60 mV at 25OC; (iii)the proton-motive force generated by the respiratory chain reverses the direction of ATPase so as to bring about net synthesis of ATP. O n the other hand, ATPase itself can function as a proton translocator, and hydrolysis of intracellular ATP leads to the ef€lux of protons into the medium and consequently establishes a proton-motive force. According to the chemi-osmotic coupling model, the proton motive force is the driving force for active transport of solutes (Fig. 15) (Mitchell 1966,1970,1973). Neutral substrates,such as lactose, will betransported via a coupled movement with protons. It is postulated that the transport proteins (the carriers) have affinity for both the substrate and the protons; the pH value gradient and the electrical potential will drive the movement of protons and charge, and consequently the active transport of solutes (i.e. symport). Anions, such as phosphate, will also &+=

4

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 229

+NADH

2Ht-

2Ht-

MEMBRANE

tH

'

fc

2H +Cytc

FIG.15. Chemi-osmoticmodel of active transport according to Mitchell (1970).

be transported by a proton symport system. However, this transport will be electro-neutral and influenced only by the pH value gradient. Uptake of positively charged substrates, such as lysine or inorganic cations, is driven by the membrane potential only (facilitated diffusion). This movement is electrogenic and does not involve protons (i.e. uniport). The electrical potential is also the driving force for the transport of lipophylic cations, such as triphenylmethylphosphonium (TPMP+1 and dibenzyldimethylammonium ion (DDA'), but this transport does not involve specific membrane proteins. The attractive feature of the chemi-osmotic coupling models is that the proton-motive force is visualized as the common factor for synthesis of ATP, for transport and for other energy-linked functions of the membrane. In addition, this model offers an explanation for the inhibitory action of uncoupling agents on transport. It has been proposed that these compounds are soluble in the membrane and act as circulating carriers conducting protons across the membrane, thereby short-circuiting the proton-motive force. Studies on mitochondria and on bacteria have supplied evidence in favour of a chemi-

230

WIL N. KONINGS

osmotic type of energy coupling; the available information has been discussed in a number of excellent reviews (Mitchell, 1966, 1973; Harold, 1972; Kaback, 1974; Lombardi et ul., 1974; Hamilton, 1975; Simoni and Postma, 1975). A completely different type of hypothesis has been presented initially by Kaback and Barnes (19711, and in a modified form by Kaback and Hong ( 1973). This hypothesis proposes a direct coupling of the carriers to specific sites of the respiratory chain. According to this model, the transport proteins (the carriers) possess a high affinity for their transport substrates only in the oxidized (disulphide) form, whereas the reduced (sulphhydryl) form has a low affinity. Active transport of a particular sugar or amino acid is associated with reduction of the appropriate carrier by the electron donor. Upon reduction, the highaffinity form of the carrier undergoes a conformational change that results in translocation of bound substrate from the outer surface of the membrane to the inner surface. The resulting low-affinity (sulphhydryl) form of the carrier then releases the substrate, and the carrier is reoxidized. By alternating oxidation and reduction of the carrier, substrate is transferred from the outside to the inside against a concentration gradient until the internal concentration is sufficient to saturate the reduced form of the carrier. At that point, the rate of efflux will equal the rate of influx, and a steady state will be achieved. This model postulates that the carriers of different specificity are coupled to specific sites in the electron- transfer chain, thereby conferring functional heterogeneity on otherwise identical electron-transfer chains. An important aspect is that the two models visualize a different utilization of the energy, released by the electron-transfer systems, for transport. According to the chemiosmotic model, the coupling of the carriers to the electron transfer chains is indirect, and all carrier proteins (A) with affinity for a solute (a) are activated by all electrontransfer chains present in the membrane. This implies that all carriers (A) participate in the transport of solute (a) will depend, therefore, on the total electron flow activity in the electron-transfer chains. According to the direct coupling model, each carrier (A) can be energized only by the electron-transfer chain to which it is coupled. The rate of transport of solute (a)will depend only on the electron flow activity in these electron-transfer chains. Information about the nature of the coupling from the carrier to the electron-transfer chain (whether it is direct or indirect) has been

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 231

supplied by experiments performed with membrane vesicles from Bacillus subtilis aro D (RBI631 (Bisschop and Konings, 1976).This mutant is defective in synthesis of menaquinone (Farrand and Taber, 19731, an essential component in the respiratory chain between NADH dehydrogenase and the cytochromes. As a consequence of this deficiency, membrane vesicles prepared from these mutant cells do not perform NADH oxidation, and NADH does not function as an energy source for amino-acid transport. The electron transfer from NADH to oxygen can be restored, partially or completely, with menadione (a menaquinone analogue) and evidence has been presented that this reconstitution results in respiratory chains which are functionally identical with the respiratory chain of the wild-type strain (Bisschop et al., 1975c; Bisschop and Konings, 1976). Before proceeding, two essential features of this system have to be taken into consideration : (i) there appears to be neither experimental nor theoretical reason for an interaction of menadione with certain incomplete respiratory chains, so that the reconstitution with menadione will occur most likely at random among the respiratory chains; (ii) the reconstitution will be the same for all respiratory chains in the membrane. All restored respiratory chains will therefore, at any NADH concentration, oxidize NADH at the same rate so that the total rate of NADH oxidation is an indication of the number of reconstituted respiratory chains. According to an indirect coupling model, such as the chemi-osmotic model, an increase in the number of functional respiratory chains (as measured by the increased rate of NADH oxidation) should result in an increase in the initial rate of transport of solute (a)until all of carrier proteins (A) are fully activated. Further increase in functional respiratory chains should not result in a further increase in the initial rates of transport of solute (a). In a directly coupled system, a random reconstitution of incomplete respiratory chains will affect the respiratory chains, coupled to carrier (A) to the same extent as the other respiratory chains present in the membrane. At a certain NADH concentration, all carrier (A)-coupled respiratory chains will energize the individual carriers to the same extent so that the total transport activity will be the sum of all individual activities of the carriers (A). Maximal activity is not obtained until all respiratory chains present in the vesicles have been reconstituted. This implies that, in a directly coupled system, a stoicheiometric relationship exists between the rate of NADH oxidation and transport activity. The

WIL N. KONINGS

232

E

: - > - -i4/[ \

-*2c n

0

8

:.a

2-

FP

I

I

I

I

I

I

NADH Oxidation (nmoler/min/mg protein)

FIG. 16. Relation between reduced nicotinamide adenine dinucleotide (NADH)oxidase activity and NADH-driven amino-acid transport. The initial rate of Lglutamate (0) or L-alanine (A)transport were determined in membrane vesicles from Ban'llus subtills aro D in which the NADH-oxidase activity was reconstitutedto different degrees by addition of different concentrations of menadione. The NADH concentration was 10 mM. Taken from Bisschop and Konings (1976).

results of the experiments shown in Fig. 16 are at variance with this prediction, but are in agreement with the predictions based on an indirectly coupled system. The data given in Fig. 16 supply also information about the efficiency of NADH oxidation in energizing amino-acid transport. For transport of one mole of amino acid, oxidation for 130-250 moles of NADH is needed. This efficiency varies for different amino acids, which indicates a variation in the energy requirement for transport of difTerent amino acids. The same results have been obtained with membrane vesicles from the wild-type B . subtilis W 2 3 (Bisschop et al., 1975c). I t is obvious from these results that only a fraction of the energy supplied by the oxidation of NADH is applied to transport of the amino acid. Similar inefficiencies in energizing amino-acid transport have been observed in membrane vesicles from E . coli (Kaback and Hong, 1973)and Staph. aureus (Short and Kaback, 1974).These observations indicate that more than 99% of the energy generated by electron transfer in the respiratory chain is not, in the membrane vesicles, available for active transport of solutes. This inefficiency can be explained if the membrane vesicles accumulate (orextrude), in ad-

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 233

dition to the amino acid, other ionic species present in the incubation mixture. However, according to Kaback (197.29, none of the ionic species in the reaction mixture, Mg2; SO:; SO:; PO:; Na+, C1-or K+ (in the absence of valinomycin) is accumulated during D-lactate oxidation by E. coli membrane vesicles. Information about the extrusion of ionic species is not available. In the concept of the chemi-osmotic coupling theory, an explanation for this inefficient use of energy for active transport could be found in a high proton permeability of the membrane vesicles. In other words, the membrane vesicles are leaky for protons. According to the chemi-osmotic coupling theory, inward movement of protons can occur via carrier proteins, or via ATPase. It appears unlikely that leakage of protons takes place via the ATPase complex since the addition of DCCD does not result in a higher efficiency of NADH oxidation in energizing transport. It is postulated that proton translocation from the outer surface of the membrane to the inside, via the carrier protein, occurs only during accumulation of solutes. Active transport of a solute therefore will increase the inward movement of protons (and/or charge) and decrease the proton motive force. This implies that the different transport systems will compete for the available energy, and that active transport of one solute will inhibit the simultaneous accumulation of another solute. This contention is supported by observations made by Schuldiner and Kaback (1975) under conditions of excess supply of energy. Membrane vesicles from E. coli ML 308-225 accumulate, in the presence of D-lactate or ascorbate-PMS, lactose at a much higher rate than proline (V- for lactose is 50 and for proline 1.3 nmoles per mg membrane protein per min) (Kaback and Barnes, 1971; Lombardi et al., 1973).In the presence of 10 mM lactose, the initial rate of proline transport is inhibited by 50%, and of triphenylmethylphosphonium transport (see p. 235) by 40%. Such an inhibitory effect of lactose was not observed in membrane vesicles which lack the lactose transport system. Similar experiments have been performed with membrane vesicles from B. subtilis aro D, incubated under conditions of limited energy supply. Even at low rates of NADH oxidation, transport of one amino acid is not inhibited by the addition of a 50 to 100-fold higher concentration of another amino acid (Bisschopand Konings, 1976).In these membrane vesicles, the energy supply for transport of one amino acid is therefore hardly affected by the simultaneous transport of another amino acid. These results, therefore, do not exclude the possibility that inward

234

WIL N. KONINGS

movement of protons occurs in the membrane vesicles via the carrier proteins, also in the absence of transportable solute. The chemi-osmotic coupling model visualizes the localization of intermediates of the electron-transfer systems partially at the outside and partially at the inside of the cytoplasmic membrane. Recently, observations have been made which indicate a localization of some components of the respiratory chain at the outside of “right-side out” membrane vesicles from B . subtilis (Bisschop et al., 1975b; Konings, 1975). In these membrane vesicles, transport can be energized under anaerobic conditions with NADH in the presence of ferricyanide as an electron acceptor (Fig. 1 7 ) and evidence has been presented that the AEROBIC

-c

10-

ANAEROBIC

5- ( b )

( 0 )

Time (min)

Time (min )

FIG. 1 7 . Effect of ferricyanide on NADH-driven uptake of L-glutamate under aerobic and anaerobic conditions by membrane vesicles from Bacillus subtilis W23. Uptake of Lglutamate was determined in the presence of NADH (10 mM) (01,NADH (10 mM)and or without potassium ferricyanide (10 mM) (A), potassium ferricyanide (10 mM) (O), electron donor or ferricyanide added (w). Taken from Bisschop et al. (1975~).

membrane-impermeable ferricyanide accepts electrons from the terminal part of the respiratory chain, most likely from cytochrome cl. Similar lines of evidence have been presented for an outside localization of anaerobic electron-transfer intermediates in membrane vesicles from anaerobically grown E . coli (Boonstra et al., 1976b). Furthermore, evidence has been obtained for a localization of electron-transfer inter-

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 235

mediates prior to the coupling site(s) of the respiratory chain at the outside of the membrane, by transport experiments in membrane vesicles from B . subtilis and E . coli with the membrane-impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulphonate (MPS). This electron donor drives transport of amino acids, as well as its lipophilic analogue reduced phenazine methosulphate (PMS) (Konings, 1975; Short and Kaback, 1975). The available evidence from studies in whole cells and membrane vesicles in favour of a chemi-osmotic type of energy coupling has been reviewed by Harold (1972) and Hamilton (1975). In this discussion we will focus our attention only on studies with membrane vesicles. (i) Reeves (197 1) demonstrated that membrane vesicles from E . coli extrude protons during oxidation of D-lactate. (ii) Electron transfer-dependent transport in vesicles from several organisms is severely inhibited by a variety of proton conductors, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), although these agents do not inhibit electron transfer (Barnes and Kaback, 1970, 197 1; Konings and Freese, 1972). Furthermore, a number of mutants of E . coli have been isolated which exhibit pleiotropic transport defects, and vesicles prepared from some of these mutants exhibit increased permeability to protons (Rosen, 1973b; Altendorf et al., 1974). (iii) Dilution of membrane vesicles, which contain internally potassium, into a medium devoid of potassium but containing valinomycin, results in valinomycin-mediated potassium emux and the generation of an electrical potential (Aq) across the membrane, interior negative. Under such conditions, lactose and amino acids are accumulated by the membrane vesicles (Hirata et al., 1973; Lombardi et af., 1974).

(iv) During D-lactate or reduced PMS oxidation, lipophilic cations such as dimethyldibenzylammonium (in the presence of tetraphenylboron) (Hirata et al., 1973; Lombardi et al., 1974; Altendorf et al., 19751,i triphenylmethylphosphonium (Schuldiner and Kaback, 19751, safranine-o (Schuldiner and Kaback, 1975) and rubidium (in the presence of valinomycin) (Lombardi et al., 1973) are accumulated (Fig. 18). There is a quantitative correlation between the steady-state levels of accumulation of the different lipophilic cations (Schuldiner and Kaback, 1975). Furthermore, steady-state levels of lactose and amino

WIL N. KONINGS

236

acid accumulation are directly related to the steady-state level of TPMP-accumulation. 20f

-24 - 18

-

-

+ =

-12

+\.s

a"

z I-

a -

I

h

-

-6

FIG.18. Uptake of triphenylmethylphosphonium(TPMPC)by membrane vesicles from Escherichia coli ML 308-225 in the presence of different electron donors. Triphenylmethylphosphoniumuptake was determined in the presence of sodium ascorbate (20 mM) and phenazine methosulphate(0.1 mM) (01,lithium D-lactate (20 mM) (4,lithium L-lactate (20 mM) (V),sodium succinate (20 mM) (01, NADH (A),or without added electron donor (0). Taken from Schuldiner and Kaback (1976).

Analogous observations have been made with membrane vesicles from anaerobically grown E. coli, during anaerobic electron transfer in the nitrate respiration system and the fumarate reductase system (Boonstra et al., 1976a) and in membrane vesicles from the phototrophic organism Rhodopseudomonas sphaeroides upon light-induced cyclic electron flow (Hellingwerfet al., 1975). (v) Strong support for a chemi-osmotic type of energy coupling comes from transport studies in membrane vesicles from Halobacterium halobium. Upon illumination, the photochemical events which occur in the membrane- bound bacteriorhodopsin result in the extrusion of protons (Bogomolini and Stoeckenius, 1974; Racker and Stoeckenius, 1974; Racker and Hinckle, 19741, and a proton-motive force is generated in the order of 200 mV (Renthal and Lanyi, 1975). Racker

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 237

and Stoeckenius (1974) observed, in a reconstituted system in which purple membranes from H. halobium and mitochondria1ATPase are incorporated into lipid vesicles, ATP production upon illumination. MacDonald and Lanyi (1975) demonstrated that these vesicles transport leucine in response to light, and presented evidence that the driving force for this transport is the electrical potential. (vi) In agreement with an indirect coupling model, as proposed by the chemi-osmotic theory, is the observation that membrane vesicles from E. coli ML 308-225 contain a large excess of lactose carriers (the product of the y-gene) relative to D-lactate dehydrogenase (Reeves et al., 1973). (vii) Convincing evidence for a chemi-osmotic mechamism of active transport in the vesicle system was supplied by Ramos et al. (1976). It was demonstrated by flow dialysis experiments that membrane vesicles from E. coli generate, in the presence of ascorbate-PMS, a large transmembrane pH-gradient which can reach two pH units at an external pH value of 5.5. Using the distribution of weak acids (Harold and Baarda, 1968), such as acetate, to measure the pH gradient (ApH) and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential across the membrane (A+),the vesicles were shown to generate a proton-motive force (A,&+) of approximately -180 mV at pH 5.5. Membrane vesicles from E. coli accumulate lactose and other substrates to apparent intravesicular concentrations which are one hundred-fold greater, or more, than those of the external medium. In order to sustain concentration gradients of this magnitude, a proton-motive force of at least 120 mV is required. Although these observations lend strong support for a chemiosmotic type of energy coupling in active transport processes, several other observations have been made which are, at this moment, difficult to explain in the framework of this theory (Lombardi et al., 1974). These are: (i) There is no correlation between rates of oxidation of various electron donors, in E. coli, and their relative effects on transport (Barnes and Kaback, 197 1 ; Kaback and Barnes, 197 1). The effectiveness in stimulating transport is much higher for ascorbate-PMS and D-lactate than for NADH or succinate (Table 2). It has been discussed before (p.200)that these observations cannot be explained by a

238

WIL N. KONINGS

specific localization of D-lactate dehydrogenase in the membrane because, in vesicles from mutants which lack D-lactate dehydrogenase, the effectiveness of succinate or NADH as an electron donor reaches similar levels as D-lactate in the wild type. Furthermore, an explanation based on the assumption that part of the membrane vesicles is inverted appears to be unlikely (p. 194). Recently, it was demonstrated that a qualitative relationship exists between the ability of various electron donors to drive transport and their ability to generate both an electrical potential (interior negative) across the membrane (Schuldiner and Kaback, 1975) and a pH-gradient (Ramos et al., 1976). AscorbatePMS and D-lactate produce maximal relative effects for each parameter, while succinate and, especially, NADH produced much weaker efTects. It appears, therefore, than an understanding is required of the role which different electron carriers have in the generation of a pH-gradient, or an electrical potential, in order to explain the different effects of the various electron donors. (ii) Electron-transfer inhibitors, which completely block D-lactate oxidation and D-lactate-dependent transport, have different effects on emux of accumulated substrates. Inhibition at sites of the E. coli respiratory chain distal to the energy-coupling site(s1 (anaerobiosis, amytal, HOQNO and cyanide) results in a rapid efflux of accumulated solutes from vesicles preloaded in the presence of D-lactate, while inhibitors of D-lactate dehydrogenase (oxalate and oxamate) cause little or no efflux from preloaded vesicles (Kaback and Barnes, 197 1 ; Lombardi and Kaback, 1972; Lombardi et al., 1974). Initially these and other observations have led to the postulation of the direct-coupling model (Kaback and Barnes, 1971; Kaback and Hong, 1973). It was postulated that, in E. coli, the carriers with different substrate specificities occupy equivalent sites in the respiratory chain between D-lactate dehydrogenase and cytochrome, b,, and that active transport of a particular sugar or amino acid is associatedwith reduction of the appropriate carrier by D-lactate dehydrogenase. In this model a specific role in the energization of transport is played by that part of the electron respiratory chain, in E. coli, lying between D-lactate dehydrogenase and cytochrome b,. It offered an explanation for the specific effects of D-lactate on transport, and also for the different effects of respiratory chain inhibitors on the emux of accumulated substrate. Inhibition beyond the energy-coupling site maintains the

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 239

carrier in a reduced state. In such a state, the carrier has a low affinity for the solute and is mobile, and thus it will allow emux to occur. Inhibition of the carriers before the energy-coupling site results in an oxidation of the carrier. In this state the carrier has a high substrate affinity and is immobile, and thus no efflux of accumulated substrate can take place. It is obvious from the previous discussion that a major objection- of this model is that it fails to explain several observations which have been discussed above, such as the uptake of sugars and amino acids upon an imposed ion gradient, electron transfer-driven uptake of lipophilic cations and the action of uncouplers. Furthermore, the different electron donors have similar effects on the transport of lipophilic cations, and respiratory chain inhibitors effect efflux of accumulated lipophilic cations in a similar way to that which has been observed for sugars and amino acids. Transport of lipophilic cations is not carrier-mediated, and an explanation for these observations must therefore be found at the level of the “energized membrane state” and not at the level of the carrier proteins. In order to explain the observations, presented above, in the context of the chemi-osmotic model of energy-coupling, Kaback et al., (1976) suggested that the membrane potential is in equilibrium with the redox state of the respiratory chain at that site between D-lactate dehydrogenase and cytochrome b, which generates the membrane potential. Inhibition of electron flow in a manner which leads to reduction of the energy coupling site results in dissipation of the membrane potential, while inhibition of electron flow in a manner which leads to oxidation of the energy-coupling site does not result in a collapse of the potential. Such an explanation reconciles aspects of the chemi-osmotic model and the direct coupling model. I t emphasizes that the site of the respiratory chain between D-lactate dehydrogenase and cytochrome 6 , plays a special role in generation of the membrane potential. In order to offer a final explanation, more insight appears to be required into the role which various components of the electron- transfer systems have in the translocation of protons and the generation of a membrane potential. C . ENERGY-DEPENDENT BINDING OF SOLUTE TO CARRIER

PROTEINS

Carrier-mediated transport of a solute through the cytoplasmic membrane requires several distinct steps: in one of the initial steps, the

240

WIL N. KONINGS

solute binds to the carrier protein at the outside surface of the membrane; subsequently the carrier-solute complex travels, or rotates, in the membrane in such a way that the solute becomes exposed to the inside surface of the membrane, and finally the solute is released at the inside. Elegant experiments performed by Schuldiner et al. (1975a, b) and Rudnick et al. (1975a, b, c) demonstrated that energy is required for the initial steps of the transport process (i.e. exposure of the carrier to the outer surface of the membrane where it is able to bind the ligand). Photoreactive p-Galactosides

k

Fluorescent /3-Galactosides

i)H

R=

I

FIG. 19. Structural formulae of various dansylgalactosides and azidophenylgalactosides. Taken from Schuldiner et al. (1976).

Schuldiner et a1 (1975a, b) used for these studies the fluorescent pgalactosides shown in Fig. 19. These compounds competitively inhibit lactose transport by membrane vesicles from E. coli ML 308-225, but are not accumulated (Reeves et al., 1973; Schuldiner et al., 1975a, b). When membrane vesicles are incubated with these fluorescent /3galactosides, an increase in fluorescence is observed upon either the addition of D-lactate, the imposition of an electrical potential (interior negative), or dilution-induced carrier-mediated lactose emux. The increase in the fluorescence, induced by D-lactate, is blocked and/or rapidly reversed by addition of /3-galactosides, sulphhydryl reagents, inhibitors of D-lactate oxidation or uncoupling agents. The increase is not observed with a danlysglucoside(Z’-(N-dansyl)aminoethyl 1-thio-

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 241

p- D-glucopyranoside), nor with membrane vesicles which lack the pgalactoside transport system, indicating that the effects are specific for the galactosyl configuration of the ligand. The affinity of the carrier for substrate is directly related to the length of the alkyl chain between the galactosidic and the dansyl moieties of the dansyl galactosides. The affinity constants of the various dansyl galactosides, as determined by fluorometric titration are in good agreement with their apparent Kt values for lactose transport. Anisotropy of fluorescence measurements with 2-(N-dansyl)aminoethyl-p-D-thiogalactopyranoside(DG,) and 6-(N-dansyl)-aminoethylp-D-thiogalactopyranoside(DG,) demonstrate a dramatic increase in polarization on addition of D-lactate which is reversed by anoxia or addition of lactose (Schuldiner et d., 1975a). These observations indicate that the changes in the fluorescence observed on “energization” of the membrane are the result of binding of the dansyl galactosides rather than binding followed by transfer into the hydrophobic interior of the membrane. The results suggest that the lac carrier protein is inaccessible to the external medium unless energy is provided, and that energy is coupled to one of the initial steps of transport. A similar conclusion was reached in studies with the photoreactive p-galactosides (2-nitro-4azidophenyl-p-D-thiogalactopyranoside (APG,) and 242-nitro-4azidopheny1)aminoethyl-p-D-thiogalactopyranoside(APG,)) (Fig. 19) (Rudnick et al., 1975a, b). Irradiation of these compounds with visible light causes photolysis of the azido group to form a highly reactive nitrene which then reacts covalently with the macromolecule to which the azido-containing ligand is bound. The /?-galactoside APG, inhibits lactose transport in membrane vesicles from E. coli ML 308-225 competitively with an apparent Kt of 75 pM. In contrast to the dansylgalactosides, APG, is actively transported by the membrane vesicles upon the addition of D-lactate, and kinetic studies revealed an apparent K, of 75 pM. Membrane vesicles devoid of lac transport do not accumulate APG, in the presence or absence of D-lactate. When exposed to visible light in the presence of D-lactate, APG, irreversibly inactivates the lac transport system, but this photolytic inactivation does not occur in the absence of D-lactate. Kinetic studies of the inactivation process yield a KD of 7 7 pM. The effects are specificfor the lac transport system, since lactose protects against photolytic inactivation and APG, does not inactivate against amino-acid transport. The p-galacto-

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side APG, behaves similarly with respect to photoinactivation, but this compound is not transported by the vesicles and has a higher affinity for the lac carrier (the Ktfor competitive inhibition of lactose transport and for the KD for photolytic inactivation in the presence of D-lactate are 35 pM). Furthermore, APG,-dependent photolytic inactivation can also be induced by an artificially imposed membrane potential (exterior positive). The studies with dansyl- and azidophenylgalactosides demonstrate the lac protein is accessible to the external medium only when energy is provided. Several possible mechanisms by which energy might lead to exposure or increased affinity of the binding site to the outside surface of the membrane have been considered (Schuldiner et al., 1975a). In view of the evidence presented in favour of a chemi-osmotic type of energy-coupling, it seems attractive to postulate that the lac carrier contains a negative charge and moves in response to a membrane potential to the outside of the membrane where it is able to bind the ligand. Studies on the effects of the sulphhydryl reagent, p-chloromercuribenzenesulphonate (p-CMBS),on APG,-dependent photoinactivation demonstrated that the lac carrier protein contains sulphhydryl groups which are not in the binding site (Rudnick et al., 1975~). Treatment of E . coli membrane vesicles with p-CMBS results in an inhibition of all carrier-mediated aspects of the lactose transport system (Kaback and Barnes, 197 1). However, p-CMBS does not block D-lactate-induced APG,-dependent photo-inactivation; in contrast p-CMBS induces APG, photo-inactivation in the absence of D-lactate. The dissociation constant of APG, for p-CMBS-treated membranes is about 20 p M , a value which is very similar to that determined for D-lactate-induced APG,-dependent photo-inactivation. Rudnick et al. ( 1 9 7 5 ~suggested ) as a possible mechanism thatp-CMBS reacts with a sulphhydryl group of the lac carrier and traps the protein at the outside surface of the membrane. In that position, substrate can bind to the carrier but cannot be translocated. The uncoupler, carbonyl-cyanide m-chlorophenylhydrazone (CCCP), does not inhibit p-CMBS-induced APG,photo-inactivation, and a membrane potential is thus not required for the p-CMBS effect. It is evident that p-CMBS does not block the binding site on the carrier, since APG, binds with high affinity and the p-CMBS-treated carrier protein is protected from APG,-binding by lactose, thiodigalactoside and melibiose. Exposure to the external

ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 243

medium of sulphhydryl groups of the lac carrikr protein appears also to occur upon energization, because inactivation of lactose transport by N-ethylmaleimide is increased two to four-fold by reduced phenazine methosulphate. The results indicate that energization of the membrane leads to an exposure, to the outer surface of the membrane, of a high-affinity binding site and a sulphhydryl group which is not in the binding site. It is suggested that the sulphhydryl group in the lac carrier protein may exist in an ionized form in the hydrophobic milieu of the membrane, and that this functional group in the protein may respond to the membrane potential (Kaback et al., 1976).

V. Conclusions Isolated bacterial cytoplamic membrane vesicles have proved to be an excellent model system for studies of integrated membrane functions. Membrane vesicles, isolated with the lysozyme-EDTA procedure, have the same orientation as the cytoplasmic membrane of intact cells, and these vesicles catalyse a number of membrane-bound functions. Observations made in studies with membrane vesicles demonstrated two types of transport systems : (i) group translocation systems which catalyse vectorial covalent reactions ; and (ii) active transport systems. The active transport systems appear to be the major mechanisms for translocation and accumulation of solutes in bacteria. The energy for active transport can be supplied by electron flow in a number of electron-transfer systems ; respiratory chains with oxygen as terminal electron acceptor; anaerobic electron transfer systems with nitrate or fumarate as terminal electron acceptor, and cyclic electron- transfer systems. Furthermore, light-dependent reactions in bacteriorhodopsin can supply the energy for active transport processes in membrane vesicles from Halobacterium halobium. I t has been demonstrated that energy released by the electrontransfer systems is not coupled to active transport via ATP. It has not yet been thoroughly established whether ATP can serve as the major source of energy for active transport in bacteria grown under glycolytic conditions it might be possible that electron-transfer systems which do not contain cytochromes supply the energy for active transport under these conditions. In view of recent studies, it appears to be beyond dispute that chemiosmotic phenomena are essentially involved in the mechanism of

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energy coupling. Electron flow in the electron-transfer systems results in the generation of a proton-motive force which is the driving force for active transport. Studies with membrane vesicles have demonstrated that the energy is coupled at least to one of the initial steps in the transport process. In order to obtain a complete understanding of the mechanism of active transport, a number of features remain to be elucidated. Among them are : involvement of electron-transfer intermediates in the translocation of protons; the role of the electrical potential, and the pH-gradient, in the energy coupling to active transport of different solutes; the molecular properties of the transport carriers and the mechanism of solute translocation. Attempts are currently in progress which hopefully, in the near future, will supply insight into these and other properties of the active transport systems.

VI. Acknowledgements I would like to express my appreciation to Dr. R. N. Campagne, Mrs. I. Kuipers-Wessels,A. Bisschop, J. Boonstra and P. A. M. Michels for their constructive criticism of the manuscript and their valuable suggestions. Dr. H. R. Kaback and Dr. J. Lanyi kindly supplied manuscripts. prior to publication. I am very grateful to Mrs. M. T. BroensErenstein, Mrs. R. G. Kalsbeek and Mrs. J. W. Schrdder-ter Avest for help in the preparation of this manuscript. The studies performed in the Laboratory of the author were supported by the Netherlands Organization of Pure Scientific Research (ZWO). REFERENCES

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Aden ine Nucleot ide Concentrations and Turnover Rates. Their Correlation with Biological Activity in Bacteria and Yeast ASTRID G. CHAPMAN and DANIEL E. ATKINSON Molecular Biology Institute and Biochemistry Division, Department of Chemistry, University of California, Los Angeles, California 90024, U.SA. I. Introduction . . . . . . . . . . . . 11. Concentrations and Fluxes of Adenine Nucleotides in uivo . . . A. Adenine Nucleotide Turnover . . . . . . . . B. Turnover of ATP . . . . . . . . . . C. Regulation of ATP Utilization and Regeneration . . . . D. Sampling of Microbial Cultures for Adenine Nucleotide Determinations . . . . E. Changes in Adenine Nucleotide Concentrations 111. Concentration of ATP, Total Adenine Nucleotide Concentration, and . . . . . Energy Charge in Relation to Cellular Activities A. Relation between ATP Concentration and Growth Rate . . . . B. Variations in Adenine Nucleotide Levels during Growth C. Adenine Nucleotides in Mutant Strains Arrested in Growth D. Correlation between Kinetics in vitro and Observations in uiuo E. Relation between Energy Charge and Total Adenine Nucleotide . . . . . . . . . . . Concentration F. Phage Infection . . . . . . . . . . . G. Other Nucleotides . . . . . . . . . . H. RNA Synthesis . . . . . . . . . . . I. Protein Synthesis . . . . . . . . . . . IV. General Discussion . . . . . . . . . . . References . . . . . . . . . . . .

253

254 256 256 26 1 268 269 272 282 282 285 286 287 289 290 29 1 293 295 297 300

254

A. G. CHAPMAN AND D. E. ATKINSON

I. Introduction

Because the adenine -nucleotides are involved in every metabolic sequence, and in most metabolic energy transductions (both chemical and mechanical), and are also intermediates in the synthesis of nucleic acids, a discussion of the roles of these compounds could include nearly all aspects of the genetics, metabolism, physiology, and locomotion of microbes, as well as much of the work reported on properties of enzymes from micro-organisms studied in vitro. Such a comprehensive treatment would, however, not only exceed the competencies of the reviewers, but also greatly exceed the available space; thus drastic and largely arbitrary limitations of the areas to be covered have been necessary. This review therefore deals mainly with reports on the concentrations of ATP, ADP, and AMP, and on the ratios of these concentrations found in intact microbial cells, and with changes in these concentrations (or concentration ratios) that occur under various conditions. Further we shall review the correlations, both stoicheiometric and regulatory, that have been reported between the concentrations of the different adenine nucleotides and the occurrence or rates of integrated functions such as growth or production of storage compounds. Even within these areas, only a sampling was possible; other equally important papers had to be omitted, with no derogatory implications. Although much of the conceptual framework within which the results will be considered was derived initially from work on enzymes in vitro, experiments of that type will not be described here. Some of the earlier results have been reviewed elsewhere (Atkinson, 1969). Moreover the whole area of cyclic AMP effects has been excluded (for a recent review of cyclic AMP in prokaryotic organisms, see Rickenberg, 1974). The roles of the adenine nucleotides may be conveniently classified under three headings. First, they are intermediates in the biosynthesis of nucleic acids and histidine, and also of nucleotide cofactors such as NAD, NADP, FAD, and coenzyme A. Second, they constitute an energy-transducing system that stoicheiometrically couples all metabolic processes and thus plays the central role in stoicheiometric correlation of metabolism. Third, they kinetically regulate the activities of a large number of enzyme reactions and probably of all metabolic sequences. In serving as intermediates, the adenylates d o not differ in principle from dozens of other compounds, but the second and third

ADENINE NUCLEOTIDE CONCENTRATlONS AND TURNOVER RATES

255

functional categories are unique. Metabolism is organized around the adenine nucleotides, and these compounds must be taken into account in the study of nearly every aspect of functional biology. Processes in which the adenine nucleotides participate stoicheiometrically (categories 1 and 2 in the above classification) are summarized in Fig. 1. Interconversions between the different adenine nucleotide species that occur in the course of energy transduction and stoicheiometric coupling between metabolic sequences (category 2) are indicated within the circle. These include the large number of reactions where ATP serves as a phosphate donor; those in which AMP is the immediate or ultimate product; the reformation of ATP from ADP by oxidative phosphorylation or by substrate-level phosphorylation; and the interconversion of ATP, ADP and AMP through the adenylate kinase reaction. The adenyl cyclase-mediated formation of CAMP from ATP, and the subsequent hydrolysis of CAMP to AMP, are also indicated in the figure, although these reactions will not be discussed in this review. All these reactions involve the removal and replacement of the y- and P-phosphate groups of ATP, and they do not affect the sum of the concentrations of the different adenine nucleotide species. Conversion of ATP to ADP or AMP, and its regeneration, will be referred to as ATP turnover. NAD FAD CoASH etc. k

Histidine, Protein

,dADP, DNA

Adenine

Adenosine

De KO

biosynthesis--,-IMP

\$.

FIG. 1. Reactions involved in adenine nucleotide formation, utilization and interconversion. Adapted from an unpublished figure by Jean. S . Swedes.

256

A. G. CHAPMAN AND D. E. ATKINSON

Reactions of category 1, in which the adenylates serve as biosynthetic intermediates, are indicated by arrows extending from or entering the adenine nucleotide circle. These reactions include the synthesis and degradation of AMP and the net utilization of adenine nucleotides in biosynthesis. The adenylate utilization and resynthesis represented by these reactions will be referred to as turnover of the adenine nucleotide pool, as distinguished from ATP turnover as already defined. 11. Concentrations and Fluxes of Adenine Nucleotides in vivo A. ADENINE NUCLEOTIDE TURNOVER

The rate of utilization of adenine nucleotides for the biosynthesis of macromolecules can be estimated from the cellular composition and the generation time of the organism. Such calculations are shown in Table 1 for Escherichia coli and Salmonella typhimurium grown in different media so as to allow a ten-fold range of growth rates to be obtained. Although the figures are compiled from different papers, and the growth rates at which total adenine nucleotides were measured differed slightly from those for the other determinations, as indicated in the footnotes, the calculated turnover rates should be approximately correct. The amount of adenylate and deoxyadenylate residues in RNA and DNA, and of histidine in'protein (ATP contributes a carbon and a nitrogen atom to the biosynthesis of histidine), is calculated from the macromolecular compostion of the cell according to the assumptions listed in the legend to the table. At rapid growth rates, net RNA synthesis represents as much as 75% of the adenylate consumption. Even at slow growth rates, where the cellular RNA content is lower, about 50% of the adenylate utilization is accounted for by RNA synthesis. The net rate of adenylate incorporation per minute into polymers is shown in the second column from the right. Figures for the two organisms are very similar. As would be expected, slower growth rates are accompanied by lower rates of net adenylate utilization. The last column shows that at very rapid growth rates the adenine nucleotide pool is replenished approximately every 40 seconds, in contrast to a turnover time of four to six minutes in slowly growing cells. The rate of adenylate utilization decreases somewhat more sharply than the growth rate because of the lower RNA content of the slowly growing cells. The difference between the rates of decrease is not great, how-

TABLE 1. Turnover of the adenine nucleotide pool' Organism

Salmonella typhimurium'

Doubling time (mid

Net adenine Sum of adenine incorporation nucleotides ( p o l e s / g into polymers DNA RNA Protein DNA RNA Protein Total dry wt) (pmoleslminlg dry wt.)

D 0 Turnover of rn adenine nucleotide pool (seconds) rn

$ z C

25 50 300

35 310 37.. 220 37 180 40 120

670 740 780 830

28 29 29 32

233 165 135 90

55 61 64 68

316 255 228 190

8.0' 3.7 6.0 4.0

12.64 5.10 2.28 0.63

38 44 158 38 1

24 40 110

26 23 32

670 750 800

21 18 25

231 172 122

55 61 66

307 251 213

9.5f 8.3 6.8

12.79 6.28 1.94

45 79 210

100

Escherichiacoli'

v o l e s adenine residuedg dry wt!

mg/g dry wt.

307 229 163

'The following assumptions were used for the calculations: the average molecular weights of nucleotide monophosphate residues in RNA, the deoxynucleotide monophosphate residues in DNA, and the amino-acid residues in protein are equal to 333, 3 1 7 and 122, respectively. Adenylates constitute 25% of the RNA bases in E. coli (Spahr and Tissieres, 1959). and it is assumed that DNA contains 25% dmxyadrnylatr rrsidurs. Histiditir cotistimtes 1% of the amino-acid residues in protein from E. coli (Roberts el nl., 1955). The net utilization of adenine nucleotidcs for thr li)rniatioti ol' lrrr histidine and pyridine nucleotides has been ignored in these calculations. "denylate residues in RNA, deoxy-adenylate residues in DNA, and histidine residues in protein. 'The composition of Sal. lyphimutium is from Maalw and Kjeldgaard ( 1966). 'The composition of E. coli is from Forchhammer and Lindahl(l97 I). 'The concentrations of total free adenine nucleotides in Sal. typhimurium at doubling times 30, 50, 105, and 240 min are from Smith and Maalee (1964). f The cellular concentrations of the adenine nucleotides at the growth rates observed were not determined by Forchhammrr i i i i t l 1.incliilil (10711. Instead, values obtained from other laboratories at similar growth rates were substituted as follows: At a doubling time of 36 min, from Dietzler tf 01. (1974~). The units are converted from jnnoleslg protein by assuming protein equal t o 67% of dry wright; iit ii doubling tinic ,4fi tiiiti. IIotti S\vc.tlrs ef 01. (1975).The units are converted fr.om pnoles/g protein by assuming protein equal to 75%of dry weight; at a doubling time of 120 min, lrom Lowry ef al. (1971).

Prn

2 #

0 0

z Gz -I n

30 c)

-I

C

3

z

2rn n

2 v)

hl

cn -J

258

A. G. CHAPMAN AND D. E. ATKINSON

ever. I t can be calculated from Table 1 that the adenine nucleotide pool turns over 30 to 50 times per generation; thus it appears that the turnover time of the adenylate pool in enteric bacteria is about 2 to 3% of the generation time. 1. Balance between Adenine Nucleotide Synthesis and Utilization

In growing cells, in the absence of exogenous nucleosides or free bases, biosynthesis de nouo must account for rLetpurine nucleotide formation (sequence 1, Fig. 1). There is also a constant influx of AMP into the adenylate pool from breakdown of unstable RNA (reaction 2, Fig. 1). The flow of ATP into mRNA is rapid, but because of the short lifetime of mRNA, only a small part of this flow, proportional to the increase in cell mass and the steady-state concentration of mRNA, represents net utilization of adenylates. For the purposes of this discussion, mRNA synthesis may be considered as an indirect conversion of ATP to AMP. The regulation of purine nucleotide biosynthesis and the maintenance of adenylate and guanylate levels have been discussed by Blakey and Vitols (1968), Stadtman (19701, Burton (19701, and Henderson and Paterson (1973). The pathway is subject to feedback control by purine nucleotides through their effects on phosphoribosyl pyrophosphate (PRPP)synthase (Atkinson and Fall, 1967; Switzer, 1967; Switzer and Sogin, 1973), and on the first committed enzyme of the sequence, PRPP amidotransferase (Nierlich and Magasanik, 1965; Rowe and Wyngaarden, 1968; Wyngaarden, 1972 (a review)). Under some conditions, nucleosides may be formed in the degradation of RNA or encountered in the environment. In such cases the “scavenger” reactions, whereby AMP or IMP is formed frcm the reaction of PRPP with adenine or hypoxanthine respectively, probably provide important routes for replenishment of the intracellular adenine nucleotide pool (reaction 3, Fig. 1). The reactions are catalysed by adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase, both of which are present in micro-organisms (Henderson and Paterson, .1973; Krenitsky et al., 1970; Hochstadt-Ozer and Stadtman, 197 1 ; Sin and Finch, 1972). Competition for a common, limiting supply of PRPP between the “scavenger” reactions and PRPP amidotransferase appears to be part of the mechanism whereby exogenous nucleotides, nucleosides, or free purine bases inhibit biosynthesis de nouo, since the “scavenger” enzymes have much greater affinity for PRPP than do the enzymes in the pathways by which nucleotides are

ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES

259

synthesized de n o w (Bagnara and Finch, 1973, 1974). Since the synthesis of purines is metabolically expensive in terms of ATP equivalents, replacement through the “scavenger” reactions represents a significant saving in energy and intermediates. Adenosine kinase appears to be missing from bacteria (Hoffmeyer and Neuhard, 1971; Yagil and Beacham, 1975)and so is not indicated in Fig. 1. Ribonucleic acid synthesis (sequence 4, Fig. 1) accounts for the major and most variable net utilization of adenine nucleotides and will be treated separately in a later section. The reduction of ADP or, in some bacteria, ATP (O’Donovan and Neuhard, 1970) to deoxyribonucleotides initiates the utilization of adenylates for DNA synthesis (sequence 5, Fig. 1). According to Table 1, DNA synthesis represents a minor ( 10 to 15%) but relatively constant adenylate consumption per cell, although the rate of adenylate utilization by this process probably fluctuates considerably during the cellular growth cycle. In exponentially growing cultures of E. coli, DNA synthesis can account for the entire turnover of the dATP pool (Neuhard and Thomassen, 197 1). The N- 1 and C-2 atoms of the purine ring of ATP are incorporated into the imidazole ring of histidine, and the remainder of the ATP molecule, 5’-phosphoribosyl-5-amino-4-imidazole carboxamide, can be re-utilized as an intermediate in purine nucleotide biosynthesis, entering the path of synthesis de novo two reactions prior to IMP formation. Biosynthesis of histidine and its subsequent utilization in protein formation (sequence 6,Fig. 1) accounts for 18 to 35% of adenylate consumption in E. coli and sul. typhimurium (Table 1). The total pyridine nucleotide content of a number of microorganisms is typically around 3 to 10 p o l e s per gram dry weight (Schon, 197 1; Brody, 1972; Wimpenny and Firth, 1972).Therefore, unless there is a very rapid flux through the pyridine nucleotide pool, which seems highly unlikely, biosyntheses of these compounds and of other cofactors containing adenylic acid contribute only negligibly to net adenylate utilization (sequence 7, Fig. 1). Lundquist and Olivera (1971) have estimated that in E. coli growing with a doubling time of 40 min there are 550 molecules of pyridine nucleotides synthesized per second per cell, or approximately 0.09 ,umoles/min/g dry weight (assuming a typical cellular mass at this growth rate of 59 x lo-” g; Franzen and Binkley, 196 1).This is equal to about 1.5% of the adenylates utilized for synthesis of macromolecules in E. coli at this growth rate according to Table 1.

260

A. G. CHAPMAN AND D. E. ATKINSON

2. Adenine Nucleotide Catabolism The conversion of AMP to IMP, adenosine or adenine (reactions 8 , 9 and 10 in Fig. 11, and subsequent conversion of these compounds to yield hypoxanthine, xanthine or inosine, is often referred to as adenylate catabolism since there is no obvious biosynthetic role for this process and since the end-products appear to accumulate or to be excreted from the cells. However, in some micro-organisms these degradation products might be converted back to purine nucleotides and might fulfil a transient biosynthetic need. Regulation of purine ribonucleotide catabolism, with emphasis on mammalian systems, has been reviewed by Fox (1974).Adenylate deaminase, which catalyses the conversion of AMP to IMP, appears to be missing from microorganisms (Zielke and Suelter, 1970; Schramm and Leung, 19731, in contrast to its obiquitous presence in mammalian cells. However, a non- specific enzyme has been isolated from Desuyouibrio desuljkicans and other organisms that can catalyse deamination of all of the adenine nucleotides (Yates, 1969). The enzyme catalysing removal of the phosphate group, 5’-nucleotidase, has been isolated from a large number of bacteria and yeasts (for review, see Drummond and Yamamoto, 1970). The enzyme has a very broad specificity, but has high affinity for AMP. A specific protein inhibitor isolated from E . coli is reported to prevent the action of the enzyme on AMP and ATP (Dvorak, 19681, and might be related to regulation of the rates of adenine nucleotide degradation. Adenosine monophosphate nucleosidase, which catalyses the cleavage of AMP to adenine and ribose 5-phosphate, has been observed in Azotobacter uinlandii, E . coli and Pseudomonas diminuta (Schramm and Leung, 1973; Schramm and Lazorik, 1975). The enzyme appears to be largely inactive during normal growth, but is activated under conditions of low adenylate energy charge and low phosphate concentration. Very little is known about the substrate specificity in uzuo, the physiological role, or the extent of these catabolic reactions in the intact cell. If degradation of AMP is appreciable in uiuo, the rates of adenine nucleotide turnover estimated in Table 1 would have to be revised upward. However, based on the lack of accumulation of adenylate degradation products during active growth, and by analogy with ascites tumor cells where adenylate catabolism accounts for a loss

ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES

261

of less than 3% from the adenine nucleotide pool per hour (Snyder and Henderson, 1973; Crabtree and Henderson, 197 11, it is assumed that little or no adenine nucleotide catabolism occurs normally during active growth, and that the enzymes involved remain largely inhibited in the cell except under circumstances that will be discussed later. 8 . TURNOVER OF

ATP

Since the rate of ATP formation is equal to the rate of ATP utilization at steady state, the rate of turnover of the y-phosphate group of ATP might in principle be estimated by measuring either process if the other could be abolished. For instance, initial rates of ATP utilization have been estimated by determining the decrease in ATP concentration following cessation of ATP generation caused by removing oxygen from obligate aerobes or light from photosynthetic bacteria, or after addition of metabolic inhibitors. In estimating the rate of ATP utilization by this approach, one must assume that no ATP is generated by any alternative path, and that the rate of utilization of ATP continues at an undiminished rate, at least initially, in the absence of ATP regeneration and in the face of a decrease in the concentration of ATP, and probably of an increase in that of ADP. Neither of these assumptions is inherently plausible, and the second is inconsistent with what is known about metabolic regulation. The probability that either assumption is even approximately valid decreases rapidly with time. A linear decrease in the concentration ofATP, suggesting an unaltered rate ofATP utilization lasting for several seconds, has however been reported by Knowles and Smith (1970) and MioviC and Gibson (1973). Even if constant, however, the rate need not be the same as that when energy is available, and these authors, as well as Slayman (19731, have emphasized that this approach can produce only a minimal estimate of the rate of ATP utilization. The reverse approach, where the rate of ATP production is estimated from the rate of recovery of the ATP concentration following, for instance, the removal of inhibitor, has been used less often, and estimates based on it are especially doubtful. The extent of simultaneous ATP utilization is unknown; it cannot be assumed that the rate of ATP regeneration during recovery from a state of energy depletion is representative of steady-state ATP production, and the concentrations of the adenine nucleotides are necessarily abnormal. If the total adenine nucleotide concentration has decreased, as

262

A. G. CHAPMAN AND D. E. ATKINSON

frequently happens during energy depletion, the rate of ATP regeneration might be limited by the low concentration of ADP, and thus may not reflect the rate- of phosphorylation of ADP under normal circumstances. Other approaches to evaluating the rate of ATP production or utilization include: calculation of the ATP yield to be expected from the amount of growth substrate oxidized or fermented; estimation of the rate of ATP formation from the rate of respiration of organisms growing aerobically; and estimation of the ATP cost of biosynthesis and growth. The first of these approaches, the estimate of microbial ATP yields from substrate utilization, requires a knowledge of the metabolic pathways involved and quantitation of the rates of substrate degradation and product formation. A very large number of such investigations have been conducted with the somewhat different objective of determining molar growth yields, YATP . This parameter was introduced by Bauchop and Elsden (1960) who proposed that there is a relatively constant yield of anaerobically grown bacterial cells (about 10.5 g of dry weight) per mole of ATP produced from substrate utilization (see reviews by Forrest, 1969; Stouthamer, 1969; Payne, 1970; Forrest and Walker, 197 1 ; Stouthamer and Bettenhaussen, 1973; Penning de Vries et al., 1974; and Rogers and Stewart, 1974). Combined with the growth rates of the cells, this information can be used to calculate the rate of ATP formation, as has been done by Stouthamer and Bettenhaussen (1973). These authors, and others, have also challenged the universality of a molar growth yield of 10.5. They have argued convincingly that the value of YATP is affected by specific growth rates, the maintenance requirement of the cell, and the resulting cell composition and extent of formation of storage material. Temperature changes may also affect the molar growth yield (Coultate and Sundaram, 1975). Jain (1972) reported different apparent cell yields of Saccharomyces cerevisiae during different stages of the cell cycle. Calculation of the rate of ATP production from the rate of respiration is dependent on reliable estimates of P/O ratios in bacteria. In contrast to the commonly observed and accepted P/O ratio of 3 for mammalian systems, the stoicheiometry of bacterial oxidative phosphorylation is a subject of considerable dispute (see discussions in the growth yield reviews cited above; also Gibson and Cox, 1973; Van der Beck and Stouthamer, 1973; Hempfling, 1970). Membrane fractions from micro-organisms normally exhibit very low P/O ratios, while

ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES

263

calculations of P/O ratios in vivo based on estimated ATP yields vary from 0.5 to 3. The value of the molar growth yield parameter, YATP, determined anerobically, is often used to estimate these aerobic ATP yields. But since the apparent value of YATp may vary with growth rate, this method entails uncertainties (Stouthamer and Bettenhaussen, 1973). Estimates of the ATP cost of biosynthesis and cell growth presuppose not only knowledge of cellular composition and of the complete set of biosynthetic pathways and polymerization reactions (and the cost of each of these processes in terms of number of ATP molecules or ATP equivalents utilized), but also an appraisal of the contribution to the total energy requirement of such cellular activities as transport, maintenance of concentration gradients, motility, and turnover of cell components. Very little information on the magnitude of the ATP demand by these latter activities is available. Therefore, such an approach cannot lead to a useful estimate of total ATP production and consumption. It may be interesting, however, to compare the calculated requirement for biosynthesis with estimates of total growth requirement obtained by using one of the other approaches as a basis for estimating the relative magnitudes of the ATP requirements for biosynthesis and for other cell functions. Atkinson (1971a, b), Forrest and Walker (1971) and Penning de Vries et al. (1974) have calculated the metabolic costs of several intermediates and of different polymerization reactions. Table 2 shows that all of these different approaches have been utilized by different authors in order to estimate rates of ATP hydrolysis and regeneration. In order to distinguish between methodological uncertainty and legitimate variations in rate of ATP turnover, it would have been interesting to see all of these different methods applied to one organism growing under one set of conditions. At present, interpretation of the tabulated results remains speculative. Three of the lowest sets of values (those for Azotobacter vinelandii, Neurospora crassa, and Proteus mirabilis)were observed in stationary-phase cells, and reflect the expected low rate of turnover in the absence of growth. Species difference is probably also a factor. The estimates based on ATP yields from respiration and substrate utilization give the highest estimated rates of ATP turnover in Table 2, while estimates based on the initial linear rate of decrease of the ATP concentrations following cessation of ATP regeneration give the lowest values. These low values probably

TABLE 2. Turnover of ATP hl

Species

Doubling time (mid

Approach

ATP

(pmoles/ g dry wt)

Rate of A T P Use or Synthesis

A T P turn- Reference

P

over time (sec)

(pnioles/niin/ g d1-v wrt)

Escherichia coli

44 78 1 I4

Aerobacter ( Klebsiella) aerogenes Klebsiella aerogenes Saccharomyces cerevisiae Neurospora crassa Proteus mirabilis Chromutiurn sp. Azotobacter vinelandii

62 208 300 97 416 C

E

300 C

6. I 6.5 4.5

10

8b Sb 6.4'

8.0 6.2'

6

Yield from respiration and glycolysis, assuming P / O = 3 From YATp, corrected for maintenance energy Yield from respiration,

assuming P / O = 3 Yield from respiration, assumingP/O = I A T P depletion following cyanide addition Yield from respiration, assuming P/O = 2 A T P increase on aeration A T P decrease on darkening Cost of synthesis A T P decrease on oxygen exhaustion A T P increase on aeration

"An ATP concentration of 6 pmoleslg dry weight is assumed. 'The average level of ATP in growing yeast, according to Weibel el al. (1974). 'Not growing. 'Converted from mmoles/kg of cell water by the factor: intracellular w a t d d r y wt = 2.54, from the same publication. An apparent typographical error caused a discrepancy of 60 between ATP turnover rates given in the text and in the summary. The value tabulated is believed to be correct.

