Advances in Microbial Physiology Volume 23

Advances in

MICROBIAL PHYSIOLOGY

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Advances in

MICROBIAL PHYSIOLOGY edited by

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

J. GARETH MORRIS Department of Botany and Microbiology University College of Wales A beryst wy th

Volume 23 1982

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British Library Cataloguing in Publication Data

Advances in microbial physiology.-Vol. I . Micro-organisms-Physiology 576'. 11'05 QR84 ISBN &12-027723-9 LCCCN 67-19850

Printed in Great Britain by Fletcher and Son Ltd, Norwich

23

Contributors P. BALL, Department of’ Microbiology, Medical School, University of Bristol, University Walk, Bristol BS8 1T D , England (present address: Pall Biomedical Ltd. Portsmouth, PO1 3PD, England) I. CHOPRA, Department of Microbiology, Medical School, University of Bristol, University Walk, Bristol BS8 1 T D , England J. H. DUFFUS, Department of’ Brewing and Biological Sciences, Heriot- Watt University, Edinburgh EH1 1H X , Scotland A. A. EDDY, Department of Biochemistry, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester M60 l Q D , England C. LEVI, Department of’ Brewing and Biological Sciences, Heriot- Watt University, Edinburgh EH1 l H X , Scotland D . J. MANNERS, Department of Brewing and Biological Sciences, HeriotWatt University, Edinburgh EHI 1H X , Scotland I. W. SUTHERLAND, Department of Microbiology, University of Edinburgh, Edinburgh EH8 9 YL, Scotland

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Contents Mechanisms of Solute Transport in Selected Eukaryotic Micro-Organisms by A. A. EDDY I. Kinetics and Energetics

.

11. Ion transport in Neurospora crassa

111.

IV.

V.

VI. VII. VIII.

.

A. ATP and electrogenesis . B. Enzymic basis of the plasma-membrane proton pump . Transport of sodium and potassium ions and protons in Saccharomyces cerevisiae and the enzymic basis of the proton pump in the yeast plasma membranes . A. Divalent ions . Amino-acid transport in yeast . A. Metabolic and physical compartments . B. Relation between amino-acid transport and metabolism . c. D-Asparagine assimilation . D. Gluthione and the y-glutamyl cycle. . E. Genetically distinct pathways of amino-acid absorption . F. Purine, pyrimidine and other yeast transport systems . G. Reversibility of solute transport: the problem of intracellular compartments . H. Transinhibition and transinactivation in feedback control of solute . uptake I. Role of the vacuolar compartment . . J. The phenomenon of shock excretion . K. Vacuolar transport systems . L. The mechanism of energy coupling during amino-acid transport in yeast . M. The extent of amino-acid concentration by yeast . N. Amino-acid efflux and the proton gradient hypothesis . 0. Gradient coupling and purine and pyrimidine transport in yeast P. Studies with yeast-membrane vesicles and amino acid-binding proteins . Carbohydrate transport in yeast . . A. Monosaccharides and the phosphorylation controversy . B. Monosaccharide transport in yeasts other than Saccharomyces cerevisiae . C. Proton symport in yeasts with monosaccharides, disaccharides and polyols' . D. Respiration-dependent proton symport in Rhodotorula gracilis . E. Other a-glucosides: intracellular or extracellular hydrolysis . . F. rx-Methylglucoside permeases of Saccharomyces spp. . . Glucose-transport systems of Neurospora crassa . The active hexose-uptake system of Chlorella vulgaris Phosphate and sulphate symports (Na' and H + ) . . A. Related effects of sodium ions .

2 7 9 11 12 16 17 17 18 19 19 20 22 24

25 27 29 30 31 37 38 38 40 42 42

46

47 50 53 53 55 57 60 61

viii

CONTENTS

IX. The steady state of solute accumulation . A. Special characteristics of the ‘slip’ models. . B. General conclusions about symport mechanisms References .

. . .

61 66 68 69

. .

Biosynthesis of Microbial Exopolysaccharides by I. W. SUTHERLAND I . Introduction: the range of microbial exopolysaccharides . . . . . A. Influence of culture medium, especially carbon substrate, nitrogen . source and ions . . B. Growth phase in batch culture . . C. Nutrient limitation in continuous culture studies . . 111. Precursors of polysaccharide . . A. Substrates . . B. Sugar nucleotides . . . C. Lipid intermediates . . IV. Control of polysaccharide production . . A. Regulatory mechanisms . . B. Changes in polysaccharides . . . V. Synthesis of polysaccharides by cell-free preparations . A. Dextrans, mutans and levans . . B. Cell-free synthesis of homopolysaccharides . . C. Cell-free synthesis of heteropolysaccharides . . VI. Relationship to other polysaccharides: shared pathways . . VII. Role of primers and secretion in polysaccharide production . . A. Primers or acceptors-are they necessary? . B. Secretion of polysaccharides . . VIII. Modification of polysaccharides . . . . A. Changes in physical properties of polysaccharides . B. Post-polymerization modification . IX. Acylation . . . A. Regular acylation on alternate repeating units . . . . B. Bacterial alginates . . . C. Xanthan-polysaccharides of Xunthomonas cumprstris . . D. General aspects of acetylation and ketalation . X. The future. . . . XI. Acknowledgements . . References . 11. Physiological aspects of polysaccharide production .

80 84 84 89 89 95 95 97 99 104 104 112 115 115 118 119 121 122 122 127 129 129 134 135 135 137 137 140 141 142 142

Yeast Cell-Wall Glucans by J. H. DUFFUS, CAROLYN LEV1 and D. J. MANNERS I . Introduction . 11. Structural analysis of yeast glucans . A. General methods . . B. Glucans in walls of Succhuromyces cerevisiue

.

151

. .

152 152 155

. .

CONTENTS

C. Glucans from other Saccharomyces species . D . Glucans in walls of Schizosaccharomyces pombe . E. Glucans from Candida species . F. Other yeast glucans . . 111. Yeast wall glucan synthesis . A. Introduction. . B. Studies with inhibitors of glucan synthesis . C. Glucan synthetases of whole and fractionated cells . D. Glucan synthesis and glucan synthetases in protoplasts IV. Physiological control of glucan content. . A. Introduction. . B. The cell cycle . C. Yeast-mycelium interconversion . D. Effects of nutrient limitation . . E. Effects of metabolic inhibitors . F. Miscellaneous . V. Acknowledgements . References .

ix

. . . . . . . . . .

. . . . .

. . .

160 162 162 165 166 166 166 167 169 171 171 171 174 174 176 177 177 178

Transport of Antibiotics into Bacteria by IAN CHOPRA and PETER BALL I. Introduction

.

11. Antibiotics: target sites and uptake . 111. Uptake and transport: some definitions.

. IV. Structure of bacterial cell envelopes in relation to antibiotic uptake: an overall picture . A. Capsules . B. Outer membranes . . C. Periplasmic space . . D. Peptidoglycans . E. Cytoplasmic or inner membranes . F. Summary . V. Aminoglycosides . A. Diffusion of aminoglycosides across the outer membrane . B. Accumulation of aminoglycosides across the cytoplasmic membrane VI. Chloramphenicol . A. Diffusion of chloramphenicol across the outer membrane . . B. Accumulation of chloramphenicol across the cytoplasmic mem. brane . VII. D-Cycloserine . A. Diffusion of D-cycloserine across the outer membrane . B. Transport of D-cycloserine across the cytoplasmic membrane . VIII. 3, 4-Dihydroxybutyl-I-phosphonate . A. Diffusion of DHBP across the outer membrane . B. Transport of DHBP across the cytoplasmic membrane . IX. Fosfomycin . A. Diffusion of fosfomycin across the outer membrane . . B. Transport of fosfomycin across the cytoplasmic membrane. . C. Transport of fosfomycin in bacteria other than Escherichiu coli .

184 185 185 188 188

188 191 191 192 193 194 194 195 200 200 200 20 1 203 203 203 204 204 205 205 208 208

CONTENTS

X

Beta-Lactams . Norjirimycin . Peptide antibiotics . Showdomycin . . A. Diffusion of showdomycin across the outer membrane B. Transport of showdomycin across the cytoplasmic membrane XIV. Sideromycins . XV. Streptozotocin . . A. Diffusion of streptozotocin across the outer membrane B. Transport of streptozotocin across the cytoplasmic membrane XVI. Tetracyclines . A. Diffusion of tetracyclines across the outer membrane . B. Transport of tetracyclines across the cytoplasmic membrane XVII. Conclusions . XVIII. Acknowledgements . References . X. XI. XII. XIII.

Author Index Subject Index

. .

. 209 . 212 . 213 . 216 . 217 . 217 . 220 . 222 . 223 . 223 . 225 . 225 . 230 . 233 . 234 . 234 24 1 255

Mechanisms of Solute Transport in Selected Eukaryotic Microorganisms A . A . EDDY Department of Biochemistry. University of Manchester Institute of Science and Technology. P.O. Box 88. Manchester M60 IQD. England . . . . . . . . . . . I . Kinetics and energetics I1. Ion transport in Neurospora crassa . . . . . . . . . A . ATP and electrogenesis . . . . . . . . . . . B . Enzymatic basis of the plasma-membrane proton pump . . . . 111. Transport of sodium and potassium ions and protons in Sacchuromyces cerevisiae and the enzymic basis of the proton pump in the yeast plasma membranes . . A . Divalent ions . . . . . . . . . . . . . . . . . . . . . IV. Amino-acid transport in yeast . A . Metabolic and physical compartments . . . . . . . . B . Relation between amino-acid transport and metabolism . . . . C . o-Asparagine assimilation . . . . . . . . . . D . Glutathione and the y-glutamyl cycle . . . . . . . . E . Genetically distinct pathways of amino-acid absorption . . . . F. Purine. pyrimidine and other yeast transport systems . . . . . G . Reversibility of solute transport: the problem of intracellular compartments H . Transinhibition and transinactivation in feedback control of solute uptake . I . Role of the vacuolar compartment . . . . . . . . J . The phenomenon of shock excretion . . . . . . . . K . Vacuolar transport systems . . . . . . . . . . L . The mechanism of energy coupling during amino-acid transport in yeast M . The extent of amino-acid concentration by yeast . . . . . . N . Amino-acid efflux and the proton gradient hypothesis . . . . . 0. Gradient coupling and purine and pyrimidine transport in yeast . . . . P. Studies with yeast-membrane vesicles and amino acid-binding proteins . V . Carbohydrate transport in yeast . . . . . . . . . . A . Monosaccharides and the phosphorylation controversy . . . . . . B. Monosaccharide transport in yeasts other than Saccharomyces cerevisiae C . Proton symport in yeasts with monosaccharides. disaccharides and polyols . . . D . Respiration-dependent proton symport in Rhodororula gracilis 1

2 7 9 11

12 16 17

17 18 19 19 20 22 24 25 27 29 30 31 37 38 38 40 42 42 46 47 50

2

A. A. EDDY

VI. VII. VIII.

IX.

E. Other rr-glucosides: intracellular or extracellular hydrolysis F. a-Methylglucoside permeases of Saccharomyces spp. . Glucose transport systems of Neurospora crussu . . . The active hexose-uptake system of Chlorellu vulgaris. . Phosphate and sulphate symports (Na' and H') . . A. Related effects of sodium ions . . . . . The steady state of solute accumulation . . . . A. Special characteristics of the 'slip' models . . . B. General conclusions about symport mechanisms . . References. . . . . . . . . .

. . .

. . .

. . .

. . .

.

.

.

.

. . . . . .

. . . . . .

. . . . . .

. . . . . .

53 53 55 57 60 61 61 66 68 69

1. Kinetics and Energetics

Yeasts belonging to the genera Saccharomyces and Rhodotorulu, other fungi such as Neurosporu crussu and Aspergillus niduluns and the green alga Chlorellu sp., have long been recognized as suitable experimental material for investigating the mechanisms and physiological roles of nutrient transport processes at the plasma membrane of micro-organisms. The relevant recent literature about some of the above systems, together with certain other examples, is the main subject of the present review, which emphasizes various aspects of the kinetics, energetics and genetic basis of solute transport. Progress on the biochemical side has been more limited, although recent work of this kind on fungal adenosine triphosphatase (ATPase) preparations is especially significant. Current notions about transport mechanisms in these systems have been greatly influenced by the growth of knowledge about nutrient transport in mammalian cells, on the one hand, and on the other by the study of similar behaviour in both bacteria and organelles such as mitochondria (Mitchell, 1967). This exchange of ideas between these related fields of study has undoubtedly been fruitful in providing analogies between different transport systems, in highlighting their differences and in emphasizing their various limitations as experimental material. The essential function of the plasma membrane, of separating cellular reactants from the environment under stabilized local conditions, is only achieved if the membrane itself is selectively permeable to ingoing and outgoing metabolites. The bulk phase of the plasma membrane, despite wide differences in composition from one cell type to another, is generally considered to be fairly permeable to water, carbon dioxide, molecular hydrogen and oxygen, and free ammonia. In contrast, ions such as NH4+, H 2 P 0 4 - , N a + , K + , MgZ+, all the common amino acids, carbohydrates such as glucose or mannitol, succinate, or citrate probably penetrate the membrane bulk phase with difficulty. The rate of diffusion of a wide range of organic compounds through the main body of the plasma membrane of various cells varies systematically with

MECHANISMS OF SOLUTE TRANSPORT

3

the chemical structure and molecular volume of the penetrating compound (Diamond and Wright, 1969). For instance, ethanol and unionized acetic acid or propionic acid appear to traverse the yeast cell membrane relatively fast (Conway and Downey, 1950), probably on account of their lipophilic character. However, the rapid utilization of acetate ions by yeast appears to involve a specific carrier mechanism (Barnett and Kornberg, 1960). Likewise, the passage of the several common nutrients listed above, to which the bulk of the plasma membrane is probably relatively impermeable, would seem to require the presence of a series of specific carriers or pores, selectively aiding the penetration of each nutrient. An analogous problem arises in connection with excretion of succinate or lactate by certain yeasts. It is widely assumed that the selectivity of such mechanisms is based on specific proteins. The relationship between transport and metabolism can be considered in the following general terms. (1) Does penetration of the solute into the cell occur as a process distinct from its chemical conversion by metabolism? For instance, does sugar uptake by yeast necessarily involve its concomitant phosphorylation (Section V.A, p. 42). The suggested role of glutathione in amino-acid transport leading initially to y-glutamyl dipeptides is a further example (Section IV.D, p. 19). (2) Given that the solute penetrates the cell as such, the mechanism might be 'passive', in the thermodynamic sense that at equilibrium it did not change the chemical potential ( p s ) of the transported species, moving from the solution outside the cell (p:) to the solution inside the cell ( p f ) . The chemical potential of the ionic species S, bearing z positive charges, in a given solution at constant temperature and pressure is conventionally defined as: ,us= ,if

+ R T In as + z FE

(1)

where ,if is the chemical potential of S in its standard reference state, as is its activity in solution, R is the gas constant, T the absolute temperature, F the Faraday and E the electrical potential of the solution relative to the standard state. The constancy of chemical potential thus corresponds to the condition that Aps = py - p: = 0 or:

+

RTln (asi/as,) zF(Ei - E,)

=0

(2)

where Ei - E, is the membrane potential, A$. A useful approximation is to replace activities by concentrations ([ S],, [SIi) giving: RTln ([S]i/[S],)

+ zFA$ = 0

(3) The values of the parameters in this equation are such that when [S]i/[S], is 10, A$ for a univalent cation is approximately - 59 mV at 25°C. Application of equation (3) in practice can be complicated (a) by the difficulty of preventing subsequent metabolism of S, and (b) by metabolic

4

A. A. EDDY

controls regulating the entry or egress of the molecule. Transport mechanisms which function so as to equilibrate the distribution of the transported solute, in the above sense, are said to involve facilitated diffusion. (3) However, in many systems, direct observation shows that p: # p? when a steady state is reached. In this event, the solute S, assumed to be metabolically inert, is said to undergo active transport. ( a ) p i > p:. As in case (2) above, rapid metabolism of S, once it had entered the cells, might obscure a tendency for S to become concentrated in the cells as it traversed the cell membrane. Maltose uptake into yeast is an example (Section V.C, p. 47). (6) p? < p i . In this case, the cells tend to exclude S as though an outwardly directed pump were operating. For instance, fungi take up N a + but also tend to exclude it in preference for K'. The outcome is that [cellular Na+] ([NaIi) is smaller than [extracellular Na+] ("a],) in a cell with a substantial negative membrane potential (Section 11, p. 7). An exit pump for Na+ has accordingly been postulated (Section 111, p. 12). In contrast, the fact that the glucose concentration in yeast fermenting high concentrations of this carbohydrate is relatively small has been attributed to its rapid metabolic transformation once it has entered the yeast cells by facilitated diffusion (Section V, p. 42). (4) The above thermodynamic criteria apply to the system as a whole and tell us little about the actual mechanisms involved. For instance, a carbohydrate that was phosphorylated as in case (1) above might subsequently be dephosphylated reforming the free sugar in the cellular pool, with pLf > p:. Such a system would qualify as an active-transport mechanism on the basis of a naive application of the above criterion. When such instances are excluded, two types of active transport have been recognized. In the first, primary active transport, the flow of solute as such across the cell membrane is coupled to an exergonic chemical reaction. In effect, the increase in free energy of S is derived from changes in chemical-bond energy associated with the reaction. Thus about 50 kJ mo1-l (12 kcal mol-') are available from hydrolysis of ATP in vivo which would be energetically equivalent, on the basis of equation (3), to a solute ratio of about lo9. Secondary active transporf is quite distinct, in that movement of S across the cell membrane is coupled, by a purely physical process, to the movement of some other specific particle S'. In the simplest case, one mole of S enters the cell only when one mole of S' leaves it:

so+ Sl = si + s:,

(4)

At equilibrium, as no covalent chemical bonds are made or broken: Aps = p; - p: = pi'

-

p:

(5)

In the terminology of Mitchell (1967), who greatly elaborated earlier suggestions (Riggs et al., 1958) of this kind, equation (4) defines an antiport

MECHANISMS OF SOLUTE TRANSPORT

5

relationship between S and S’. A symport relationship with n equivalents of S’ corresponds to: So + nSA*Si

+ nSf

(6)

The corresponding form of equation (5) is: - Aps = nAp”

(7) Clearly, the free-energy increment derived from coupling passage of S to that of n equivalents, as opposed to one equivalent of S’, is n times larger. The above examples assume that the coupling of the flows of S and S’, either by means of a symport or an antiport relationship, is strictly stoicheiometrical. Whether real systems behave in that way is problematical (Section IX, p. 61). (5) Discussion of the mechanism of solute transport, either of the passive type or that based on secondary active transport, is aided by carrier models. It is convenient, in the first instance, to regard these as mathematical models describing how a binding site for the substrate S alternately presents itself to the intracellular aqueous phase and the extracellular solution. The detailed study of one particular system, namely that catalysing facilitated diffusion of glucose into the human ,erythrocyte, has led to the recognition both of the extreme complexity of such mechanisms in practice and their likely molecular complexity (LeFevre, 1975; Holman, 1980). However, a general mathematical treatment (Regen and Morgan, 1964) shows that the net flux (J) through such a system, in which S carries no electrical charge, is of the form:

where A, B, C and D are explicit functions of the rate constants used in Fig. 1 , and the inward and outward fluxes are conveniently represented as J and f,respectively. Hence = % when [S], = [Sli, Aps being zero. The problem of describing the effect of the membrane potential A$ on the kinetics of the simple carrier is considered in an elementary way in Section 1V.N (p. 38). The effect of A$ on the equilibrium distribution of a charged solute is described by equation (3) in the form of the Nernst equation. Several studies have shown that the influx or efflux of S represented by J‘ and f, are frequently hyperbolic functions of [S], or [S]i respectively. It will be observed that the apparent K, value with respect to [S] and the corresponding V,,, value for these two fluxes vary with the trans concentration of [S]. Similar considerations are involved in the steady-state kinetic analysis of an active membrane-transport system, to which the scheme shown in Fig. 1 might also apply (Schachter, 1972; Cuppoletti and Segel, 1974, 1975a). In this instance, # fswhen [S], = [SIi; equation (8) then takes the form:

x

2

x

A. A. EDDY

6

ES [s]kcl

.3$

[slk +3

E FIG. 1. A simple kinetic model of a carrier. The membrane is regarded as two superficial phases separated by a bulk phase, through which diffuse or otherwise translocate the carrier E and its complex ES with the substrate. It is sometimes assumed that [S] and [El combine sufficiently rapidly to be in equilibrium with [ES] on the same side, the rate-determining steps in the transport of S then being simply passage of ES and E through the bulk phase of the membrane. However, this particular simplification is not an essential part of the scheme, which is shown here with separate rate constants assigned to the binding and dissociation steps as well.

where the various a and parameters are explicit but complex functions of the rate constants used in Fig. 1. The maximum accumulation ratio ( [ S ] i / [ S ] , )maintained by the system is cc,/az and is independent, therefore, of the actual magnitude of [ S ] , . It can be shown, further, that the separate fluxes and 's, are each hyperbolic functions of [S], and [S]i respectjvely. Let K L and V k , values be the Michaelis-Menten parameters fEr J, and KL and V i a , values be the corresponding parameters for the efflux J assayed in the absence of S in the trans compartment. Substitution in equation (9) then shows (e.g. Cuppoletti and Segel, 1974, 1975a; Heinz, 1978) that, when the distribution of the solute comes to equilibrium through the carrier:

Discussions in the literature of active solute transport (see Section IX, p. 6 1) have frequently been based on the notion that energy coupling serves to modulate the values of K , and V,,, appearing in equation (10) by specifically influencing the magnitude of one or more of the parameters representing the rates of the various steps in the sequence of reactions illustrated in Fig. 1. The importance of this type of approach is that it attempts to relate accessible kinetic parameters, K , and V,,,,,, to the ratio [ S ] i / [ S ] , . I defer until Section IX (p. 61) further consideration of equation (9) and Fig. 1 in terms of a more detailed model of secondary active transport. (6) The reader will observe several formal parallels between the above treat-

MECHANISMS OF SOLUTE TRANSPORT

7

ment and the brilliant expositions of the chemiosmotic interpretation of proton-dependent transport processes by Mitchell ( 1 967,1970,1977). Several recent reviews also cover this ground (Harold, 1977; Hamilton, 1977; Eddy, 1978; West, 1980). It is not, however, the purpose of this article to consider the chemiosmotic interpretation of the synthesis of ATP which takes place in the mitochondria and chloroplasts of eukaryotic micro-organisms. The evidence that is available indicates that the ionic pumps of the plasma membranes of these organisms are not involved in ATP synthesis. Thus the plasma membrane of yeast lacks a typical electron-transport chain, resembling in this respect the plasma membrane of anaerobic organisms such as Streptococcus faecalis rather than the plasma membrane of facultative aerobes like Escherichia coli which carry out oxidative phosphorylation. A further comparison concerns the extensively studied transport systems of mammalian cells which include the sodium pump based on an Na/K-ATPase (Ullrich, 1979; Crane, 1977; Eddy, 1977; Heinz and Geck, 1978). A wide range of such cells involve Na' ions in secondary active transport of various carbohydrates and amino acids. Similar coupled mechanisms are found in certain bacteria (Lanyi, 1979). Their occasional occurrence in eukaryotic micro-organisms is considered below.

11. Ion Transport in Neurospora crassa

The study of ion transport in Neurospora crassa has been especially significant in providing compelling evidence, based on classical electrophysiological techniques, for the presence of an electrogenic proton pump at the plasmalemma (Slayman, 1970; Gradmann et al., 1978).The giant algal cells of Nitella sp. also extrude protons electrogenically (MacRobbie, 1975; Raven, 1980) as do various plants (Smith and Raven, 1979). In contrast, Acetabularia sp. absorbs chloride ions electrogenically, a process which may represent the main component of A$ (Mummert and Gradmann, 1976). A technical limitation of the Neurospora sp. preparations needs to be borne in mind (Slayman et al., 1973). Whereas the micro-electrodes used for studying the membrane potential are implanted in hyphal preparations containing large mature cells of colonial origin, chemical measurements of ATP content, for instance, or assay of potassium fluxes are done on small cells from shaken liquid cultures. Typical shake-flask preparations of Neurospora sp. normally contain about 180 mM of K + and little Na'. However, cellular K + can be partly replaced by N a + when extracellular [K'] is lowered below 0 . 3 m ~in the presence of excess N a + . Such preparations extrude N a + rapidly when extracellular [K'] is raised again. Subsequent uptake of K + exceeds the loss of Na', charge balance being maintained

8

A. A. EDDY

by the simultaneous extrusion of hydrogen ions (Slayman and Slayman, 1968). The K + content of the cells eventually reaches a dynamic steady state in which extracellular K + exchanges with intracellular K + (Slayman and Slayman, 1968). The apparent K,,, value for net K + uptake (10 mM) is larger than that for K + / K + exchange (1 mM). There are other interesting aspects of the kinetics, including a pH-dependence indicating that two binding sites for K + are involved in its uptake (Slayman and Slayman, 1970). An important aspect of the interpretation of the above observations concerns the effect of Ca 2+ on the membrane potential of the system. When Ca 2 + was present, the value for A$ was relatively insensitive to changes in [K'], the main contribution to the potential being made by a process

Time (minutes) External potassium concentration (mM)

0.1

0.3

I

3

I

10

30 1

w- -160 -

0 .&

c a, +

x -200 -240L

FIG. 2. Factors influencing the membrane potential of hyphae of Neurospora crassa (Slayman, 1970). In a, a voltage record from a single hypha is shown. Azide (0.1 mM or 1 mM) was introduced at time indicated by arrow 1 and washed away at arrow 2.

The experimentwas repeated about 10 minutes later. The presence of azide depolarized the cell membrane by about 0.2V. In b, the membrane potential is shown as a function of external potassium ion concentration ([K+],). In the presence (0)of ImM-CaCI,, the internal potential (A$) was little affected by changes in the external potassium ion concentration, whereas in the absence ( 0 ) of CaZ a systematic dependence on this concentration was shown. +

MECHANISMS OF SOLUTE TRANSPORT

9

that was rapidly blocked by azide (Fig. 2) and by oxygen deprivation or by cyanide. This behaviour and a great deal of other evidence (Gradmann et af., 1978) favours the view that the main contribution to the membrane potential in the presence of C a 2 + is made by an electrogenic proton pump, rather than by the ionic diffusion potential associated with the transmembrane gradient of K'. Indeed, (1) the measured membrane potential of - 160 to - 240 mV greatly exceeds the K + equilibrium potential of about - 50 mV which applies when the fungi are immersed in 10 mM K + (see Slayman, 1965 a, b) and (2) A$ is little affected by the magnitude of the intracellular concentrations of K', Na' and NH: or by the presence of anions such as C1- (Slayman, 1977). Furthermore, the cellular pH in N . crassa is about pH 6.5, so that the spontaneous efflux of H + is evidently not the source of the elevated membrane potential. The bearing of these important observations on the mechanism of K + transport as such is problematical. Clearly, uptake of K + does not, in general, proceed to equilibrium with the membrane potential. However, the factors governing the steady state have not been determined. Slayman (1977) proposes that a large fraction of the H + ejection through the proton pump is neutralized by a process in which ( I ) K + and H + entry are coupled to the exit of either N a + or of K' when the cells lack Na' and (2) H + re-entry would be inhibited by lowering A$. This suggestion appears to be related to the possible presence of a K + / H + antiporter (Fig. 3) but clear evidence for such proton recycling seems to be lacking. The possibility of anion excretion associated with active proton efflux in the absence of K + is a relevant matter (Slayman and Slayman, 1970). So too is the possible effect of Ca2+ on the movements of Na' and K' which Slayman and Tatum (1965) studied in different conditions than were used for the membrane potential determinations on hyphae. There are certain circumstances in which the addition of K + to the hyphae caused a rather small and slow depolarization (Slayman, 1977). It is important to know whether K + is in fact then being absorbed. Interestingly, the addition of relatively small concentrations of NHd to the nitrogen-starved hyphae indicated that NH,f was absorbed electrogenically. The absorption of NH:, like the electrogenic absorption of H + with glucose, is presumably initially compensated by an electrogenic outflow of ions other than H + (Section IV.L, p. 31). In yeast, the compensating ion is K + (Eddy, 1978). This aspect needs to be borne in mind in considering the mechanism of K + transport in Neurospora.

A. A T P A N D E L E C T R O G E N E S I S

Study of the relation between the fall in values for A$ during oxygen depriva-

A. A. EDDY

10

4FP A-

ADP + Pi

K+, Na', Li+

H+

(proton conductor)

FIG. 3. Coupling of the ionic flows across the yeast plasma membrane. The tentative scheme relates: ( I ) the electrogenic proton pump which appears to be accelerated at low cellular pH values; (2) excretion of anions ( A - ) such as succinate (no details of the mechanism are known); (3) the postulated electrogenic absorption of K + , N a + or Li+; (4) the postulated electroneutral exchange of these ions with protons, which appears to be inhibited at low cellular pH values, and (5) the uptake of protons through a proton conductor such as dinitrophenol. Observations showing that these separate processes may occur are discussed in the text.

tion and the fall in cellular ATP content strongly indicates, together with other evidence, that the voltage generator is fueled by ATP (Slayman et af., 1973). A subtle analysis, based on a cable theory of the current voltage relationships of the plasma membrane, assayed by means of a three-electrode voltage clamp technique, led to the view that the extrapolated membrane potential required just to balance the proton current through the pump was about - 0.4V. As the total energy available from hydrolysis of ATP in vivo was estimated to be equivalent to about - 0.5V, Gradman et af. (1978) inferred that the pump ejected one hydrogen ion for each ATP hydrolysed. However, there were some grounds for thinking that the proton stoicheiometry is in fact variable and can rise, in certain circumstances of partial energy restriction, to two hydrogen ions per ATP with a reversal potential of -0.2V (Slayman et af., 1973; Slayman, 1980; Warncke and Slayman, 1980). Slayman (1980) concluded that these observations imply that the starved fungus uses about half as much ATP as the energy-replete cells in absorbing a nutrient such as glucose whose entry depends on the circulation of protons through the proton pump (Section VI, p. 55). The matter of

MECHANISMS OF SOLUTE TRANSPORT

11

the proton pump stoicheiometry is discussed in the wider context of other plant and algal systems by Smith and Raven (1979).