1895 1740 2040 1100 800 500

0.19 0.22 0. I3

.f

0.3" 0.4" 1.2

g

o

h

%

66 7 167 67 158

0.7 2.9 6 2.4

i

z

z

j

I13

105 50'

4.6 7.5

k 1

79' 2 12

4.6 180 30

m

.Converted from nmoles/mg protein by the factor: protein dry wt. from the same publication. 'Helms et d.(1972). 'Stouthamer and Bettenhaussen ( 1973). *Harrison and Maitra (1969). 'Rogers and Stewart (1974). 'Slayman (1973). %an der Beek and Stouthamer (1973). 'MioviCand Gibson(l971). Knnwlm and Smith [ 1870).

?

n I

P

m

x3z m

0

z

= 60% of

ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES

265

reflect regulation of ATP utilization under ,these conditions. The ATPlADP concentration ratio and the adenylate energy charge (defined on page 268) are strongly stabilized in vivo, and as a consequence of regulatory interactions the decrease in ATP concentration comes to a halt well before total depletion ofATP (Knowlesand Smith, 1970; Miovid and Gibson, 197 1 ; Slayman, 1973). Most of the errors to be expected will cause the estimated turnover times to be too long. Thus, despite the uncertainties of the estimates, it seems likely that in growing bacteria the entire ATP pool is utilized and regenerated in a second or less, in contrast to the much slower turnover of the total adenine nucleotide pool shown in Table 1. 1. ATP Turnover at Dgerent Growth Rates. Maintenance Requirement

Holms et ul. (1972) found no significant variation in the rate of ATP regeneration in E . coli cultured at five different growth rates (doubling times ranging from 114 to 44 mid, whereas Stouthamer and Bettenhaussen ( 1973) reported a linear relationship between the rate of ATP regeneration (720 to 1200 pmoleslminlg dry weight) and rate of growth (doubling time 360 to 53 min) in Klebsiellu (Aerobacter) aerogenes. Such a linear relationship was also observed in cultures of Sacch. cerevisiae (doubling time 786 to 97 min; ATP regeneration 62 to 667 pmoleslminlg dry weight) and Candida parupsilosis (doubling time 1050 to 208 min; ATP regeneration 50 to 266 pmoleslminlg dry weight) by Rogers and Stewart (1974). The implication of a constant molar growth yield, of course, is that the rate of ATP regeneration is proportional to the growth rate. This assumption presumably cannot be precisely correct; the degree of error involved will depend on the magnitude of the maintenance energy requirement of the cell. Gunsalus and Shuster ( 196 1) calculated that the expected maximum yield of cells per mole of ATP is 33 g dry weight. The implication of molar growth yield of about 10 g is that two-thirds of the ATP produced is consumed by processes other than biosynthesis. Stouthamer and Bettenhaussen ( 19731, by extrapolating to conditions of no growth (dilution rate = 01, estimated a rate of ATP production equal to 645 pmoleslminlg dry weight, which they termed the maintenance coefficient. According to this value, net biosynthesis and formation of macromolecular cell constituents account for 10 to 50% of the ATP utilization, depending on the growth rate. Other recent estimates of maintenance coeficients include

?

n TABLE 3. Steady state. values of ATP concentration, adenine nucleotide pool and energy charge in exponentially growing microorganisms Organism

Conditions

Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Enterobacter aerogenes Salmonella typhimurium Klebsiella aerogenes Hydrogenomonus eutropha Rhodospirillum rubrum Clostridium kluyueri Acetobacter aceti Bacilllus subtilis

Different carbon sources Different carbon sources

2.9-5.8 2.9-8.5 -

Different carbon sources

Different carbon sources PO, above 0.2 mm H g Light and dark aerobic Different carbon sources

-u

pmoles/g dry wt ATP

10.8* 55-73' 5.0' 6.7' 2.0-5.3 6.1-6.5 5.7 3.0-4.3' 4.5-7.0 7.6 0.4

2D

Sum of adenine nucleotides 3.6-6.8 3.8- 1 0.9 10' 12.8* 4.2-9.Oc.* 9.5' 7 .3'

-

3.7-8.0 8.6-9.1 8.1 4.7-8 .O' 8.3-1 1.2 8.0

-

Energy charge value

Reference

z

D

z D

z 0

P 0.9 1-0.94 0.75-0.85 0.80 0.90 0.94 0.74 0.90 0.80-0.95 0.46-0.73 0.8 1-0.85 0.82 0.54-0.80 0.76-0.79 0.87 0.70

m Frauen and Binkley ( 196 1 ) L o w r y e t a / . (1971) Chapmanetal. (1971) E z Mathews (1972) v) Bagnara and Finch (1973;1974) Dietzler et a/. (1974~) Swedes et al. (1975) Wiebe and Bancroft (1975) Smith and Maalse (1964) Harrison and Maitra (1969) Bowien and Schlegel(1972) Schon (1969) Decker and Pfitzer (1972) Bachi and Ettlinger (1973) Hutchison and Hanson ( 1974)

3

Azotobacter vinlandii Peptococcus prevotii Myxococcus xanthus Chromatium Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Neurospora uassa

Different light intensities Steady-state condition in second half of growth phase Different carbon sources Steady-state conditions in late exponential phase

'Units are converted from pmoles/absorbance unit to jmoleslg dry weight by assuming an A.sonmof 1.0 corresponds to 0.317 mg dry weighdrnl for E . coli (Franzen and Binkley, 1961). or an Amurn of 1.0 corresponds to 0.189 mg dry weighthl for yeast (Talwalkar and Lester, 1973), or using any other dry weight information given in the publication cited. *Units are converted kom pmoles/g wet weight or estimated mM concentration to pmoleslg dry weight on the assumption that dry weight equals 25% of wet weight. For E . coli dry weight is equal to 22 to 27% of wet weight, whereas the average value for a large number of bacteria is 20% (Luria, 1960).

-

-

5.4" 0.85 2.9-3. lC 6. 1" 7.6-8.5

7.9" 1.06 4.0-4.1" 6.9" 11.5-12.3

0.84 0.82 0.86 0.8 1-0.85 0.88 0.78-0.86

Liao and Atkinson (197 1) Montague and Dawes (1974) Hanson and Dworkin (1974) Miovik and Gibson (1973) Talwalkar and Lester (1973) Weibel el al. (1974)

6.5

8.0-9.6* 8.9

0.80-0.90 0.82

Ball and Atkinson (1975) Slayman(l973)

? I

rn

zz rn

z

C

0, rn

0

2

=Units are converted from pmoleslg protein to pnoles/g dry weight rn U on the assumption that protein represents 67% of the dry weight, as C) reported for E. cob (Forchhammer and Lindahl, 1971) and Sal. lyphimurium (Maaloe and Kjeldgaard, 1966) at fast growth rates. At slower C) rn growth rates the protein content increases to 80% of dry weight. z "The AMP concentration was assumed by the authors to equal 0.2.5 of the ADP concentration.

2

30 z v)

D

z 0 -I

268

A. G. CHAPMAN AND

D. E. ATKINSON

315 pmoleslminlg dry weight (Hempfling and Mainzer, 1975), and 680 pmoleslminlg dry weight for E . coli and 40 pmoleslminlg dry weight for Klebsiellapneumoniae (Brice et al., 1974) and 80 ,umoles/min/g dry weight for Bacillus megaterium (Downsand Jones, 1974). These values were con-

verted from the reports of moles/h/g cells by assuming dry weight equal to 25% of wet weight. Rogers and Stewart (1974) estimated lower values for maintenance energy of Sacch. cerevisiae and C . parapsilosis (3.5 to 31 pmoleslminlg dry weight) and reported that the values differed under different growth conditions. The relationship between ATP turnover and bacterial growth rate would therefore depend on the magnitude of the maintenance requirement and on the growth rates. At slow growth rates where maintenance processes apparently account for a very sizable fraction of the ATP utilization, the observed variation of total ATP requirement with changes in growth rate would be less pronounced than at higher growth rates. The amount of growth substrate needed to maintain bacteria viable but with no net growth has been a subject of earlier reviews (Marr et al., 1963, Mallette, 1963; Dawes and Ribbons, 1964). C . REGULATION OF

ATP

UTILIZATION A N D REGENERATION

1. The Adenylate Energy Charge

The turnover time of ATP in growing bacteria is short (one second or less) and the reactions in which ATP is hydrolysed or regenerated are numerous, yet the ATP concentration is kept at a relatively stable value in the cell. This can only be accomplished if ATP-utilizing and ATP-regenerating processes are kept in balance through precise and fast-acting regulation of their rates. The adenylate energy charge, (ATP + 1/2 ADPMATP + ADP + AMP), is a linear measure of the amount of metabolic energy stored in the adenine nucleotide pool. The responses to variations in the energy charge of enzymes catalysing certain ATP-utilizing or ATPregenerating reactions and the roles of these responses in stabilizing the cellular energy charge have been discussed previously (Atkinson, 1968, 1969, 1971a, b, 1972). The predictions, from enzyme response patterns observed in uitro, that the energy charge in viuo must be stabilized in the range 0.8 to 0.95 during growth and normal metabolism have been verified in nearly all organisms and tissues examined

ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES

269

(see Chapman et al., 1971 for a compilation). The ATP and total adenine nucleotide concentrations observed in a number of microorganisms during active growth are shown in Table 3, along with the calculated energy charge values. With the exception of the low value for Myxococcus xanthus, the total adenine nucleotide levels observed during growth are all within an approximate three-fold range (about 4 to 12 ,umoles/g dry weight). The large majority of the energy charge values fall within the predicted range, 0.8 to 0.95. It is quite remarkable that this degree of stabilization is maintained in the cell, when it is considered that during energy imbalance the entire ATP pool could be depleted in less than a second in the absence of any form of adenylate control. D. SAMPLING OF MICROBIAL CULTURES FOR ADENINE NUCLEOTIDE DETERMINATION

Many of the reported estimates of adenine nucleotide concentrations in intact cells published before 1960 lead to calculated energy charge values below 0.8, while the great majority of those published since about 1960 correspond to energy charge values of 0.8 or above (Chapman et al., 197 1). The low values from many earlier papers, and a few more recent ones, probably result from insufficient attention being paid to speed of sampling and to avoidance of ATP degradation in the sample. Indeed, it is our present belief that values around 0.8 published in, or derived from, a number of recent papers, including one of our own (Chapman et al., 19711, are slightly low because of an unrecognized loss of ATP during handling, and that the true value of the energy charge is probably near 0.9 in most, if not all, normally metabolizing cells. A brief comment on sampling requirements when adenine nucleotides are to be determined seems appropriate. Since no feasible sampling technique is more rapid than the rate of ATP turnover, estimations of adenine nucleotide concentrations can be valid only if these concentrations remain unchanged during sampling and processing. Thus enzyme activity must be abolished before the environment of the cells in the samples comes to differ significantly from that in the bulk culture as a result, for example, of depletion of substrate or especially of oxygen in the sample. It is also necessary that the inactivating agent act rapidly, so that all enzymes are destroyed essentially

270

A. G. CHAPMAN AND D.

E. ATKINSON

simultaneously. It is evident that if several enzymes that catalyse reactions in which ATP is used remained active, even for very short periods, after enzymes catalysing ATP regeneration were inactivated, the concentrations of ATP, ADP, and AMP observed would not reflect those in the intact cell, but would correspond to an energy charge value lower than the true value. Enzymes are usually inactivated and the cells destroyed (promoting extraction of the nucleotides) by addition of perchloric acid either directly to the cell suspension, as removed from the culture, or after filtration or centrifugation. The effect of varying the time between sampling and inactivation will of course depend on the species and the conditions (aerobic or anaerobic and high or low cell density, and others). Sampling requiring more than six seconds was found to cause a decrease in the ATP concentration in Neurospora c~assa(Slayman, 1973). A 3 to 30 second delay in the sampling of E. coli does not usually affect the ATP level. The oxygen tension in samples was not substantially lowered until 40 secor,ds at the cell densities employed by Holms et al. (1972).Decreases in ATP levels after a one-second delay have been reported in Sacch. cereuisiae (Weibel et al., 1974). Filtration at 4 O C decreases ATP values in Sacch. cereuisiae more than 50% (Weibel et al., 1974). However, the same ATP level was observed following direct sampling or filtration of Myxococcus xanthus (Hanson and Dworkin, 19741, and E . coli (Lowry et al., 1971), where high energy charge values have been observed even after a 10min delay imposed by filtration (Franzen and Binkley, 1961). Freshly filtered E . coli cells exhibited a lowered energy charge following resuspension, however, and two to five minutes was required for this parameter to return to control values (Chapman et al., 197 1). Cole et al. (1967) showed that centrifugation caused a 50% decrease in the ATP level in E . coli, and washing the cells caused a 90% decrease. This has been confirmed by Lowry et al. (1971). Lundin and Thore (1975) showed that centrifugation decreased the amount of adenine nucleotides extracted by 10 to 50% in five different species of bacteria ( E . coli, Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, and Klebsiella pneumonia) and led to energy charge values in the range of 0.2 to 0.5. The ATP content of Halobacterium halobium is also sharply lowered by centrifugation (Danon and Stoeckenius, 19 74). Prolonged centrifugation of Nitrobacter winogradskyi leads to a decrease in the ATP level. The concentrations of ATP and the energy charge values were very low,

ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES

271

compared to those observed in other growing bacteria, even after attempts to inhibit ATP hydrolysis during centrifugation by addition of glutaraldehyde (Eigener, 1975; Eigener and Bock, 1975). Knowles and Smith (1970) reported that centrifugation and washing did not affect the ATP level in Azotobacter, but this treatment was followed by resuspension and aeration for two minutes, which probably allowed time for recovery of the ATP levels, as has been shown by Strange et al. ( 1963) with suspensions of Aerobacter aerogenes. Centrifugation and resuspension under strictly anaerobic conditions led to lower nucleotide levels than direct sampling of cultures of the anaerobic organism Clostridium Kluyveri (Decker and Pfitzer, 19 72). Centrifugation of Bacillus lichenformis (Leitzmann and Bernlohr, 1965) and Bacillus subtilis (Chow and Takahashi, 1972) that were in the exponential growth phase resulted in very low energy charge values, whereas Sacch. cereuisiae (Ball and Atkinson, 1975) and Polytomu uvella (Mangat, 1971) maintained high energy charge values after centrifugation. Higher energy charge values were observed in E. coli (Swedes et al., 1975) and in Bacillus brevis (Davison and Fynn, 1974) if acidprecipitable material was removed by centrifugation before neutralization of the perchloric acid extract. Failure to carry out this procedure resulted in a moderate decrease in the observed energy charge in E. coli, and a large change in B . brevis, presumably due to re-activation of ATP-destroying enzymes after neutralization. Addition of ethylenediamine tetra-acetic acid (EDTA) during extraction of several bacterial species with various acids and solvents likewise inhibited ATP hydrolysis (Lundin and Thore, 19 7 5 ). Freezing Sacch. cereuisiae before acid extraction was found to lower the observed ATP value by 60% (Weibel et al., 1974). Low energy charge values in Sacch. cereuisiae treated in a similar fashion have also been observed by Akbar et al. (1974) and Polakis and Bartley (1966). During certain phases of growth, a portion of the adenine nucleotides, largely AMP, have occasionally been shown to be excreted into the growth medium by E . coli (Chapman et al., 1971; Moses and Sharp, 19721, Chromatium sp. (MioviC and Gibson, 19731, Acetobacter aceti (Bachi and Ettlinger, 1973) and Myxococcus xanthus (Hanson and Dworkin, 1974). In such cases the adenine nucleotide determinations on cell suspensions must be corrected for nucleotides in the medium in order to estimate intracellular nucleotide concentrations.

272

A. G. CHAPMAN AND D. E. ATKINSON E. CHANGES IN ADENINE NUCLEOTIDE CONCENTRATIONS

Typical reports of changes in the concentrations of the adenine nucleotides in response -to changes in environmental conditions, or relation of these concentrations to biological capacities or functions, such as viability, glycogen synthesis, or sporulation and germination, are tabulated in this section. 1. Depletion $Energy Source

Reports of changes in energy charge or concentrations of the nucleotides on depletion of the carbon and energy source are presented in Table 4. Results obtained with E . coli in several laboratories are essentially in agreement in that all measurements of ATP concentration, or of the total adenylate pool, show a sharp drop when the substrate is exhausted. In marked contrast, the value of the energy charge changes much less drastically. Both of these changes are rapidly reversed if the energy source is restored. Although consistent for E . coli, this pattern does not hold for all micro-organisms. In two obligate aerobes, B . subtilis and Acetobacter aceti, depletion of the energy source was reported to cause a large decrease in the concentration of ATP and in the energy charge, with a much smaller change in the total adenylate pool. In baker’s yeast, growing under various conditions, the energy charge may either be maintained at relatively high values or fall sharply on substrate exhaustion, but changes in the size of the adenylate pool are small. Saccharomyces cerevisiae does not ordinarily synthesize hnctional mitochondria in the presence of glucose, and it appears that the ability to maintain a high value of the energy charge during aerobic starvation may depend on the presence of functional mitochondria (Ball and Atkinson, 1975).

2. Re-addition o f Substrate Table 5 summarizes the effects of the re-addition of substrate to starving cells of various microbial species. A rapid increase in the energy charge to normal values is seen in both yeast and E . coli. In Peptococcus prevotii, which catabolizes the ribose moiety of nucleotides during starvation, and thus decreases the adenylate pool to very low levels, the total adenylate concentration as well as the energy charge returns fairly rapidly to values typical of growing cells. Addition of an oxidizable

TABLE 4. Effects of depletion of energy source Species

Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Peptococcus preuotii Bacillus subtilis Acetobacter aceti Saccharomyces cereuisiae Saccharomyces cereuisiae Saccharomyces cereuisiae Saccharomyces cerevisiae

Conditions

Glucose depletion Glucose depletion Resuspended minus glucose Glycerol depletion Glucose depletion Resuspended minus glucose Serine depletion Glucose depletion Acetate depletion Glucose depletion Glucose depletion Glucose depletion Ethanol depletion Glucose depletion Resuspended minus glucose

(

.Chapman et d.(197 1 ) . bSwedesetal. (1975). cHolms et d.(1972). 'Lowly et 01. ( 197 1 ). 'Coleelal. (1967). JMontague and Dawes (1974). 'Hutchinson and Hanson (1974).

Energy

(aerobic) (aerobic)

ATP

+

+ (0.8 -0.7)

+

+

+

(0.9 -0.8)

(aerobic) (aerobic) (aerobic) (aerobic) (anaerobic) (anaerobic) (aerobic) (aerobic) (aerobic) (aerobic) (aerobic) (aerobic) (anaerobic)

N o change + (0.8- 0.7) N o change + (0.85- 0.7) + (0.8 0.5)

(aerobic)

+

Sum ot'adenine nucleotide concentrations

+

(0.8 -0.7)

+

+ +

-

(0.85 0.6) (0.7 - 0 . 3 ) (0.87- 0.1)

+ 45%

+

-50% -55%

90%

>75% >90%

-

Littlechange +-75% N o change N o change N o change i -15%

+ -25%

(0.9 -0.2)

rn rn

Oxygenuptaker

+

> 0

zZ

-50%

+-75%

+ +

Reference

-50%

4 -90%

+ (0.84 --c 0.78)

Comments

During 10 h During 10 h Catabolizes purines

b

rn

2

b

2

c

d

o

f g h

z

i

0

J

W

k k k

> c +

2 d z

z

C W

"0 <,

*Bachi and Ettlinger(l973, 1974). W 'Akbar et d. (1974). W . JWeibel ef ~ l(1974). 'Ball and Atkinson (1975). u) 'Talwalkar and Lester (1973). The symbol indicates a decrease in energy charge value or in the conh) centration of adenine nucleotides.

5

+

4

W

274

A. G. CHAPMAN AND D.

E. ATKINSON

TABLE 5 . Effects of re-addition of energy source o n energy charge Species Escherichia coli Peptococcus preuotii Bdellouibrio bacteriouorus Saccharomyces cereuiriae Saccharomyces cereuisiae Saccharomyces cereukiae Saccharomyces cerevisiae Saccharomyces cereuisiae

Substrate added

0bservation

Glucose Serine

Reference

Rapid t EC and ZAdN ATP t 50-1OOX in 10 min EC t (0.5 to 0.85) Peptone medium EC t (0.48 to0.72 in 15 min) ZAdNt EC t (0.1 to 0.8 in 2 min) Glucose Ethanol EC t (0.87 to 0.95 in 2 m i d Glucose EC t (0.73 to 0.9) Glucose EC t (0.5 to 0.8) EC t (0.4 to 0.8) Ethanol

a b c

d e

f g h

‘Chapmanetal. (1971). bMontague and Dawes (1974). =Gadkariand Stolp (1975). ’Talwalkar and Lester (1973). eSomlo (1970). fMaitra(1971). ‘Kopperschlager el al. (1967). ‘Maitra and Estabrook (1967). XAdN indicates sum of adenosine nucleotide concentrations; EC indicates adenylate energy charge. The symbol t indicates an increase in the parameter indicated.

substrate (peptone medium) to a saprophytic mutant of Bdellovibrio bacteriovorus causes a rise in the total adenylate level and the energy charge value. 3 . Depletion of Nitrogen

Depletion of available nitrogen prevents growth but does not interfere directly with ATP regeneration. Thus the energy charge would not be expected to change in this situation, or might reasonably rise slightly. The results presented in Table 6 show that changes were generally small, as predicted. Unusually low values for the energy charge of the growing culture were reported in one paper dealing with yeast (Rothman and Cabib, 1969) and in one dealing with E . coli (Dietzler et al., 1974~). No explanation for these values is apparent. 4. Depletion $Phosphate

The effects of depletion of available phosphate are shown in Table 7. It is evident that further study of this situation is needed. At an unspecified time after phosphate depletion, the energy charge of E . coli

had fallen to 0.67, well below the range normally observed in growing

275

ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES

TABLE 6. Effects of depletion of nitrogen source on energy charge Species Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Saccharomyces cerevisiae Saccharomyces cerevisiae

Conditions Time after depletion not given Aerobic, excess glucose Aerobic, excess glycerol Two levels of oxygen Aerobic, excess glucose Resuspended, minus nitrogen source Aerobic, excess glucose

Observation

Reference

ATP 4 65%, EC + (0.84to 0.75)

a

CAdN + slightly; EC stable ATP stable EC stable EC t (0.74to 0.87) EC stable; ZAdN t 100%

b C

d

;

EC and CAdN stable

g

‘Lowry et al. ( 197 1). bChapmanelal. (1971). CHolmsel al. (1972). ’Dieuler elal. (1974b). ‘Dieuler el af. (1974~). mothman and Cabib (1969). @Balland Atkinson ( 1975). ZAdN indicates the sum of adenosine nucleotide concentrations. EC indicates adenylate energy charge. The symbols T and 1 indicate increase or decrease in the parameters indicated.

cells, and the concentration of ATP was about 50% of normal. In B. subtilis, both the concentration of ATP and the energy charge fell to very low values, as was also observed with this species on exhaustion of the energy source. In this case (Hutchison and Hanson, 1974) the extent of the change in the concentration of ATP was less, and the TABLE 7. Effects of depletion of phosphate source on energy charge Species Escherichia coli Escherichia coli Escherichia coli Escherichia coli Bacillus subtilis

Conditions Unspecified time after phosphate depletion Media containing low or high concentrations ot’phosphate Resuspended in medium lacking phosphate

0bservation

Reference

ATP + ATP i 50%;EC i (0.84to 0.67) More NDP + NMP secreted in low phosphate medium ATP i 70% in 40 min ATP i 90%; EC + (0.7to 0.3)

“Damoglou and Dawes (1967). bLowryetal. (1971). CMosesand Sharp (1972). ‘Nazar el al. (1972). .Hutchison and Hanson (1974). EC indicates adenylate energy charge. NDP and NMP are nucleoside diphosphates and monophosphates, respectively. The symbol i indicates a decrease in the parameter indicated.

a b C

d e

276

A. G. CHAPMAN AND D. E. ATKINSON

decrease in energy charge almost totally prevented, by simultaneous tryptophan depletion. This effect may result from the prevention of protein synthesis with a consequent decrease in energy demand. If so, it appears that macromolecular synthesis in B . subtilis is less strongly inhibited when the energy charge is low than seems to be the case in E. coli. 5. Glycogen Synthesis

Table 8 summarizes correlations that have been found between the rate of glycogen synthesis and the concentrations of some possible modifiers of ADP-glucose synthetase. The observations are all from the same laboratory. Preiss and colleagues have shown that this enzyme is activated by fructose diphosphate (Preiss, 1969;and the review by Dawes and Senior, 1973). The enzyme also gives the sharpest response to variation in the adenylate energy charge that has yet been observed (Shen and Atkinson, 1970).Dietzler and colleagues have attempted to account for changes in rates of glycogen synthesis in intact E. coli cells by measuring the concentrations of ATP, glucose 6-phosphate, fructose diphosphate, and ADP, and in some cases the value of the adenylate energy charge. When different rates of glycogen synthesis were obtained by the use of various carbon sources, a plot of [FDPI2/v against [FDP12was linear. Since the exponent of substrate concentration that linearizes such reciprocal plots is algebraically identical with the slope of a Hill plot (Kuehn et al., 197 11, this result is consistent with the hypothesis that a strongly co-operative modifier effect of fructose diphosphate on the synthetase is the primary limiting factor in synthesis of glycogen under these conditons. However, when the rate of glycogen synthesis was increased by exhaustion of the nitrogen supply, no such relationship was observed. In fact, a decrease by a factor of four in the concentration of fructose diphosphate was associated with a four-fold increase in the rate of glycogen synthesis, rather than a decrease by a factor of 16, as would be expected on the basis of the second-order dependence of fructose diphosphate concentration seen in the other experiment. In the nitrogen-deficient suspension it seems likely that an increase in the adenylate energy charge may have been a factor promoting the increased rate of synthesis. A number of observations have been made on intact cells that do not lend themselves conveniently to formal tabulation; they are summarized in the following sections.

D ? l

z z

TABLE 8. Glycogen synthesis and associated changes in metabolite concentrations

rn

Species Escherichia coli Escherichia coli Escherichia coli

Conditions Depletion of nitrogen source Depletion of nitrogen source Addition of 2,4-diniaophenol Depletion of nitrogen source High or low aeration

Escherichia coli

Addition chloramphenicol Depletion of nitrogen source

Escherichia coli

Aerobic, various carbon sources

.Dietzler eta[. (1973). *Dietzler etal. (1974a, 1975~). cDietzler et al.(1974b). 'Dietzler etal. (1974~). 'Dietzleretd. (1974d, 1975a, 1975b). FDP indicates frucrose diphosphate; G6P, glucose 6-phosphate; ZAdN, sum of adenosine nucleotide concentration EC, energy charge. The symbols t and i indicate increase or decrease of the parameter indicated.

Observation Glycogen synthesis t 4.2Xi ATP t 50%; FDP 4 76% Glycogen synthesis t ; ATP t ; FDP & ;G6P stable Glycogen synthesis t ;ATP & ;FDP stable; G6P & Glycogen synthesis & alow t aeration; FDP t ; G6P t ; ZAdNr; ECstable Essentially same as low aeration Glycogen synthesis t ; EC t (.74 to .87); L4dN stable; FDPJ 77% 1OX range in glycogen synthesis; ATP stable, G6P stable, glycogen synthesis related to (FDPY

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6. Aerobic to Anaerobic Transition or vice versa

Escherichia coli: Aerobic to anaerobic transition caused the growth rate and the level of ATP to decrease; the energy charge was not determined (Cole et al., 1967). With the same organism, aerobic to anaerobic transition caused the total adenylate pool and the energy charge to decrease transiently. During the reverse transition the energy charge remained stable but the total adenylate pool decreased (Chapman et al., 197 1).

Klebsiella aerogenes: When a glucose-limited chemostat culture was changed from aerobic to anaerobic conditions, the energy charge decreased transiently and the total adenylate pool decreased by approximately 20%(Harrison and Maitra, 1969). Proteus mirabilis: Aeration of washed anaerobically grown cells caused the energy charge to increase from 0.1 to 0.74, but the total adenylate pool remained constant (Van der Beek and Stouthamer, 1973).

Saccharomyes cerevisiae : Aerobic to anaerobic transition of a culture incubated in the presence of ethanol caused the energy charge to decrease rapidly from 0.84 to 0.6, while the total adenine nucleotide pool remained constant (Maitra and Estabrook, 1967). When cells of this yeast growing exponentially were changed from aerobic to anaerobic conditions, both the energy charge and the total adenine nucleotide pool remained essentially unchanged (Betz and Moore, 1967). Change from anaerobic to aerobic conditions in a turbidostat culture of Sacch. cerevisiae caused both the total adenylate pool and the energy charge to decrease. The energy charge values of 0.5 before, and 0.4 after, the transition are extremely low in comparison to those seen elsewhere in growing yeast (Akbar et al., 1974). When cells of Sacch. cerevisiue were changed from aerobic conditions in a glucose-exhausted medium to anaerobic conditions with excess glucose, the adenylate pool and energy charge both increased. The transition from starvation to glucose excess is probably more important in this case than that from aerobic to anaerobic conditions (Chapman and Bartley, 1969). When aerobically grown cells of this yeast were incubated anaerobically for 75 minutes in the presence of glucose, the energy charge remained constant, while the adenylate pool decreased. The pool rose when oxygen became available (Ball and Atkinson, 1975). Methanobacterium: After an anaerobic to aerobic transition, produc-

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tion of methanol and the value of the energy charge fell sharply (Roberton and Wolfe, 1970). The significance of a change in the availability of oxygen will depend on the cell’s metabolic capabilities (obligate aerobe or facultative anaerobe) and, in the case of facultative organisms, on whether a fermentable substrate is available. An interruption in regeneration of ATP leads, as expected, to a decrease in the value of the energy charge, but the extent of the decrease depends on the species. The duration of the depression depends on whether a substrate is available that can be metabolized anaerobically with regeneration of ATP. For example, when an E . coli culture is made anaerobic in the presence of glucose, the energy charge is below its normal value only during a short period of adaptation, and then recovers fully. 7. Light to Dark Transitions:Photosynthetic Organism

Two papers dealing with Rhodospirillum rubrum (Schon, 1969) and Rhodopseudomonm spheroides (Fanica-Gaignier et al., 19 7 1) report concentrations of adenine nucleotides under a variety of conditions and suggest that bacteriochlorophyll synthesis is regulated in part by ATP inhibition. G&t (1972) reviews this work and results from his laboratory. Chromatiurn D : The total adenylate pool remained constant and the energy charge decreased when the light was turned off. The charge recovered when the cells were again illuminated. Different levels of illumination caused different rates of growth and different levels of bacteriochlorophyll, but the energy charge values were essentially identical (MioviCand Gibson, 1971). A decrease in the level of illumination caused a transient decrease in the energy charge and an increase in the rate of bacteriochlorophyll synthesis (MioviE and Gibson, 1973). A similar rapid reversible decrease in the concentration of ATP has been observed followinga transition from light to dark in anaerobically incubated Halobacterium halobium (Danon and Stoeckenius, 19 74). 8. Sporulation and Germination

In studies of sporulation in Bacillus lichenqormis (Leitzmannand Bernlohr, 1965) and B . subtilis (Chow and Takahashi, 1972) the cells were centrifuged and washed before disruption for analysis. The very low values of energy charge that were observed probably do not reflect the

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A. G. CHAPMAN AND D. E. ATKINSON

values in cells before this treatment, so that comparisons of values at different stages of sporulation are of doubtful significance. Bacillus megaterium: During germination of heat-shocked spores, the value of the energy charge increased rapidly from 0.1 to about 0.85. The total adenylate concentration also increased (Setlow and Kornberg, 1970a, b). Bacillus subtilis: The value of the energy charge decreased rapidly on cessation of growth. The energy charge was low during the presporulation phase, and increased to about 0.6 on the appearance of spores (Hutchison and Hanson, 1974). Myxococcus xanthus: The sum of the adenine nucleotide concentrations remained constant during sporulation and germination. The energy charge also remained essentially constant, at a value of about 0.84 in the spores (Hanson and Dworkin, 1974). N o consistent pattern is evident. The very low value of energy charge observed in spores of B. megaterium is not seen in B. subtilis or M . xanthus. Changes in nucleotide levels or in the value of the energy charge must be seen in the context of changes in many other metabolite levels, and as part of a programmed pattern of differentiation, not yet understood. Further work on metabolic regulation during sporulation and germination may be expected, and should be valuable for its own sake, and more broadly in terms of biological differentiation. 9. Viability Klebsiella aerogenes: At a high rate of growth in an aerobic glucoselimited chemostat, the value of the energy charge was 0.90 and the viability 100%.At a low growth rate, the value of the energy charge was 0.84 and about 70% of the cells were viable (Harrison and Maitra, 1969). Escherichia coli: During stationary phase, aerobic cells remained fully viable for 70 hours during which time the energy charge fell from 0.8 to 0.5. The viability decreased as the energy charge fell below 0.5. Cells, when resuspended in the presence or absence of glucose, remained fully viable when the energy charge value was greater than 0.6. The viability decreased as the energy charge fell below 0.6 (Chapman et al., 1971). Peptococcus prevotii: During anaerobic starvation, the value of the energy charge was about 0.5 and the total adenine nucleotide pool

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level was about 1% of the initial value when the viability began to decrease. This organism is able to utilize purine nucleotides as an energy source (Montague and Dawes, 1974). Saccharomyces cerevisiae: Full viability was observed during stationary phase, or in resuspended cultures lacking an energy source, until the energy charge value fell to 0.1-0.3 (Ball and Atkinson, 1975). Polytoma uvella: The value of the energy charge in stationary-phase cells gradually decreased from 0.85 to 0.4. The level of the total adenine nucleotide pool decreased by 75%, and the viability remained constant for 80 h (Mangat, 197 1). The relation between the value of the intracellular energy charge and viability (defined as the ability to form a colony on solid medium) has not been extensively studied, and reported results are too few to support any generalizations as to the relationship between viability and energy charge. An early paper from this laboratory (Chapman et al., 1971) reported 100% viability in E . coli as long as the energy charge remained above 0.5 or 0.6 (typically from one to several days, depending on conditions) with a decrease in viability commencing as the charge declined below that range. Further work has shown that modifications in the conditions of aging and in the composition of the plating medium can lead to rather high viability at somewhat lower energy charge values. In both B . subtilis and yeast, shown in Table 1 not to maintain high values of energy charge duration starvation, cells remain viable after the energy charge has reached quite low levels. 10. Addition oflnhibitors or Phosphoryl Traps

Several papers have reported effects of inhibitors (usually respiration inhibitors) or of deoxyglucose, which serves as a substrate for hexokinase and thus traps phosphoryl groups in a virtually unmetabolizable compound. Escherichia coli : The addition of chloramphenicol to nitrogen-starved cells caused a decrease in the rate of glycogen accumulation and in the level of the adenylate pool, while the energy charge value remained constant (Dietzler et al., 1974b). Methanobacterium: Following the addition of chloroform, pentachlorophenol, or carbonylcyano-m-chlorophenylhydrazone to growing cells, the energy charge decreased to a value below 0.4, while

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the adenylate pool level remained nearly stable (Roberton and Wolfe, 1970).

Chromatium: Addition- of arsenate caused the growth rate to decrease by about 50% while the energy charge value and concentrations of the adenine nucleotides remained stable (MioviCand Gibson, 1973). Saccharomyces cerevisiae: When starved cells were incubated aerobically in the presence or absence of p-nitrophenol, it was observed that the energy charge was lower in poisoned cells than in controls, and that this value rose to 0.85 on addition ofglucose (Kopperschlager et al., 1967). When deoxyglucose was added to a culture of aerobic cells of this yeast in the presence or absence of ethanol or to starving anaerobic cells, the value of the energy charge decreased strongly (Feldheim et al., 1966). Also with this yeast, when oxygen was depleted, the decrease in energy charge was greater in the presence of deoxyglucose than in its absence (Maitra and Estabrook, 1967). Saccharomyces cerevisiae respiratory mutant : When deoxyglucose was added to cultures growing on glucose, the energy charge decreased transiently (Talwalkar and Lester, 1973). Neurospora crassa: When cyanide, azide, or deoxyglucose was added to a mycelial suspension in nitrogen-free buffer, the adenylate pool level decreased 20 to 50% and the energy charge fell to 0.4. The charge recovered to 0.8 on removal of the inhibitor (Slayman, 1973). 111. Concentration of ATP, Total Adenine Nucleotide Concentration,

and Energy Charge in Relation to Cellular Activities A. RELATION BETWEEN

ATP

CONCENTRATION AND GROWTH

RATE

A number of studies have been carried out to establish the relationship between the growth rate of a micro-organism and the corresponding cellular concentrations of ATP, total adenine nucleotides, or the energy charge parameter. Two conflicting types of results have been reported: those in which the ATP concentration and growth rate appear to be independent, and those in which ATP concentration and the rate of growth varied in the same direction (for a summary see Hobson and Summers, 1972). This is partially an unnecessary discrepancy arising from the choice of units used to express the ATP concentration. Since mass per cell is an exponential function of the growth rate in E . coli (Franzen and Binkley, 1961), Sal. typhimurium

ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES

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(Schaechter et ul., 1958) and B. lichenformis (Van Dijk-Salkinoja and Planta, 197 1) it follows that, even when there is no change in ATP per unit of cell mass at different growth rates, the same results, when expressed as ATP per cell, will show a logarithmic dependency on growth rate. Cellular concentrations that are expressed as amounts per gram of protein are similarly, although to a much smaller extent, affected by the reported variation in cellular protein content in E. coli

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A. G. CHAPMAN AND D. E. ATKINSON

(Silver and Mateles, 1969) ranging from 67% of dry weight at rapid growth to 80% of dry weight in cells growing slowly (Forchhammer and Lindahl, 197 1). Salmonella typhimurium and Acinetobacter calcoaceticus (Abbott et al., 1974) are similar to E . coli in this respect (Maaloe and Kjeldgaard, 1966), whereas a constant protein content of 55% was reported for B . lichenifrmis over a three-fold variation in growth rate (Van Dijk-Salkinoja and Planta, 197 1). The relationship between growth rate and ATP concentrations in E. coli as reported by different groups of investigators is presented in Fig. 2a. Variations in growth rates were obtained by varying the carbon source or the availability of oxygen. The corresponding relationships between the growth rates and the concentrations of the total adenine nucleotides, expressed as pmoles/g dry weight, and the energy charge are presented in Fig 2b and 2c. These figures reveal that although there is some variation in the ATP concentrations when cells are grown on Doubling time ( h 1

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FIG. 3. Contents of total adenine nucleotide concentrations (01, ATP ( A ) , ADP (A)and AMP (01 and energy charge ( 0 )values in an adenine-limited steady state culture of an adenine-requiring mutant of Ejcherichia coli (PC 0294). The cells were grown aerobically in a standard growth medium containing supplements and 0.5% glucose. The influx medium contained 7.5 pg adenine/ml. Each point on the graph represents the mean of three samples taken at hourly intervals after the cell density had reached steady state.

ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES

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different substrates, there is no simple direct relationship between the rate of growth and the concentrations of ATP or total adenine nucleotides. Within each of the sets of experiments shown in Figure 2a there appears to be a trend towards lower ATP conceno-ations at very low growth rates, but at higher growth rates the reported variations in ATP concentrations may be caused largely by factors such as the composition of the growth medium or whether growth was aerobic or anaerobic. In the two cases in which they were determined, energy charge values were shown to be constant, within experimental error, over the entire range of growth rates. Similarly, a decrease in ATP concentration was observed only at very low growth rates when Klebsiella aerogenes (Harrison and Maitra, 1969) or Selenomonas ruminatium (Hobson and Summers, 1972) was grown in glucose-limited chemostats. The ATP concentration in Klebsiella was constant over a five-fold variation in growth rate, but decreased to 50%at very low growth rates, whereas the energy charge value remained within the range 0.85 to 0.9 over the entire 15-fold range of growth rates. In the special case of adenine limitation, the energy charge in an adenine-requiring mutant of E. coli has been shown to remain at 0.89, within experimental error, over a five-fold range of growth rates in adenine-limited chemostat cultures, while the concentrations of ATP and of total adenine nucleotides decreased to 30%of normal (Fig. 3; Swedes et al., 1975). B. V A R I A T I O N S I N A D E N I N E N U C L E O T I D E LEVELS D U R I N G

GROWTH

In the majority of papers cited in Table 1 , as well as in other studies where only the concentration of ATP has been reported, the ATP concentration per unit mass appears to be essentially constant during the exponential growth phase. However, there are a number of studies in which this constancy was not observed. In some cases the variation apparently resulted from peculiar experimental conditions, such as utilization of AMP present in rich growth medium, or diluting out large stationary-phase inocula. Large variations in ATP levels, and in some cases also in total adenine nucleotide levels and energy charge values, have been reported to occur in E. coli during the exponential growth phase (Cole, et al., 1967) in Sacch. cereuisiae (Weibel et al., 1974; Bailey and Parks, 1972), Streptococcus faecaelis (Forrest, 1965) and in Neurospora crassa (Slayman, 1973). Changes in growth conditions

286

A. G. CHAPMAN AND D. E. ATKINSON

(mixed substrate utilization or change in effective aeration of culture, for example) may be responsible for most of these observed variations. I t has also been reported that there is a cyclic variation in the ATP concentration of synchronously dividing E . coli ( H y k and Clark, 1971). The reported gradual 40 or 50% increase in ATP prior to cell division is less dramatic than the cyclic variation in the deoxyribonucleoside triphosphates, probably a consequence of the continuous synthesis of RNA as contrasted with the stepwise synthesis of DNA during the cell cycle (Khachatourians and Huzyk, 1974). The energy charge value and the concentration of AMP have been reported to oscillate with a 22 hour period in the growing front of a Neurospora crassa surface culture (Delmer and Brody, 1975). The energy charge varied between 0.65 and 0.93. C . ADENINE NUCLEOTIDES IN MUTANT STRAINS ARRESTED IN GROWTH

Several studies have been conducted on mutant strains that not only are unable to grow on the carbon sources normally utilized by the wild-type strain, but are inhibited if these compounds are added to growing cultures. Attempts have been made to correlate cessation of growth with changes in adenine nucleotides or other metabolites Hydrogenomonas eutropha can utilize gluconate via the EntnerDoudoroff path (Bowien and Schlegel, 1972). Gluconate does not support the growth of a mutant lacking 2-keto-3-deoxy-6-phosphogluconate aldolase. Growth of the mutant in an acetate medium is suppressed completely by addition of gluconate. High levels of ketodeoxyphosphogluconate (which cannot be metabolized further by the mutant) accumulate under these conditions, and there is a rapid decrease in the energy charge to a new steady-state value of 0.69. The concentration of the total adenine nucleotides decreases about 70% during the continuous abortive phosphorylation of gluconate. The low energy charge value was assumed to be the reason for the cessation of growth, since accumulation of ketodeoxyphosphogluconate without a decrease in the energy charge value had little effect on growth under other conditions. An “oxidative phosphorylation” (op,) mutant of Sacch. cerevisiae is unable to grow on an oxidizable, non-fermentable carbon source despite the presence of a normally functioning respiratory apparatus (Somlo, 1970, 1971; Kolarov et al., 1972). This mutant, when incubated

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287

with ethanol, was reported to contain a normal steady state concentration of ATP, but an elevated AMP concentration (20%of total adenine nucleotides, as compared with 5% of total in the wild type), and consequently a lowered energy charge value. During growth on glucose, the energy charge in the mutant was approximately normal (0.80),while during ethanol incubation the energy charge value was decreased to 0.67 to 0.75, which evidently is too low to support growth despite there being a normal level of ATP. In later work with the same mutant, Kolarov et al. (1972) observed values of energy charge in wild-type and mutant cells metabolizing ethanol that were similar to those reported by Somlo, but found a marked decrease in the ATP concentration and the total adenylate pool, with a relative but not absolute increase in the concentration of AMP. These workers attributed the discrepancies to differences in extraction procedures. In a mutant strain of E. coli possessing a temperature-sensitive adenylate kinase, a shift to the non-permissive temperature resulted in sharp decreases in the rates of synthesis of protein, RNA, and phospholipid. The level of ATP declined to about 10% of normal, and the energy charge fell to 0.24, within 20 min (Glaser et al., 1975). Acetobacter aceti can utilize ethanol but not glucose as a carbon source during aerobic growth. Addition of glucose to a glucose-sensitive mutant arrests growth and prevents utilization of acetate accumulated during ethanol utilization (Bachi and Ettlinger, 1973, 1974). However, the energy charge value remained high (0.87 ) after glucose addition both in the wild type and the mutant. Only a transient 20% decrease in adenine nucleotide concentration in the mutant was associated with the cessation of growth. In this mutant, therefore, changes in factors other than the adenine nucleotide system appear to be responsible for the arrest of growth. D . C O R R E L A T I O N BETWEEN K I N E T I C S I N V I T R O A N D

OBSERVATIONS I N V I V O

One of the challenging questions in the field of metabolic regulation is the extent to which the inhibition-activation patterns observed when studying the kinetics of isolated enzymes reflect precisely those occurring in intact cells. Among the different approaches used is the study of enzyme activities in cells that have been rendered permeable. For instance, Reeves and Sols (1973) have reported that the regulatory properties of phosphofructokinase measured in E. coli made per-

288

A. G. CHAPMAN AND D.

E. ATKINSON

meable by treatment with toluene are very similar to those reported for the isolated enzyme. Another approach is to determine the concentrations of substrates and known effectors of an enzyme in uiuo under varied conditions and compare them with the relative rate of the reaction catalysed by the enzyme, when this can be estimated from product formation, or indirectly from the formation of the end product of a sequence in which the enzyme in question is believed to catalyse the rate-limiting step. This approach has been used, for example, in studies o f (i) the correlation between substrate and modifier concentrations and the activity of glucose 6-phosphate dehydrogenase in Hydrogenomonas eutropha under conditions of rapid or slow synthesis of poly-P-hydroxybutyrate (Bowien et al., 1974); (ii) AMP nucleosidase activity in Azotobacter uinelandii under conditions of active degradation of AMP (Schramm and Lazorik, 1975); and (iii) of pyruvate kinase activity in yeast during glycolysis or gluconeogenesis (Barwell and Hess, 1971). Some of the problems involved in using the concentrations of intermediates to identify regulatory reactions have been discussed in reviews by Sols and Marco (19701, Rolleston (1972)and Stebbing (19741,and with reference to Sacch. cereuisiae specifically by Gancedo and Gancedo (1973). These include the unknown extent of compartmentation, complexing of metabolites, enzyme-enzyme or enzyme-membrane interactions, and variations in intracellular pH value or in free inorganic ion concentrations. Reactions competing for substrates, cofactors or effectors have to be taken into account. Initial levels of metabolites (and probably rates of enzyme-catalysed reactions) following a transition are likely to be different than the new steady-state levels. Nevertheless, several attempts have been made to translate kinetic control patterns observed in uitro to metabolic situations occurring in uiuo. 1. Glycogen Synthesis

Attempts to correlate changes in metabolite concentrations accompanying changes in the rate of glycogen accumulation in E . coli with the regulatory properties of ADP-glucose synthase observed in uitro were mentioned earlier (Table 5, Dietzler et al., 1973, 1974a, b, c, d, 1975a, b,. c). Under one set of conditions, glycogen synthesis appeared to be a second-order function of the intracellular concentration of fructose diphosphate. However, under other conditions a four-fold decrease in

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289

the concentration of fructose diphosphate was associated with a fourfold increase in glycogen synthesis, rather than the decrease by a factor of about 16 that might have been expected on a simplistic basis. This approximately 64-fold discrepancy between observation and “prediction” merely illustrates that metabolic processes will be controlled by different factors under different conditions, and that even a close correlation between the concentration of a possible modifier compound, or some other parameter, and the rate of a metabolic process does not mean that the same relationship is to be expected when conditions are different. In evolving to meet a wide variety of conditions, cells have necessarily developed complex patterns of regulatory interactions, and it is an essential feature of a viable metabolic system that each of several inputs may exert the primary regulatory influence under appropriate conditions. In many cases, the rates of component reactions will be affected simultaneouslyby several control parameters. E. RELATION BETWEEN ENERGY CHARGE A N D T O T A L ADENINE NUCLEOTIDE CONCENTRATION

A sudden fall in the energy charge value is often accompanied by a decrease in the sum of the total adenine nucleotides (see Tables 1 (p. 257), 2 (p. 264) and 4 (p. 273) for examples). I t has been suggested that, at least in some cases, this decrease results from the operation of control systems that have evolved to limit the extent of the fall in energy charge resulting from accumulation of AMP when there is a sudden increase in the energy demand on the adenine nucleotide pool (Chapman and Atkinson, 1973). Activation by low energy charge of any of the enzymes catalysing degradation of AMP would tend to stabilize the energy charge at the expense of the total adenine nucleotide concentration. Removal of AMP would obviously increase the value of the energy charge, since the,concentration of AMP appears only in the denominator of the expression for energy charge; in view of the presence of adenylate kinase in all cells, catalysing the reaction 2 ADP +ATP + AMP, it is obvious that removal ofAMP will cause conversion of ADP to ATP and thus tend also to increase the ATP/ADP ratio. Adenosine monophosphate nucleosidase, which catalyses cleavage of AMP to adenine and ribose 5-phosphate, has been demonstrated in Azotobacter vinlandii, E. coli and Pseudomonas sp. (Schramm and Leung, 1973; Schramm and Lazorik, 1975). Kinetic studies in vitro have shown

290

A. G. CHAPMAN AND D. E. ATKINSON

that the rate of the enzyme reaction increases sharply as the energy charge decreases from its normal value of approximately 0.9. This is a consequence primarily of an increase in the concentration of the substrate for the reaction, AMP (the reaction is of kinetic order greater than one with respect to AMP). During the aerobic-anaerobic transition of a culture of Azotobacter vinlundii, there is a sharp deccease in ATP concentration, an increase in AMP concentration, and also an increase in the concentration of the adenine breakdown product hypoxanthine. The path of degradation of [I4C]ATPin cell-free extracts, and its inhibiton by antibodies to AMP nucleosidase, suggests that this enzyme is probably responsible for the first step in the formation of hypoxanthine. If ffee inorganic phosphate accumulates, the enzyme is apparently very rapidly inhibited before the energy charge value has a chance to recover to control values. Hypoxanthine accumulation does not appear to cause depletion of the total adenine nucleotide level, which implies a continuous flux into the adenylate pool during this process. Both of these observations contrast with the otherwise very similar protection against transient sharp drops of energy charge that apparently results in mammalian liver from the action of AMP deaminase (Chapman and Atkinson, 1973). The deaminase is less sensitive to phosphate, and in liver or ascites cells a marked decrease in the total adenine nucleotide level accompanies a sudden increase in phosphorylation demand, and apparently results from the action of adenylate deaminase. F. P H A G E I N F E C T I O N

When E. coli, growing at a doubling time of 50 minutes, is infected with T, phage, there is a three- to five-fold increase in the rate of DNA synthesis within five minutes and a 60 to 80%reduction in the rate of RNA synthesis. About 60 min after infection there is a release of about 250 phage equivalents per infected cell (Mathews, 1968, 1972). Uninfected E. coli growing at a doubling time of 60 min contains 4.4 x lo6 DNA base pairs per cell (Manor et ul., 1969). The DNA content in 250 phage equivalents, at 4 x lo5 nucleotides each, would correspond to about 5 x lo7 base pairs of phage DNA. Bremer and Yuan (1968)have reported 1.8 x lo7 base pairs of phage DNA per infected cell. Therefore, there is a 4- to 10-fold increase in the amount of DNA per cell. Normally, at a similar doubling time, DNA synthesis represents

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about 7% of adenylate utilization in E. coli (Table 1, p. 257). Following a four- to ten-fold increase in DNA synthesis, 30 to 70%of the adenylates would be utilized for DNA synthesis, assuming that the rate of production remained unchanged. During the same period the RNA synthesis is decreased by about 60 to 80%. Since the rate of protein synthesis is also decreased by about 50% during infection (Mathews, 1968) it follows that the increased rate of DNA synthesis can be accomplished without any major change in the flux through the adenine nucleotide pool, but rather by a rechannelling of the majority of the nucleotides formed into deoxyribonucleotides and subsequent DNA synthesis. During this major shift in cellular biosynthesis there is little, if any, change in the energy charge value or in the concentrations of the deoxyribonucleoside triphosphates, and a gradual 25% decrease in the levels of all the ribonucleoside triphosphates in phage-infected wildtype E. coli. G. OTHER NUCLEOTIDES

Much more is known about the adenine nucleotide concentrations and ratios in micro-organisms than about the other nucleotides. The impressive amount of information on the adenine nucleotide levels in cells growing under different environmental conditions has been generated partially because of special interest in the adenylates (since they participate in all metabolic sequences),but also partly because the levels of these compounds in viuo are much more easily measured than are those of the other nucleotides. Not only are the other nucleotides present in the cell in lower concentrations than the adenine nucleotides, but the assay procedures for them are less sensitive and convenient than for the adenylates. The triphosphate is the major component of each nucleotide pool under normal circumstances (Frauen and Binkley, 1961; Salser et ul., 1968; Nazar and Wong, 1972; Bagnara and Finch, 1973). Triphosphates have been successfully determined by the use of chromatographic methods, but much less information has been obtained on the levels of the monophosphates and diphosphates. The adenine nucleotides are the subject of this review, but it should be noted that conditions causing a change in the concentration of ATP appear generally to cause a similar change in the levels of the other nucleoside triphosphates (Mangat, 1971; Fiil et al., 1972; Irr, 1972; Mathews, 1972; Nazar and Wong, 1972; Nazar et al., 1972; Bagnara

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and Finch, 1973; Beck et al., 19731, probably as a consequence of the nucleoside diphosphate kinase reaction which catalyses the transfer of a y-phosphate group between any of the nucleoside diphosphates and triphosphates (Parks and Agarwal, 1973). I t may be assumed that the triphosphate-diphosphate ratios in the other pools tend to follow the ATP/ADP ratio. Under specific conditions, as when nucleosides or free purine or pyrimidine bases are added to growing cultures or to a cell suspension, or when cells defective in nucleotide interconversion activity are starved for precursors, the resulting variations in nucleoside triphosphate concentrations need not be in parallel (Thomas et al., 1970; Bagnara and Finch, 1974; Erlich et al., 1975). A shift to a nonpermissive temperature in a mutant of E . coli with a temperaturesensitive adenylate kinase results in a sharp decrease in the concentrations of ATP and GTP, while the concentrations of the pyrimidine triphosphates remain relative constant (Glaser et al., 1975). The guanylate nucleotides have been shown to play a unique role in the regulation of RNA and protein synthesis. Guanosine triphosphate is required as a cofactor in both the initiation and elongation steps in protein synthesis, and the guanine nucleotide, guanosine 5'diphosphate 3' diphosphate (ppGpp), which was first identified by Cashel and Gallant ( 19691, inhibits ribosomal RNA synthesis (Lazzarini et al., 197 1; Winslow, 197 1; Harshman and Yamazaki, 197 1; Fiil et al., 1972). The suggested mechanism is based on the correlation of ppGpp levels and rates of RNA accumulation in rel' and rel- strains of E . coli and on effects in uitro of ppGpp. Results of recent nutritional shiftdown experiments (decreased uptake of glucose; Hansen et al., 1975) and shift-up experiments (enrichment of the growth medium by amino acid or glucose addition; Friesen et al., 1975) support the involvement of ppGpp in modulation of ribosomal RNA biosynthesis, although the level of this nucleotide cannot, of course, be the sole determinant of the rate of RNA synthesis. It has also been shown that ppGpp inhibits synthesis de nova of purine nucleotides (Gallant et al., 197 1) or uptake of purine by E. coli membranes (Hochstadt-Ozer and Cashel, 19721, which would agree well with a decreased flux through the adenine nucleotide pool (and the pools of the other nucleotides) under conditions of inhibited RNA accumulation (Table 1, p. 257). Inhibition of protein synthesis has also been attributed to elevated ppGpp levels in E . coli (Laffler and Gallant, 1974).

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RNA

293

SYNTHESIS

The observation that the RNA content is proportional to the growth rate of a bacterial cell, and that the initial response to a change in the nutritional composition of the medium is frequently a changed rate of RNA accumulation (see Table 1 (p. 257), and reviews by Neidhardt and Fraenkel, 196 1; Neidhardt, 1964; Maaloe and Kjeldgaard, 1966; Edlin and Broda, 1968; Ryan and Borek, 1971; Koch, 1971; Nierlich, 1974) suggests than an understanding of the control of RNA synthesis will be essential to understanding the regulation of bacterial growth. Nucleoside triphosphates are substrates for RNA biosynthesis, and substrate availability has been discussed as a possible mechanism for regulating rate of RNA synthesis (Neidhardt and Fraenkel, 1961; Cashel and Gallant, 1968; Edlin and Stent, 1969; Gallant and Harada, 1969; Winslow, 1971; Nazar and Wong, 1972; Nazar et d., 1972; Beck et al., 1973; Erlich et al., 1975; Kudrna and Edlin, 1975). However, it has been shown that the cellular steady-state concentration of ATP (and in some cases also of the other nucleoside triphosphates) in E . coli remains essentially constant over a ten-fold range of growth rates with the corresponding wide range of rates of RNA accumulation (Fig. 2, p. 283). During different types of nutritional shift-down experiments, inhibition of RNA accumulation could not be accounted for by depletion of nucleoside triphosphates (Nazar and Wong, 1972). Only during starvation for inorganic phosphate in E . coli is there a relationship between the rate of net RNA synthesis and nucleoside triphosphate levels that might suggest limitation by substrate concentration under these conditions (Nazar et al., 1972). Indeed, during nutritional shift-up experiments, the rate of RNA accumulation has been shown to increase rapidly despite a large transient decrease in the concentrations of the nucleoside triphosphates (Kjeldgaard, 196 7 ; Irr, 19 7 2 ; Beck et al., 1973).

The results just summarized suggest that the concentrations of the free nucleoside triphosphates are not important determinants of the rate of RNA synthesis under normal conditions. This conclusion is consistent with present views on regulation of the biosyntheses of the monomer building blocks from which biological macromolecules are constructed. Since the pioneering papers of Umbarger (1956) and of Yates and Pardee (19561, the role of product negative feedback in

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regulation of the syntheses of amino acids and nucleotides has come to be recognized as general. Such control systems are suited to stabilization of the concentrations of these building blocks, and thus they will cause an increase in the rates of their syntheses when the pools tend to be depleted by an increased rate of utilization in macromolecular synthesis. By the same token, these feedback control systems are so inappropriate for control of macromolecular synthesis by precursor concentrations that their existence argues strongly against such control. The rate of the RNA polymerase reaction has been shown to be maximal at nucleotide concentrations well below those normally observed in the cell (Anthony et al., 1969; Rhodes and Chamberlin, 1974). Likewise, the chain elongation rates for messenger RNA have been observed to remain fairly constant under different conditions (see summary in Dennis and Bremer, 19731, whereas the rate of ribosomal RNA synthesis appears to increase with increasing growth rate (Dennis and Bremer, 1974; Bremer and Dennis, 1975). The afinity of the RNA initiation process for nucleoside triphosphates is about one-tenth that of the elongation process, and may affect the rate of RNA synthesis at very low nucleotide concentrations (see discussion in Nierlich, 1974). Variations in this afinity would be an attractive means of regulating the rate of general RNA synthesis, but very specific individual controls are of course needed, and have been the subject of extensive study. Since the differential rate of RNA synthesis in bacteria increases with increased growth rate, and since RNA synthesis accounts for the majority of net adenylate utilization (see Table 1 , p. 257), it follows that the rate of purine and pyrimidine nucleotide biosynthesis must increase with the increased RNA synthesis. As noted earlier, purine nucleotide synthesis might be inhibited by ppGpp accumulating during inhibition of RNA synthesis. Conversely, Beck et al., (1973) have shown that the activities of two enzymes in the pathway for pyrimidine biosynthesis (aspartate carbamoyl transferase and dihydro-orotate dehydrogenase)increase rapidly several-fold following nutritional shift up of an E . coli culture. Extrapolating from Table 1 , (p. 257), it can be estimated that during the shift-up experiment of Beck et al. (1973) the net adenylate incorporation into stable RNA increases about 30 to 35% froin a pre-shift rate of approximately 2.5 pmoles/min/g dry weight to a post-shift rate of 3.3 pmoleslminlg dry weight. The 60% decrease observed in the ATP concentration during the first six minutes after the nutritional tran-

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sition can be accounted for by this increased rate of RNA synthesis along with the reported five minute lag in the change of rate of nucleotide synthesis de novo. Of the total RNA content of E . 6012, about 3% is messenger RNA and 97% stable RNA, of which 80 to 88%, depending on the growth rate, is ribosomal RNA (Forchhammer and Lindahl, 197 1; see summaries in Maalse and Kjeldgaard, 1966; Nierlich, 1974). However, due to the rapid turnover of mRNA, stable RNA synthesis accounts for only 30 to 60% of total RNA synthesis at any given time. Ribosomal RNA synthesis as a fraction of total RNA synthesis increases markedly with increasing growth rate (Pato and von Meyenburg, 1970; Bremer et al., 1973). The rapid increase in RNA accumulation following nutritional shift up is accomplished with only a minor change in rate of RNA synthesis by shifting the RNA production almost entirely to ribosomal RNA synthesis (see discussion in Nierlich, 19741, along with a gradual, modest increase in the chain elongation rate for rRNA (Dennis and Bremer, 1974). It has been suggested that ppGpp influences this selectivity by inhibiting a protein factor that stimulates ribosomal RNA gene transcription (see Nierlich, 1974). The formation of ppGpp from ATP and GDP (Haseltine et al., 1973; Pedersen et al., 1973; Sy, 1974; Block and Haseltine, 1975) could be sensitive both to the energy state of the cell (although there is no apparent correlation between rate of ppGpp formation and variations in physiological substrate concentrations) and to precursor availability for protein synthesis through the requirement of uncharged transfer RNA for its formation. I. PROTEIN SYNTHESIS

Of the many biological activities of the cell that can be tested as a function of changed energy state, the ability to synthesize proteins (i.e. the incorporation of radio-active amino acids into polymers or the induction of a specific enzyme activity)is among the most commonly employed. In a culture of an adenine-requiring mutant of E . coli, depletion of both glucose and adenine caused a 90% decrease in the rate of [14Clleucineincorporation (Swedes et al., 1975). The energy charge value under these conditions was maintained at 0.8 and the adenine nucleotide level was close to normal (Fig 4). Following addition of glucose with the cells still starved for adenine, the energy charge rose

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Time (min)

FIG. 4 . Protein synthesis (v),contents ofATP ( v )and of total adenine nucleotides (O), and energy charge (0)in an adenine-requiring mutant of Eschel-ichiu coli (PC 0294) resuspended in medium lacking adenine and glucose. Exponentially growing cells (doubling time 2.7 h at 27OC) were harvested by centrifugation (4OC)and resuspended at 27OC in a medium containing standard supplement concentrations, except that leucine concentration was 5 mg/litre and neither adenine nor glucose was present. Glucose was added as indicated o n the figure. The rate of protein synthesis was calculated as the number of nanomoles of [14Clleucineincorporated per mg of protein during a 1 min pulse. From Swedes et uf. ( 1975).

rapidly to about 0.9 and there was a simultaneous ten-fold increase in the rate of protein synthesis. During and after this initial period, there was a continuous decline in the concentration of ATP and of total adenine nucleotides, presumably because of resumption of RNA synthesis. The energy charge was maintained at 0.9 until the total adenine nucleotide concentration dropped below 30%of the control value, after which there was also a gradual decrease in the energy charge. At the same time the rate of protein synthesis also began to decline slowly. This experiment demonstrates that the rate of P4Clleucine incorporation is not determined by the concentration ofATP over a considerable range of variation below the normal physiological level. In contrast, the rate of protein synthesis declines approximately in parallel with the decrease in energy charge. Many factors are potentially limiting for a process as complex as protein synthesis. Before the addition of glucose, the levels of all intermediates must have been low, and synthesis of amino acids was limited by the inavailability of precursors as well as of an energy so-urce.After addition of glucose, the levels of metabolic intermediates must have risen rapidly, and the only primary limiting

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factor was the supply of adenine nucleotides. Thus, although many factors contributed to the rise in the rate of leucine incorporation when glucose was added, the course of incorporation after that addition allows for a clear comparison of the effects of variation in the absolute concentration of ATP and in the value of the adenylate energy charge. I t is evident that protein synthesis, as measured by leucine incorporation, is much more dependent on the value of the energy charge than on the concentration of ATP. Shift down of illumination caused a prolonged (2.5to 3 h) decrease in overall protein accumulation by 90% in Chromatium sp., and a small transient decrease in the energy charge value (MioviC and Gibson, 1973).During this same period, there was a large increase in the rate of bacteriochlorophyll synthesis. Thus, the decrease in level of illumination caused a shift to preferential synthesis of photosynthetic pigment without any prolonged alteration of the energy charge value of the cell. Selective syntheses of specific proteins have for a long time been known to be of regulatory importance. This process is probably controlled largely by specific inducers or compounds associated with the particular process under consideration. Numerous examples of induction of specific enzymes under conditions when there is no net synthesis of protein have been reported. The selective induction of p-galactosidase in starved E . coli cultures is the most thoroughly studied of these cases (Beckwithand Zipser, 1970). IV. General Discussion

The unique role of the adenine nucleotides in stoicheiometric coupling and energy transduction between metabolic sequences has been recognized for over thirty years, and was first emphasized in the classic review by Lipmann (1941).During the last decade, it has become apparent that the associated role of these nucleotides in kinetic correlation and regulation is equally unique, ubiquitous, and important. All metabolic pathways are stoicheiometrically related through the adenine nucleotide system, since all either utilize or regenerate ATP. I t is a logical necessity, if these sequences are to be mutually regulated so as to maintain biological homeostasis, that all sequences must also be kinetically regulated, directly or indirectly, by the same coupling system. Evidence for control by the energy charge of the adenine nucleotide system has been obtained for every sequence for which it

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has been sought, and there is no reason to doubt the generality of such regulation. Most of the evidence is in the form of responses to variation in energy charge observed in vitro in studies on key enzymes of the sequence in question. Such experiments have been excluded fiom this review. The conclusion is re-inforced, however, by the stability of the energy charge in normally metabolizing cells of a wide variety of types. As documented in this review, and previously (Chapman et al., 197 11, evidence that the energy charge in intact metabolizing cells is maintained at values above 0.75 is extensive and convincing. O n the basis of trends in the results reported as sampling and analytical methodologies improve, we consider it a reasonable working hypothesis that the energy charge under normal conditions will not lie outside the range 0.87 to 0.95. When it is remembered that the turnover time for ATP is probably less than one second, the tight stabilization of the energy charge appears to be the most striking aspect of biochemical homeostasis presently known. A small imbalance, persisting for a very short time, would suffice to pull the energy charge far from its normal range. Evidently the controls are sensitive, general, and fast. Any regulatory system is circular in operation, and control of all metabolic sequences by the value of the adenylate energy charge is equivalent to control of the energy charge by its own momentary value. The experiments on enzymes in uitro, cited on p. 268, lead to a clear distinction between enzymes that participate in ATP-regenerating sequences, and are strongly inhibited by increasing values of energy charge in the range above about 0.7, and those that participate in ATP-utilizing sequences, which respond oppositely to variation in energy charge in the same range. Responses of these two types appear to be the primary basis for stabilization of the energy charge, which in turn underlies all other types of biological homeostasis. I t should be realized that each sequence will be regulated also by one to several other inputs related to the metabolic function of the sequence-for example, product negative feedback in the case of biosynthetic sequences. For any given sequence, the energy charge is only one regulatory input, and it is no more important than any of the others. I t is only when general metabolism-the correlation of sequences-is considered that the unique importance of energy charge regulation is seen. I t is not to be expected that the value of the energy charge (or of any other input) will be the sole regulatory influence on any sequence, but it may be proposed with confidence that a study of

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regulation of any metabolic sequence cannot be complete unless it takes the effect of variation in energy charge into account. Both the thermodynamic coupling role and the kinetic regulatory role of the adenine nucleotides are so general that neither can be ignored in consideration of any biochemical events in uiuo. Accumulation of indirect but convincing evidence for participation of the adenylate energy charge in regulation of all metabolic sequences (the stability of the charge alone seems irrefutable in this connection) is one thing, but evaluation of the quantitative significance of variation in energy charge for a given sequence under specific conditions is quite another, and is presently beyond our reach. The flux through any sequence responds in a complex way to the values of several parameters, and it is difficult to isolate or evaluate the effect of any one of them in uiuo, even aside from sampling and analytical difficulties. In addition, flux rates in uiuo are hard to estimate reliably except in the few cases where the product can be expected to accumulate nearly quantitatively, as in synthesis of storage compounds. The excellence of the energy charge-stabilizing system itself works against the investigator; the more sensitive a regulatory system, the more difficult it is to measure changes in the primary control parameter. I t is quite possible that changes in energy charge of 0.01 unit or less may have significant effects, and these cannot presently be measured in a functioning cell. Further, local variations of this magnitude cannot be ruled out,. and analysis of disrupted cells can give only an average value. It seems clear that attempts at quantitative correlation of changes in energy charge in vim with changes in metabolic fluxes would at the present time be premature and unproductive. Not only may the concentrations of other important effectors change simultaneously with change in energy charge (or be caused to change by the change in energy charge), but the enzymic capabilities of the cell may be quite different under different conditions because of covalent modification of enzymes (adenylylation of glutamine synthase (Shapiro and Stadtman, 1970) or phosphorylation of mammalian pyruvate dehydrogenase (Reed, 1969; Hucho, 19751, for example) or because of differential control of the synthesis or degradation of enzymes or enzyme systems. As an illustration, Sacch. cereuisiae growing on glucose lacks functioning mitochondria. Soon after glucose exhaustion, mitochondria are synthesized. The cell is as metabolically different under these two conditions as if it were two different organisms Quantitative predictions thus seem pointless, but qualitative tests of

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some predictions are possible in intact cells. Comparison of the relative importance of variation in the absolute concentration of ATP and in the value of the energy charge is an example. Storage of chemical energy in any system means maintaining the reactant/product concentration ratio for some chemical reaction far from its equilibrium value. Metabolic energy transduction is built around the maintenance of the ATP/ADP ratio very far from the ratio that would be at equilibrium in aqueous solution at physiological phosphate concentration. In any such system, the ratio is much more important than the absolute concentrations of the reactant and product, and it could be predicted that the properties of regulatory enzymes would have evolved on that basis. The view that ratios rather than absolute concentrations should be studied is foreign to the standard concepts of metabolic chemistry. Where experiment is possible, it is to be preferred to the perpetuation of standard concepts, and the results presented in Fig. 3 and 4 (p. 284 and p. 296, respectively) provide strong evidence that the energy charge (or equivalently, the ratios among the adenine nucleotides) is more important than the absolute concentrations of ATP in actual functioning cells. After addition of glucose to cells starved for glucose and adenine, leucine incorporation proceeds at very low ATP concentrations, but drifts downward roughly in parallel with the decline in energy charge (Fig. 4, p. 296). A more general result is presented in Fig. 3 (p.2841, where growth itself is seen to be compatible with a decrease in ATP concentration by a factor of about three, but to occur only when the energy charge is in its normal range. I t thus appears that maintenance of the energy charge within a very narrow range, by operation of the metabolic regulatory systems of the cell, is a necessary condition for growth. I t should be re-emphasized that, in responding primarily to the energy charge or the ATP/ADP ratio, while being relatively insensitive to the absolute concentrations of the nucleosides, a cell is not doing something strange or mystifying, but is merely exhibiting the end result of responses that have been tailored in the course of evolution to the chemical realities of energy transduction. REFERENCES

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Physiology of Mating in Three Yeasts MARJORIE CRANDALL School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506, U.S.A. and

RICHARD EGEL lnstitut fur Biologie Ill der Universitat Freiburg, 0 - 7 8 0 0 Freiburg, Schanzlestr. 9-1 I , Federal Republic of Germany

and

VIVIAN L. MACKAY Waksman Institute of Microbiolog y, Rutgers University, N e w Brunswick, N e w Jersey 08903, U S A . I. Introduction . . A. Ecology . . B. General Characteristics C. Lifecycles . . 11. Hansenula wingei . A. Mating-Type Locus B. Haploid Functions C. DiploidFunctions 111. Schizosaccharoqces pombe A. Mating-Type Locus B. Haploid Functions C. Diploid Functions IV. Saccharomyces cerevisiae A. Mating-Type Locus

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B. Haploid Functions . . . . . C. Diploid Functions . . . . . V. Comparative Discussion . . . . A. Steps in Yeast Conjugation Compared . B. Evolutionary Aspects of Sexual Reproduction . C. Comparison with Mammalian Systems VI. Acknowledgments . . . . . . . . . . . . . References

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I. Introduction

Sexual reproduction in yeasts has a survival function by providing an alternative to the vegetative processes when conditions are no longer conducive for growth. If both sexes are in the correct physiological state (usually under starvation conditions), then initiation of copulation involves the mutual induction of a sexual response. This response is mediated by diffusible compounds and by physical contact. Initial cell contacts between opposite mating types can be disrupted easily, but stronger intercellular bonds form later that result in the fusion of two cells into one. Union between mates involves mixing of parental gene pools. The new diploid organism or its subsequent offspring might be better equipped to survive in a new environment because they may contain new combinations of parental genes. Hence, sex is more advantageous to the survival of the species than it is to the individual organism. The purpose of this review is to compare the steps in the mating process in three species of yeasts. The various physiological factors, events and regulatory phenomena that are part of the mating process will be described for Hansenula wingei, Schizosaccharomyces pombe and Saccharomyces cereuisiae in Sections 11, 111 and IV, respectively. Then, in Section V, the similarities and differences among these yeast systems will be discussed. Emphasis in this article will be on recent observations since reviews of earlier work are available for each mating system (for H . wingei: Crandall and Brock, 1968; Crandall and Caulton, 1975; for Schizosacch. pombe: Leupold, 1970; Gutz et al., 1974; for Sacch. cerevisiae: Fowell, 1969a, b; Bilinski et al., 1975; Sena et al., 1975).For a comprehensive review of conjugation in all yeasts, fungi and other micro-organisms, consult Crandall(l97 7). The three yeasts to be reviewed here are quite diverse in terms of their ecological niches, metabolism, morphology and life cycles. Therefore, for a better understanding of the physiology of sexual

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reproduction in these organisms, it is necessary first to consider these characteristics. A more detailed description of each yeast is given in Lodder (1970). A.

ECOLOGY

All three yeasts are found in Nature growing saprophytically on plant sugars and related compounds but their symbiotic associations are significantly different. The ecology of H. wingei has been reviewed previously by Crandall and Brock (1968).This yeast is carried by bark beetles (species of Ips) and is inoculated under the bark of coniferous trees where it grows on the tree sap and also serves as food for the insect larvae. In turn, the yeast depends on the insect for dissemination from one tree to the next. Thus, this is a true symbiotic relationship. The correlation between the biochemical characteristics of species of Hansenula and their natural environment, ploidy in Nature and degree of evolution is discussed by Wickerham and Burton ( 1962). Strains of Schizosacch. pombe have been isolated mainly from tropical or subtropical areas where they are associated with native and industrial brewing (Sloof, 1970). In fact, the specific epithet “pombe” refers to a native Bantu beer in East Africa. Other strains of Schizosacch. pombe have been isolated from grape or apple musts in temperate regions. Different strains of Sacch. cerevisiae have evolved in commercial establishments by selection for desirable properties; hence, the terms “brewer’s yeast” and “baker’s yeast”. The heterothallic strains studied in genetics laboratories were derived originally from isolates of baker’s yeast by repeated selection and subculturing to obtain strains with good viability, high sporulation capacity, regular growth characteristics and stable haplo- and diplophases. Strains of Sacch. cerevisiae similar to these laboratory strains can be isolated from ripening grapes, but are found rarely on unpicked, immature fruit with a low sugar content (Lastand Price, 1969). Thus, the common physiological denominator for asexual (vegetative) reproduction in these three yeasts is the requirement for sugars or sugar derivatives in their ecosystems. In order to understand the physiology of mating in yeasts, it must be borne in mind that sexual reproduction occurs optimally under non-growing conditions, i.e.

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the processes of conjugation and sporulation are triggered by nitrogen starvation and/or by low molecular-weight sex-specific pheromones that inhibit cell division. Pheromones (from the Greek, therein, to carry, and hormun to excite; Karlson and Luscher, 1959)are used in this review to mean sex-specific chemicals secreted by one mating type that induce a sexual response in the opposite mating type. 8. G E N E R A L C H A R A C T E R I S T I C S

Both Schizosacch. pombe and Sacch. cerevisiae are facultative anaerobes. While their industrial importance results from their fermentative abilities, their mating processes occur optimally under aerobic conditions. In contrast, H. wingei is an obligate aerobe, lacking the ability to ferment (Wickerham, 1970). Obviously, then, its sexual processes also require respiratory activity as with the other two yeasts discussed here. Cells of these three yeasts differ strikingly in morphology. Hansenula wingei is dimorphic; the yeast phase is a small ( 3 x 5 pm) elongated oval cell, and the pseudohyphae result from cell elongation which occurs under starvation conditions. Cells of Schizosacch. pombe are cylindrical (about 3 jm in width and from 4 to 15 pm in length). Saccharomyces cerevisk cells are large (6 x 8 pm) rounded ovals. C . LIFE CYCLES

Figure 1 illustrates the life cycles of the three yeasts. Although each yeast may exist in either the haplo- or diplophase, diploids of Schizosacch. pombe sporulate at the end of the vegetative growth phase and, therefore, are difficult to maintain in culture. Both H. wingei and Sacch. cerevisiae divide by budding, but Schizosacch. pombe divides by binary fission, as do most bacteria. All three yeasts agglutinate prior to conjugation; however, events at the level of the zygote differ. Hansenula wingei undergoes nuclear fusion immediately, forms a diploid bud, and arrests at this stage until transferred from conjugation buffer to growth medium. Zygotes of Schirosacch. pombe usually undergo meiosis and sporulate immediately following nuclear fusion. However, under special conditions, the zygote will divide to form a diploid culture. Rare diploid matings result in tetraploids if nuclear fusion occurs or in twin meiosis if only cytoplasmic fusion takes place. In Sacch. cerevisiae, cytoplasmic fusion is usually followed immediately by nuclear fusion after which diploid buds are produced. However, there are a few

31 1

PHYSIOLOGY OF MATING IN THREE YEASTS Storvotion

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M. CRANDALL, R. EGEL AND V. L. MACKAY Diploid budding

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FIG. 1. Life cycles of three yeasts: (a)Hansenula wingei, (b)Schizosaccharomycespombe, and (c) Saccharomyces cerevisiae. The cycles include information summarized from references cited in Sections 11, 111 and IV, respectively, and from unpublished data from the laboratories of M. Crandall, R. Egel and V. L. MacKay, respectively. The dashed lines indicate processes observed with a low frequency or under special conditions. The heavy lines on Fig. 1b indicate that Schizosaccharomyces pombe exists primarily in the haplophase.

reports of delayed nuclear fusion resulting in a transient heterokaryon that produces haploid buds. Sporulation of a diploid cell in all three yeasts occurs under starvation conditions, and results in the production of a tetrad containing four haploid spores surrounded by an ascal wall that is the original cell wall of the diploid. The ascospores are oval-shaped in Schizosacch. pombe, round in Sacch. cerevisiae and shaped like Derby hats in H. wingei. The ascospores germinate when conditions become favourable for growth thereby regenerating the haplophase. Each haploid can either divide indefinitely or mate if a suitable partner is available and conditions are conducive for sexual activity. Hunsenula wingei is strictly heterothallic but Schizosacch. pombe and Sacch. cerevisiae have both heterothallic and homothallic strains. In Schizosacch. pombe, homothallism (self-diploidization) is effected by frequent switching

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from one mating-type potential to the other during cell division. Thus, mature homothallic colonies are mixed and contain cells expressing one or the other mating-type activity, which can mate and then sporulate. In contrast, spores of Sacch. ceretrisiue carrying homothallism genes (HOor HTH) convert early after germination to stable diploid colonies. However, the initial event causing homothallism, i.e. a directed genetic change or “paramutation” (Brink et al., 1968)of one mating type to the other is comparable in both.of these yeasts (for brevity, only one direction is indicated in Fig. lc). The complex morphogenetic changes that occur during the alternation of generations involve ordered sequences of biochemical reactions each requiring an enzyme which is the product of a separate gene. Thus, a discussion of the physiology of mating must also include a discussion of the various genetic loci involved in this process. While many genes affecting sexual processes have been identified, probably more remain undetected. Yet the most important conclusion from these preliminary genetic studies is that the mating-type locus plays a central role in controlling genes governing both haploid and diploid functions of the life cycle. For this reason, the mating-type locus is a valuable system for the study of genetic regulation of cellular differentiation in eukaryotes. 11. Hansenula wingei A. MATING-TYPE LOCUS

1. Muting- Type Alleles

The two opposite mating types described originally in H. wingei are called strains 5 and 21 because they were the 5th and 21st haploids selected from a sporulated culture of a diploid isolated from Nature (Wickerham, 1956).Strains 5 and 21 appear to be identical with respect to all characteristics studied thus far except mating type-related functions. Other isolates will hybridize with these original strains; strains V l B (Herman et al., 1966)and 72 (Wickerham, 1956)are of mating type 5 , and strains V1A and 73 are of mating type 21. Diploids resulting from crosses between complementary types always yield asci containing two spores of type 5 and two spores of type 21, indicating that the mating types of the above strains are allelic at the mating-type locus (mat).The suggested nomenclature for the two mating types would be

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matl-5 and matl-21 but, since the mat locus has not been mapped, just the strain numbers will be used in this review. 2. Homothallism versus Heterothallism

The species H . wingei is classified by its strictly heterothallic mating ability (Wickerham, 1961). However, many species of Hansenula are homothallic. The significance of the heterothallic nature of H. wingei is that the haploid cultures are stable and never diploidize by mutation to the opposite mating type followed by conjugation with a sister cell. This lack of self-mating was investigated further and it was found that the frequency of spontaneous mutation from mating type 21 to 5 is less than 3 x 1 P and from mating type 5 to 21 is less than 6 x lV (R.W. Wood and M. Crandall, unpublished observations). I t therefore appears that, in these heterothallic strains, the mat locus does not include both 5 and 21 alleles in the same haploid genome. This is in contrast to the situation in both Schizosacch. pombe and Sacch. cerevisiae in which both alternative mating-type alleles can be present in a haploid genome but only one is expressed (see Sections 111 and IV). 3. Functions Controlled by the Mating- Type Locus

Both haploid and diploid functions are determined by the matingtype locus, as discussed in Sections IIB and IIC, respectively. The subheadings under B and C are a composite of the processes studied in the three yeasts being reviewed in this chapter. Where research has not been done in a given area for one yeast, either this will be indicated as not studied or the section will be omitted. B. H A P L O I D F U N C T I O N S

When the cell is in the haplophase, the functions regulated by the mat locus are concerned primarily with mating. Each haploid matingtype cell requires the expression of only one complement of conjugation-specificgenes. Alternative (compatible) alleles for mating are expressed in opposite mating-type cells. This is in contrast to the diploid condition where both alternative alleles at each locus are carried in the same cell and they interact to repress mating functions and promote diploid functions.

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31 5

1. Initiation of Conjugation

The process of conjugation occurs between haploid cells of opposite mating type. Conjugation (or sexual reproduction) may be viewed as an alternative to budding (or asexual reproduction). Commitment to either conjugation or vegetative growth is determined by both physiological conditions and the stage in the cell cycle. a. Physiological Conditions Inducing Conjugation. In H . wingei, conjugation,proceeds best in a buffer lacking a nitrogen source, salts, trace elements and the three required vitamins (biotin, thiamin and pyridoxine). This conjugation buffer contains 0.5% (w/v) glucose as an energy source, 0.1% (w/v) MgSO,. 7H,O which promotes agglutination, and 0.01 M KH,PO, buffer adjusted to pH 5.5. Equal numbers of cells of strains 5 and 21 are mixed together at about lo8 cells/ml in 10 ml of conjugation buffer in an Erlenmeyer flask ( 125 ml) and aerated at 250 rev/min on a rotary shaker (Brock, 1961; Crandall and Brock, 1968). Another medium devized for hybridizing yeasts (Herman, 197 la) is called restrictive growth medium because it contains growth-limiting concentrations (w/v) of nutrients (0.1%glucose +0.02% yeast extract +0.02% Bactopeptone +2.0% agar). Opposite mating types mixed together on this medium grow together and mate. Cells of opposite mating type carrying complementary auxotrophic markers can also be hybridized on a minimal-agar medium lacking the growth factors required by both haploids. The only growth on this medium will be that of the prototrophic diploid hybrid. In the above three examples, mating occurs or is facilitated in media in which essential nutrients are limiting or absent. Evidently H . wingei has a large intracellular pool of amino acids (Brock, 1961),purine and pyrimidine bases and vitamins because auxotrophs will mate in the absence of growth factors (Crandall and Brock, 1968). However, extensive conjugation also occurs in complete growth medium (Brock, 1961). b. Responsive Stage in the Cell Cycle. Cells from stationary-phase cultures, which are probably in G1, mate better than cells from exponential-phase cultures. Typically 7 6 8 0 %conjugation is obtained with non-synchronized stationary-phase populations of strains 5 and 21 (Fig. 2). Presumably, mating could be synchronized and increased to 95% or better if competent non-budding cells in G 1 were selected on sucrose gradients as has been reported for Sacch. cerevisiae (Sena et al., 1973, 1975; see Section IV, B.l, p. 355).

31 6

1

0

FIG. 2. Effect of growth stage on conjugation ability (-0-1 in Hunsenulu wingei. Cultures of strains 5 and 21 were grown overnight in medium containing 3.0% (w/v) glucose, 0.7% (w/v)yeast extract and 0.5% (w/v) KHpPO,, then 1 ml was added to 25

ml fresh medium in a 125-ml Erlenmeyer flask. Cultures were aerated on a rotary shaker at 250 rev/min at 30OC. The increase in absorbance at 640 nm for both strains was the same so only one curve is presented (--O--). The one-day, two-day and three-day cultures were prepared in advance so that all of the conjugation assays could be done on the same day. The conjugation assays of the zero time, and 4-, 6- and 8 h cultures were done using cell suspensions of the same density as the stationary-phase cultures. Percentage conjugation was assayed according to standard procedure (Crandall and Brock, 1968). Conjugation was allowed to proceed for five hours. Note that the zero-time sample is simply a one-day culture diluted into fresh medium, harvested immediately and assayed for conjugation. It gave the same extent of conjugation as the 24-h sample.

c. Inhibitors $Conjugation. For mating to occur, both sexual partners must be able to synthesize protein. Prior ultraviolet irradiation of either strain 5 or 21 destroys conjugation ability (Brock, 196 1) as does addition of cycloheximide, fluorophenylalanine or ethionine to the conjugating mixture. Metabolic inhibitors (potassium cyanide, sodium arsenite, dinitrophenol, sodium azide) as well as boric acid and nystatin also inhibit conjugation.

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2. Cellular Recognition

The first step in conjugation in H. wingei is recognition of the opposite cell type by formation of a neutralized complex between complementary macromolecules on the respective cell surfaces. These complementary agglutination factors may be located on surface filaments which extend From the cell wall. a. Constitutive versus Inducible Agglutination. In haploid strains 5 and 21 of H. wingei, sexual agglutination is constitutive, i.e. cells clump immediately when mixed (Wickerham, 1956). This clumping is sexspecific since suspensions of individual strains are stable, nonflocculent and settle out only slowly. Haploids derived From crosses of sexually agglutinative x non-agglutinative strains of H. wingei show a wide range of inducible agglutination from immediate to latent. Neither strain 5 nor 21 clumps with the diploid. Thus, the nonagglutinative diploid is used as the control to test for specificity in sexual agglutination. b. D@sible Sex Factors. Since the agglutination factors are present at all times in the respective haploids, it would be predicted that sex pheromones such as those which induce agglutination in Succh. cerevisiue (see Section IV, B.2., p. 356) would not be involved in the agglutination reaction in H. wingei. In agreement with this prediction, evidence for diffusible inducers of agglutination has not been obtained from experiments with mixtures of agglutinable cells containing either the non-agglutinative diploid or non-agglutinative mutants (M. Crandall, unpublished data). Similarly, evidence was not found in H. wingei (Brock, 1965a) for the sex-specific formation of protuberances (“shmoos”, see Section IV, B.2, p. 357) originally reported in Sacch. cerevisiae by Levi (1956). However, two reports describe a sex-specific budding response in H. wingei. Brock (1961)found a sex-specific increase in bud formation attributable to cell contact in mixtures of ultraviolet-inactivated cells and unirradiated cells of opposite mating type. Herman (1971b) found that, when single cells of complementary mating types were positioned about 15 pm from each other on agar blocks, the cells budded preferentially toward one another. This sex-specific budding was attributed to the action of diffusible substances. Intergeneric budding responses were observed between complementary mating types of Sacch. cerevisiae and H . anomula indicating that the diffusible constituents possess broad specificity.

31 8

M . CRANDALL, R. EGEL AND V. L. MACKAY

c. Complementary Agglutination Factors. The cell-surface macromolecules on strains 5 and 21 that determine the complementarity or specificity of mating are called 5-factor (50 and 21-factor (210. The properties of these two agglutination factors are compared in Table 1. TABLE 1. Comparison of the properties of complementary glycoprotein mating factors from Hansenula wingei Properties Activity

Agglutinates cells of strain 21 and forms a soluble complex with 2 1fin nitro Six or more

Number of combining sites Isolation Cytoplasmic extracts of a subtilisin digestion of whole cells

-

Molecular size

Molecular weight Chemical composition Carbohydrate content Inactivated by

2 1 -Factor

5-Factor

Inhibits the agglutination activity of 5f by forming a neutralized 5 f 2 lfcomplex One Cytoplasmic extracts or trypsin digestion of whole cells Homogeneous: 2.9S2,,,

Heterogeneous-the sedimentation co-efficient varies with the preparation. Values reported: 3.5, 6.5,9.0, 15.4, 16.7,31S2,,, Varies from 15.000 to lo8 daltons

Homogeneous: 2.9S2,,, About 40.000 daltons

Phosphomannan-protein

Mannan-protein

50-96%

25-35%

Reducing agents

Heat, alkali and other chemical denaturants

These data are summarized from results and literature presented in Crandall el Taylor and Orton (197 1). Yen and Ballou (1974)and Sing el d. (1976).

QL. (1974),

5-Factor (5-agglutinin) causes a sex-specific clumping of cells of strain 22 and hence is considered multivalent (Taylor, 1964; Brock, 1965b). Taylor and Orton (1968) presented evidence that there are six combining sites per molecule of 5-agglutinin (about lo6 daltons) isolated by subtilisin diges;ion of strain 5 cells. These six binding sites are joined to the core by sulphide bonds and are liberated by treatment with a reducing agent. Each has a molecular weight of about 12,000 daltons. Reduction of radioactively labelled 5-agglutinin was repeated by Yen and Ballou (1974)who separated three small protein fragments from the 5f core on a Bio-Gel A-5 m column. One of the labelled fragments adsorbed to strain 21 cells but did not cause

&

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agglutination and hence was considered univalent. This fragment contained 28 amino-acid and about 60 mannose residues. Thus, the molecular weight of the combining site was estimated to be 12,500 daltons, in close agreement with the estimate of Taylor and Orton (1968). Molecules of 5f of different sizes, present in cytoplasmic extracts, were estimated to have the number of combining sites proportional to their molecular weights (Crandall et al., 1974).Yen and Ballou (1974) showed that the central core of 5-agglutinin consists of about 10% protein to which are bound short manno-oligosaccharides (average chain length being eight mannose units) through linkage to a serine or threonine residue. These two amino acids constitute, respectively, 55%and 6 8 %of the core protein. Hence, 5-agglutinin is a novel glycoprotein in which most of the amino acids are hydroxyamino acids and most of these are substituted by carbohydrate. Taylor and Tobin ( 1966)determined that 5f is a branched, randomly coiled polymer. This lack of a-helical structure may explain the heat stability of 5f which retains activity even after being heated to 100°C for 10 min (Crandall and Brock, 1968). Taylor and Orton (1971)have proposed a model for 5f in which the six disulphide bonds holding the combining sites to the central core are arranged at the corners of a regular octahedron. The binding energy of each individual site on the 5f molecule is from -5 to -9 Kcal/mole but the standard free energy of association for the molecule as a whole with strain 21 cells is -14.5 Kcal/mole (Taylor and Orton, 1970). This value is high for free energies of reversible biological reactions and is due to co-operativity between several combining sites on the 5f molecule when combined with the same strain 21 cell.

( a )

( b )

FIG. 3. Illustration of two possible structures for 5-agglutinin. (a) A single glycoprotein chain; and (b) several glycoprotein subunits held together by carbohydrate interactions or phosphodiester bridges. In both cases, the active sites are represented by segmented circles bound to the glycoprotein core by disulphide bridges. The short lines represent oligomannose units branching from the heavier line(s)which represents the core protein backbone (Yen and Ballou, 1973).

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M. CRANDALL, R. EGEL AND V. L. MACKAY

It is not known whether the central core in the model of Taylor and Orton (1971) consists of a single peptide chain to which the six combining sites are linked through disulphide bridges (Fig. 3a) or is composed of six polypeptide chains each linked to one binding fragment (Fig. 3b; Yen and Ballou, 1973).Interestingly, 5f released by subtilisin also contains 5% phosphate. Therefore, this molecule should be called a phosphomannanprotein (Yen and Ballou, 1974) even though the phosphate is not required for activity (Sing et al., 1976).5-Factor is inactivated by both pronase and a bacterial exo-a-mannanase. These results, considered together with other results discussed by Yen and Ballou ( 1974), suggest that the specificity of the binding sites resides in the structure of the protein, but that both the protein and the carbohydrate moieties of the whole molecule are important for agglutination activity. The complementary agglutination factor, 2 1f, does not agglutinate cells of strain 5 but does inhibit the agglutination activity of 5f and, hence, is considered univalent (Crandall and Brock, 1968).Just as with 5f, 21f can be prepared either by digestion of cells with a proteolytic enzyme (e.g. trypsin) or by disintegration of cells with glass beads and recovery of the soluble extract. In contrast to 5f, however, 21f is homogeneous with respect to molecular size (2.9s; about 40,000 daltons; Crandall and Brock, 1968; Crandall et al., 1974). Both mating-type factors are mannanproteins, but 2 If has less carbohydrate (25-.35%) than 5f (90%).In contrast to the stability of 5f, 21f activity is easily destroyed by various physical and chemical denaturants. The role of these mating factors in viuo is to bring the two cell types together into intimate contact, allowing them to initiate the process of cell fusion. The mating factors do this by virtue of their complementary nature. Solubilized 5f and 21f form a stable neutralized 5f-21f complex which can be purified using classical methods in protein chemistry (Crandall et al., 1974).As expected, this complex is soluble since 21f is univalent and, therefore, networks of crosslinks cannot be formed as in the case with precipitating antibodies. The complex is stable probably because the free energy of binding is high (Taylor and Orton, 1970).During purification, the neutralized complex was detected using a biological assay developed by Crandall and Brock (1968) which is based on the fact that 21f is unstable in alkaline solution whereas 5f is stable. The complex may be detected by treating extracts with alkali and measuring recovered 5f activity. Using this assay, the

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5f-2lf complex was found to be heterogeneous with respect to molecular size, reflecting the heterogeneity of the 5f component isolated fiom the cytoplasm. Three peaks of complex were detected, with estimated molecular weights of 0.5, 1.2 and 3.8 x lo6 daltons, respectively. For these three peaks, the calculated number of combining sites were 6, 16 and 63, roughly in proportion to molecular weight (Crandall et al., 1974). Heterogeneity of cytoplasmic 5f is not understood, but it is thought that these molecules are precursors of cellwall agglutination factors. These same assays for 5f, 21f and the complex were used to search for the presence of free or neutralized 5f and 2 If in the non-agglutinative diploid. Since none of these activities was found in either cytoplasmic extracts or on the cell wall, it was proposed that synthesis of both mating factors is repressed in the diploid (Crandall and Brock, 1968; Section 11, C.2, p. 328). But, interestingly, zygotes remain clumped during conjugation indicating that the surface agglutination factors are not destroyed during the mating process. Evidently, the haploid agglutination factors are passively diluted out with growth of the diploid that ordinarily does not synthesize either mating factor. d. Surface Filaments. preliminary electron-microscope studies of H. wingei have demonstrated the presence of filaments on the cell surfaces of strains 5 and 21 (K.Aufderheide, personal communication; Day et al., 1975; see electron micrographs by A. Day reproduced in Crandall and Caulton, 1975). Based on electron-microscope observations, these workers suggest that the agglutination factors might be located on these surface filaments. In fact, fibrillar material external to the thick cell wall can be seen in all of the electron micrographs presented in Fig. 4 which came from the electron-microscope study of conjugation performed by Conti and Brock (1965).In Fig. 4a, the surface filaments are seen to overlap forming a region of greater electron density between the opposite cell types during an early stage of agglutination. Thus, the suggestion that the 5f and 21f might be located on these filaments is attractive and merits further study. 3 . Cell Fusion

a. Initial Cell Binding. In the first stage of mating, strong interactions between the complementary agglutination factors result in deformation of the cell walls at the contact surfaces (Figs.4a and b). I t is

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M . CRANDALL, R. EGEL AND V. L. MACKAY

FIG. 4. Electron micrographs of thin sections through cells from a conjugating mixture of Hunsenulu wingei strains 5 and 21 (reproduced by courtesy of S. Conti; photomicrographs (b),(c),(el, (0and (g)were published in Conti and Brock, 1965). (a) Initial cell agglutination showing overlapping of surface filaments. (b) Strong cell binding causing cell-wall deformations.

PHYSIOLOGY OF MATING IN THREE YEASTS

FIG. 4(c). Protuberances formed at the site of cell contact.

FIG. 4(d). Early stage in the formation of the conjugation tube.

323

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M. CRANDALL, R. EGEL AND V. L. MACKAY

Fig. 4(e). Cytoplasmicfusion.

PHYSIOLOGY OF MATING IN THREE YEASTS

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FIG. 4(0. Nuclear fusion.

FIG. 4(g). First diploid bud.

seen that deformation also occurs between like cell types which are forced together by virtue of the geometric relationships of the cells in the agglutinated clump. b. Formation of the Conjugation Tube. Localized cell-wall extension at the site of cell contact causes the formation of protuberances (Fig. 4c)

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M. CRANDALL, R. EGEL AND V. L. MACKAY

which eventually fuse (Fig. 4d) to form a conjugation tube between the two cells (Fig. 4e). Figure 4d shows a very early stage in formation of the conjugation tube where the two cell walls are apparently being dissolved at the point of contact as indicated by curvatures at the inner surfaces of the walls. The juncture between the two original outer surfaces of the walls is indicated by a line of low electron density. This juncture line is still visible in the flap of remaining cell-wall material (Fig. 4e). The wall changes at the point of fusion result in a distortion of the shapes of both cells producing the typical dumbbell-shaped zygote (seen in Figs. 4e, f and g). Formation of the conjugation tube must involve a localized increase in the activities of cell-wall degradative and synthetic enzymes because the absolute levels of several enzymes in conjugating mixtures of strains 5 and 21 were the same as in non-conjugating controls with only one cell type present (Brock, 1965a). While there is no evidence that diffusible pheromones function in wall extension in H. wingei as discussed in Sacch. cereuisiae (see Section IV. B. 2, p. 3571, there is evidence that cell contact stimulates localized cell-wall enzyme activities, as shown by the increase in bud formation in mixtures of ultraviolet-inactivated cells in admixture with unirradiated cells of opposite mating type (Brock, 1961). c. Cytoplasmic Mixing, Mitochondria1 Fusion and Recombination of Cytoplarmic Genes. Figure 4e shows mixing of cytoplasmic organelles in the conjugation tube but genetic studies of mitochondria1 recombination have not been performed in H. wingei. 4. Nuclear Fusion versus Heterokaryosis

Figure 4 illustrates the initial stages of nuclear fusion during conjugation. There is no evidence for growth and cell division of a heterokaryon in H. wingei but there is evidence that a heterokaryon exists transiently (Herman, 1959). 5. Zygotic Budding

In Fig. 4g, the fusion nucleus is seen entering the first diploid bud. This bud usually comes off at the mid-point of the conjugation tube presumably because this is the thinnest part of the wall (Brock, 1961; however, see Section IV, B.5, p. 371). A small percentage of zygotes have a polar bud instead of the usual central bud. However, it is not known whether this bud is diploid or whether it was on the original

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haploid parent before mating was initiated. It is also possible that this zygote resulted from melange a trois or represents a transient dikaryotic stage. A pedigree analysis performed by micromanipulation would determine which of these four alternatives obtains. Primary budding of the zygote occurs in conjugation buffer even though it lacks a nitrogen source, salts, trace elements and the three vitamins required for growth. 6. Mutant Genes Aflecting Conjugation Non-mating mutants of strain 5 were isolated (Crandall and Brock, 1968)using a selection technique based on the fact that cycloheximide resistance (cyh) is recessive (Herman, 1959). When strain 5 cyh-I was crossed to wild-type strain 21 (cycloheximide sensitive), the resulting heterozygous diploid did not grow on medium containing cycloheximide. In fact, only those cells of strain 5 which did not mate grew on this medium. The disadvantage of this selection technique is that, even if a 10-fold excess of strain 22 were used for mating, still about 10%of the strain 5 cells did not mate for trivial reasons. Nevertheless, several spontaneous mutants that had lost both the ability to mate as well as to agglutinate were isolated using this method (Crandall and Brock, 1968). A non-mating mutant that was still agglutinative was isolated without selection following nitrous acid mutagenesis. Since the non-agglutinative non-mating mutants were not conditional, they could not easily be analysed genetically and, therefore, the nature of this double loss is not understood. I t is probably not a double mutation since this double defect occurs spontaneously with a frequency of about (Crandall and Brock, 1968). From these preliminary mutant studies and fi-om studies of induction of agglutination in the nonmating diploid (Section 11, C.2, p. 329), it may be concluded that agglutination in H. wingei is a necessary condition but not sufficient to induce the later steps in conjugation. C . D I P L O I D FUNCTIONS

When the cell is in the diplophase, the functions regulated by the mat locus are concerned primarily with sporulation and repression of haploid functions. For regulation of mating and for promotion of sporulation and other diploid-related processes, it is thought that alternative or compatible genes must be present together in the same diploid cell instead of being separated as in the haploid cells.

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1 . Heterozygosity versus Homozygosity at the Mating- Type Locus

All of the diploid studies in H . wingei have been with the heterozygous 5 x 21 hybrid.-Diploids homozygous at the mat locus could arise from diploid mitotic recombination or rare illegitimate matings between the same mating types. Since 5 x 5 and 21 x 21 hybrids would probably behave like haploids (i.e. would agglutinate and mate but would not sporulate), detection of the homozygous diploid state would have to depend on other criteria such as phenotypic expression, DNA content, radiation survival or difficulty in isolating recessive mutations.

2. Repression $Haploid Functions Haploid cells are constitutively agglutinative and capable of conjugating, whereas diploid cells are non-agglutinative and non-maters, suggesting the existence of regulatory genes that function in the diploid to repress haploid functions (Crandall and Brock, 1968). a. Sexual Agglutination. Although the 5 x 21 hybrid has the genes for both agglutination factors, neither 5f nor 21f is synthesized in the diploid (Crandall and Brock, 1968; Section 11, B.Z., p. 321).Yet, under certain physiological conditions, the diploid can be induced to synthesize either 5f or 2 If. Details of these inducing conditions have been summarized recently (Crandall and Caulton, 1975) and, therefore, will be reviewed here only briefly. Induction of 5f occurs in the diploid when cells are grown: (i)to late stationary phase in a medium containing 0.67% yeast extract; or (ii)to late stationary phase in synthetic minimal medium containing 0.4 mM V+ or V5+ (induction of 5f by vanadium is enhanced by oxygen limitation); or (iii)to limiting cell density in Ca2+-and Mg2+-freesynthetic minimal medium; or (iv) to limiting cell density in vitaminlimited medium, then resuspended in a buffer containing phosphate, Mg2+,glucose and pyridoxine and aerated for 2-4 h. Induction of 21f occurs in the diploid when cells are grown: (i) to late exponential phase in a medium containing 0.67% yeast extract + 1 mM Na,EDTA; or (ii) to stationary phase in a medium containing yeast extract, then washed, resuspended and grown in a synthetic minimal medium lacking trace metals. I t is apparent that the differential induction of 5f or 2 I f is controlled by the metal-ion concentration; high concentrations of metal ions in-

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duce synthesis of 5f whereas low concentrations induce synthesis of 2 1f. Since these inducing conditions have no effect on haploid agglutination (Crandall and Caulton, 1975), they must affect the diploid regulatory mechanisms rather than synthesis of the factors themselves. On the basis of these observations, the model proposed by Crandall and Brock (1968) for mutual regulation of haploid genes in the diploid is still favoured. In this model, regulatory genes carried by each haploid genome operate in apposition to repress mating functions in the diploid. For example, strain 21 is visualized as carrying a structural gene for synthesis of 21f and a regulatory gene for repression of synthesis of 5f and vice versa. This mutual repression in the diploid may be disrupted by the non-physiological conditions already listed in which growth limitation induces either 5f or 2 1fin the diploid. b. Conjugation and Triploid Formation. Although the induced diploid can agglutinate with one or the other haploid, it cannot continue with the rest of the steps in conjugation which apparently remain repressed. Even though the diploid does not usually either agglutinate or conjugate, rare diploid x haploid matings do occur (at frequencies of about lo-* to Crandall and Caulton, 1975).As expected, triploids are non-agglutinative and non-maters just like the diploid. At present it is not known whether these rare diploid matings have occurred following mitotic segregation of a diploid homozygous for mating type or as a result of a physiological breakdown in the regulatory mechanisms governing haploid conjugation genes in a normal diploid. Tetrad analysis of these triploids is being performed to distinguish between these possibilities.

3 . Meiosis and Sporulation

a. Physiotogical Conditions Inducing Sporulation. Hansenula wingei sporulates after 7-9 days at 25OC on 2.5% (w/v)malt-extract medium containing 3.0% agar (Wickerham, 1951). At the same time, pseudohyphae are produced at the edges of the colonies. If the diploid is precultured on restrictive growth medium (0.1%glucose + 0.02% yeast extract + 0.02% Bacto-peptone + 2.0%agar) for two days at 25OC prior to transfer to malt-extract agar, sporulation is enhanced (Herman, 197 la). Neither of these two media supports vigorous growth. For the first gene, two complementary alleles, mat]' and matl-, are postulated.

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M. CRANDALL, R. EGEL AND V. L. MACKAY

Strains derived fiom the original 5 x 21 diploid (N.R.R.L. Y2340) sporulate poorly (0.1-1.0%) and will sporulate only on agar. In contrast, strains that _sporulate well yield a higher percentage of sporulation (30%)in a shorter time ( 2 days) in 2.5% malt extract broth. Good sporulating strains have been selected (R. W. Wood and M. Crandall, unpublished observations) from crosses between genetically-marked strains 5 and 21 and wild type strains V I A and V l B (Herman et al., 1966) derived fi-om a diploid that sporulates with a high frequency. The standard sporulation procedure is as follows (L. J. Lawrence and M. Crandall, unpublished observations). A stationary-phase diploid culture is diluted 1 : 100 into fresh growth medium containing 2.0%glucose, 0.7%yeast extract and 0.5% KH,PO,. This preculture is aerated at room temperature (23-27OC) for 20-24 hours (midstationary phase). The cells are harvested, washed twice and resuspended in malt-extract broth at the same cell density. Usually 2 ml of this suspension are aerated in an 18 mm-diameter test tube on a roller drum at room temperature. After 12 hours, the cells are committed to sporulation; after 2 days, the asci are mature; after 3 days, the asci rupture. The substance in Difco malt extract that induces sporulation in H. wingei may be a metal ion since sporulation is inhibited by Na,EDTA (2 mM). The inducer is dialysable, stable to autoclaving and not extracted into petroleum ether. Two peaks of sporulation are observed, at pH 5 and pH 9. The inducer is removed from malt extract broth by the diploid but not by the haploids. However, if the diploid culture is autoclaved, the inducer is released into the spent broth. An electron-microscope study of ascosporogenesis in H. wingei has been published by Black and Gorman (1971). b. Responsive Stage in the Cell Cycle. Sporulation occurs best with stationary-phase cultures. The diploid cells are probably in G1 at this time.