B. E N Z Y M I C B A S I S O F T H E P L A S M A - M E M B R A N E P R O T O N P U M P

Scarborough (1 976) devised an ingenious method for isolating closed plasmamembrane vesicles from N . crassa, a significant fraction of which contained an exposed ATPase activity. Such material is assumed to represent closed elements of the membrane with the cytoplasmic face outwards. In the presence of MgZ+ and ATP, the vesicles accumulated thiocyanate ions (CNS-) by a mechanism involving hydrolysis of ATP. This process was inhibited by proton conductors, as though an electrogenic proton transfer was involved. Furthermore, the fluorescence of 1-anilinonaphthalene-8-sulphonicacid was enhanced in the presence of ATP and the vesicles. Scarborough (1976) concluded that hydrolysis of ATP generated an electrical potential across the membrane of the everted vesicles with their interior positive relative to the solution. The natural orientation of the membrane would thus correspond to the normal sense of A$ in vivo. Recent work makes it likely that this electrogenic process drives accumulation of CaZ+ into the vesicles, possibly by a mechanism dependent on coupling of the electrogenic proton pump with an electroneutral Ca2+/2H+ exchanger (Stroobant and Scarborough, 1979). Such a system would serve to expel cellular C a 2 + in vivo (compare Fig. 3). The permeability of these vesicles to K + merits study. An important development has been to implicate protons as the charged species expelled by ATPase (Scarborough, 1980). In the presence of both MgZ+ and ATP and the permeant anion CNS-, the isolated plasmamembrane vesicles concentrated imidazole, a base which is believed easily to penetrate through the bulk phase of the membrane, whereas its conjugate protonated form fails to do so. Hydrolysis of ATP thus expelled protons into the vesicles, where the protonated base accumulated, to an extent indicating that the pH value there was about two units lower than that in the surrounding medium. The sensitivity of this process to orthovanadate, to diethylstilbestrol and to the presence of a proton conductor, its dependence on Mgz+ and ATP, together with other evidence excluding a likely role for C a z + , CI-, K + or N a + , indicated that the ATPase is probably the electrogenic proton pump. The plasma membrane Mg2+-ATPase is an integral membrane protein which is inhibited neither by oligomycin (a mitochondria1 ATPase inhibitor) nor ouabain (an inhibitor of N a + / K + ATPase). A number of salts, including K +,stimulate the activity. However, the physiological significance of this effect for K + transport is doubtful (Bowman el al., 1980). The matter is important because certain bacteria carry both a K+-activated ATPase, directly energizing uptake of K + and an

12

A. A. EDDY

electrogenic ATPase expelling protons (Brey et al., I Y80). Vanadate ions, which also depolarize the hyphae, diethylstilbestrol, or NN-dicyclohexylcarbodiimide each inhibit the fungal peripheral enzyme (Bowman and Slayman, 1979; Scarborough, 1977; Bowman et al., 1978, 1980) which differs in several respects, including substrate specificity and pH optimum, from the fungal mitochondrial ATPase (Bowman et al., 1978). Dame and Scarborough (1980) have recognized a single protein of about lo5 daltons which is inactivated by trypsin, in the absence of Mg2+ and ATP, with a marked loss of ATPase activity. They propose that hydrolysis of ATP proceeds by phosphorylation and dephosphorylation of this protein, which may accordingly more closely resemble the animal-cell transport ATPase systems involved in N a + / K + , Caz+ and K + / H + translocation (Sachs, 1977) than the mitochondrial, bacterial and chloroplast ATPases which appear not to involve a phosphorylated intermediate.

111. Trsnsport of Sodium and Potassium Ions and Protons in Succhuromyces

cerevisiae, and the Enzymic Basis of the Proton Pump in the Yeast Plasma Membrane Yeasts have long been known both to extrude protons or N a + , apparently against their electrochemical gradients, in exchange for K + and to absorb K + in preference to N a + (Conway and Brady, 1950; Rothstein and Enns, 1946; Rothstein, 1974). In the absence of extracellular K + , yeast cells excrete large amounts of H + together with bicarbonate and succinate (Conway et al., 1950). A related property is that cell preparations loaded with Na', and suspended in the absence of K + , excrete Na+, organic anions and bicarbonate (Conway et al., 1954).The mechanism of these complex processes is controversial. It has been analysed in terms of (1) two distinct systems regulated by the cellular pH (Ryan and Ryan, 1972), one involved in K + / N a + or K + / K + exchanges and the other involved in K + / H + exchange, or (2) in terms of a single system mediating the various exchanges (Rothstein, 1974). Conway and Downey (1950) suggested that the yeast plasma membrane contained vectorial redox pumps driving the efflux of protons by an electrogenic mechanism (see Conway, 1953). However, the apparent absence of redox components (Fuhrmann et al., 1974) from such membrane preparations is a major difficulty with this hypothesis. An especially important question is whether proton pumping is indeed electrogenic. It was early envisaged that the yeast plasma membrane might contain a distinct ATPase activity involved in solute transport (Eddy and Indge, 1962; Matile et al., 1967), but the matter has only received serious attention with the recognition of the role of ATP in electrogenic proton pumping in N .

MECHANISMS OF SOLUTE TRANSPORT

13

crassa. Recent studies confirm the presence of such an enzyme, with properties distinct from those of the mitochondria1 ATPase, in Schizosaccharomyces pombe (Delhez et al., 1977; Dufour et al., 1980) and Sacch. cerevisiae (Peters and Borst-Pauwels, 1979; Willsky, 1979; Serrano, 1980). Serrano (1 980) has shown that diethylstilbestrol and dicyclohexylcarbodiimide each inhibit both the plasma-membrane ATPase and proton pumping in a respiratory deficient yeast mutant. His observations are consistent with the view that the plasma-membrane Mg2+-ATPase corresponds to the proton pump in these cells. Foury and Goffeau (1975) reached a similar conclusion in work with the ATPase inhibitor Dio-9 and Schiz. pombe. Roon et al. (1978) have sounded a cautionary note about the interpretation of the actions of Dio-9, which can damage the plasma membrane causing release of cellular contents. It is possible that impurities present in some commercial preparations of the antibiotic are responsible for this effect (Dufour et al., 1980). The vanadate-sensitive plasma-membrane ATPase preparations undergo vanadate-sensitive phosphorylation in the presence of ATP, three phosphoprotein components being involved. These may be involved in the mechanism of the Mg2+-ATPase,although the matter has not been proved (Fuhrmann, 1977; Willsky, 1979; Malpartida and Serrano, 1980). Dufour and Goffeau (1980) have reported considerable progress towards the important objective of purifying and reconstituting the plasma membrane ATPase of Schiz. cerevisiae in defined lipids. Optimal expression of ATPase activity required the addition of fluid phospholipid micelles or liposomes. The longer term objective is to demonstrate, with characterized molecular species, the proposed proton movements that ATP hydrolysis causes. The molecular weight of the monomeric form of the enzyme is lo5. Dufour et al. (1980) have shown that the purified reconstituted enzyme was inhibited noncompetitively by N N-dicyclohexylcarbodiimide,competitively by the purified antibiotic Dio-9 and competitively by two synthetic drugs, octylaminolester and miconazole nitrate, among other compounds. Each of these four compounds also caused an efflux of K + and an equivalent influx of H + when added to a suspension of the yeast fermenting glucose. It was concluded that the plasma membrane ATPase controls these fluxes. The mechanism of these latter effects is not understood. Interpretation depends on knowing whether the inhibited ATPase allows protons to flow back into the yeast electrogenically and what other pathways through the plasma membrane are open for the passage of H + and K + , either as separate or linked processes (see p. 15). What is the relationship of the uptake and efflux of K + to the putative electrogenic proton pump? Peiia (1975) has shown that, in the presence of a proton conductor, Rb' (a substitute for K') can flow into the yeast in response to the outflow of H + through the proton conductor and he

14

A. A. EDDY

gave some reasons for thinking that ATP as such was not involved in the exchange. Furthermore, Seaston et al. (1976) showed that Sacch. carlsbergensis cells thoroughly depleted of ATP lose K + in exchange for H + when these ions are absorbed through a proton conductor (Eddy, 1978).These processes are illustrated in Fig. 3 which includes the possibility of an electrogenic uptake or efflux of K + as envisaged by several authors (Riemersma and Alsbach, 1974; Pefia, 1975; Seaton et al., 1976; Foury et al., 1977; Hoeberichts et al., 1980). Preparations of yeast plasma membrane vesicles appeared to be permeable to K + but whether the process was electrogenic was not established (Fuhrmann et al., 1976). A further part of the scheme shown in Fig. 3 is the proposed direct exchange (antiport) between cellular Na+ and extracellular H + (Eddy, 1978) and, in view of the work of Rodriguez-Navarro and Sancho (1979) between cellular K and extracellular H + . The Spanish workers have demonstrated these overall processes in Mg2'-depleted yeast and hinted that they are (1) independent of phosphate-bond energy, and (2) markedly slowed by lowering the cellular pH value from 6.6 to 5 . 5 (see also Ryan and Ryan, 1972). Dufour et al. (1980) have proposed that MgZ+ deprivation might permit proton uptake through the ATPase, a possibility that cannot be ignored altogether but implies a degree of deficiency of Mg2+ that would stop fermentation among other processes. Rodriguez-Navarro and Asensio (1 977) considered in an earlier study of the factors controlling uptake of Li+ by yeast that, as in the case of N a + (Conway et al., 1954; Conway and Duggan, 1958), the distribution of Li+ depended on two opposing processes, namely (1) an electrogenic absorption of the ion, in competition with K + , through the K+-carrier and (2) subsequent ejection of Li+ through the Na+-pump. As Rodriguez-Navarro and Asensio (1977) noted, somewhat similar ideas have been put forward for Escherichia coli, in which ejection of Na+ probably depends on a N a + / H + antiporter (see Brey et al., 1980, for a recent account) and may be relevant to the behaviour of K + as well. Raven (1980) has also considered some of the possible functions of a N a + / H + antiporter and K + / H + antiporter in species of Chlorella and Scenedesmus. In Fig. 3 , efflux of Na+ or Li' reaches equilibrium through the antiporter with the pH distribution across the cell membrane when (equation 5, Section I, p. 4): +

[H]o/[H]i = [Nalo/[Nali

( 1 1)

At the typical extracellular pH value of 4.5, the cellular pH value is near 6.1 (Eddy et al., 1980). Thus the antiporter mechanism would normally tend to exclude Li+ or Na' from yeast cells. In contrast, the electrogenic uptake of Na+ or Li+ through the K+-carrier would tend to concentrate these cations in the yeast, equilibrium being reached when (equation 3, Section 1, p. 3):

15

MECHANISMS OF SOLUTE TRANSPORT

[Na]i/[Na], = e-A*F'RT

(12)

The latter exponential factor may be as large as lo3 judging from the maximum gradient of K + that yeast cells form (Eddy et al., 1980). The outcome of the competition between these two processes, respectively concentrating and excluding Na' or Li+, would depend on their relative rates; these would be governed, among other factors, by the cellular pH value (Ryan and Ryan, 1972; Theuvenet et al., 1977; Rodriguez-Navarro and Sancho, 1979). The evidence these workers obtained implies, in terms of the above hypothesis, that a low cellular pH value tends both to inhibit entry of H + linked to expulsion of N a + and to accelerate restoration of the cellular pH value by increasing the rate of proton ejection through the proton pump. These ideas can be extended to the behaviour of yeast towards K + and the relationship of the K + distribution ([K+]i/[K+],) to A$. Clearly, the mechanism illustrated in Fig. 3 requires that when the cellular pH value is greater than the extracellular value [K]i/[K]o < e-A*F'RT.Conway (1960) tentatively estimated that the A$ value was - 0.07V in his yeast preparations, on the basis of a limited study with micro-electrodes. Direct evidence is otherwise lacking. However, systematic work with N . crassa indicates that the K + distribution is unlikely to be in equilibrium with A$, especially when [K], is large, and that voltages of at least - 0.2V might be found in yeast. Indeed, Eddy et al. (1980), as other investigators have also found, observed a maximum ratio of about 2. lo3 for the K + distribution when [K'], was 0.08 mM and the cellular pH value was about 6. The K + distribution thus corresponded to a predicted membrane potential in the order of at least - 0.2V. It may be relevant that methylamine is also concentrated by about a factor of lo3 (Roon et al., 1975a) but whether this is simply in response to the membrane potential is not known (Roon et al., 1977). It is tempting to suggest that, under the above conditions, the K + / H antiporter functioned slowly and K + distribution was determined mainly by the A$ value. Raising [K'], to, for instance, 10 mM might lead to: (1) a faster net uptake of K + , through the electrogenic route; (2) expulsion of protons and therefore an increase in the cellular pH value (Rothstein, 1960); and (3) an appropriate non-electrogenic efflux of K + through the antiport mechanism, associated with acceleration of the proton pump. As the latter process is electrogenic, the net consequence might be that, although [K+]i/[K+], was no more than about 20, the A$ value was not lowered very much below the value found when [K'], was 0.1 mM. Recent work by Barts et al. (1980) supports the notion that, at large concentrations of K + , the A$ value is much larger than the potential corresponding to K + distribution. These workers used the distribution of the lipophilic cation dibenzyldimethylammonium as an estimate of A$ value. Interestingly they found that, in their preparations, contrary to previous views, it and possibly +

16

A. A. EDDY

other such lipophilic cations were more rapidly absorbed through a physiological transport system of the yeast, namely the thiamin carrier, than through the bulk membrane phase. Thiamin itself appears to be concentrated, at least by certain yeast strains, by a factor of about lo4 (Iwashima et al., 1975). Barts et al. (1980) suggest that the lipophilic cation distribution nevertheless reflects the membrane potential in a way that is at least qualitatively correct. The A$ value at 1 mM K + appeared to be - 0.1 1V falling to about - 0.07V at 100 mM K + . As cellular K + was presumably in the range of 0 . 1 - 0 . 2 ~ ~ these observations suggest that the distribution of K + was near equilibrium with the A$ value at the lowest concentration studied. It is nevertheless clear that the estimates of A$ near 0.1V are much too small to maintain the maximum gradients of K + that have been reported in the literature. Further study of this matter is evidently required. A very different approach to understanding interactions between the transport of Na+, K + and other monovalent ions has involved detailed kinetic analysis of the initial rate of uptake of a given alkali cation in the presence of a selected concentration of a second such cation. Earlier work by Armstrong and Rothstein (1967) suggested that H + and the alkali-metal cations combine with two sites, one of which was a transport site and the other a site that modified the activity of the first. Subsequent studies by Borst-Pauwels and his colleagues have greatly extended these findings and have led to a scheme in which at least two independent sites on a carrier or pore (Theuvenet et al., 1977) participate in the translocation process (Derks and Borst-Pauwels, 1980). A two-site translocation mechanism leads to a quadratic rate equation which can be distinguished by various subtle criteria from two separate single-site carrier mechanisms (Borst-Pauwels, 1976) and from the influence of surface charge on simpler kinetic models (Theuvenet and Borst-Pauwels, 1976). No direct connection between these models and the overall processes of ion exchange in yeast can yet be made, but the possibility that the antiport function (K+/H+) and the electrogenic translocation of K + (Fig. 3) occur through the same molecular complex does not appear to be excluded.

A. D I V A L E N T I O N S

Various yeasts absorb Mg2+, C o 2 + , M n Z + and C a 2 + , from dilute solutions of these ions at about pH 5, by an energy-dependent mechanism which interacts in a complicated way with both K + and H,PO;(Rothstein, 1960; Boutry et al., 1977; Peiia, 1978; Roomans et al., 1979a). Other divalent cations are also absorbed, the relative affinities being Mg2+> Zn2+ > Mn2+> C a 2 +> Sr2+ (Fuhrmann and Rothstein, 1968a, b). Conway and Beary (1958) detected a second system for absorbing Mg2+ at higher pH

MECHANISMS OF SOLUTE TRANSPORT

17

values which is antagonized by K + and exhibits a very low affinity for Mg2+. The high-affinity physiological Mg2 carrier is activated by allowing the yeast first to absorb phosphate. During uptake of Zn2 +,Co2 and Ni2+,electroneutrality was maintained by excretion of either two equivalents of K or Na (Fuhrmann and Rothstein, 1968a, b; see, however, Norris and Kelly, 1977). Certain of the stimulatory effects of phosphate on uptake of Mg2 and M n Z + may be related to the fact that these cations are stored in cellular vacuoles (Okorokov et al., 1977) which are known to contain large amounts of polyphosphate (Indge, 1968). Boutry et al. (1977), Peiia (1978) and Roomans et al. (1979a) have argued that uptake of Ca2 increases with the magnitude of the membrane potential and that depolarization of the cells, in the presence of K + or during phosphate absorption, accounts for the slower uptake of Ca2+ in these conditions. Further progress may depend on the study of these complex processes at a subcellular level, where a start has now been made by Fuhrmann (1977). +

+

+

+

+

+

IV. Amino-Acid Transport in Yeast A. M E T A B O L I C A N D P H Y S I C A L C O M P A R T M E N T S

The size of the amino acid pool in Sacch. cervisiae varies over a wide range from about 0.2-2.0 pmol (mg yeast dry weight)-'. A recent study by Watson (1976) showed that cultures growing in a chemostat, under conditions where glutamate was the limiting source of nitrogen, contained 0.06 pmol mg- ' of free amino acids at a specific growth rate of 0.04h-', and 0.6 pmol mg-' at a growth rate of 0.36h-'. Glutamate comprised almost half of the pool, irrespective of the nitrogen source, in keeping with its central metabolic role. When one of a series of 19 amino acids was used as the sole source of nitrogen, that amino acid was either the major pool component or the most abpndant constituent next to glutamate. This behaviour during growth contrasts with the marked tendency of nitrogen-starved preparations of yeast rapidly to deaminate exogenous hydrophobic amino acids, such as tyrosine or isoleucine, when glucose is present. The carbon skeletons of the amino acids accumulate in the medium as keto acids and fusel-oil derivatives, a circumstance that might give the superficial impression that the 14C-labelled amino acids were not absorbed by the yeast (Woodward and Cirillo, 1977). Another relevant factor, where amino-acid uptake is assayed with radioisotopes, is endogenous synthesis of amino acids. For instance, during nitrogen starvation, Watson (1976) observed that the concentrations of aspartate and ornithine rose while other amino acids were depleted. Similarly, Jones et al. (1969) found that, during the first 12 hours of fermentation of a complex nutrient solution

18

A. A. EDDY

containing [14C]~-glutamate and other amino acids, only about 40% of the glutamate residues in the yeast protein were derived directly from that source. Moreover, the nitrogen atoms and carbon atoms of glutamate were metabolized by different pathways, as only 17% of the nitrogen atoms in the glutamyl residues of the protein were derived from added [lSN]~-glutamate. Further discussion of these interesting matters is outside the scope of the present article, but one related issue is directly relevant to the mechanism of amino-acid transport in yeast, namely the role of cellular compartments. For instance, Wipf and Leisinger (1977) and Jauniaux et al. (1978) concluded that (1) anabolic formation of ornithine from glutamate occurred in the yeast mitochondrion; (2) arginase degraded arginine to ornithine in the cytosol; and (3) arginine itself was synthesized from ornithine in the cytosol, with citrulline as an intermediate. Wiemken and Durr (1974) had previously concluded that most of the cellular complement of ornithine, citrulline and arginine was segregated in the large central vacuole of the yeast cells and was metabolized relatively slowly in comparison with the smaller pool engaged in the above reactions. It followed that transfer of these amino acids across the vacuolar membrane was an important aspect of the kinetics of uptake of these amino acids from the medium (Wiemken and Durr, 1974; Boller et al., 1975). Cowie and McClure (1959) earlier concluded that free amino acids of Candida utilis were contained in two functionally distinct pools, namely a concentrating expandable pool, fed from the surrounding medium, and a smaller conversion pool in which interconversions of the amino acids occurred and amino acids were synthesized from sugar and N H 4 + (Halvorson and Cowie, 1960). Protein synthesis appeared to involve the amino acids of the conversion pool (Cowie and McClure, 1959) although, paradoxically, exogenous phenylalanine was preferentially used for this purpose by Sacch. cerevisiae (Halvorson and Cowie, 1960). As Subramanian et al. (1973) have noted, the interpretation of this very complex behaviour is problematical. A particular difficulty is that Halvorson and Cowie (1960) describe the expandable pool as being extracted by cold water, whereas the common experience of earlier and subsequent workers in this field is that yeast cells retain absorbed amino acids tenaciously (see, for instance, Seaston et al., 1976). It is possible that this role for an expandable pool relates to a physiological artefact, the phenomenon of shock excretion (Section IV.J, p. 29). A further complication is that the kinetics of phenylalanine uptake and efflux described by Halvorson and Cowie (1960) are likely to have been influenced by its metabolic degradation (Woodward and Cirillo, 1977).

B. R E L A T I O N B E T W E E N A M I N O - A C I D T R A N S P O R T A N D M E T A B O L I S M

Here we consider aspects other than provision of ATP or ionic gradients

MECHANISMS OF SOLUTE TRANSPORT

19

(Section IV.L, p. 3 1). As already noted, certain amino acids, including valine and tyrosine, are rapidly absorbed and, in the presence of glucose, the carbon chain is excreted into the medium. During growth on glutamate, large amounts of 2-oxoglutarate are excreted in certain circumstances for somewhat similar reasons (Lewis and Rainbow, 1963). In all of these examples, it is commonly assumed that the amino acid is absorbed and possibly concentrated as such through a permease, or carrier, and then undergoes metabolic conversion. A clear exception is D-asparagine.

C.

D-ASPARAGINE

ASSIMILATION

Certain yeasts contain a constitutive L-asparaginase, they neither grow on D-asparagine as a sole source of nitrogen, nor do the whole cells hydrolyse the amino acid. Utilization of L-asparagine appears to involve its absorption and subsequent intracellular hydrolysis. Other yeast strains use D-asparagine slowly as a sole source of nitrogen for growth. They hydrolyse both the D- and the L-amino acid, releasing NH4+ and aspartate, in a manner indicating that a second asparaginase is located outside the permeability barrier represented by the plasma membrane. This asparaginase is a glycoprotein, like external invertase and acid phosphatase of yeast (Dunlop et a[., 1976; Pauling and Jones, 1980). Growth on D-asparagine is relatively slow because the D-aspartate released inhibits growth once it is absorbed through the general amino-acid permease (Rykta, 1975). Thus mutants lacking this permease grow faster on D-asparagine (Dunlop et al., 1976). There appears to be no evidence that D-asparaginase serves a transport function; the two products of its action are released into the medium and may then be absorbed.

D. G L U T A T H I O N E A N D T H E y - G L U T A M Y L C Y C L E

A very different view of amino-acid transport than is implicit in the chemiosmotic mechanisms (Section IV.L, p. 31) is due to Meister (see Meister and Tate, 1976). The basic idea is that amino-acid transfer across the plasma membrane may involve transfer of the y-glutamyl group of intracellular glutathione (y-glutamylcysteinylglycine) to an amino-acid or peptide acceptor, initially located outside the cell, with formation of intraceilular cysteinylglycine and the transpeptidation product which, in the course of the reaction, becomes displaced into the cells. The enzyme responsible for the transpeptidation reaction is y-glutamyltranspeptidase, a well-defined enzyme which is associated with the brush border of the kidney proximal tubule and with other mammalian-cell membranes involved in amino acid

20

A. A. EDDY

transport. Subsequent steps in the proposed y-glutamyl cycle involve firstly conversion of the y-glutamyl amino-acid derivative into 5-oxoproline and the free (absorbed) amino acid, a process catalysed by y-glutamylcyclotransferase. A further set of reactions would regenerate glutathione itself. Yeast contains relatively large amounts ( 5 pmol g-l) of glutathione and also contains the enzyme glutathione synthetase catalysing condensation of y-glutamylcysteine with glycine in the presence of ATP. Yeast also contains y-glutamyl transpeptidase, y-glutamylcyclotransferase, dipeptidase and 5oxoprolinase (Mooz and Wigglesworth, 1976). It is therefore feasible that the y-glutamyl cycle functions in yeast (Mooz and Wigglesworth, 1976; Penninckx et al., 1980). However, there have as yet been no convincing arguments that the cycle functions in vivo to translocate amino acids either in yeast or other systems. Nevertheless, highly circumstantial evidence relates aspects of the cycle to amino-acid absorption. Firstly, Mooz (1979) observed that a yeast lacking the general amino-acid permease contained a smaller amount of glutathione than did the wild-type strain and lower amounts of glutathione synthetase and y-glutamylcysteine synthetase. Secondly, Penninckx et al. (1980) found that the y-glutamyltranspeptidase activity of the above series of yeasts was repressed by NH4+ and unaffected by mutations leading to loss of the general amino-acid permease, or of either the specific lysine permease or the arginine permease. However, the apfmutation, which lowers the activity of several amino-acid permeases, lowered the y-glutamyltranspeptidase activity. Penninckx et al. (1980) accordingly suggested that the transpeptidase might be a common element involved in a group translocation mechanism for the amino acids. Clearly, none of these observations establishes a direct relationship between the enzyme and amino-acid transport. A recent attempt to relate amino-acid uptake to glutathione turnover suggests, in contradiction to an earlier claim, that the hypothetical y-glutamyl cycle accounts for an insignificant fraction of the uptake (Robins and Davies, 1980). Furthermore, the major routes for amino-acid absorption by yeast appear to be energized by linkage to the stoicheiometrical uptake of protons (Section IV.L, p. 31) in a manner that seems irrelevant to the current formulation of the y-glutamyl cycle in which the reactions are supposed to be maintained by a supply of ATP.

E. G E N E T I C A L L Y D I S T I N C T P A T H W A Y S OF A M I N O - A C I D A B S O R P T I O N

Work by Grenson and her collaborators in Belgium first clarified the genetic basis of amino-acid uptake in a series of strains based on the wild-type strain Z1278b of Sacch. cerevisiae. Earlier workers had noted the wide range of amino acids that yeasts absorbed and the fact that these competed with each other for entry (Halvorson and Cohen, 1958; Surdin et al., 1965). Indeed

MECHANISMS OF SOLUTE TRANSPORT

21

Surdin et al. (1965) detected a recessive allele (aap)in another family of yeast strains which lowered the rate of uptake of all the amino acids, as though these entered through a common system (permease). A similar locus ( a p f ) was subsequently detected in the Belgian strains (Grenson and Hennaut, 1971). A general amino-acid permease, handling a wide range of common amino acids except for proline, had meanwhile initially been recognized in the yeasts based on strain C1278b by the fact that the activity of the permease was quite repressed during growth with N H 4 + as sole source of nitrogen (Grenson and Hou, 1972). Derepression of the general permease occurred during growth with either proline or glutamate as the sole source of nitrogen, the rate of uptake of many amino acids increasing by a factor of at least 10 (Grenson et al., 1970). Repression of the general amino-acid permease by ammonia is relaxed in mutants (gdh-A) lacking the anabolic NADP+linked glutamic dehydrogenase activity (Grenson and Hou, 1972; Roon et al., 1975b) when nitrogen catabolite repression of various other functions, such as synthesis of arginase, is also abolished (Dubois et al., 1974). Another gene amc has been recognized as conferring NH, +-sensitivity on the general amino-acid permease (Rytka, 1975). Derepression of the general amino-acid permease also occurs when yeast grown in the presence of NH4+ is subsequently starved of nitr0ge.n in the presence of glucose (see, for instance, Indge et al., 1977). A similar treatment, or growth with a nitrogen source other than N H 4 + such as urea or proline, enhances the activity of the proline permease of Sacch. cerevisiae (Grenson et al., 1970; Brandriss and Magasanik, 1979), the proline permease of Sacch. chevalieri (Magaiia-Schwenke et al., 1973) and one of the glutamate permeases of the Belgian yeast series (Darte and Grenson, 1975). Studies with selective inhibitors suggest that derepression of the proline permease by nitrogen starvation involves biosynthesis of a specific mRNA (Kuznar et al., 1973). The mechanism of NH,' regulation in fungi has been extensively investigated and Pateman et al. (1973) have suggested that NADP'-glutamate dehydrogenase serves two distinct regulatory functions. One involves combination of the enzyme at a special site in the membrane which senses the extracellular concentration of NH,' . The other function is based on a combination of the enzyme with intracellular NH,'. The hypothesis is designed to explain the behaviour of a series of mutants affecting different enzyme and uptake systems in Asperxiflus nidulans. When the wild-type yeast of the Belgian series was grown with NH,' as a source of nitrogen, amino-acid uptake depended on various permeases that are not subject to nitrogen catabolite repression. For instance, lysine uptake under these conditions exhibited two kinetic components in the concentration range up to 1 mM. Arginine inhibited uptake of lysine by one of these components, but not by the other. A mutant strain (arg-p) was accordingly selected by virtue of its resistance to L-canavanine, an arginine

22

A. A. EDDY

antagonist. This strain failed to absorb arginine or lysine by their shared route but absorbed lysine through the second of the above routes (Grenson, 1966). A similar approach involving the lysine antagonist, L-thiosine, was used to isolate the mutant lys-p, gene, governing this second mode of entry of lysine. Two specific permeases each for methionine and histidine have been recognized, and two for dicarboxylic amino acids (Table I). When uptake of a series of amino acids is studied in the absence of N H 4 + , as compared with its presence, there is in general not only a large increase in the rate of amino-acid uptake but also an apparent loss of specificity of the system. This is because many more ofthe common amino acids, including the D-enantiomorphs, can compete for the fast general permease formed in the absence of NH4+,whereas competition for each slower specific permease is restricted to a small group of amino acids (Grenson et al., 1970). Methylamine and NH4+ also share a common concentrative permease in Sacch. cerevisiae (Roon et al., 1975a). Nitrogen starvation failed to increase the activity of this system. The transport systems listed in Table 1 refer to specific families of yeast strains. An interesting paper by Keenan and Rose (1979) shows that an unrelated strain of Sacch. cerevisiae absorbed arginine with an apparent K , value of about 20 p ~similar , to the value of 10 ~ L found M for the product of the arg-p gene, when the yeast lipids were enriched in oleyl residues. Both this system and a second arginine-transport system, with an apparent K , value of 21 mM with respect to arginine, exhibited quite different K , values when the cell lipids were enriched in the doubly unsaturated linoleyl residues. Keenan and Rose ( 1 979) proposed that the carrier systems responded to changes in the fluidity of the cell membrane.

F. P U R I N E . P Y R I M I D I N E A N D O T H E R

YEAST-TRANSPORT

SYSTEMS

Suggestions that purine transport in bacteria might occur by a grouptranslocation mechanism (Section I, p. 3) involving the purine phosphoribosyltransferases, led to similar work with yeast and other systems. Housset and Nagy (1977) observed that the rate of uptake of adenine and guanine by Schiz. pombe increased with the specific activity of their respective phosphoribosyltransferases (see Cummins and Mitchison, 1967). Mutant strains devoid of one or other enzyme nevertheless concentrated the respective purine though at a slower rate than the wild type, showing that a grouptranslocation mechanism was probably not involved. Moreover, in Sacch. cerevisiae and in C . albicans, adenine, cytosine and hypoxanthine share the same uptake system, namely cytosine permease, which is distinct from known enzymes involved in the metabolism of these substrates (Grenson, 1969;Polak and Grenson, 1973). A distinct guanine permease which also accepts

TABLE 1. Genetically defined yeast permeases for nitrogenous substrates

General amino acid permease

Gene

Representative substrates

Reference

gap

Various amino acids (not proline), D-amino acids, citrulline L-Lysine L-Arginine, L-lysine

Grenson et al. (l970), Rytka ( 1 975)

Lysine permease Arginine permease" Histidine permease" Methionine permease" Dicarboxylate permease"

his-p, met-p dic-p

,

L-Histidine L-Methionine L-Glutamate

Proline permease

L-Proline

Ammonia permease Urea permease" Allantoin permease

amt-l dur-3

A gene affecting the activity of several of the above systems Ammonia. methylamine Urea Allantoin

S-Adenosylmethionine permease Cytosine-purine permease

sump

Uracil permease Uridine permease

ura-p urid-p

dal-4 cyt-p

Adenosylmethionine Cytosine, adenine, hypoxan t hine Uracil Uridine

"Other systems exist but have not been characterized genetically.