4. Mitotic Recombination Heterozygous diploids segregate homozygous recombinants during vegetative growth with a frequency which is locus dependent (about lo-,; Crandall and Richter, 1973). These high frequencies of mitotic recombination may be a consequence of somatic pairing of chromosomes observed in the diploid of this yeast (Crandall and Robinow, 1973). The influence of mat on this process has not been studied.

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III. Schirosaccharomycespombe A . MATING-TYPE LOCUS

1. Mating- Type Alleles

Most of our present knowledge about the mating types of Schizosacch. pombe comes from work by Leupold (1950, 1955, 1956 and 1958)with Schizosacch. pombe strain liquefaciens isolated by Osterwalder (cited in Sloof, 1970). Basically, three mating types can be distinguished. One mating type is homothallic, showing both conjugation and sporulation in a clonal culture and is designated h9’ for 90% sporulation on malt agar. Two other mating types are heterothallic and of complementary mating types h+ and h-. Both h+ and h- are self sterile but hybridize with each other and also with hm (Leupold, 1950). In later studies, Leupold (1958)discovered two subtypes among the heterothallic strains with “+” activity. These two strains, termed h+N for “normal” and h+R for “recombination-derived”, can be distinguished by the different mutations they undergo. “Normal” h+ strains have the wild-type genetic configuration at the mating-type locus (mat) whereas in “recombination-derived” h+ strains the mat locus is altered as a result of recombinational events during meiosis or mitosis. Owing to these differences in constitution at the mat locus, hfN mutates to h9’ whereas hfR mutates to h- (see p. 332). The h- mating type has been renamed h-‘ by Gutz and Doe ( 1973a)because it is stable (whether it is wild type or derived from h+R)and does not mutate to any other mating type. In contrast, hg0canmutate to h+Nor to h-‘. Such complicated genotypic interconversions between mating types can be rationalized in terms of a model in which mat is visualized as being a complex locus consisting of several alternative or compatible genes which are closely linked. Thus, the above-mentioned mating types map together in a narrow region on linkage group 11. Within this mating-type locus, two genes are recognized which are 1.1 map units apart (Leupold, 1958).These genes are now called matl and mat2 (Gutz m d Doe, 1973a)and the following allele combinations are assigned to the original mating types described by Leupold (1958):

hfN : matl+ mat2+ h-’ : matl- mat? h+R : matP mat2” h90

:

matl- mat2+

M. CRANDALL, R. EGEL AND V. L. MACKAY

332

For the second gene, a functional allele, mat2+(which resembles matl+) and an inactive allele, mut2O, are postulated. Both conjugation and meiosis are promoted- by the complementary interaction between mutl- and either matl+or mut2+.The presence of both a "+" allele and a "-"allele in the same cell explains the homothallic behaviour of the h9' strain. A new term, k+, is proposed for h9' (Egel, 1976) to signif) the homothallic mating type but the original designation, h9', will be used in this review. These allele combinations are consistent with the observed mutational and recombinational behaviour of the mating types. For example, it was stated that h+N mutates to hM whereas h+R mutates to h-S. Both of these mutations are thought to result from the same change at mutl as follows :

1

1

1

I

h-S : Similarly, these schemes can account for the mutation of h9' to PNor to kS. However, the combination of mutl- mat2O is apparently stable does not mutate to other mating types. since Interconversion between mating types by recombination between mutl and mat2 is envisaged as follows :

In this diagram, hybrids between the mating types indicated on one side of the double-headed arrow yield recombinant spores indicated on the other side of the arrow. These mutational and recombinational interconversions are explained well by this two-gene model at the mat locus. However, the fact that mutation from to h+ is irreversible is not understood. It could be explained if the genetic information of matl- is present in a concealed

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fashion in h+R in addition to mat]+ and is uncovered by some unusual genetic event which also results in the loss of matl+. An unusual genetic structure at mat1 may also explain the hot spot ofmitotic recombination associated with matl-. Strains homozygous for this allele, such as hgO/h-‘, undergo mitotic crossing over in the vicinity of mat1 approximately once in every 45 cell divisions (Angehrn and Gutz, 1968; Gutz et al., 1974).Further evidence for genetic complexity at the mat locus is indicated by the observation that some diploid strains give rise to recessive lethal factors associated with mat2 (h90/h-S -t h90/h-’ or h+N/h90 h+N/h90’;1 = lethal factor). Such mutations were observed about once every 360 cell divisions (Gutz and Angehrn, 1968; Gutz et al., 1974).These lethal factors were recognized by the regular segregation of two spores that germinate and undergo one or two divisions but do not form colonies. Their nature remains to be resolved, but the possibility of a loss of chromosome I1 or an extensive deletion was excluded. While the original h-S strains are stable, Gutz and Doe (1973a) described unstable h-u strains which mutate to homothallism or to heterothallism with “+” activity. In deviation fi-om the original authors, the instability of h-U strains has been ascribed to altered mat2+ alleles (Meade, 1975; Egel, 1976). Based on new data (Egel, 19761, the mat locus of Schizosacch. pombe has been re-interpreted as follows: m u t P ( = m a t l - ) is the gene for 2 ~ma@+) is the basic genetic entity specifying mating type o ; m ~ t (= mating type o ; the latter segment can be transposed and inserted into m a t P , thereby abolishing the former o function; the insertion is notated as m a t P : : m a W (= matl+);usage of matlp for this insertion is possible though not encouraged; precise excision of the inserted segment, restoring o function of ma@, explains reverse mutations; at either position, mat2P can undergo “changes of state”, resulting in partial reversible inactivation. The unrevised nomenclature is still used throughout this review. Other mutations, also mapping at the mating-type locus, will be referred to later in this review (see Section 111, A.2, p. 335 and Section 111, B.6, p. 344).

-

2. Homothallism versus Heterothallism Schizosaccharomyces pombe can exhibit either homothallic or heterothallic mating behaviour, depending on which alleles of the mating-

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M. CRANDALL, R. EGEL AND V. L. MACKAY I3

-

"+"

I,

ACTIVITIES

ACTIVITIES

mlo)

m=

Agglutination

Cell wall deformations

Prezygotic fission

@

Conjugation

t ---t

C T j2 n

Cell wall deformations

Meiotic commitment Meiosis (zygotic or azygotic)

me14

FIG. 5. Genes affecting conjugation, meiosis and sporulation in Schizosaccharomyces p m b e . Key: (a)Wild-type genes. mat I-: mating-type allele determining "-"specificity; mot]+or mut2+: mating-type alleles determining "+" specificity. (b)Mutant genes and their defects. mmol to mmo4: affect switching between mat1 and mat2 expression in h" cells; m m l : converts h w to pseudo-h+a which does not self-agglutinate and does not agglutinate with h+ but is cross fertile with h-; maml also converts h- to sterile; meiosis is observed in homozygous mamllmaml diploids; mapl: converts h" to pseudo-h-" which does not self-agglutinate and does not agglutinate with h- but is cross fertile with h+; map! also converts h+ to sterile; when map1 is present in the homozygous condition, meiosis is blocked in diploid cells but this block is reversed by cell contact with h + or h" cells; mam2: converts hw to pseudo-h+ which is still self-agglutinable and

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type locus are present. The wild type h90 strain is homothallic because it contains both the m t l - and mat2+alleles. However, only one of these genes appears to be active in any haploid cell (Gutz et al., 1975; Egel, 1976; see below and Section 111, C.3, p. 348). Switching of gene expression fiom matl- to m t 2 + activity or vice versa must occur during cell division since conjugation between sister cells is observed frequently in hW strains (Leupold, 1950). When h9’ colonies are exposed to iodine vapour (a treatment that stains an amylose-like substance in the ascospores a dark blue colour; Leupold, 19551, the colonies are homogeneously stained blue. This uniformity of sporulation results from frequent switching of mating-type expression (that does not allow the development of either “+” or “-” sectors) followed by self-mating and zygotic meiosis. The hequencies of switching between compatible mating-type potentials in h90 are decreased by certain mutations called “speckled” and “mottled”. Homothallic strains carrying these mutations produce colonies containing sectors of cells expressing one or the other matingtype activity resulting from several generations of growth of “+” or - cells without a change in mating type (H. Gutz, personal communication). In these mutant colonies, the few sporulated areas between the “+” and “-” sectors appear blue against an iodinenegative background. H. Gutz and J. Meade (personal communication) have analysed these two phenotypes genetically. The “speckled” mutation maps at the mat locus, but its precise position is not certain. The “mottled” iodine reaction can result from the action of four unlinked genes, termed mmo (mating type modifiers; Gutz et al., 1975). Their normal gene products seem to mediate the spontaneous alternation between mtl and mat2 expression, which leads to homothallism 66

9,

agglutinates with both h+ and h- but does not conjugate with h+; cross fertile with h-; mam2 also converts h- to sterile; meiosis observed in homozygous mam2/mam2 diploids; m a p : converts hW to pseudo-h- which is still self-agglutinable and agglutinates with both h+ and h- but does not conjugate with h-; cross fertile with h+; map.? also converts h+ to sterile; meiosis observed in homozygous rnap2lmap2 diploids; &sI: no dissolution of separating walls in prezygotes; extended growth of pre-conjugation tubes; haploid fission resumed on fresh medium; m e i l : maps at md2; meil to mei3: no premeiotic DNA synthesis; extended growth of zygotes; diploid fission resumed on fresh medium; mil: no meiotic division; diploid fission potential lost; mesl:* no second meiotic division; spol to spol8: no spore formation. ‘The terms pseudo-h+ or pseudo-h- refer to heterothallic mating behaviour resulting from the presence of map or mam genes in an hWcellwhich contains both matl- and mt2+genes. The gene symbols mei and me5 replace mel and mell, respectively (Egel and Egel-Mitani, 1974).

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M. CRANDALL, R. EGEL AND V. L. MACKAY

(Fig. 5).They can act independently since, in double mutants carrying two mmo mutations, the frequencies of mating-type changes were decreased below the levels in the single mutants. Conceivably, the regulatory region where-the switching between mat1 and mat:! activation occurs is the site of the “speckled” mutation and is also the target of the mmo gene products. Alternative models to explain homothallism in Schizosacch. pombe do not consider mat2 as a second matingtype gene but instead suggest modifier action (Fincham and Day, 1963) or mutator specificities (Meade, 1975)of mat2+ affecting matl-. Gutz and Doe (1973b, 1975)have extended their studies on matingtype regulation to three other Schizosacch. pombe strains from different geographical origins. Two of these wild-type isolates were apparently homothallic of “mottled” colony type, i.e. they changed frequently from “+” to “-” activity and vice versa whereas the third strain was found to be heterothallic, although rare mating-type changes were also observed with this strain. Hence, it appears that the aforementioned mutant phenotypes are not just laboratory freaks; rather they represent different attempts to ensure sexual reproduction, each of which is adaptable in Nature. In concluding this section, it should be noted that mutations from homothallism to pure heterothallism can also occur at additional genes unlinked to the mating-type locus. Such mutants will be discussed in the context of conjugation (see Section 111, B.6, p. 344). 3. Functions Controlled by the Mating- Type Locus

The key events of sexual reproduction, conjugation and meiosis, are under the control of the mating-type locus in Schizosacch. pombe. This species exists primarily in the haplophase because conjugation and meiosis occur under identical conditions and usually in close succession. Therefore, the separation of haploid uerJus diploid functions, as discussed in Sections I1 through IV of this review, is not the best organization for Schizosacch.pombe. However, the advantages of a common outline in the review are thought to outweigh this possible source of confusion. B . HAPLOID FUNCTIONS

1. Initiation ofConjugation

a. Physiological Conditions Inducing Conjugation. Both conjugation and sporulation in fission yeasts occur when conditions cease to favour

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growth. Genetic crosses are performed routinely on malt-extract agar. A nitrogen-fkee agar-containing medium has also been used occasionally (Leupold, 1970; Gutz et al., 1974). Liquid media, however, are better suited for physiological analyses of conjugation and sporulation. The following observations on conditions influencing conjugation have been made using h+ and h-, or h9' strains. Aeration is required. Zygotes are not formed in yeast extract broth. Malt-extract medium yields 1 0 4 0 % conjugation. The optimal pH value for mating is between 4 and 6. In a synthetic medium containing low concentrations of nitrogenous nutrients (Egel, 197 11, conjugation starts 2.5 hours after the nitrogen source (aspartate) has been consumed. The percentage conjugation is better in this medium, proceeding to a final 3040%. Zygote formation can be stopped by adding aspartate. Depletion of nitrogenous compounds is accentuated by shift-down experiments from a nitrogen-enriched to a nitrogen-free synthetic medium (Egel and Egel-Mitani, 1974). In contrast, starvation for sugar, vitamins, phosphate, required amino acids or nucleotides does not induce conjugation. However, the carbon source does exert some influence on conjugation. A distinct optimal zygote yield is reached at 0.8-1.0% glucose concentration. Below a concentration of 1%, maltose is comparable to glucose but zygote yield is not decreased in higher concentrations of maltose. The highest yield of zygotes (80%) was obtained in 1%fructose medium (R. Egel, unpublished observation). Apparently, glucose repression occurs at concentrations higher than 1%, and the effects of other sugars are dependent on the metabolic rates at which these are converted to glucose or glucose metabolites within the cells. While working with a different Schizomcch. pombe strain (N.C.Y.C. 132), which is homothallic but unable to mate with any of Leupold's standard mating types, Calleja and Johnson (1971) and Calleja (1973) observed conjugation in stationary-phase cultures grown in malt extract, especially after a shift from anaerobic to aerobic conditions. b. Responsive Stage in th Cell Cycle. Vegetative cells of Schizosacch. pombe lack a measurable G1 phase during exponential growth (Bostock, 1970) since DNA synthesis for the next nuclear division is over by the time the cells separate. However, if a random population of cells, predominantly in G2 phase, is shifted to nitrogen-free sporulation medium, this shift is followed by another round of mitotic cell division, not accompanied by DNA synthesis, before agglutination and conjugation commence (Egel and Egel-Mitani, 1974). The emerg-

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M . CRANDALL, R. EGEL AND V. L. MACKAY

ing cells accumulate in G1 phase. In agreement with this observation, prezygotes of a mutant blocked at cell fusion (fusl;Bresch et al., 1968; see Fig. 5, p. 334) are held at this stage. Hence, only G1-phase cells are able to conjugate. Cells physically separated according to their cellcycle position have not been studied. However, it is known that cells of Schizosacch. pombe are capable of mating over a period from 0.1 to 0.7 of the cell cycle (Streiblova and Wolf, 1975). c. Inhibitors of Conjugation. Conjugation is inhibited by nitrogencontaining compounds such as ammonium salts, amino acids or nucleotides. Glucose causes some repression, especially at concentrations above 1% (Egel, 197 1; R. Egel, unpublished observations). Calleja ( 1974b) observed a slight stimulation at intermediate concentrations (lo+ M) of cyclic AMP, but conjugation was inhibited at higher concentrations. Cycloheximide at low concentrations ( 10 pglml) is a potent inhibitor of conjugation, if it is added before agglutination has started. At the time of agglutination, about one hour before zygote formation, cells are no longer sensitive. However, higher concentrations of cycloheximide ( 100 pglml) were inhibitory until 10 minutes before conjugation (Friedmann, 1974). Other inhibitory agents are temperatures above 3 l0C, proteolytic enzymes and detergents (see Section 111, B.2, p. 345). Conjugation is not inhibited by 2-deoxyglucose at a concentration that causes lysis during cell division (250 pglml; Kroning and Egel, 1974). Calleja (1973) found that conjugation in Schizosacch. pombe is inhibited by respiratory poisons such as potassium cyanide, sodium azide and dinitrophenol and by the antibiotics cycloheximide, nystatin, neomycin and puromycin. However, polymyxin and chloramphenicol were ineffective when added at the time of induction (shift to aerobic conditions), but were inhibitory when added earlier. Several inhibitors of bacterial wall growth were ineffective at all times. 2. Cellular Recognition a. Constitutive versus Inducible Agglutination. Conjugation in Schizosacch. pombe is preceded by a sexual agglutination or flocculation reaction (Egel, 197 1; Calleja and Johnson, 197 1). Neither hf nor hcells agglutinate in clonal cultures. These strains acquire the ability to agglutinate with cells of the opposite mating type only after growth in a mixed culture- for at least one generation (Egel, 1971). The homothallic h90 strain, on the other hand, self-agglutinates under sporula-

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tion conditions since both mating-type activites are inherent in such a culture. Therefore, agglutinability is clearly inducible in the heterothallic strains of Schizosacch. pombe but cells of homothallic strains are self-agglutinable either because both agglutination factors are synthesized by each cell or because different cells express one or the other agglutination activity. b. Dzfusible Sex Factors. The chemical basis of the cellular interactions occurring during induction of agglutination has not been determined, nor has complementary induction of agglutinability been achieved in the absence of cell contact. Apparently constitutively synthesized, diffusible sex factors are not involved in induction of agglutination. However, there is evidence for the existence of diffusible compounds inducing some sexual responses in Schizosacch. pombe. For example, the exchange of culture medium from h+ to h- cells and vice versa had two effects : (i) conjugation occurred sooner with cells pretreated with medium from the opposite mating type; and (ii) electrophoretic patterns of proteins from pretreated cells revealed degradation of soluble proteins (Friedmann, 1974; K. Friedmann, unpublished observations). Another mating-type effect became noticeable when DNA synthesiswas compared in separate and mixed cultures of h+ and h- cells after a shift to nitrogen-free sporulation medium (Egel and Egel-Mitani, 1974). Synthesis of DNA was retarded in the mixed culture. In Sacch. cerevisiue, a similar effect is mediated by diffusible pheromones (see Section IV, B.2, p. 359). c. Complementa?y Agglutination Factors. Agglutination is complementary in the sense that cells of both heterothallic mating types are required. But, since agglutinability is not constitutive, it might be difficult to demonstrate that complementary agglutinins are located on the surfaces of opposite mating-type cells as in H . wingei (see Section 11, B.2, p. 318). Such evidence would be even more difficult to obtain in homothallic strains and therefore no attempts have been made to study the agglutinins of Schizosucch.pombe. Some data are, however, available on the aggregation reaction itself (Calleja, 1974a). Agglutinability is resistant to washing in de-ionized water, temperatures up to 5OoCand changes in pH value down to 2 or up to 1 1. Reversible deflocculation is effected by brief ultrasonic treatment, by heating to 80°C, by lowering the pH value to 1 or by raising the pH value to 13, by hydrogen bond-breaking agents such as

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M. CRANDALL, R. EGEL AND V. L. MACKAY

guanidinium chloride or urea, or by strong detergents such as sodium dodecyl sulphate. However, reflocculation of cells dispersed by any of these treatments occurs slowly. Free counterions seem not to be essential for reflocculation, since cells packed by centrifugation agglutinated in de-ionized water. Yet, spontaneous reflocculation was enhanced by CaZ+ions. An acceleration in re-agglutination of ultrasonicallydispersed zygotes has also been observed in 0.05 M sodium citrate (R. Egel, unpublished observations). This treatment has been used to purify zygotes initially present at 1&30% to a final purity of greater than 90%after 2-3 repetitions. Zygotes are at least twice as large as unmated cells, and may agglutinate faster for this reason alone, but they may also have accumulated higher numbers of agglutinin molecules per unit surface area. Deflocculation becomes irreversible after treatment with proteases or with sulphhydryl reagents (Calleja, 1974a). This is evidence that the agglutinins from Schizosucch. pombe are proteins whose activity depends on disulphide bonds as is the case for the agglutinins from H . Wingei (see Section 11. B.2, p. 318). (d) Suface Filaments.Day and Poon ( 1975) describe hair-like hollow filaments that penetrate cell walls and cytoplasmic membranes seemingly as communication channels between sexually agglutinating cells of Ustilago violacea. Similar surface filaments have been observed on cells of Schizosacch. pombe (Poon and Day, 1975; Y o 0 et al., 197 1 ; Calleja et al., 1976). These surface organelles might be involved in flocculation and might also serve to transmit mating type-specific signals during induction of mating. Another sex-related induction phenomenon presumably mediated by these surface filaments will be discussed in Section 111. B.6 (p. 345). I t involves a mutant affected in mating-type expression ( m a p l , see Fig. 5 , p. 334).

3. Cell Fusion

a. F o m t i o n Ofthe Conjugation Tube. Electron micrographs of early stages in conjugation in fission yeasts have been published only for Schizosacch. octosporus (Conti and Naylor, 1960) but these agree with observations made in other species of Schizosaccharomyces as well as in other yeasts. Cells first adhere to one another with a portion of the stainable, probably proteinaceous, fringe surrounding the electrontransparent cell wall and then develop protuberances at the site of con-

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tact. The separating walls between adjacent cells become thinner at the site of the protuberances and eventually dissolve, usually starting from the central portion. The fused protuberances then form the conjugation tube, which widens until it reaches a diameter similar to those of the conjugating cells. This is also seen in the photomicrographs in Fig. 6

(p. 342). In Schizosacch. pombe, both cells usually initiate conjugation at their ends, especially during the early phase of a synchronized mating. Thus, the resulting zygotes are extended or crescent-shaped. Occasionally, T-shaped. zygotes arise by initiation of conjugation at the long side of a cell. Cell ends, which also serve as the sites of wall extension during vegetative growth (Johnson, 1965; Streiblovi and Wolf, 1972; Biely et al., 19731, are evidently preferred as recognition sites for conjugation. Streiblovi and Wolf (1975) have analysed zygotes of Schizosacch. pombe by cell-wall fluorescence staining (Fig. 6). This technique permits distinction between growing and non-growing cell poles. In their material, 62.5% of the cells had conjugated at the “secondary pole”, which is derived from the last division septum, and 37.5% at the ”primary pole” which is derived from one of the poles of the mother cell. Hence, both poles are able to initiate conjugation but the choice is biased in favour of the more recently synthesized wall material. Several enzyme activities must be involved in formation of the conjugation tube. The most striking alteration is dissolution of the separating walls. This reaction alone would be destructive to the conjugating cells unless the remaining walls were firmly connected by concurrent synthesis. Interestingly, certain glycoside hydrolases display glycoside transferase activities as well (Nisizawaand Hashimoto, 19 70). If such a bifunctional enzyme were involved in cell fusion, it could dissolve part of the walls by hydrolysis and cross-link the apposed remainders by its transferase activity. How the lytic activities of both partner cells are localized at the fusion zone is not understood. One possibility is that an enzyme has to be activated by mating type “+” and “-”-dependent substances simultaneously. In a search for conjugation-specific enzyme activities, Fleet and Phaff (1974) observed a four-fold increase in p-glucanase activity following induction of agglutination in the homothallic hW strain. This enzyme may be responsible for the carbohydrate-releasing autolytic activity formed during conjugation (Kroning and Egel, 1974). Since carbohydrate-releasing activity was also present during prezygote for-

W

P N

a

FIG. 6. Successive stages (a, b, c) of conjugation in Schizosaccharomyces pombe. Cell walls were stained with primulin and visualized by fluorescence photomicrography. Reproduced by courtesy of E. Streiblovi.

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macion in a fusion-defective homothallic strain ( f u s l ; see Fig. 51, it may be involved in the initial wall extension rather than in cell-wall dissolution during fusion. In agreement with this idea, wall extension in fusl prezygotes is seen to be exaggerated with respect to wild-type zygotes. These mutant prezygotes, therefore, resemble the shmoos seen in Sacch. cereuisiue which are physiologically blocked at this same early stage of conjugation when local wall extension is also seen (see Section IV. B.2, p. 357). In Schizosacch. pombe, prezygotic cells of mutant fusl can be easily detached from one another because their walls are not connected permanently. It is not known whether dissolution depends on prior connection, whether progressive dissolution is a prerequisite to stable connection (by disclosure of cross-linkable sites), or whether both processes share a common mechanism. It is clear however that, during conjugation, lytic activities are normally counteracted by synthesis of new wall material. When glucan synthesis was inhibited by 2-deoxyglucose (250 pglml, a concentration sufficient to cause lysis of dividing cells), lysis of the prezygote did not occur but, rather, wall extension was augmented in that a swelling in the area of the conjugation tube was induced in wild-type zygotes as well as in fusl prezygotes (Kroning and Egel, 1974). Thus, the cross-linking activity in conjugation is more resistant to 2-deoxyglucose than the same activity involved in growth. 4. Nuclear Fusion versus Heterokaryosis In prezygotic cells, the haploid nuclei move into the initial protuberances thereby facilitating nuclear fusion once the conjugation tube is formed. However, when diploid cells conjugate (Fig. lb, p. 3 1 11, nuclear fusion is not always accomplished. Instead, both diploid nuclei can undergo meiosis independently (“twin meiosis”; Gutz, 1967a, b ) leading to eight-spored asci with no genetic exchange between the partner nuclei. The fact that twin meiosis is observed in crosses of h’lh’ and h-lh- strains, each of which is incapable of sporulation, suggests the operation of some cytoplasmic interactions inducing meiosis in both the nuclei without fusion. The frequency of twin meiosis is particularly high when conditions become marginally in favour of conjugation (e.g. on yeast-extract agar of certain batches; Gutz, 1967a). Conceivably, the conjugation tube widens too slowly, thereby preventing nuclear fusion but allowing cytoplasmic mixing which promotes meiosis in the separate diploid nuclei.

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5 . Zygotic Fission

In wild-type strains of Schizosacch. pombe, zygotes are formed only in depleted medium and,- since this same medium induces sporulation, the zygote usually undergoes meiosis immediately. If, however, zygotes are transferred to fresh medium, a few of them divide and diploid fission will continue as long as the medium sustains vegetative growth (see Section 111, C. 1, p. 347). Liquid medium containing yeast extract is usually used for diploid growth since yeast extract apparently contains an inhibitor of sporulation and also the distribution of components of the medium is more uniform in liquid. On agar-containing medium, diploid clones become contaminated with haploid spores probably because nutrients are distributed unevenly throughout the colony. Diploids containing mutations that block before meiotic commitment (e.g. mei3; see Fig. 5 , p. 334) undergo zygotic fission with a high frequency. In such zygotes, division proceeds according to the usual growth pattern of fission yeasts; elongation starts at the poles of the conjugated cells, and a transverse septum marks the first diploid division (Egel, 1973a). 6. Mutant Genes AJecting Conjugation Mutations at the mating-type locus have been discussed (see Section 111, A . l , p. 331) and mutations regulating the expression of the mating-type alleles (termed mmo) were presented in Section 111, A.2 (p. 335). Other mutations affecting conjugation fall into several classes. For example, the majority of non-sporulating isolates are also sterile (Bresch et al., 1968). Unless conditionally sterile mutants could be isolated, such strains are not suitable for genetic analysis (see however, Section IV, B.6, p. 374 for a discussion of low-frequency mating of sterile mutants in Sacch. cerevisiae). Strains of another mutant group in Schizosacch. pombe have changed from homothallism (h9? to heterothallism, although the mating-type locus is unifnpaired. These strains are defective at additional genes, termed mating type auxiliary genes, which affect either the plus (map)or the minus activity (mum) exclusively (Egel, 1973b; Fig. 5 , p. 334). Mutations in these genes abolish one mating-type activity, but leave the other operative in homothallic strains. Therefore, they lead to sterility in heterothallic strains that contain only one mating-type allele. Two of these mutations (map1and

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maml) prevent homothallic self-agglutination, and may therefore affect either agglutinin expression or earlier induction steps (Fig. 5, p. 334). Two other mutants (map2 and mam2) are still able to self-agglutinate, but later stages of conjugation do not occur (Fig. 5, p. 334). Hence, after agglutination, a new set of mating type-specific interactions are essential for initiation of conjugation. These interactions, governed by rnap2, mum2 as well as maml, are not essential for meiosis in diploid cells. The functioning of mapl is, however, needed for meiosis. This block in meiosis is unlikely to result from a defective agglutination factor. Moreover, the missing map1 function can be restored by cell contact with mapl+h+ or mapl+ h90 cells (Fig. 5, p. 334). Induction of meiosis and sporulation in mapllmapl h9O/hW cells by mapI+ h+ or mupl' h90 cells takes place even if both strains carry thefu.rl marker to prevent conjugation. Therefore, the mapl -specific induction signal can be transmitted across walls and membranes. Induction of meiosis in diploid map1lmapl cells was observed only in mixed culture with h+ or h90 cells. Asci were not detected if both strains were separated by membrane filters on agar plates, or by dialysis tubing in liquid culture. When closely spaced colonies were treated with iodine vapour to detect sporulation, only a thin reaction line appeared between adjacent colonies where they actually touched. Hence, cell contact seems to be necessary for induction. The sensitivity of this induction phenomenon to various treatments was tested in comparison to conjugation and to meiosis in wild-type strains. Meiosis in diploid strains is more resistant than conjugation to inhibition by proteolytic (pronase) digestion, detergent (sodium dodecyl sulphate) or elevated temperatures. The mupl-specific induction of meiosis is as sensitive as conjugation to these treatments (Fig. 7). Conceivably, a common component is involved in the initiation of conjugation and in the induction of meiosis. This may be a diffusible pheromone or a transmission organelle (possibly a surface filament), or even one of the agglutination factors. Observations concerning the map and mum mutants are best explained by assuming that two parallel pathways of complementary "+" or "-"reactions lead to conjugation, each one being restricted to cells expressing one of the heterothallic mating types. These reactions concern initial induction of agglutination, wall extension and looselybound pair formation. A common reaction is needed for both initial

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Concentration of pronose or sodium dodecyl sulphate (mg/ml)

Temperature ("C)

FIG. 7. Sensitivity of meiotic induction in diploid map1 cells compared with zygotic or azygotic meiosis in haploid or diploid wild-type cells of Schizosaccharomyces pombe. Abscissa: (a) pronase; (b)sodium dodecyl sulphate; (c) temperature; ordinate: zygotic or zygotic sporulation (asci/ml). Cells of the strains mapllmupl fusIIJiLs1 h W / h W +fusl h+ ( V ; induction of azygotic meiosis), h+/h- (0;zygotic meiosis) and hm ( 0 ;conjugation and zygotic meiosis) were shifted to nitrogen-free sporulation medium (Egel and Egel-Mitani, 1974). Pronase or sodium dodecyl sulphate was added at the indicated concentrations, the samples were incubated at 3OoC, or at the indicated temperature, and asci were counted microscopically.

induction of conjugation and meiosis, whereas the other reactions are conjugation-specific. The normal fusl+ gene product (see Section 111, B. 3, p. 343) is usually expressed by both heterothallic mating types, although its expression by only one partner is sufficient to accomplish cell fusion. If both partners carry the fwI mutation, conjugation is arrested at the wall-extension stage. Hence, the fusl mutation can be considered recessive, even in the context of haploid gene expression, because the phenotype of the other cell can reverse the mutational defect. Conjugation is also modified by mil, mei2 and met3 mutations (see Section 111, C.3, p. 349) that block meiosis in the early stages. Zygotes of these mutants do not become committed to the meiotic pathway, and the cell-wall changes associated with formation of conjugation tubes are not halted as usual (Egel, 1973a). Instead, elongation continues and additional conjugation tubes are occasionally formed. Zygotes of +is group will frequently undergo fission and form diploid colonies after being transferred to fresh growth medium.

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DIPLOID FUNCTIONS

1. Heterozygosity versus Homozygosity at the Mating- Type Locus

The occurrence of diploid cells is the exception rather than the rule in Schizosacch. pombe because conjugation is usually followed immediately by meiosis and sporulation. However, methods are available for establishing diploid clones (see Section 111, B.5, p. 344). Primary diploid clones selected from zygotes are conserved by suppressing sporulation by growth in liquid yeast-extract medium. Diploid clones are also generated by self-diploidization, which can occur in any haploid strain. This process provides an explanation for the rare occurrence of sporulating segregants in the conjugation-deficient fusl mutant (Egel, 1973a). In addition, rare two-spored asci may contain diploid spores. In a temperature-sensitive mutant affected early in nuclear division ( c d c S 3 3 ;Nurse et al., 19761, frequencies of both endodiploidization (diploidization without conjugation) and two-spored asci are increased. After cells of a cdc2 h90 strain have been incubated at 35OC, they produce diploid colonies if plated on growth medium, or two-spored asci containing diploid spores if kept in sporulation medium at 3OoC (R. Egel, unpublished observations). Thus, the adverse effect of this mutation is comparable to the consequence of colchicine inhibition in higher eukaryotic cells, i.e. chromosomal duplication occurs without nuclear or cellular division. Meiosis and sporulation, the 'most conspicuous diploid functions, are promoted by the same combinations of mating-type alleles which promote conjugation between haploid cells. These compatible combinations are h'lh-, h90/h90, h9O1hf and h90/h-. On the other hand, diploid cells homozygous for one of the heterothallic mating types, h'lh+ or h-lh-, resemble haploid cells in that they cannot initiate meiosis without prior conjugation. 2. Repression of Haploid Functions

a. Sexual Agglutination. In any strain of Schizosacch. pombe, sexual agglutination is repressed during vegetative growth. Yet, when shifted to sporulation conditions, diploid strains become self-agglutinative. Thus, self-agglutination is never fully repressed in the diploid when heterozygous for one of the compatible mating-type combinations. The lowest degree of agglutination is expressed by h+lh- strains.

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b. Conjugation and Triploid Formation. Triploid zygotes may arise occasionally by hsion of a diploid with a haploid cell (Leupold, 1956). Conjugation between -two diploids is also possible. The resulting zygote may undergo tetraploid meiosis after nuclear fusion or twin meiosis if karyogamy is impeded (Section 111, B.4,p. 343, Fig. lb, p. 31 1 ) . A low frequency of zygotes is observed in h+lh- cultures. These zygotes may have formed by matings between h+lh+ and h-lh- cells that arose by mitotic recombination or possibly also by matings between two h'lh- cells. Higher zygote frequencies are observed in strains containing one or two h90 alleles, and may be explained in krms of active and inactive mat alleles. Apparently, mat1 - and mat2+ alleles in cis position cannot be active simultaneously (see Section 111, A.2, p. 335). Therefore, the sexual activity of a diploid h9O/hW cell (matlmat2+/matl- mat2+) would depend on which of its mat alleles is activated in trans position. If the active alleles are matl- on one chromosome and m t 2 + on the other, the diploid cell will sporulate azygotically. If, however, both matl- or both mat2+ alleles are activated, then the respective diploid cells express one or the other heterothallic mating potential. If these diploids meet partner cells expressing the opposite mating potential, they conjugate and the tetraploids sporulate zygotically. 3. Meiosis and Sporulation a. Physiological Conditions Inducing Sporulation. Sporulation and conjugation in Schizosacch. pombe are favoured by the same conditions, namely nitrogen starvation (see Section 111, B.1, p. 337). b. Responsive Stage in the Cell Cycle. When diploid strains containing compatible mating-type alleles are shifted to a nitrogen-free sporulation medium, the cells divide once mitotically, thereby passing from G2 to G1 phase with respect to DNA synthesis. This division is followed by premeiotic DNA synthesis, meiosis and sporulation (Egel and Egel-Mitani, 1974). Hence, diploid cells initiate meiosis and sporulation at the G1 phase of the cell cycle, where there is only one copy of the diploid chromosomal complement. c. Znzuence of the Mating-Type Locus on Meiosis. Meiosis, like conjugation, is initiated only by an interaction between complementary matl- and either matl+ or mat2+ gene products (see Section 111, A. 1, p. 332). Conjugation and meiosis cannot, however, be initiated by the

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same reaction, since these processes are inactivated by different mating-type mutations. For example, m i l mutations map at mat2 of the mating-type locus and, in fact, were derived from the ma&'+ allele of the original h90 strain. One of these mutations ( m i l - B 1 0 2 ; Bresch et al., 1968)is still able to self-conjugate like its hw parent, but meiosis in the resulting diploid homozygous for meil -B102 is blocked. This haploid mutant conjugates but does not sporulate with h- (matlmat2") cells, yet it sporulates normally in crosses with h+ (matl+ mat2+) cells. Sporulation by the latter hybrid may occur as a result of an interaction between the matl- allele from the original h90 strain and the m t l + allele fiom h+ strains. Thus m i l - B I 0 2 was derived from mat2+, maps at mat2 and represents a defect in mat.?+ function in meiosis but not conjugation. Meade and Gutz (1976)and Meade (1975) have isolated additional mutations at the mat2 locus. These mutations block either conjugation or meiosis, or formation of the map1 gene product or all three functions simultaneously. Thus, it is apparent that mat2 is a complex locus responsible for at least three different sexual activities. d. Mutant Genes AJecting Sporulation. The aforementioned mil mutations in the mat2+ gene abolish meiosis when homozygous without preventing conjugation in the haploid. Another gene, mapl, affects +" function in conjugation and blocks meiosis when homozygous, but its deficiency can be supplemented by cell contact with mapl+ cells (either h+ or h90; see Section 111, B.6, p. 345).Recessive mutations in the "early" genes for sporulation, for example mil, met3 and probably also mei2, block meiosis before premeiotic DNA synthesis (Egel and Egel-Mitani, 1974). Hence, zygotes containing these mutations in the homozygous condition usually produce diploid colonies (Egel, 1973a). Mutants of a fourth gene, mek, are blocked before the first meiotic division, but premeiotic DNA synthesis proceeds normally. Mutations at mesl affect meiosis I1 and mutations at genes spol through spol8 affect spore formation. All of these mutations affecting sporulation are recessive. Their isolation was accomplished by mutagenesis in the haploid homothallic h90 strain, and scoring for non-sporulating albeit self-conjugatingcolonies (Bresch et al., 1968). Mutations in Schizosacch. pombe affecting conjugation, meiosis and sporulation can be arranged in a sequence of sexual reactions which is branched before conjugation to include the heterothallic reactions of either "+" or "-"mating-type specificity (Fig. 5, p. 334). I t is en66

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M. CRANDALL, R. EGEL AND V. L. MACKAY

visioned that these mutations represent only a few of the many enzymes or gene products involved in the life cycle. Conceivably, many of the hnctions involved in sexual reproduction are also involved invegetative reproduction and, therefore, mutations at these loci would be lethal. Future work on the genetics of conjugation, meiosis and sporulation would best be done with conditional mutants. 4. Mitotic Recombination

Apart from the matl-lmatl- -dependent mitotic recombination confined to the mating-type locus (see Section 111,A. 1, p. 3331, data do not exist on other mating-type effects. IV. Saccharomyces cerevisiae A. M A T I N C - T Y P E L O C U S

1. Mating- Type Alleles

In Sacch. cerevisiae, the mating-type locus (mat)regulates functions in both the haploid and diploid phases of the life cycle. The mat locus consists of two alternative mating-type alleles, designated a and a (Lindegren, 19451, which segregate 2 :2 in tetrad analysis and which determine the a and a mating types of haploid cells. Genetic mapping has located the mat locus on chromosome 111, about 20 map units from the centromere (Lindegren, 1949; Hawthorne and Mortimer, 1960). Although a and a have been renamed matl-A and matl-B, respectively (Plischke et al., 19761, the older terminology will be retained in this review. The following observations suggest that mat is a complex locus. Hawthorne ( 1963a) obtained a deletion in an a strain which extended from a point in mat distally about 20 map units. Although the deletion caused lethality, it could be maintained in a heterozygous state in diploid cells. Haploid ascospores carrying this deletion died after germination unless crossed immediately with wild-type cells; in all crosses, such spores mated as a cells. The mutation of a to a as a consequence of this deletion suggests the complex nature of mat in Sacch. cerevisiae. Hawthorne ( 1963a)proposed that a distal a cistron was inactivated by the deletion, thereby allowing the proximal a cistron to be expressed. Similarly, D. Hawthorne and R. Mortimer (unpublished

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observations) isolated a lethal mutant from an a strain that mates as a and appears to carry a pericentric inversion or deletion extending into the mat locus fiom the proximal side, and may also possess a deletion distal to the mat locus. Based on the structure of the mat locus proposed from these observations, occasional recombinational events between a and a would be expected, as occurs in Schirosacch. pombe (Section 111, A.l, p. 332). Reciprocal recombination at the mat locus has not been reported for Sacch. cerevisiae, although gene conversion at the mat locus occurs with a frequency of about 0.3-0.4% (Fogel and Mortima, 197 1; R. K. Mortimer, cited in Roman, 1963). Recent evidence suggests that the mat locus is asymmetric, i.e. that there are more or different mutable sites in a than in a. Of 48 nonmating haploid mutants isolated from an a strain (MacKay and Manney, 1974a), 11 showed no recombination between the mat locus and the sterile defect (MacKay and Manney, 1974b). However, in an analysis of 100 sterile mutants derived from an a parent, none of the lesions was linked to the mat locus (MacKay and Manney, 1974b; T. Manney, personal communication). Thus, there appears to be a fundamental difference in the genetic structure of a and a. The hypothesis that a and a exist as independent cistrons with only one expressed in a given haploid cell is strengthened by the observations made with the homothallism system (see below). 2. Homothallism versus Heterothallism

Early studies by Winge (1935) indicated that some species of Saccharomyces are homothallic, i.e. haploid cells derived from a single spore mate with each other within the first few generations after spore germination, giving rise to clones that are ala diploids capable of sporulating. Even after the demonstration of heterothallism, i.e. two mating types, by Lindegren and Lindegren ( 19431, genetic studies in Saccharomyces spp. were plagued with confusion resulting from experiments performed with strains that gave rise to mixtures of haploid and diploid cells, probably as a result of the action of homothallism genes. Self-mating between sister cells of a spore clone arises by directed mutation of the alleles at the mat locus caused by specific homothallism genes. These include the D gene observed originally by Winge (1935)in Sacch. chevalieri, and studied later by Hawthorne (1963b1, the HM,, HM,, and HM, loci in Sacch. cerevisiae (Takahashi, 1958, 1961;

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M. CRANDALL, R. EGEL AND V . L. MACKAY

Takahashi and Ikeda, 19591, the Ha and Ha loci in Sacch. lactis (Herman and Roman, 1966), the HO, H M a and HMa genes in Sacch. ov$nmis (Takano and Oshima, 1967, 1970; Oshima and Takano, 1972; Takano et al., 1973; Harashima et al., 19741, and the Hp and Hq system of Sacch. norbasis (Santa Maria and Vidal, 1970).All of these genes are nuclear and unlinked to each other or to the mat locus, with the possible exception that H M a may be linked to the mat locus (Harashima et al., 1974). Both D and HO are dominant alleles (Hawthorne, 1963b; Hopper and Hall, 1975a)and are probably analogous (Oshima and Takano, 1972). Genes HO (and D ) , HMa and HMa have been renamed HTHI, HTH2 and HTH3, respectively (Plischke et al., 19761, but the older terminology has been retained in this review. Only the homothallism system in Sacch. ovijbmis will be discussed here since it has been studied in the most detail, and Takano and Oshima ( 1970)have presented evidence that the other homothallic species of Saccharomyces are very similar. As proposed by Oshima and Takano (19721, the combination of a non-specific gene for homothallism (HO) with a mating type-specific homothallism gene (HMa) causes a to mutate to a in haploid cells within the first several divisions after gemination of the ascospore. This heritable change from a to a, caused by the homothallism genes, apparently results from a genetic change at the mat locus. As soon as the new phenotype is expressed, the a cell mates with one of the surrounding a cells (Fig. lc, p. 312).Although the resulting a/a diploid is homozygous for H O and HMa, further mutation of a does not occur, and both the a and a alleles are stable in the diploid. The new a allele is indistinguishable phenotypically and genetically from a alleles in heterothallic strains (Takano and Oshima, 1970; Hicks and Herskowitz, 1976a).The homothallic conversion of a to a occurs analogously, requiring the presence of both HO and HMa genes and also yielding d a diploids homozygous for the homothallic genes. Recently, Harashima et al. (1974)suggested that H O may act on either mating type when H M a and HMa are either both present or both absent (the homothallic genotypes are summarized in Fig. 8). Using strains of Sacch. cerevisiae carrying the D gene, Hicks and Herskowitz (1976a) demonstrated that the switch from a to a occurs within two divisions after spore germination, and that the switch from a to a occurs within five generations. Furthermore, expression of the new phenotype generally appears in the oldest cell (the original spore

PHYSIOLOGY OF MATING IN THREE YEASTS a

8 t

t

0

353

o+a

HO hma hma HO HMa hmo HO HMa HMO

FIG. 8. Schematic representation of the effect of homothallism genes in Saccharomyces cerevisk. S indicates the original spore cell; D-1, D-2, etc.: the first, second, etc., daughters of S;D-1-1, D-1-2, etc.: the first, second, etc., daughters ofD-1; D-1-1-1: the first daughter of J L 1 - 1 . See text for further explanation. The figure is based on the work of Takano and Oshima (1970), Oshima and Takano (1972), Harashima et al. (1974)and Hicks and Herskowitz (1976a).

cell) and its most recent daughter (Fig. 8). Mating between cells expressing the new mating type and cells of the original mating type is facilitated by the close physical contact that exists between the spore mother cell and its progeny during initial development of the spore clone. Self-diploidizationcan be delayed by transferring spores to sodium acetate agar (Palleroni, 196 1). Haploid cells from microcolonies that develop on this agar can then be hybridized with haploid strains of opposite mating type. Recently, Hopper and Hall (1975a) isolated a mutant carrying a recessive nuclear gene that also causes directed mutation at the mat locus. This gene is designated cmt (changeof mating type) or hthl (in the revised nomenclature of Plischke et al., 1976). While both cmt and HO change mating-type expression, they are not allelic and, furthermore, cmt is recessive whereas HO is dominant. Dominance is an important point to consider for any model explaining the action of these two genes and other similar genes for homothallism. Another significant question is whether these homothallism genes should be more correctly called heterothallism genes, since they result in changes of heterothallic mating behaviour (L.J. Wickerham, personal communication).

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M. CRANDALL, R. EGEL AND V. L. MACKAY

3. Functions Controlled by the Mating- Type Locus In haploids, mating-type specificity is determined by which allele at the mat locus is expressed. The alleles at the mat locus also control the various steps of conjugation, including the ability of cells to produce and respond to diffusible mating type-specific pheromones (see Section I.A, p. 3 10)and the ability of cells to agglutinate in the early stages of conjugation. In diploids, the mat locus governs such diverse functions as inhibition of mating, promotion of meiosis and sporulation, survival following X-irradiation, mitotic recombination induced by ultraviolet radiation and the pattern of budding. B. HAPLOID FUNCTIONS

1. Initiation of Conjugation

a. Physiological Conditions Inducing Conjugation. A few of the parameters affecting conjugation in Sacch. cerevisiue are summarized in the following paragraphs. i. Culture medium. In general, complex media (containing yeast extract, glucose and, in some cases, peptone) have been used for conjugation studies (Lindegren and Lindegren, 1943; Pomper and Burkholder, 1949;Jakob, 1962; Haefner, 1965; Fowell, 1969a; Bilinski et al., 1973).However, as discussed in Fowell(1969a), Iguti and h u b u (1964) found that only glucose and inorganic salts are necessary for mating; although the presence of yeast extract in the conjugation medium enhances visible zygote formation, it appears to inhibit nuclear fusion, as determined by the recovery of prototrophic diploids. Sena et al. ( 1975)point out that, while a rich medium can stimulate sexual agglutination, the rapid growth possible in this medium may lower the frequency of mating. In a yeast extract-glucose medium, zygote formation is optimum at pH 4.5 (Bilinski et al., 1973). Lee et al. (1975) reported that high concentrations of glucose ( 10%) yield maximal mating eficiency in a medium also containing 1%yeast extract and 2% Bactopeptone (Difco). ii. Aeration. Aerobic growth is obviously not required for conjugation since mating also occurs between strains that are respiratorydeficient (Ephrussi and Hottinguer, 1951;Jakob, 1962). However, for the respiratory-proficient strains used in most studies, aeration

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enhances diploid formation (Jakob, 1962; Sena et af., 1975). Adequate aeration can be achieved by incubating the mating mixture on an agar surface (Haefner, 1965; Fowell, 1969a). iii. Cell ratios and concentration. The highest percentage of zygotes is obtained when a and a cells are present in equal numbers (Bilinski et al., 1973; Sena et af., 1973). In addition, both of these groups reported that cell concentrations greater than 107/ml lower zygote frequencies. To obtain synchronous zygote populations, Bilinski et al. (1973) employed higher cell concentrations (108/ml),a suboptimal pH value (8.5) and sonication to restrict zygote formation to a later time period when conditions were adjusted to maximize conjugation. Mating mixtures containing 30 to 40%zygotes were achieved by this method. b. Responsible Stage in the Cell Cycle. Sexual pairing and mating in Sacch. cereuisiae occur between single unbudded cells (Campbell, 1973; Sena et af., 1973) in the G1 stage of the cell cycle which is prior to initiation of the next round of DNA synthesis (see Hartwell, 1974 for a review of the yeast cell cycle). Hartwell (1973) demonstrated that, in mixed a and a cultures, cells of both mating types accumulate as unbudded cells, as a result of the action of diffusible sex factors which are produced by each mating type and which arrest cells of the opposite mating type in G1 (Bucking-Throm et al., 1973; see Section IV. B.2, p. 359). Furthermore, in studies using different temperature-sensitive cell division cycle (cdc) mutants (B. Reid, cited in Hartwell, 19741, the only mutant exhibiting continued mating ability at the restrictive temperature was cdc-28, whose lesion causes the cell to arrest in G 1. All of these results indicate that mating is restricted to the G1 interval when haploid cells contain a single complement of the genome. This mechanism would seem optimal for subsequent nuclear fusion, replication and mitosis or meiosis of the resultant diploid. The ability of cells to mate only in G 1 has facilitated the isolation of synchronous and nearly pure populations of zygotes (Sena et af., 1973, 1975). Using zonal centrifugation of a mixture of a and a haploid cells, these workers isolated homogeneous fractions containing unbudded cells that yielded up to 65% zygotes. In a modification of this procedure, the a and a cultures were fractionated separately, and the unbudded fractions then mixed. Following conjugation, a fraction containing 9 0 4 5 % zygotes was obtained by density-gradient centrifugation of this mating mixture. Such a centrifugation step would be

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M. CRANDALL, R. EGEL AND V. L MACKAY

useful to improve the purity of the synchronous zygote populations of Bilihski et al. (1973). c. Inhibitors of Conjugation. Sakai and Yanagishima ( 1971) demonstrated that cycloheximide, but not chloramphenicol, inhibits induction of sexual agglutination in Sacch. cerevisiae. Thus, cytoplasmic but not mitochondria1 protein synthesis is required for this early step in conjugation. As expected, constitutive strains can agglutinate in the presence of cycloheximide (Sakai and Yanagishima, 1972).Sakai and Yanagishima (1971)also found that ethionine and 8azaguanine did not affect agglutination in the inducible.strains. They suggested that this lack of inhibition may be attributable to large intracellular pools of metabolites. For later steps in conjugation, energy metabolism is necessary (Yanagishima, 1973). Cell fusion can be inhibited specifically with little inhibitory effect on agglutination by 2deoxyglucose (Shimoda and Yanagishima, 1974). Using temperaturesensitive mutants, Zuk et al. (1975) found that, at the restrictive temperature, conjugation was inhibited in mutants defective in RNA or protein synthesis but was not inhibited in mutants defective in DNA synthesis. 2. Cellular Recognition In mating mixtures of asynchronous cultures, several preliminary steps appear to be required prior to cell fusion; these include phasing of cells in G1 and weak mating type-specific aggregation followed by mating type-specific cell agglutination. A number of hormone-like factors have been associated with such interactions. These diffusible pheromones as well as the large molecular-weight agglutination factors are discussed below and summarized in Table 2 (see also Yanagishima, 1973 and Duntze, 1974). a. Mating- Type Spec@ Substances i. a Hormone and a hormone. Yanagishima (1969a, b) described a cell-expansion response in cells of one mating type of Sacch. cerevisiae when exposed to culture filtrates of the opposite mating type. While retaining their nearly spherical shape, a cells underwent a 30 to 40%increase in volume in response to u culture filtrates or extracted “u hormone”. The analogous reaction was noted with a cells and a filtrate or “a hormone”. Preliminary chemical and physical characterization of the substances suggested that both hormones were steroids (Takao et

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TABLE 2. Mating type-specific substances produced by Saccharomyces cerevisiae Substance

Made by

Acts on

a Hormone

a

a Hormone

a and a

a a

a-Factor a Substance-I

a

a-Factor I a-Factor I1

a

Barrier factor

a

a Agglutination factor a Agglutination factor

a

a

a

Chemical Nature

Effects

(Sterol?) Cell volume expansion n-Octanoic Cell volume expansion acid a Peptide G 1 arrest, cell-wall changes and production of shmoos a Peptide Induction of agglutinability in a cells (probably identical with a-factor) Transient G1 arrest a Protein Cell-wall changes and a Protein production of shmoos a cells or Unknown Inhibition ofa-cell response to a-factor a-factor a GlycoBinding to a cell surface protein Binding to a cell surface a Glycoprotein

al., 19701, although recently Yanagishima (1973) and Sakurai et al. ( 1974a)reported that a hormone is probably n-octanoic acid. Although n-octanoic acid promoted cell expansion only in a cells, it was found in filtrates of both a and a cultures and, therefore, may not be sexspecific. As yet, confirmation of the nature of a hormone has not been achieved (N. Yanagishima, personal communication). ii. Alph-Factor. Levi (1956) first suggested the existence of diffusible substances associated with mating in Sacch. cerewisiue, after observing a morphological change in a cells when placed on an agarcontaining medium on which a mating mixture had previously been incubated. The morphologically altered forms which arise are referred to as “shmoos” (Fig. 9) and are distinct from the enlarged cells formed after exposure to a and a hormones. A diffusible peptide called “afactor”, responsible for this reaction, is synthesized constitutively by a cells and can be isolated from filtrates of a cultures (Duntze et al., 1970). A&a-Factor is an oligopeptide (about 1700 daltons molecular weight) with the following primary amino-acid sequence: H,N-(Trp)His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-T~-COOH (Duntze et al., 1973; Duntze, 1974; Stotzler et al., 1976). In some derivatives of

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FIG. 9. Formation ot'shmoos in a cells of Saccharomyces cereuisk treated with a-factor. (a) Stationary-phase cells of mating type a exposed for 4 h to 8 units of a-factor/ml prepared from a culture filtrate of mating-type a cells by the method of Duntze et al. (1970). (b) Untreated stationary-phase cells of mating type a. From MacKay and Manney (1974a).

this peptide, the amino-terminal tryptophan residue is lacking, and the methionine residue can be present as a methionine sulphoxide (Stotzler and Duntze, 1976). Since all of these related peptides are biologically active, the amino- terminal tryptophan residue and the penultimate carboxy-terminal methionine residues are probably not part of the active site. It is likely that these derivatives of a-factor are oxidation and degradation products since they are recovered from cultures that have been well aerated and have produced enough acid during growth to lower the pH value of the medium to 2.5 to 3.0. Cupric ion copurifies with a-factor, but it is not known whether these ions are required for activity. Synthesis of a-factor is inhibited by cycloheximide or by different conditional mutations that prevent

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protein or RNA synthesis (Scherer et al., 1974). This implies that afactor synthesis requires ribosomal translation of a specific mRNA. The small size of the molecule suggests that it might result from translation of a larger mRNA molecule or from cleavage of a larger polypeptide. Although a shmoos arise either in the presence of a-factor or in mating mixtures, such altered cells are probably not premating forms since, in general, they appear in mating mixtures only after the onset of zygote formation. Thus, it seems likely that a shmoos represent cells which had been stimulated by specific sex factors secreted by cells of the opposite mating type, but had not established effective cell contact with a potential partner. In addition to inducing the shmoo reaction in a cells, a-factor also arrests the cell cycle in late G1 (Bucking-Thromet al., 1973). Thus, subsequent nuclear DNA replication, and hence cell division, are blocked but synthesis of mitochondria1 DNA, protein and bulk RNA are unaffected (Throm and Duntze, 1970; Petes and Fangman, 1973). The mechanism of action of a-factor has not been elucidated, but it could be either transported into the cell where it might trigger initiation of mating or it may stimulate a cells by interacting with a surface receptor. The possibility that a-factor acts via alterations in the intracellular concentrations of cyclic 3‘,5’-adenosine monophosphate (CAMP)was examined, but differences were not found either shortly after treatment of a cells with a-factor or after prolonged incubation (V. L. MacKay, unpublished observations). Furthermore, addition of CAMP, its dibutyryl derivative, or dibutyryl cGMP did not induce the shmoo response (W. Duntze, personal communication). Thus, the mechanism of G1 arrest does not appear to be analogous to that reported for many mammalian cell-culture systems in which CAMP levels are higher in resting G1 cells (Abell and Monahan, 1973). iii. a Substance-I. Inducible a strains become sexually agglutinative after a lag period in mixed culture with a strains, after treatment with filtrates from a-cultures, or treatment with a partially purified fraction also from a culture filtrates (Sakai and Yanagishima, 1972; Sakurai et al., 1974b; Yanagishima et al., 1974). The active compound present in a culture filtrates which specifically induces agglutinability in a cells is called a substance-I, and appears to be a peptide (Sakurai et al., 1975; see Table 2, p. 357).However, D. Radin (personal communication) has shown that the inducer of agglutinability

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M. CRANDALL, R. EGEL AND V. L. MACKAY

produced by a cells (presumably a substance-I) shows the same response to heat, changes in pH value and inactivation by protease as a-factor ( s e e Section IV, B.2, p. 357). Since purified a-factor is also a strong inducer of agglutinability in a cultures, and since non-mating a mutants which fail to produce detectable a-factor (see Section IV, B.6, p. 372) also do not make the agglutinability inducer (D.Radin, personal communication), it appears likely that a substance-I and afactor are identical. iv. a-Factor. Although a cells treated with culture filtrates from a strains do not form shmoos, the existence of an a-factor, analogous in action to a-factor, has been suggested by the presence of both a and a shmoos in mating mixtures incubated with agitation in liquid medium (Ahmad, 1953; Levi, 1956; Bucking-Throm et al., 1973).A@ha shmoos are also formed on complex agar-containing medium by placing fresh a cells close to a heavy overnight growth of a cells (Fig. 10).In general, only a small percentage of the challenged a cells in this agar assay respond to form shmoos, and the morphological change is less pronounced than that seen with a-factor treatment of a cells (MacKayand Manney, 1974a). However, all wild-type a strains tested evoked some response in a cells, while most non-mating (sterile) mutants derived from a cells did not evoke a shmoos (MacKay and Manney, 1974b; see

FIG. 10. Formation of shmoos in a cells induced by a cells of Saccharomyes cereuisiae. aCells in the heavy streak were grown overnight o n yeast extract-peptone-dextrose medium, then a cells were lightly streaked close to the a cells. From MacKay and Manney (1974a).

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Section IV, B.6, p. 372, for further description of these mutants). The insensitivity of the a-factor assay on solid medium may reflect a low level of constitutive a-factor synthesis, poor diffusion of the substance, a decreased or transient response by a cells or instability of the a-factor. Wilkinson and Pringle (1974) detected a substance synthesized constitutively by a cells that transiently accumulates a cells in G 1 as unbudded cells within two hours after addition. However, a shmoos are not generated and, after four hours, the a cells overcome the block as shown by the development of buds and initiation of DNA synthesis. This factor (designated a-factor I ) may be responsible for the retardation of growth of a cells in mating mixtures reported by Shimoda and Yanagishima (1973). The existence of an a-factor I that causes G1 arrest of a cells in mating mixtures is also suggested by the experiments of Biliriski et al. (1974). Inhibition of DNA synthesis in a cells from a conjugating mixture was demonstrated by taking advantage of the differential adsorption of a and a cells to Dowex 1 x 4 resin (Biliriski and Litwihska, 1974). Purified a and a cell fractions from the resin were tested for the ability to incorporate radioactive adenine into DNA, and it was found that both cell types were mutually inhibited with respect to both DNA synthesis and budding as a result of a short period of incubation together in conjugation medium. Recently, another a-factor (designated a-factor 11) has been detected that causes a cells to shmoo. This factor is present in concentrated culture filtrates when a strains are grown in the presence of either a cells or a-factor, and is also present in concentrated culture filtrates from one a strain even when it is grown in the absence of either a cells or a-factor (MacKay, 1976). All wild type a cells tested respond to this a-factor I1 activity, but a cells, a/a diploids and non-mating a mutants do not (MacKay, 1976). Alpha cells form shmoos only at pH values between 4 and 6. As found with a-factor, the two a-factor activities (G1 arrest and shmoo formation) may reside in one molecular species (R. Betz, V. L. MacKay and W. Duntze, unpublished observations). Both afactor I and a-factor I1 are sensitive to proteases and resistant to RNAse and DNAse. Both a-factors adhere to and can be eluted from the same cation-exchange resins in the same fractions. Attempts to estimate the molecular weight of the a-factors indicated that they are either quite large or are aggregates of small subunits. Thus, although it has not been determined whether both the G1-arrest activity and the shmooing activity reside in the same molecule, it appears that more than one activity is expressed by a cells, namely a-factor I that accumulates a cells in G1 and the a-factor I1 activity that gives rise to a-shmoos.

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v. Barrier factor. When a cells are placed on solid medium near a cells, they form shmoos unless “barrier” a cells are interposed between the a-factor-producing cells and the a-assay cells (Hicks and Herskowitz, 1976b). Only a cells and some a-ste mutants exhibit this barrier effect on the further difision of a-factor through the agar to the assay a cells. The barrier phenomenon results from the constitutive production of a diffusible factor by a cells that inhibits the response to a-factor. This barrier factor acts either by protecting a cells or by inactivating a-factor. Hicks and Herskowitz (197613) concluded that barrier factor is distinct from a-factor 11, since some mutant strains that produce the inhibitor are a-factor-deficient and vice versa. However, with better assay and purification techniques now available for a-factodd, this question should be re-examined. The barrier phenomenon appears to be distinct from the observation of R. Chan (personal communication) in which a cells act directly to remove a-factor from the medium without any diffusible barrier substances being involved. vi. Other sex-specific responses. Herman ( 197 lb) reported that a and a cells bud toward one another when placed on agar slabs close together but not touching. In this assay, a cells expand in volume about two-fold before budding, whereas a cells form numerous buds. In contrast, cells of like sex exhibit avoidance patterns by budding in a direction away from the other cell. Avoidance budding patterns were also observed between diploid and haploid cells. Budding responses between presumptive complementary mating types of Sacch. cerevisiae and H. anomla indicated some intergeneric cross-reactions. vii. a and a Agglutination factors. In a cells, two modes of sexual agglutinability have been described, namely a constitutive capacity and an inducible one (Sakai and Yanagishima, 1972; see Section IV, B.2, p. 359). All a strains tested by these investigators were constitutively agglutinative. When a cells are mixed with constitutive a strains, agglutination occurs rapidly even in the presence of cycloheximide (a protein synthesis inhibitor) or after heat killing. With inducible strains, however, agglutinability occurs only after a lag period in mixed culture, and can be blocked by either of the above treatments. Thus, Sakai and Yanagishima ( 1972)postulated that agglutination factors are present on the surfaces of both a strains and constitutive a strains but, in inducible a strains, the synthesis, activation, or accessibility of the

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agglutination factors must be promoted (Yanagishima, 1973).Following an earlier observation (Sakai and Yanagishima, 1972) that protease treatment destroys agglutination capacity in both mating types, Yanagishima et al. ( 1975) succeeded in releasing the sex-specific agglutination factors from the wall by treating cells with Glusulase (a crude preparation of snail digestive enzymes available from Endo Labs, Garden City, New Jersey, U.S.A.). When cells of each mating type are pretreated with the crude agglutination factor from the opposite mating type, subsequent mixed-cell agglutination is inhibited. Since the isolated factors do not cause agglutination of the opposite cell type but do inhibit agglutination, they are considered univalent (Shimoda et al., 1975).The biological activities of both agglutination factors, either in vitro or in situ, are susceptible to certain proteases. Preparations of both factors contain sugar. Therefore, the a and a agglutination factors from Sacch. cerevisiae may be glycoproteins (Shimoda and Yanagishima, 1975) like the 5 and 21 agglutination factors from H . wingei (see Section 11, B.2, p. 3 18). b. Ear& Events in Conjugation. Several of the pheromones already described are associated with early events in mating, including synchronization of the opposite mating type in G1 and induction of sexual agglutination. The proposed functions of these substances are illustrated in Fig. l l and are discussed in the following paragraphs. Bucking-Throm et al. (1973)proposed a model for conjugation in which the first step was reciprocal arrest of cells of the opposite mating type in G1 caused by a-factor and a-factor. However, if high concentrations of a-factor are added to mating mixtures of unbudded a and a cells, shmoos are formed and subsequent cell fusion is nearly abolished (Sena et al., 1973). Thus, the shmoo form may be in a physiological condition unfavourable for mating. This observation is in agreement with the observation made in several laboratories (Bilinski et al., 1973; Sena et al., 1973)that mating has an optimal cell concentration ( lo7 cells/ml) and, above this density, zygote formation is decreased presumably due to supra-optimal concentrations of afactor. The second interaction between cells of opposite mating types is cellular recognition by formation of weak bonds between unbudded cell pairs (Campbell, 1973; Sena et al., 1973).This weak binding can occur between boiled cells or cell-wall fragments and live cells of op-

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a substance- I

agg Iutinot ion

(probably a - f a c t o r )

Tight binding

FIG. 1 1 . Proposed scheme for cell interactions leading to conjugation in Sacchuromyces cereuisiae. See text for details.

posite mating type (Sakai and Yanagishima, 19721, as well as in cell mixtures incubated in saline (Campbell, 1973). Development of larger agglutinated clumps occurs in mating mixtures in growth medium, and requires protein synthesis (Sakai and Yanagishima, 197 1, 19 7 2 ; Sena et al., 1973; Yanagishima, 1973). These clumps exhibit tighter intercellular binding (Radin et al., 1973) and are presumably the result of induction of glycoprotein agglutination factors on a cells by a-factor (a substance-I).There is suggestive evidence in other yeast systems that initial cell recognition occurs via the mediation of surface filaments (see Sections 11, B.2, p. 321 and 111, B.2, p. 340). In Sacch. cerevisiae, surface fuzz is seen on both haploids during conjugation (Osumi et al., 1974) and such surface hairs are also implicated in the non-sexual flocculation reaction of brewer’s strains (Day et al., 1975). The initial weak binding between cells during mating may be due to interactions between agglutination factors present on the surface material extending from the cell wall, and the tighter cell binding may be due to an increased synthesis of the agglutination factors, above the basal level, that

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is induced by cell contact. Since surface filaments are involved in cell recognition in bacteria, algae, ciliated protozoa and other yeasts (reviewed in Crandall, 19771, it seems worthwhile to investigate this aspect of conjugation in more detail in Sacch. cerewisiae. While the mating-type specific pheromones appear to regulate the interaction between opposite mating types, the specificity of cellular recognition is not absolute since “illegitimate” fusions between cells of the same mating type do occur, albeit at a low frequency (about Hopper and Hall, 1975b). Fusion of like cell types is recognized by selecting for prototrophic diploids resulting from complementation between multiple nutritional markers carried by each haploid strain. Although Friis and Roman (1968) observed these rare matings only between a cells, diploids have also been isolated from mixtures of two multiply marked a strains (V. L. MacKay, unpublished observations). Since most of these hybrid clones retained the ability to mate as a, they were thought to arise by direct fusion of two a cells. However, these hybrids may contain chromosomal aberrations or imbalances. The prototrophic clones that do not mate are capable of sporulating and are, therefore, probably ala diploids. The low frequency of heterozygous diploids formed probably reflects the low frequency of mutation to a in a strains. Although the interaction between complementary agglutination factors on the walls of opposite mating types promotes cell fusion, walls of the same mating type do not interact and, hence, act as barriers to fusion. In agreement with this idea, it was found that protoplasts of two different a strains of S u c h . cerewisiae (prepared by removal of the walls with Glusulase) fused with a higher frequency (about than the frequency of fusion of intact cells of the same two a strains (about D. Malone, S. Fogel and D. Radin, personal communication). These workers found that concanavalin A and nitrate together facilitated fusion of the protoplasts. This technique of protoplast fusion might be useful in enhancing hybridization of sterile mutants (see Section IV, B.6, p. 374).

3 . Cell Fusion

a. Formation ofthe Conjugation Tube. Breakdown of the thick cell wall may be initiated by physical contact or by pheromonal interactions

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between cells of opposite mating type. In electron micrographs of conjugating pairs (Osumi et al., 19741, the parental walls became progressively thinner as the area of contact between the cells enlarged. Wall dissolution was most pronounced in the electron-transparent region thought to be glucan. Changes in cytoplasmic membrane structure occurred simultaneously until both the wall material and the membrane became sufficiently fragmented to allow cytoplasmic fusion. An increase in the release of sugar and protein into the medium from mating mixtures was detected by Shimoda and Yanagishima (1972). This release could be attributable to an increase in autolytic activity or to leakage of cytoplasmic constituents as a result of fi-agmentation of the wall and membrane. Virus particles from species of Penicillium and Aspergillus were reported to infect Sacch. cerevisiae only during conjugation (Lhoas, 1972; Border, 1972) when the breakdown of the surface layers is extensive. However, it is not clear whether viruslike particles were already present in the yeast strains used in these experiments. In similar experiments (Mitchell et al., 1976), it was not possible to transduce killer virus during conjugation between sensitive yeast strains lacking this double-stranded RNA virus. In no system has it been possible to demonstrate an infective cycle for any mycophage even though virus-like particles are found in many fungal cells (Lemke and Nash, 1974). Although pronounced alterations of walls may require extended physical interaction between cells of opposite mating types, changes in wall composition can be initiated in a cells by treatment with a-factor (Lipke et al., 1976). In the shmoos that are formed, budding and the cell cycle are, of course, blocked in the presence of a-factor but cellwall synthesis continues. The new wall material differs structurally from walls of budding cells by containing more glucan and less mannan with the latter present in a less highly branched configuration. In addition, shmoos are more susceptible to glucanases, meaning that, in mating mixtures, breakdown or restructuring of the wall may begin as a result of pheromonal action before cells have established physical contact. Lipke et al. ( 1976) also observed that the wall at the tip of the shmoo is thinner, as was seen for walls of mating pairs in the early stages of interaction (Osumi et al., 1974). Small vesiclesvisiblein the tip region of shmoos (see Fig. 12; W. Duntze, personal communication) may be involved in this localized wall synthesis. Further evidence for localized,

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mating-type specific alterations in the cell surface were obtained by fluorescence microscopy (J. Tkau and V. L. MacKay, unpublished observations; cited in MacKay, 1976).Mating-type a or a cells were incubated for two hours with pheromone fi-om the opposite mating type and then treated with concanavalin A conjugated with fluorescein isothiocyanate. The portion of the walls of the shmoos synthesized in response to pheromones had a greater fluorescenceand, hence, a greater affinity for the lectin than the rest of the surface which stained uniformly as in the control cells. When zygotes are taken from a mating mixture (without exogenous pheromones) and stained, the appearance of a more intense fluorescent band at the conjugation bridge demonstrates that zygotes are formed by fusion of two cells at the region of altered wall structure, i.e. at the tips of the developing shmoos. Corroborating this conclusion, peg-shaped cells are seen in agglutinated clumps in mixed cultures prior to fusion with the tips directed toward one another (Osumi et al., 1974). b. Cytoplasmic Mixing, Mitochondria1 Fusion and Recombination of Cytoplasmic Genes. Disorganization or dedifferentiation of the parental mitochondria occurs early during zygote formation (Smith et al., 1972). This presumably promotes the extensive recombination of mitochondrial DNA that occurs during mating, which results in diploid clones with recombinant phenotypes for parental mitochondrial genes. Generally, the cytoplasmic markers studied confer resistance to antibiotics, such as to erythromycin, chloramphenicol and oligomycin. In an effort to discern the nature of the recombinational events and the time during zygote formation at which they occur, several groups have performed pedigree analyses to follow the inheritance patterns of the first fav zygotic buds (Wilkie and Thomas, 1973; Waxman et al., 1973; Callen, 197413). As already discussed, the first diploid bud usually emerges at the neck of the zygote where fusion took place; the next two buds customarily develop at either end, with no apparent pattern for subsequent buds. From zygote lineage data, Callan (1975) has proposed that mitochondrial mixing and recombination are limited mainly to the neck, so that buds from this region can contain either a parental genome or a recombinant one, whereas buds from the ends would possess mainly one or the other parental type. After a few rounds of bud formation, the mitochondrial genotype becomes stabilized in the zygote, so that subsequent buds from the zygote

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FIG. 12. Freeze-etched electron micrographs of a shmoos of Saccharomyces cereulszae, formed in response to treatment with a-factor. (a)A whole shmoo, showing thinningof the cell wall at the tip and the presence of small vesicles under the thin areas. (b) Higher magnification of the tip area showing small vesicles. The micrographs are unpublished observations of W. Duntze.

possess a single genotype (Wilkie and Thomas, 1973). The time of stabilization and the mitochondrial genotype selected appear to be strain-dependent. Wilkie and Thomas ( 1973) suggested that stabilization could depend on whether or not the mitochondrial DNA molecule was bound to a membrane-attachment site, thereby allowing

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replication of the associated DNA in the zygote. Unattached DNA molecules would not be replicated until incorporation into a mitochondrion. Waxman et al. ( 1973) suggested that asymmetry of transmission of mitochondrial markers from one of the haploid parents to the diploid progeny may be determined also by a nuclear gene. Their interpretation was based on genetic analysis and on the observation that inhibition of cytoplasmic protein synthesis after zygote formation altered the inheritance patterns in clones derived from zygotic buds, whereas inhibition of mitochondrial protein synthesis did not affect the observed asymmetry.

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The possibility that cellular mating type influences mitochondrial marker transmission has not been resolved, since differing results have been obtained with different strains. Thus, mitochondrial inheritance of a particular gene has been found to be affected inconsistently or not at all by the mating type of the contributing parent (Bolotin et al., 1971; Wilkie and Thomas, 1973; Trembath et al., 19731, to be more frequent if the contributing parent is an a strain (Suda and Uchida, 1972; Rank and Bech-Hanson, 1972)or to occur more frequently if the parent is an a strain (Coen et al., 1970; Saunders et al., 1970; Kleese et al., 1972a, b; Bunn et al., 1970; Waxman et al., 1973; Callen, 1974a). However, in all cases, widespread variation in transmission and recombination frequencies was noted when different strains were used. Although some of these examples of preferential inheritance may result from action of unidentified nuclear genes in conjunction with or instead of mat, the data obtained in certain studies (notably, Callen, 1974a) may represent a direct mating-type effect, since care was taken by this latter researcher to use isogenic strains. Further investigations, using different sets of isogenic strains, will be required to resolve these apparent contradictions. 4. Nuclear Fusion versus Heterokalyosis Prior to cytoplasmic fusion, the ultrastructure of the parental cells appears typical of haploid cells in G1. The nuclear membrane displays a single spindle plaque (Byers and Goetsch, 1973) and remains intact, although the nucleus itself may have started to move toward the region of subsequent fusion (Osumi et al., 1974). At the time that wall and membrane fragmentation occur, proliferation of the endoplasmic reticulum can be seen (Osumi et al., 1974)as well as elongation and dedifferentiation of mitochondria, previously observed by Smith et al. (1972)in youngzygotes. The duration of the transient heterokaryotic state following cytoplasmic fusion is unknown, although fusion is generally complete before emergence of the first bud. However, Fowell( 1951) and Wright and Lederberg ( 1957) observed, in microcolonies derived from isolated zygotes, the existence of cells carrying auxotrophic markers of the haploid parents. More recently, another instance of extended heterokaryosis may have been observed. Wilkie and Thomas ( 1973) found that some clones derived from early buds of isolated zygotes

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were haploid. This result could be attributed to delayed fusion of the two haploid nuclei (D. Wilkie, personal communication). Byers and Goetsch ( 1973)explored the mechanism of nuclear fusion in mating cells by electron microscopy. Nuclear membranes of vegetative cells in the G1 phase contain a single spindle plaque which duplicates approximately at the same time as bud emergence. Premating haploid cells arrested in G1 likewise possess a single plaque, but this does not undergo duplication during the mating process. In addition, these plaques carry a unique “half-bridge” structure found also in a cells treated with a factor and in a temperature-sensitive cell division cycle mutant (cdc-28) blocked at the same point in G1. As the haploid parents approach cytoplasmic fusion, microtubules project in an outward extranuclear direction and, after cytoplasmic fusion, the microtubules extend into the cytoplasm of the other parent and connect to the plaque of the other nucleus (perhaps drawing the nuclei together). Nuclear fusion occurs at the plaques, initiated at the distal ends of the half bridges. The half bridges fuse forming a complete bridge between the two plaques which originated in the two haploid parents. Development of nuclear fusion at a limited region of the membrane was similarly noted by Osumi et al. (1974).Following fusion of the two haploid nuclear membranes, replication of the diploid genome probably begins. 5 . Zygotic Budding The formation of the new double plaque may coincide (and interact) with the emergence of the first zygotic bud, as in mitotic cycles. Thus the first diploid bud probably originates in proximity to the double plaque which is formed in the constriction between the two original haploid cells. As noted by several groups (Byers and Goetsch, 1973; Waxman et al., 19731, most of the first (and often the second) buds develop at this site. One prediction of this double-plaque model would be a greater occurrence of extended heterokaryosis among the minority of zygotes that develop their first bud at an end rather than in the middle of the zygote. If the appearance of the first bud were delayed, then the probability of nuclear fusion before bud development would be increased. As the first zygotic bud enlarges, the pronounced endoplasmic reticulum and the disorganized mitochondria (Smith et al., 1972)begin

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L. MACKAY

to revert to their normal appearance (see also Osumi et al., 1974).At this point, the state of the cytoplasm is not significantly different from that of vegetative cells, and the subsequent events of bud development, completion of DNA synthesis, and nuclear division probably proceed as in vegetative cells. See Hartwell (1974) for a review of the vegetative cell cycle in yeasts. 6. Mutant Genes Affecting Conjugation Our understanding of the conjugation process in Sacch. cerevisiae and, in particular, of the various elements involved in its early stages has been enhanced by the isolation of mutants defective in one or more mating characteristics. Many of these have been analysed both phenotypically and genetically. a. Isolation and Phenotypic Characterization of Mutants Defective in Mating. A large-scale isolation of mutants affected in the mating process was undertaken to determine the roles of a-factor and afactor in mating. Most of the non-mating mutants isolated were defective in pheromonal production as well as not being responsive to the pheromone from the opposite mating type (MacKay and Manney, 1974a). In contrast to the self-sterile but cross-fertilefwl mutant in homothallic strains of Schizosacch. pombe (see Section 111, B.6, p. 5461, these sterile mutants of Sacch. cerevisiae will not conjugate with wildtype partners of opposite mating type (p. 374). Sterile mutants were selected in an a strain which carried multiple auxotrophic markers and a recessive allele (can11 for resistance to canavanine, an arginine analogue. After mutagenesis, the a cells were mixed with a 1000-fold excess of a cells, carrying different nutritional markers as well as the dominant allele ( C A N I ) for sensitivity to canavanine. The cell mixture was incubated on a non-selective rich medium, to allow mating, then resuspended and plated on a minimal medium containing canavanine with only the growth factors of the a strain. The excess a cells were, therefore, prevented from growing by the presence of canavanine and the absence of their required nutrients. Likewise, canavanine prohibited growth of a/a CANllcanl diploids formed during the mating period. Therefore, the only cells capable of forming colonies on the selective medium were a cells that either did not mate or could not mate. Subsequent screenings identified haploid

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mutants that had original markers and were still non-sporulating but were unable to mate with tester strains of either mating type. A modified procedure allowed the selection of sterile mutants of the a mating type; also some of these were temperature sensitive.This method for isolation of sterile mutants selects against certain types of lesions. For example, mutants that form non-viable zygotes would not be isolated. Similarly, non-mating mutants still able to respond to the G 1-arresting sex factor of the opposite mating type would be prohibited from dividing and, therefore, would be lower in frequency because the other cells in the mixture would grow on the rich medium used for mating. As predicted, only one sterile a mutant out of 107 was still responsive to sex-factor arrest, and none of the 66 sterile a mutants was responsive to the sex factor of the opposite mating type (MacKayand Manney, 1974a). Both sex-factor production and mating ability were lacking in a majority of the mutants isolated. For example, of 383 a sterile (ste) mutants obtained, 5 1% had simultaneously become defective in synthesis of a-factor. Similarly, 83% of the 66 a ste mutants failed to produce detectable a-factor, although this percentage may be artificially high due to the relative insensitivity of the assay. Genetic analyses of these mutants (see p. 374) showed that the double phenotype of non-mating together with defective sex-factor production always resulted from single gene mutations (MacKay and Manney, 1974b). These data indicate that the ability to produce the appropriate sex factor is necessary, but not sufficient, for conjugation. Recently a new method has been devised for screening large numbers of colonies for a-factor production (T. Manney, personal communication). In this procedure, an a culture is mutagenized and then plated onto a non-selective medium seeded with an a indicator strain that is temperature sensitive for growth, such as ts-187 (Hartwell, 1974). After 24 h incubation at the restrictive temperature, at which only the a cells can form colonies, the plates are shifted to the permissive temperature for an additional 24 h, allowing both strains to propagate. Around wild-type a colonies that secrete a-factor, clear zones result due to inhibition of a cell growth while, around mutant a colonies that synthesize less a-factor, smaller zones of inhibition appear due to a decreased level of a-factor production. When these partially defective mutants are mixed with a cells in dense mixtures on a solid medium, mating frequencies are normal, yet at least one of these a-factor-

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synthesis mutants mates poorly in liquid medium at lower cell densities (T. R. Manney, unpublished observations).These apparently conflicting results may be resolved if it is assumed that a threshold level of a-factor is necessary to divert a cells into a “premating” condition. In a dense cell mixture on solid medium, the level of a-factor produced by these partial mutants apparently is sufficient to achieve this premating condition in a cells, and then cell fusion is promoted by the effective cell contact on agar. However, in liquid culture at low cell density, where cell contact is minimal due to agitation and a-factor is at low concentrations, zygote formation is decreased. From this discussion, it would be predicted that complete loss of the ability to produce a-factor would result in sterility even on solid medium and, in fact, this is what is observed with the a mutants selected for their inability to mate. The association of a-factor with the mating process has been further substantiated by the selection of a mutants that fail to respond to a-factor, i.e. that continue to divide in its presence (Manney and Woods, 1976). All of these unresponsive mutants studied are sterile, indicating that the condition induced in a cells by a-factor is a prerequisite for mating. b. Genetic Analysis $Sterile Mutants. Approximately 60%of the sterile mutants mate with wild-type cells of the opposite mating type at low frequency to lo-’; MacKay and Manney, 1974a). Most of the sterile x wild-type hybrids were able to sporulate and the sterile mutation segregated 2 : 2, indicating that the low frequency of mating was not due to reversion of the sterile mutation. Based on genetic analysis (MacKayand Manney, 1974b), the mutants were assigned to five loci. i. The stel locus. Of the 48 a ste mutants analysed genetically, eleven carry defects (designated stel) that are located at the mat locus or within 0.94 to 5.0 map units of it, since recombination between stel and mat was never observed. In contrast, none of the 100 a ste mutants analysed to date possesses defects at the mat locus. Thus, a functional genetic locus analogous to stel may not exist in a cells. Although the gene product of stel has not been identified, it appears to have a pleiotropic effect on the cell, since none of the stel mutants agglutinates with a cells (D.Radin, personal communication), produces a-factor or, with one exception, forms shmoos in response to a cells. Mutations with phenotypes identical to stel but unlinked to the mat locus have also been identified (stel and ste5 ; see p. 375). ii. The ste2 locus. Two a ste mutants contain lesions which are unlinked to the mat locus but specific for the a mating type; i.e. a asco-

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spores, carrying such a mutation, mate at wild-type fi-equencies with a cells and fail to exhibit its presence in any way. Both a-specific ste defects map at a single locus, designated ste2, which has not been located on the genetic map. Cells of mating type a, containing the ste2 marker, agglutinate with a-tester strains (D. Radin, personal communication), and retain the ability to cause a cells to shmoo. However, a ste2 cells are not induced to shmoo, but they still either inactivate a-factor or remove it from the medium (T.R. Manney, personal communication; Hicks and Herskowitz, 197613). iii. The ste3 locus. An a-specific defect, not expressed in a cells, was identified in five a ste mutants. All five lesions map at a single site (ste3) which is unlinked to either ste2 or the mat locus. Analogous to the a ste2 mutants, all a ste3 strains agglutinate with a cells (D. Radin, personal communication) and produce a factor. Their ability to deplete the test medium of a-factor has not been determined but they do not form shmoos in response to a-factor. iv. The stel and ste5 loci. The remaining sterile a and a mutations do not exhibit mating-type specificity; any cell carrying such a nonspecific ste mutation is sterile, independent of the original mating type. Thus, the defective gene product is presumably required in both mating types. Some of these non-specific ste mutants are still capable of producing their respective sex factor while others are defective. None of the mutants tested is agglutinative with cells of the opposite mating type (D.Radin, personal communication) and, therefore, these pleiotropic mutations are similar to stel. Attempts to establish the number of genes represented by this group have been hampered by the difficulty in obtaining diploids from crosses between two mutants. However, by mating temperature-sensitive non-specific ste strains, it was possible to identify two loci designated stel and ste5. Furthermore, three temperature-sensitive non-specific ste mutants isolated by A. Teriba and L. Hartwell (personal communication) carry defects at three different loci, none ofwhich corresponds to ste5 (MacKay, 1972). Even if one were allelic with ste4, there are at least two more nonspecific ste genes. Whether any of these is allelic with the sterile gene nu& mapped by Mortimer and Hawthorne (1978)has not been determined. I t seems likely that more ste loci will be identified since only four of the 58 non-specific mutants have been analysed genetically. Estimating the number of ste loci would be facilitated by determining the map locations of the individual non-specific mutations by recom-

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binational analysis to avoid the difficulty of crossing two non-mating mutants. v. Putative regulatory loci. Other ste mutants isolated in both a and a strains mate at a low frequency with wild-type cells of the opposite mating type to form diploids that do not sporulate. This phenotype is not the result of a dominant mutation preventing sporulation, but rather is thought to arise from inactivation of mating type-specific regulatory loci at the mat locus that control mating ability in haploids and one or more gene products in diploids that turn off the mating ability of the other haploid parent, induce sporulation and activate the da-mediated DNA repair pathway (see Sections IV, C.2, p. 378 through IV, C.4 for a discussion of these diploid functions). This interpretation is based on the following observations : ( 1) most of the nonsporulating diploids were still able to mate, possessing the mating type of the non-mutant parent; (2) the diploids may possess an increased sensitivity to X-rays (T. R. Manney, personal communication); (3) the original mutants did not produce their respective sex factor or respond to the sex factor of the opposite mating type; and (4) preliminary genetic analysis is consistent with the lesions mapping at the mat locus. However, further investigations of these mutations have been hampered by their unstable nature. Similar non-sporulating diploids (thought to be d a ) were reported by Kassir and Simchen (1976) to carry a defect which prevents both induction of meiosis in the diploid and mating in the haploid; the defect maps at the a allele in the mat locus. c. Additional Mutations Affecting Mating. The genotypic and phenotypic expression of mating type can also be influenced by the homothallism genes (discussed in Section IV, A.2, p. 35 1) and by the deletion and inversion that extend into the mat locus (discussed in Section IV, A. 1, p. 3501, all of which change the mating type of the cell. Wygal and Haber (1975) isolated, from an a parent strain, mutants that now mate only as a, as a result of defects that appear to map at the mat locus. For four of the mutants, the diploids formed between the mutant “a” and a normal a were capable of sporulating unless the “a” mutation was suppressed by an amber nonsense suppressor. Suppression of the “a” haploid, however, yielded a non-mating phenotype rather than a return of a character. Thus, the efficiency of suppression or the amino acid inserted into the polypeptide may be sufficient to prevent induction of sporulation but not to activate structural genes for the a mating pathway. In any case, their results suggest that muta-

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tion of a to a can occur by production of a nonsense codon at one or more points in the mat locus. Haploid a mutants with altered mating integrity have been isolated as dual mating type (dmt) (Blamire and Melnick, 1975). Although the haploid mutants do mate with either mating type, the a mating-type clearly predominates. However, ala dmtldmt diploids mate equally well with cells of either mating type. From these diploids, new mutants were isolated which now mate with only one mating type, either a or a. Blamire and Melnick (1975) suggested that, in wild-type diploids, the gene products of mcat and DMT might interact to inhibit expression of a and a genes under their control, while in ala dmtldmt diploids, both mating types are expressed. In the “single mating type” diploid mutants derived from the ala dmtldmt diploids, the new lesions might reside in “structural” genes such as the ste genes described by MacKay and Manney ( 1974a, b) that may specify products required for mating. Mutations affecting mating type in Sacch. cerewisiae have also been isolated by Vezinhet et al. (1975). These include point mutations for mating type, lethal deletions located in the chromosome carrying the mat locus, illegitimate copulation benveen cells of the same mating type and plasmogamy without karyogamy. The ability of an a cell to mate is also dependent on a seemingly unrelated gene Rex2 (hiller expression) that is not linked to mat (Leibowitz and Wickner, 1975).Mutants of Rex2 carry the normal double-stranded RNA plasmid that is associated with the killer phenotype (i.e. the secretion of a toxin that kills cells lacking the plasmid), yet they are nonkillers. These hex2 strains do not mate at normal frequencies nor do they secrete a-factor, whereas decreased mating ability has not been noted for a Rex2 cells. One possibility is that the Rex2 mutation interferes with synthesis or secretion of certain extracellular substances, such as the killer toxin or a-factor. Obviously further genetic crosses and biochemical studies are necessary to determine whether the various mutants isolated affect mating through positive or negative control, or whether they represent a gain or loss in function at structural genes. C.

DIPLOID FUNCTIONS

1. Heterorygosity versus Homozygosity at the Mating- Type Locus

The extensive role played by the mat locus in the diploid phase of the Sacch. cerewisiae life cycle is disclosed by a comparison of the properties

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of cells heterozygous at mat ( a / d with those homozygous at mat (either a/a or a / d . Although the homozygous cells mate with the same high frequency of haploid cells, a/a cells are unable to mate, except rarely. Other properties of a/& diploids that distinguish them from diploids homozygous at the mat locus include the ability to undergo meiosis and sporulation, increased survival after X-irradiation, higher frequencies of mitotic recombination after induction with ultraviolet radiation and different budding patterns. Thus, heterozygosity at the mat locus results in the apparent inhibition of certain haploid functions and the implementation or enhancement of other functions, some of which are observed only in the diplophase. 2. Repression of Haploid Functions Diploid cells homozygous at the mat locus have the same mating reaction as their haploid counterparts (Roman and Sands, 1953; Mortimer, 1958). For example, production of a-factor by a/a cells and the shmooing response of a/a cells to a-factor are the same as in haploids (Duntzeet al., 1970). In a/a diploids, however, the ability to produce or respond to either a- or a-factor is absent (Duntze et al., 1970; T. R. Manney, personal communication). In addition, a/a diploids normally do not agglutinate in mixtures with haploid cells (Sena et al., 1975). The rare matings of a/a diploids with a or a cells, giving rise to triploid cells, apparently involves a/a or a/a diploids that arose in the population by mitotic recombination (Gunge and Nakatomi, 1972). Also, genes conferring homothallism are inactive in ala but are active in a/a or a/a diploids as they are in a or a haploids. Thus pheromone production, shmooing, agglutination and conjugation are deficient as a result of heterozygosity at the mat locus. Additional evidence for this interpretation is provided by recent analysis of an a/a diploid strain showing unusually high frequencies ( 1-2%) of mating with haploids of one or the other mating type (Haber, 1974). Acquiring the ability to mate is due to the loss of one or the other homologue of chromosome 111, yielding a few percent of monosomic diploids (2n - 1) that are haploid for the mating-type locus. This sporadic event in the diploid culture was attributed to the action of a recessive nuclear gene, designated htr (hermaphrodite), which may cause the loss of other chromosomes as well. Similarly, T. R. Manney (personal communication) has observed in another ala diploid a high

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frequency of non-disjunction involving chromosome 111, giving rise to an increased occurrence of mating with haploids and to abnormal segregations of mat in tetrad analysis. 3. Meiosis and Sporulation

The works of Fowell (1969a1, Moens and Rapport (197 1) and Piiion et al. (1973) are suggested for discussions of metabolic, cytological and molecular studies of sporulation, respectively. This review will be limited to observations concerning the involvement of the mat locus in sporulation. a. Physiological Conditions Inducing Sporulation. Recent studies of early steps in meiosis have been facilitated by the isolation of strains that sporulate quickly and by methods to obtain rapidly synchronous populations of meiotic cells. Cells are pregrown, either in a complex glucose-containing medium to the end of the log phase (Esposito et al., 1969) or in an acetate-containing vegetative medium to the mid-log phase (Roth and Halvorson, 1969; Simchen et al., 1972), and then transferred to acetate-containing sporulation medium (pH 5). The latter regime yields a high degree of synchrony with 65 to 70% asci by 15-16 h. b. Responsive Stage in the Cell Cycle. Limited experimental evidence suggests that d a diploids become committed at G 1 either to another mitotic cycle or to meiosis and sporulation. Cells at other stages of the cell cycle, when transferred to sporulation medium, complete their mitotic cycle (although without growth of the bud) before entering meiosis (D.Milne, cited in Harhvell, 1974). Therefore, the G 1 stage seems to function as a decision point where the cell’s next activity is determined (Hartwell, 1974). c. Influence ofthe Mating- Type Locus on Meiosis. The ability of a diploid cell to sporulate is predetermined by the combination of alleles at mat; homozygous diploids cannot sporulate, while d a diploids can (Lindegren and Lindegren, 1943; Roman and Sands, 1953). When diploid cells are transferred to sporulation medium, many profound metabolic and morphological changes take place, but it is difficult to determine whether these changes are directly linked to sporulation or are simply the consequence of the particular conditions of the medium in which the cells are sporulating. To control these variables, M. Pellecuer (cited in Vezinhet et al., 1974) isolated isogenic diploids that

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M . CRANDALL, R. EGEL AND V. L. MACKAY

differed only in the alleles at the mat locus. When placed in potassium acetate medium (pH 71, both a/a and a/a diploids increased identically in dry weight, protein, carbohydrate and lipid contents, and both showed the same variation in respiration rate with time (Vezinhet et al., 1974). It is concluded that these changes are not the factors that trigger sporulation but are, rather, the consequences of the action of the sporulation medium on the cell, i.e. nitrogen starvation in the presence of an exogenous energy source. In agreement with this conclusion, Hopper et al. ( 1974) reported that, in sporulation medium, synthesis of glycogen, RNA and protein is similar in ala, a/a and a/a cells but breakdown of pre-existing RNA and protein is more extensive in a/a cells. The earliest macromolecular change reported is an increase in RNAse activity (Tsuboi and Yanagishima, 1976) that precedes the breakdown of vegetative RNA into precursors that may be used for premeiotic DNA synthesis (Simchen et al., 1972). The configuration at the mat locus is critically important for premeiotic DNA synthesis which occurs between four and eight hours after transfer to sporulation medium (Piiion et al., 1973). Diploids homozygous at the mat locus do not replicate their DNA in sporulation medium (Roth and Lusnak, 1970) whereas ala diploids undergo a round of DNA replication and then continue into the subsequent stages of meiosis. Related studies by Simchen et al., (1973) identified a period, possibly co-inciding with meiotic prophase, in which sensitivity to ultraviolet radiation is acquired by sporulating ala cells but not by a/a or a/a cells or by a sporulation-defective mutant. That meiosis is directly regulated by the mat locus has also been shown by studies of Roth and Fogel ( 197 1 ) and Fogel and Roth ( 19741, using haploid cells that are disomic ( n + 1 ) for chromosome 111. These cells undergo premeiotic DNA synthesis and genetic recombination only if they are heterozygous at the mat locus. Thus, it appears that both a and a alleles contribute to meiotic DNA synthesis and that, in cells containing only a or a alleles, meiotic DNA synthesis is inhibited or is lacking a required component. However, control by the mat locus is not absolute since, in some strains, a low level of sporulation can be detected in a/a or ala cells (Zakharov and Kozhira, 1967; Hopper et al., 1974). Although premeiotic DNA synthesis is one of the earliest molecular landmarks of sporulation and mat locus control, the regulation exerted by the mat locus may occur at an earlier step. I n sporulating a/a diploids and in a sporulation-defective mutant, synthesis of mitochondrial DNA is initiated before the onset of nuclear DNA synthesis (Piiion et ul., 1973). Since a/a and a/a diploids exhibit only a fraction of

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the mitochondria1 DNA synthesis seen in ala cells, the initial control imposed by the mat locus probably precedes such synthesis. Thus, it appears that, within three hours after transfer to sporulation medium, cells complete their mitotic cycle and return to G 1. Then, they may be influenced by the altered nutrition of the medium and by the presence of both a and a gene products to enter a meiotic cycle. However, cells in this stage are not fully committed to sporulation; although they will continue on the meiotic pathway if transferred to water, they will revert to vegetative growth if transferred to growth medium (Simchen et al., 1972).Full commitment to sporulation is not achieved until after 7 to 8 hours in sporulation medium, after the termination of DNA synthesis. d. Mutations Asfecting Mating-Type Regulation $Meiosis. Gerlach ( 1974) described diploid mutants, homozygous for mating type (ala),that mate as a, as expected, but are capable of sporulating. All four spores derived from meiosis are of the a mating type. Tetraploid genetic analysis indicated that this phenotype in the diploid resulted from homozygosity of a single recessive mutation. This lesion, designated sca (sporulation capable), is unlinked to the mat locus and is similarly expressed in ala diploids, resulting in four haploid a spores. Sporulating ala diploids have also been isolated by Hopper and Hall (1975b),who demonstrated that a dominant mutation, CSP (control of sporulation), was responsible for the phenotype. The ala csplCSP diploid can, however, still mate and has the X-ray survival and ultraviolet-induced mitotic recombination frequency typical of normal ala cells (Hopper et al., 1975).The CSP phenotype is not the result of a genetic alteration at the mat locus, since the CSP mutations are unlinked to the locus and are expressed in ala cells as well. Hopper and Hall (1975a) proposed two alternative models for control of sporulation by mat and the wildtype allele, csp. In both of these models, regulation of expression of sporulation genes is proposed to be mediated by a sporulation inducer (SI) whose activity (if csp is the structural gene for SI) or synthesis (if csp is an operator for the structural gene) is governed by the genetic configuration at the mat locus.

4. Mitotic Recombination

The configuration at the mat locus influences genetic recombination during mitosis as well as meiosis. Friis and Roman ( 1968) compared the frequencies of intragenic mitotic recombination, after ultraviolet

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M. CRANDALL, R. EGEL AND V.

L. MACKAY

induction, in a/a, a/a and a/a cells. Although the results were influenced somewhat by the origins of the strains, a/a diploids clearly were more recombination proficient (10- to 100-fold) than a/a or a/a cells. Thus, heterozygosity at the mat locus stimulates mitotic recombination. 5 . Radiation Survival

Diploids heterozygous at mat (a/a) are more resistant to lethal damage caused by X-rays than a/a or a/a diploids (Mortimer, 1958; Laskowski, 1960). Therefore, heterozygosity at the mat locus may decrease the initial damage to the cell or it may increase the efficiency of repair mechanisms. A similar influence of the mat locus on survival after ultraviolet irradiation has not been detected. Some X-ray sensitive (rad) mutants appear to be deficient in matmediated X-ray repair. For example, when a/a diploids are homozygous for rud-52 or rad-54, they are no longer more resistant to X-rays than a/a diploids (J. Game and R. Mortimer, unpublished observations). Since haploid cells that acquire the rad-52 and rad-54 mutations also become more sensitive to X-rays, these genes probably act in both mt-independent and mat-mediated repair. Other X-ray-sensitive mutations (rad-50, 5 1 and 57) still exhibited the m t effect seen in wildtype cells, i.e. homozygous rudlrad diploids were more resistant when heterozygous at the mat locus. Therefore, J. Game and R. Mortimer (personal communication) suggest that rad-52 and rad-54 govern early steps in X-ray repair that precede the repair step mediated by the mat locus, whereas rad-50, 5 1 and 57 govern later steps in a matindependent branch pathway. The only mutant that might be defective specifically in the mat-mediated branch pathway is the stel mutant isolated as a temperature-sensitive, non-mating mutant (MacKay and Manney, 1974a, b; described in Section IV, B.6, p. 375). Although Xray survival is normal in the stel haploid, a/a diploids homozygous for ste4 exhibit temperature-sensitive X-ray survival as well as abnormal sporulation (MacKay et al., 1972). A further effect of the mat locus on X-ray survival has been reported recently by E. Hunnable and B. S. Cox (unpublished observations). If, after X-ray treatment, cells are kept in saline for a period prior to plating onto -growth medium, the fraction of diploid cells that survive increases. After ultraviolet treatment, both haploid and diploid cells

PHYSIOLOGY OF MATING IN THREE YEASTS

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exhibit similar liquid-holding recovery. However, successful holding recovery after X-irradiation requires both a diploid nucleus and heterozygosity at the mat locus; neither haploids disomic for chromosome I11 and heterozygous at the locus, nor homozygous mat diploids, exhibit liquid-holding recovery. Thus, Hunnable and Cox concluded that there are two separable X-ray-repair mechanisms in diploid cells, namely the mat-dependent system that governs liquidholding recovery and a mat-independent process functional in diploids and cells with higher ploidies that is responsible for the “shoulder” seen at low doses in survival curves of these cells. That the processes of meiosis and sporulation, mitotic recombination and X-ray repair are related is indicated from mutant studies. For example, a number of the many radiation-sensitive mutants have decreased levels of induced mitotic recombination and abnormal sporulation (Mori and Nakai, 1968; Resnick, 1969; Game et al., 1975). In addition, some, but not all, of the mitotic recombination-defective mutants of Rodarte-Ramon and Mortimer (1972) are sensitive to Xrays andlor ultraviolet radiation. Thus, it seems likely that at least some of the polymerases, nucleases, ligases and binding proteins required for meiosis and recombination (both meiotic and mitotic) are also required for DNA repair. Since each of these processes is regulated to some extent by the mat locus, perhaps the locus could also affect the activity or synthesis of common gene products. 6. Budding Pattern The budding patterns of a/a or ala diploids and ala diploids differ in the location of a bud relative to the previous one (R. K. Mortimer, personal communication). An ala mother cell and its mature daughter (F,) both form buds of the next generation (F,) synchronously. These F, buds occur at opposite ends of the doublet (mother and F,). In diploids homozygous for the mat locus, F,. buds develop asynchronously at the joint between the mother and F, cell. Similar observations of budding patterns were made by B. Weinstein (personal communication) in which 10 out of 10 ala doublets exhibited polar budding, whereas 8 out of 9 ala and 18 out of 20 ala doublets budded medially. The budding pattern of haploid a or a cells is the same as for ala or ala diploids indicating yet another influence of the mat locus on cellular behaviour.

M. CRANDALL, R. EGEL AND V. L. MACKAY

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V. Comparative Discussion A.

STEPS I N -YEAST C O N J U G A T I O N C O M P A R E D

The basic steps in conjugation are the same for the three yeasts reviewed in this article, but the steps differ in detail (Table 3). These differences may be real or they may reflect biases in experimental procedures. Therefore, more comparative studies are necessary to further our understanding of yeast-mating physiology. Yeast-cell mating involves both temporal and spatial organization of biochemical reactions which lead to new form and function. The organization and sequence of reactions are controlled genetically, not only by the genes governing individual reactions but also by a central locus, the mating-type locus (mat),that governs expression of conjugation-specific genes. The picture that has emerged from genetic analyses is that the mat locus is a cluster of genes, some of which may be regulatory, others structural. These genes are involved in different steps leading to cell fusion and also in regulating the expression of both haploid and diploid functions. Furthermore, it appears that, in some yeasts, haploids of opposite mating type carry the structural genes for both alternative mating types in the Cir configuration but only one is expressed. The evidence for this is that some strains can switch from one mating type to another. Certain of these mutations in mating type are irreversible, due to deletions, but others are reversible and are under the influence of homothallism genes. The latter reversible genetic changes should be called “paramutations” since they probably do not result from alteration of the genetic information at each mating-type allele but rather result from changes in gene expression. Paramutation is defined by Brink et al. (1968) as directed genetic change that occurs in somatic cells, that heritably alters the functional state of a locus, and that is exerted by factors located within the chromosome itself. Therefore, this term appropriately describes the action of homothallism genes. Strictly heterothallic yeasts may be viewed as more primitive in that they have not evolved the complex genetic mechanisms for homothallism. Since the complementary regulatory genes that govern expression of each mating-type allele in the respective haploids are both present in the diploid, neither mating type is expressed. But now a new series of reactions is initiated as a result of heterozygosity at the mat locus,

TABLE 3. Comparison of conjugation in three yeasts

Strains are:

Hansenula wingei Heterothallic only

Schizosaccharomyces pombe Heterothallic o r homothallic

Saccharomyces cerevisiae Heterothalk or

Stage of cell cycle for optimal conjugation

Not studied; probably G1 in stationary phase

G1

G1

Growth stage for optimal con,jugati on Con,jugation occurs best in

Stationary phase

Stationary phase

Exponential phase

z

Buffer lacking both a nitrogen source and growth factors Undetected

Medium lacking a nitrogen source

Complete growth medium

c)

Undetected for agglutination; sex-specificpheromones induce proteolytic activity and stimulate cell fusion Inducible in haploids

Sex-specificpheromones cause G1 arrest and induce sexual agglutination

rn

Diffusible sex pheromones

Sexual agglutination

Constitutive in haploids; inducible in diploid

homothallic

W

I

3

g

c)

<

%

lnrormation summarized from references discussed in Sections 11,111, and IV.

3z

z 2 ; 2

Constitutive or inducible in haploids

g i!