Grenson ( 1 966) Grenson et a/. (1 966). see also Chan and Cossins (l976), Keenan and Rose (1979) Crabeel and Grenson (1970) Gits and Grenson (1967) Darte and Grenson (1979, Joiris and Grenson ( 1969) Seaston et al. (l973), Magaiia-Schwencke et ul. (1973). Brandriss and Magasanik (1979) Grenson and Hennaut (1971). Surdin et a/. ( 1965) Roon et u/. (l975a, 1977), Dunlop et al. (1976). Sumrada et al. (1976) Sumrada and Cooper (l977), Sumrada et al. (1978). Turoscy and Cooper (1979) Petrotta-Simpson et a/. (1975) Grenson (l969), Polak and Grenson (l973), Chevallier et al. (1975) Grenson (l969), Jund et al. (1977) Grenson ( 1 969)

24

A. A. EDDY

hypoxanthine has been proposed (Pickering and Woods, 1972). Mutant alleles governing the cytosine permease (cyt-p), a uracil permease (ura-p) and a uridine permease (urid-p) have been described (Grenson, 1969). The dependence of the rate of uptake of uracil, uridine, adenine or guanine on their utilization appears to be due to feedback inhibition of the individual permease by the absorbed substrates (Cummins and Mitchison, 1967) rather than to utilization of the substrates being part of the transfer mechanism taking these compounds into the yeasts. Several other transport systems are listed in Table 1 and are considered further below.

G . R E V E R S I B I L I T Y O F S O L U T E TRANSPORT: T H E P R O B L E M O F INTRACELLULAR COMPARTMENTS

Inspection of simple carrier models (Fig. 1) leads one to expect that the relatively large amounts of amino acids inside yeast cells would tend to leak out of the cells when these were transferred to a medium lacking amino acids. Taylor (1949) observed, however, that yeast retained absorbed glutamate tenaciously, and a great deal of subsequent work with other amino acids has confirmed this observation. It is not always clear from published work whether an apparent loss of, for instance, cellular leucine (Surdin et al., 1965) was due to its efflux or to its metabolic degradation (Woodward and Cirillo, 1977). Indeed, Magaiia-Schwenke et al. (1973) detected an insignificant efflux of the proline analogue sarcosine, through the proline permease, unless either azide or proline was present in the medium when a slow efflux of sarcosine did occur. Efflux was much slower, however, than normal influx of the amino acid. Azide, like dinitrophenol, inhibits amino-acid uptake and therefore might help to reverse it, whereas extracellular proline would prevent re-entry of sarcosine leaving the cells and might alternatively accelerate the exit process. Kotyk et al. (1971) and Karst and Jund (1976) similarly observed a very slow rate of efflux of glycine and 2-aminoisobutyrate, respectively, from yeast and their observations are in general agreement with those of Seaston et a f . (1976) and Indge et a f . (1977) who estimated that the fraction of the large ninhydrin-positive amino-acid pool leaking out of yeast was about 0.01 min- under the most favourable conditions. Furthermore, histidine uptake (Crabeel and Grenson, 1970) appeared to be virtually irreversible, the absorbed amino acid being displaced neither by exchange with the extracellular amino acid nor following resuspension of the yeast in a medium without added amino acids. Such behaviour contrasts with that of uracil (Grenson, 1969; Jund et af., 1977) and cytosine (Chevallier et af., 1975) which are excreted relatively rapidly in certain circumstances and form a cellular pool representing the balance between substrate influx and efflux. However, in these two cases significant differences in efflux kinetics were

MECHANISMS OF SOLUTE TRANSPORT

25

observed. Whereas dinitrophenol stimulated cytosine efflux and inhibited cytosine uptake, it inhibited both uptake and efflux of uracil. Allantoin transport in Succh. cerevisiue appeared to be almost irreversible and accumulated allantoin was not exchanged at all readily with extracellular allantoin, nor was it displaced in the presence of dinitrophenol which nevertheless inhibited allantoin absorption (Sumrada and Cooper, 1977). Allantoate behaved similarly (Turoscy and Cooper, 1979).

H. T R A N S I N H I B I T I O N A N D T R A N S I N A C T I V A T I O N I N F E E D B A C K CONTROL OF SOLUTE UPTAKE

Several distinct factors probably contribute to the apparent irreversibility, or at least kinetic asymmetry, of these transport systems. In a simple reversible system, the maximum pool size would be reached when solute uptake and efflux were both taking place at the same rate comparable with the initial rate of uptake of the solute. Alternatively, the action of the relevant permease might genuinely be almost irreversible in the prevailing physiological circumstances. Such a system would be expected to approach equilibrium in a characteristic way (Hunter and Segel, 1973). The entry rate would progressively decline, in the limit to zero, as the intracellular substrate concentration rose. This behaviour is to be distinguished, in principle, from the possibility of allosteric control of a permease, unrelated to its equilibrium behaviour and exerted through the cellular concentration of its substrate or another effector (see, e.g., Cuppoletti and Segel, 1974). However, the progressive fall in entry rate is conveniently termed transinhibition in both instances. A further possibility is that the permease might be inactivated (Hunter and Segel, 1973) by a secondary mechanism related to the increase in cellular substrate concentration (transinactivation) so that influx and efflux necessarily both became slow. Another distinct factor is that absorbed solute might be sequestered in an intracellular organelle and therefore not be accessible to the permease. The main yeast vacuole has been especially considered from that standpoint. Cummins and Mitchison (1967) proposed that the main pool of purine derivative plays an important part in regulating adenine uptake, and that accumulation of adenine lowered its rate of further uptake by a factor of about four. Similar behaviour was encountered with guanine (Housset and Nagy, 1977). As adenine efflux was very slow, Cummins and Mitchison (1967) concluded that cellular adenine probably lowered the V,,, value of the transport system without altering the K, value, but more complex behaviour has also been observed (Housset and Nagy, 1977). Cummins and Mitchison (1967) show that although adenine uptake was slowed it did not stop when the adenine pool reached its maximum size. Slow efflux of adenine in this

26

A. A. EDDY

instance thus appeared to be a kinetic property of the adenine permease. The uridine and uracil permeases are also regulated by an analogous feedback inhibition (Grenson, 1969).An example of a similar phenomenon is the progressive inhibition of leucine uptake in Sacch. sp. by cellular leucine. Leucine uptake was initially fast, declined as leucine accumulated and was then restored to its original value during a short period spent without leucine in which the pool of cellular leucine was depleted. Whether new permease molecules were synthesized during this interval or latent ones reactivated is not known (Bussey and Umbarger, 1970). Feedback inhibition of the two histidine permeases was studied by Crabeel and Grenson (1 970) who showed that preloadingcells with histidine led to a rapid inhibition of histidine uptake through the high-affinity system, whereas preloading with several other amino acids failed to do so. The inhibition, presumably by cellular histidine itself, took place within 0.5 of a cell generation and was reversed relatively slowly. The second histidine permease retained a large part of its activity at high cellular histidine loads. Similar behaviour was observed with methionine. Likewise, Morrison and Lichstein (1976) correlated the decline in activities of the lysine and arginine permease systems with the corresponding cellular amino acid contents. Such studies have shown that the feedback inhibition is too rapid to be due to repression of formation of new permease molecules, and the dilution of the existing activity. Whether the inhibition can be reversed is not clear. Indge et al. (1977) made certain observations, with non-growing yeast suspensions, which suggest that the rapid fall in rate of uptake of glycine as it accumulated in cells was due to factors ancillary to the amino-acid pump itself. The yeast preparations were initially starved of nitrogen in the presence of glucose for 60 minutes and then exposed to glycine. Starvation increased the initial rate of uptake of glycine. It also dramatically increased the amount of glycine that was absorbed. Thus, unstarved suspensions absorbed about 1 pmol mg- l, the net influx of glycine at that stage having fallen to a small fraction of its initial value. Starved preparations, however, absorbed glycine at a rate that was scarcely diminished when the cellular glycine content had reached 1 pmol mg-'. Their glycine content continued to increase to about 2 pmol mg-I, by which time the swollen yeast cells started to burst. The existence of this phenomenon suggests that feedback regulation in this instance depends on factors which are not an essential part of the pumping machinery. An explanation based on transinactivation thus seems preferable to one based on transinhibition and including allosteric feedback control by glycine. The rate of efflux of glycine from yeast suspensions that contained only a small fraction of the maximum amount that they could absorb was found to be very slow indeed (Seaston et al., 1976). In this case, there was no question whether the permease was inactivated because the cells were capable of absorbing more glycine. Efflux of glycine was also very slow in the presence

MECHANISMS OF SOLUTE TRANSPORT

27

of dinitrophenol. I t was concluded that efflux of glycine through the general amino-acid permease was inherently slow because of the kinetic properties of the system. In work with the histidine permease already referred to, the yeast eventually contained a relatively large amount of histidine and continued to absorb histidine rapidly. The efflux of histidine from these suspensions was nevertheless too small to be detected. The permease thus exhibited a marked asymmetry in its kinetic behaviour. The proline permease, also already referred to, behaves rather similarly.

I. R O L E O F T H E V A C U O L A R C O M P A R T M E N T

Some workers have taken the different view that a very slow rate of solute efflux is best explained in terms of segregation of the absorbed material in an intracellular compartment such as the main yeast vacuole. However, this approach leads to further question as to how the vacuole can absorb material irreversibly. There is, nevertheless, clear evidence that amino acids, purines and other compounds accumulate in the main vacuole in vivo. A number of studies with C. utilis and Sacch. cerevisiae indicated that more than half of the amino-acid pool was contained in the vacuoles, where it appeared to be labelled more slowly during a pulse label with [ 14C]glucose than was the cytosolic fraction (Wiemken and Durr, 1974). These authors estimated that the average amino-acid concentration in the vacuole was five times greater than in the remainder of the cell. The vacuolar pool was rich in basic amino acids, in citrulline and glutamine, but deficient in glutamate relative to the cytosol (see also Zacharski and Cooper, 1978). A favoured method for extracting the cytosolic amino-acid pool is to disrupt the plasma membrane, with cytochrome c (Durr et al., 1975) or a polybase such as diethylaminoethyl-dextran (Huber-Walchli and Wiemken, 1979), under isoosmotic conditions. The osmotic pressure is then lowered, thereby rupturing the vacuolar membrane and releasing its contents. Distribution of amino acids between the two compartments, as defined by these extraction procedures, depends on the total cellular amino-acid content (Huber-Walchli and Wiemken, 1979). For instance, growth of C . utilis with NH,' as the source of nitrogen resulted in a cytosolic pool that was slightly larger than the vacuolar pool. During growth on glycine, similar amounts of glycine occurred in both compartments. However, growth with arginine, ornithine or citrulline resulted in these being concentrated mainly in an enlarged vacuolar pool. These recent obsekvations establish very clearly that the non-vacuolar compartments contain a large concentration of the amino acids other than arginine, ornithine and citrulline. On present evidence, the cytosolic pool can therefore be assumed to be accessible to the permeases of the plasma membrane which evidently restrict their efflux as already discussed. Boller

28

A. A. EDDY

et al. (1975) had earlier asked “whether active transport of amino acids in yeast occurs at the level of the plasma membrane, or at that of the vacuolar membrane, or at both levels”. The new results suggest that only basic amino acids occur in the vacuole at much larger concentrations than in the cytosol. Arginine, for instance, was concentrated about 30-fold relative to the cytosol, for which purpose some type of ‘pump’ is evidently required at the vacuole membrane, in addition to those at the plasma membrane (see Section IV.K, p. 30). Indge et al. (1977) observed that suspensions of Sacch. cerevisiae absorbed small amounts of glycine mainly into the cytosol, whereas uptake of larger amounts caused the vacuole to swell, roughly equal amounts of glycine then being found in both compartments. Huber-Walchi and Wiemken (1979) criticized the assay procedure used by Indge et al. (1977) on the grounds that amino acids in the vacuoles may have leaked into the cytosolic fraction during their separation. Nevertheless, Huber-Walchli and Wiemken (1979) themselves found a similar distribution of glycine in preparations of C . utilis grown in the presence of glycine. Indeed the two studies are not incompatible. The Swiss work does not involve cells which have been allowed to accumulate glycine for short intervals of time without growing, when according to Indge et al. (1977) the amino acid is not taken mainly into the vacuole but into the cytosol. Certain of the suspensions used by Indge et al. (1977) were thoroughly depleted with respect to energy metabolism, and glycine transport was driven by ionic gradients acting across the plasma membrane. As entry of glycine into the vacuole caused it to swell, it is interesting that Indge et al. (1977) observed swelling of the vacuole in the presence of lysine or arginine, in suspensions that absorbed glycine mainly into the cytosol. This observation is consistent with the evidence of Huber-Walchli and Wiemken (1979) that, during growth, basic amino acids were concentrated into the vacuole. The bearing of this work and similar studies (Zacharski and Cooper, 1978) with lysine and arginine on the apparent irreversibility of their transport at the plasma membrane must now be considered. First, the cytosolic concentration of arginine and lysine in growing yeast cells is probably only about 5-1 0% of its apparent value. Zacharski and Cooper (1978) have pointed out that this may explain why endogenous arginine, an inducer of yeast arginase, cannot induce more than a very low level of the enzyme in the absence of extracellular arginine. If the average steady-state cellular concentration of lysine, for instance, following extended exposure to lysine was 70 mM, the corrected cytosolic concentration would be of the order of 5 mM. Morrison and Lichstein (1976) observed no significant efflux of lysine under these conditions, either in the presence or absence of extracellular lysine. However, they concluded that lysine influx had also terminated in these suspensions, which were presumably not growing (see Sumrada and Cooper, 1978). The lack of efflux in this system was therefore principally

MECHANISMS OF SOLUTE TRANSPORT

29

due to the very low amounts of active permease rather than to segregation of lysine in the vacuole. Clearly nothing can be inferred from these particular experiments about reversibility of the lysine permease. Similar considerations apply to the efflux kinetics exhibited by urea (Cooper and Sumrada, 1975; Sumrada et al., 1976) and allantoin as well as allantoate (Zacharski and Cooper, 1978). An assay involving polybase treatment indicated that, like arginine, allantoin was segregated to the extent of about 90% in the vacuolar fraction, whereas more than 60% of the urea was located in the cytosolic fraction. Now, efflux of urea, either in the presence of extracellular urea or following addition of dinitrophenol, was readily demonstrated, whereas efflux of allantoin in these circumstances was very slow indeed. Although this difference might be attributed to their different intracellular distributions (Turoscy and Cooper, 1979), other factors may be more important. Thus, rapid efflux of urea may be related both (1) to the presence of an energy-independent facilitated diffusion system which is known to function in parallel with the urea permease, and (2) to reversal of the urea permease. Allantoin transport, however, occurs through a permease which is not by-passed by a facilitated diffusion pathway. Even if 90% of the cellular load of allantoin was not immediately available for efflux, the observations of Sdmrada and Cooper (1977) and Sumrada et al. (1978) indicate that efflux of allantoin was exceptionally slow in a system which evidently contained a functional permease that could absorb allantoin. The conclusion then, is that the allantoin permease resembles the general amino-acid permease in exhibiting a low rate of efflux both in the presence and absence of extracellular substrate or of dinitrophenol. The low-affinity histidine permease appears to have similar properties, although the action of dinitrophenol on the system has not been reported. Efflux through the proline permease was also relatively slow. This behaviour contrasts with that of the cytosine permease (and possibly the urea permease) which, albeit briefly, catalysed rapid efflux of cytosine in the presence of dinitrophenol, at a rate similar to the initial rate of cytosine uptake (Chevallier et al., 1975). Analogous behaviour has been encountered with the extensively studied 8-galactoside permease of E. coli (Kepes and Cohen, 1962). These two types of kinetic behaviour are considered further in Section 1V.N (p. 38) in connection with the mechanism of energy coupling a specific permeases.

J. T H E P H E N O M E N O N O F S H O C K E X C R E T I O N

Lewis and Phaff (1965) discovered that certain suspensions of yeast rapidly leaked up to 30% of their amino-acid content, together with other constituents of the cell pool, when they were exposed to fermentable carbohydrates. The amino acids were subsequently re-absorbed. The phenomenon was associated

30

A. A. EDDY

with exposure of a latent ATPase activity (Lewis and Stephanopoulos, 1967) and was attributed to membrane damage causing temporary non-specific leakage of yeast contents (see also Hagler and Lewis, 1974). Lewis and Phaff (1965) suggested that the excreted amino acids may correspond to the expandable pool described by Cowie and McClure (1959). It seems unlikely that the high rate of amino-acid efflux observed, which occurs only with certain yeast strains grown and tested in a particular manner, reflects normal kinetic behaviour of the amino-acid permeases. A rapid efflux of cellular amino acids is also caused by nystatin (Lampen, 1966).

K. V A C U O L A R T R A N S P O R T SYSTEMS

Recognition of the role of the vacuole in accumulating certain amino acids in vivo has led to attempts to reproduce this phenomenon with preparations of isolated vacuoles. Boller et al. (1975) demonstrated that such preparations accumulated arginine but only in exchange for arginine already present in the vacuoles. Arginine uptake was inhibited by histidine and by various arginine analogues in a way different from that found with intact sphaeroplasts. Durr et al. (1979) explored the notion that arginine might be associated with various polyphosphates that are known to occur in the yeast vacuole (Indge, 1968). Some evidence for this was obtained both in terms of the relative number of equivalents of each compound that accumulated in the yeast in various circumstances, and in the ability of vacuolar extracts or authentic polyphosphates to bind arginine during equilibrium dialysis. Ludwig et al. (1977) also observed that basic amino acids stimulated accumulation of low molecular-weight polyphosphates in yeast but they did not associate this behaviour with the vacuole. The Swiss work indicates that arginine may be retained in the vacuole in response to the electric field associated with a Donnan equilibrium. It is possible that strict coupling of arginine influx and efflux arises in that way if movement of other ions across the vacuolar membrane is severely restricted. There are also some grounds for thinking that anions other than polyphosphate may participate. As Durr et al. (1979) have hinted, in putting forward a number of simple possibilities, the mechanism of translocation of phosphate residues across the vacuolar membrane is a key aspect of the problem. Divalent cations such as Mg2+ and Mn2+ also accumulate in the vacuole, so that interactions between the basic amino-acid transport system and these divalent cations might be expected to occur (Okorokov et al., 1977). The yeast vacuole accumulates in vivo large amounts of the sulphonium cation S-adenosyl-L-methionine when cells are suspended in a solution of the compound. Work with isolated vacuoles showed that S-adenosylmethionine was accumulated by a saturable mechanism which was not inhibited by azide

MECHANISMS OF SOLUTE TRANSPORT

31

or dinitrophenol. Nor was it stimulated by addition of ATP or similar compounds. If metabolic energy is involved it is therefore supplied within the vacuole. Transport of S-adenosylmethionine into sphaeroplasts, on the other hand, was strongly inhibited by either uncoupling agent (Schwenke and de Robichon-Szulmajster, 1976). In contrast with arginine, uptake of S-adenosylmethionine into the vacuole did not occur by self exchange, and the mechanism involved has not been established. Guanosine and adenosine were also absorbed by isolated vacuoles by a saturable mechanism (Nagy, 1979). Inosine and hypoxanthine were absorbed at rates proportional to their concentrations. A limited study suggested that these processes did not involve energy metabolism that was inhibited by azide. It is interesting that a fast exchange of adenosine across the vacuolar membrane occurred, even though net efflux of adenosine into a solution lacking the compound was very slow. Whether uptake of adenosine involves exchange with a component of the vacuolar pool is not at present clear.

L. T H E M E C H A N I S M O F E N E R G Y C O U P L I N G D U R I N G A M I N O - A C I D T R A N S P O R T I N YEAST

Further discussion of the kinetic behaviour of the amino-acid permeases requires an understanding of the mechanism of energy coupling involved. The most important advances have been concerned with demonstrating that uptake of amino acids is intimately associated with uptake and efflux of H + and, in certain circumstances, of K + . When glutamate or lysine are absorbed by yeasts or bacteria and retained as such, compensating ionic shifts occur and in certain instances principally involve Na' or K f (Davies et af.,1953). However, Conway and Duggan (1958) found that yeast containing a relatively large amount of Na' excreted more Na', during a two-hour interval, not only when lysine and certain other basic amino acids were present but also when one of a series of neutral amino acids was present. Eddy et a/. (1970a) were unable to repeat this observation with a different strain of yeast. Their work focussed attention on K + instead of Na'. The yeast suspension absorbed one equivalent of K + with aspartate, excreted an equivalent of K during uptake of lysine and, especially significantly, excreted roughly two equivalents of K + during the very early stages of uptake of glycine. Charge neutralization was maintained by uptake of protons (Eddy and Nowacki, 1971). As uptake of glycine was both accelerated by lowering the pH value of the yeast cell suspension and inhibited by raising the extracellular concentration of K +,I was impressed by the analogy between glycine transport in yeast and Na+-dependent glycine transport in mouse ascites tumour cells, a process which was likewise stimulated by Na' and inhibited by extracellular K + (Eddy, 1966; Eddy et af.,1970a, b; Eddy and Nowacki, +

32

A. A. EDDY

1971). At that time, the membrane potential of the mouse ascites tumour cells was believed to be relatively small and coupling with K + appeared to involve an antiport function for K + , although later work with valinomycin and other ionophores (Gibb and Eddy, 1972; Reid et al., 1974) and with fluorescent probes sensing the membrane potential (Philo and Eddy, 1978a, b) disproved this, showing instead that efflux of K + from mouse tumour cells is probably electrically coupled to influx of Na+ with amino acids. Possible functions of K + , which have been suggested in the literature, are illustrated in Fig. 4, the crucial question being whether the proton symport with the substrate S is electrogenic. If it is electrogenic, absorbed protons would be recycled through the proton pump, as in Mitchell's classical

+

(C) I

P%P

p

-t-

"SH'

+

K+

FIG. 4. Charge neutralization during operation of symport mechanisms for substrates bearing different formal charges. In general, one or a small number n of cosubstrate ions are absorbed with the substrate S. In (a), (c) and (d) the mechanism is inherently electrogenic. Neutralization might initially be achieved by membrane depolarization leading to efflux of K + or by ejection of protons through the proton pump. The true stoicheiometry of the symport is most clearly revealed when the proton pump is not functioning (see Fig. 5). However, it is not always feasible to make such assays because the symport may not then function sufficiently rapidly. The examples given are only some of the possibilities (Table 2, p. 35). In (b), efflux of K + (antiport) through the symport itself neutralizes uptake of H + with S. An antiport function for K + has been proposed in two Na+-dependent renal systems transporting glutamate (Burckhardt et a/., 1980; Schneider and Sacktor, 1980; Schneider et al., 1980). Other roles for K + have also been postulated (West, 1980).

MECHANISMS OF SOLUTE TRANSPORT

33

formulation of the mechanism of lactose transport in E. coli (Mitchell, 1963, 1967). In all of the schemes illustrated in Fig. 4, amino-acid uptake is envisaged as a physical process which might, in principle, occur independently of the proton pump and a supply of ATP. Eddy et al. (l970b) depleted yeast suspensions of ATP, in the presence of antimycin and deoxyglucose, finding that the cells retained the ability to absorb a small amount of glycine and to concentrate it relative to the extracellular medium, provided the extracellular concentration of K + was small and the pH value relatively low. This process did not involve exchange with existing cellular amino acids or metabolic conversion of the absorbed amino acid (Eddy et al., 1970b; Seaston et al., 1976). It was accompanied by uptake of about two H + and expulsion of about two K + from the yeast (Eddy and Nowacki, 1971; Seaston et al., 1976). All of these aspects are consistent with the notion that the concentration of glycine was driven by the flow of H + and K + down their ionic gradients acting across the plasma membrane. However, somewhat ambiguous results were obtained in the first attempts to decide whether K + served an antiport function (Fig. 4b) or a facultative role (Fig. 4a). First, glycine uptake in the presence of glucose did not accelerate the turnover of K + , an observation most consistent with the scheme in Fig. 4 (a) (Eddy et al., 1970a). A facultative role for K + was also indicated by the fact that glycine uptake in the presence of K + was smaller when the yeast was fermenting glucose instead of being starved. The A$ value might then be less dependent on [Kl0 (Section 111, p. 15). Second, whereas replacement of cellular K + by N a + first led to an obligatory dependence of amino-acid uptake on energy metabolism in which no net transfer of H + occurred (Fig. 4a), uptake of various amino acids could also be associated with absorption of one equivalent of H + rather than two equivalents once the cell suspension had been kept for 10 minutes in the ion-titration apparatus. The explanation of this behaviour is not known (Eddy, 1978). Third, Eddy er al. (1970b) observed that glycine uptake was only partically inhibited by dinitrophenol, a circumstance which Eddy and Nowacki (1971) concluded supported the antiport scheme (Fig. 4b). Subsequent work resolved this latter ambiguity and showed clearly that dinitrophenol prevented concentration of glycine in ATP-depleted yeast suspensions, probably by short-circuiting the proton gradient (Mitchell, 1963; Seaston et al., 1976). Important confirmation has come from studies of the effect of proton conductors on leucine transport in rho respiratory-deficient strains of various Saccharomyces species (Ramos et al., 1977, 1980). All of these workers favoured the Mitchell type of scheme in which the amino acid was concentrated at the expense of a proton gradient and K + served simply to neutralize inflow of protons when the proton pump was not functioning. It is apparent that the development of a reliable means of assaying yeast membrane potential would strengthen these arguments. The role of protons as a cosubstrate of several of the main amino-acid ~

A. A. EDDY

7 0.5 p m o l

t

f

-

9

0.1 p m o l 0.2p m o l 0.5pmol Gly

Gly

0.5 minute

GlY

+

(C (C 1

I

2

+

1.j Y

0

0.5 minute

[$ a,

0 0

\

+ Y

+

I

0

0.5 p m o l

0

+

0.5 minute

Y

In

4

I

0.5 pmol Glt

0 c (

0.5 minute

FIG. 5. Proton and potassium-ion movements following addition of small amounts of glycine, lysine or glutamate to suspensions of Candidu utilis (Eddy et al., 1977 and unpublished work by P. Hopkins). The lines represent recordings from the specific ion electrodes placed in the yeast suspension. In a, 0.1 pmol, 0.2 pmol and 0.5 pmol of glycine were added to the suspension (50 mg dry wt) suspended in 5 mM Tris solution brought to pH 4.8 with tartaric acid. About one equivalent each of H t and K + was displaced. The solution contained 1 mM KC1, 15 pg of antimycin and 10 pmol of 2-deoxyglucose to stop energy metabolism. The cells had previously been grown in a chemostat with glutamate as the limiting source of nitrogen. From Cockburn et al. (1975). In b, the cells were from an 18-hour batch culture grown on glucose-mineral salts medium containing 2.5 mM glutamate as the sole source of nitrogen. Addition of 0.5 pmol of lysine caused uptake of about one equivalent of H f and about two equivalents of K + appeared in the medium. In c, the metabolic inhibitors were absent and the yeast suspension was aerated. Lysine in

35

MECHANISMS OF SOLUTE TRANSPORT

TABLE 2. Proton symport mechanisms in eukaryotic micro-organisms Proton equivalent

System

Saccharomyces spp. General amino acid permease (Seaston et a/.. 1973)

Specific amino acid permeases (Seaston et a/., 1973; Cockburn et a/., 1975) Other permeases (Brocklehurst er al., 1977) (Seaston et a/., 1973; Serrano, 1977; Eddy rt al., 1977) (Cockburn at a/., 1975; Roomans and Borst-Pauwels, 1979) (Reichert and Forst. 1977) (Roomans er al., 1979b) Candida spp. (Eddy et a/., 1977)

(Eddy et a/., 1980) (Deak, 1978) Rhodotorula sp. (Hofer and Misra, 1978; Deak, 1978)

Chlorella sp. (Komor and Tanner, 1974; Gruneberg and Komor, 1976) Neurospora sp. (Slayman and Slayman, 1974)

Glycine L-Methionine L-Lysine L-Citrulline L-Phenylalanine L-Leucine L-Lysine L-Methionine L-Proline L-Glutamate

2 2 2 2 2 2 1 1

cr-Methy lglucoside Maltose

1 1

1

2 or 3

Phosphate

2 or 3

H ypoxanthine Sulphate

3

1

1 1 1 2 3

Glycine L-Lysine L-Arginine L-Glutamate Phosphate D-Xylose, glucose D-Xylose D-Galactose 2-Deoxyglucose Xylitol

1

1 -1

6-Deox yglucose Glucose 1 -Deoxyglucose

Glucose

1 1

2 -1

this instance caused the ejection of protons and of some K' which together roughly balance the uptake of lysine cations. In d, the yeast was grown in a nitrogen-limited chemostat, and the suspension was given 0.5 pmol of glutamate in the absence of metabolic inhibitors. The initial influx of H + and efflux of K + were subsequently partially compensated by ejection of protons from the yeast. The ratio H +:glutamate absorbed by these preparations was about two in the presence of the metabolic inhibitors.

36

A. A. EDDY

permeases of yeast is now well established (Table 2). In each case, the observations were made with energy-depleted suspensions of yeast in order to minimize the possibility of proton recycling through the proton pump. The importance of this factor is illustrated in Fig. 5 which compares the electrode traces for H + and K + observed when preparations of C . utilis were given successive small amounts of glycine, or a single dose of lysine or glutamate. The observations with glycine conform to the pattern of one H + absorbed per equivalent of glycine and one K + displaced (Table 2 ) . Similarly, yeast-suspensions depleted of ATP absorbed about one H with lysine and expelled about two K'. However, omission of the metabolic inhibitors led to quite a different pattern of ionic movements involving excretion of K' and a varying amount of H + . Glycine uptake by semistarved suspensions of Sacch. cerevisiae could be shown initially to involve successive absorption of H and their subsequent expulsion by a process that appeared to require ATP (Eddy and Nowacki, 1971). Roon et a/. (1978) have drawn attention to the fact that the potential rate of working of the proton pump required to neutralize amino-acid uptake must be assumed to be much larger at pH 4.5 than its apparent rate, as assayed by titration with sodium hydroxide. It is therefore interesting that lysine uptake can stimulate proton ejection at pH 5 (see p. 34). Studies with dicyclohexylcarbodiimide, quercetin and diethylstilbestrol, acting as inhibitors of the plasma-membrane ATPase of rho mutants of Saccharomyces species (Ramos et al., 1980), all show that inhibition of the presumptive proton pump retards amino-acid uptake. This behaviour supports the validity of the proton-cycling mechanism which is basically a cryptic process. Other work with Dio 9 and Schiz. pombe (Foury and Goffeau, 1975) was interpreted in a similar fashion, but is open to the objection that commercial preparations of the drug, though possibly not purified preparations (Dufour et a/., 1980), have a lytic action (Roon et al., 1978). Acid excretion induced by uptake of lysine by Sacch. cerevisiae was first demonstrated by Eddy and Nowacki (1971). Such observations indicate that, even by studying the initial pH changes in the system, the intrinsic proton stoicheiometry of the amino-acid carrier is only clearly revealed when proton cycling, implicit in the chemiosmotic mechanism (Fig. 4), is prevented. The nature of the factors initiating fast ejection of protons in the presence of lysine, presumably through the proton pump, is unknown. The behaviour of glutamate shown in Fig. 5 illustrates the concurrent displacement of K + and influx of H f that took place immediately following addition of that amino acid. A subsequent phase of acid ejection occurred and, as other work has shown, this can eventually be accompanied by uptake of K + in amounts stoicheiometrical with the glutamate absorbed (Eddy et a/., 1970a; Eddy, 1980). One interesting aspect of glutamate absorption is that the anion is absorbed with more than one equivalent of H + , probably with two in +

+

MECHANISMS OF SOLUTE TRANSPORT

37

Candida utilis and with up to three in Saccharomyces spp. (Table 2 ) . Whether these ratios are always strictly integral is uncertain. The possibility that more than one permease is involved, when the amino-acid concentration is varied in such assays, needs to be borne in mind. The excess of positive charge entering the glutamate would mean that the anion depolarized the yeast. This is the suggested explanation of why efflux of K + occurs simultaneously.

M. THE EXTENT O F A M I N O - A C I D C O N C E N T R A T I O N B Y YEAST

It is important to know whether the concentration gradients of amino acids developed by yeast are consistent with the magnitude of the driving forces represented by the proton gradient, ApH. Seaston et al. (1976) showed that the maximum value of the ratio [gly]i/[gly], formed by ATP-depleted yeast, apparently at the expense of the gradients of H + and K', varied about a mean value of 4. lo4 when [gly], was near 0.1 p ~ A. similar ratio was observed in the presence of glucose in the range of glycine concentration from 2-10 p~ (Indge et al., 1977). The choice of these very small amino acid concentrations was based on the need to employ conditions in which the cellular pool was not apparently limited by feedback regulation (Section IV.H, p. 25). It is therefore clear that the maximum accumulation ratio observed during energy metabolism is similar to that maintained by energydepleted suspensions. The magnitude of the ApH value maintained during energy metabolism at very small extracellular concentrations of K', corresponds to a factor of 104-105,if it is correct (Fig. 3 ) to assume that K + is then distributed in equilibrium with A$ (Seaston et al., 1976; Eddy et al., 1980). As two H + are absorbed with glycine through the general amino-acid permease, these estimates lead to a predicted maximum value for the glycine gradient of an unrealistically large 108-1010-fold at thermodynamic equilibrium. The notion that the concentration of glycine is driven by a proton gradient is therefore feasible on energetic grounds, though clearly the system is far from thermodynamic equilibrium. Raising [gly],., above 10 p~ leads to a marked fall in the maximum ratio for glycine accumulation. As it seems unlikely that the ApH value falls very much under these conditions, an explanation is probably to be found in terms of feedback inhibition of further amino-acid uptake as the cellular content rises (Section IV.H, p. 25). Reference to Table 2 (p. 35) shows that various specific aminoacid permeases function with one H + per amino-acid molecule. In these instances, the energy input per mole of amino acid absorbed is half that associated with the general permease. It seems possible that the latter system is designed to bring about a fast absorption of the common amino acids which are then metabolized.

38

A. A. EDDY

N. AMINO-ACID EFFLUX A N D THE PROTON G R A D I E N T HYPOTHESIS

The likelihood that amino-acid uptake in yeast is limited by feedback mechanisms, the natures of which are at present far from clear, means that the system based on intact cells is probably not one in which it is profitable to study in detail the relations between Ap" and the magnitude of the amino-acid gradient. However, the question arises whether the near irreversibility of amino-acid uptake can be explained in terms of the proton gradient hypothesis. Eddy et a/. (1970a) observed that the starved yeast absorbed glycine about 10 times faster at pH 4.5 than at pH 7, and that the rate fell by a further factor of about 9 when [K],, was raised to 0.1 M at pH 7, an effect that might now be attributed to a fall in the A$ value. The work showed that this fall in the rate of amino-acid uptake was due to a decrease in V,,, value with only a small change in K , value. Seaston er al. (1976) suggested that this behaviour may mean that amino-acid efflux, which takes place from a pH value near neutrality and against the electric field, would resemble the kinetics at pH 7 in depolarized cells and therefore also exhibit a relatively small V,,, value. A kinetic analysis indicated that this behaviour was most likely to be found if ( I ) the amino acid (S) and H C bound in that order on the carrier (E), and (2) the complex ESH' carried a single formal charge, with E uncharged. The general notion underlying this scheme is that the electric field greatly assists the inwards movement of ESH and hinders its exit (Fig. 6). An important prediction, which is not easy to test, is that efflux would be relatively fast if the natural sense of the electric field could be reversed. Such a system would also exhibit a relatively slow rate of aminoacid exchange and a low rate of uptake in the presence of dinitrophenol. These considerations show that the strikingly slow efflux of amino acid from yeast is not incompatible with simple concepts of how such carrier systems may function (Fig. 4, p. 32). +

0. G R A D I E N T C O U P L I N G A N D P U R I N E A N D P Y R I M I D I N E T R A N S P O R T I N YEAST

Although active transport of urea (Sumadra er a/., 1976), allantoin (Sumadra and Cooper, 1977), allantoate (Turoscy and Cooper, 1979) and S-adenosylmethionine (Murphy and Spence, 1972) is each inhibited by proton conductors and may, therefore, be driven by the proton pump of the yeast plasma membrane, no other information at present implicates proton gradients in the activity of the respective permeases. Allantoin is concentrated up to about 104-fold and allantoate up to about 3. 103-fold, whereas urea is concentrated up to about 200-fold (Sumadra and Cooper, 1977). It seems

MECHANISMS OF SOLUTE TRANSPORT

(a) @

EHS

,0140 ,EHsO I

,

(b)

1 E

OUT

39

I

EHS

1'

0.1

E

0

z

, I 'EHS 10

,E

0

. . . . . .

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

PH 7

pH6 . . .. .. .. . . . . . . . . . .. .. . . . . . . . . .. . .. ... ...

FIG. 6. Simple ways of representing the effect of an electric field on the rate constants of the carrier model. In (a), the unloaded carrier E is assumed to carry no formal electrical charge and to acquire a formal unit positive charge when the cosubstrate H + and the substrate S bind. The converse case is illustrated in (b). A simple theory discussed by Britton (1965) supposes that the rate coefficient, here taken as unity in the absence of an electric field, is changed by the factor in the presence of a membrane potential. The values shown in the diagram correspond to the case where the exponential term corresponds to a factor of 10, that is where the A$ value is about 0.12V negative. The concept of a 'proton well', in which the electric field is wholly converted into an equivalent pH gradient (Mitchell, 1977). was used by Schwab and Komor (1978) as the basis of the gated pore model illustrated in (c). The combination of E, H + and S is envisaged as occurring at the centre of the membrane. The separate movements of S and H', towards and away from the centre of the membrane are not illustrated. They can be regarded as being relatively slow processes, or at least as occurring no faster than the voltage-insensitive re-orientation of EHS or of E that corresponds to the translocation steps in models (a) and (b). Thus binding and dissociation of one or both of the ligands could be among the rate-limiting processes in model (c), whereas in (a) and (b) the translocation steps are conventionally assumed to be rate limiting. Hopfer and Groseclose (1980) have made a penetrating analysis of one form of gated-pore model.

significant, in this connection, that the last system is bypassed by a second carrier, catalysing urea transfer by facilitated diffusion. Uracil transport in Succh. cerevisiue involves a distinct permease operating, probably, in parallel with a second system (Jund et ul., 1977) which has some of the characteristics of facilitated diffusion. This dual system maintains only a small uracil gradient of up to about 40-fold. Losson et al. (1978) observed that dinitrophenol and azide inhibited the initial rate of uracil uptake, whereas the ATPase inhibitors Dio-9 and chlorhexidine failed to do so, even though all

40

A. A. EDDY

four compounds more or less inhibited uptake of uridine and cytosine. It would be useful to know whether uracil transport over longer intervals is quite insensitive to Dio-9 before concluding that there is a fundamental difference in the mode of energy coupling with uracil compared with uridine and cytosine. Indeed, cytosine and uracil uptake in rho- respiratory-deficient strains was strongly inhibited by dinitrophenol, as though both processes depended on the proton gradient acting across the plasma membrane. Foury and Goffeau (1975) also inferred that uridine uptake in Schiz. pombe was coupled to proton ejection through the plasma-membrane ATPase. Direct evidence for participation of H + as cosubstrate in the cytosine permease was obtained by showing that hypoxanthine uptake was associated with uptake of about one equivalent of H + and the ejection of one equivalent of K + (Reichert and For& 1977). Reichert et al. (1975) and For&t et al. (1978) concluded that several substrates of the cytosine permease, including adenine, guanine, hypoxanthine and cytosine itself, exhibited an apparent affinity for the system that roughly correlated with their tendency to become protonated to the positively charged form. However, evidence was obtained that the substrate associated with a group with an acid dissociation constant pK, = 5.1. This group, possibly a protein carboxyl group, together with a proton and the substrate itself would form an uncharged complex on the carrier. Large concentrations (0.1-0.6~)of Na+ or K + inhibited absorption of the bases competitively, especially at pH 6 compared with pH 3. Fori3 et al. (1978) favour the view that K + and H + serve antiport and symport functions respectively, in this system, but the observations do not appear to exclude the possibility that efflux of K + was the result of membrane depolarization caused by symport of hypoxanthine with protons (Fig. 4, p. 32). Whichever mechanism applies, the competitive relationship between Na+ or K + and the purine contrasts with the non-competitive effect of K + on glycine uptake. There are a number of differences between the kinetic behaviour of these two systems, including those that concern their reversibility (Section IV.G, p. 24) in the presence of proton conductors, but insufficient information is available to carry the analysis further in terms of schemes like those shown in Fig. 6.

P. S T U D I E S W I T H Y E A S T - M E M B R A N E V E S I C L E S A N D A M I N O ACID-BINDING PROTEINS

Only limited success has been achieved in attempts to prepare plasma membrane vesicles that transport amino acids, even though the yeast vesicles obtained selectively transported glucose and galactose (Christensen and Cirillo, 1972; Fuhrmann et al., 1976) and contained some at least of their Mg-ATPase, apparently on the inside of the vesicles, in its natural location

MECHANISMS OF SOLUTE TRANSPORT

41

(Fuhrmann, 1977). Merkel et al. (1980) separated, by sucrose-gradient centrifugation, a material enriched in the plasma-membrane markers, chitin synthetase activity and concanavalin A binding, together with a Mg-ATPase activity that was not associated with the mitochondria1 marker succinate dehydrogenase. This material formed vesicles which took up leucine, glycine and proline, by a mechanism that was more or less inhibited in the presence of a proton conductor. The vesicles were prepared and assayed in the presence of sodium chloride, and when this compound was omitted from the assay medium, amino-acid uptake was impaired. Further work is needed, however, to show whether ( I ) N a t has a direct effect on the process of amino-acid uptake by vesicles (see Merkel et al., 1980) and (2) whether Na' interacts with amino-acid transport into intact yeast cells from which the vesicles were prepared. Such interactions have not hitherto been reported. Opekarova et al. (1 975) obtained a protein fraction, presumably associated with the cell surface, from the wild-type yeast of the Belgian series (C1278b), by means of a simple osmotic treatment. The preparation bound arginine and lysine and exhibited a molecular weight of about 5000. Hypo-osmotic treatment of the yeast impaired arginine transport which, however, was not restored when the separated protein was added back to the system. The protein was also present in a 'mutant carrying the arg-p, allele. Opekarova et al. (1975) proposed that the protein is located in the periplasmic space, on the membrane surface, and that it functions in conjunction with other components buried in the membrane. Other evidence for the presence of labile binding proteins for phenylalanine (VofiSek, 1972) and leucine (Bussey and Umbarger, 1970) has been obtained. The difficulties encountered in this kind of work have been emphasized by VofiSek (1973). A promising approach, on different lines, has been to use S-chloroacetyl-L-ornithine as a site-specific reagent for the general amino-acid permease. Larimore and Roon (1 978) found that this compound inhibited tryptophan transport competitively in initial-rate studies in the presence of glucose. In longer term assays, it appeared to inactivate the general permease selectively and irreversibly, especially in the absence of glucose. As Larimore and Roon (1978) point out, these observations may provide a means of assaying and fractionating the relevant component of the general amino-acid permease and studying it in relation to nitrogen catabolite repression, transinhibition and transinactivation (Section IV.H, p. 25). The cytosine permease has been the subject of a significant study by Parlebas and Chevallier (1976) who labelled permease' and permeasestrains independently with [ 3H]leucine or [ 14C]leucine,mixed them and used the classical double labelling procedure, based on 3 H : 14C ratios, to detect a polypeptide of about 80,000 daltons that only the permease' strains contained. A single haploid yeast cell was estimated to contain rather more than lo4 permease molecules, representing about 1% of the protein in the

42

A. A. EDDY

separated membrane fraction. Intragenic complementation tests indicated that the permease is probably based on the monomeric protein. In contrast, apparent interallelic complementation was observed in a single gene that appears to specify the S-adenosylmethionine transport protein which Petrotta-Simpson et at. ( 1 975) suggest may therefore contain more than one copy of the monomer. V. Carbohydrate Transport in Yeast

A.

MONOSACCHARIDES A N D THE PHOSPHORYLATION CONTROVERSY

A prescient comment by Sobotka et al. (1936) indicates the early attention paid to the concept, allied to modern views about mobile carriers, that diffusion of carbohydrates into baker’s yeast is based on their transient combination with constituents of the cell wall. However, later work focussed attention on the possibility that carbohydrate entry was not simply a diffusion process but involved phosphorylation of the compound (Rosenberg and Wilbrandt, 1952). The debate between proponents of the mobile-carrier hypothesis, on the one hand, and those, on the other hand, who believe that a group-translocation mechanism transfers phosphate from polyphosphate to the incoming sugar, has still not been entirely resolved. The main issue is whether ir, Sacch. cerevisiae monosaccharides such as glucose or galactose are only transported by facilitated diffusion (Cirillo, 1961; Heredia et af., 1968; Kotyk, 1967; Kuo and Cirillo, 1970; Kotyk and Michaljanicova, 1974) or whether a second process leading to their phosphorylation (Van Steveninck, 1972; Meredith and Romano, 1977) operates in conjunction with the facilitated diffusion carrier (Fig. 7). All parties agree that fast uptake of galactose involves an inducible membrane component, governed by the GAL 2 gene, formed during growth in the presence of galactose (Kuo et af., 1970), whereas fast uptake of glucose is not adaptive but constitutive (Cirillo, 1968). When fermentation by glucose-grown yeast is suppressed with iodoacetate, glucose, galactose or L-sorbose are absorbed and appear to compete for a common mechanism (Cirillo, 1962; Van Steveninck and Rothstein, 1965; Heredia et al., 1968; Van Steveninck, 1972) which can function so as (1) to transfer the carbohydrate either inwards or outwards and (2) gives rise to counterflow phenomena (Cirillo, 1961; Kotyk, 1967). Polyphosphate groups at the cell surface were originally implicated in glucose uptake on the basis of metal-binding studies. Glucose uptake in the absence of iodoacetate was assayed in terms of the fermentation rate and was completely inhibited by low concentrations of uranyl ions, and impaired by Ni2+, both of which were believed to bind to a small number of superficially located polyphosphate groups (Van Steveninck, 1966; Van

MECHANISMS OF SOLUTE TRANSPORT

43

FIG. 7. Different ways of representing the phosphorylation hypothesis of carbohydrate uptake. Brocklehurst et al. (1977) represented the phosphate donor as situated either outside the permeability barrier (a) or inside it (b). In the former case, a proton might accompany the carbohydrate moiety across the cell membrane. In both (a) and (b) the carbohydrate (SOH) is phosphorylated as it traverses the cell membrane, whereas (c) shows how phosphorylation might be associated with a facilitateddiffusion carrier. Depending on the availability of the phosphate donor, the carbohydrate would enter either as the free sugar (S) o r as (SP) the phosphorylated derivative (Van Steveninck, 1969).

Steveninck and Booij, 1964; Van Steveninck and Rothstein, 1965). The number of these metal-binding groups diminished during glucose fermentation and was restored when the glucose became exhausted. Furthermore, after poisoning with iodoacetate, the amount of glucose taken up from dilute solutions was found to be equivalent to the number of uranyl ions the yeast bound before iodoacetate was added. Van Steveninck (1969) observed that the glucose absorbed under these conditions accumulated as glucose 6phosphate and fructose 6-phosphate. Provision of larger concentrations of glucose, in the presence of iodoacetate, led to the appearance of free glucose inside the yeast. The schemes shown in Fig. 7 summarize the suggested mechanisms underlying these observations, which have also been treated as a mathematical model (Van Steveninck, 1968a; Jaspers and Van Steveninck, 1977a). The scheme envisages a passive carrier-mediated diffusion process associated with a metabolically linked transport mechanism, the so-called active transport of glucose (Van Steveninck, 1969). According to Van

44

A. A. EDDY

Steveninck, glucose uptake by facilitated diffusion, with low concentrations of sugar, is a relatively slow process. The contrast between the rates of active and passive transport is even more pronounced with galactose. ‘Active’ galactose uptake was also selectively impaired by Ni2 +; it exhibited a characteristic dependence on pH value and a 100-fold lower K,,, value with respect to galactose than facilitated diffusion of galactose (Van Steveninck and Dawson, 1968; Van Steveninck, 1972). Van Steveninck (1972) claimed that, during induction of galactose metabolism, a stage was reached in which galactose was partly phosphorylated and the concentration of free galactose in the yeast exceeded that outside by a factor of 50. Yeast adapted to utilize galactose also concentrated 2-deoxygalactose both in a phosphorylated form and as the free sugar. During uptake of galactose, studies with a pulselabelling technique showed that the specific activity of intracellular galactose phosphate increased many times more rapidly than that of the fraction containing intracellular galactose. An analogous study with 2-deoxyglucose led to a similar conclusion, namely that the intitial product of absorption of the sugar was the phosphorylated compound (Van Steveninck, 1968b). In this case too, the cellular concentration of the free sugar appeared eventually to exceed the extracellular concentration. Thus, active transport of both deoxyglucose and galactose appeared to lead to concentration of these sugars in yeast, presumably by the action of phosphatases on the initial phosphorylated derivatives. Although the scheme illustrated in Fig. 7c is broadly consistent with the observations of Van Steveninck and his colleagues, other work both challenges the interpretation of some of the above observations and provides a coherent picture of absorption of galactose as a process in which phosphorylation is not involved. Cirillo and his colleagues studied galactose uptake into yeasts lacking galactokinase (gal I ) , galactosyl phosphate uridyltransferase (gal 7) or the inducible galactose carrier (gal 2). No indications were obtained that cells without galactokinase either accumulated galactose against a concentration gradient, or formed phosphorylated galactose, whether small or large concentrations of galactose were present. In addition the kinase-less cells were unable to use galactose for growth. These observations are thus inconsistent with the essential part of the scheme in Fig. 7 according to which phosphorylation of galactose occurs by two distinct routes. Furthermore, the kinase-less cells transported galactose by a passive carrier-mediated facilitated diffusion mechanism catalysing exchange of galactose between the two compartments at a rate several times larger than the rate of utilization of galactose by the wild-type yeast (Kuo et al., 1970). The net rate of carbohydrate uptake by this mechanism, however, is probably smaller if the behaviour of glucose and mannose is a guide. Uptake of both of these sugars is regulated in terms of the Pasteur effect (Serrano and De La Fuente, 1974). It is interesting that in the absence of the kinase, net uptake

MECHANISMS OF SOLUTE TRANSPORT

45

of galactose, as opposed to the rate of exhange, declined drastically within one minute, well before the cellular galactose concentration had reached the extracellular value. This behaviour, which also occurs with glucose and 2-deoxyglucose (Heredia et al., 1968), is possibly due to recycling of the sugar by the carrier and can easily lead to the potential rate of transfer of the sugar through the system being underestimated. When proper account was taken of this factor, Kuo et al. (1970) were able to conclude that the facilitated diffusion system alone could support the observed rate of metabolism. Kuo and Cirillo (1970) also reached a firm conclusion on the issue whether tracer galactose labelled the cellular galactose pool or the galactose phosphate pool first. They found that the specific radioactivity of the free galactose pool was initially about seven times higher than that of the phosphate fraction in yeast lacking the uridyltransferase enzyme. In yeast suspensions containing the latter enzyme, the interpretation of the pulse-labelling experiments with galactose is complicated by formation of a series of compounds, including trehalose and uridine diphosphate hexoses, which were not taken into account in the early work of Van Steveninck (1972) and which may account for the apparent concentration gradient of neutral carbohydrates that developed during induction of the ‘active’ galactose-transport system (Kotyk and MichaljaniEova, 1974; Meredith and Romano, 1977). Indeed Kotyk and MichaljaniEova (1974) found no evidence that their yeasts concentrated galactose as such. They also demonstrated that incoming galactose first entered the pool of free sugar rather than the sugar phosphate pool, just as Kuo and Cirillo (1970) found in their mutant yeast. The balance of evidence thus favours the view that, in Sacch. cerevisiae, galactose uptake occurs by facilitated diffusion and is not concentrative. The galactose permease is subject to catabolite inactivation in the presence of glucose (Holzer, 1976). Facilitated diffusion has also been demonstrated in yeast plasma membrane vesicles although the experiments that have been done do not, by themselves, exclude an ancillary role for a phosphorylation mechanism. These vesicles exhibited counter transport with glucose and mannose and provided evidence that prior adaptation of the yeast to galactose led to formation of a specific galactose carrier in the plasma membrane (Fuhrmann et al., 1976). Entry and exit of glucose or galactose was inhibited by uranyl ions but not by NiZ+ (Fuhrmann, 1977). However, uranyl ions also inhibit transport of amino acids and various other solutes (Maxwell et al., 1971) and the existence of this effect need not imply that polyphosphate is involved in all or any of these processes. The effect of Ni2+ on sugar fermentation, which was formerly attributed to the association of NiZ+ with superficial polyphosphate involved in sugar transport (Van Steveninck, 1966), has now been shown to result from its absorption into the yeast cell, where it probably inhibits alcohol dehydrogenase (Fuhrmann and Rothstein, 1968a, b). The outcome of the above work seems to provide no firm grounds for implicating

46

A. A. EDDY

polyphosphate in sugar transport in Succh. cerevisiae (see Jaspers and Van Steveninck, 1976, and Dubbelman and Van Steveninck, 1979, for a different view). It is therefore interesting that a recent study of 2-deoxyglucose uptake, which takes into account some at least of the complex products of its metabolism, concluded that this sugar first appeared in the cell in the phosphorylated form and was not concentrated as such (Meredith and Romano, 1977). There is rather convincing evidence that monosaccharide uptake into yeast is closely regulated in relation to energy metabolism. The rate of uptake of glucose, fructose, mannose or the non-phosphorylated pentose, xylose (Sols, 1967) is faster anaerobically than aerobically (Pasteur effect). This behaviour is associated with an increase in the apparent Michaelis constant for sugar entry under aerobic conditions, compared with either anaerobic conditions or with aerobic conditions in the presence of dinitrophenol (Kotyk and Kleinzeller, 1967; Serrano and De La Fuente, 1974). Though the mechanism is unknown, it has been discussed in relation to the plausible possibility of feedback inhibition by the concentration of intracellular sugar phosphate (Sols, 1967; Serrano and De La Fuente, 1974). The evidence for this view, however, is mainly circumstantial and has been disputed (Kuo and Cirillo, 1970). In Aspergillus niduluns, Romano and Kornberg (1969) have suggested that endogenous acetyl-CoA inhibits glucose uptake. The ability of hydrocarbons to inhibit glucose uptake in a Candida strain has been explained in similar terms (Gill and Ratledge, 1973). Some progress has been reported with attempts to detect protein components of the galactose carrier in baker’s yeast by two distinct methods. The first involved techniques based on incorporation of 14C and 3H during carrier induction and the second, binding studies based on equilibrium dialysis (HaSkovec and Kotyk, 1969). A lipoprotein fraction binding glucose and other substrates of the constitutive glucose carrier has also been isolated (Horak and Kotyk, 1973).

B. M O N O S A C C H A R I D E T R A N S P O R T I N Y E A S T S O T H E R T H A N SACCHAROMYCES CEREVISIAE

Although utilization of sugars by yeasts is an important taxonomic criterion, sugar transport has been studied in only a few yeast species with a limited range of substrates (Barnett, 1976). Relatively little information is available either about the genetic basis of these systems or the numbers of distinct carrier systems involved. One interesting aspect concerns the stimulatory effect of oxygen on carbohydrate uptake and the so-called Kluyver effect. (The more familiar Pasteur effect involves lowering of the anaerobic rate of sugar uptake or utilization during respiration.) Certain yeast species can

MECHANISMS OF SOLUTE TRANSPORT

47

use D-galactose or certain glycosides aerobically but cannot metabolize them anaerobically, i.e. ferment them. The same yeast strains nevertheless ferment glucose. Barnett (1968) suggested, as one possibility, that this Kluyver effect might involve a direct or indirect requirement of oxygen for solute transport. Sims and Barnett (1978) have now provided evidence supporting this interpretation. For instance, they showed that D-fucose was absorbed, apparently through the galactose carrier of Torulopsis dattila, about four times faster aerobically than anaerobically. No such effect was observed with 2-deoxyglucose, illustrating the important fact that the Kluyver effect is shown only with certain transport systems in a given yeast or fungus (FloresCarreon et al., 1969) and with different transport systems in different yeasts. A quite distinct line of enquiry has shown that Rhodotorula gracilis (synonym Rhodosporidium toruloides) and certain Candida species (Gill and Ratledge, 1973) are obligatory aerobes in which concentrative monosaccharide transport is apparently tightly coupled to aerobic metabolism (Kotyk and Hofer, 1965). Whether the effect of anaerobiosis involved in the selective Kluyver phenomenon is related to its general effect on sugar uptake in the Rhodotorula and Candida species is not known. Glucose uptake in R . gracilis was barely concentrative (Kotyk and Hofer, 1965) and, in Kluyveromyces lactis, involved an inducible system (Royt and MacQuillan, 1976). The latter yeast absorbed 2-deoxyglucose without concentrating it, the sugar subsequently being phosphorylated intracellularly. In contrast, 2-deoxyglucose appeared to be phosphorylated and concentrated 3-5-fold during its uptake into an unnamed strain of Saccharomycesfragilis (Jaspers and Van Steveninck, 1975).

C . PROTON SYMPORT IN YEASTS WITH MONOSACCHARIDES, DISACCHARIDES A N D POLYOLS

Proton symport with carbohydrates (Table 2, p. 35) was first demonstrated in yeast with a strain of Sacch. carlsbergensis (Sacch. uvarum) which, after adaptation to the sugar, absorbed protons with maltose, sucrose or a-methylglucoside but not with 2-deoxyglucose, galactose or glucose. Similarly a strain of Sacch. fragilis absorbed protons with lactose by an adaptive mechanism but did not absorb them with glucose (Seaston et al., 1973). The circumstances in which these effects occurred with maltose showed that two distinct phenomena were involved. Firstly, maltose was absorbed along with protons by a mechanism that evidently did not involve ATP since it persisted when glycolysis and respiration were stopped by the presence of antimycin, deoxyglucose and iodoacetamide. This mechanism is the proton symport. Secondly, when ATP was available, the proton pump ejected acid rapidly about 0.5 minute after maltose was added (Fig. 8). The maltose permease

48

A. A. EDDY

in yeast strains lacking maltase activity absorbed maltose as such (Okada and Halvorson, 1963) and free maltose was also found in certain specific circumstances in yeast fermenting maltose (Harris and Thompson, 1961; Brocklehurst, 1976; Serrano, 1977).The genetic basis of the maltose permease is complex (Zimmermann et al., 1973) and no structural gene for the system has yet been defined. The maltose permease of certain yeast strains is inactivated by glucose, an example of the phenomenon of catabolite inactivation (Holzer, 1976). Seaston et al. (1973) compared the amount of [ 14C]maltose absorbed by yeast, by assaying the amount of 14C retained, with the number of proton equivalents absorbed in the presence of metabolic inhibitors. The ratio H + : maltose was inferred to be 2-3, but later work showed that a large fraction of the absorbed maltose was hydrolysed and released from the yeast as glucose. The corrected stoicheiometrical ratio was close to one H + per maltose both in the presence and absence of metabolic inhibitors (Eddy et al., 1977; Serrano, 1977). The question arises whether the proton symport is electrogenic. Charge neutralization during maltose uptake was maintained by efflux of K + when the proton pump was not functioning owing to the presence of metabolic inhibitors. In their absence, the fast initial net efflux of K + caused by maltose slowed in about two minutes. Rapid proton ejection started within about one minute and was accompanied by succinate excretion (Brocklehurst et al., 1977). Factors regulating this sequence of events have not been studied, and nothing is known about influx and efflux of K + as opposed to its net movements (compare Section IV.L, p. 31). Certain arguments nevertheless indicate that the proton symport may be electrogenic. Firstly, Serrano (1 977) observed that dinitrophenol lowered the initial rate of uptake of maltose by about 95% without lowering the cellular ATP content. This behaviour is consistent with a role for the proton electrochemical gradient as the driving force, rather than the operation of an electroneutral exchange of H + and K + coupled to uptake of maltose. However, Serrano (1977) added dinitrophenol 20 minutes before the assay of maltose uptake, so that the extracellular pH value had risen and the cellular pH value would have fallen in the meanwhile. Another interpretation of the inhibitory effect of dinitrophenol in these circumstances is, therefore, that maltose uptake was retarded by the fall in cellular pH value rather than by lowering of the membrane potential. Such effects are known to occur with the yeast general amino-acid permease (Seaston et al., 1976) and in Chlorella sp. (Komor et al., 1979). Secondly, Serrano (1977) showed that inhibition of maltose uptake, caused by raising the extracellular concentration of K and ostensibly depolarizing cells, was not observed in the presence of the ostensibly hyperpolarizing thiocyanate ion. Thirdly, uptake of maltose initially caused changes in the fluorescence signal from certain carbocyanine dyes which may be due to electrical depolarization at the plasma membrane although this +

(a1

+ Y

2 U

a,

a

-

0.5 minute

5 minutes

FIG. 8. Flow of protons and of K + during uptake of maltose (mal) or a-thioethylglucoside (TEG) by Saccharomyes carlsbengensis. The lines represent recordings from specific-ion electrodes. Carbohydrate was added at the time indicated by the arrow. In (a), 20 mM maltose was added to the anaerobic yeast suspension at pH 5; in (b), 4 mM a-thioethylglucoside was added (adapted from Brocklehurst et a/.. 1977). In (c), pH traces obtained when 0.25 mM, 0.5 mM or 4 miv maltose was added to a yeast suspension depleted of ATP in the presence of antimycin, deoxyglucose and iodoacetamide (adapted from Seaston et al., 1973). The traces are displaced on the pH scale for the purpose of display. The response increased with maltose concentration.

50

A. A. EDDY

is by no means certain (Brocklehurst, 1976; Brocklehurst et al., 1977). In this system, the problem of eliminating fluorescence signals due to metabolic events in mitochondria has not been solved. Such effects might be smaller in rho- mutants, but these frequently take up maltose slowly (Brocklehurst, 1976; Khan and Greener, 1977).

D.