386

M. CRANDALL, R. EGEL A N D V. L. MACKAY

namely the reactions leading to meiosis and sporulation. The alternative mating-type alleles must be present in the trans configuration at the mat locus to initiate reductional division; these alleles also govern such diverse processes as the production of sex-specific pheromones for sporulation, the functioning of repair enzymes involved in recombination, as well as cell-division patterns in the diploid. Yet, even if a cell has the correct genetic constitution, sexual functions cannot be expressed unless the physiological conditions are permissive. For this reason, studies of haploid and diploid functions have centred on determining the stage in the cell cycle or phase of growth of the culture as well as the medium which yields the maximal sexual response. The unifying finding in all three yeasts discussed in this article is that cells mate when they are in G 1 of the cell cycle (Table 3,p. 385) and have only one complement of each chromosome. If cells hybridize when they are not replicating their genome, the resulting diploid will have an even number of chromosomes. Euploidy ensures balanced diploid growth, good spore survival following meiosis, and successful regeneration of the haplophase. How is mating restricted to G1 ? In the laboratory, a population of cells in G1 may be obtained by a variety of procedures, including allowing cells to enter stationary phase, growing cells under conditions of nitrogen limitation, selecting unbudded cell fractions by densitygradient centrifugation, or mixing opposite mating types together. In the mating mixture, cells are synchronized in G 1 through the mediation of pheromones secreted by each cell type. In Nature, mating is probably also stimulated by these pheromones and/or by limitation of the growth processes (Table 3, p. 385).Diffusible pheromones have not been detected in some yeasts, yet they may exist and be induced only after effective cell contact is established via sexual agglutination. Recognition of the opposite sex is accomplished by the interaction of 'cell-surface agglutination factors which are complementary and which form complexes between cell surfaces to bind the cells together. It has been suggested that these agglutination factors are on surface filaments that extend from the thick wall and that make initial contact before the walls touch. Agglutination factors determine the specificity of mating and are probably under the control of the mating-type alleles since mutants or recombinants have never been isolated which agglutinate with one mating type but conjugate with the other. Some

PHYSIOLOGY OF MATING IN THREE YEASTS

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strains of the same species are inducible for sexual agglutination whereas others are constitutive (Table 3 , p. 385). Constitutively agglutinable strains may be more primitive in that they lack an inducible mechanism for regulating expression of the agglutination factors. However, constitutively agglutinable strains do have some regulatory genes since neither agglutinin is expressed in the diploid under normal growth conditions. When the diploid is grown under abnormal or stress conditions, the agglutination factors are synthesized (Table 4). The fact that the diploid is inducible for agglutination implies the existence of regulatory genes that are carried by each haploid but are active only in apposition. Induction of sexual agglutination may have specific metal-ion requirements. For example, vanadium ions specifically induce the 5/21 hybrid of H . wingei to produce 5-agglutinin (Crandall and Caulton, 1975) and the a-factor of Sacch. cereuisiae copurifies with cupric ions (Duntze et al., 1973). These metal ions may be required for synthesis of conjugation-specificmacromolecules. Cell contact is mandatory for triggering later steps in conjugation and may involve the exchange of chemical inducers between conjugal partners. Unfortunately, not much is known about these later steps in cell fusion because of the lack of sophisticated technology allowing study of localized biochemical events. The development of such technology could be aided by useful model systems available in each yeast. For example, the processes of wall extension and formation of copulatory-like protuberances that occur during cell fusion can also be induced under non-mating conditions. More specifically, in H . wingei the diploid produces elongated cells when it is induced to synthesize one of the agglutination factors (Table 4); in Schirosacch. pombe, cells carrying thefusl gene are blocked at cell fusion but still respond to the opposite mating type by forming elongated extensions at the site of contact with the opposite sex (Fig. 5, p. 334); in Sacch. cereuisiae, similar elongated forms, called shmoos, are formed in haploid cells either in mating mixtures or in pure culture if stimulated with an extract containing pheromones from the opposite mating type (Table 4).In all of these situations, agglutination is induced, the cell elongates but mating is blocked. The discovery of conditions allowing separation of initial wall changes from later events in cell fusion will aid further studies of the formation of the conjugation tube. In addition to these sexual responses, similar elongated cells are observed in H . wingei (pseudohyphae are produced under starvation conditions) and in

TABLE 4. Summary of the physiological conditions that induce sex-specilic interactions in three yeasts Hansenula wingei

Induced cell

Schizosaccharomyces pombe

Diploid

Haploid h+ and h-

Haploid a

Occurs only in mixed cultures under nitrogensource limitation

a-Factor (an oligopeptide pheromone) or in mixed

5/21

Inducing conditions

Yeast extract Yeast extract or with a chelating high agent concentrations or of vanadium trace-metal ion or limitation vitamin limitation, then nitrogen source limitation, or Ca2+and MqP+ limitation

0)

Haploid a

a-Factor (a proteinaceous pheromone) z or in mixed cultures 0

E 0

F I-

?J rn G)

rn I-

5

Induced cell agglutinates with

21

5

h+ and hin mixed culture

a

Associated morphological change

None

Cell elongation and lack of bud separation

Normal zygote formation

G1 Arrest-and cell

Not tested

or irregular cell wall and lack of bud separation

Q)

cultures

Inducing pH value Range of pH values

3-8 (broad)

W

Saccharom.yces crreiiisiap

Not tested 4-6

Information summarized from references discussed in Sections 11,111 and IV.

elongation producing shmoos

5 4-5.5 or 6 (narrow1 a

G1 Arrest and cell

elongation producing shmoos

40 5 r

PHYSIOLOGY OF MATING IN THREE YEASTS

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Sacch. cerevisiae (in cdc mutants blocked in cell division; Hartwell, 1974). Furthermore, the elongated diploid cells of H . wingei produced after induction of 21f (Crandall and Caulton, 1975) look very similar to the cdcl mutant of Sacch. cerevisiae' reported by Hartwell et al. (1974). Therefore, it is likely that similar events are occurring in these diverse systems. The yeast-to-hyphal change observed in higher fungi is also analogous to these examples of cell elongation without division and, therefore, a comparative biochemical study of these separate systems is deserved. What little is known about the biochemical reactions in cell fusion comes from inhibitor studies. New protein, and hence probably RNA, synthesis is required for induction of conjugation, as is energy metabolism but later stages in conjugation are more resistant to inhibitors. There may be many biochemical reactions in common in both cell division, where one cell becomes two, and in cell fusion, where two cells become one, but the fact that sterile mutants divide normally provides evidence that conjugation-specific enzymes or functions exist that are not required for growth. The existence of conjugation-specific enzymes is also suggested by the finding that cell fusion in Schizosacch. pornbe is resistant to a concentration of 2-deoxyglucose that causes lysis of dividing cells. Therefore, the crosslinking enzyme involved in synthesis of the conjugation bridge either is not sensitive to this growth inhibitor, or the site of action is extremely localized so that lysis of zygotes does not occur. Even less is known about fusion of the nuclei following formation of the conjugation tube, because it involves intracellular events that are more difficult to study. The methods that have been used to study events at this level are electron microscopy and genetic recombination (of both nuclear and cytoplasmic markers). Reflecting the interest in these latter events, there has been an explosion of articles on mitochondrial inheritance in Sacch. cerevisiae in recent years. It is not known whether mixing of cytoplasmic organelles during conjugation is followed by fusion of mitochondria or whether recombination of parental mitochondrial genes occurs between naked mitochondrial DNA molecules released from disintegrated mitochondria. Electronmicroscope evidence indicates that mitochondria partially break down following cell fusion and, therefore, it is conceivable that higher levels of membrane degradative enzymes are present intracellularly at this time. These membrane-active enzymes could be involved in formation

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M . CRANDALL, R . EGEL AND V. L. MACKAY

of the conjugation tube as well as in breakdown of mitochondria1 components and in nuclear fusion. Disorganization of the two nuclear membranes at the point .of contact in the cytoplasmic bridge is seen in Fig. 4 (p. 325). After nuclear fusion is completed, the zygote will either divide or, if conditions are not favourable for growth, it will sporulate. In either situation (mitosis or meiosis), genetic recombination can take place. I t has been discovered that heterozygosity at the mat locus also influences recombination. Recently, studies of the effect of the mat locus on DNA repair have overlapped with studies of X-ray-sensitive Crud) mutants and of sterile (ste) mutants. Now it is possible to construct a branched pathway for DNA repair represented by a sequence of genes (mat, rad, ste) that were previously thought to be unrelated. These merged studies of mating and X-ray survival will undoubtedly produce new insights into the enzymic reactions involved in DNA repair and genetic recombination. B. E V O L U T I O N A R Y A S P E C T S O F S E X U A L R E P R O D U C T I O N

The morphological variations (Fig. 1, p. 31 1) and physiological variation (Tables 3 and 4, pp. 385 and 388) in sexual reproduction in these three yeasts imply differences in biochemistry and hence genetic constitution. The fact that these yeasts have evolved similar life cycles suggests a selective advantage in being able to reproduce sexually as well as asexually. The advantages of the greater metabolic capacity and growth rate of the diploid in Nature have been discussed by Fowell (1969b) and Kreger-van Rij (1969). The diploid also has an increased resistance to X-rays, a selective advantage compared with the haploid sensitivity to recessive lethal damage and the increased probability of dominant lethal damage in higher ploidies (Owen and Mortimer, 1956; Mortimer, 1958). Such a balance is likely to extend to other harmful agents as well. Under starvation conditions, diploids undergo meiosis and sporulation, yielding haploid ascospores that are slightly more resistant to environmental conditions than vegetative cells (Fowell, 1969a). After germination under more favourable conditions, spores can fuse giving rise again to diploids. Since genetic recombination occurs at a high frequency during meiosis in yeast (Fogel and Mortimer,

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197 1 ), the spores are likely to carry new combinations of the original parental genes and, therefore, may form diploid hybrids that are more vigorous. The existence of complementary mating types with their concomitant sex-specific factors would seem to promote diploid formation, and the existence of regulatory genes would prohibit further mating that would yield higher ploidies. This transition from haploid spore to diploid cell can be achieved even in the absence of a conjugal partner via the homothallism genes that are present in many species of Hansenula, Schizosaccharomyces and Saccharomyces (Kreger-vanRij, 1969). If' a spore has not mated within the first several divisions following germination, then it or its progeny can mutate to the opposite mating type and mate with a cell from a previous generation (see Section IV, A.2, p. 35 1). Although this diploid would be homozygous for all genes except those at the mat locus, it would still have a selective advantage because of its diploid genome. The fact that Schizosacch. pombe exists primarily in the haplophase in Nature may indicate that this yeast is more primitive in that it has not evolved genetic mechanisms that allow conjugation under growth conditions or that separate the process of mating from sporulation. C . C O M P A R I S O N WITH M A M M A L I A N S Y S T E M S

Mammalian organisms consist of many interacting systems involving cell contact analogous to yeast cell mating. Only a few examples need be cited, including recognition between egg and sperm in fertilization, cell adhesion in tissue formation and B-cell and T-cell interaction with macrophages in antibody formation. In all of these systems, specificity of cellular recognition is determined by complementary cell-surface macromolecules (see references and recent reviews cited in Crandall el al., 1974 and in Crandall, 1977). Perhaps cell-surface agglutination factors of eukaryotic yeast systems became primitive tissue aggregation factors or forerunners of immunoglobulin molecules that evolved further in more advanced eukaryotic systems. Similarly, the diffusible oligopeptide pheromone that induces both initial cell contact and the later steps in yeast mating may be represented in the mammalian system by the thymus factor thymin, a polypeptide hormone (Basch and Goldstein, 1974) that stimulates precursor T-cells to differentiate into cells competent to recognize immunologically and selectively destroy

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M . CRANDALL, R . EGEL AND V. L. MACKAY

(neoplastic, graft or bacterial) cells. In both of these systems, a small molecular-weight peptide synthesized by a different cell type acts on a target cell causing it both to differentiate and to synthesize a new cellsurface macromolecule. The nature of the antigen-binding receptors on T cells is uncertain, whereas the cellular recognition factors on opposite sexes of yeast cells are glycoproteins. In both cases, these receptors are complementary to a foreign macromolecule. However, this macromolecule cannot be completely foreign, or the target cell would not have the genetic information for synthesizing a complementary macromolecule. In mammalian cells, this genetic information may have been conserved during evolution from simpler aggregating systems. In sexually interacting microbial cells, the genetic information may have evolved and been selected because it codes for molecules that promote mating efficiency. Cells able to mate efficiently would be favoured for survival and hence would pass onto their progeny, genes coding for complementary mating substances and diffusible compounds that induce sexual differentiation. The chemical nature of recognition factors and pheromones produced by all microbial systems studied is reviewed by Crandall ( 197 7). VI. Acknowledgments

Unpublished work of M. Crandall and the preparation of this manuscript were supported by U.S.P.H.S. grant GM21889 and by the Biomedical Science Grant awarded to the University of Kentucky. Unpublished work of R. Egel was supported by the Deutsche Forschungsgemeinschaft (SFB 46) and performed with the technical assistance of Janet Walsh. Unpublished work of V. L. MacKay was supported by U.S.P.H.S. grant GM22149 and by the Biomedical Science Grant awarded to Rutgers University, and performed with the technical assistance of Madeline van de Burgt-Goui. We thank all of the researchers who contributed figures and unpublished data. We also thank the numerous persons who have critically read sections of this manuscript. REFERENCES

Abell, C. W. and Monahan, T. M . (1973).Journal of Cell Biology 59,549. Ahmad, M. (1953). Annals ofBotany 17, 329. Angehrn, P . and Gutz, H . (1968).Genetics 60, 158 (abstract).

PHYSIOLOGY OF MATING IN THREE YEASTS

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AUTHOR INDEX Numbers in italics r e f i to thr pages on which references are listed al thr end ofeach a r t i h .

A Abbott, B. J., 284,300 Abeles, R.H., 202,215,250 Abell, C.W., 359,392 Abelson, P. H., 257,304 Adams, B. G.,138,140,166 Adesnik, M.,151,168 Adler, L. W., 227,249 Aganval, R. P.,292,304 Ahmad, F.,50,80 Ahmad, M., 360,392 Ahmed, S.A., 67,76 Aiba, S., 12,76 Ainsworth, G.C.,28, 76 Aitken,D. M.,101,103, 104, 116 Akbar, M.D., 27 1,273,278,300 Alais, J., 139,166 Alberghina, F. A. M., 75,83 Aldanova, N.A. 2 17,249 Alexander, J. K.,50.80 Alexander, J. V., 32.81 Alroy, Y.,72, 76 Altendorf, K. H.,179, 183, 196, 226, 227, 235,244,246 Ames, G.F.,210,244 Andersen, K.,90,92,93,107,168,I16 Anderson, B., 192,244 Anderson, J. C.,61,81 Anderson, J. G.,3, 15,61,67,76,81,82 Anding, C.,138,171 Andreasen, A. A., 146, 166 Andreoli, T.E., 2 1 7,250 Andrews, J. F., 42,80 Angehrn, P.,333,392,394 Anthony, C.,52.82 Anthony, D. D., 294,300 Antonov, V., 217,249 Apple, J. L., 155,158, 166 Aranachalam, T.,151, 167 Ark, R.,6,83 Armitt, S., 63.64; 76,82

Arnaud, M., 140,169,379,394 Amow, P. M.,147,I71 Arsenault, G.P.,149, 152,166,171 Arst, H.N.,52,76 Asano, A., 206,225,246 Ashburner, M., 127,166 Atallah, A. M., 139, 166 Atkinson, D. E., 65, 80, 254, 257, 258, 263. 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 280, 281, 285, 289, 290, 295, 296, 298, 301,303,305

Augustin, H. W., 282,302 Austin, S., 51, 79 Axtell, J. D.,313,384,393

B Baarda, J. R., 237,246 Bachi, B., 266,27 1,273,287,301 Bachofen, R.,66,81 Baehr, M-L., von, 274,282,303 Bagnara, A. S., 259, 266, 283, 291, 292, 301

Bagni, N., 75,83 Bailey, R. B., 139,166,285,301 Bainbridge, B. W., 47,48,61,76 Bal,A. K.,151, 171 Ball, W.J., Jr, 267, 271, 272, 273, 275, 278,281,301 Ballou, C. E., 97, 98, 118, 318, 319, 320, 366,367,396,397,398 Bancroft, K., 266,306 Bangham, A. D., 153,167 Baniecki, J. F., 141, 148, 167 Bard, M.,144,145, 167 Barksdale, A. W.,149, 151, 152, 156, 166, 167,169,171 Barnes, E. M., 183, 185, 186, 197, 198, 203, 204, 205, 206, 207, 208, 212, 213, 216, 230, 233, 235, 237, 238, 242,244,245,247

399

400

AUTHOR INDEX

Barnes, I. J., 50.80 Blanco, R., 206,213,251 Barnes, L.D., 276,303 Blenden, D. C., 99,117 Barninova, S . E., 63,76 Block, R.,295,301,303 Ban, R.M., 137,167 Bloom, S. J., 65,77 Barratt, R. W., 34,83 Bloss, H.E., 141,148,167 Blumenthal, H.J., 59.61.77 Bartlett, K., 138,139,167 Bartley, W., 57,81,271, 278,301,304 Block, E., 271,302 Bartnicki-Garcia, S., 15,76,137,167 Boehlke, K. W., 74,77 Barton, D. H.R., 145,167 Boggs, J. M., 132,170 Barwell, C. J.,288,301 Bogomolini, R.A., 236,245 Basch, R.W., 391,393 Bohlcek, J., 87,I18 Bauchop, T., 262,301 Bolotin, M., 370,393 Bauer, S . , 341,393 Bolton, E.T., 257,304 Bayley, S. T.,94,95,98,103,116, 117, Bond, F. T., 145,171 119 Boonsaa, J., 180,187,188,200,204,215, 218, 219, 220, 221, 222,223, 227, Beacham, I. R., 259,306 Beadle, G. W., 32,82 234,236,245,247,248 Bean, G. A., 123,167 Boos, W., 177,209,217,226,245,248 Bech-Hanson, N. T., 370,396 Border, D. J., 366,393 Beck, C., 58,76,292,293,294,301 Borek, E., 293,305 Beckett,A., 140,141,170 Borgii, P. T., 366,393 Beckwith, J. R.,297,301 Bornefeld, T., 46,77 Beever, R.E., 64.65,76 Borowitzka, L.J., 89,90,I17 Bengali, Z. H., 60,76 Borrow, A. E., 47,77 Benko, P.V., 50.76 Borstel, R.C., von, 350.352.353.396 Bennett, R.D., 136,154,170 Bostock, C. J., 337,393 Berg, L.A., 121,169 Bowien, B., 266,286,288,301 Bager, E.A., 210,217,218,226,227,245 Boyer, P. D., 290,217,226,247 Bergey'sManual, 86,87,102,115,117 Boze, H., 58,78 Bagquist, A., 69.70,76,77 Bracker, C. E., 79 Bergstein, P.E., 277,288,302 Bragg, P. D., 188,250 Banlohr, R.W., 271,279,303 Brasier, C. M., 155, 158, 161,167 Berry, L.,295,301 Bremer, H., 190,294,295,301,302 Bettenhaussen C., 262,263,264,265,305 Brennan, P. J., 165,167 Betz, A., 278,301 Brennerman, J.A., 157, 172 Betz, H., 67,79 Bresch, C., 338,344,349,393 Bevan, E. A., 366,396 Bret, J. P.,34,77 Bhattacharwa, F., 204,217,245 Brice, J. M., 268,301 Biely, P.,341,393 Brierley, M.R.,38,77 Biemann, K., 149,166 Brillard, M., 139,168 Biggs, D . R.,147,170 Brink, R.A., 313,384,393 Bilinski, T., 308,354,355, 356, 361,364, Britten, R. J.,257,304 393,398 Brock, T. D., 308,309,315,316,317,318, Binkley, S. B., 259, 266, 267, 270, 282, 319, 320,321, 322, 326,327,328, 283.29I , 302 329,393 Bisschop,A., 178,179,180,181,182,183, Brockerhoff, H., 131,167 195, 196, 199, 200, 206, 209,211, Broda, P.,293,302 214,215,231,232,233,234,247,248 Brodie, A. F., 1 1 1, 118,206,225,246 Black, S. H., 330,393 Brody. S . , 46,70, 77, 78,259,286,301, Blakley, R.L.,258,301 302 Blamire, J., 377,393 Bronner, F., 205,216,245

AUTHOR INDEX

Bronner, H. S., 185,245 Brown, A. D., 87, 89, 90, 101, 102, 103, 104,116,117,118 Brown, C. E., 50, 77 Brown, C. M., 56.57, 77, 79, 80 Brown, R. H., 94,117 Bruckdorfer, K. R., 134, 167 Bruckforfer, K. R., 133, 146, 154,168 Brunner, H., 39, 77 Brunschede. H., 301 Brunsting, A. H. M., 262,263,304 Brushaber, J. A., 141, 143,167 Bucking-Throm, E., 164, 167, 168, 355, 357,359,360,363,387,393,394 Buckland, B., 97, 117 Bull, A. T., 8, 10, 11,36,39,40,41,42,43, 45, 47, 48, 57, 58, 60, 61, 62, 64, 66, 73, 75, 76, 77, 78, 82 Buller, A. H. R., 24.26, 77 Bu’Lock, J. D., 62, 73, 77, 166, 167 Bungay, H. R. 12,38,44,77, 79 Bunn, C. L., 370,393,398 Burkholder, 0. R., 354,396 Burnett, J. H., 48, 77 Burton, K., 258,292,301,305 Burton, K. A . , 309,398 Bushell, M. E. 8, 11,36,39,40,41,43,57, 64.66, 72, 73, 75, 77 Butler, G. M., 24,25,26,77 Butler, K. W., 132, 133, 167 Butlin, J. D., 221,226,245,249 Button, D. K., 53, 77 Byers, B., 370, 371, 379, 380, 381, 393, 396 Byrne, P. F. S., 165, 167

C Cabib, E., 274,275,305 Calam, C. T., 4,37, 77 Calderone, R. A,, 143, 171 Caldwell, I. Y., 18, 19.2 1.77 Caldwell, J., 140, I72 Calleja, G . B., 337,338,339,340,393,398 Callen, D. F., 367, 368,370,393 Callow, D. S., 40.81 Camici, L. G., 8, 77 Campbell, D. A., 355,364,393 Campbell, W. A., 156, 160, 171 Cantino, E. C., 63, 77

40 1

Carlberg, D. M., 111, 117 Carter, B. L. A., 10, 11,39, 42.45.58, 60, 6 1,62,63, 77, 78 Carter, H. E., 146, 169 Carter, J., 193, 248, 257, 266, 270, 273, 275,283,303 Carter, J. R., 212,246 Cashel, M., 292,303 Cashel, M. B., 292,293,301 Cass, A., 133, 169 Cadton, J. H., 321, 328, 329, 387, 389, 393 Cazzulo, J. J., 103, 117 Cha, Y. A., 205,247 Chain, E. B., 8, 77 Chamberlin, M. J., 294,304 Chance, B., 60, 78 Chapman, A. G., 266,269,270,271,273, 274, 275, 278, 280, 281, 289, 290, 298,301 Chapman, C., 278,301 Chapman, D., 132,167,170,171 Chappel, C., 163,167 Charney, W. 164,167 Chassang, A., 393 Chassang-Douillet, A., 58, 78 Cheah, K. S., 107,117 Chee, K. H., 142,167 Cheney, J. C., 43,72,79 Chevaugeon, J., 25, 78 Chignell, C. F., 110, 117 Chignell, D. A., 110, I I 7 Child, J . J., 141, 143, 145, 153, 160, 167, 168

Chlebowiu, E., 356,398 Cho, K.Y., 87.95.98, 117 Chojnacki, T., 54,81 Chow, C., 95,98, I19 Chow, C. T., 271,279,301 Christensen, C. M., 163, 171 Christian, J. H. B., 88, 89, 117 Cicmanec, J. F., 5 1.78 Cirillo, V. P., 176, 191, 192,245 Clark, D. J., 268,286,303,304 * Clark, D. S., 8, 78 Clarke, G. A., 65.78 Clark-Walker, G . D., 58,59, 70, 7 1,82 Clausen, E., 111, 117 Clayton, C. W., 157, 172 Clement-Metral, J., 279,302 Cleverdon, R. C., 131, I72

402

AUTHOR INDEX

Clutterbuck, A. J., 31,32,78 Cochrane, J. C., 74.78 Cochrane, V.W., 74,78 Coen, D., 370,393 Cohen, G. N.,225,245 Cohen, S. S., 75, 78 Cohen-Bazire, G.,96,117, 189,245 Cohn, W.E.,350,352,353,396 Cole, H. A., 270,273,278,283,285,301 Cole, J. A., 187,245,251 C o l h g e , A., 9, 19, 21, 23, 24, 26, 32,33, 34.83 Colvin, H. J., 70,78 Combs, L.E.,99,120 Connelly, C. M., 60,78 Conner, R.L., 130,168 Constantinides, A., 10,78 Conti, S. F.,32 1,322,340,393 Cook, A. M.,288,301 Coole, P.,21 7,250 Cooper, R.A., 135,173 Corrie, J. E.T., 145, 167 Coultate, T.P.,262,301 Cove, D. J., 52.62, 76,79 Cowie, D. B., 257,304 Cox, G. B., 186, 187, 221, 226,245,249, 262,303 Cox, G. S., 184,245 Crabbendam, K.J., 54.80 Crabtree, G.W.C., 261,301 Crandall, M., 308, 318, 319, 320, 321, 328, 329, 330, 365, 387, 389, 391, 392,393,394 Crandall, M. A., 308, 309, 315, 316, 319, 320,321,327,328,329,393 Culotti, J., 389,394 Cutter, V. M.,34,83

D Daatselaar, M. C. C., 204,214,247 Dabes, J. N.,6,12,71, 78 Dalton, B. P., 104,117 Damoglou, A. P.,301 Danon, A., 109, 110, 116, 117, 270, 279, 301

D'Aoust, J., 92,117 Dark, F.A., 271.305 Darke, A., 132,168 Darnell, J. E., 151,168

Davison, J. A., 271,301 Dawes, E. A., 267,268,273,274,276,281, 301,302,304 Dawson, P. S. S., 13.39, 78 Day, A. W., 321,340,365,394,396 Day, P.R.,336,394 Dayhoff, M.O.,101,117 Dayhoff,R. E., 101,117 Decker, K.,266.27 1,302 DPfago, G., 141, 143, 144, 145, 153, 164, 167,168 De Gier, J., 133, 168 De Greef, W.J., 133, 168 De Hertogh, A. A., 61,80 De Kruyff, B.,132,133,168 Delmer, D. P.,70,78,286,302 Demel, R. A., 132, 133, 134, 146, 154, 167,168 DeMoss, J. A., 187,220,248,249 Dennis, P.P.,294,295,301,302 Desi, J. D., 5 1, 78 De Sombre, E.R., 127,170 Detroy, R.W.,73,77 Deuel, T. F., 183,211,247 Deutsch, J.,370,393 Devor, K. A., 209,245 Dickerson, A. G.,54, 78 Dickson, L. G.,163, 168 Dietz, G.W.,185,204,207,213,245 Dietzler, D. N.,257, 266, 274, 275, 277, 281,288,302 Dingle, S. L., 191,249 DiTullio, N.W.,162,170 Divjak, S.,67,78 Dod, B. J., 135,168 Doddema, H., 204, 209, 211, 214, 215, 245 Doe, F.J., 33 1,333,336,394 Donovick, R., 37, 78 Dorpema, J. W.,180, 189, 206,223, 224, 236,246 Downs, A. J., 268,302 Drabble, W.T.,52,82 Drews, G., 189,248 Drummond, G. I., 260,302 Dubuc, J.. 163,167 Ducharme, L.,102, 117 Dujon, B.,370,393 Dundas, I. E. D., 102, 103, 104, 1 1 1 , !IS, 117, I19 Dunker, S . S.,53,77

403

AUTHOR INDEX

Dunn, E., 52.81 Duntze, W., 164, 167, 168, 173, 355, 356, 357, 358, 359, 360, 363, 378, 387, 393,394,397,398 Dupkon, P., 139,168 Duperon, R., 139,168 Dutton, P. L., 185,245 Dvorak, H. F., 260,302 Dvornik, D.,'163, 167 Dworkin, M., 267,270, 27 1,280,303

E Eagon, R. G., 178,186,205,213,246,250 Eakin, E. A., 70,77 Eakin, R. A., 69, 70.76 Eakin, R. T., 70, 76 Eaton, N., 367,369,370,371,398 Eberhard, S. J., 191,249 Edlin, G., 293,302,303 Edward, D. G. ff., 130,168 Edwards, D. L., 70.78 Edwards, J. A,, 149, 151, 152, 167, 168, I69 Edwards, P. A., 131, 134, 167, 168 Egel, R., 332, 333, 335, 337, 338, 339, 341, 343, 344, 346, 347, 348, 349, 393,394,395 Egel-Mitani, M., 335, 337, 339, 346, 348, 349,394 Eidia, G., 101, 117 Eigener, U., 27 1,302 Eimhjellen, K., 88,91, 117 Elliott, C. G., 121, 122, 135, 136, 137, 138, 140, 141, 142, 144, 153, 154, 155, 162, 163, 164, 168,169, 170 Ellis, E. A., 151, 171 Ellis, S. H., 4, 77 Elsden, S. R., 262,301 Elmavergi, H., 38,40, 78 Emerson, R., 10,25,67,78,79 Emmens, M., 206,248 Engelman, D. M., 132,172 Enoch, H. G., 188,245 Ephrussi, B., 354,394 Eposito, M., 379,380, 381,396 Epstein, A., 59, 79 Epstein, W., 204, 21 7,245 Erlich, H., 292,293,302 Esposito, M. S., 140,169,379,394 Esposito, R., 379,380,381,396

Esposito, R. E., 140,169,379,394 Esser, K., 45,80 Estabrook, R. W., 274,278,282,304 Ettlinger, L., 266.27 1,273,287,301 Eustanov, A. V., 2 17,249 Eveleigh, D. E., 146, 172 Even, H. L., 57.82 Even-Shoshan, A., 210,246

F Falkenberg, P., 96.98, 117 Fall, L., 258, 266, 269, 270, 271,273, 274, 275,278,280,281,298,301 Fangman, W., 379,380,38 1,396 Fangman, W. L., 359,396 Fanica-Gaignier, M., 279,302 Fantes, P. A., 54, 78 Farrand, S. K., 23 1,245 Fazeli, A., 60, 80, 164, 168 Fein, J. E., 205,245 Feinstein, M. B., 132, 169 Feir, H. A., 65, 78 Feldheim, M. E., 282,302 Fencl, Z., 7,39,60,78, 80,81 Ferguson, A. R., 67,79 Fernandez, G. M., 132,169 Fevre, M., 34.78 Fiddy, C., 11, 15, 28,34,41, 78 Fiechter, A., 58, 78, 264, 267, 270, 271, 273,285,305 Fiil, N. P., 291, 292,302,303 Finch, L. R., 258, 259, 266,283,291,292, 301,305 Fincham, J. R. S., 64, 78,336,394 Finean, J. B., 131, 169 Finer, E. G., 132, I68 Finkelstein, A., 133, 169 Finn, R. K., 6, 12, 71, 78 Firth, A., 259,306 Fisher, J., 196,201,250 Fitt, P. S., 103, 112, 113, 118, 119 Fitzgerald, W. A., 130, 168 Flavell, R. B., 63.64, 78 Fleet, G. H., 341,394 Flook, A. G., 132, 168 Fogel, S., 308, 315, 351, 354, 355, 363, 364,378,380,390,394,396,397 Forbes, E., 52.81 Forchhammer, J., 74, 78, 257, 267, 284, 295,302

404

AUTHOR INDEX

Forrest, W. W., 262,263,285,302 Gancedo, J. M., 288,3CU Foster, J. W., 2, 78 Ganesan, A. K.,213,246 Fournier, R. E., 2 16,246 Garnjobst, L., 32, 79 Fowell, R. R., 308, 354, 354, 370, 379, Gaudry, R., 163,167 390,394 Geiger, K. H., 38, 80 Fox, C. F., 212,246 Gelpi, E., 102, 119 Fox, I. H., 260,302 Gerlach, W. L., 38 1,394 Fraenkel, D. G., 293,304 Gershfeld, N. L., 135, 169 Francis, G. W., 102, 119 Gest, H., 188, 189,250,279,303 Frank, M. E., 99, 117 Ghei, 0. K.,216,246 Franzen, J. S., 259, 266, 267, 270, 282, Ghosh, S., 191,248 283.29 1,302 Gibbons, N . E., 88, 91, 105, 116, 117, Fredrickson, A. G., 5, 6, 12, 78, 80,81,83 118,119 Frear, D. S., 122, 170 Gibson, F., 186, 187,245,262,303 Freeland, J. A., 138, 140, 164, 169 Gibson, J., 261, 264, 265, 267, 271, 279, 282,297,304 Freese, E., 180, 182, 185, 186, 190, 197, 198, 199, 204, 207, 208,210,211,212, Gibson, K. D., 189,246 225,235,246,247 Gier, J. de, 133, 134, 167, 171 Freidmann, K. L., 338,339.394 Gilley, J. W., 44, 79 Frerman, F. E. 19 1,246 Gillie, 0.J., 26,29,34, 79 Fried, J. H., 149, 168 Gingold, E. B., 370,397 Friedenthal, M., 59, 79 Givner, M., 163,167 Friedman, M., 380,395 Glaser, L., 193, 248, 257, 266, 270, 273, Friend, J., 121, 171 275,283,303 Fries, E. R., 162, 169 Glaser, M.,287,292,303 Fries, N., 40,41, 79 Glasgow, J. E., 46,79, 81 Friesen, J. D., 74, 77,29 1,292,302 Gleason, F. H., 59,67, 79,82 Friis, J., 365,38 1,394 Glover, J., 131, 169 Fryberg, M., 145, 169 Gochnauer, M. B., 87, 90, 91, 101, 117, Fuentes, M., 185, 204, 207,247 118 Fukushima, H., 130,171 Goetsch, L., 370,371,393 Fuller, M. S., 67, 79 Goldberg, H. S., 99, 177 Futai, M., 179, 189, 190, 199, 200, 201, Goldstein, D., 21,22,23,31, 79 246 Goldstein, G., 39 I , 393 Fynn, G. H., 271,301 Goldthwait, D. A., 294,300 Gollub, E. G., 144, 169 Golub, E. E., 205,216,245 G Gooday, G. W., 155,166,169 Goodman, D., 290,304 Gaden, E. L., 10, 78 Goodwin, T. W., 123, 169 Gadkari, D., 274,302 Gordon, A. S . , 204,213,246,247 Gain, R. E., 136, 169 Gajewski, W., 54, 81, 308, 354, 355, 356, Gorman, C., 330,.393 Gorts, C. P. M. 57, 79 361,364,393 Sottlieb, D., 9, 82, 122, 138, 141, 142, Gale, E. F., 146, 169 145, 146, 169, 172, 173 Galindo, A. J., 157, 159, 169 Graham, J. M.,134, 167 Gallant, J., 292,293,301,302,303 Grant, J. K.,127, 169 Gallegly, M. E., 157, 169, 172 Gray, B. M., 135, 168 Galun, E., 30, 79 Green, C., 131, 134, 167, 168 Galzy, P., 58,78,377,379,380,393,398 Green,D. M.,149, 151, 152, 167, 169 Game, J. C., 383,294 Green, R. J., 156, 172 Gancedo, C., 288,303

AUTHOR INDEX

Grenson, M., 51.79 Gries, E. M., 216,251 Griffen, D. H., 72, 79 Griffen, D. M., 4 3 , 6 3 , 8 0 Griffin, P., 313,330,395 Grifiths,E.,95, 103, 113, 116, 117 Grindle, M.,145, 169 Groen, A., 205,246 Grotbeck, R. C., 370,395 Grove, S. N., 79 Gruber, H., 110,119 Grunwald, C., 134, 154, 169 Gull, K., 34, 83 Gunatilaka, A. A. L., 145.167 Gunge, N., 378,394 Gunsalus, I. C., 265,303 Gutnick, D. L., 204, 217, 226, 227, 245, 248

405

Hanson, C. W., 267,270.27 1,280,303 Hanson, R. S., 266,273, 275,280,303 Hara, M., 12, 76 Harada, B., 293,302 Harashima, S.,352,353,394 Harder, W., 205,246 Hare, J. F., 189, 199,246 Harnish, W. N., 121, 169 Harold, F. M., 217, 226, 227, 230, 235, 237,244,246,249

Harper, S. H. T., 39, 80 Harris, E. J., 217,249 Harris, P., 19 1,246 Harrison, D. E. F., 264, 266, 278, 280, 285,303

Harshman, R. B., 292,303 Hartman, R. E., 63, 64, 65, 78, 79, 135, 169

Guttman, S-M., 153, 155, 170 Guttowski, S.J., 221, 226.249 Guntz, H., 308, 331, 333, 335, 336, 337, 343,349,392,394,396

Guymon, L. F., 178, 186, 205, 213, 246, 25 0

H Haag, G . ,359,397 Haagen-Smit, A. J., 149, 172 Haarasilta, S.,67, 79 Haber, J. E. 376,378,394,398 Haddock, B. A., 217,226,249 Haefner, K., 354,355,394 Hagerby, B., 70,79 Hagiya, M., 164, 172,357,359,397,398 Halaban, R.,46, 79 Hall, B. D., 352,353,365,380.38 1,395 Hallermeyer, C., 138, 169 Halpern, Y.S., 177,210,211,246 Halvorson, H. O., 140,141,169,170,379, 394,397

Hamer, G., 38, 83 Hamilton, D., 62, 77 Hamilton, I. D., 264, 265, 270, 273, 275, 283,303

Hamilton, J. A., 186, 187,245 Hamilton, W. A., 177,230,235,246 Hampton, M.L., 198,199,246 Hankinson, O., 62, 79 Hansen, M. T., 292,303 Hansford, G. S.,38, 79

Hartwell, L. H., 164, 167, 355, 359, 360, 363,372,373,379,389,393,394

Hartzell, R., 135,172 Harvey, R.,63.79 Harvey, R.J., 73, 79 Haseltine, W. A., 295,301,303 Hashimoto, Y.,341,396 Haskins, R. H., 121, 136, 141, 142, 143, 145, 146, 153, 160, 167, 168, 169, 172, 173 Haslam, J. M., 144,167 Hasnain, S.,95, 119 Haug, A., 133, 170 Hawker, L. E., 30.79 Hawthorne, D. C., 350, 351, 352, 375, 394,395,396 Hayflick, L., 133,154, 172 Haynes, R. H., 383,394 Heap, R. B., 153, 169 Heather, W. A., 161, 171 Hebb, C. R., 147,169 Heftmann, E., 127, 136, 154,169,170 Held. A. A., 67.79 Hellingwerf, K. J., 180, 189, 206, 223, 224,236,246 Hemming, F. W., 122, 137, 167, 172 Hempling, W. P., 60, 76,262,268,303 Henderson, J. F., 258,261,301,303,305 Hendrie, M.R., 121, 122, 153, 168, 169 Hendrk, F. F., 156, 160,171 Hendrk, J. W., 121, 122, 123, 136, 142, 153, 154, 169, 170 Hengstenberg, W., 191, 192,246

406

AUTHOR INDEX

Henry, S. A., 140, 141, I 7 0 Heppel, L. A., 183, 210, 218, 226, 227, 245,246,250

Herman, A,, 313,315,317,326,327,329, 330,352,362,395

Herring, A., 366,396 Herskowitz, I., 352,353,362,375,395 Hertzberg, E. L., 190,246 Herzog, H. L., 164, I 6 7 Hescox, M. A., 111. 117 Heslot, H., 308,333,337,394 Hess, B., 224,248,288,301 Hickman, C. J.. 161, 171 Hicks, J. B., 352,353,362,375,395 Higgins, J., 60, 78 Highberger, J. H., 63.82 Hinckle, P. E., 236,249 Hinkelmann, W., 59,82 Hinkle, P. C., 190,246 Hirata, H., 206,225,235,244,246 Hirose, T., 38.80 Hobson, P. N., 282,285,303 Hoch, H. C., 141,170 Hochstadt-Ozer, J., 193, 194, 205, 246, 258,292,303

Hochstein, L. I., 88, 101, 102, 104, 117, 119

Hockenhull, D. J. D., 6,79 Hodgkiss, I. J., 63, 79 Hoffman, H., 99, I I 7 Hoffmeyer, J., 259,303 Hofmann, E., 274,282,302,303 Hofsten, A., von, 40.41, 79 Hofsten, B., Von. 40.41, 79, 104, 119 Hohl, H. R., 141, 170 Holden, J. T., 177,246 Holliday, P., 12 1, I 7I Holligan, P. M., 5 1.62, 79 Holmes, W., 60, 78 Holmes, W. L., 162, I 7 0 Holmlund, C. E., 135, 169 Holmes, W. H., 264, 265, 270, 273, 275,

Horecker, B. L., 213,246 Horenstein, E. A,, 63, 77 Horgen, P. A., 151, I 7 2 Horlick, L., 163, I 7 0 Horowirz, D. K., 151, 170 Hossack, J. A., 137, 146, 154, I 7 0 Hostalek, S., 73, 77 Hottingua, H., 354,394 Hsia, J. C., 93, 117, 132, I 7 0 Hsiung, H. M., 163,171 Huang, L., 133, I 7 0 Huang, M., 147, I 7 3 Huang, M. Y., 12, 79 Hubbard, J. S., 103, 117 Hucho, F., 299,303 Hueting, S., 54,80 Hughes, D. E., 270, 273, 278, 283, 285, 301

Huguenin, B., 157,158, 170 Huisingh, D., 162, 171 Hulme, M. A., 62, 77 Humber, L., 163, I 6 7 Humphrey, A. E., 38.79 Hunt, L. T., 101, 117 Hunter, J. H., 157, I 7 2 Hutchinson, S. A., 122, 123,171 Hutchison, K. W., 266,273,275,280,303 Hutter, R., 50, 79 Huttunen, M. T., 188, 218,220,221, 222, 223,245

Huzyk, L., 286,303

I

Holt, S. C., 189,246 Hob, R. B., 165, I 7 0 Holzer, H., 67, 79 Hong, J. S., 198, 199, 201, 203, 204, 205,

Iguti, S., 354,395 Iida, H., 130,I 7 1 Ikeda, Y.,352,397 Illingworth, R. F., 140, 141, 170 Ingraham, J., 292,293,294,301 Ingram, M., 103, 117 Irr, J. D., 291,292,303 Isaac, P. K., 9, 79 Itagaki, E., 187, 188,250 Ivanov, V. T., 21 7,249 Iwadare, T., 149, 168 Iyengar, C. W. L., 130,168 Izaki, K., 204,216,248

208, 214, 215, 225, 226, 227, 228, 230,232,248,246,247,249 Hopper, A. K., 352, 353, 365, 380, 381, 395

Jachvmczyk, W., 361,393

283,303

J

AUTHOR INDEX

407

Jackson, L. L., 122,170 Katz, D.,21,22,23,31,79 Jackson, R. M.,161,172 Kawase, Y.,148,170 Jagger, W.S.,217,249 Kay, W.W., 216,246 Jain, V.K., 262,303 Keen, N.T.,63,64,65,79 Jakob, H., 354,355,395 Keith, A., 132,170 Janacek, K., 48.79 Kellerman, G.M.,147, 170 Janin, J., 29 1,305 Kemp, R.B.,45,81 Jarrnan, T.R.,145,167 Kennedy, E. P.,189, 199,212,246 Jasne, S.J., 144,173 Kepes, A.,212,247 Jeffreys, E. G.,47.77 Keradjopoulos, D.,103, 118 Jennings, D.H.,48,51,62,79 Kern, H.,143,144,168 Jensen, E.V.,127,170 Kerwar, G. K., 213, 225, 226, 227, 240, Joenje, H.,184,185,247 24 1,242,247,249 Johansson, M.,70,79 Kessell, R. H.J., 47, 77 Johnson, B., 57,79 Khachatourians, G . C.,286,303 Johnson, B. F., 337, 338, 340, 341, 354, Khaki, I. A., 159,170 393,395,398 Kidby, D. K., 123,172 Johnson, C. L., 205,247 Kiltz, H.H.,357,397 Johnson, M.J., 37,65,77,81 Kim, W.K., 122,171 Johnson, T.H., 217,249 Kinghorn, J. R.,5 2 , 8 1 Jollow, D.J., 146,172 Kinghorn, N. R., 52, 79 Jones, C. W., 268,301,302 Kinoshita, S., 12.80 Jong, L.,de, 231,232,234,245 Kinsky, C. B., 133,168 JOO, C. N.,102, 105,I18 Kinsky,S. C.,133,145,168,170 Kirsch,J., 38 1,395 Kitano, S.,363,397,398 K Kjeldgaard, N.O.,21, 72,80,82,257,267, Kaback, H. R., 176, 177, 178, 180, 182, 282,284,293,295,303,304,305 183, 184, 185, 186, 188, 189, 190, Kleese, R. A., 370,395 192, 193, 196, 197, 198, 199, 200, Klein, W.L.,190,217,226,247 201, 202, 203, 204, 205, 206, 201, Knight, R. H.,74,82 208, 209, 210, 211, 212, 213, 214, Knights, B. A., 121, 122, 135, 136, 137, 215, 217, 218, 219, 220, 221, 222, 138, 140, 144, 153, 154, 164, 168,169, 170,I73 223, 225, 226, 227, 228, 230, 232, 233, 235, 236, 237, 238, 239, 240, Knoche, H. W., 122,123,171 241, 242, 243, 245, 246, 247, 248, Knowles, C.J., 261,264.265.271,303 Kobayashi, H.,8,79 249,250 Kauorowski, G., 185, 196,201,204,207, Koch, A. L.,7 1, 79,293,303 Koch, T.K., 102,119 2 4 5 250 Kalbitzer, S., 164,168,357,387,394 Kocur, M.,87,118 Karnen, M.D.,279,302 Koepsell, H.J., 39,80 Kane, B. E., 151, 170 Kogane, F., 8.24,21,31,84 Kanner, B. I., 92,118,217,226,248 Kohn, L.,201,247 Kaosiri, T.,142,170 Kohn, L. D.,202,215,250 Kaplan, J. G.,103, 104, 118, 119 Kolarov, J., 286,287,303 Karlson, P.,310,395 Kornatsu, Y.,204,247 Karst, F., 144, 145, 170 Kon, M.,114,119 Kassir, Y.,376,395 Koncewiu, M.,93,118 Kates, M.,86.87, 102, 105, 106,118,119 Konings, W.N.,178, 179, 180, 181, 182, Kato, T.,148, 170 183, 184, 185, 186, 187, 188, 189, Katsunuma, T.,67.79 190, 195, 196, 197, 198, 199, 200,

400

AUTHOR INDEX

204, 205, 206, 207, 208, 209, 210, 211, 212, 214, 215, 216, 218, 219, 220, 221, 222, 223, 224, 225, 226, 231, 232, 233, 234, 235; 236, 245, 246,247,248 Kopperschlager, G., 247,282,303 Kornberg, A., 280,305 Kornberg, H. L., 63, 76, 79, 191, 192, 193,246,248 Kostellow, A. B., 21 1,247 Kotyk, A., 48,79 Kouyeas, V.,155,157,170 KovaC, L., 286,287,303 Kowarik, J., 341,393 Kozhira, T.N.,380,398 Kraepelin, G . 59.82 Kraml, M.,163, 167 Krana, M.J.. 97.98, 118 Kreger-van Rij, N.J. W., 390,391,395 Krenitsky, T.A., 258,303 Kritchevsky, D.,135,I72 Kroes, J., 132,170 Kroning, A., 338,341,343,395 Krulwich, T.A., 206,213,248,251 Kryff, B., de, 132, 173 Kubitschek, H.E.,19, 79 Kudrna, R., 293,303 Kuehn, G. D.,276,303 Kuenen, J. G.,206,248 Kunau, W.H.,109, I19 Kundig, W.,19 1 , 192,244,248 Kung, H., 240,249 Kunisawa, R., 96, 117 Kushner, D.J., 86, 87, 90, 91, 92,94,95, 101,103,104,105,114,I17,I18,119 Kushwaha, S. C., 87,118 Kusumi, T., 352,398 Kwiencinski, R.,70,78

L Lablache-Combier, A., 139,166 Lacoste, L., 139, 166 Lacroute, F., 144,145, I70 Ladbrooke, B. D.,132, I70 Ladet, J., 58,78 Lamer, T.,292,303 Lagarde, A. E.,2 13,248 Laine, I. A., 2 1 7.249 Lais, C. J., 257, 266, 274, 275, 277, 281, 288,302

Lambowia, A. M., 70,79 Lampen, J. O.,145, 146,170 Landrey, J. R., 130, 168 Langcake, P., 122, 135, 137, 142, 144, 165,171 Lange, R., 216,250 Langenbach, R. S., 123,I71 Lanyi, J. K., 90.93, 94, 98, 100,102, 103, 104, 107, 108, 109, 118, 224, 237, 248,249 L a r h o r e , F.. 57,82 Larsen, H.,86,87,90,91,92,93,96,101, 102,107,1 1 1 , 115,117,118,119 Laskin, A. I., 284,300 Laskowski, W.,382,395 Last, F.T.,309,395 Lasure, L. L., 149,152, 156,167 Laviola, C., 157,I72 Law, J. F., 268.301 Lawrence, L. M.,318,319,320, 321, 391, 394 Lazorik, F. C., 260,288,289,305 Lazzarini, R. A., 292, 293,302,303 Leal, J. A., 121, I71 Leckie, M.,275,281,288,302 Leckie, M. P., 257, 266, 274, 275, 277, 28 1,288,302 Lederberg, J., 370,398 Lee, E.H.,354,395 Leibowitz, M.J., 377,395 Leitzmann, C., 271,279,303 Lemke, P. A., 366,395 Leonian, L. H.,122,177 Leopold, L. B. 25.80 Lepierre, C., 91,118 Lerner, S. A., 192,250 Lester, R. L., 187, 188,245,248,267,273, 274,282,305 Leung, H.,260,289,305 Leung, H., 260,289,305 Leupold, U.,308,331,333,335,337,348, 394,395 Lever, J. E., 210,244 Levi, J. D.,3 17,357,360,395 Levinson, S. L., 206,Z13,248 Levinthal, C., 291,305 Lhoas, P., 30,31,32,80,366,395 Liao, C-L., 267,303 Lichstein, H. C., 50,51,78, 82 Lieberman, J. R., 164,173 Lieberman, M.M.,103, 104,118

409

AUTHOR INDEX

Liebl, V., 103,104,I18 Light, P.A.,68,80 Lilly,V. G.,121, 122,169, 171 Lima Costa, M. E., 231,232,234,245 Lin, E. C. C., 177, 188, 192,248,250 Lin, H. L., 123,171 Lin, H-K., 122,171 Lindahl, L., 74, 78, 257, 267, 284, 295, 302 Lindegren, C. C., 350,351,354,379,395, 396 Lindegren, G., 351,354,379,396 Lindenmeyer, A.,68.70,80 Lingappa, B. T.,59,83 Linnane, A. W.,146, 147, 170, 172, 173, 370,393,397,398 Linton, A. H., 30,79 Lipke, P. N.,366,367,396 Lipmann, F.,297,303 Little, J. G.,383,394 Litwinska, J., 54, 81,308, 354, 355, 356, 361,364,393,398 Liu, P. K., 144, 169 Lloyd, D., 45,81 Lloyd, E. C., 47, 77 Lloyd, P.B., 47, 77 Lo,T. C. Y.,204,216,248,249 Lockridge, O.,202,215,250 Lodder, J., 309,396 Lombardi, F. J., 196, 204, 205, 207, 209, 210, 211, 212, 217, 230, 233, 235, 237,238,246,248 Long, M., 63,79 Loprieno, N., 308,333,337,394 Louis,B. G.,103, 112,113,118 Lowendorf, H.S.,55,56,80 Lowry, 0.H., 193, 248, 257. 266, 270, 275,283,303 Lowry, R. J., 24,80 Lozier, R.,244.250 Lukins, H. B., 370,393,397,398 Lund, F., 295,304 Lundin, A.,270,271,303 Lundquist, R.,259,304 Luria, S. E., 267,304 Luscher, M., 310,395 Lusena, C. V.,354,395 Lusnak, K.,380,397 Lutsky, B. N.,163,171 Lynch, J. M., 39,80 Lynn, R. J.. 130, 133, 173

Lysek, G., 45,46,77,80

M Maalse, O., 21, 72, 80,82,257,266, 267, 282,284,292,293,294,301,304,305 McCann, M. J.. 4.77 McCarthy, B.J.,87, 109, 111, 112,119 Macaulay, B. J., 63,80 McClees, J. S., 196,204,216,226,249 McCorkindale, N.J., 122, 123,171 McCoy, C. J., 284,300 MacDonald, R. E., 108, 109, 114, 118, 119,224,231,248 Macdonald-Brown, D. S.,56,77 McDowell, L. L., 61,80 McDowell, T.D., 191,249 McElhaney, R. N.,133,171 Machek, F., 7, 78, 80 McIntosh, A.F.,39,60,81 MacKay, V., 164,168 MacKay, V. L., 351, 357, 358, 360, 361, 362, 367, 372, 373, 374, 375, 377. 378,382,394,396 M'Kendrick, A. G., 580 MacKenzie, R. M., 6,79 McKillen, M. N.,216,246 MacLennan,D. H., 93,117 MacLeod, R.A.,204,205,245,248,250 McMorris, T. C., 149, 152, 166,167, 169, 171 Madyastha, P. B., 138,171 Magasanik, B., 258,304 Magee, P. T., 380,395 Magnani, J. L., 275,277,281,288,302 Mailer, C.,132, 171 Mainzer, S. E., 268,3a3 Maitra, P. K., 264, 266, 274, 278, 280, 282,285,303,304 Malenkow, G . G.,217,249 Mallette, M. F., 268,304 Malloly, F. B., 130, 168 Mandels, G.R.,9,80 Mandersloot, J. G.,133. 168 Mangat,'B. S.,271,281,291,304 Manney, T. R., 164, 167, 168, 351, 355, 357, 358, 359, 360, 361, 363, 372, 373,574,377,378,382,393,394,396 Manor, H., 290,304 Marco, R., 288,305 Marquez,E.D., 111, 118

410

AUTHOR INDEX

Marr, A. G., 189,246,268,304 Marshall, C. L., 102, 118 Marth, E. H., 62,82 Manluf, G., 55, 81 Manluf, G. A., 55,80,81 Mason, H. R. S., 4 2 , 8 0 Massey, V., 202,215,250 Mateles, R. I., 284,305 Matheson, A. T., 95.98, 102,117, 119 Mathews, C. ,K., 266,290,29 1,304 Matin, A., 204, 205, 206, 214, 215, 216, 248 Matsche, N. E., 42,80 Maxon, W. D., 37.83 Maxwell, D. P., 141, 170 Maynard, Smith, J., 6 , 8 0 Meade, J. H., 333,335,336,349,394,396 Means, A. R., 127,171 Means, C. W., 39.80 Meek, G. A., 57,81 Meers, J. L., 56, 77,80 Megee, R. D., 5, 12, 78,80 Mellano, H. M., 143, 171 Melnick, L., 377,393 Melnik, E. J., 217,249 Memmen, K. F., 144,168 Mercer, E. I., 138, 139, 167 Mescher, M. F., 93, 118 Mevel-Ninio, M., 226,25 1 Meyer, D. J., 268,301 Meyenberg, H. K., yon, 49, 58. 76, 78,80, 29 1,292,295,302,303,304 Mialhe, H., 135, 172 Mian, F. S.,60, 80 Micetich, R. G., 121, 153, 169 Michels, P. A. M., 180, 189,206,223, 224, 236,246 Miki, K., 187, 188,248 Miller, A. B., 103. 117 Miller, A. L., 65, 80 Miller, R. J., 258,303 Mills, J. S., 149, 168 Milner, L. S., 186.200, 203,204,208,225. 24 7 Miovic, M. L., 261, 264. 265, 267, 27 1, 279,282,297,304 Mirkes, P. E., 74,80 Mirocha, C. J., 163, 164, 171, 173 Mitchell, C. H., 370,393 Mitchell, D., 366;396 Mitchell, J. E., 160, 161, 172

Mitchell, J. L. A., 76, 80 Mitchell, P., 228,229,230,248 Mizuguchi, T., 10,83 Mjelde, A., 93, 119 Moat, A. G., 50.80 Modi, V. V., 51, 78 Moens, P. B., 379,396 Mohagheghi, A., 60.80 Molin, S.,292,303 Montague, M. D., 267,273,274,281,304 Molzahn, S. W., 145, 171 Monahan, T. M., 359,392 Monier, R., 73,83 Monod, J., 5,80,213,225,245,246 Moor, H., 137, 171 Moore, C., 278,301 Moore,R. L.,87, 109,111. 112, 119 Moo-Young, M., 38, 78,80 Moore, D. J., 79 Mor. J-R., 67, 78,264,267,270,271,273, 285,305 Mori, S., 383,396 Morrison, K.B., 19,21,22,33,80 Morse, M. L., 53.77 Morse, S.D., 185,245 Mortimer, R. K.,350, 351,352,353,375, 378,382,383,390,394,395,396 Morton, R. A., 131, 169 Moses, V.,271,275,283,304 Moss, F. J., 27 1,273,278,300 Motta, J. J., 123,167 Mullakhanbhai, M. F., 90, 102, 119 Muller, G., 338,344,349,393 Mullins, J. T., 151, 170, 171, 173 Munim-al-Shakarchi, A., 73, 77 Munkres, K. D., 70, 78 Munnecke, D. E., 143, 171 Murakawa, S., 204,216,248 Murali, D. K.,69,70, 77 Musgrave, A., 152, 177

N Nagasaki, S.,9, 80 Nakai, S., 383,396 Nakatomi, Y.. 378,394 Nash, C. H., 366,395 Nash, W. E., 205, 216,245 Nasmyth, K.,347,396 Naylor, H. B., 340,393 Nazar, R. B., 275,291,293,304

41 1

AUTHOR INDEX

Ne’eman, Z., 133, 172 Neidhardt, F. C., 293,304 Neijssel, 0. M., 54,80 Neil, S. N., 258,303 Nelson,R. R., 162, 163, 169, 171 Nes, W. R., 130, 131,133, 136, 171 Netter, P., 370,393 Neuhard, J., 259, 292, 293,294,301,303, 304 Neupert, W., 138,169 Neville, M. M., 53, 80 Newton, N. A., 186, 187,245 Ng, A. M. L., 39,60,81 Ng, W. S., 60,61,81,82 Nicholas, H. J., 139, 166 Niederpreum, D. J., 34,84 Nieuwenhuis, D., 152, 171 Nierlich, D. P., 258,293,294,395,304 Nilson, E. H., 268,304 Nisbet, L. J., 54,81 Nisizawa, K., 34 1,396 Nixon, D., 47,77 Nobles, M. K., 29.81 Nogi, Y., 352,353,394 Nolan, R. A., 151,171 Noon, J. P., 161, 171 Norberg, P., 103, 104, 119 Norman, C., 143,171 North, M. J., 54.81 Novak, M., 7,39,78,81 Nowak, R., 122, 171 Nozawa, Y., 130,171 Nulty, W., 287,292,303 Nurse, P., 347,396

0 Oda, G., 95,119 O’Donovan, G. A., 295,304 Oehlschlager, A. C., 145, 169 Oehr, P., 216,251 Oelze, J., 189,248 Oesterhelt, D., 109, 110, 119,224,248 Okunuki, K., 187,248 Olden, K., 189, 199,246 Oldfield, E., 132, 171 Olivera, B. M., 259,304 O’Malley, B. W., 127, 171 Omang, S . , 96, 118 Onubu, T., 354,395 Or, A., 21 7,226,248

Orchard, P. O., 70.82 Oro, J., 102, 119 Orton, W. L., 3 18.3 19,320,398 Osagie, A U., 166, 167 Oshima, Y., 352,353,394,396,398 Osmond, C. B., 62,81 Ostwald, R., 132, 170 Osumi, M., 365, 366,367,370,371,372, 396 Oura, E., 67, 79 Ourisson, G., 138, 171 Overath, P., 209,245 Overman, S.A., 65,66,81 Ovichinnikov, Yu. A., 2 17,249 Owen, M. E., 390,396 Owens, N. F., 132,167 Oxender, D. L., 177,248

P Pai, M. K., 5 , 8 0 Palameta, B., 105,118 Palleroni, N. J., 353,396 Palumbo, S. A., 36.81 Pangborn, W. A., 132,172 Papa, K. E., 156, 160,171 Pardee, A. B., 177, 216, 246, 248, 250, 293,306 Park, D., 15,24,27,81 Parker, W., 121,122,169 Parks, L. W., 138, 139, 140, 145, 147, 166, 171,173,285,301 Parks, R. E., 292,304 Parnes, J. R., 217,226,248 Pasero, J., 377,398 Passerson, S.,59, 79 Paszewski, A., 54, 81 Patel, L., 227,248 Pateman, J. A., 52, 79,81 Patterson, A. R. P., 285,303 Pato, M. L., 292,295,303,304 Patterson, G. W., 123, 163, 167, 168 Pavlasova, E., 226,249 Payne, W. J., 262,304 Pearce, R. F., 102, 117 Peat, A., 40,42,43, 81 Pedersen, F. S.,295,304 Peduzzi, R., 59, 83 Pellecuer, M., 377,379,380,398 Penning de Vries, F. W. T., 262,263,304 Perrjtt, A. M., 146, 171

412

AUTHOR INDEX

Peterkin, P. I., 103, 112, 118, 119 Petes, T., 379,380,381,396 Petes, T. D., 359,396 Pethica, B. A., 132, 168 Petrochilo, E., 370,393 Petty, K. M., 185,245 Pfendt, E. A., 133, 154, 172 Pfennig, N., 96, 117 Pfitzer, K., 266.27 1,302 Phaff, H. J., 341,394 Philipson, L., 151, 168 Phillips, A. W., 146, 171 Phillips, D. H., 37, 81 Phillips, K. L., 13, 78 Phillips, M. C., 132, 167, 168 Pillai, C. G. P., 138, 171 Pifion, R., 379,380,38 1,396,397 Pirt, S.J., 10, 29,30,35,36,39,40,42,43, 47, 48,58,61, 76, 78,81, 8 2

Planta, R. J., 282,284,305 Plaut, B. S., 74, 81 Plischke, M. E., 350,352,353,396 Plomley, N . J. B., 26,81 Po, L., 191,250 Pohkis, E. S.,57.81, 271,304 Pomper, S.,354,396 Poole, R. K.,45.81 Poon, N. H., 321,340,365,394,396 Popplestone, C. R., 151, 171 Postma, P. W., 1 7 7 , 189, 190, 199, 218, 225,226,227,230,250

Powell, A. J., 62, 77 Powell, E. O., 43.81 Pratt, B. H., 157, 161, 171, 172 Pratt, R. G., 156, 160, 161, 172 Preiss, J., 276,304 Pressman, B. C., 217,249 Prezioso, G., 225,226,227,249 Price, D., 309,395 Pringle, S. R., 36 1,389,394,398 Prokop, A., 60,80 Proudlock, J. W., 146, 172 Pugh, E. L., 105,119 Purdon, A. D., 132, 172 Pursey, B. A., 122,123,171 Pye, K. E., 45,81

Rader, R. L., 194,205,246 Radin, D., 364,396 Radin, D. N., 308, 315, 354, 355, 363, 364,378,397

Rado, T. A., 74,78 Ragsdale, N. N., 148, 172 Ramkrishna, D., 5.81 Ramos, S., 237,238,249 Rand, R. P., 132, 172 Rank, G. H., 370,396 Raper, J. R., 149,172 Raper, K. B., 29,81 Rapport, E., 379,396 Rast, D., 6 6 , 8 1 Rattray, J. M. B., 123, 172 Rauser, W. E., 95, 118 Rayman, M. K.,204,216,248,249 Raymond,.J. C., 88, 119 Razin, S., 131, 132, 133, 135, 169, 172, 173

Reed, L. J., 299,304 Rees, D. C., 70,82 Rees, T., 62.81 Reeves, J. P., 201, 205,207, 217,230,233, 235,23 7,238,240,248,249

Reeves, R. C., 161, 172 Reeves, R. E., 287,304 Reichle, R. E., 32, 8 1 Reid, B. J., 389,394 Reinert, W. R., 55,8 1 Reiskind, J. B., 151, 170 Reissig, J. L., 46,79, 81 Reistad, R., 91,92, 100, 119 Renthal, R., 237,249 Renz, D., 209,245 Resnick, M. A., 383,396 Reusser, F., 39,80 Rhodes, G., 294,304 Ribbons, D. W., 268,301 Richards, J. B., 122, 172 Richter, R. H., 330,393 Rickard, P. A. D., 27 1,273,278,300 Rickenberg, H. V., 254,304 Rieck, J. T., 36.81 Riggs, T. R., 130, 172 Righelato, R. C., 19,21,22,24,33,39,40, 42,43,47,48,80,81,83

R Racker, E., 92, 110, 118, 119, 236, 237, 24 9

Roberton,A. M., 279,281,304 Roberts, C. F., 54.63, 76, 78, 81 Roberts, K. R., 55,81 Roberts, R. B., 257,304

AUTHOR INDEX

41 3

Robertson, A, B., 264,265,270,273,275, Ryan, A. M., 293,305 Ryan, F.J., 32.82 283,303 Robinow, C. F. 330,393 Robinson, J. H., 52.82 Robinson, P. M., 14,23,24,27,81,82 S Robinson, T., 146, 171 Rockwell, E.,63,82 Rodarne-Ram&, U.S., 383,396 Safe, S.,139, 140, 172 Roels,J. A., 38,82 Safferman, R. S., 146,171 Roger, M., 393 Sakai, K.,356,359,363,364,397 Rogers, H. J., 204,248 Sakurari, A., 164, 172,357,359,397,398 Rogers, P.J., 58, 59, 70,71, 82,262,264, Salmond, W.,149,168 265,268,305 Salser, W., 29 1,305 Rogers, T.O.,50.82 Salts, T.,379,380,381,396 Roheim, J. R., 74,82 Salts, Y.,379,380,381,397 Rohr, M., 39.77 Sanders, W.M., 38, 77 Sands, S. M., 378,379,397 Rohringer, R., 122, I77 Sansome, E., 158,172 Rolleston, F. S., 288,305 Santa, Maria, J., 352,397 Rollin, F.,95,98,119 Roman, H., 351, 352,365,378,379,381, Sanwal, B. D.,204,248,249 Sasny, P. S., 105,118 394,395,397 Sauer, B. L., 70.78 Roman, H.L.,379,380,381,396 370,397 Romano, A. H., 50, 63,65.66, 77,8I,82, Saunders, G.W., Saunders, R. M., 318, 319, 320,321,391, 191,249 394 Roon, R. J., 57,82 Savage, E. J., 157,172 Roper, J. A., 3 1,32,78 Savage, G. M., 39.80 Rose,A. H.,137, 140,141,146,154, I70 Roseman, S., 53, 80, 191, 192, 244, 248, Scammell, G. W.,39.82 Scarborough, G. A., 53,82 249 Rosen, B. P., 190. 196,204,210,216,225, Schaechter, M., 21,82,282,305 Schairer, H. K.,209,245 226,227,235,249,250 Schairer, H . U.,217,226,249 Rosenberg, H., 22 1,226,249 Scharrer, J. M., 40,78 Rosenberger, R. F.,21,22,23,31,79 Schechter, E.,201,237,240,249 Roth, R., 379,380,394,397 scherer, G.,359,397 Roth, R. M., 382,396 Schibeci, A., 132,172 Rothblat, G.H., 131, 135, 172,173 Schisler, L. C.,165, 170 Rothman, J. E.,132, 172 Schlegel, H . G.,266,286,288,301 Rothman, L.B., 274,275,305 Schlosser, E., 122, 141,142, 145,172 Rothstein, A., 48,49,82,83 Schneider, H.,132, 133, I67 Roman, B., 213,246 Schbn, G., 259,266,279,305 Rottem, S., 133,154,I72 Schormdler, J., 65,83 Rowe, P.B., 258,305 Schou, E. G., 67.79 Rowen, R., 87,92,119,120 Rowley, B. I., 10,39, 42, 47, 48, 58, 61, Schramm, V. L.,260,288,289,305 Schreier-Muccillo, S., 132,171 76,78,82 Rudnick, G., 239,240,241,242,243,247, Schroepfer, G. J., 163,I71 Schronbrunn, A., 202,250 249 Schuhmann, L.,. 110,119 Ruiz-Herrera, J., 187,220,249 Schuldiner, S., 227, 233, 235, 236, 237, Rusch, J. P., 76,80 238, 239, 240, 241, 242, 243, 247, Russell, P.J., 151, I70 248,249 Ryabova, I. D.,21 7,249

41 4

AUTHOR INDEX

Schulz, B. E., 59,82 Schweizer, H., 164, 168 Scott, W.T., 122,123. 171 Scotti, T., 8, 83 Scrutton, M. C., 64, 82 Sedo, R. J., 257, 266, 271, 273, 285. 295, 296,305 Sedgley, J. H., 161, 171 Segal, I. H., 50, 55, 76, 83 Sehgal, S. N., 105, 119 Sekuzu, I., 187,248 Sena, E., 364,396 Sena, E. P., 308, 315, 354, 355, 363, 364, 378,397 Senior, P. J., 276,302 Sermonti, G., 8, 77 Seshadri, R., 151, 152, 167, 171 Sessa, G., 153, 172 Setlow, P., 280,305 Setlow, R. B., 148, 173 Sha’afi, R. I., 132, 169 Shallenberger, M. K., 227,250 Shapiro, B. M., 229,305 Sharp, P. B., 27 1,275,283,304 Shaw, D. S., 159,170 Shaw, L., 185,204,207,247 Shaw, P. D., 122,172 Shemyakin, M. M., 217,249 Shen, L. C., 276,305 Shepherd, C. J., 157, 161, 171, 172 Shepherd, D., 62, 77 Sherald, J. L., 148, I72 Shih, C-N., 62,82 Shimoda, C., 164,172, 173,356,357,359, 361, 363, 365, 366, 367, 370, 371, 372,396,397,398 Shinmyo, A., 8 , 8 2 Shkrob, A. M., 217,249 Short, S. A., 178, 185, 186, 189, 190, 196, 200, 201, 202, 205, 208, 210, 211, 212, 215, 225, 230, 232, 235, 237, 238,239,243,247,248,249,250 Shuster, C. W., 265,303 Siekevitz, P., 135, 172 Sietsma, J. H., 136, 141, 142, 143, 145, 146, 172, 173 Silver,J. C., 151, 172 Silver, R. A., 284,305 Silver, S., 204,217,245 Silverman, M. P., 90, 118 Silverstein, S.C. 196, 201,250

Simchen, G., 376, 379,380,381,395,396, 397 Simoni, R. D., 1 7 7 , 225, 226, 227. 230, 235,244,250 Sims, J. J., 143, 171 Sin, I. L., 258,305 Sing, V., 318,320,397 Singh, A. P., 188,250 Sips, H. J., 200, 204, 215, 219, 222, 234, 24 5 Sisler, H. D., 148, 172 Sistrom, W. R., 88, 119 S.jogren, R., 63.82 Skinner, V. M., 64.82 Skowronski, B. S.,9.82 Slater, J. H., 11, 77 Slayman, C. L., 55.56.69, 70, 80, 82,261, 264,265,267,270,282,285,305 Slayman, C. W., 55,56, 70, 79, 80,82 Sledbodnik, J.. 147, 169 Sloneker, J. H., 146, 169 Slonimski, P. P., 370,393 Sloof, W. C., 309.33 1,397 Smalley, H. W., 62, 77 Smith, D. G . , 367,370,37 1,397 Smith, G. N., 62, 77 Smith, I. C. P., 132, 133, 167, 171 Smith, J. E., 3, 15, 39, 60, 61, 67, 76, 81, 82 Smith, J. H., 30.82 Smith, L., 186, 250, 261, 264, 265, 271, 303

Smith, N., 95, 119 Smith, P. F., 130, 131, 133, 135, 172, 173 Smith, R. C., 257,266,305 Snoswell, A. M., 186, 187,245 Snyder, F. F., 261,305 Snyder, J. R., 370,395 Sobel, M. E., 206,213,251 Sogin, D. C., 258,305 Solomons, G. L., 37,38,39,42,43,82 Sols, A., 287,288,304,305 Somlo, M., 274,286,305 Spanr, P. F., 257,305 Spahr, P. F., 257,305 Spencer, J. L., 10,78 Sprinson, D. B., 144, 169, 173 Sproston, T., 148, 173 Sprott, G. D., 205,250 Sprivastava, K. C., 367,370,371,397

AUTHOR INDEX

Stadtman, E. R., 193, 211,246,247, 258, 299,303,305 Staehelin, L. A., 179, 183, 196,244 Stamps, D. J., 155, 157, 158, 173 Stan, H-J., 65,82,83 Standish, M. M., 153, I67 Stanley, S. O., 56, 77 Starr, P.R.,140,145, 171, 173 Sauble, E. J., 66,BI Stebbing, N., 42,83,288,305 Steel, R., 37, 38, 77, 83 Steele, G. C., 13, 16, 17, 83 Steensland, H., 28, 41, 83, 87, 91, 92, 93, 96,118, I19 Stent, G. S., 290,293,302,304 Stern, J. R., 205,247 Steveninck,J., van, 49,83 Stevenson, J., 94,100, 102,104,117, I18 Stewart, G. G., 321,365,394 Stewart, P. R., 58,59, 70, 71,82,262,264, 265,268,305 Stier, T. J. B., 146,166 Stinnett, J. D., 178, 186,213,250 Stoeber, F. R., 213,248 Stoeckenius, W., 92, 109, 110, 116, 117, 119, 224, 236, 237, 245, 248, 249, 250,270,279.301 Stolp, H., 274,302 Storck, R., 59,83 Stotzky, G., 87, 120 Stoaler, D., 164, 168, 357, 358,387,394, 397 Stouthamer, A. H., 262, 263, 264, 265, 278,305 Strange, N., 305 Strange, R. E., 271,305 StreiblowA. E., 338,341,397 Strsm, A. R., 95, I19 Strominger, J. L., 93, 118 Stroobant, P., 239,245,247 Sturani, E., 75,83 Styles, E. D., 313,384,393 Jubic, J., 286,287,303 Suda, K., 370,397 Suelter, C. H., 260,306 Sughrue, M. J., 277,288,302 Sugimoto, M., 12,83 Summers, R., 282,285,303 Sundaram, T. K., 262,301 Sundeen, J., 149, 151,167,168 Suskind, S. R., 53.80

415

Sussman, A. S., 24,80 Suzuki, H., 8, 79 Suzuki, I., 65,78 Swedes,J. S., 257,266.27 1,273,285,295, 296,305 Switzer, R. L., 258,305 Sy, J., 295,305 Symonds, A. M., 153,169 Sypherd, P. S.,366,393

T Taber, H. W., 231,245 Takahashi, H., 204,216,248 Takahashi, I., 27 1,279,301 Takahashi, J., 10.83 Takahashi, T., 164,173,351,352,397 Takano, I., 352,353,396,398 Takao, N., 164, 172, 173, 356, 357, 359, 397,398 Talwalkar, R. T., 267,273,274,282,305 Tamura, S., 164,172,357,359,397,398 Tanaka, H., 10,83 Tanaka, K., 204,247 Tanaka, S., 148,170, 192,250 Taniguchi, S., 187, 188,250 Tannenbaum, S. R., 72.76 Tanner, W., 56, 83 Tatum, E. L., 32,34, 79, 82,83, 141,173 Tavorrnina, P.A., 146,173 Taylor, A., 366,367,396 Taylor, C. P. S., 132, 171 Taylor, N. W., 318,319,320,398 Tempest, D. W., 54.56, 75.80, 83 Tepperman, H. M., 130, 173 Tepperman, J., 130, 173 Terenzi, H. F., 59,83 Terui, C., 8.82 Terui, G., 12,83 Thiersault, M., 139, 168 Thom, C., 29,81 Thomas, D des S., 151, 173 Thomas, D. Y.,367,368,369,370,398 Thomas, E., 184,245,250 Thomas, E. I., 184,250 Thomas, G. A., 292,305 Thomas, J., 213,246 Thomassen, E., 259,304 Thompson, E.D., 138,145,147,171,173 Thore,A., 270,271,303

41 6

AUTHOR INDEX

Throm, E., 164,173,359,398 Thuriaux, P., 347,396 Thurmann, R., 204,248 Tiefenberg, M., 217,250 Timberlake, W. E., 43, 72, 79 Tinker, D. 0.. 132, 172 Tissikres, A., 257,305 Tobin, R., 319,398 Tomlinson, G . A . , 88. 101,102,119 Tonolo, A., 8.83 Topiwala, H. H., 4,38,83 Tornabene, T. G., 102,119 Torsvik,T., 113, 114, 119 Tosteson, D. C., 217, 250 Tremblath, M. K., 370,397,398 Trinci, A. P. J., 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 23, 24, 25, 26, 28, 30, 31, 32, 33, 34, 35, 36, 37, 40, 41, 42, 43,47,48,61, 76, 77, 78, 81,83 Trocha, P., 144,169 Trocha, P. J.. 144, 173 Tsuboi, M., 359,398 Tsurhiya, H. M., 5 , 6 , 12, 78, 80, 81, 83 Tsuchiya, T., 190,204,216,225,226,250 Tsuda, S . , 141, 173 Tulloch, A. P., 121, 153, 169 Tully, J. G., 131, 173 Turian, G., 59,83 Turner, N. A., 142, 167 Turnock, C., 74,81 Tuttle, A. L., 188. 189,250 Tweedie, J. W., 55.83 Tfield, L. A,, 275,291, 293,304

Van den Berg, J., 38,82 Van den Ende, H., 155,173 Vandenheuvel, F. A., 131, I 73 Vander Beek, E. G., 262,264,278,305 Vander Neut-Kok, E. C. M., 133,171 Vandewalle, B., 139, 166 Van Dijk-Salkinoja, M. S., 282,284,305 Van Etten, J. L., 74,82, 138, 145, 173 Van Eyk, R. W. V., 133, 168 Van Laar, H. H., 262,263,304 VanThienen, G., 189, 190, 199,218,226, 227,250 Varney, N. F., 292,305 Varricchio, F., 73,83 Veenhuis, M., 178, 179, 180, 181, 182, 183,195, 196,199,206,248 Venema, G., 184,185,247 Verkleij, A. J., 132, 173 Vermeulen, C. A., 178, 179, 180, 181, 182, 183, 195, 196, 199,206,248 Vero-Barcellona, L., 8.83 Vevergaert, P. H. J. Th., 132, 173 Vezinhet, F., 377,379,380,393,398 Vidal, D., 352,397 Vinopradova, E. J., 2 17,249 Viotti, A., 75, 83 Visentin, L. P., 95,98, 102, 117, 119 Vitols, E., 258,301 Vogel, H. J., 28,83 Voith, K., 163, 167 Voncken, R. M., 38, 82 Vranda, D., 43,83 Vries, W., de, 180, 188, 221,248

U

w

Uchida, A., 370,397 Uden, N., van, 48, 49.83, 89, I I 9 Ueda, K., 10.83 Ueda, M., 148. 170 Umbarger, H. E., 293,305 Unrau,A. M., 145, 151,169, 171

V Vagelos, P. R., 287,292,303 Valentine, R. C., 226.25 I Valenzuela-Pera, J., 61, 82 Van Deenan, L. L. M., 133, 168 Van Deenen, L. L. M., 132, 133, 134, 146, 154, 167, 168, 173

Wade, H. E., 271,305 Wagner, R. P., 69, 70, 76, 77 Wak, A. C., 114, 119 Walker, D. A., 132, 167 Walker, D. J., 262,263,302 Walker, S., 335,394 Wall, R., 151, 168 Wallace, P. G., 147, 173 Walsby, A. E., 96,97, 120 Walsh, C., 185,204, 207,247 Walsh, C. T., 196,201,202,215,250 Waltho, J. A., 88,89, 117 Ward, J. B., 193, 248, 257, 266, 270, 273, 275,283,303 Wassef, M. K., 105, 118, 119

41 7

AUTHOR INDEX

Watanabe, N., 191,250 Watkins, J. C.,153,169 Watson, K.,68,83 Watson, T.G.,42,83,89,120 Waxman, M.F., 367,369,370,371,398 Wean, R. E.,40.83 Weber, K.,295,303 Weber, M.M.,91, 102, 116,120 Webster, R. K.,162, 171 Weete, J. D.,123, 138, 171, 173 Weibel, K. E., 264, 267, 270, 271, 273, 285,305 Weihe, G. R., 152,171 Weil, R.,201,237,240,241,242,249 Weiner, J . H.,200,210,250 Weissbach, H.,184,245,250 Weissmann, G.,153, 167, 172 Welch, J.. 308,315,354,355,378,397 Welch, S . K.,380,395 Wergel, N.,192,244 Werkman, B. A., 155,173 Weston, G.0.. 37.82 Wheeldon, L.W., 146,172 Wheeler, G.E., 137, 170 Wheeler, R., 122, 123, 171 Whelen, W. J., 38, 77 White, D. C.,178,186, 196,205,208,210, 21 1,212,225,250 Wickerham, L.J., 309,310,313,314,317, 329,330,395,398 Wickner, R. B.,377,395 Widdowson, D.A., 145,167 Wiebe, W. J., 266,306 Wijk, R.,van, 58,84 Wiley, J. S., 135,173 Wilke, C.R.,6, 12,71, 78 Wilkie, D.,367, 368, 369, 370, 371,397, 398 Wilkinson, L. E., 361,398 Willecke, K.,216,246,250,251 Willey, W. R.,50,84 Williams, R.M.,132,170 Wilson, R.W., 34,84 Wilson, W. T.,99, 120 Wimpenny, J. W. T., 187, 245, 251,259, 270,273,27a,283,285,301,306 Winfree, A. T., 45.46.84 Winge, O.,351,398 Winslow, R. M.,292,293,306 Wong, J. T-F., 275,291,293,304 Wong, P. T.S . , 93,I17

Wood, T. C., 50.76 Woods, R.A.,144,167,171, 173 Woods, V.,374,396 Woodward, D.O.,63,78 Wormser, M.,135,169 Witta, L.D.,36.81 Wolf, A., 338,341,397 Wolf, J. C.,164,173 Wolfe, R. S.,279.28 1,304 Wolfson, E. B.,206,213,251 Wright, R. E.,370,398 Wu, C.W., 294,300 Wu, H. C.P., 213,251 Wu, L., 146, 169 Wulff, K.,103,118 Wygal, D.,376,398 Wygaarden, J. B.,258,305,306

Y Yagil, E., 259,306 Yaguchi, M.,95,98,102,117, 119 Yamamoto, M.,260,302 Yamamoto, T.H.,226,251 Yamazaki, H.,292,303 Yanagishima, N., 164, 172, 173,356,357, 359, 361, 363, 364, 365. 366, 367, 370.37 1,372,396,397,398 Yanagita, R., 8,24,27,31,84 Yashouv, J., 133,172 Yates, M.G.,260,306 Yates, R. A., 293,306 Yeh, Y-F., 318,320,397 Yen, P. H., 318,319,320,398 Yengoyan, L. S., 105, I18 Yoo, B.J., 340,398 Young, J. E.,40.83 Yu, F. M.,217,249 Yuan, D.,290,301

Z Zaborowska, D., 356,398 Zakharow, I. A., 380,398 Zalokar, M.,3 1.84 Zentmyer, G.A., 159, 169 Zielke, C.L.,260,306 Zipser, D.,297,301 Zonneveld, B.J. M., 27,84 Zuk, J., 308,354,355,356,361,364,393, 398 Zwilling, B. S., 87,120 Zygmunt, W. A., 146,173

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SUBJECT INDEX

A

Accretion of fungal growth in chemostats, 38 Accumulation of secondary metabolites by fungal colonies, 29 Acetate, effect of, on glucose transport into Aspergillus nidulans, 50 incorporation into yeast sterols, 140 Acetobacter aceti, effect of depletion of energy source on energy charge of, 272

Active transport, coupled to electron transfer in bacterial membrane vesicles, 203 in bacteria, mechanism of energy coupling to, 225 in bacterial membrane vesicles, 194 of solutes in bacterial membrane vesicles, 175 Adenine nucleotide, catabolism, 260 concentration in microbes, and energy charge, 289 concentrations, changes in, in microbes, 272 determinations on microbial cultures, 269

energy charge of, 266,28 7 Achlya bisexualis, hormone A of, 149 maximum growth rate of, 43 protein synthesis in, 72 Achlya heterosexualis,hormones of, 152 Achlya spp., sexual hormones of, 149 sterol synthesis by, 123 Acholeplasm laidlawii, adsorption of sterols by, 135 sterol incorporation by, I3 1 Acholeplasma spp., carotenoid synthesis by, 131 Acidic amino-acid residues in halobacterial proteins, 100 Acidic proteins from halobacterial ribosomes,95 Acidity, effect of, on fungal colony growth, 28 effectof, on fungal growth, 18 sugar metabolism by Aspergillus nidulans, 54 Aconitase, effect of cholesterol on level of, in fungi, 143 Actinomucor repas, hyphal growth unit of, 20

Actinomycin D, effect of, on antheridiolinduced branching, 15 1 41 9

pool in microbes, measurement of, 268 pools of fungi, oscillations in, 70 Adenine nucleotides, concentrations in bacteria and yeasts, 253 in Escherichia coli, and protein synthesis, 296 in mutant strains arrested in growth, 286

Adenine phosphoribosyl transfer system in bacteria, 193 Adenine phosphoribosyl transferase in bacterial membrane vesicles, 194 Adenosine diphosphate-glucose synthetase, activity of microbes, 276 Adenosine monophosphate, excretion of, from microbes, 27 1 nucleosidase activity, regulation of in bacteria, 289 nucleosidase in bacteria, 260 Adenosine triphosphatase, activity of bacterial membrane vesicles, 189 complex, role of in bacterial active transport, 190 of bacterial membrane vesicles, 199 role of, in halobacterial transport, 108 Adenosine triphosphatase-deficient bacteria, transport into, 22 1

420

SUBJECT INDEX

Adenosine triphosphate, breakdown during sampling of microbes, 269 concentration, relation of to growth rate, 282 content of fungi, 69 exogenous, effect of on bacterial active transport, 225 levels in bacteria, 285 permeability of bacteria for, 225 role of, in bacterial active transport, 225 synthesis by bacterial membrane vesicles, 190 synthesis of, in bacteria, 229 Adenylate energy charge, definition of, 268 Adenylate charge in Neurospora crmsa, 69 microbial, stabilization of, 265 Adenylate kinase mutant of Escherichia coli, 287 Adenylate levels in microbes, 258 Adsorption of sterols by fungi, 135 Aeration, effect of, on conjugation of Saccharomyces cerevisiae, 354 of anaerobically grown Saccharomyces cerevisiae, effect of, on enzyme synthesis, 146 Aerobacter aerogenes, energy charge of, 27 1 turnover of ATP in, 264 Aerobiosis, effect of, on microbial adenylate charge, 278 Agaricus b i s p w , germination of spores of, 66 sterols of, 165 Aged zone in fungal hyphae, 27 Ageing of fungi, I 2 Agglutination, factors, in Saccharomyces cerevisiae mating, 357 factors of Saccharomyces cerevisiae, operation of, 362 in Schizosaccharomycespombe, 338 sexual, in Hansenula Wingei, 328 in Schizosaccharomyces pombe, 347 Agglutinins, and conjugation in Hansenula wingei, 3 18 Hamenula wingei, possible structures of, 319 in Hamenula wingei, glycoprotein nature of, 31.9 Alanine uptake by bacteria, eftect of light on, 224

Agitation in fungal liquid cultures, 38 Alleles, mating- type, in Schizosaccharomyces pombe, 33 1 mating-type, in Saccharomyces cerevisiae, 350 of Hamenula wingei, 3 13 Allumyces spp., growth of colonies of, 25 Alpha factor, as an inducer of agglutinibality in Saccharomyces cerevisiae, 360 production by Saccharomyces cerevisiae, 373 role of, in mating in Saccharomyces cereuisiae, 357 yeast, mode of operation of, 359 Alpha factors in Saccharomyces cerevisiae, multiple nature of, 36 1 Alternation ofgenerations in yeasts, 3 13 Amino-acid, accumulation by Rhodopseudomanus sphroides, 223 residues in membranes from gas vacuoles, 97 synthesis in fungi, effect of electron transport on, 69 transport, into Bacillus subtilis, and reduced nicotinamide nucleotides, 232 into bacterial vesicles, 204 into fungi, 50 systems in bacterial vesicles, 209 uptake by bacteria under anaerobic conditions, 219 Amino acids, accumulation by bacterial vesicles, 207 accumulation of halobacterial vesicles, 108 Amino-acyl tRNAs from halobacteria, 103 Ammonia assimilation in fungi, 56 Ammonia catabolite repression in fungi, 52 Amytal, effect of, on bacterial membrane vesicles, 18 7 lack of effect of, on amino-acid uptake by bacteria, 224 Anubenaflos-aguae, properties of vacuoles from, 97 Anaerobic electron transfer, in bacterial membrane vesicles, 18 7 systems, coupling of transport to, 2 1 7 Anaerobic growth of Saccharomyces cereuisiae, lipid requirements during, 146

SUBJECT INDEX

h a e r o b i c phosphorylation in halobacteria, 110 Anaerobic sugar metabolism by fungi, 57 Anaerobiosis, effect of, o n microbial adenylate charge, 278 effect of, on transport into bacterial membrane vesicles, 208 Anaplerotic metabolism in fungi, 62 Anion transport into fungi, 55 Antheridial branches, interaction between, in Achlya spp., 156 Antheridial structures, induction of, in Achlya spp., 149 Antheridiol, formula of, 150 hormone nature of, 149 Antibiotics, effect of, on halobacteria, 93 Antimycin A, effect of, on amino-acid transport into bacteria, 223 Antimycin, effect of, on fungal respiration, 70 Apical transport and fungal growth, 15 Applicability of classic models to fungal growth, 7 Approaches to modelling of fungal growth, 4 Aqualinderelle fernentans, lack of a tricarboxylic acid cycle in, 67 Arabinose transport into bacterial membrane vesicles, 2 13, 2 14 Arabitol, uptake by fungi, 5 1 Arsenate, effect of, on adenylate charge of Chromatium sp., 282 Arginine metabolism by halobacteria, 102

Arrest of G 1 in Saccharomyces cerevisiae, 359 Arthrobacter pyridinolis, fructose accumulation by, 213 transport coupled to electron transfer in vesicles of, 206 Ascomycetes, sterols and sexual reproduction in, 162 Ascospore germination in Saccharomyces cerevisiae, 3 12 Ascospores, shape, in yeasts, 3 12 Ascosporogenesis in Hansenula wingei, 330 Ascus formation by Saccharomyces cereviszae, sterol formation during, 140 Asexual reproduction, of fungi, effect of sterols on, 14 1 in fungi, effect of sterols on, 148

42 1

Aspartate, effect of, on fungal pyruvate carboxylase activity, 65 Aspergilli, hyphal growth units of, 20 Aspergillw nidulans, chemostat culture of, and RNA synthesis, 8 effect of ammonia o n metabolism of, 52 effects of starvation on, 4 7 extension rates of hyphae of, 16 oscillations in chemostat behaviour of, 44

regulation of glucose catabolism in, 60 transport of solutes into, 48 Aspergillus niger, distribution of RNA synthesis in, 7 phosphorus uptake by parts of colonies Of, 3 1 polarization of hyphal growth in, 14 subcellular distribution of sterol in, 138

Auto-inhibition of fungal spore germination, 66 Autolysis of pellets of Penicillium chrysogenum, 8 Autotrophism, negative, in fungal hyphae, 23 Avenasterol, occurrence of, in fungi, 153 8-Azaguanine, effect of, on conjugation in Saccharomyces cerevisiae, 356 Azidophenylgalactosidae, formulae of those used in bacterial transport studies, 240 Azotobacter vinelandii, Ah4 P nucleosidase activity in, 289 AMP nucleosidase activity of, 288 dehydrogenase activities of membrane vesicles of, 186 glucose transport in, 2 13 membrane vesicles from, 2 16 transport coupled to electron transfer in vesicles of, 205 turnover ofATP in, 263 Azygotic meiosis in yeasts, 3 1 1

B Bacillus cereus, energy charge 01; 270

Bacillus lichenijormis, energy charge during sporulation of, 279

422

SUBJECT INDEX

Bacillus lichen~o fonis-continued transport coupled to electron transfer in vesicles of, 204 Bacillus subtilis, amino-acid transport into membrane vesicles of, 2 10 effect of ferricyanide on glutamate uptake by membrane vesicles of 234 effect of phosphate depletion on energy charge of, 275 electron micrographs of thin section through membrane vesicles of, 18 1 energy charge of, 266 glutamate uptake by vesicles of, 207 membrane vesicles of, 178 Michaelis constants for amino-acid transport in membrane vesicles of; 210 orientation of membrane vesicles of, 196

oxidation of substrates by membrane vesicles of, 185 solute transport in a menaquinone-deficient mutant of, 231 transport coupled to electron transfer in vesicles of, 204 Bacillus megaterium, energy charge during sporulation of, 280 membrane vesicles from, 2 16 transport coupled to electron transfer in vesicles of, 204 Bacteria, concentration and turnover of adenine nucleotides in, 253 extreme halophilic, classification of, 86

Bacterial cytoplasmic membrane, vesicles, as models in membrane function studies, 242 Bacterial membrane vesicles, active transport coupled to electron transfer in, 203

active transport of solutes in, 175 orientation of, 194 Bacteriorhodopsin, and proton extrusion in halobacteria, 236 chromophore in, 110 leucine uptake by halobacterial vesicles containing, 108 presence of, in halobacteria 87, 224 Bacteriorhodopsin-mediated phosphorylation in halobacteria, 109

Balance between adenine nucleotide synthesis and utilization, 258 Bark beetles, occurrence of Hansenula wingez in, 309 Barrier factor, operation of, in mating of Saccharomyces cerevisiae, 362 Basidiobolus ranarum, growth of colonies of, 33 Basidiomycetes, sterols and sexual reproduction in, 162 sterols in, 123 Bauchop-Elsden values, nature of, 26 1 Bdellovibrio bacteriovorus, energy charge of, 273

Beta-Galactosidase activity of Aspergillus nidulans, 54 Beta-Galactosides, transport of, into bacterial membrane vesicles, 2 12 Beta-Glucanase activity in conjugation tube formation in Schizosaccharomyces pombe, 34 1 Binding between cells of Saccharomyces cerevisiae during conjugation, 364 Binding of sterols in fungi, 135 Biological activity of bacteria and yeasts, and concentration and turnover of adenine nucleotides, 253 Biomass production of Aspergillus nidulans in chemostat culture, 4 1 Biomass production of Geotrichum candidurn, 1 I Biosynthesis of fungal membranes, role of sterol esters in, 139 Biotin deficiency, effect of, in fungi, 5 1 Biotin in fungal pyruvate carboxylases, 64 Blackman kinetics as applied to fungal growth, 12 Branch initiation, regulation of, in fungal growth, 15 Branching, fungal, effect of sterols on, 143

Brassicasterol, properties of, 125 Budding pattern in Saccharomyces cerevisiae, 383 Budding, zygotic, in Saccharomyces cerevisiae, 37 1 Buffering capacity, effect of, on fungal colony growth, 28 Butyrate uptake by bacterial membrane vesicles, 19 1

SUBJECT INDEX

C Caesium uptake by bacterial membrane vesicles, effect of valinomycin on, 217

Calcium accumulation by Escherichia coli, 216

Calcium-activated ATPase in bacterial membrane vesicles, 189 Calcium transport in bacterial membrane vesicles, 196 Calcium transport into bacteria, 2 16 Calcofluor, effect of, o n Phytophthora palmivora, 15 7 Campasterol, formula of, 124 Candida parapsilosis, turnover of ATP and growth rate in, 265 Candida utilis, diffusion transport in, 49 macromolecule synthesis in, 72 Carbohydrate transport in bacterial membrane vesicles, 19 I Carbohydrates, utilization of, by halobacteria, 101 Carbonyl cyanide, effect of, o n transport into bacterial membrane vesicles, 235

Carbon dioxide fixation in fungi, 63 Carboxylic acid transport systems in bacteria, 2 14 Carotenoids, effect of, o n membrane permeability, 133 protective effect of, in halobacteria, 110

Carriers, bacterial membrane, oxidation and reduction of, 230 Carrier proteins, energy-dependent binding of solutes to in bacteria, 239 Catabolism of adenine nucleotides in micro-organisms, 260 Catabolite repression in Saccharomyces cerevisiae, 58 Cation transport into bacteria, 2 16 Cell age, and modelling of fungal growth, 12 Cell concentration, effect of, o n conjugation of Saccharomyces cerevisiae, 354 Cell cycle, and conjugation in Hansenula wingei, 3 15

42 3

and conjugation in Schizosaccharomyces pombe, 337 and conjugation of Saccharomyces cerevisiae, 354 Cell envelopes of extreme halophiles, structure of, 9 1 Cell fusion in conjugation in Hansenula wingei, 32 I and sporulation in Saccharomyces cerevisiae, 3 79 Cell cycle stage for sporulation in Schirosaccharomyces pombe, 348 Cell fusion in mating of Saccharomyces cerevisiae, 366 Cell fusion in Schizosaccharomyces pombe, 340

Cell membrane, dissolution of, in halobacteria, 93 Cellulase activity, distribution of, in fungal colonies, 9 Cellulase production in fungi, 15 1 Cellular recognition, in conjugation in Hansenula wingei, 3 I 7 in conjugation in Saccharomyces cerevisiae, 356 in Saccharomyces cerevisiae mating, 356 in Schizosaccharomyces pombe, 338 Chaotropic agents, effect of, on bacterial membranes, 227 Chemi-osmotic coupling in bacterial solute transport, 228 Chemostat growth of mycelial fungi, limitations of, 39 Chloramphenicol, effect of, o n energy charge of Escherichia coli, 28 1 Chlorate, effect of, on amino-acid uptake by bacteria, 2 19 Chlorella ellipsoidea, effect of inhibitors of sterol synthesis on, 163 Chloroform, effect of, on energy charge of Methanobacter sp., 28 1 Cholestanol, effect of, on growth of Phytophthora infestans, 142 Cholestanol, formula of, 124 Cholesterol, effect of, on growth of Phytophthora infestans, 142 effect of, o n mating of phytophthoras, 160

formula for, 124 Cholesterol-lecithin interaction, 13 1

424

SUBJECT INDEX

Cholesterol metabolism by fungi, 154 Cholesterol, model of structure of, 128 presence of, in Achlya bisexualis, 15 1 uptake by Phytophthora infestuns, 135 Cholesteryl oleate, uptake of, by fungi, 136 Chromutium D, energy charge in, 279 Chromatium sp., turnover of ATP in, 264 Chromogenesis in halobacteria, 87 Chytridiomycetes, sterol synthesis by, 123 Circadian rhythm of fungal conidiation, 46 Citrate, cycle enzymes in halobacteria, 10 1 transport in bacterial vesicles, 205 transport into Bacillus subtilis, 2 14 synthase, properties of, in halobacteria, 103 Cladosporium sp., hyphal growth unit of, 20 Classic models, applicability of, to fungal growth, 7 Classification of extreme halophilic bacteria, 86 Claviceps purpurea, sucrose assimilation by, 54 Clostridium kluyveri, energy charge of, 266, 27 1 Cochliobolus carbonum, effect of zearalenoneon, 163 sterols and sexual reproduction in, 162 Cokeromyces poitrassi, sugar metabolism by, 58 Colonies, fungal, differentiation in, 9 Colonization of solid substrates by moulds, 35 Colony differentiation in fungi, 23 Colony expansion as a parameter of mould growth, 33 Colony expansion, kinetics of, with fungi, 29 Colony growth of fungi, 23 Colourless mutants of halobacteria, effect of light on, 1 1 1 Complementary agglutination factors in Hansenula wingei, 3 18 Complementary agglutination factors in Schizosaccharomycespombe, 339 Complementary alleles at the matingtype locus in Schirosaccharomyces .. pombe, 332

Complexing of polyene antibiotics and sterols, 146 Concanavalin A, and fusion of yeast protoplasts, 365 Condensing effect of sterols o n phospholipid monolayers, 132 Configurations of sterol molecules, 127

Conidiation, fungal, circadian rhythms in, 46 Conidiation in Aspergillus n i p , 6 1 fungal, effect of sterols on, 148 Conjugation, early events in Saccharomyces cerevisiae, 363 initiation of, in Hansenula win& 3 15 in Saccharomyces cerevisiae, 354 in Schizosaccharomyces pombe, 336 Conjugation in Hansenula wingei, inhibitors of, 3 16 Conjugation in Schizosaccharomyces pombe, 348

Conjugation in Schirosaccharomyces pombe, inhibitors of, 338 Conjugation in yeasts, trigger for, 3 10 Conjugation in yeasts, comparative aspects of, 384 Conjugation tube formation in Hansenula wingei, 325 Conjugation tube formation in Schizosaccharomycespombe, 340 Constitutive agglutination in Hansenula wingei, 3 1 7 Consumption of adenosine triphosphate, estimates of, 262 Control of metabolism in fungi, 5 7 Coprinus disseminutus, leading hyphae in colonies of, 25 Coprinus lagopw, sterols of, 164 Coprostanol, effect of, o n erythrocyte permeability, 134 model of structure of, 128 Cordycepin, effect of, on fungal branching, 151 Coupling of transport to anaerobic electron-transfer systems, 2 1 7 Coupling of transport to cyclic electrontransfer systems, 223 Crystals, ergosterol, in Neurospora crassa, 14 1 Cunninghamella spp., hyphal growth unit of. 20

SUBJECT INDEX

Cupric ions, and a k h a factor in Saccharomyces cereviskze, 358 Cyanide, effect of, on active transport into bacterial membrane vesicles, 238

effect of, on bacterial membrane vesicles, 18 7 on fungal respiration, 70 Cyanobacteria, proteins from gasvacuole membranes of, 97 Cyclic-adenosine monophosphate, synthesis of, 255 Cyclic electron- transfer systems, coupling of transport to, 223 Cyclic electron-transfer systems in bacterial membrane vesicles, 188 Cyclic variations in ATP levels in microbes, 286 Cycloartenol content of potato leaves, 144

Cycloheximide, effect of, on hrphal growth in fungi, 2 1 effect of, on conjugation in Hansaula wingei, 3 16 on production of metabolite A, 152 Cyclopropane fatty acids, synthesis of, by bacterial membrane vesicles, 184 Cytochrome oxidase, effect of aeration of anaerobically grown yeast on, 146 Cytochrome oxidase, in halobacteria, 107 Cytochromes in bacterial membrane vesicles, 185 Cytochromes in fungi, 68 Cytochromes of halobacteria, nature of, 107

Cytochromes, role of, in transport into bacterial membrane vesicles, 208 Cytoplasmic enzymes, absence of, from bacterial membrane vesicles, 183 Cytoplasmic membrane, bacterial, as an osmotic barrier, 175 Cytoplasmic mixing in Hansenula wlngei, 326

425

Dead Sea, halophiles in the, 114 Deamination of adenine nucleotides by bacteria, 260 Deceleration phase of growth of fungal colonies, 30 Degradation of adenine nucleotides in micro-organisms, 260 Degradative enzymes in wall, action of, in conjugation tube formation in yeasts, 326 Dehydrogenase-coupled solute transport into bacteria, 230 Dehydrogenase-deficient mutants of Escherichia coli, 199 Dehydrogenases in bacterial membrane vesicles, 185 Dendryphiella salina, sugar uptake by, 5 1 Density of fungal hyphae, 25 Deoxyantheridiol, formula of, 150 Deoxyglucose, effect of, on adenylate charge of Sacchuromyces cereuisiae, 28 2 effect of, on microbial energy charge, 28 1

inhibition of conjugation in Saccharomyces cerevisiue by, 356 Deoxyribonuclease, use of, in preparation of bacterial membrane vesicles, 179 Deoxyribonucleic acid, halobacterial, base composition of, 11 1 polymerase activity in halobacteria, 112

repair in Saccharomyces cerevisiae, 383 synthesis during yeast sporulation, 380 Depletion of energy source, effect of, on energy charge, 272 Depletion of nitrogen, effect of, on microbial energy charge, 273 Detergents, inhibitory effect of, on conjugation in Schizosaccharomyces pombe, 338

Diameter of bacterial membrane vesicles,

Cytoplasmic mixing in mating Saccharomyces cereuisiae, 367

in

D Dansylgalactosides, formulae of those used in bacteria transport studies, 240

180

Diauxic growth of Aspergillus niger, 7 Dichloros, effect of, on sexual reproduction in fungi, 164 Dichotomous growth in fungal colonies, 25

Dicyclohexylcarbodiimide, effect of, on mutants of Escherichia coli, 227

426

SUBJECT INDEX

Di-ether lipids, occurrence of, in halobacteria, 87 Diethylstilbesterol, effect of, o n fungi, 153

Differentiated fungal mycelia, differences from undifferentiated; 13 Differentiation, biochemical, in the lungal mycelium, 7 Differentiation of fungal colonies, 23 Diffusible sex factors in Schizosaccharomyces pombe, 339 Diffusible sex hormones in Hansenula wingei, 3 1 7 Diffusion transport into fungi, 49 Dihydroxyacetone as a precursor of the phytanol moiety in halobacterial lipids, 106 Dimorphism, yeast-mycelium in fungi, and sugar metabolism, 59 Dinitrophenol, effect of, on transport into bacterial membrane vesicles, 235

Diploid functions, in Hansenula wingei, 327

in Saccharomyces cerevisiae, 3 7 7 in Sctuzosaccharomycespombe, 34 7 Disaggregation of halobacterial flagella, 99

Distribution of metabolic activities in mycelial systems, 7 Double-stranded DNA in halobacteria, 113

Doubling time, effect of, o n turnover of adenine nucleotides in bacteria, 257 Drosophila, effect of, ecdysone on, 127 Drug resistance in Phytophthora drechsferi, segregation for, 159 Drugs that inhibit sterol synthesis in fungi, 162 Dunaliella vitidis, intracellular salt concentrations in, 89

E Early events in conjugation in Saccharomyces cerevisiae, 363 Ecdysone, effect of, on Drosophila, 127 Ecology of halobacteria, 1 14 Ecology of yeasts, 309 Efficiency of protein synthesis in fungi, 72

Electric potential across the bacterial plasma membrane, 228 Electrogenic movement of solutes into bacteria, 229 Electron carriers, and generation of a pH gradient across bacterial membranes, 238 Electron donors, and transport in bacterial membrane vesicles, 197 oxidation of, in Escherichia coli and transport, 237 role of, in transport into bacterial vesicles, 208 Electron spin resonance, application of, to membranes, 132 Electron transfer, active transfer coupled to, in bacterial membrane vesicles, 203

Electron-transfer systems in bacterial membrane vesicles, 185 Electron-transport chains in fungi, 68 Electron-transport chains in halobacteria, 106 Embden-Meyerhof pathway, operation of, in fungi, 59 Endonuclease activity of bacterial membrane vesicles, 184 Endoplasmic reticulum, role of, in zygotic budding ofyeast, 37 1 Endothia parasitica, glucose catabolism in, 61

Energetics of active transport in bacteria, 203 Energy charge and protein synthesis, 295 Energy charge and total adenine nucleotide concentration in microbes, 289

Energy charge, definition of, 268 value for, in different microorganisms, 266 Energy conservation sites in fungal mitochondria, 68 Energy coupling in bacterial transport, mechanism of, 228 Energy coupling, mechanism of, to active transport, 225 Energy-dependent binding of solute to carrier proteins in bacteria, 239 Energy depletion, and rate of regeneration of adenosine triphosphate, 26 1

SUBJECT INDEX

Energy, need for, to effect contact between lac carrier protein and galactosides, 24 1 Energy-transducing systems in microbes, and adenine nucleotides, 254 Enterobacter aerogenes, energy charge in, 266 Entner-Doudoroff pathway in Hydrogenomonas eutropha, operation of, 286 Envelopes, cell, structure of; in extreme halophiles, 9 1 Environmental conditions, effect of, on hyphal growth in fungi, 20 Enzyme activities and conjugation tube formation in Schizosaccharomyces pombe, 341 Enzyme activity, inactivation of, during sampling of microbes or adenine nucleotide determinations, 269 Enzyme I in bacterial phosphotransferase systems, 192 Enzyme orientation in bacterial membrane vesicles, 196 Enzymology of nucleic acids in halobacteria, 1 1 1 Episterol, formula for, 125 Episterol in fungi, 138 Ergosterol, effect of, on growth of Phytophlhora infestans, 142 properties of, I25 Ergosterol-requiring mutants of yeast, 126 -<Ergosterol uptake by Phytophthora infestans, 136 Erythritol permeability in mycoplasmas, 133 Erythrocytes, effect of sterols on permeability of, 134 Escherichia coli, adenylate kinase mutant of, 287 anaerobic electron-transferase system of, 187 ATPase activity of membrane vesicles of, 189 effect of aerobic to anaerobic transitions on energy change of, 278 effect of growth rate on energy charge of, 283 effect of nitrogen depletion on energy charge of, 274

42 7

effect of phosphate depletion on energy charge of, 275 energy charge and viability of, 280 energy charge of, 270 glycogen synthesis in, 277 ion transport into, 233 localization of D-lactate dehydrogenase in membrane vesicles of; 200 Michaelis constants for transport of amino acids into membrane vesicles of, 210 Michaelis constants for transport of glucose into, 2 14 mutants of defective in adenosine triphosphatase, 226 orientation of membrane vesicles of, 196 oxidation of substrates by membrane vesicles of, 185 phage infection of and energy charge in, 290 preparation of membrane vesicles of, 180

proline transport into membrane vesicles of, 198 respiratory chain of, 186 synthesis of pyridine nucleotides by, 259 sugar transport in, 19 1 transport coupled to electron transfer in vesicles of, 204 turnover of adenine nucleotides in, 256 turnover of ATP in, 264 Esterified sterol, location of, in fungi, 136 Esterified sterols in fungi, 136 Ethanol, effect of, on fungal conidiation, 148 energy charge of yeast when using as a source of energy, 273 Ethanol utilization by Saccharomyces cerevisiae, 28 7 Ether-linked lipids, biosynthesis of; 105 Ether-linked lipids in halobacteria, 105 Ethionine, effect of, on conjugation in Hansenuh w'ngei, 3 1 6 Ethylene glycol, effect of, on fungal conidiation, 148 Exonuclease activity of bacterial membrane vesicles, 184 Expansion of colonies as a parameter of mould growth, 33

42 8

SUBJECT INDEX

Exponential phase of growth of fungal colonies, 30 Extension rates of fungal hyphase, 16 Extracellular salt concentrations, and growth of halophiles, 88,90 Extremely halophilic bacteria, physiology of, 86

F

Fructose accumulation by Arthrobacter pyridinolis, 2 13 Fructose diphosphate supply and microbial glycogen synthesis, 289 Fruiting zone in fungal colonies, 26 Fruits, association of Saccharomyces cerevisiae with, 309 Fucosterol, formula for, 125 presence of, in Achlya bisexuulis, 15 1 Fumarate as an electron acceptor in bacteria, 22 1 Fumarate, effect of, on anaerobic uptake of lactose by bacteria, 222 Fumarate reductase, role of, in solute transport in bacteria, 222 Fumarate transport into BnCillw subtilis,