RESPIRATION-DEPENDENT P R O T O N

SYMPORT IN

RHODOTORULA GRAClLlS

This organism concentrates D-xylose, L-rhamnose and D-arabinose, but not D-ribose, by a mechanism with which D-galactose interacts (Kotyk and Hofer, 1965; Janda et af.,1976; Alcorn and Griffin, 1978). L-Rhamnose is not metabolized by the yeast and D-ribose is only metabolized after a period of adaptation (Hofer, 1970; Janda et al., 1976). This yeast transport system has been studied in some detail because of its seeming strict dependence on energy metabolism. Thus the presence of proton conductors, or of certain other inhibitors of energy metabolism, or the absence of oxygen each leads to virtual cessation of net carbohydrate uptake or efflux, although a relatively slow exchange of cellular for extracellular carbohydrate persists (Hofer, 1971a, b). Such behaviour is in marked contrast to that of the better known b-galactoside permease of E. coli which has some of the properties of a facilitated diffusion carrier when energy metabolism is prevented (Mitchell, 1967). Misra and Hofer (1975) showed that respiring yeast extruded protons by a mechanism that accelerated in the presence of K + and was inhibited by preliminary treatment of the yeast with dicyclohexylcarbodiimide. The respiring yeast accumulated the lipophilic cations tetraphenylphosphonium and triphenylmethylphosphonium in a manner that can plausibly be attributed to functioning of an electrogenic proton pump (Hauer and Hofer, 1978). For instance, proton conductors, anaerobiosis or the presence of K + each lowered the amount of lipophilic cation accumulated in the steady state. When the observed accumulation ratio of the cation was inserted into the Nernst equation (see equation 2, p. 3), the apparent membrane potential varied systematically with extracellular pH value, from about 0.08V at pH 8 to values near zero at pH 4.5. Other work has shown that the pH gradient across the plasma membrane was about two units at pH 4.5 and about 0.5 pH unit at pH 8 (Hofer and Misra, 1978). Thus Ap", expressed as a ratio, appears to be of the order of 10'-102 in this system, if it is correct, although this is uncertain, to compute the membrane potential from lipophilic cation distribution (see Hauer and Hofer, 1978). Hofer and Misra (1978) showed that uptake of D-galactose or D-xylose, from concentrations in the range 1-10 mM, by respiring yeast suspensions

MECHANISMS OF SOLUTE TRANSPORT

51

was accompanied by about one equivalent of protons in the pH range 3-5. Proton uptake failed to occur under anaerobic conditions, when sugar uptake was also abolished. Furthermore, uptake of D-galactose or D-xylose diminished the rate of uptake of the lipophilic phosphonium cations, as though carbohydrate absorption depolarized the cells (Hauer and Hofer, 1978). The system thus appears to be an electrogenic symport. Hofer (1970) has shown, further, that it is probably based on a mobile membrane carrier. Nothing is known about the mechanism of the respiratory dependence of carbohydrate uptake. When uptake of D-xylose in the steady state was studied as a function of its extracellular concentration ( [ S ] , ) , the cellular concentration ([Sli) tended to approach a constant value of about 0.7 M. Thus [S]i/[S], was about lo3 at the smallest xylose concentrations that were studied, and about three at the highest concentrations near 0.2 M (Kotyk and Hofer, 1965; Hofer and Misra, 1978). Although metabolism of xylose complicates the detailed interpretation of these observations, similar behaviour has been found with other symport mechanisms absorbing metabolically inert sugars, and this is discussed in Section IX (p. 61). It is relevant in this connection that a second mechanism appears to be involved in the uptake of D-xylose from concentrations much larger than the 1-2 mM solutions required to halfsaturate the proton symport (high-affinity system). This second low-affinity system exhibited an apparent K , value of about 18 mM near pH 5 (Alcorn and Griffin, 1978) and 15 mM at pH 6.5 (Hofer and Misra, 1978). The former workers suggested that whereas D-galactose was a competitive inhibitor of the high-affinity system, in agreement with other observations (Janda el al., 1976), it interacted only weakly with the low-affinity xylose carrier. Starving yeast, moreover, appeared selectively to derepress the activity of the highaffinity carrier. These and other observations are consistent with the notion that two distinct xylose carriers are involved. In contrast, Hofer and Misra (1978) have proposed that the low-affinity carrier corresponds to the proton symport absorbing xylose without a proton. The pK, value of the carrier was estimated to be 6.7 and, at pH 8 . 5 , it appeared simply to equilibrate xylose with cellular water. However, later work of Alcorn and Griffin (1978), with the same yeast strain, strongly indicates that two distinct xylose carriers are involved and that their activities are additive near pH 5 where the symport mechanism would be expected to be fully protonated (Fig. 9). It seems possible that the xylose proton symport functions in parallel with a second carrier system which is not itself concentrative. As the low-affinity carrier catalysed roughly the same rate of uptake of D-xylose from a 2 0 m ~solution as did the high-affinity system (Alcorn and Griffin, 1978), one must suppose further that respiration controls the activity of both systems. In contrast, xylose would be absorbed from a 60 PM solution about 13 times faster through the proton symport than through the low-affinity system (Model 1 1 1 in Table

52

A. A. EDDY T

01

0

I

5

I

I

1

10

15

20

D-Xylose concentration ( m M )

FIG. 9. Initial rate (v) of D-XylOSe uptake by Rhodotorula gracilis (glutinis) as a function of extracellular xylose concentration. The 105 observations made with a series of cell preparations were pooled. The interrupted line is the best fit to a single Michaelis-Menten function, whereas the continuous line represents the sum of two Michaelis-Menten functions. Redrawn from Alcorn and Griffin ( 1 978).

1 of Alcorn and Griffin, 1978). These pump and leak models are discussed further in Section IX (p. 61). A number of polyols including D-mannitol, D-xylitol and D-arabinitol were relatively slowly concentrated by a constitutive mechanism present in preparations of R. grucilis (Kloppel and Hofer, 1976). The process was inhibited by D-xylose or glucose, possibly because these utilized the same carrier as the polyols, although the point was not established rigorously. A second carrier system, exhibiting a much lower apparent K , value, was induced in the presence of specific pentitols, the uptake of which was associated with simultaneous absorption of protons (Kloppel and Hofer, 1976). The marked concentration dependence of the accumulation ratio ( [SIi/ [ S],) for polyols was emphasized by Kloppel and Hofer (1 976) who consider this ratio decreases below unity in the presence of high concentrations of, for instance, mannitol or sorbitol. Although the experimental evidence they present supports this possibility, further work is needed to show whether the apparent exclusion of large concentrations of polyols from the yeast, after relatively long periods of incubation, is really due to their active transport out of cells, as Kloppel and Hofer (1976) propose, or to some other mechanism (Deak and Kotyk, 1968). The latter workers have also discussed the analogous behaviour of a Cundidu species towards D-xylose in terms of

MECHANISMS OF SOLUTE TRANSPORT

53

an active transport of carbohydrate operating either into or out of the yeast cells. The existence of this phenomenon is at present, however, no more than an interesting possibility (see Section IX, p. 61).

E. O T H E R

X-GLUCOSIDES:

I N T R A C E L L U L A R OR E X T R A C E L L U L A R

HYDROLYSIS

Mitchison et al. (1973) showed that maltose utilization in a certain strain of Schiz. pombe involved its hydrolysis by an externally located maltase. Two other strains of this yeast possessed a similar system and exhibited no induced proton uptake in the presence of maltose (Brocklehurst, 1976). This type of behaviour is better known in the cases of sucrose hydrolysis by invertase in Sacch. cerevisiae and melibiose hydrolysis by a-galactosidase in Sacch. carlsbergensis (De La Fuente and Sols, 1962). The distinction between extracellular and intracellular modes of cleaving oligosaccharides has been elegantly demonstrated by using protoplast preparations which lack the periplasmic hydrolases (Sutton and Lampen, 1962). Trehalose and sucrose uptake in R. gracilis were shown in that way to involve exo-enzymes (Janda and Hedenstrom, 1974): A number of other a-glucosides are known to be absorbed by yeasts and utilized for growth (Barnett, 1976). Proton symport mechanisms are involved in certain instances, now to be discussed (Table 2, p. 35).

F.

a-METHYLGLUCOSIDE

P E R M E A S E S OF SACCHAROM YCES S P P

Work by Hawthorne (1958) and by Terui et a f . (1959) and later studies by tenBerge (1 972) established that a-methylglucoside fermentation depended on the presence of one of three pairs of dominant genes. One of these genes probably governed permeability to the substrate. Rapid hydrolysis of a-methylglucoside also depends on the presence of isomaltase. Analysis of the transport system has been helped by use of the glucoside analogue athioethylglucoside which is absorbed but not metabolized by yeast. Studies with strains of defined genotype carrying the MGL2 gene demonstrated that, after growth in glucose-containing medium, they absorbed athioethylglucoside by facilitated diffusion, the K , value with respect to a-thioethylglucoside being about 5 0 m ~ On . the other hand, growth with a-methylglucoside, or with glucose in the presence of a-thioethylglucoside as a gratuitous inducer, led to the expression of an active transport system concentrating a-thioethylglucoside as such up to about 100-fold and exhibiting a K , value of about 2-3 mM (Okada and Halvorson, 1964a, b). Proton conductors inhibited this mechanism. Accumulated sugar was displaced from

54

A. A. EDDY

the yeast cells in the presence of maltose, a-methylglucoside or cr-thioethylglucoside itself, behaviour in marked contrast to that of the amino-acid permeases discussed on p. 38. Uptake of a-thioethylglucoside by the active transport mechanism was inhibited competitively by a-methylglucoside, trehalose, maltose and by glucose. As the inhibition constant for a-methylglucoside was very similar to the apparent K,,, value for its own uptake, this glucoside and a-thioethylglucoside probably share the same carrier. Whether maltose, glucose and trehalose were absorbed by the same system was not established. A gene (ssf) governing penetration of sucrose has been defined in another series of yeast strains lacking the extracellular invertases specified by the SUC genes (Khan et a/., 1973). The specificity of this system in relation to the above substrates has not been defined in detail. However, further progress has come with the recognition that various a-glucosides, in addition to maltose, can stimulate uptake of protons during their absorption (Brocklehurst et ul., 1977). These include a-methylglucoside and a-thioethylglucoside itself, where the ratio H + : carbohydrate absorbed was approximately unity (Brocklehurst et a/., 1977; P. Hopkins, D. Gardner and A. A. Eddy, unpublished work). Furthermore, sucrose, trehalose, a-phenylglucoside, glucose and L-sorbose each stimulated proton uptake in yeast suspensions exhibiting the adaptive a-methylglucoside permease that concentrated a-thioethylglucoside. These observations suggest that all of the various a-glucosides are substrates of one or more proton symports which also interact with glucose or sorbose. Proton absorption with sorbose was also detected by Jaspers and Van Steveninck ( 1 977b) in Succh. frugilis. Brocklehurst ( 1 976) observed in these laboratories that trehalose uptake occurred along with one equivalent of protons, whereas Kotyk and Michaljanizova (1 979) recently reported that in certain types of yeast, neither trehalose nor maltose caused a fast absorption of protons and yet the former sugar, at least, was absorbed by the yeast. It may be relevant that Brocklehurst et a/. (1977) observed that, after growth of a certain yeast strain in the presence of a-methylglucoside, no accelerated proton uptake occurred in the presence of this compound, although growth with maltose led to expression of the a-methylglucoside proton symport, which earlier had been shown to concentrate a-thioethylglucoside (Kroon and Koningsberger, 1970). These observations are consistent with the work of Okada and Halvorson (1964a, b) who showed that a-thioethylglucoside was absorbed by two distinct mechanisms, namely facilitated diffusion and active transport. On the basis of the study already referred to Kotyk and Michaljanizova (1979) have proposed that, in certain circumstances, yeast concentrates trehalose, possibly into some cell organelle, by a mechanism that is insensitive to dinitrophenol and does not involve a proton symport acting at the plasma membrane (see also Burger et al., 1959). Concentration of sucrose, by a dinitrophenol-sensitive route, into a

MECHANISMS OF SOLUTE TRANSPORT

55

segregated compartment of the yeast cell was postulated by Avigad (1960). Transport of a-methylglucoside into the yeast cell was formerly considered by Van Steveninck (1970) to involve its simultaneous phosphorylation, but this view was later challenged on various grounds concerned with characterization of the products of a-methylglucoside catabolism (Kotyk and MichaljaniEova, 1974; Brocklehurst et al., 1977). If the hypothetical phosphorylation involved transfer of phosphate groups into the yeast from the outer surface of the cell membrane, then an accelerated uptake of protons might occur during sugar entry (Brocklehurst et al., 1977; Jaspers and Van Steveninck, 1977b). In that event, transfer of phosphate groups would neutralize the flow of protons whereas, according to the symport hypothesis, charge neutralization was likely initially to be effected by efflux of K + . In fact, Brocklehurst et al. (1977) found that proton uptake during entry of a-thioethylglucoside or a-methylglucoside was balanced by an equivalent outflow of K +.Thus there appear to be no grounds at present for believing that transport of a-methylglucoside in yeast involves its phosphorylation, nor is there any evidence that a-thioethylglucoside itself undergoes phosphorylation. Okada and Halvorson (1964b) showed that the cellular concentration of a-thioethylglucoside in the steady state, reached after incubating yeast for up to 90 minutes was about 150 times the extracellular concentration when this was about 0.1 mM. Raising extracellular a-thioethylglucoside systematically lowered the accumulation ratio from the above value to one of about 10-fold in the presence of 32 mM thioethylglucoside. Similar observations were made by Kroon and Koningsberger (1970) with another yeast strain. Possible explanations of this important phenomenon are considered in Section IX (p. 61).

VI. Glucose-Transport Systems of Neurospora crassa Shake cultures of N . crassa grown with large concentrations of glucose (50 mM) absorb this sugar by facilitated diffusion, the K , value with respect to glucose being about 8 mM (Scarborough, 1970a). A similar, or the same, system (system I) absorbs 3-O-methylglucose, apparently without intervention of a phosphorylation step or formation of any other transitory intermediate compound (Neville et al., 1971). In contrast, growth with low concentrations of glucose or in the presence of other appropriate substrates, or starvation of conidia, leads to derepression of a second transport system (system 11) exhibiting a small K , value for glucose of about 1 O p ~(Scarborough, 1970b; Neville et al., 1971). The presence of high concentrations of glucose inactivated system I1 (Schneider and Wiley, 1971), an example

56

A. A. EDDY

possibly of catabolite inactivation better known in yeast (Holzer, 1976). System I1 absorbs and concentrates, besides glucose, 3-0-methylglucose, 2-deoxyglucose and L-sorbose by a mechanism that is inhibited by proton conductors (Schneider and Wiley, 1971; Neville et al., 1971; Scarborough, 1970a, b). No detailed study of factors influencing the magnitude of the substrate accumulation ratio in derepressed cultures has been made and the maximum value of the ratio [S]i/[S], for 3-0-methylglucose has been reported to vary from lo2 (Neville et al., 1971) to 6 . lo3 (Slayman and Slayman, 1975). It seems possible that the low-K, inducible system I1 normally operates in parallel with the high-k;, constitutive system I and that the accumulation ratio varies with the relative activities of the two systems. Neville et al. (1971) found that these had similar V,,, values for 3-0-methylglucose in their derepressed cells. Work by Slayman and Slayman (1974, 1975) first clearly established that system I1 is an electrogenic proton symport. Uptake of graded concentrations of 2-deoxyglucose, glucose or 3-0-methylglucose caused a graded rapid depolarization of the fungal hyphae of up to about 0.12V at the highest carbohydrate concentration, which was followed by a slower partial repolarization which was attributed to delayed acceleration of the proton pump. The K, values with respect to sugar concentration deduced from the magnitude of the initial depolarization were similar to those corresponding to the concentration dependence of the rate of uptake of the sugars by fungal cell suspensions. Uptake also caused a transient uptake of protons to occur when 3-0-methylglucose or 2-deoxyglucose was added to the cells taken from shaken liquid cultures. The effect of glucose on proton uptake was not readily separated from the concomitant induced efflux of protons. However, whereas approximately one equivalent of H + was absorbed along with 3-0-methylglucose (Slayman and Slayman, 1975) an indirect estimate suggests that about 0.5 equivalent of H f was absorbed along with glucose (Warncke and Slayman, 1980). Cyanide or proton conductors, such as azide, greatly retard sugar uptake through system I1 for reasons that are not understood (Hansen and Slayman, 1978) and recall the similar behaviour of R. gracilis referred to on p. 50. It is therefore interesting that diverse treatments, including addition of 3-0methylglucose or proton conductors which depolarize the plasma membrane of N . crassa, produce rapid increases in the cellular content of cyclic adenosine 3’: 5’-monophosphate (Trevillyan and Pall, 1979). These workers have suggested that this compound serves as a signal controlling homoeostatic mechanisms that decrease cell membrane permeability and bring about other changes that may help to preserve the integrity of the plasma membrane when this is being stressed. The yeast cell membrane contains proteins that bind cyclic adenosine 3’ : 5’-monophosphate (Jaynes et al., 1980).

MECHANISMS OF SOLUTE TRANSPORT

57

VII. The Active Hexose-Uptake System of Chlorella vulgaris

This unicellular green alga possesses an inducible active-uptake system for hexoses such as glucose, 1-deoxyglucose, 3-0-methylglucose and 6-deoxyglucose (Komor and Tanner, 1971, 1980). There is compelling evidence that a proton symport is involved and that phosphorylation of the sugar during entry probably does not occur. A membrane-bound protein was formed during induction of hexose transport and disappeared following withdrawal of the inducer (Fenzl et al., 1977). Active transport was maintained either by respiration or by light energy (Tanner, 1969). Proton conductors markedly inhibited translocation of sugar in either direction across the cell membrane, an effect that has been attributed to the concomitant fall in cellular pH value by one unit from optimal values near pH 7, rather than to the accompanying fall in the proton electrochemical gradient acting across the membrane (Komor et al., 1979). As in the other cell systems discussed above, the electrically polarized plasma membrane of Chlorella sp. appears to be equipped with an electrogenic proton pump and various, as yet, ill-defined systems for accumulating K + in preference to Na'. Raven (1980) has lucidly reviewed these matters which are not discussed further here. Near pH 6, addition of various sugars to suspensions of Chlorella vulgaris caused an immediate rise in pH value (Komor, 1973) corresponding to the absorption of 1.5-2 protons per sugar molecule with 1-deoxyglucose, 2-deoxyglucose or 3-0-methylglucose (Komor and Tanner, 1974a) and absorption of about one equivalent of protons with glucose or 6-deoxyglucose (Komor and Tanner, 1974a). Proton and sugar uptake, respectively, showed a similar dependence on sugar concentration. Komor and Tanner (1974b) showed that the uptake of 6-deoxyglucose as a function of its concentration exhibited composite kinetics. A high-affinity system ( K , 0.3 mM) functioning at pH 5.7 was identified with the proton symport that absorbed one equivalent of H + , whereas a low-affinity system ( K , 50 mM), which was detected at higher pH values up to 8.5, was attributed to absorption of 6-deoxyglucose without a proton through the same carrier system. In keeping with this view, the two systems were induced together and were both absent from uninduced cells. Moreover, raising the extracellular pH value appeared reversibly to inactivate the high-affinity system in the same proportion as it activated the low-affinity system. The proposed explanation is that the hexose carrier functioned either with or without a proton as cosubstrate dependent on the prevailing pH value (Komor and Tanner, 1974b). When the extracellular pH was raised from 6.3 to 8.3, accumulation of 6-deoxyglucose from a 4.7 mM solution diminished from almost 50-fold to 15-fold (Komor and Tanner, 1974b). N o quantitative

58

A. A. EDDY

interpretation of these observations has been made but the notion that the carrier functions in two modes is important (see Schultz and Curran, 1970). Thus Komor and Tanner (1974b) stressed the similarity between the kinetics of sugar efflux and the properties of the unprotonated carrier, a suggestion which has an immediate bearing on the important problem of defining how the carrier functions when a steady state of sugar distribution is achieved. Three other lines of evidence are relevant. Firstly, studies with the lipophilic tetraphenylphosphonium cation indicated that the alga generated a negative membrane potential of about 0.13V, which was transiently lowered by up to 0.07V in the presence of sugar substrates. The estimated magnitude of Ap" acting across the plasma membrane corresponded to a factor of almost 2. 103-fold, which would be sufficient to account for the maximum accumulation ratio of 6-deoxyglucose observed of about 1.5. lo3 at very low carbohydrate concentrations of 1-10 PM (Komor et al., 1973; Komor and Tanner, 1976). The symport thus brought the proton gradient and the sugar distribution almost to equilibrium in these circumstances (equation 6, p. 5). However, in general, such an equilibrium is probably not achieved. When , accumulation the concentration of 6-deoxyglucose was raised above 10 p ~ the ratio was progressively lowered to a factor of about 120 with a 1 mM solution (Komor et af.,1973). Furthermore, accumulation of the different sugar, 1deoxyglucose, from a 0.4 mM solution, or of 3-U-methylglucose from a 1 mM solution (compare Fig. 11, p. 62), correspond to a ratio of about 2. lo2, rather similar indeed to that formed by 6-deoxyglucose (Komor and Tanner, 1971). As the different sugars were absorbed initially with different numbers of protons (Komor and Tanner, 1974a, b; Gruneberg and Komor, 1976), the proton gradient is unlikely to have been in equilibrium with the sugar gradient in each instance, unless the symport mechanism functions by adjusting the effective proton stoicheiometry in the steady state of sugar distribution to an appropriate value different from its initial value. This rather remote possibility is made unlikely by the following evidence. Decker and Tanner (1 972) found that glucose and 6-deoxyglucose stimulated respiration to an extent that corresponded, on certain reasonable assumptions, to consumption of about one ATP molecule per sugar absorbed, whereas 3-0-methylglucose or 1-deoxyglucose caused a larger relative oxygen uptake (Komor and Tanner, 1974a). The difference between the above two groups of sugars corresponds to their different proton stoicheiometries, and it seems likely that respiration is stimulated when absorbed protons are expelled, possibly through a plasma-membrane ATPase (Gruneberg and Komor, 1976). If this view is correct, the fact that the increase in respiration caused by 10 mM 6-deoxyglucose persisted into the steady state of its distribution (Decker and Tanner, 1972) may mean that a proton current persists through hexose symport under these conditions. In other words, the steady hexose distribution, corresponding to the relatively small accumulation ratios observed

MECHANISMS OF SOLUTE TRANSPORT

59

FIG. 10. The perplexing behaviour in Chlorellu sp. of transport of I-deoxyglucose compared with 6-deoxyglucose. Griineberg and Komor (1976) observed that influx of one equivalent of the former sugar caused firstly, uptake.of two proton equivalents into unloaded cells and secondly, efflux of two equivalents of 6-deoxyglucose when the cells were loaded with this compound. As in (a), they suggested that the carrier complex EHS containing 1-deoxyglucose dissociated prematurely into EH in about half of the instances it moved to the inner location. This would give a ratio of two H + for 1-deoxyglucose and one H + for 6-deoxyglucose which was supposed not to dissociate prematurely. The unloaded carrier then bound 6-deoxyglucose which was .thus returned to the medium twice as fast as 1-deoxyglucose entered. Another way of considering these stoicheiometrical ratios is shown in (b). S is 1-deoxyglucose and S’ is 6-deoxyglucose. The carrier is supposed to have two binding sites for H + and two potential sites for hexoses, translocation occurring when the sites are occupied as shown. Further study of this interesting phenomenon seems worthwhile. Griineberg and Komor (1976) concluded that, in the presence of I-deoxyglucose, a considerable proportion of the carrier catalysed transport of protons without concomitant transport of hexose.

at the higher sugar concentrations, was probably not in equilibrium with the proton gradient. Finally, Gruneberg and Komor (1976) loaded Chforeflasp. with 6-deoxyglucose and found that the subsequent influx of 6-deoxyglucose stimulated an equivalent efflux of intracellular 6-deoxyglucose. In contrast, influx of 1-deoxyglucose stimulated an efflux of 6-deoxyglucose that was about twice as large as the influx of the former sugar. They proposed the explanation illustrated in Fig. 10a, according to which 1-deoxyglucose brings the carrier, together with a proton, into the inwards facing position without the hexose itself necessarily being transferred into the cell. If transfer of the sugar molecule occurred only half as frequently as transfer of H + , then two H would be absorbed for each hexose. Furthermore, the inwards facing carrier would transfer 6-deoxyglucose out of the cell twice as fast as l-deoxyglucose influx occurred. Another type of model is illustrated in Fig. lob, which assumes that the carrier is in fact bivalent. Direct evidence for this is lacking. Indeed Gruneberg and Komor (1976) found no clear evidence for a second proton-binding site, although the observations they made do not settle the matter. The steady state of sugar distribution is discussed further in Section IX (p. 61). +

60

A. A. EDDY

VIII. Phosphate and Sulphate Symports (Na' and H')

Concentrative sulphate transport in N . crassa involves two distinct permeases one of which is found in mycelia and the other in conidiospores (Marzluf, 1974). The activity of these systems is regulated by feedback inhibition by a product of sulphate metabolism; their synthesis is repressed by a metabolite derived from methionine and the permease itself is inactivated under certain conditions. Marzluf (1974) showed that sulphate efflux could occur in the presence of azide, especially in the presence of extracellular sulphate or related ions such as chromate. Somewhat similar systems occur in yeast (McCready and Din, 1974) and Penicilliurn notaturn (Cuppoletti and Segel, 1975b). Cuppoletti and Segel(1975b) found that uptake of sulphate was promoted by H + ions and by various divalent ions including Ca2'. A carrier complex containing Ca2+,S 0 4 2- and H was proposed. Indeed, sulphate uptake promoted Ca2 uptake, but not in a stoicheiometrical manner. The uptake of H + was not assayed. Roomans and Borst-Pauwels (1978, 1979) have suggested the different hypothesis that these multivalent cations stimulate uptake of anions in P . notaturn and in yeast by lowering the electrostatic potential at the membrane-solution interface. This potential (not to be confused with A$), in certain circumstances, hinders uptake of anions by slowing their rate of passing into the interfacial region where the membrane carrier may be supposed to associate with the transported ligands. Roomans et al. (1979b) found that a strain of Sacch. cerevisiae absorbed about three equivalents of H + along with one equivalent of sulphate, about one equivalent of K being expelled simultaneously. It seems possible, therefore, that is absorbed, in effect, as a positively charged complex with protons, influx of which depolarizes the yeast causing efflux of K'. One interesting aspect of the assays of ionic fluxes was that they were done in the presence of glucose. Although the proton pump was functioning, apparently its activity was not accelerated by addition of sulphate (compare Fig. 5 , p. 34). Both in Candida utilis (Eddy et al., 1980) and in Saccharomyces spp. (Cockburn et al., 1975; Roomans and Borst-Pauwels, 1979), phosphate uptake caused simultaneous uptake of more than one equivalent of protons per H,PO,- absorbed. Cockburn et al. (1975) and Eddy et af. (1980) observed a limiting stoicheiometrical ratio of 3H':2K' per phosphate equivalent at low concentrations of phosphate, or its analogue arsenate, although a ratio of 2H':lK' per phosphate applied at higher phosphate concentrations according to Roomans and Borst-Pauwels (1979). The fact that phosphate uptake both transiently stimulated efflux of a lipophilic cation from the yeast and inhibited Rb+ uptake is consistent with the idea that +

+

+

MECHANISMS OF SOLUTE TRANSPORT

61

phosphate uptake depolarized the cell membrane (Roomans and BorstPauwels, 1977). As noted by Cockburn et al. (1975), the very large apparent gradients of phosphate concentration, of the order of 106-fold (Button et al., 1973) that yeast can form in the presence of dilute solutions of the ion, require large driving forces in order to be maintained. It is presumably this latter factor that accounts for the stoicheiometrical ratio of two or three for H + uptake with H,PO,-, absorption of which would accordingly be aided both by the electric field and the pH gradient acting across the yeast plasma membrane. A study of uptake of phosphate from dilute solutions near pH 7 showed that Na' or Li', in contrast to K + , stimulated the process. As the uptake of N a C itself was stimulated by phosphate, Roomans et al. (1977) have suggested that N a + is a cosubstrate of a separate phosphate carrier functioning under these conditions. At higher phosphate concentrations, a second phosphate carrier was in evidence. Its activity was independent of Na' and can probably be identified with the proton symport detected at pH5. Some of the other complex interactions of various cations with phosphate uptake are discussed by Rooman and Borst-Pauwels (1979) in terms of the surface potential of the yeast. Yeast depleted of phosphate can absorb K + and phosphate together when both are presented (Eddy et al., 1980).

A. R E L A T E D E F F E C T S O F S O D I U M I O N S

Phosphate uptake in a biflagellate marine fungus exhibited a specific requirement for N a + (Belsky et d., 1970). Uptake of nitrate, urea, glucose and amino acids by marine diatoms was also stimulated by N a + (Rees et al., 1980).The mechanism of these effects has not been established (see Whiteman et al., 1980 for a revealing analysis). It is interesting in this connection that although correlations were found between uptake of nutrients and extracellular N a + in the freshwater ciliate Tetrahyrnena sp. absorption of amino acids and glucose continued in the absence of N a + (Rasmussen et al., 1978). Transport of a-aminoisobutyrate into a trypanosomatid flagellate Crithidia species was almost independent of Na' and showed a number of characteristics of a proton symport (Midgley, 1978).

IX. The Steady State of Solute Accumulation Although the mechanism is not understood, the behaviour of the yeast amino

A. A. EDDY

62

acid permeases discussed in Section 1V.M (p. 37) indicates that substrate accumulation in these systems may eventually be limited by inactivation of the permeases themselves. In contrast, the various carbohydrate symports of yeast and species of Chlorella and Neurospora that have been studied in any detail appear to establish a steady state of carbohydrate distribution in which influx and efflux of carbohydrate are equal and the activity of the symport itself can be regarded, to a first approximation, as constant. In all of these examples, the steady state of cellular solute concentration ([Sli) tends to increase with the magnitude of [S],, ostensibly towards a limiting value, as illustrated in Fig. 1 1 for the particular case of 3-0-methylglucose in Chlorella vulgaris. Clearly the very survival of the cell depends on such a limitation being imposed. Similar behaviour is indeed found in a wide variety of solute transport systems. It was clearly defined in the early work on the b-galactoside permease of E. coli (see Eddy, 1978).

or 0

I

I

I

3

6

9

Outside c o n c e n t r a t i o n (c,-103

M)

FIG. 11. Cellular concentration of 3-0-methylglucose as a function of the extracellular concentration in the steady state of hexose distribution formed by Chlorella vulgaris (Komor and Tanner, 1971). Inspection of the observations shows that [C],/[C], fell markedly as [C], increased.