Facilitated diffusion of solutes through the bacterial cytoplasmic membrane, 176 Fatty acids, biosynthesis of, in halobacteria, 105 214 Fermentative metabolism of fungi, 57 Ferricyanide, effect of, on bacterial mem- Functional properties of bacterial membrane vesicles, 184 brane vesicles, 200 effect of, on glutamate uptake by mem- Functions controlled by mating-typing brane vesicles of Bacillus subtilis, 234 locus in Schizosaccharomyces pombe, 336 Filaments, surface, and cellular recognition in Saccharomyces cerevisiae, 364 Functions controlled by the mating-style locus in Hansenula Wingei, 3 14 surface, and conjugation in Hansenula Fungal biochemistry, status of, 2 wingez, 32 1 Fungal growth in submerged liquid culon Schizosaccharmycespombe, 340 ture, 36 Fish, use of salt to preserve, 85 Fission, zygotic, in Schizosaccharomyces Fungal growth, mathematical modelling of, 3 pombe, 344 Fungal growth modelling, examples of, 9 Fixation of carbon dioxide in fungi, 63 Fungal growth, physiology and ccntrol Flagella of halobacteria, 99 of, 1 Fluidity, membrane, role of lipids in, 132 regulation of branch initiation in, 15 Fluorescent galactosides, use of, in bacFungal mycelial form, regulation of, 14 terial transport studies, 240 Fluxes of adenine nucleotides in mic- Fungal transport processes, modulation of, 53 robes, 256 Formate as an electron donor for Fungicides, effect of, on sterol biosynthesis, 148 anaerobically grown Escherichia coli, 187 Fungi, colony growth of, 23 Formate dehydrogenase in bacterial hyphal growth units in, 19 membrane vesicles, 188 subcellular distribution of sterols in, Formate, effect of, on transport of pro135 line into bacterial membrane Fusarium aqwductuum, effect of glucose vesicles, 2 18 on colony growth of, 28 Form, mycelial, regulation of, in fungi, kinetics of growth of, in chemostat cul14 ture, 4 1 Formes a n o s w , fluctuations in the ATP Fusmum spp., hyphal growth unit of, 20 content of, 70 Fusion, cellular, in conjugation in HunFragility of halebacterial cell envelopes, senula Wingei, 32 1 92 Fusion of cells in mating of Saccharomyces Freeze drying of halophilic bacteria, 9 1 cerevisiae, 366

SUBJECT INDEX

Fusion of protoplasts of Saccharomyces cerevisiae, 365

429

Glucose depletion, effect of, on energy charge of microbes, 273 Glucose, effect of, on zygote formation in Schizosaccharomycespombe, 33 ? Glucose metabolism of Pythium ultimum, G effect of cholesterol on, 142 Glucose 6-phosphate dehydrogenase activity of Hydrogenomoncrc eutropha, Galactosaminoglycan in walls of Neuro288 spora crassa, 46 Glucose 6-phosphate transport into bacGalactose, presence of, residues in gasterial membrane vesicles, 2 13 vacuole membranes, 97 Glucose ultake mechanisms of NeuroGalactose transport into bacterial memspora crmsa, 53 brane vesicles, 2 13 Glucuronate transport into bacterial Galpermease system in bacteria, 2 13 membrane vesicles, 2 13 Gametangia formation in fungi, effect of Glutamate dehydrogenase activity of sterols on, 152 fungi, effect of ammonia in, 52 Gas vacuoles in halobacteria, 96 Glutamate dehydrogenases in fungi, 56 Gene conversion at the mat locus in Glutamate, transport of, into haloSaccharomyces cerevisiae, 35 1 bacteria, 107 Genes affecting conjugation in SchzzoGlutamate uptake by membrane vesicles saccharomycespombe, 334 of Bacillus subtilis, effect of ferriGenes, homothallism, in Saccharomyces cyanide on, 234 cerevisiae, 353 mutant, affecting conjugation in Glutamate uptake by bacterial membrane vesicles, 182, 220 Saccharomyces cerevisiae, 3 72 affecting conjugation in Schizo- Glutamate uptake by vesicles of Bacillus subtilis, 207 saccharomycespombe, 344 Genes that affect conjugation in Hm- Glutamine-oxoglutarate amino transferase system in fungi, 56 senula Wingei, 32 7 Genotypic interconversions in Schizo- Glutamine synthase activity in fungi, 57 Glutamine synthetase, absence of, from saccharmyces pombe, 33 1 bacterial membrane vesicles, I83 Geotrichum candidum, biomass proGlycerate transport into bacteria, 214 duction of, 1 1 Glycerol-assimilation pathways in bacextension rates ofhyphae of, 16 teria, 54 maximum growth rate of, 43 Glycerol kinase synthesis by Neurospora Geotrichum lactis, batch cultivation of, 10 crassa, 54 Gibberellafujikuroi, ribosome efficiency in, Glycerol permeability in mycobacteria, 73 133 Gibberella zeae, zearalenone production Glycerol phosphate dehydrogenase actiby, 163 vity of bacterial membrane vesicles, Glucanase, effect of, o n mating in 188 Saccharmyces cerevisiae, 363 Gluconate transport in bacterial vesicles, L-a-Glycerol phosphate, effect of, o n 205 anaerobic uptake of lactose by bacGluconate transport into bacterial memteria, 222 brane vesicles, 2 13 Glycerol phosphate, role of',in aminoGluconate utilization by Hydrogenomonas acid uptake by bacterial vesicles, eutropha, 286 208 Gluconeogenic enzymes of fungi, 67 Glycerol production by Saccharomyces cereGlucose concentration, effect of, on vzsiae, as affected by sodium chloride, 89 growth of fungal colonies, 28

430

SUBJEC3 INDEX

Glycogen synthesis and energy charge of microbes, 276 Glycogen synthesis, and microbial adeuylate charge, 288 Glycolate transport into bacteria, 2 14 Glycolysispathways in fungi, 59 Glycoprotein mating factors in Hansenula wingei, 3 18 Glyoxylate cycle enzymes in halobacteria, 101 Glyoxylate cycle, operation of, in fungi, 62 Great Salt Lake, extreme halophiles in the, 114 Group translocation in bacterial membrane vesicles, 190 Group translocation in fungal transport,

Growth zone width, peripheral, determination of, with fungi, 32 Guanine plus cytosine content of bacterial deoxyribonucleic acid, 1 1 1 Guanylate nucleotides, and energy regulation, 292 Guanylate.levels in microbes, 258 Gulosamino-uronic acid residues, presence of in envelopes of halobacteria, 92

H

Haem, deficiency in synthesis of an sterol requirement, 144 Halobacteria, effect oflight on, 109 Halobacteria, electron-transport chains 50 in, 106 Group translocation through the bacHalobacteria spp., physiology of, 87 terial cytoplasmic membrane, 176 Growth-arrested mutants, adenine Halobacteriaceae, lipids of, 104 physiology of, 85 nucleotides in, 286 Growth, fungal, physiology and control Halobacterial flagella, isolation of, 99 Halobacterium cutirubrum, electronof, 1 Growth inhibitors, effect of, on fungal transport chains in, 107 fatty-acid synthesis by, 105 colony expansion, 34 proteins from ribosomes of, 95 Growth, mycelial in liquid culture, proteins in gas vacuoles of, 98 measurement of, 37 Halobacterium halobium, chemi-osmotic Growth of fungi and sterols, 12 1 Growth of fungi in submerged liquid culenergy coupling in transport into, 236 ture, 36 Growth of halobacteria, effect of light on, classification of, 87 110 effect oflight on energy charge of, 279 Growth of microbes, and concentration effect of light on transport into, 224 energy charge of, 2 70 of adenine nucleotides, 254 purple membranes of, 109 Growth of undifferentiated fungal Halobacterium salinarium, classification of, mycelia, 13 87 Growth phase, synthesis of sterols in with properties of flagella of, 99 Saccharomyces cereuisiae, 139 Growth rate, and microbial energy Halobacterium spp., vacuolation in, 96 Halobacterium uokanii, effect of salt concharge, 282 centration on growth of, 90 effect of, on ATP turnover in microHalococcus spp., intracellular salt concenorganisms, 265 tration in, 89 on turnover of adenine nucleotides Halophilic bacteria, physiology of, 85 in bacteria, 256 Growth stage and conjugation of Han- Halophilic enzymes, 102 Halophilic phages, 114 senula wingei, 3 16 Growth unit, definition in analysis of Halophilic proteins in bacteria, 100 Halophilism, extreme, ecological confungal growjh, 19 siderations of, 114 Growth zone, peripheral, in fungal Haploid budding in yeasts, 3 1 1 colonies, 24

SUBJECT INDEX

Haploid functions in Hansenula wingei, 3 14 Haploid functions in Saccharomyces cerevisiae, 354 Haploid functions in Saccharomyces cerevisiae, repression of, 378 Haploid functions in Schizosaccharomyces pombe, 336 Haploid functions, repression of, in Schizosacchurmycespombe, 34 7 Hansenula wingei, ecology of, 309 growth stage and conjugation in, 3 16 life cycle of, 3 10 mating-type alleles in, 3 13 physiology of mating in, 308 Helminthosporium cynodontis, pyruvate metabolism in, 65 2- Heptyl-4- hydroxyquinoline- N -oxide, effect of, on lactose transport into bacterial membrane vesicles, 209 Hermaphroditic strains of Achlya spp., 152 Heterokaryosis in Sacchuromyces cereuisiae, 370 Heterokaryosis in Schizosaccharomyces pombe, 343 Heterothallic species, of fungi, effect of sterols on reproduction in, 160 Heterothallism in Hamenula wingei, 3 14 Heterothallism in Saccharomyces cereuisiae, 35 1 Heterothallism in Schizosaccharomyces pombe, 333 Heterozygosity at the mating-type locus in Hansenula wingei, 328 Heterozygosity at the mating-type locus in Saccharmyces cereuisiae, 3 7 7 Heterozygosity in Schizosaccharomyces pombe, 347 Hexose monophosphate pathways, operation of, in fungi, 59 Hexose transport in Staphylococcus aureus, 192 Hides, use of salt to preserve, 85 Homothallic species of fungi, effect of sterols on, 152 Homothallism in Hansenula wingei, 3 14 Homothallism in Saccharomyces cerevisiae, 35 1 Homothallism in Schizosaccharomyces pombe, 333

43 1

Homozygosity at the mating-type locus in Saccharomyces cerevisiae, 3 7 7 Schizosaccharomyces Homozygosity in pombe, 347 Hormones, mating, in Saccharomyces cerevisiae, 356 sexua1,'ofAchlyaspp., 149 steroid, function of, 127 Hydrogenomonas eutropha, adenine nucleotide content of, 286 energy charge of, 266 Hydrolysis of adenosine triphosphate, estimates of rate of, 263 Hydrolysis of ATP, inhibitors of, 27 1 Hydrophobic amino-acid residues in halophilic enzymes, 103 Hydrophobic nature of halobacterial proteins, 100 Hydroxybutynoate, effect of, on bacterial lactate dehydrogenase, 202 Hydroxysterols, occurrence of, in membranes, 135 Hypertrophic growth of fungi, 43 Hyphae, fungal, extension rates of, 16 Hyphal growth in fungi efrect of environmental conditions on, 20 Hyphal growth, polarization of, in fungi, 14 Hyphal growth unit, fungal definition of, 18 Hypochytridiomycetes, sterol synthesis in, 123

I Illegitimate fusion in mating of Saccharomyces cerevisiae, 365 Immunology of halobacteria, 88 Inducible conjugation agglutination in Hansenula Wingei, 3 17 Induction of conjugation in Hansenula wingei, 3 15 Infection with phage and energy charge, 290 Inhibition of conjugation in Saccharomyces cerevisiae, 354 Inhibitors, effect of, on hyphal growth in fungi, 2 1 Inhibitors of conjugation in Hansenula wingei, 3 16

SUBJECT INDEX

432

Inhibitors of’ conjugation in Schizosaccharomycespombe, 338 Initiation of con.jugation in Schizosaccharomycespombe, 336 Initiation of RNA synthesis, regulation of;294 Inner surface enzymes in bacterial menibrane vesicles, 196 Interaction events in conjugation of‘ Saccharomyces cerevisiae, 364 Intracellular salt concentration in halophiles, 8 8 Invagination in preparation of bacterial membrane vesicles, 196 Inversion of bacterial membranes in vesicles, 238 Inversion of membrane vesicles from barreria, 199 Iodine vapour, use of, to isolate yeast mutants, 335 Ion transport in halobacteria, role for lipids in, 108 Ion transport into Escherichia coli, 233 Isocitrate lyase activity of fungi, 62 Isocitrate lyase, effect of; cholesterol on levels of in fungi, 143 Isolation of bacterial membrane vesicles, I77

Iso-osmotic internal salt concentrations in halophiles, 89

K Kex niutants ofSaccharomyces cerevisiae, 37 7 Kinetics ofcolony expansion by fungi, 29 Kinetics of fungal growth in submerged liquid culture, 40 Kinetics of growth of Geotrichum candidum, I8

Kinetics ofsolute transport in fungi, 48 Klebsiella aerogenes, efrect of aerobiosis o n energy charge of, 278 energy charge and viability of, 280 transport coupled to electron transport in vesicles of, 205 Klebsiella pneurnoniae, ATTP turnover by, 268

energy charge of, 270 Kluyveromyces jugilis, catabolite pression of respiration in, 58 Kryptogenin, formula of, 155

re-

L Lac carrier protein in Escherichia coli, access of, to galactosides, 24 I Lactate as an electron donor in proline transport membrane vesicles of Escherichia coli, 198 Lactate as an energy source in transport into bacterial vesicles, 207 Lactate dehydrogenase, density of, in membranes of EJcherichia coli, 237 Lactate dehydrogenase, localization of, in membrane vesicles from Escherichia coli, 200 Lactate dehydrogenase of Escherichia coli, outer surface location of in membrane vesicles, 202 Lactate dehydrogenase of Eschehchia coli, properties of, 200 Lactate, effect of, on glutamate uptake by bacteria, 220 effect of, on proline uptake by membrane vesicles of Escherichia coli, 203 Lactate oxidation by bacterial membrane vesicles, 185 Lactate transport into Bacillus subtilis, 2 14 Lactate transport mechanism in bacterial membrane vesicles, 197 Lactose accumulation by bacteria under anaerobic conditions, 218 Lactose accumulation by membrane vesicles of Escherichia coli, 233 Lactose carriers in membrane of Escherichia coli, density of, 23 7 Lactose transport into bacterial mcmbrane vesicles, 2 12 Lactose transport into Escherichia coli, Michaelis constants for, 214 Lactose transport, role of proton-motive force in, 228 Lactose uptake by Escherichia coli, effect of anerobic electron donors on, 222 Lag phase in growth of fungal colonies, 30 Lanosterol, effect of, on growth of Phytophthora infestam, 142 formula for, 125 Leading hyphae in fungal colonies, 24 Leakage from fungal cells, effect of sterols on, 14 I Lecithin-cholesterol interaction, 13 1

SUBJECT INDEX

Leptospheria typhae, sterol distribution in, 139 Leucine accumulation by halobacterial membrane vesicles, 224 Leucine uptake by halobacterial vesicles, 108 Light-dependent transport into halobacteria, 242 Light, effect of, on alanine uptake by bacteria, 224 effect of, on halobacteria, 109 on microbial energy charge, 279 Linear colonies of Neurospora crmsa, growth of, 29 Lipid in fungal mycelia, 26 Lipid synthesis by Pythium acanthium, effect ofcholesterol on, 143 Lipid metabolism in halobacteria, 102 Lipids of Halobacteriaceae, 104 Lipophilic cations, effect of, on transport into bacterial membrane vesicles, 235 Lipopolysaccharide, presence of, on surface of Gram-negative membrane vesicles, 178 Liposomes, use of, to research sterol function in membranes, 132 Liquid culture, submerged, of fungi, 36 Localization of D-lactate dehydrogenase in membrane vesicles from Escherichia coli, 200 Location of carriers in bacterial membranes, 234 Locus, mat, in Hansenula wingei, 3 14 Low-density vesicles in Saccharomyces cerevisiae, composition of, 137 Lysozyme, use of, in preparation of bacterial membrane vesicles, 178

M Macroregulation of fungal growth, 46 Magnesium-activated ATPase in bacterial membrane vesicles, 189 Magnesium ions, and ribosomes from halobacteria, 94 Maintained states in fungal physiology, 47 Maintenance coefficient of fungi, 42 Maintenance coefficient of microbes, definition of, 265

433

Maintenance energy in fungal cultures, 38 Maintenance energy of yeasts, 268 Maintenance requirement of microorganisms, 265 Malate transport into Bacillus subtilis, 2 14 Malt extract, effect of, on fungal hyphal growth unit length, 22 Manganese active transport of’, into vesicles, 2 1 7 Manganese transport into bacteria, 2 16 Mannan proteins, mating factors of Hansenula wingei, 320 Mannan, yeast, binding of sterol to, 138 Mannitol uptake by fungi, 5 1 Marine pseudomonads, transport coupled to electron transfer in vesicles of, 205 Mat locus in Saccharomyces cerevisiae, 350 Mat locus in Saccharomyces cerevisiae, mutation at, 353 Mat locus in Schizosaccharomyces pombe, 333 Mathematical modelling of fungal growth, 3 Mating-defective mutants of Saccharomyces cerevisiae, 3 7 2 Mating in Phythiaceae, 156 Mating mutants of Sacchuromyces cerevzsiae, 376 Mating of phytophthoras, effect of cholesterol on, 160 Mating processes in yeasts, 308 Mating-type locus and sporulation in Saccharomyces cerevisiae, 3 7 9 Mating-type locus in Hansenula wingei, 3 13 Mating-type locus in Saccharomyces cerevisiae, functions controlled by, 354 Mating-type locus in Saccharomyces pombe, 33 1 Mating-type regulation of meiosis in Saccharomyces cerevisiae, 38 1 Mating type-specific substances in Saccharomyces cerevisiae, 354 Mating types in Phytophthora spp., 157 Maximal transport rates for amino acids into bacterial vesicles, 2 1 1 Maximum growth rates of filamentous fungi, 42 Mean cumulative age in fungal colonies, 12 Meat, use of salt to preserve, 85

434

SUBJECT INDEX

Mechanism of energy coupling to active transport in bacteria, 225 Mechanism of energy coupling in bacterial active transport, 228 Medium composition, effect of, on conjugation in Saccharomyces cerevisiae, 354 effect of, on fungal hyphal growth unit length, 21 Meiosis and sporulation in Saccharomyces cerevisiae, 3 7 9 Meiosis in Hansenula wingei, 329 Meiosis in Schizosaccharwnyces pombe, 348 Meiosis in Schirosaccharomyces pombe, effect of mating-type locus on, 348 Meiosis in Schizosaccharomyces pombe, induction of, 345 Meiosis mutants in Saccharomyces cerevisiae, 38 1 Melanin, extracellular, synthesis of by fungi, 10 Membrane fluidity, role of lipids in, 132 Membrane stretching in Saccharomyces cereuisiae, and sterol composition, 146 Membrane vesicles, bacterial, active transport of solutes in, 175 Membrane vesicles, bacterial, composition of, 184 Membrane vesicle-defective mutants of Escherichia coli, 22 7 Membrane vesicles from Escherichia coli, localization of D-lactate dehydrogenase in, 200 Membranes, halobacterial, transport across, 107 Membranes, vacuole, from halobacteria, 97 Menadione, as a substitute for menaquinone in deficient bacteria, 23 1 Menadione reductase from halobacteria, properties of, 100 Menaquinone-deficient mutant of Bacillus subtilis, solute transport in, 23 1 Menaquinone, presence of, in halobacteria, 11 1 Menaquinone-8, occurrence of, in halobacteria, 94Menaquinone synthesis, mutants defective in, and vesicle transport, 209

Metabolic activities, distribution of, in mycelial systems, 7 Metabolic control of fungal growth, 1 Metabolic control in fungi, 57 Metabolic pathways in halobacteria, 101 Metabolism, fungal, effect of sterols on, 141 Methanobacterium sp., effect of aerobiosis on energy charge of, 278 Mevalonate, incorporation of, into fungal sterols, 122 Michaelis constants for amino-acid transport into bacterial membrane vesicles, 2 10 Michaelis constants for sugar transport in Escherichia coli, 2 14 Michaelis constants for transport of carboxylic acids into bacteria, 2 15 Michaelis-Menten kinetics of solute transport into fungi, 49 Michaelis-Menten relationships in microbial growth, 6 Microbacterium phlei, transport coupled to electron transfer in vesicles of, 206 Microbial cultures, sampling of, for adenine nucleotide determinations, 269 Micrococcus denitnfcans, transport coupled to electron transfer in vesicles of, 205 Minimum specific growth rate of fungi, 43 Mitochondria, energy conservation sites in fungal, 68 Mitochondria, proton-motive force in, 229 Mitochondria, fungal, sterol in, 137 Mitochondrial fusion in Hansenula wingei, 326 Mitchondrial fusion in mating of Saccharomyces cereuisiae, 36 7 Mitochondrial profiles of yeast, and anerobic growth, 147 Mitotic recombination in Hansenula wingei, 330 Mitotic recombination in Saccharomyces cereuisiae, 38 1 Mitotic recombination in Schizosaccharomyces pombe, 3 1 1, 350 Modelling, mathematical, of fungal growth, 3 Modelling of fungal growth, examples of, 9

435

SUBJECT INDEX

Models, valid use of, in analysis of fungal growth, 4 Modulation of fungal transport processes, 53 Molar growth yields, definition of, 26 1 Models of fungal sterols, 128 Molecular spacing in membranes, and sterols, 133 Molybdate transport into fungi, 55 Monod equations for microbial growth, 6

Monolayers, lipid, use of, to research sterol function in membranes, 132 Monopodial branching pattern in fungal colonies, 25 Morphological zones in mature fungal colonies, 24 Mould growth, colony expansion as a parameter of, 33 Mould-induced changes in the substrate, 27 Mucor genevemis, sugar metabolism by, 58

Mucor hiemulis, extension rates of hyphae of, 16 Mucor mucedo, productive zone in colonies of, 26 Mucor rowcii, effect of temperature on growth of, 15 sterols in, 139 Mucor spp., hyphal growth units of, 20 Muramidase, use of, in preparation of bacterial membrane vesicles, 178 Mutant genes affecting conjugation in Saccharomyces cerevisiae, 3 7 2 Mutant genes affecting conjugation in Schizosacchuromycespombe, 344 Mutants, respiratory chain, lactose transport into vesicles from, 209 Mutations in marine bacteria, and extreme halophilism, 115 Mycelia, fungal undifferentiated, growth of, 13 Mycelial fungi, problems in the study of, 2

Mycelial systems, distribution of metabolic activities in, 7 MycoPlasma spp., sterol requirements of, 130

Mycoplasmas, requirement for sterols in, 130

Mycoplasmu mycodes, sterol requirement 01; 133

Myxococcus xanthus, ATP content of, 270 energy charge during sporulation of., 280

energy charge of, 267,269

N Negative autotrophism in fungal hyphae, 23

Neurospora crassa, adenine nucleotide concentration in, 270 adenylate charge in, 69 circadian rhythm in conidiation by, 46 distribution of sterols in, 140 effect of cyanide on energy charge of, 282

effect of L-sorbose o n hyphal growth unit length in, 2 1 effects of sterols on sterile mutants of, 164

ergosterol crystals in, 141 extension rates ofhyphae of, 16 growth kinetics of, 10 multiplicity of glucose-uptake mechanisms in, 53 operation of glyoxylate cycle in, 63 polyene-resistant mutants of, 14.5 transport of solutes into, 48 turnover of ATP in, 263 Nicotinamide nucleotides, reduced, role of in bacterial solute transport, 232 Nitrate, and fusion of yeast protoplasts, 365

effect of, in regulating glucose metabolism in fungi, 62 on transport of proline into bacterial membrane vesicles, 2 18 on glutamate uptake by bacteria, 220

Nitrate reductase activity of bacterial membrane vesicles, 188 Nitrate reductase, role of, in solute transport in bacteria, 223 Nitrate reduction by halobacteria, 8 7 Nitrobacter winogradskyi, energy charge of, 270

Nitrogen, effect of depletion of, on energy charge of, 273

SUBJECT INDEX

436

Nitrogen source starvation, and conjugation in Hansenula wingei, 3 15 Nuclear fusion in Haruenula wipgei, 326 Nuclear fusion in mating of Saccharomyces cereuisiae, 3 70 Nuclear fusion in Schizosaccharomyces pombe, 343 Nuclear membranes in mating of Saccharomyces cereuisiae, 3 7 1 Nucleases, use of, in preparation of bacterial membrane vesicles, 1 7 7 Nucleic acid synthesis in microbes, and turnover of adenine nucleotides, 257 Nucleic acids, adenine nucleotides in biosynthesis of, 253 Nucleic acids of halobacteria, 1 1 1 Nucleotide metabolism by bacterial membrane vesicles, 184 Nutrient concentration, effect of, on fungal colony expansion, 34 Nutrient concentration, effect of, on growth of fungal colonies, 27 Nutrient limitation in chemostat cultures of filamentous fungi, 39 Nystatin, affinity of, for sterols, 145

0

Ornithine carbamoyl transferase, properties of, from halobacteria, 104 Oscillatory phenomena in fungal cultures, 43 Osmolarity, effect of, on uptake of glutamate by bacterial membrane vesicles, 182 Osmometer, behaviour of bacterial membrane vesicles as, 183 Osmotic barrier of the bacterial cytoplasmic membrane, 175 Outside location of anaerobic electrontransfer intermediates in bacterial membranes, 234 Oxalate, effect of, on transport into bacterial membrane vesicles, 238 Oxamate, effect of, on transport into bacterial membrane vesicles, 238 Oxidation, terminal, of substrates by fungi, 67 Oxidative metabolism of fungi, 57 Oxidative phosphorylation, and active transport in bacteria, 225 Oxidative phosphorylation-deficient mutants of Escherichia coli, 226 Oxidative phosphorylation in bacterial membrane vesicles, 190 Oxygen, response of fungal hyphae to, 23 Oxygen tension, effect of, on fungal colony growth, 28

Obligate halophilism, physiological basis of, 86 Oestradiol, effect of, on fungi, 153 Oligomycin, effect of, on bacterial adenosine triphosphatase, 225 P Oligopeptide nature of alpha factor in Para-Nitrophenol, effect of, on energy Saccharomyces cereuisiae, 35 7 charge of yeast, 282 One-step isolation of bacterial memParamutation in yeasts, 3 13 brane vesicles, 179 Oogonia production by fungi, effect of Particle density on bacterial membrane vesicles, 196 sterols on, 155 Oogonia delimitation, effect of hor- Passive diffusion through the bacterial cytoplasmic membrane, 176 mones on, in Achfya spp, 149 Pasteur effect in yeasts, 58 Oogoniol, formula of, 150 Pathogenicity of fungi, role of sterols Oomycetes, sterol synthesis by, 123 in, 143 Oospore formation by Phytophthora Pathways, metabolic, in halobacteria, 101 capsin', effect ofcholesterol on, 161 Oospore production by fungi, effects of Pellet growth of fungi in chemostats, 40 Pellets, fungal, distribution of metabolic sterols on, 154 activities in, 8 Ophiostoma multiannulatum, kinetics of Penicillia, hyphal growth units of, 20 growth of, m chemostat culture, 40 Orientation of bacterial membrane Penicillin, use of, in preparation of bacterial membrane vesicles, 1 7 7 vesicles. 194

SUBJECT INDEX

Penicillium camemberti, pyruvate metabolism in, 65 Penicillium chysogenum, autolysis of pellets of, 8 effects of starvation on, 47 extension rates of hyphae of, 16 Penicillium roquefrtii, sterol-polysaccharide fractions in, 138 Pentose phosphate pathway, operation of, in fungi, 59 Peptococcus prevotii, energy charge and viability of, 280 energy charge of, 267, 272 Perchloric acid, use of, in determination of adenine nucleotides, 270 Peripheral growth zone, effect of, on radial expansion of fungal colonies, 30 Peripheral growth zone in fungal colonies, 24 Peripheral growth zone width in fungal colonies, determination of, 32 Periplasmic binding proteins, and aminoacid transport into bacterial membrane vesicles, 209 Periplasmic enzymes, presence of, in bacterial membrane vesicles, 183 Perithecial development in Cochliobolus carbonum, effects of sterols on, 162 Permeability of fungal cells, effect of sterols on, 141 Permeability of membranes, role of lipids in, 133 Perturbations of conditions in fungal chemostat cultures, 44 Petite mutants, and sterol requirement in yeasts, 144 Phage-host relationships in halobacteria, 113 Phage infection and energy charge, 290 Phage particles in halobacteria, 113 Phenethanol, effect of, on Neurospora crassa, 59 Phenethanol-induced morphogenesis in fungi, 140 Pheromones, and mating in Saccharomyces cereuisiae, 361 in yeast conjugation, 3 10 Phosphate, effect of depletion of, on microbial energy charge, 273 Phosphate uptake by Neurospora craJsa, 5.5

43 7

Phosphate uptake by Rhodotorula rubra, regulation of, 53 Phosphatidylethanolamine synthesis by bacterial membrane vesicles, 184 Phosphatidylglycerol synthesis by bacterial membrane vesicles, 184 Phosphoenolpyruvate carboxylase activity of fungi, 64 Phosphoenolpyruvate phosphotransferase system in bacterial membrane vesicles, 190 Phosphofructokinase activity of Eschrichia coli, 28 7 Phospholipid content of bacterial membrane vesicles, 183 Phospholipid synthesis by bacterial membrane vesicles, 184 Phosphorus uptake by colonies of Aspergillus niger, 3 1 Phosphoryl traps, effect of inhibitors of, 28 1

Photophosphorylation in halobacteria, 109

Photosynthesis in bacterial membrane vesicles, 188 Photosynthetic halophilic bacteria, isolation of, 88 Photosynthetic microbes, energy charge of, 279 Phototrophic bacteria, membrane vesicles of, 188 Phycomyces blabesleenus, sterol distribution in, 138 Phymtotrichum omnivorum, effect of sterols on conidiation in, 148 sterol crystals in, 141 Physical properties of bacterial membrane vesicles, 180 Physiological conditions inducing sporulation in Saccharomyces cereuisiae, 379 Physiological factors affecting conjugation in Saccharomyces cerevisiae, 354 Physiology and control of fungal growth, 1

Physiology of Halobacteriaceae, 85 Physiology of mating in yeasts, 307 Phytanyl moieties in ether-linked lipids of halobacteria, biosynthesis of, 106 Phytophthora cactorum, effect of sterols on, 121

438

SUBJECT INDEX

Phytophthora cactorum, incorporation of mevalonate into fungal precursors in, 122 uptake of cholesteryl oleate-by, 136 Phytophthora capsici, effect of cholesterol on oospore formation by, 16 1 Phytophthora drechsleri, selfing in, 159 Phytophthora infestans, conversion of lanosterol into cholesterol by, 122 effect ofsterols on growth of, 142 Phytophthora palm'vora, relative sexuality in, 158 Phytophthora pararitica, oospore formation by, 155 Phytophthora spp., cholesterol metabolism by, 154 effect of nystatin on, 145 effects of sterols on, 141 effect of sterols on sexual reproduction in homothallic, 152 effect of sterols on sporangium formation by, 148 sterol requirements of, 123 Phytylglycerol derivatives in halobacteria, 102 Pigments, accumulation of, by fungal colonies, 29 Plant cells, effect of sterols on permeability of, 134 Plasmodiophora brassicae, sterols in spores of, 122 Ploidy of Hansenula wingei, 309 Plugging of septa1 pores in growth of fungal hyphae, 33 Polarity, hyphal, regulation of, 14 Polarization of hyphal growth, 14 Polyamines, effects of, on halophilic enzymes, 104 Polyamines, metabolic role of, in fungi, 74

Polyene-resistant strains ofyeast, 145 Polyisoprenoid chains, presence of, in halobacterial lipids, 105 Polynucleotide phosphorylase, presence of, in halobacteria, 112 Polyol uptake by fungi, 5 1 Polyphosphate reserves in Neurospora crassa, 69

Polytomu uvella, energy charge and viability of, 28 1 Polytomella uvella, energy charge of, 27 1

Pool, adenine nucleotide, size of in micro-organisms, 266 ATP, consumption of, in bacteria, 265 Population dynamics of microbial growth, 4 Poriferasterol, formula for, 125 Potassium ions, and ribosomes from halobacteria, 94 effect of, on physiology of halophiles, 91

requirement for transport of glutamate into halobacteria, 108 Potassium uptake by bacterial membrane vesicles, effect of valinomycin on, 217 Pressure, effect of, on halobacterial gas vacuoles, 96 Primulin staining of conjugation in Schizosaccharmyces pombe, 342 Productive zones in fungal colonies, 26 Proline accumulation by membrane vesicles of Escherichia coli, 233 Proline transport into membrane vesicles of Escherichia coli, 198 Proline uptake by bacterial membrane vesicles, effect of formate on, 2 18 Properties, physical, of bacterial membrane vesicles, 180 Propylene glycol, effect of, on fungal conidiation, 148 Protease, effect of, on agglutination in Saccharomyces cerevisiae, 363 Protective effect of carotenoids in halobacteria, 110 Protein, in bacterial membranes, 2 I2 Protein synthesis, and energy charge, '

295

Protein synthesis during sporulation of Saccharomyces cerevisiae, 380 Protein synthesis, efficiency of, in fungi, 72

Protein synthesis in microbes, and turnover of adenine nucleotides, 257 Proteins, envelope, in halophilic bacteria, 93

gas-vacuole membrane, comparison of properties of, 98 removal of, from halobacterial ribosomes, 95 Proteolytic enzymes, effect of, on bacterial membrane vesicles, 198

SUBJECT INDEX

439

effect of, on conjugation in Schizo- Pyruvate carboxylase-less mutants of Aspergillus nidulans, 64 saccharomyces pombe, 345 Protiolytic inactivation of yeast trypto- Pyruvate kinase activity of Saccharomyces cerevisiae, 288 phan synthase, 67 Proteus mirabilis, effect of aerobiosis on Pyruvate transport into Escherichia coli, 215 energy charge of, 278 transport coupled to electron transfer Pythiaceae, reproduction in, 156 Pythium acanthium, effect of cholesterol on in vesicles of, 206 turnover ofATP in, 263 lipid synthesis by, 142 Proton gradient, generation of large, in effect of polyene antibiotics on, 153 Escherichia coli, 23 7 sterol location in, 137 Proton gradients across halobacterial Pythium paroecandrum, transport of sterol membranes, 108 in, 143 Pythium spp., cholesterol metabolism by, Proton-motive force in bacteria, 228 Proton translocation across the bacterial 154 plasma membrane, 228 effect ofpolyene antibiotics on, 145 effect ofsterolson, 121, 141 Proton uanslocators, effect of, on halobacteria, 110 effect of sterols on sexual reproduction in homothallic, 152 Protoplasts, Saccharomyces cerevisiae, fusion Pythium sylvaticum, mating in, 156 of, 365 Pseudomow aeruginosa, dehydrogenases in Pythium ultimum, effect of cholesterol on glucose metabolism by, 142 membrane vesicles of, 186 energy charge of, 270 gluconate transport into, 213 membrane vesicles of, 178 R transport coupled to electron transfer in vesicles of, 205 Rad loci in Saccharomyces cerevisiae, 382 Pseudomow oxalatinw, transport coupled Radial expansion of fungal colonies, to electron transfer in vesicles of, 205 effect of peripheral growth zone on, Pseudomow putida, transport coupled to 30 electron transfer in vesicles of, 204 Radial growth rate of fungal colonies, 29 Purification of halophilic enzymes, prob- Radiation survival in Saccharomyces cerelems in, 104 visiae, 382 Purine nucleotide biosynthesis, regu- Rates of growth of fungi in submerged lation of, in microbes, 258 liquid culture, 40 Purine nucleotide catabolism, regulation Re-annealing of membrane in preparaof, 260 tion of bacterial vesicles, 1 7 8 Purine uptake by bacteria, 193 Recognition, cellular, in conjugation of Purity of bacterial membrane vesicles, Hamenula wingei, 3 1 7 183 cellular, in Saccharomyces cerevisae Puromycin, effect of, on fungal cellulase mating, 356 production, 15 1 Recombination, mitotic, in Hansaula Purple membranes of halobacteria, 109 Wingei, 330 Purple membranes of Halobacterium mitotic, in Saccharomyces cerevisiae, 38 1 hnlobium, 237 in Schizosaccharomycespombe, 350 Puuescine, metabolic role of, in fungi, Reconstitution of bacterial membranes, 75 as vesicles, 201 Pyridine nucleotide content of micro- Regeneration of adenosine triphosphate organisms, 259 in micro-organisms, 261 Pyrimidine transport in bacteria, 194 Regulation of ATP utilization and reP w v a t e carboxylase activity in fungi, 64 eeneration. 268

440

SUBJECT INDEX

Regulation of branch initiation in fungal growth, 15 Regulation of fungal mycelial fo-rm, 14 Regulation of RNA synthesis in microbes, 293

Regulation of solute transport in fungi, 50 Regulation of spatial distribution of fungal hyphae, 23 Regulatory activity of adenine nucleotides in microbes, 254 Regulatory loci, mating, in Saccharmyces cereuisiae, 376 Relative sexuality in Achlya spp., 156 Renaturation ofhalobacterial DNA, 112 Repair mechanisms for DNA in yeasts, 383

Repression of haploid functions in Saccharomyces cereuisiae, 3 7 8 Reproduction of fungi, and sterols, 12 1 Reproductive structures, zone of, in fungal colonies, 27 Respiration by membrane vesicles of Escherichia cob, 198 Respiratory activity, and yeast life cycles, 3 10

Respiratory-chain carriers, outside location of, in bacterial membranes, 234 Respiratory chain coupling of carriers in bacterial solute transport, 230 Respiratory-chain inhibitors, effect of, on bacterial membrane vesicles, 18 7 Respiratory chains in bacterial membrane vesicles, 185 Respiratory enzymes, catabolite repression of synthesis of, in yeasts, 58 Rhamnose accumulation by Arthrobacter pyridonolis, 2 13 Rhizopus arrhizw, polysaccharide-sterol fractions in, 138 Rhodopseudomonnr sphaeroides, cyclic electron transfer in, 189 energy charge of, 2 7 9 transport coupled to electron transfer in vesicles of, 206 preparation of membrane vesicles of, 180

transport of salutes into, 223 Rhodospirillum rubrum, energy charge of, 219

Rhodotorula rubra, regulation of' phosphate uptake by, 53 Rheology of fungal liquid cultures, 38 Rhizoctonia solani, differentiation in colonies of, 9 Rhizopus stolonijer, hyphal growth unit of, 20

Rhizopus nigricans, pyruvate metabolism in, 65 Rhizopus stolonijer, colonial radial growth, of, 35 Rhythmic mycelial growth of fungi, 45 Ribonuclease, use of, in preparation of bacterial membrane vesicles, 179 Ribonucleic acid polymerase in halobacteria, 113 Ribonucleic acid polymerase, properties of, from halobacteria, 103 regulation of activity of, 294 Ribonucleic acid synthesis and energy charge, 293 Ribonucleic acid synthesis and function in fungi, 7 1 Ribosomes of halobacteria, properties of, 94

Ribosomes of halobacteria, 94 Right-side out orientation of bacterial membrane vesicles, 197 Rubidium uptake by bacterial membrane vesicles, effect of valinomycin on, 217 Rust-infested leaves, sterols in, 122

S Saccharomyces cereuiske, ATP content of, 270

budding pattern in, 383 changes in molar growth yield during cell cycle of, 26 1 composition of low-density vesicles in, 137 D gene in, 352 distribution of sterol in, 138 ecology of, 309 effect of aerobiosis on energy charge of, 278 effect of depletion of glucose on energy charge of, 273 effect of ergosterol on ultrastructure of, 147

SUBJECT INDEX

effect of nitrogen depletion on energy charge of, 275 effect of sodium chloride on physiology of, 89 energy charge and viability of, 28 1 energy charge of, 267 fermentative metabolism of, 57 glycolysisby, 6 7 hormone secretion by, 164 kinetics of biotin uptake by, 5 1 life cycle of, 3 10 lipid requirements of anaerobically grown, 146 mutants of, with altered sterols, 144 mating-type locus in, 350 oscillations in size of a chemostat population of, 44 polyene-resistant strains of, 145 physiology of mating in, 308 sterol synthesis during ascospore formation by, 140 Saccharomyces lactis, D gene in, 352 Saccharomyces mrbensis, D gene in, 352 Saccharomyces oviformis, D gene in, 352 Saccharomyces spp., sterol mutants of, 126 sterols in, 144 Salmonella typhimurium, energy charge of, 283

membrane vesicles of, 184 pyrimidine transport into, 194 transport coupled to electron transfer in vesicles of, 205 turnover of adenine nucleotides in, 256 Salt concentration, intracellular, in halophiles, 88 Salt, use of to preserve food, 85 Salterns, occurrence of halobacteria in, 115

Sampling of microbial cultures for adenine nucleotide determinations, 269

Saponin content of host tissue, and fungal pathogenicity, 144 Saprolegnia spp., sterol synthesis by, 123 Satellite DNA in halobacteria, 112 Scavenger reactions in microbial nucleotide synthesis, 258 Schizosaccharomyces pombe, ecology of, 309 life cycle of, 3 10 mating in, 33 1 oscillations in respiratory activity of, 45

44 1

physiology of mating in, 308 Shmoos, formation of, by Saccharomyces cerevisiae, 358 nature of, in mating of Saccharomyces cerevisiae, 357 Shmoos of Saccharomyces cerevisiae, electron micrographs of, 368 Sea water, as a source of halophiles, 90 Secondary metabolites, accumulation of, by fungal colonies, 29 Segregated models of microbial growth, 5 Segregation for drug resistance in Phytophthora drechslezi, 159 Selenomonas ruminatium, energy charge of, 285

Self diploidization in Saccharomyces cerevisiae, 353 Self mating in Saccharomyces cerevisiae, 35 1

Selfing in mating of fungi, 158 Septa1 pores in fungal hyphae, 26 Septate hyphae, peripheral growth zone of colonies of, 32 Serine transport into bacteria, effect of ATP on, 226 Sex factors, diffusible, in Hansenula zuingei, 3 1 7 Sex factors, diffusible, in Schizosaccharomyces pombe, 339 Sex-specific budding in Hansenula wingei, 317

Sexual agglutination in Hansenula w'ngei, 328

Sexual agglutination in Schizosaccharomyces pornbe, 347 Sexual agglutination in yeasts, 3 1 1 Sexual hormones of Achlya spp., 149 Sexual reproduction, fungal, role of sterols in, 123 Sexual reproduction in homothallic fungi, effect of sterols on, 152 Sexual reproduction in yeasts, 308 Shape of ascospores in yeasts, 3 12 Shape of sterol molecules, 126 Shearing shock o n fungal mycelia, 10 Sitosterol, effect of, o n growth of Phytophthora infestans, 142 formula for, 125 Sitosterol uptake by Phytophthora infestans, 135

442

SUBJECT INDEX

Sodium amytal, effect of, on lactose transport into bacterial vesicles, 208 Sodium chloride, tolerance of, with extremely halophilic bacteria, 86 Sodium cyanide, effect of, on lactose transport in bacterial vesicles, 208 Sodium dodecyl sulphate, effect of, on conjugation in Schirosaccharomyces pombe, 346 Solar irradiation, effect of, on halobacteria, 109 Solid substrates, colonization of, by moulds, 35 Solutes, active transport in bacterial membrane vesicles, 175 energy-dependent binding of; to carrier proteins in bacteria, 239 Sorbose, effect of, on fungal colony expansion, 34 effect of, on hyphal growth in fungi, 21 Sordaria jmicola, inhibitors of sterol synthesis in, 162 Specificity of amino-acid transport proteins in bacteria, 2 1 1 Spatial distribution of fungal hyphae, regulation of, 23 Speckled colonies of Schizosaccharomyces pombe, 335 Spermidine, metabolic role of’, in fungi, 75

Spermine, metabolic role of; in fungi, 75

Sphaeroplasts, bacterial, use of., in preparation of membrane vesicles, 1 7 7 Spoilage caused by halobacteria, 88 Sporangium formation in Phytophthora spp., effect of sterols on, 148 Spores, growth of undifferentiated fungal mycelium from, 1 7 Sporidia of Ustilago maydis, effect of triarimol treatment on, 148 Sporulation and meiosis in Saccharomyces cerevisiae, 3 79 Sporulation, bacterial, energy charge changes during, 279 Sporulation in Hansenula wzngei, 329 Sporulation in Saccharomyces cerevisiae, physiological conditions inducing, 379

Sporulation in Schirosaccharomyces pombe, 348

Sporulation in Schirosaccharomyces pombe, mutant genes affecting, 349 Sporulation in yeasts, 3 1 1 Spreading colonial mutants of Neurospora crassa, 23 Stability of messenger RNA in bacteria, 295

Stabilization of energy charge value in microbes. 268 Staphylococcus aurew, energy charge of, 270

evidence for dehydrogenase-coupled solute transport in, 232 Michaelis constants for amino-acid transport into membrane vesicles of, 210 ~~

orientation of membrane vesicles of, 196

oxidation of substrates by membrane vesicles of, 185 preparation of membrane vesicles of, 178

sugar transport by, 192 transport coupled to electron transfer in vesicles of, 204 Starvation of fungi, and effect on sterol ester content, 139 Starvation states in fungal physiology, 47 Starving cells, effect of addition of substrate to energy charge of, 272 Stigmasterol, effect of, on growth of Phytophthora infestans, 142 Stigmasterol uptake by Phytophthora infestans, 135 Ste loci in Saccharomyces cerevisiae, 374 Stemphylium solani, effect of sterols on asexual reproduction in, 148 Sterile mutants of Saccharomyces cerevisiae, 372

Sterile mutants of Saccharomyces cerevzszae, genetic analysis of, 374 Steroids, functions of, in living organisms, 127 Sterol esters, extraction of, from fungi, 138

Sterol esters in low-density vesicles of yeast, 137 Sterol, membrane, source of, in yeast, 138

Sterol methyl transferase activities in nvstatin-resistant veast mutants. 147

443

SUBJECT INDEX

Sterol requirement for anaerobic growth of Saccharomyces cereuisiae, 146 Sterol-requiring mutants of yeast, 144 Sterol structure of nystatin-resistant mutants, 147 Sterol synthesis, effect of inhibitors of., on yeast metabolism, 147 Sterol synthesis in Ustilago maydis, effect of triarimol on, 148 Sterols and sexual reproduction in fungi, 162

Sterols, effect on, of asexual reproduction in fungi, 148 effect of, on fungal metabolism, 14 1 on reproduction in heterothallic fungi, 160 functions of in cells, 127 functions of in fungi, 123 fungal, lists of, 124 Sterols in fungi, 12 1 Sterols in model systems, 130 Sterols, pools of, in the yeast cell, 140 Sterols, subcellular distribution of, in fungi, 135 Structural role for membrane adenosine triphosphatase, 227 Structured models of microbial growth, 5 Subcellular distribution of sterols in fungi, 135 Subcellular structures in halophiles, nature of, 9 1 Substrates, changes in, induced by fungi, 27

Succinate as an electron donor in proline transport into membrane vesicles of Escherichia coli, 198 Succinate dehydrogenase, effect of aeration of anaerobically-grown yeast on, 146 Succinate oxidation by bacterial membrane vesicles, 185 Succinate,synthesisin fungi, 63 Succinate transport into Bacillus subtilis, 2 14

Sucrose assimilation by Clauiceps purpurea, 54 Sugar phosphates in bacterial sugar transport, 193 Sugar transport systems into bacterial membrane vesicles, 2 12 Sulphate transport into fungi, 55

Sulphhydryl groups in the lac carrier protein, 24 1 Surface filaments and cellular recognition in Saccharomyces cerevisiae, 364 Surface filaments and conjugation in Hansenula wingei, 32 1 Surface filaments in Schizosaccharomyces pombe, 340 Survival at extreme temperatures, effect of sterols on fungal, 142 Survival, radiation, in Saccharomyces cereuisiae, 382 Synthesis and utilization of adenine nucleotides, balance between, 258

T Temperature phages in halobacteria, 114 Temperature, effect of, on fungal colony expansion, 33 effect of, o n growth of fungal conidia, 14

on hyphal growth in fungi, 20 on microbial molar growth yields, 26 1

on synthesis of glycerol kinase in Neurospora crassa, 54 incubation, effect of, on ribosome eficiency, 73 Tension, oxygen, effect of, on fungal colony growth, 28 Terminal oxidation of substrates by fungi, 67 Testosterone, effect of, on Saccharomyces cereuisiae, 164 Testosterone transport in bacterial membrane vesicles, 19 1 Tetrahymena pyrformis, presence of tetrahymenol in, 1.30 Tetrahymenol, as a substitute for sterols, 130

Tetraploid meiosis in Schizosaccharomyces pombe, 3 1 1 Thallus, fungal, colonization by, 14 Thermophilic nature of some halophilic enzymes, 103 Thiobacillus neopolitanus, transport coupled to electron transfer in vesicles of, 206

444

SUBJECT INDEX

Thraustotheca spp., effect of antheridiol on, 149 Three-constant growth m o d d for microorganisms, 6 Threonine deaminase, properties from halobacteria, 103 Toluene, effect of, on phosphofructokinase activity of Escherichia coli,

Triphenylmethylphosphonium, transport of into bacterial membrane vesicles, 236 Triploid formation in Hansenula wingei, 329

Triploid formation in Schizosaccharomyces pombe, 348 Trisporic acid, effect of, o n zygomycetes, 155 28 7 Torula utilis, electron- transport chain in, Triton, effect of, on halobacterial envelopes, 94 68 Transient GI arrest in mating ol'Saccharo- Tropical ecology of Schizosaccharomyces myces cereuisiae, 35 7 pombe, 309 Transient heterokaryosis in Saccharomyces Tryptophan, effect of, o n fungal hyphal cereuisiae, 3 I2 growth unit length, 22 Transient phenomena in fungal cultures, Tryptophan synthase, proteolytic inacti43 vation of, in Saccharomyces cereuisiae, Translocation of solutes through the 67 bacterial cytoplasmic membrane, Tryptophan transport into Neurospora 176

Transport across halobacterial membranes, 107 Transport, active, in bacterial membrane vesicles, 194 active, of solutes in bacterial membrane vesicles, 175 Transport-controlled features of fungal growth, 48 Transport coupled to electron transfer in bacterial vesicles, 204 Transport-limited growth of fungi, 48 Transport, membrane, effect of hormoneson, 127 Transport of amino acids into bacterial membrane vesicles, 209 Transport of sugars into bacterial membrane vesicles, 2 12 Transport processes, fungal, modulation of, 53 Transport regulation in fungi, 50 Transport, requirement of, energy for initial steps in bacteria, 240 Transfer-RNAs of halobacteria, 1 13 Triarimol, effect of, on sterol synthesis in Ustilago maydis, 148 Tricarboxylic acid cycle operation in fungi, 67 Trichoderma spp., ability of, to produce metabolites that induce oospore formation, 161 Trichoderma uiride, hyphal growth unit of, 20

crassa, 50

Tube, conjugation, formation of, in Hansenula wingei, 325 conjugation, in Saccharomyces cereuisiae, 366

Tube, conjugation, formation of, by Schizosaccharomycespombe, 340 Turnover of adenine nucleotides in bacteria and yeasts, 253 Turnover of adenosine triphosphate in microbes, 26 1 Turnover of ATP at different growth rates, 265 Turnover time of ATP in microbes, length of, 268 Twin meiosis in Schizosaccharomyces pombe, 31 1

U Ultraviolet radiation, effect of, on halobacteria, 1 1 1 Uncouplers, effect of, o n halobacteria, 110 Undifferentiated fungal mycelia, growth of, 13 Undifferentiated fungal mycelium, growth of, from spores, 1 7 Unsaturated fatty-acid requirement of anaerobically-grown Saccharomyces cerevisiae, 146 Unsegregated models of microbial growth, 5

SUBJECT INDEX

Unstructured models of microbial growth, 5 Urea, effect of, on envelopes of halobacteria, 94 Uridine transport into bacterial vesicles, 205 Ustilago may&, effect of triarimol on sterol synthesis in, 148

v Vacuolation in leading hyphae in fungal colonies, 24 Vacuoles from halobacteria, 96 Vacuoles, properties of, from halobacteria, 96 Valinomycin, effect of, on cation uptake by vesicles, 2 1 7 Valinomycin-mediated efflux of potassoim ions from bacterial membrane vesicles, 235 Vanadium, effect of, on sexual agglutination in Hansenula wingei, 328 Variations in adenine nucleotide levels during growth, 285 Vegetative growth of fungi, effect of sterols on, 141 Veillonella alcalescens, transport coupled to electron transfer in vesicles of, 206 transport into membrane vesicles of, 22 1

nitrate respiration in membrane vesicles of, 188 Verticillium albo-drum, effect of carbon dioxide on metabolism of, 6 3 Verticillium sp., hyphal growth unit of, 20

Vesicle, bacterial membrane, orientationof, 194 Vesicle formation from halobacteria, 92 Vesicles, bacterial membrane, active transport of solutes in, 175 bacterial membrane, isolation of, 176 halobacterial, use of, to study electron-transport chains, 107 effect ofantheridiol o n fungal, 15 1 Vesicles in shmoos of Saccharomyces cereuisiae, 361 Viability, microbial, and energy charge, 280

445

Viability of halobacteria, effect of light on, 110 Viscosity offungal liquid cultures, 37 Vitamin deficiency, effect of, on fungi, 5 1 Volume expansion of yeast in mating, 357

w Wall fragments, absence of, from bacterial membrane vesicles, 182

X- Ray- sensi tive mutants of Saccharomyces cerevisiae, 3 8 2

Y Yeast hormones, nature of, 164 Yeast-mycelium dimorphism in fungi, and sugar metabolism, 59 Yeast, oscillations in glycolytic behaviour of, 45 Yeasts, concentration and turnover of adenine nucleotides in, 253 operation of glutamine-oxoglutarate aminotransferase systems in, 57 physiology of mating in, 307 Yield factor in microbial growth, 6 Yield of adenosine triphosphate to be expected from a substrate, 261

Z Zearalenone, effects of, o n sexual reproduction in fungi, 163 Zearalenone, formula of, 163 Zygote formation by yeasts, 3 10 Zygote formation in Saccharomyces cereuisiae, 361 Zygote formation in Schirosacchromyces pombe, 337 Zygotic fission in Schizosaccharomyces pombe, 344 Zygotic budding in Hansenula wingei, 326 Zygotic budding in Saccharomyces cereuisiae, 3 7 1 Zymosterol, formula for, 124 Zymosterol, methylation of, 139

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