As several of these systems are evidently proton symports, the question arises as to the extent to which these are expected to concentrate different concentrations of solute. In this connection, an interesting general account of recent work on the mechanism of energy coupling in cosubstrate systems has been given by West (1980). The b-galactoside transport system of E. coli in particular has been studied with a view to defining the way in which energy metabolism affects the kinetic parameters defining entry and exit of lactose or its analogues. Komor et al. (1973) used a similar approach in a study of the K,,, values for 6-deoxyglucose influx and efflux and their contribution, together with that of the carrier translocation constants, to hexose accumulation in Chlorella vulgaris. The work leads to an interesting

MECHANISMS OF SOLUTE TRANSPORT

63

paradox. The maximum accumulation ratio of about 1.5. lo3 observed with dilute hexose concentrations could be accounted for in terms of the measured values of K , and V,,, when these were inserted into the equivalent of equations 9 and 10 (p. 6). However, the use of this type of kinetic analysis predicts that [S]i/[S], is constant when [S], varies, in contradiction to a wide range of experimental evidence. The existence of this problem has long been recognized and has recently been stressed by Kotyk and Stru2insky (1977). There appear to be two main ways (Eddy, 1980) of resolving the above paradox which arises because the substrate distribution is supposed to reach thermodynamic equilibrium through the actions of the carrier mechanism, the proton symport. The possibility put forward by Kotyk and Stru2inskL (1977), namely that, at large values of [S],, the local supply ofenergy available to the carrier in the membrane is insufficient to maintain the larger substrate accumulation ratios observed at small values of [ S],, seems unlikely to provide a general solution to this problem. Two other possibilities are the following. Firstly, the driving forces represented by the pH gradient and the electrical field acting across the plasma membrane might vary systematically with So so that [S]i/[S], varied in the observed manner and remained in equilibrium with the proton gradient. This type of explanation is not attractive in general in that it fails to provide independent regulation of the accumulation of different types of substrate. One might also suppose that the number of protons (n of equation 6, p. 5) absorbed with each mole of S was appropriately regulated, but I have not been able to devise a carrier model which behaves in that way. The more likely possibility is that the carrier system functions out of equilibrium with the proton gradient. In other words, the steady distribution of substrate S is associated with a finite net flow of protons through the system. Various ways in which the respective flows of H + and of S might in principle become dissociated are illustrated in Fig. 12. The possibility that S might traverse the plasma membrane as ES or ESH+ seems more important than a scheme in which EH' causes proton translocation without S. These non-equilibrium models are of two types. Passage of S without a proton occurs either through the proton symport itself (single carrier, two channel model) or through a separate carrier (two carrier, two channel model). Some relevant systems are listed in Table 3. The notion that active transport of S may represent the action of a pump operating in parallel with a leak has sometimes been taken to imply that the leak is a simple diffusion process. This is unlikely, for molecules such as glucose and, where two pathways are in fact present, both are likely to be carrier mediated (see Kotyk and StruiinskL, 1977). There is fragmentary evidence that the steady state of solute absorption is indeed associated with energy dissipation through the proton gradient. Firstly, hexose uptake in Chlorella vulgaris was associated with a continuing accelerated respiration (Griineberg and Komor, 1976). Secondly, when accumulation of u-thioethylglucoside by a certain yeast reached a steady state,

A. A. EDDY

64

net proton absorption did not fall to zero and was detected in the presence of metabolic inhibitors that deprived the proton pump of ATP (Eddy et al., 1979). In this particular system, two separate carrier systems for the glycoside appear to be involved. When the activity of the proton symport was varied systematically, by graded derepression of the system over a period of time, [S]i/[S], also varied systematically (Eddy et al., 1979). Thirdly, a preliminary attempt to establish the current-voltage relationships for the electrogenic symport of 3-0-methylglucose in N . crassa showed that, when

(d 1

ES

ES

E‘S

E’S

SES-

SES

FIG. 12. ‘‘Slip” and “leak” models describing coupling of flow of solute S to flow of protons. A single carrier E is involved in models (a), (c) and (d), and a further carrier E’ in (b). In (a), passage of S depends on formation of the ternary complex EHS on one face of the membrane, its translocation to the inner face and subsequent dissociation to form the free carrier E. This re-orientates to become available again at the outer membrane face. The line joining any two carrier species represents a reversible process with a characteristic rate constant in the forward ( k “ , k f 2 etc as indicated) and reverse ( E l , k - 2 etc) directions. As movements of H C and S are indissolubly linked in model (a), the steady-state distribution of S is necessarily inequilibrium with theproton gradient. In the“leak”model(b),movement ofS, without H’, occurs through the parallel channel based on E’. This arrangement comprises a reversible pump driven by the proton gradient through E and a “leak” occurring through E’. Recent work on the Na+-dependent amino-acid symport of mouse ascites tumour cells is consistent with such a model (Eddy et al., 1980; Hacking and Eddy, 1981). In the “slip” model (c), translocation of S mediated by the carrier E occurs through two channels. In one of these a proton is simultaneously translocated. In the “slip” model (d), considered for reasons outlined in the text, the carrier bears a second binding site for S and this site must be occupied if translocation of S without a proton is to occur. Clearly a bivalent carrier, with two sites each for both H + and S, might operate in various other ways as well. Schemes (a), (c) and (d) are considered further in Fig. 13.

TABLE 3. Tentative assessment of the number of carrier systems cooperating in the steady state of solute transport. The limitations of the evidence are emphasized in the text System

Numher of carriers

Glucose in Neurosporu crassa Xylose in Rhodotorulu grucilis a-Thioethylglucoside in Succhuromyces spp.

2 2 2

Urea in Saccharomyes spp. Uracil in Succhuromyces spp. Cytosine in Succharomjlces spp. Lactose in Escherichia coli Thiomethyl-B-galactoside in Escherichiu coli Hexoses in Chlorellu vulguris

2 2 2 1 2 1

Reference Neville et al. (197 l), Scarborough ( 1 970a, b) Alcorn and Griffin (1978), Hofer and Misra (1978) Okada and Halvorson (1964b), Kroon and Koningsberger (1970), Eddy et al. (1979, 1980) Cooper and Sumrada (1975) Jund et a/. (1977) Chevallier et al. (1 975) Booth and Hamilton (1980) Maloney and Wilson (1973) Komor et al. (1973), Komor and Tanner (1974b)

66

A. A. EDDY

the hyphae had accumulated 3-O-methylglucose, the membrane potential lowering the current through the system to zero was more positive than was expected on the basis of model a of Fig. 12 (Slayman et al., 1977). It seems possible that, if confirmed, this behaviour is due to an inward flow of protons that is larger than would be expected if hexose efflux was obligatorily associated with proton efflux. Finally, heat dissipation and accelerated respiration in the steady state of methylthio-p-galactoside accumulation in E. coli had been demonstrated (Long et al., 1977). It will be apparent that the models illustrated in Fig. 12 would not necessarily exhibit simple Michaelis-Menten kinetics. An example of the rather small deviations that may be important in accounting for the concentration dependence of the accumulation ratio is given in Fig. 9. The precise way in which the flow of material occurs between the competing pathways of course determines the magnitude of the accumulation ratio. This aspect has been examined for pumpleak models of cytosine accumulation (Chevallier et al., 1975) and a-thioethylglucoside accumulation (Kroon and Koningsberger, 1970; Eddy et al., 1979, 1980) in yeast. The problems posed by the so-called “slip” models shown in Figs 12 c and 12 d are more complex. First, there is a requirement to demonstrate passage of S through the symport by the alternative routes, respectively with and without an accompanying proton. It is only in Chlorella vulgaris that there is, at present, a strong presumption that 6-deoxyglucose behaves in that way (Komor and Tanner, 1974b). An important case is that of lactose transport in E. coli where Booth and Hamilton (1980) have concluded that lactose transfer, as opposed to that of methylthio-p-galactoside, occurs exclusively through the lac permease. Evidence that transfer of lactose in this system can occur without the concomitant passage of a proton is at present lacking. Nor is it clear how far strains of these bacteria lacking p-galactosidase tolerate the presence of high concentrations of lactose.

A. S P E C I A L C H A R A C T E R I S T I C S O F T H E “ S L I P ”

MODELS

It has been known for many years (see, for instance, Eddy, 1968) that the substrate accumulation ratio ([S]i/[S],) is independent of the magnitude of [S], in kinetic models in which (a) the carrier E effectively equilibrates with both the substrate S and the cosubstrate H + , on the opposing faces of the cell membrane, and (b) both EHS and ES traverse the membrane, in addition to E itself. The basic assumption made in such models is that binding and dissociation of the ligands are rapid processes in comparison with their translocation through the membrane. A model calculation is illustrated in Fig. 13, 11. It is also known that the same- assumptions lead to simple

I IO-~

IO-~

lo-'

I

101

102

10

Substrate concentration ([Sl,)

FIG. 13. Predictions based on "slip" models of the dependence of the solute accumulation ratio ([S],/[S],) on solute concentration ([S],). In each case [H+], was lo2 and [H+Ii was 1. Line I is the equilibrium case based on scheme (a) of Fig. 12. Line II is based on scheme (c) of Fig. 12. The assumption made was that H + and S were in virtual equilibrium with the carrier on either side of the membrane, and that translocation ot EHS and of' ES was relatively slow. To this end the rate constants k+' = k + z = k + 3 = k'4 = k-1 = k - 2 = k-3 = k - 4 were set at 100 and k+5 = k+6 = k+7 = k-5 = k-6 = k-7 were set at unity. The positive index of the above parameters corresponds to the respective conversions: E to EH or ES; and ES or EH to EHS. If [HI, refers to the left-hand side of scheme (c) of Fig. 12 and [HIi refers to the right-hand side, then k + 5 , k + 6 and k t 7 refer to the translocation from left to right. The negative indexes refer to the respective reverse processes. The required solution, corresponding to a steady-state distribution of S , is accordingly that k + 5 [EHS], k + 6 [ES], - k - 5 [EHS]i - k - 6 [ESIi = 0. Each of these terms was evaluated from the appropriate combination of the rate constants (160 terms/node) derived by the method of Indge and Childs (1976). Dr. K. J. Indge kindly made available his general program for listing these coefficients and I am greatly indebted to Miss A. Seaston for assistance with the further tedious computation involved. It will be observed that [ S]i/[S], in these circumstances was almost constant, a result in agreement with Eddy (1968). Line 111 is a different case based on scheme (c) of Fig. 12 in which the equilibrium assumption was now abandoned. The rate constants were chosen so as to favour the passage of S via ES. The values used were k + ' = k + * = k + 3 = k + 4 = k + S = k-' = k+' k-7 = 1, k - ' k - 2 = k-3 k - 4 = 0.1. Also k+6 = k - 6 = 100. It will be observed that, in these circumstances, the accumulation ratio declined markedly as [S], increased. Line IV. This case corresponds to scheme (d) of Fig. 12. The ligands H + and S were assumed to equilibrate with the carrier, with [El [S] = [ES] and [ES] [H'] = [EHS] and [ES] [S] = [SES]. The distribution of S in its steady state was then determined for the condition k'' = k-' = k + 2 = k - 2 = k + 3 = k b 3 = I . The relationship [ESH], + [SES], - [EHSIi [SESIi = 0 then holds when only one of the bound molecules of S is translocated. An analytical solution for the value of [SIi at a given value of [S], was found using the procedure outlined in Eddy et al. (1980). It will be observed that [S]i/[S], varied significantly with [S],. Line V. The same procedure as for line IV was used except that both binding sites for S were assumed to translocate. The relationship [EHSJ, 2[SES], - [EHS], - 2[SESIi = 0 then holds. This model leads to substrate exclusion at large values of S.

+

1

+

1

68

A. A. EDDY

Michaelis-Menten kinetics with respect to [S],, and to rate equations of the general form of equation 9 (Eddy, 1968; Heinz, 1978). One possible mathematical condition, that [S]i/[S], varies with [S], thus appears to be that the rate equations are higher than first order with respect to [S],. For instance, such terms arise when the translocation steps are not the sole ratelimiting steps because binding and dissociation of H + and S are also relatively slow (see Segel, 1975). This behaviour could occur if these two ligands combined with the carrier within the membrane, as in the gated-pore models of symport mechanisms (Tanner and Komor, 1979; Crane and Dorando, 1979; Komor and Tanner, 1980; Hopfer and Groseclose, 1980). An appropriate selection of kinetic parameters gave the result illustrated in Fig. 13, trace 111, according to which [S]i/[S], fell markedly as [S], was raised. A similar result was achieved by retaining the concept of equilibrium binding of the ligands and introducing a second binding site for S on the carrier (Wong, 1965) which might, on this view, be dimeric (Fig. 13, traces IV and V). One of these models leads to large substrate concentration ratios at small values of [S], and to substrate exclusion at large concentrations of S. The reason appears to be that the mechanism can either function as a proton symport or as a proton antiport. The possibility that certain yeasts may behave in that way is discussed on p. 52, and was considered theoretically by Kotyk and Struiinskjl (1977). These computations illustrate the diverse range of behaviour implicit in symport mechanisms obeying the ordinary rules of enzyme kinetics. It is very striking that the concept of “slip” permits substrate accumulation to approach thermodynamic equilibrium with the Ap” value at very small values of [S],, and yet allows a marked degree of ‘uncoupling’ from the proton gradient to occur at large values of [S],. This behaviour contrasts with the pump-leak models which operate further from thermodynamic equilibrium at low concentrations of substrate.

B. G E N E R A L C O N C L U S I O N S A B O U T S Y M P O R T M E C H A N I S M S

Sufficient is now known about many of the systems listed in Table 2 to make it clear that the solutes involved traverse the cell membrane in association with protons and, at least in certain instances, by an electrogenic mechanism. Detailed kinetic analysis of these mechanisms, including their efflux characteristics, has not progressed very far in terms of schemes like those illustrated in Fig. 12. Indeed, the fact that the membrane potential is commonly an adventitious variable in initial rate studies probably limits their usefulness (Hopfer and Groseclose, 1980). There is also a dearth of information available about the actual flow of protons and of electric charge in the steady state of solute distribution. In the analogous Na f -dependent aminoacid symport of the mouse ascites tumour cells, the corresponding current

MECHANISMS OF SOLUTE TRANSPORT

69

of Na’ depolarizes the cells to a predictable extent (Hacking and Eddy, 1981). This phenomenon defines the magnitude of the relevant ‘‘leak’’or “slip” pathways which were considered in connection with Fig. 12. Detection of such currents of cosubstrate ions would provide important confirmation of the validity of these chemiosmotic schemes in which ionic currents are coupled over the plane of the cell membrane rather than within it, as Williams (1978) has advocated in a related context. For note added in proof see puge 269

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Biosynthesis of Microbial Exopolysaccharides I. W. SUTHERLAND Department of Microbiology, University of Edinburgh, Edinburgh, Scotland I. Introduction: the range of microbial exopolysaccharides . . . . . 11. Physiological aspects of polysaccharide production , . . . . . A. Influence of culture medium, especially carbon substrate, nitrogen source and . . . '. . . . . ions . B. Growth phase in batchculture . . . . C. Nutrient limitation in continuous culture studies . 111. Precursors of polysaccharide . . . . . A. Substrates . . . . . . . . B. Sugar nucleotides . . . . . . . C. Lipid intermediates . . . . . . IV. Control of polysaccharide production . . . A. Regulatory mechanisms . . . . . B. Changes in polysaccharides . . . . . V . Synthesis of polysaccharides by cell-free preparations. . . . . A. Dextrans, mutans and levans B. Cell-free synthesis of homopolysaccharides . . . C. Cell-free synthesis of heteropolysaccharides . . . V1. Relationship to other polysaccharides: shared pathways . VII. Role of primers and secretion in polysaccharide production A. Primers or acceptors-are they necessary? . . . B. Secretion of polysaccharides . . . . . . VIII. Modification of polysaccharides . . . . . A. Changes in physical properties of polysaccharides . B. Post-polymerization modification. . . . . IX. Acylation . . . . . . . . . . A. Regular acylation on alternate repeating units . . B. Bacteria alginates . . . . . . . . C . Xanthan-polysaccharides of Xanthomonas campestris . D. General aspects of acetylation and ketalation . . X. The future . . . . . . . . . XI. Acknowledgements . . . . . . . . References . . . . . . . . . .

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I. W. SUTHERLAND

I. Introduction

Numerous micro-organisms produce exopolysaccharides, i.e. polysaccharides found outside the cell wall, either attached to it in the form of capsules or secreted into the extracellular environment in the form of slime. Such polymers vary considerably in their chemical structures. To date, most attention has been paid to polysaccharides from microbial species of medical significance. The structures of a number of these polymers have been elucidated. The three groups of exopolysaccharides which have been most studied are those produced by Enterobacter aerogenes, Escherichia coli and Streptococcus (Dipfococcus)pneumoniae. From these examples, it is possible to see the variety of chemical structures represented in the polysaccharides synthesized by a small discrete group of bacteria. Some components such as D-glucose, D-mannose, D-galactose and D-glucuronic acid occur very frequently, others such as L-rhamnose or L-fucose are slightly less common, D-mannuronic acid and L-guluronic acid are rare. At the same time, it has also now become clear that some of the earlier reports of polysaccharide composition and structure, made before current sophisticated procedures were available, should be regarded with caution. Even recently, some reports have indicated compositions or structures which, because of the analytical procedures employed or through use of material of doubtful purity, differed from the published reports of other laboratories. It is also surprising that so little attention has been paid to the structure of polysaccharides of potential commercial importance. Only in the last few years have such polymers been scrutinized in an effort to determine the basis of their unique rheological properties. For the present review, exopolysaccharides will be considered as belonging to five distinct groups. (i). The first group is comprised of the dextrans and related polysaccharides. This group is formed entirely of one monosaccharide type (i.e. they are homopolysaccharides), produced by bacteria utilizing sucrose as a specific substrate. In the absence of sucrose (or a few closely related substrates), the bacteria can grow, but are unable to form the polysaccharide which may be a dextran, “mutan” or levan (Fig. 1). The bacteria that synthesize such polymers include species of Streptococcus and Leuconostoc. One should probably regard with caution some earlier reports of dextran or levan synthesis by a wide variety of bacterial genera in which formation of an exopolysaccharide from sucrose was claimed, but no characterization of the polymer was reported. (ii). The second group of polymers is also produced by bacteria metabolizing a specific carbon substrate. Instead of dextrans or levans, a heteropolysaccharide is formed, i.e. a polysaccharide containing more than

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

(a)

_ _ _ -0 - C H G

O

&’O HO

OH

(c)

81

OH

I

HO H,

@,:&&I-

H OH H

OH H CH20H o Q 2 0 H $ ;

OH H

FIG. 1. The structure of some representative homopolysaccharides. (a) Portion of the structure of a dextran, (b) part structure of “mutan”, i.e. a polysaccharide produced by Streptococcus mutans, and (c) structure of a levan.

one component monosaccharide. As the process of polymer formation is intracellular rather than extracellular, the reason for the specific substrate requirement is not clear. To date, one such system has been studied, viz. production of a heteropolysaccharide by a yellow pigmented pseudomonad (Sutherland and Williamson, 1979). This process is additionally interesting in that a specific nitrogen source appears to be needed for optimal polysaccharide production, although not for growth. (iii). A number of micro-organisms produce extracellular homopolysaccharides from any of several carbon substrates, the intracellular formation being followed by secretion of the homopolysaccharide into the extracellular environment. Some of these homopolysaccharides are composed

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I. W. SUTHERLAND

solely of carbohydrate, e.g. bacterial cellulose, or pullulan from species of the fungus Aureobasidium; others additionally contain acyl groups. Some of the acylated polymers which are produced by certain species of Agrobacterium and by related bacteria are of particular interest in that the polysaccharides contain a mixture of succinyl and pyruvyl substituents (Hisamatsu et al., 1978b). Also among polymers of this type is sialic acid, a polymer of N-acetyl neuraminic acid with N- and 0-acetyl groups. (iv). Probably the largest, and certainly the most heterogeneous group of (a)

PYR=:B,Goi I

PYR=:PGAL or

(b)

-

P P +4 G l c A I + 4 G I c 1

I

I

P

+3GlcAI+2MonI

I Gal

AC

(d)

+2

3 Man I 4 6

3 3 G l c I

I Go I

a

Gal 1-3

-

PYr

a

Monl +2Mon

1-

a

2Glc I

P -

7

I GlcA

(el

a -2Rha

I -3Rha

a I +4

GlcA I

B

RGlc

2 Rho I

a P + 3 Rha I + 3 Gal I -

FIG. 2. Structures of some representative extracellular heteropolysaccharides. (a) Colanic acid from Escherichia coli K12, Salmonella spp. and Enterobacter cloacae; (be) exopolysaccharides synthesized by various strains of Enterobacter aerogenes, (b) type 5 , (c) type 7, (d) type 28 and (e) type 81; (f) the exopolysaccharide of Xanthomonas campestris (Jansson et al., 1975).

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

83

exopolysaccharides is composed of those heteropolysaccharides formed from repeating unit structures. These are widely distributed among eukaryotic and prokaryotic genera, but, as yet, relatively few have been thoroughly studied to elucidate their chemical structures. Interest has been concentrated primarily on heteropolymers formed by micro-organisms of medical importance, together with a small number of other polymers. Most are formed from relatively simple structural units ranging in size from disaccharides to octasaccharides, but one or more types of acyl group may also be present. Some of these structures are illustrated in Fig. 2, but a more comprehensive list of the heteropolysaccharides produced by a single bacterial species can be found in an earlier review (Sutherland and Ellwood, 1979). (v). An example of the final type of microbial exopolysaccharide is provided by bacterial alginate. This is a heteropolysaccharide composed of two types of monomer viz. D-mannuronic acid and L-guluronic acid, together with 0-acetyl groups (Fig. 3). However, unlike the heteropolysaccharides of the previous group, there is no repeating unit. The polymer molecule appears to contain segments composed of one or other monomer type, interspersed with sections in which both monomers are present in irregular sequences (Haug et al., 1974). COO H

COOH

COOH

COOH

COOH

O\

Polyguluronic a c i d

o\

Mixed block structures

COOH

FIG. 3. The carbohydrate structures found in bacterial alghates.

The aim of this review is to attempt to survey current knowledge of the production and synthesis of microbial exopolysaccharides. Although the main emphasis is on bacterial polymers, relevant information is included on similar products from yeasts, fungi and other eukaryotes. There is no attempt to provide a comprehensive treatise, and readers wishing to discover additional information about microbial polysaccharides should refer to one of the

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I. W. SUTHERLAND

reviews that have appeared in the past few years. Some of these have covered specific aspects such as capsule synthesis (Troy, 1979), regulation of biosynthesis (Sutherland, 1979aj and industrial applications of the products (Sandford, 1979; Sutherland and Ellwood, 1979); other more general reviews include those by Sutherland (1977a, b) and by Tonn and Gander (1979).

11. Physiological Aspects of Exopolysaccharide Production A. I N F L U E N C E O F T H E C O M P O S I T I O N O F T H E C U L T U R E M E D I U M , ESPECIALLY CARBON SUBSTRATE, NITROGEN SOURCE AND IONS

Most of the exopolysaccharide-producing micro-organisms which have been studied utilize carbohydrates as their carbon and energy source and either an ammonium salt or amino acids as their source of nitrogen. Yet as different ecological environments have been studied, it has become clear that a great variety of substances can be converted into polysaccharides. Amino acids may be used both as the source of carbon and of nitrogen, as exemplified by Myxococcus xanthus, a bacterium that can be grown in carbohydrate-free medium and which only utilizes carbohydrate very poorly, if at all (Bretscher and Kaiser, 1978). When nutrient limitation leads to formation of fruiting bodies, polysaccharides are produced (from amino acids) and are found surrounding the cells and thereafter the microysts in the fruiting bodies (Sutherland, 1979b). Again, hydrocarbons are utilized by various polysaccharideproducing micro-organisms. Not all such polysaccharides have been examined, but some of the substrates used and the composition of some of the polymers are listed in Table 1. Nitrogen-fixing species of bacteria are generally found to produce various amounts of exopolysaccharide and some of these have been the subject of structural and other investigations (Jarman, 1979; Kang and McNeely, 1977). However, such bacteria, although using atmospheric nitrogen as their nitrogen source, are unexceptional in their carbon sources which are carbohydrates such as glucose, sucrose or mannitol. There is no apparent correlation between the composition of the polymer synthesized and the ability of the producer organism to utilize, or not, any particular substrate except in the case of levan- and dextran-producing bacteria. These micro-organisms will be discussed separately. Early studies by Wilkinson and his colleagues (reviewed by Wilkinson, 1958)elucidated certain basic features of bacterial polysaccharide production, though these may not necessarily hold good for all species of bacteria let alone other micro-organisms. In strains of E. coli and Enterobacter aerogenes, polysaccharide production increased under conditions where growth was limited by the nitrogen, phosphorus, sulphur or potassium content of the

TABLE 1. Polysaccharide production from hydrocarbons and related substrates Micro-organism Pseudomonas sp. Alcaligenes faecalis var. myxogenes Methylococcus rapsulatus Methylocystis parvus Methylomonas mucosa Nocardia sp.

Substrate(s)

Polymer composition

Reference

Ethanol, ethylene glycol, propan- 1-01 Ethylene glycol

Glucose, mannose

Tanaka et al. (1974)

Glucose, galactose, succinate

Harada and Yoshimura (1964)

Methane, methanol

Rhamnose, arabinose, glucosamine Glucose, rhamnose

Wyss and Moreland (1968)

Methanol Methanol n-Alkanes

-

-

Hou et al. (1978) Tam and Finn (1977) Raymond and Davis (1960)

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I. W. SUTHERLAND

medium. The composition of the polysaccharide was independent of the nature of the nutrient limitation or of the identity of the carbohydrate substrate. Similar results were reported for a strain of Pseudomonas although in this bacterium, unlike the enterobacteria, the production of polysaccharide commenced late in the exponential phase of growth (Williams and Wimpenny, 1977). The ratio of the polysaccharide produced to the cell yield was greatest for phosphorus or nitrogen deficiency, lower for sulphur deficiency and lower still for potassium deficiency (Wilkinson, 1958). The failure of the bacterium to yield as much polysaccharide under conditions of potassium limitation was probably due to inhibition of nutrient uptake resulting from competition between potassium and ammonium ions for a common transport system (Dicks and Tempest, 1967). Various ions are known to be required either for substrate uptake or as cofactors in polysaccharide synthesis. Polysaccharide production in washed suspensions of Enterobacter aerogenes was stimulated by Mg2+, K + and Ca2 (Wilkinson and Stark, 1956). Chromobacterium violaceum in defined media produced more polysaccharide in the presence of a lowered concentration of Fe3 + (Corpe, 1964), and Ca2+ also stimulated polysaccharide formation by this strain. Nitrogen limitation in the presence of excess carbohydrate led to high yields of polysaccharide in cultures of Rhizobium meliloti (Dudman, 1964).A notable difference between Ent. aerogenes and R . meliloti was in the effect of aeration. With the former organism, high aeration was necessary for optimal polymer yields, but with the latter organism both growth and polysaccharide production were best in fermentors with low aeration. In the case of Acetobacter xylinum, less bacterial cellulose was formed in shaken than in static batch cultures (Dudman, 1960). Alginate production in Azotobacter vinelandii was also affected by the respiration rate of the bacteria (Deavin et al., 1977). By altering the impeller speed in the fermentor, the rate of oxygen transfer in the medium was altered and the bacteria could be grown at different specific respiration rates (Fig. 4). At higher respiration rates, the amount of polysaccharide produced fell, due to more of the carbohydrate substrate being converted into carbon dioxide. As these bacteria are capable of nitrogen fixation, molybdate limitation was used as an effective method of limiting the available nitrogen. Under the conditions tested, molybdate limitation and nitrogen limitation yielded the highest specific rates of polysaccharide synthesis. Polysaccharide production is not confined to aerobic micro-organisms. Anaerobic bacteria such as Clostridium perfringens produce exopolysaccharide when cultured in suitable media under strictly anaerobic conditions (Baine and Cherniak, 1971). The conditions under which polysaccharide is produced by Xanthomonas campestris have been studied in various laboratories because of the industrial applications of this polymer (Sutherland and Ellwood, 1979). Using 20-litre +

87

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES (u

+ 0

t

I

.c

I

50 E

0 -!

0

2

U

40 0

60 0

\

0

0

\

-0

0-

\

\.

I

C 0

10

’E

0

;

a,

>

0

10

20

30

40

50

S p e c i f i c r e s p i r a t i o n r a t e ( p m o l O2 h-’ mg cell-’)

s

FIG. 4. Exopolysaccharide production by Azotobacter vinelandii at various respiration rates. n indicates conversion of sucrose into sodium alginate, o polysaccharide concentration and cell mass. Reproduced with permission from Deavin et al. (1977).

fermentors, Cadmus et a f . (1978) observed that 50-60% conversion of substrate (glucose) into polymer was obtained in nitrogen-limited medium. With relatively low aeration (0.25 vol. of air I-’ min-’) and nitrogen limitation, the polymer that was obtained was low in pyruvate, whereas the polysaccharide recovered from more highly aerated cultures (1.5 vol. of air I-’ min- l ) supplied with increased available nitrogen, had a normal pyruvate content. Samples of the polymer produced early in the fermentation also had a lower pyruvate content. Because of the scale of xanthan production and the economics of using “waste products” from other industries, the possible use of substrates such as whey which are complex in their carbohydrate and amino acid composition has been tested (Charles and Radjai, 1977). Surprisingly, both glucose and galactose were simultaneously utilized and no diauxic effect was observed. It is possible that one sugar was primarily used as a substrate for exopolysaccharide production whereas the other provided cell carbon, but the authors indicated that polysaccharide was derived from both monosaccharides. The concentration of the carbon substrate employed has ranged from 1 to 4% or more and there has been no significant inhibition of growth at the highest substrate concentrations. In methanol-utilizing. bacteria, this is not the case. When methanol concentrations over 1% were examined, growth and polysaccharide production were inhibited, although methanol continued to be utilized (Tam and Finn, 1977). Although alginate synthesis by Az. vinelandii can be demonstrated in various media, the composition of the polysaccharide is affected by the Ca2 +

88

I. W. SUTHERLAND

concentration in the growth medium. This reflects the dependence on Ca2 for activity of the polymannuronate epimerase involved in the synthesis of the alginate (Haug and Larsen, 1971). The addition of EDTA to batch-culture media effectively removed C a 2 + and led to an increased proportion of D-mannuronic acid to L-guluronic acid in the alginate produced (Couperwhite and McCallum, 1974). Typical increases were from 0.43 to 1.5 and from 0.33 to 4.0 when glucose and sucrose respectively was the substrate. Eukaryotic systems have also been studied. Polysaccharide was obtained from Cryptococcus laurentii var. Javescens cultured in complex media with glucose as the carbon source (Cadmus et al., 1962). Polymer yields increased from approximately 0.6% to 1.2% when the level of available glucose was raised from 3% to 5% (w/v). The optimal concentrations of available nitrogen and phosphorus and the optimal temperature and aeration conditions were determined; the best yields were obtained under conditions of nitrogen limitation. Pullulan synthesis in Aureobasidium pullulans was also favoured by nitrogen limitation (Catley, 1971). A number of fungal exopolysaccharides are phosphorylated and the effect on their production of phosphorus limitation might thus be expected to differ from the effect of such limitation on polysaccharide production by prokaryotes or on the biosynthesis of non-phosphorylated polysaccharides in general. Production of a phosphate-containing polysaccharide by Aspergillus nidulans was stimulated by increasing the phosphate content of the medium from 0.05% to 0.2% (Leal and Ruperez, 1978). When Hansenula holstii, Hansenula capsulata and a strain of Sporobolomyces were compared, phosphorus limitation was found to yield differing results (Slodki et al., 1970). Phosphate deprivation caused the two Hansenula spp. to produce nonphosphorylated polymers, whereas the Sporobolomyces sp. formed a polymer whose phosphate content was greatly decreased, whose acetate content was increased and whose hexose composition was altered from 79% D-galactose and 21% D-glucose to 46% galactose and 54% glucose. As with many prokaryotes, aeration favoured polysaccharide production in cultures of Monilinia fructigens (Santamaria et al., 1978). This was also observed for pullulan synthesis, forcibly aerated cultures producing more polymer than did cultures incubated in shaken flasks (Catley, 1979). Aureobasidium pullulans showed greater variation in polymer yield when different substrates were used than did many other fungal species, though not all strains produced pullulan under comparable conditions. Of key importance to the commencement of polysaccharide formation were the pH and the concentration of nitrogen in the culture medium. In cultures grown at 28°C for 7 days, up to 6% of substrate was converted into polymer (Yuen, 1974); this is similar to some of the extents of conversion obtained with prokaryotes. +

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

a9

B. G R O W T H P H A S E I N B A T C H C U L T U R E

In their studies of polysaccharide production by Enterobacter aerogenes, Duguid and Wilkinson (1953) noted that, under the conditions used, bacterial growth ceased after 24 hours. Much of the polysaccharide was found after bacterial growth had ceased, but the rate of polymer production was greatest in the logarithmic growth phase. Thereafter, the production rate declined. In washed cell suspensions (non-growing cells) of Enterobacter aerogenes, a uniform rate of polysaccharide production was sustained over a 4-hour period (Wilkinson and Stark, 1956). It is probable that the rate of polysaccharide synthesis remained relatively constant in non-proliferating bacteria until the integrity of the cell surface was lost. In this context, limitation of Mg2+, with its role in biosynthesis and maintenance of the cell envelope, may have a more injurious effect than other limitations. Careful washing of bacteria prior to use in such experiments is also necessary or other important surface components might be lost. In a strain of Pseudomonas examined by Williams and Wimpenny (1 977), polysaccharide production was apparently a feature of the late logarithmic and early stationary phases of batch-culture growth. In contrast, in Pseudomonas aeruginosa the maximum rate of polysaccharide synthesis was noted during exponential growth and little more polymer was formed when growth ceased (Mian et al., 1978). In eukaryotic micro-organisms, exopolysaccharides may be produced at different stages of the growth cycle. When differentiating into the hard-walled resting stage, the myxomycete Physarum polycephalum synthesized copious amounts of polysaccharide (Zaar, 1978). The polymer formed in non-nutrient media closely resembled that produced by cultures in nutrient media at the end of the growth phase. Another Physarum species, Phys. flaviconum also secreted polysaccharide as the growth rate declined (Cheung et al., 1974). It was suggested that the microplasmodium became highly vacuolated and that extracellular slime was secreted by vacuolar exocytosis. Pullulan synthesis may also be a feature associated with morphological changes in Aureobasidiurn pullulans (Catley, 1979). Polymer synthesis appeared to start at the same time as hyphal budding and other evidence has been presented associating pullulan formation with the yeast form of the micro-organism.

C. N U T R I E N T L I M I T A T I O N I N C O N T I N U O U S C U L T U R E S T U D I E S

To an increasing extent, continuous culture has been used as a technique for the study of exopolysaccharide synthesis. Mian et al. (1978) noted that in chemostat cultures of Pseudomonas aeruginosa alginate production and

90

1. W. SUTHERLAND

cell concentration were essentially independent of dilution rate under nitrogen limitation and dilution rates of 0.05-0.1 h -l. A feature of this and other studies of polysaccharide production in continuous culture was the high yield of polymer obtained (Tables 2 and 3). The specific rate of polysaccharide production increased with increasing dilution rate. However, a drawback observed with Ps. aeruginosa was the appearance of non-mucoid variants. This bacterial species appears to be particularly prone to evolving these variants in continuous culture (Piggott, 1978), perhaps reflecting the highly selective environment from which the original polysaccharide-producing strains were derived (i.e. the cystic fibrosis patient undergoing intensive antibiotic therapy). Exopolysaccharide was produced even under conditions of carbon limitation, but the rate of emergence of non-polysaccharideproducing variants was then very high indeed. TABLE 2. Bacterial production of alginate Data from Mian et al. (1978) and Jarman et al. (1978) Rate of alginate production (g polymer h(g dry wt. cells-’)) by:

’

Limiting nutrient

Azotobacter vinelandii

Pseudornonas aeruginosa

Nitrogen Carbon Molybdate Phosphate Sulphate

0.017 0.0 19 0.026 0.022 0.020

0.27 0.19

Alginate production has also been studied in the nitrogen-fixing species A . vinelandii in continuous culture (Deavin et al., 1977; Jarman et al., 1978; Jarman, 1979). As with Ps. aeruginosa, some polysaccharide was produced in carbon-limited cultures of this bacterium. The rate of polysaccharide synthesis was largely independent both of the specific growth rate, in the range 0.05 h- to 0-25 h - l, and of the nutrient limitation. This contrasts with Ps. aeruginosa in which the specific rate of alginate synthesis increased with growth rate in nitrogen-limited cultures (D = 0.05 h- ‘-0.1 h- l ) (Mian et al., 1978). At low respiration rates, 40% of the substrate was converted into alginate, but at very high specific respiration rates much of the sucrose was “wasted” as carbon dioxide and only 8% yielded polysaccharide (Jarman, 1979). Polysaccharide was produced with a range of growth-limiting nutrients, but it was not reported whether changes in the ratio of mannuronic acid to guluronic acid were noted (Table 2 ) . Davidson (1978) showed that in continuous culture the ratio of monosaccharide components in the heteropolysaccharide from Xanthomonas cam-

TABLE 3. Efficiency of microbial conversion of various substrates into polysaccharides Micro-organism

Growth conditions

Carbon substrate

Conversion into polymer (%I

Reference

~~~

Pseudomonas aeruginosa Arthrobacter sp. Xanthomonas campestris

Xanthomonas juglandis Corynebacterium autotrophicum Leuconostoc mesenteroides Hansenula sp.

Nitrogen limitation Batch culture Synthetic medium nitrogen limitation plus urea/distiller’s solubles plus urea/distiller’s solubles Sulphur limitation Batch culture Batch culture

56-64

Glucose Corn sugar Glucose

44

50-60

Mian et al. (1978) Cadmus et al. (1 963) Cadmus et al. (1978)

Glucose Glucose Glucose Fructose Sucrose Glucose

68 81-89 60 50-70 29-55 58

Silman and Rogovin (1970) Silman and Rogovin (1972) Evans et al. (1979) Andreesen and Schlegel (1974) Jeanes et al. (1954) Slodki et al. (1 970)

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I. W. SUTHERLAND

pestris was essentially independent of the nutrient limitation, although the degree of acetylation and pyruvylation did vary. Silman and Rogovin ( I 970)

using medium with glucose as the carbon source noted increases in the rate of substrate utilization and of xanthan production when the dilution rate was increased from 0.023 h-’ to 0.0285Y’. This work was later extended to other dilution rates when further correlation between dilution rate and the rate of production of polysaccharide was observed (Silman and Rogovin, 1972). Because of the reported tendency to suffer contamination of continuous cultures, Silman and Bagley (1 979) sought to overcome such problems through the development of a “viscostat”. The viscosity of the culture was continuously sensed and the rate at which fresh medium was supplied was automatically adjusted so as to maintain the contents of the fermenter at constant viscosity. This procedure assumes that the product is of constant chain length and also that, if contamination does occur, the contaminant does not itself produce a polysaccharide which contributes to the total viscosity. This need not necessarily be the case. The probe used in the “viscostat” was of the vibrating reed type capable of viscosity measurement at high shear rate (above 200 cP) and set to a viscosity of about 2400 cP. The problem with such sensing systems is the tendency of many exopolysaccharideproducing cultures, held in vessels with baffles, to show regions of localized polymer accumulation and increased viscosity together with wall growth within the fermenter. Adhesion to the baffles may also occur and, with both eukaryotic and prokaryotic micro-organisms, non-uniform growth of cultures may be a normal feature. Evans et al. (1 979) whose continuous-culture experiments extended over 37 days or more, appear to have had fewer problems with contamination in cultures of Xanthornonas juglandis (a host-specific variant of X . carnpestris). In tests of various nutrient limitations at the same dilution rates, it became clear that nitrogen or sulphur limitation yielded most polymer (23-27 g 1- ’) and the highest viscosities as ascertained from K values (i.e. the apparent viscosity of a 1% solution extrapolated to zero shear). The values reported were in the range 11,500 to 18,000cP. Significantly, in nitrogen-limited defined salts medium, there was no evidence of culture deterioration after 900 hours of growth, thus disposing of one of the main alleged drawbacks of continuous culture at least for this bacterium under the particular conditions employed. Yet when the carbon source was limiting, culture variants were found. The sulphur-limited cultures were maintained for 12 weeks with yields of 27 g polysaccharide 1- and approximately 60% conversion of the supplied substrate. Associated with the high yields of polymer was high culture stability. This was also found with other strains of X . carnpestris and X . juglandis, although considerable variations in polysaccharide yield and solution viscosity were also noted. One strain in particular produced high yields of acetone-precipitable polymer (33 g IF1), but the calculated K value was

B IOSY NTH ESIS 0 F MICROBIAL EX0 PO LYSACC HAR ID ES

1

50

93

c .-0

-

L -

40

30 20

-10

t

5

0.

t

3 -

0:

a

'i

Ei L

o

&7

0

x

0.5

u r n v 0

In

h -

0

n

4/ I 0 0.03

I

I

I

'0

0.04 0.05 0.06 Dilution r o t e ( h - ' )

FIG. 5. Effect on polysaccharide production of dilution rate in sulphur-limited cultures of Xanthomonas juglandis. o Indicates amount of polysaccharide produced, concentration of cells, glucose consumption, rn ratio of polysaccharide produced to glucose consumed, n culture viscosity (K value) and A rate of polysaccharide production. Reproduced with permission from Evans et al. (1979).

only 4400 cP. The effect of different dilution rates in medium containing 24 mg S 1-1 and 50 g glucose 1-' was examined (Fig. 5). Little variation in the amount of polysaccharide was found with dilution rates in the range 0.03-0.05 h - l , but higher dilution rates caused a considerable decrease in the yield of polysaccharide. These results are thus not dissimilar from those reported by Jarman (1979) for bacterial alginate production. Evans et al. (1979) also found relatively little variation in the viscosity of polysaccharide from X . jugfundis produced at different dilution rates. The composition of

TABLE 4. Production of polysaccharide by Xanthomonas campestris in chemostat culture Data from Davidson (1978) Limiting nutrient

Cell yield (g dry wt. 1 - I ) Polymer yield (g dry wt. 1 ') Ratio of polymer: cells (g g - l ) Composition of polymer Ratio of mannose: glucose Content of uronic acid (%) Content of acetate (%) Content of pyruvate (%)

Glucose

Nitrogen

Sulphur

1.1 2.7 2.45

1.6 7.0 4.38

1.8 6.4 3.56

1.1 21 4.9 5.8

1.1 21 3.1 6.6

1.2 18 -

83

Magnesium 1.5 5.7 3.8 1.1 16 5.2 1.1

Potassium 1.7 5.3 3.12 1.1 23 4.5 5.5

Phosphorus 2.9 5.0 1.72 1.2 17 -

0.9

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

95

the polymers showed greater variation than that reported by David’son(1978) for X . campestris (Tables 4 and 5). Of particular interest were the products obtained in potassium and magnesium-limited media. These contained little uronic acid, no acetate or pyruvate and less mannose than did other preparations. It seems possible that these products resemble the polysaccharides from mutants of X . campestris described by Whitfield (1979) and may lack much of the normal side-chain structure. This phenomenon has apparently not been noticed in studies of other exopolysaccharides of simpler structure. It may be restricted to types such as xanthan and colanic acid, which have trisaccharide or larger side chains. The differences between the results of Davidson (1978) and Evans et al. (1979) may possibly stem from the different media used to obtain the respective limitations. Despite the similarities noted in polysaccharide production by the different species alluded to above, differences can also be expected. The stability of strains varies considerably, but, in general, carbon limitation is most likely to yield mutants deranged in polysaccharide synthesis (e.g. Andreesen and Schlegel, 1974). It should be remembered that, as yet, only a relatively small number of bacterial species have been studied in continuous culture. Production of dextrans by Leuconostoc mesenteroides in continuous culture depended on the carbon substrate employed when the carbon and energy source was limiting (Lawford et al., 1979). Cultures limited in glucose or maltose failed to synthesize extracellular dextransucrase. Under sucrose limitation, after a critical sucrose concentration was reached, total extracellular enzyme activity increased with increasing dilution rate. Thus, as already found for other heteropolysaccharide-producing bacteria, under conditions of carbon limitation, substrate is diverted from cell synthesis to extracellular polymer production.

111. Precursors A. S U B S T R A T E S

In the case of three of the five types of polysaccharide outlined in the introduction, polymer composition is independent of the nature of the carbonaceous substrate. Provided an uptake mechanism exists, the substrate would be converted, at least in part, into polymer. In the two other groups, a specific substrate is required. One bacterium studied in our laboratory was unusual in requiring sucrose as a specific substrate for the production of a heteropolysaccharide containing D-glucose, D-mannose, D-galactose and D-galacturonic acid (Sutherland and Williamson, 1979). It was assumed that the biosynthetic mechanisms were similar to those needed for the production

TABLE 5. Composition of the polysaccharide produced by Xanthomonas juglandis XJ107 grown in chemostat culture under conditions of different nutrient limitations Data of Evans et al. (1979) reproduced with permission. The culture medium was in each case chemically defined, save in (a) when a complex medium was employed Limiting element

Nitrogen'"' Nitrogen Carbon Potassium Magnesium Phosphorus Sulphur

Content (%) in polysaccharide of:

Dilution rate (h-l)

0.03 0.03 0.045

0.03 0.03 0.03 0.035

Glucose

Mannose

Glucuronic acid

Rhamnose

Acetate

Pyruvate

36.3 34.0 38.0 75.0 63.3 29.9 34.3

23.2 26.3 24.1 5.6 10.7 29.0 30.8

18.0 18.0 15.2 2.0 7.1 17.0 18.1

5.0

3.0 3.0 0.5

1.6 1.7 1.8

5.7

15.0 7.5 7.5 2.0 1.4

~

-

-

-

3.0 1.6-3.2

3.7 1.5-5.4

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

97

of other heteropolysaccharides and that the sucrose requirement was associated with specific uptake mechanisms. A mutant strain was capable of forming polymer from glucose. Associated with the specific sucrose requirement in the wild-type bacterium was a further requirement for nitrate; in the presence of other nitrogen sources growth, but not polysaccharide synthesis, occurred. In a strain of Achromobacter mucosum, starches and dextrin were similarly needed for the production of a polysaccharide (Ozawa et al., 1972). Dextrans, mutans and levans can only be synthesized from sucrose. Although the bacteria that synthesize these polymers can grow in the absence of sucrose, no exopolysaccharide is formed. This has been very elegantly demonstrated by electron microscopy for one strain of Leuconostoc mesenteroides (Brooker, 1977). Cells harvested from exponential phase cultures totally lacked extracellular polymer when no sucrose had been provided. After 120 minutes exposure to sucrose, a layer of polysaccharide 1 10-130 nm thick was revealed. Insoluble dextran appeared as short filaments radiating from the surface coat and continued to accumulate. The conversion of sucrose into polymer proceeds at the cell surface so does not involve the intermediates required in heteropolysaccharide synthesis. There is also no competition for precursors used in wall formation. It should, however, be remembered that some bacteria may produce both levans and dextrans (Gibbons and Nygaard, 1968) or levans and mutan (Scales et al., 1975). In such systems, the absolute requirement for sucrose remains, but polymer synthesis may be affected by provision of other oligosaccharides and dextrins.

B. S U G A R N U C L E O T I D E S

With the exception of dextrans, levans and mutan, exopolysaccharide synthesis is dependent on the availability of sugar nucleotides. These compounds which are mainly nucleotide diphosphate monosaccharides, provide an activated form of the monosaccharide and represent the immediate precursors of the oligosaccharide repeating units of the heteropolysaccharides. As such, the potential exists for regulation of polysaccharide synthesis via control exercised on the supply of sugar nucleotides. Some of the sugar nucleotides are precursors of exopolysaccharides only (e.g. UDP-galacturonic acid, GDPmannuronic acid), whereas others may also be used in the formation of the wall polymers, lipopolysaccharides and teichoic acids (e.g. UDP-galactose). The anabolism of glucose in the prokaryotic cell (in contrast to its catabolism) proceeds via the formation of glucose 1-phosphate and thereafter of either UDP-glucose or ADP-glucose (Fig. 6). Other sugars are not metabolized by such divergent pathways. In the eukaryotic microbial cell, UDPglucose is the precursor of extracellular and wall polymers and also of

98

1. W. SUTHERLAND GLUCOSE.

-

GLC-6. V

-

GLC - 1.P

----_-----ADP- GLUCOSE PYROPH 0 s PHORY LASE

PYRO PHOSPHORYLASE

I

UDP- GLUCOSE~

- GLC

(GLC 15 4 GLC 1 GLYCOGEN

/ ExoPoL-saccHamns

ADP

L VS

FIG. 6. The possible anabolic fates of glucose. Abbreviation: LPS, lipopolysaccharide

glycogen; ADP-glucose formation is not the prelude to the specific synthesis of carbon and energy storage polysaccharides in contrast to other glucosecontaining macromolecules, as is the case in prokaryotes. The sugar nucleotides function both as glycosyl donors and as the precursors of many monosaccharides. The latter role was reviewed by Ginsburg (1964). Thus, UDP-glucose represents not only an activated precursor for glycosyl moieties, but also a precursor in the formation of D-galactose, D-glucuronic acid and other monosaccharides. It was found in relatively high concentrations, both in enteric bacteria forming exopolysaccharides and in mutants incapable of polymer formation (Grant et al., 1970). GDP-mannose, either as a direct precursor of mannose or by way of GDP-fucose as the precursor of fucose, was also found at relatively high concentrations in these bacteria whereas UDP-glucuronic acid, solely employed in these organisms as a precursor of glucuronic acid, was present at concentrations of 1 pmol (g dry weight of cells)- l . Almost without exception, the presence of a particular monosaccharide in an exopolysaccharide requires that the microbial cells form a nucleotide precursor of that monosaccharide. The one known exception in microorganisms is t-guluronic acid found in bacterial alginate. This sugar derives from GDP-mannose and GDP-mannuronic acid (D-mannuronic acid is the C-5 epimer of L-guluronic acid), but is not formed at the nucleotide level. Instead, an epimerase functioning at the polymer level converts some of the D-mannuronosyl residues into L-guluronic acid (Haug and Larsen, 1971). So far, this is the only mechanism of this type that has been discovered in the synthesis of microbial exopolysaccharides. Yet a number of monomers have been found in microbial exopolysaccharides for which, as yet, no corresponding nucleotide derivatives have been found. Some of these are amino-uronic acids and complex deoxysugars of the type found in the polymer from Achromobacter georgiopolitanum (Smith,

B IOSY NTH ESI S 0 F MICRO 6IAL E X 0 PO LYSACCHAR ID ES

99

1968). Others are simpler compounds-methyluronic acids are found in Rhizobium polysaccharides (Dudman, 1978), as are methylhexoses (Dudman, 1976; Kennedy, 1978); L-guluronic acid is present in polymers from Beijerinckia indica (Haug and Larsen, 1970). The mode of synthesis of these monomers is not clear. It is possible that they are methylated by mechanisms similar to those operating on other polymers and probably, as in most C, transfers, involving folic acid derivatives. Al+.ernatively,a biosynthetic route accomplished by mechanisms similar to those involved in the formation of pyruvylated sugars might be postulated. An ethylidene derivative of Dgalactose was detected in the polysaccharide from a strain of Salmonella typhimurium (Garegg et al., 1971b). It is thus possible that after addition of a pyruvate ketal to form the carboxyethylidene monosaccharide, decarboxylation could occur. Obviously, this aspect of polysaccharide synthesis demands further investigation. Similarly, L-guluronic acid in polymers from which D-mannuronic acid is absent is assumed to come directly from a nucleotide precursor rather than by post-polymerization modification as in bacterial alginate (Haug and Larsen, 1971). C. L I P I D INTERMEDIATES

Following the identification of isoprenoid lipid intermediates in the synthesis of lipopolysaccharides and peptidoglycan (Weiner et al., 1965; Osborn and Weiner, 1968; Wright et al., 1965), Troy et al. (1971) demonstrated their involvement in the synthesis of an extracellular bacterial heteropolysaccharide. The lipid involved was indistinguishable from that required for the formation of the other polymers, being undecaprenyl phosphate. Membranes from which the lipid had been removed by extraction with an organic solvent regained their activity on the addition of undecaprenyl phosphate. The sequence of reactions involved in production of the polysaccharide repeating unit was recognized by Troy et al. (1971) as: UDP-Gal + P.lipideGal.P.P.lipid

+ UMP

+ GDP.Man + Man-Gal-P.P.lipid+ GDP Man-Gal-P.P.lipid+ UDP.GlcA GlcA-Man-Gal-P.P.lipid+ UDP GlcA-Man-Gal-P.P.lipid+ UDP.Gal + Gal-Man-Gal-P.P.lipid+ UDP

Gal.P.P.lipid

+

I

GlcA

A similar series of reactions was postulated for the production of another polysaccharide of Enterobacter (Klebsiella) aerogenes (Sutherland and Norval, 1970) on the basis of the properties of mutants defective in the different enzymes in the sequence. These produced lipid intermediates to which 2 moles, 1 mole or no galactose was attached after the initial transfer

I. W. SUTHERLAND

100

TABLE 6. The transfer of monosaccharides to lipid in Enterabacter aerogenes Type 8 mutants Presumed intermediate

Monosaccharide

Transferase Wild type Mutant 027, 034, 036 031, 032 029, 038 037

Glucose 1-Phosphate

+ + + + -

Disaccharide Trisaccharide Galactose I

Galactose I1

-

-

-

-

+ + +

+ +-

of glucose 1-phosphate from UDP-glucose. Mutants lacking the glucose 1phosphate transferase were also identified (Table 6). Cell-free systems such as these from strains of Enterobacter aerogenes functioned at pH 7.6-7.9 in the presence of MgZ . The sequential transfer of sugar 1-phosphate, followed by sugars, from sugar nucleotides to isoprenoid lipid phosphate is analogous to the process of 0-antigen heteropolysaccharide synthesis in Salmonella species (Wright and Kanegasaki, 1971). In both systems, enzyme specificity is such that one sugar must be transferred to make a suitable acceptor for the next in the sequence. In both of the Enterobacter aerogenes systems studied, the repeating unit of the heteropolysaccharide was a tetrasaccharide. The largest intermediate attached to lipid by the cell-envelope fraction was a lipidpyrophosphateoctasaccharide, i.e. two repeat units. Yields of this intermediate were much lower than those of the lipidpyrophosphate tetrasaccharide (Troy et al., 1971). No evidence for oligosaccharides intermediate in size between one and two repeat units has been found. This too suggests a close similarity to lipopolysaccharide synthesis in which, after attachment of the repeat unit to lipid, multiples of the repeat unit are formed by transfer to the reducing end of a growing chain: +

R-0-H-H-P-P-lipid + H-.-0-R-P-P-lipid + .-H-R-H-W-M-H-R-P-P-lipid + P-P-lipid + 0 - 0 - 0 - n - P - P - l i p i d + .-R-.-.-H-R-R-M-n-0-O-O-p-p-lipid + P-P-lipid

The involvement of lipid intermediates in the biosynthesis of a homopolysaccharide was reported by Garcia et al. (1974) using cell-free preparations from Acetobacter xylinum; although Colvin (1959) had suggested that such intermediates were involved in production of bacterial cellulose, the nature of the ethanol-soluble intermediates was not established. The preparation used by Garcia et al. (1974) was frozen and thawed, then treated with EDTATris at pH 8.0 to remove some of the high UDP-galactose 4-epimerase activity which was initially present. Incorporation of 14C-labelled glucose from UDP-glucose into butanol-soluble material was observed and, as with

6 I OSYNTH ESlS

0F MICRO 6 IAL E X 0 POLYSACC HAR I D ES

101

the Enterobacter aerogenes systems, an absolute requirement for Mg2 was noted. The lipid intermediates presumed to be cellulose precursors were characterized as lipidpyrophosphate glucose and lipidpyrophosphate cellobiose; the intermediates were not found in strains of Acetobacter xylinum that failed to form cellulose. In a recent study using membrane preparations from Acetobacter xylinum, Sandermann and Dekker (1979) reported the formation of a D l +2 linked polymer rather than cellulose, but they did not indicate whether any lipid-linked intermediates were involved in its formation. As the production of this polymer was unaffected by bacitracin, the involvement of a lipidpyrophosphate glucose intermediate appears unlikely. However, inhibition by moenomycin was observed. This antibiotic has been reported to inhibit reactions involving lipidphosphate hexose and an intermediate of this type may, therefore, be involved. As well as the lipid intermediates reported by Garcia et al. (1974) from Acetobacter xylinum preparations, this bacterium has been the source of acid-labile lipid-linked maltose (Sandermann, 1977). The role of this compound has not been elucidated and it is clear that Acetobacter xylinum may form a number of different products under laboratory conditions, the relative roles of which require further study. Although there is good evidence, therefore, for the involvement of lipid intermediates in some microbial systems, they are not required for dextran or levan synthesis. It is also possible that they are not needed for the formation of some other exopolysaccharides. There may be some analogy to the production of polyribitol phosphate by Staphylococcus aureus (Fiedler and Glaser, 1974a, b). In this system, unlike peptidoglycan or lipopolysaccharide synthesis, no evidence was obtained for the involvement of the isoprenoid lipid pyrophosphate type of intermediate. Examination of cell-free preparations of Azotobacter vinelandii by Scott ( 1 979) failed to reveal lipidlinked precursors of bacterial alginate. Similarly, C . L. Saunderson (unpublished work) in our laboratory failed to obtain transfer of mannose from GDP-mannose to lipid-linked material using cell-free preparations of Xanthomonas campestris; glucose from UDP-glucose was transferred by such preparations into lipid-soluble material and into “polymer”. Thus the expectation that all bacterial extracellular heteropolysaccharides would be formed in a manner similar to those of Enterobacter aerogenes has not been fulfilled. The possibility exists of the involvement in exopolysaccharide synthesis of mechanisms similar to those recently detected in the synthesis of some enterobacterial lipopolysaccharides. Although the “side chains” of many lipopolysaccharides are probably formed using isoprenoid lipid pyrophosphate intermediates (Wright et al., 1965; Wright and Tipper, 1979), some homopolymeric side-chains are produced by a different mechanism. Kopmann and Jann (1975) studied the mannan-synthesizing system of E. coli 09. The polymer was formed from GDP-mannose, but did not utilize lipid-linked +

102

I. W. SUTHERLAND

intermediates of the isoprenyl alcohol type in the mannose transfer reactions. In contrast to lipopolysaccharide (heteropolysaccharide) side-chain production in many species of Salmonella, the homopolysaccharide 0 antigens of E. coli 09 and related strains were found to grow at the reducing end of the molecule (Flemming and Jann, 1978a, b). It was necessary to supply glucose in the growth medium as glucose was present at the reducing end of the mannan chain. The mannose from GDP-mannose was transferred to a hydrophobic carrier extractable with butan-I -01 and presumed to be a glucolipid intermediate in the biosynthesis of the E. coli mannan (Kanegasaki and Jann, 1979). The possibility that such precursors may also play a role in those biosyntheses of exopolysaccharides wherein isoprenoid lipids cannot be identified as intermediates should now be investigated. The likelihood that more than one lipid intermediate might be involved in the synthesis of an exopolysaccharide was raised by Johnson and Wilson (1977). In a study of colanic acid (Fig. 2a) production in E. coli, a lipid intermediate of the isoprenylpyrophosphate sugar type was identified on the basis of its chromatographic behaviour and the inhibition of synthesis of the lipid intermediate by bacitracin. In addition, undecaprenyl phosphate glucose was tentatively identified. That the formation of more complex repeating units such as the acetylated and pyruvylated hexasaccharide of colanic acid requires more than one lipid-linked intermediate thus remains a possibility. Again, an analogy can be found in the route of synthesis of Salmonella lipopolysaccharide. In some of these polymers, repeating units composed of pentasaccharides are found. Formation of the main chain involves the assembly of the monosaccharides on a lipid-pyrophosphate intermediate, but addition of the glucose side-chains (Fig. 7) requires a lipid-monophosphate glucose as donor (Wright, 1971; Nikaido and Nikaido, 1971). One exopolysaccharide is definitely formed from isoprenoid phosphate intermediates. In E. coli K235, the exopolysaccharide is sialic acid, poly N-acetylneuraminic acid. Extraction of the sugar-linked lipid from this strain indicated that 95% was composed of the C,, isoprenoid alcohol and the remainder was the CG0isoprenologue. The undecaprenyl phosphate was essential for sialic acid synthesis (Troy et al., 1975) although ficaprenylphosphate also partially restored activity. The lipid was involved in the reaction: CMP-N-acetylneuramic acid

+ P-undecaprenol& N-acetylneuraminyl-P-undecaprenol + CMP

As was to be expected, bacitracin failed to inhibit this reaction. In intact membranes, addition of exogenous lipid stimulated polysaccharide synthesis (Vijay and Troy, 1975). The incorporation of the extra lipid was temperaturedependent, requiring the membrane fluidity found at 37°C. At 20°C, which is below the membrane-lipid transition temperature, exogenous lipid was not

BIOSYNTHESIS

OF MICROBIAL EXOPOLYSACCHARIDES A be

LI P I D-P-P - G a l - R h a - P i a n

’

Abe

- Gal-

Abe

Glc

Rha-han-(hal-

103

Rha-dan)n

LIPID-P-Glc

1 Abe LIP1 D - P-P-Gal -,ha-dan

Abe

Glc

GlC

Abe

-La1 -Rha-dan~dal-Rha-dan)”

+LIPID-P

FIG. 7. Glucosylation of the lipopolysaccharide of Salmonella typhimurium.

inserted. Through growth of E. cofi at 15”C, it was possible to uncouple synthesis of the polymer from the synthesis of the lipid-linked intermediate (Troy and McCloskey, 1979). The cells grown at 15°C either failed to synthesize an endogenous acceptor or failed to assemble it; the sialyl transferase was synthesized. The acceptor was characterized as a homopolymer of sialic acid with a degree of polymerization about 12. The whole process of polymer formation probably required a second enzyme catalysing the formation of an oligomer linked to undecaprenol: N-acetylneuraminic acid-P-undecaprenol

+

(N-acetylneuraminic acid).-P-undecaprenol

+ P-undecaprenol

together with a transferase: N-acetylneuraminic acid-P-undecaprenol

+ acceptor +

N-acetylneuraminic acid-acceptor

+ P-undecaprenol

Thus even a homopolymer such as sialic acid requires at least three enzymes for the jinaf specific stages of its synthesis; additional enzymes, specific to synthesis of the polysaccharide, form the sugar nucleotide CMP-Nacetylneuraminic acid. Sialic acid is not the only exopolysaccharide which is formed by mechanisms insensitive to bacitracin. Hyaluronic acid can be synthesized by particulate preparations from a species of Streptococcus using UDPglucuronic acid and UDP-N-acetylglucosamine as precursors (Stoolmiller and Dorfman, 1969). No evidence was found for the involvement of lipid intermediates nor was synthesis affected by the addition of bacitracin. The mechanism of chain growth was considered to be through the addition of

1 04

I. W. SUTHERLAND

monosaccharides to the non-reducing end of the polymer chain. Unlike certain other systems in which sugar transfer from UDP-sugar nucleotides was detected, UMP was not a reaction product. It is not clear why certain polymers are formed from lipidmonophosphate precursors, whereas others require as precursors lipidpyrophosphate derivatives. The possibility of separate isoprenoid lipid pools for peptidoglycan, lipopolysaccharide and exopolysaccharide synthesis appears to be unlikely because of the fluidity of the lipid (Wright and Tipper, 1979). Whereas lipid monophosphate derivatives are utilized for modification of polysaccharides in lipopolysaccharide synthesis, their role in exopolysaccharide formation is in formation of the actual polymer. In this respect, the process of sialic acid production resembles that described by Scher and Lennarz (1969) for synthesis of a cell-bound mannan by Micrococcus lysodeikticus. The only eukaryotic exopolysaccharide in which the involvement of a lipid intermediate has been reported is pullulan (Taguchi et al., 1973). Isotopicallylabelled sucrose incubated with preparations from Aureobasidium pullulans yielded glycosylated lipid which appeared to have the characteristics of a lipid-pyrophosphate glucose.

IV. Control of Polysaccharide Production A . REGULATORY MECHANISMS

1. The Microbial Surface

'

Numerous factors influence the rate and the amount of polysaccharide synthesis. The physiological effects of various growth conditions have already been considered, but the actual mechanisms involved are much less clear. In effect, the initial site at which control mechanisms can influence polysaccharide production is nutrient uptake. As heteropolysaccharide-producing micro-organisms require intracellular substrate for polymer production, any system regulating substrate uptake is likely to affect polysaccharide synthesis. Ideally, as much substrate as possible should enter the microbial cell and be converted into polysaccharide rather than be converted into cell material or catabolized to products excreted from the cell. In fact, very high conversion of substrate into polysaccharide can be achieved, so nutrient uptake may not normally be a limiting factor. The presence of an outer-membrane protein associated only with capsulate strains of E. coli raised the possibility that such a protein might be responsible

BI OSY NTH ESI S 0 F MICROBIAL E X 0 PO LYSACCHAR I D ES

105

for some aspect of capsule synthesis or structure (Paakanen et al., 1979). The protein had a molecular weight of approximately 40,000 and was apparently different from previously described outer-membrane proteins. Gayda et al. (1979) also reported the identification of an outer-membrane protein which was regulated by the cap R gene product. Wild-type bacteria grown at suboptimal temperatures contained less of the protein (designated a or 36) and it was postulated that this might lead to enhanced polysaccharide synthesis under these conditions. A 2 megadalton fragment of E. coli K-12 DNA when cloned in plasmid pSClOl was found to specify seven polypeptides, one of which was protein a; the synthesis of capsular polysaccharide was inhibited in cup R mutants into which the hybrid plasmid (pMC44) had been introduced by transformation. Plasmid mutants unable to repress capsule synthesis either failed to synthesize the 40K outer-membrane protein a or were defective in synthesis of other (25K or 14.5K) polypeptides. The protein of Gayda et al. (1979) thus has a negative correlation with capsule synthesis in E. coli, whereas the production of a protein of the same molecular weight which was observed by Paakanen et al. ( 1 979) was positively associated with capsulation. As polysaccharide synthesis primarily involves processes at the cytoplasmic membrane or within the cytoplasm, it is difficult to advance a role for outer-membrane proteins in polymer producrion. It would seem more likely that such protein would have a role in either secretion or attachment (see pp. 127 and 125). Control is, therefore, most probably exercised over the manufacture and availability of intermediates.

2 . Regulation of Homopolysaccharide Synthetases The regulation and control of homopolysaccharide synthesis has, like most polysaccharide synthesis, received relatively little attention. Caulfield et al. (1979) studied the control and secretion of levansucrase in Bacillus subtilis. The enzyme was excreted independently from cell division, as nalidixic acid stopped cell division, but did not affect levansucrase formation and release from the bacteria. The enzyme was inducible. When bacteria were induced with sucrose, an additional membrane protein with a molecular weight of 5.6. lo4 was observed. This protein had not been noticed in non-induced cells. The protein was associated with lipid and addition of 0.2 mM quinacrine inhibited release of the enzyme although it did not affect lipid synthesis. Cerulenin, a compound that specifically affects lipid synthesis, was found when added to B. subtilis to inhibit both the synthesis and the release of levansucrase. There was thus a close similarity between levansucrase export and the process of penicillinase secretion. Surprisingly, the dextransucrases of Leuconostoc r n ~ ~ s e n t ~ ~ r are o i c ~in~~s ducible enzymes (Kobayashi and Matsuda, 1974), whereas the corresponding

106

I. W. SUTHERLAND

enzymes from Streptococcus sanguis and Streptococcus mutans (Carlsson and Elander, 1973; Guggenheim and Newbrun, 1969) are constitutive and are freely produced in the absence of sucrose. In those species that produce the constitutive enzyme, its formation is associated with the logarithmic phase of bacterial culture growth. Addition of inhibitors of protein synthesis caused cessation of dextransucrase synthesis. Although various workers have postulated a possible role for plasmids in the control of dextransucrase production by Strep. mutans, no strain totally lacking plasmids is completely incapable of glucan synthesis (Montville et al., 1978) and few strains (4%) from human cariogenic isolates carried plasmid DNA.

3. Isoprenoid Lipids In those exopolysaccharides which are formed through the mediation of isoprenoid lipid intermediates, the availability of isoprenoid lipid pyrophosphate may provide a means of regulating polysaccharide synthesis. This possibility has been considered already in the light of some indirect evidence (Sutherland, 1977b, 1979a). Isoprenoid lipids are involved in glycosylation mechanisms in both prokaryotic and eukaryotic cells. In eukaryotic microorganisms, the isoprenoid lipids are of the dolichol type, C,,-C, compounds present in small amounts. Hemming (1977) proposed that in mammalian tissue the concentration of dolichol phosphate is probably the rate-limiting step for glycosylation processes. The situation in eukaryotes is probably more complex than that found in prokaryotes as Lehle and Tanner (1978) detected a number of glycolipids, the major one being dolicholmonophosphate mannose. The only alkali-insensitive component detected was tentatively identified as a dolicholdiphosphate oligosaccharide. The oligosaccharide appeared to have a degree of polymerization (DP) of 12 or more and the probable structure of the compound was dolichol-P-P-(GlcNAc),Man,. In the capsulate yeast Cryptococcus laurentii, transfer of galactose to an endogenous acceptor was detected but clearly did not involve a lipid (Raizada et al., 1974). This system was almost certainly a component of the means of synthesis of cell envelope glycoprotein rather than of the exopolysaccharide capsule. However, as in the yeast system described by Lehle and Tanner (1978), a relatively large oligosaccharide, probably a dodecasaccharide, was formed. Better evidence for the involvement of isoprenoid lipids in the control of polysaccharide synthesis has been found in prokaryotes, but in these cells too, a family of lipids is found. The predominant isoprenoid lipid found in bacteria is the C, alcohol bactoprenol, but others of C5,, C,, and other chain lengths have been detected. In the cell-free system which synthesizes Salmonella typhimurium lipopolysaccharide, the formation of lipid-linked galactose (the first step in the sequence) is stimulated

,,

,

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

,

101

less by C,, and C,, polyprenylphosphates than by C, polyprenylphosphate; the effect of C,, or C,, polyprenylphosphates was much less (Mankowski et al., 1977). Systems that synthesize exopolysaccharides do not appear to have been similarly tested, but it seems unlikely that the control of polymer synthesis is achieved through the utilization of polyprenylphosphates of different chain length. In the yeast system involving lipid-P-P-GlcNac, synthesis, c,,, c,, and c,,, dolichyl phosphates were all functional acceptors (Palamarczyk et al., 1979). More probably, control is exerted through the availability of lipid phosphate which is required for the synthesis of wall and extracellular polymers. As outlined by Wright and Tipper (1979), the existence of separate pools of lipid for each polymer is unlikely. After synthesis from isopentenylpyrophosphate (Christenson et al., 1969), the lipid is in the pyrophosphate form. This is also true after completion of polysaccharide synthesis except for sialic acid (p. 102). To permit entry of lipid into a fresh synthetic cycle, a specific phosphatase is required. This enzyme has been identified in Micrococcus lysodeikticus (Goldman and Strominger, 1972). It is assumed that a similar enzyme exists in other microbial species in which isoprenyl pyrophosphates are formed. The concentration of lipid phosphate could be controlled by dephosphorylation to yield the free alcohol (Willoughby et a[., 1972). Rapid degradation of C, lipid phosphate appeared to occur in preparations obtained from Streptococcus pyogenes (Reusch and Panos, 1976) and the frequent discovery of free isoprenoid alcohol, as for example in Streptococcusfaecafis (Umbreit et al., 1972) and other bacteria, indicates that such specific phosphatases may be of widespread occurrence. To return the lipid to a functional state, an ATP-dependent phosphokinase is necessary and such enzymes have been identified in both Gram-positive (Higashi et al., 1970) and Gram-negative bacteria (Poxton and Sutherland, 1974). A system of regulation through lipid phosphate availability, of the type indicated in Fig. 8, can thus be postulated. In most cells, there is presumed to be sufficient isoprenoid lipid present to permit the simultaneous synthesis (in the Gram-negative bacterium) of peptidoglycan, lipopolysaccharide and exopolysaccharide, as all these polymers are normally produced in the growing bacteria (Sutherland, 1979a). In other species, exopolysaccharide is a product of the late logarithmic phase or early stationary phase of growth in batch cultures. It is possible that in such bacteria, there is insufficient isoprenoid lipid to allow polysaccharide synthesis in the growing cell, but once there is no further requirement for the lipid cofactors to be utilized for lipopolysaccharide and peptidoglycan synthesis, isoprenoid alcohol becomes available. In mutant strains of Sulmonella unable to transfer 0-antigens from lipid to acceptors in the core structures, exopolysaccharide synthesis was not observed (Sutherland, 1975), presumably because insufficient lipid was available. Other types of mutant defective in lipopolysaccharide formed the exopolysaccharide colanic acid.

I. W. SUTHERLAND

108

ISOPENTENYL PYROPHOSPHATE

I PP

+

FARNESYL PYROPHOSPHATE

POLYMER

-

MUCOPEPTIDE LPS orTEICHOIC ACID EXOPOLYSACCHAR IDE

INTRACELLULAR and MEMBRANE- BOUND PRECURSORS ATP

I PA

FIG. 8. A scheme for the possible regulation of carrier lipids involved in polysaccharide synthesis. IPP represents C, ,-isoprenyl pyrophosphate, IP represents C, ,-isoprenyl phosphate and IPA represents C,,-isoprenyl alcohol.

The production of exopolysaccharide is frequently enhanced at suboptimal incubation temperatures and this too could be a manifestation of increased lipid availability, less being needed under these conditions for production of wall polymer. Some unusual mutants of several strains of Enterobacter aerogenes isolated in our laboratory several years ago failed to form any polysaccharide at low temperature (20°C) and also formed less lipopolysaccharide at that temperature. However, at 37"C, the bacteria produced normal lipopolysaccharide and exopolysaccharide (Norval and Sutherland, 1969). The possibilities of insufficient isoprenoid lipid being synthesized in these mutants when grown at 20°C to permit wall and exopolysaccharide synthesis and of priorities in its utilization, have been discussed earlier (Sutherland, 1977a, b, 1979a). In this respect, there is a similarity to the isoprenoid lipid limited L-forms of Streptococcus pyogenes studied by Reusch and Panos (1976) in which wall synthesis and isoprenoid lipid availability were directly correlated. A means of testing the role of isoprenoid alcohol kinase in regulating the availability of lipid intermediates for exopolysaccharide synthesis has been made available by the discovery of moenomycin. This antibiotic was shown by Sandermann (1976) to act as an inhibitor of C,, isoprenoid alcohol kinase. Inhibition is probably due to the close chemical similarity of part of the antibiotic molecule to isoprenylphosphate (see facing page). The only problem with regard to its use, may be the differences known to exist between the kinase of Staphylococcus aureus described by Sandermann and Strominger (1 972) and the corresponding enzyme studied in Enterobacter aerogenes (Poxton et al., 1974).

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

109

0

Ib’

CH3

I

CH n--C=CH--CH2--CH

CHn

I

CH2

I1

CH 3

I

CH3

I

2 4 = C H - - C H 2 4 4 H 2 4 H 2- C--CH= C H - - C H 2 - C H 2 4 = C H 4 H 2 - O -

I

r,s

CH3

Structures of (a) C55-isoprenylmonophosphage and (b) of the Cz5-moenocino! moiety

4. Genetic and Enzyme Regulation

Less is known about the regulation of exopolysaccharide synthesis than of lipopolysaccharide formation, but control of the means of production of the two polymers cannot be entirely separated. Several of the precursors may be common to both types of molecule and loss of lipopolysaccharide synthesis may also involve inability to produce exopolysaccharide. Genetic studies on polysaccharide regulation have mainly been confined to Streptococcus pneumoniae and enterobacteria. Consequently, our understanding of the genetic controls exercised on polysaccharide production is essentially limited to these two dissimilar groups of bacteria (see review by Markovitz, 1977). Austrian et al. (1959) used transformation to restore polysaccharide production to mutants of Strep. pneumoniae defective in UDP-glucose dehydrogenase activity and consequently unable to synthesize UDP-glucuronic acid. Twenty different single-site mutations were recognized in the gene controlling the dehydrogenase. The studies on Strep. pneumoniae revealed the interesting phenomenon of binary capsulation, in which bacteria simultaneously produced type I and type I11 polysaccharides after transduction of DNA from type I cells to type I11 recipients (Bernheimer and Wermundsen, 1969). The only enzyme common to the biosynthetic pathways of the two polysaccharides was UDP-glucose dehydrogenase. The binary capsulation was ascribed to the presence in the recipient cells of non-homologous loci for the enzyme derived from each type. Various models for the genetic overlap were later proposed (Bernheimer and Wermundsen, 1972). Segments of DNA from donor (type I) bacteria were thought to carry the capsular gene at one end and the area for homologous pairing at the other. In contrast, segments leading to type I material only being synthesized, were considered to carry the capsular genome near the middle of the segment. The presence of a locus controlling capsulation in several K serotypes of

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I. W. SUTHERLAND

E. coli has been recognized (Stocker and Makela, 1978). The gene or genes forming the kps locus located near serA are well separated from genes known to control lipopolysaccharide production. Other genes controlling capsular production mapped near his to which they were closely linked. Strains of E. coli with capsular polysaccharide and the O(lipopo1ysaccharide) antigenic types 08 and 09, simultaneously expressed two groups of genes linked to his-one determining the anionic polymer, the other an uncharged 0 antigen. Stocker and Makela (1978) proposed that, in strains of this type, the neutral polymer might be assembled sequentially on a non-isoprenoid carrier and controlled by the rfe gene whereas the exopolysaccharide was assembled on the isoprenoid carrier under the regulation of his-linked genes. Polymerization would then be determined by a gene between trp and gal independent of rfe function. Certainly, Schmidt et al. (1976) noted that the synthesis of K antigens was unaffected by the loss of rfe function in several 0 serotype 08 and 09 strains, but the involvement of isoprenoid lipid intermediates in production of these capsular polysaccharides remains to be investigated. In E. coli strains that produce colanic acid, mutations in the capR locus cause overproduction of the exopolysaccharide as well as other effects. Several enzymes involved in the synthesis of precursors were formed at a higher rate in capR mutants. Such mutants also showed 2 to 4-fold derepression of the three enzymes controlled by the gal operon (Mackie and Wilson, 1972). One of these three enzymes is UDP-galactose 4-epimerase which is also required to produce a precursor of colanic acid. The effect of the capR mutation on the gal operon was independent of CAMP or of glucose-mediated repression; the product of this locus thus acted as an additional negative control affecting expression of the gal operon. Lieberman et al. (1970) demonstrated that the mucoid bacteria resulting from mutation at the capR locus were derepressed for several enzymes, including GDPmannose pyrophosphorylase. In the episomal state, the capR allele was dominant in controlling synthesis of the two pyrophosphorylase enzymes. Additionally, Gayda et al. (1979) reported the control of a surface protein by the capR gene product (p. 105). Examination of the nucleotide pools in colanic acid-producing bacteria indicated that the concentrations of UDPglucose and UDP-galactose were essentially the same in mucoid and repressed bacteria (Grant et al., 1970). Levels of UDP-glucuronic acid and GDP-fucose wwt much higher in the mucoid strains. Renotypic changes can be induced in some E. coli strains and in some &r colanic acid-producing bacteria through growth in media containing DL-p-fluorophenylalanine (Kang and Markovitz, 1967; Grant et al., 1970). The effect of the fluorophenylalanine was thought to be mediated by its incorporation into the product of the capR gene, causing sufficient alteration in this protein to permit derepression of a number of enzymes involved in colanic acid synthesis.

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As well as the capR regulator gene, a second control site, caps, was recognized (Lieberman and Markovitz, 1970). Mutation at this site also led to derepression of synthesis of GDP-mannose pyrophosphorylase and of UDP-glucose pyrophosphorylase (Buchanan and Markovitz, 1973). The capR or lon locus is responsible for the control of ten enzymes considered to be involved in colanic acid synthesis (Fig. 9) and in the wild-type bacteria represses four spatially distinct operons as well as having a further role in cell devision (Gayda et al., 1976). Ultraviolet- or X-ray-irradiated cupR mutants formed non-septate filaments which lost viability on incubation in complex media; rescue could be achieved by anaerobic growth or by the addition of pantoyl lactone. The relationships between the different functions of the capR locus have still to be elucidated, but a 2.106 dalton DNA fragment with most of these functions has been cloned (Berg et al., 1976). GDP-FUC

UDP-GlcA~UDP-Glc

'0," /

G D P -Man /r7

Man-I-P 1\5

Man-6-P

-

7 -

U D P - Gal

1\6

Glc- I - P ?2

Glc- 6- P

t

Glc

Fruct - 6 - P ?3

FIG. 9. Enzymes involved in the synthesis of colanic acid. 1, Hexokinase; 2, phosphoglucomutase; 3, phosphoglucose isomerase; 4, phosphomannose isomerase; 5, phosphomannomutase; 6, UDP-glucose pyrophosphorylase; 7, GDP-mannose pyrophosphorylase; 8, UDP-galactose 4-epimerase; 9, UDP-glucose dehydrogenase; 10, GDP-fucose synthetase; 11, glucose transferase; 12, galactose transferase 1; 13, galactose transferase 11; 14, glucuronosyl transferase; 15, fucosyl transferase I; 16, fucosyl transferase 11; 17, polymerase(s); 18, ketalase; 19, acetylase. Whether other polysaccharide-synthesizing systems are under such complex regulatory mechanisms as is that which produces colanic acid remains to be investigated. As yet, it has not been possible to undertake suitable genetic analysis of most of the strains of greatest interest. An exception is the alginate-producing strain of Pseudomonas aeruginosa in which a chromosomal locus controlling polysaccharide production has been postulated (Govan and Fyfe, 1978). In Rhizobium leguminosurum a single mutation led to decreased polysaccharide production and to inability to nodulate legume seedlings (Sanders et al., 1978).

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B. C H A N G E S I N P O L Y S A C C H A R I D E S

Some bacterial strains may produce more than one polysaccharide as, for example, those E. coli strains that secrete both colanic acid and a “capsular” polymer (Qrskov et al., 1963). It is clearly possible for such strains to lose the ability to secrete one of the polysaccharides while retaining the capacity to form the other. However, there are also reports of bacteria which normally form one characteristic exopolysaccharide, replacing this by another. Less common have been reports of variations within a polysaccharide structure. This may be due to failure to recognize alterations such as the production of mixtures of polysaccharides some having “complete” structures and others lacking side-chains or other components. Alcaligenesfaecalis var. myxogenes and related bacteria have the potential to produce two polysaccharides, termed succinoglucan and curdlan respectively. Harada et al. (1968) recognized four distinct types among mutants from one such culture. The first group of strains produced considerable amounts of succinoglucan, but little curdlan; the second type produced similar amounts of each polymer. The third group produced little or no succinoglucan, but relatively large amounts of curdlan, whereas the fourth group did not form curdlan and were also poor producers of succinoglucan. A mutant from the first group was unstable and spontaneously yielded colonies forming more curdlan than succinoglucan (Amemura et al., 1977). This derived strain was stable and did not revert spontaneously. Another isolate from the same strain produced only the repeating unit of succinoglucan, an oligosaccharide with an apparent degree of polymerization of 9, in contrast to the normal polymer weight of approximately 3. lo5 (Hisamatsu et a[., 1978a). This mutant may, therefore, correspond to the mutants in lipopolysaccharide synthesis incapable of forming side-chains greater than one repeat unit in length because of deficiencies in the polymerase controlled by the rfc gene-the so-called SR mutants (Wright and Tipper, 1979). Although the possibility exists of there being such mutants in exopolysaccharide synthesis, the absence of selection procedures for mutants of this type has almost certainly led to their being overlooked until now. As curdlan is a /?I + 3-linked glucan, whereas succinoglucan is a heteropolymer composed of glucose and galactose in the approximate molar ratio of 8 : 1; synthesis of the latter requires an extra sugar nucleotide and presumably a number of specific glycosyl transferases. Curdlan probably requires fewer enzymes for its synthesis. Both polymers are also pyruvylated and succinylated. The mechanism for switching production from one polymer to another remains unclear and its elucidation must probably await genetic studies of Alcaligenesfaecalis or Agrobacterium species. The presence of large numbers of mutants derived in the main from a single parent has provided

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113

Harada and his colleagues with a considerable amount of information concerning the synthesis of curdlan and succinoglucan. A recent example of variations within a single polymer was reported by Jansson et al. (1979) in a study of an exopolysaccharide of Rhizobium trifolii. This polymer possessed a heptasaccharide repeating unit (Fig. 10). Some of

-p-

D

-Glc-(1+4)

- p - D-Glc

FIG. 10. The structure of the extracellular polysaccharide of Rhizobium trijolii.

the oligosaccharides (about 33%) were incomplete and lacked the terminal portion of the structural unit, the side-chains terminating instead in the glucose ketal. It is not clear whether the material examined contained a single molecular species with varying terminal sugars or a mixture of chain types, completely galactosylated and non-galactosylated in the ratio 2 : 1 respectively. In this respect, a similar mutation exists in certain derivatives of Xanthomonas campestris in which the repeating unit is a pentasaccharide. Several mutants were isolated on the basis of their differing colonial appearance and tendency to adhere to the surface of the growth medium (Whitfield, 1979). The polysaccharides produced by these mutants appeared to retain the normal cellulosic backbone structure, but lacked some of the side-chain components, including both carbohydrates and acyl groups (Fig. 2f). Similar results have been obtained when wild-type bacteria were grown in the presence of certain inhibitors of acylation (G. Carolan, unpublished work). These observations suggest that a much higher degree of flexibility exists in the synthesis of xanthan than had been expected from the earlier studies on Enterobacter aerogenes (Troy et al., 1971; Sutherland and Norval, 1970). Thus the backbone of cellulose was apparently synthesized, but either fewer or incomplete side chains were added. In lipopolysaccharides, the 0 antigenic chains are shorter than the exopolysaccharide molecules and variations in them are more readily recognized. McConnell and Wright (1979) observed that the lipopolysaccharide from Salmonella anatum grown at 20°C carried fewer side chains than normal, i.e. not all core molecules carried side chains. A further finding was that those side chains which were formed were longer than normal. This could result from longer residence time on the isoprenoid carrier lipid, but also indicates some flexibility in the translocation or ligase

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mechanisms. By analogy, similar variability can be expected in exopolysaccharide synthesis. The variability of polysaccharide structure may be more pronounced in polymers which have relatively large repeating unit. Thus, colanic acid (hexasaccharide units) was not completely digested by phage-induced fucosidases (Sutherland, 1971); the incomplete digestion was ascribed to the lack of glucuronosyl residues on some of the side chains. More recently, Johnson and Wilson (1977) observed that when cells of E. coli were harvested from mid-logarithmic phase batch cultures, washed in saline then transferred to fresh medium, the polysaccharide then formed lacked 25% of the normal side chain galactose and glucuronic acid. The incomplete polymer may be the result of removal of enzymes by the saline washing procedure. Alternatively, the product of older cells may be less regular. Although Acetobacter xylinum normally forms bacterial cellulose and numerous studies have considered various aspects of this, Colvin et al. (1 977) noted the presence of a water-soluble glucan in cultures of this species. The polymer could be precipitated with ethanol to yield material with an average sedimentation constant Szo,w of 11.1 k 0.3. It was characterized as a polydisperse p1 + 4-linked glucan in which every third residue (on average) was substituted with a glucose at the 2 position. Whether this is a separate polymer or an intermediate stage in bacterial cellulose synthesis is not clear, nor is it known whether other strains of Acetobacter xylinum form the same soluble polymer. It appears likely that this polysaccharide was quite distinct from cellulose and it was not susceptible to the action of a P-glucosidase preparation (Colvin et al., 1979). Unlike bacterial cellulose, the new polysaccharide was acetylated with an acetyl content of 8%. The location of the 0-acetyl groups was not determined so it remains uncertain whether they were linked to the main chain residues or to the pl + 2-linked branches. The formation of a high molecular-weight non-cellulosic polymer by membranes of Acetobacter xylinum was also reported by Sandermann and Dekker (1979). The disaccharide product of acid degradation of this polysaccharide was sophorose, indicating that the major linkage was p1,2. Thus, Acetobacter xylinum can apparently synthesize bacterial cellulose, or an acetylated polymer with mixed j1,2 and p1,4 linkages or a Pl,Zlinked glucan. There may, therefore, be some similarity to those bacteria that form both curdlan and succinoglucan, but the conditions under which Acetobacter xylinum forms non-cellulosic polysaccharides require adequate definition. Although it has been recognized that Rhizobium spp. produce high molecular-weight acidic polysaccharides, the presence of a second type of polymer in culture fluids of these bacteria, has only recently been reported (York et al., 1980). After preferential precipitation of the acidic polysaccharides, p-linked glucans remained in solution. They were separated from any residual acid polymer through the use of an ion-exchange cellulose.

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115

Unlike the P1,2-linked glucans from Agrobacterium tumefaciens and other sources, those from Rhizobium leguminosarum, Rhizobium phaseoli and Rhizobium trifolii were relatively low molecular-weight compounds with an apparent degree of polymerization of 20 or less. They were also unusual in that no reducing terminal carbohydrate could be identified. What relationship production of this low molecular-weight polymer bears to the synthesis of the normal acid polysaccharides has not yet been studied. If both are formed simultaneously, it should prove a useful system for the investigation of regulatory mechanisms controlling the production of the two polymers.

V. Synthesis of Polysaccharides by Cell-Free Preparations A. D E X T R A N S , M U T A N S A N D L E V A N S

In contrast to heteropolysaccharide production, dextran synthesis occurs at or outside the cell surface. Thus cell-free preparations which catalyse the synthesis of dextrans are not difficult to obtain and there are numerous reports of such dextransucrase enzymes. The dextran formed in cultures of Streptococcus sanguis was compared with that obtained from partially purified enzyme systems using sucrose as substrate (Arnett and Mayer, 1975). Both dextran preparations contained residues substituted at the C(6)and C(3) positions. It was reported that the proportion of residues substituted at the C(3)position was lower in the enzymically synthesized polymer than in the native dextran (6.7 mol %compared with up to 13.6 mol %). From this, it was concluded that this dextransucrase was an enzyme complex consisting either of two distinct enzymes which specifically forged 1 -+ 3 and 1 -+ 6 linkages respectively, or of a dextransucrase forming one type of linkage together with a transglucosylase catalysing an internal rearrangement of the molecule. Further purification (Huang et al., 1979) yielded two active proteins, one of which appeared to be homogeneous. Both proteins were of the same approximate molecular weight viz. 1.02. los. When the dextrans produced by the two active enzyme fractions were studied, both 1 -+ 3 and 1 -+ 6 bonds were detected, the product of one enzyme containing slightly more 1 3 linkages than the other. The authors did not consider this difference significant. No evidence was obtained to indicate that either enzyme catalysed rearrangement of 1 -+ 6 to I + 3 linkages. Robyt and Tanaguchi (1976) used a different approach with cell-free preparations from Leuconostoc mesenteroides. Dextransucrase was partially purified then immobilized by attachment to beads of Bio-Gel P2. When [ 14C]sucrose was incubated with the particulate enzyme and unlabelled dextran was then added, further incubation led to the release of the radioactive material in the form of a slightly branched -+

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polymer. It was suggested that interaction of the enzyme-bound radioactively labelled polymer with low molecular-weight dextran resulted in the formation of 1 + 3 branch linkages. A branching mechanism was proposed in which a C(3)-OH on the acceptor acted as a nucleophile on C(1)of the reducing end of a dextran-dextranase molecular complex displacing the dextran from the enzyme and forming a 1 + 3 branch linkage (Fig. 11).

-0-0-0-0-9

+ 0-0-0 ____-- 0 - 0 - 00-+ -

t

0-0-0 FIG. 11. A possible mechanism of branching in the biosynthesis of polyglucose structures. Both 0 and 0 represent non-reducing glucose residues, whereas ,#’ represents a reducing glucose residue.

Because of its proposed role in dental caries, “mutan”, the a1 .+ 3-linked glucan formed by Streptococcus mutans, has been the subject of numerous studies. Unfortunately, as Montville et al. (1978) pointed out, the tendency “to use different strains, different growth media and different purification procedures . . . . . yields different and conflicting results”. Most purification procedures have employed adsorption of the enzyme to hydroxylapatite followed by elution with phosphate buffer. The most noticeable feature of this procedure is the poor recovery of active material; much protein appears to be irreversibly adsorbed to the hydroxylapatite. Considerably different molecular weights have also been reported for the dextransucrase preparations so obtained (Table 7), but this is probably partly due to the interconversion of soluble and insoluble forms of the enzyme as well as to differences between the producer strains of Strep. mutans employed. In any culture the dextransucrase is found in soluble and insoluble forms and the enzyme activity may be cell-associated or present in the culture supernatant. Transition from insoluble to soluble enzyme may be effected by the presence of dextran (Montville et a f . , 1978). The equilibrium between the enzyme associated with the bacterial surface and that present in the supernatant is affected by sucrose (which enhances the cell-associated form) or by 1M NaCl (which releases more enzyme into the supernatant fluid). Ethanol precipitation was used to purify glycosyltransferases from the culture supernatant of Strep. mutans strain Ingbritt (Baird et al., 1973). When

TABLE 7. Some Dextran Sucrase Preparations from Streptococcus mutans Strain

Purification method

OMz176 67 15

Hydroxylapatite (HA) HA, isoelectric focussing

HS-6

Gel filtration, ion-exchange chromatography, HA Gel filtration, HA

GS5

Recovery 21% 13%

0.3% 32%

Composition

7 proteins 4 proteins (2 active) 1 protein -

1 protein?

Molecular weight

Reference

9.4 .lo4

Guggenheim and Newbrun (1969) Chluzdinski et al. (1974)

1.7 .105

Fukui et al. (1974)

1.9 . l o 5 and 2.35.105 4.5 .lo7

Kuramitsu (1975)

-

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I. W. SUTHERLAND

incubated with sucrose the product yielded glucan and fructan, the former being separable into water-soluble and insoluble material. The soluble glucan contained predominantly ul,6 linkages, whereas the insoluble polysaccharide was highly branched and composed of u1,3 and ~ 1 , linkages. 6 It was not a mixture of polymers of each linkage type. The homopolymer levan (polyfructose) is synthesized by a number of bacterial species. Enzymes from Bacillus subtilis strains or from Aerobacter levanicum catalyse the reaction:

n sucrose e fructose,

+ n glucose

The enzyme from B. subtilis was purified to homogeneity (Dedonder et al., 1963; Dedonder, 1972) and crystallized as a protein of molecular weight 40,000. When constitutive strains grown on glycerol were used as the source of the enzyme, it was free from levan. The products obtained from the purified enzyme were levans containing a number of branch points. In wild-type bacterial isolates, levansucrase is inducible, but constitutive mutants have been isolated. Two groups of mutants showed derepressed enzyme synthesis (Dedonder et al., 1972). Further mutants were isolated which produced up to 100 times more levansucrase than did the wild-type organism. Cell-free levansucrases from B. subtilis which were prepared by other workers showed differences in their molecular weights. That studied by Caulfield et al. (1979) had a molecular weight of 5.4. lo4, whereas a similar enzyme isolated from B. subtilis var. Marburg was shown to have a molecular weight of 3. lo5 (Petit-Glatron et al., 1980). The enzyme was prepared from a strain which constitutively hyperproduces levansucrase to the extent of approximately 5% of total protein synthesis. The cell-associated, phenolextractable enzyme differed from the normal extracellular form in displaying activity only in the presence of the detergent Triton X100. The normal enzyme showed no such requirement and was inactivated by phenol treatment. The phenol-extracted enzyme also differed in being extremely susceptible to attack by proteolytic enzymes in the absence of detergent. It was assumed that the cell-associated enzyme formed part of a complex trapped at the cell surface. The levans formed by B. subtilis enzymes are b2,6-linked polymers whereas a fructan prepared from Streptococcus mutans was b(2 -,1) linked (Baird et al., 1973).

B. C E L L - F R E E S Y N T H E S I S O F H O M O P O L Y S A C C H A R I D E S

From ultrasonically produced extracts of Rhizohium japonicum, the membrane fraction sedimented by centrifugation at 105,000 g was capable of polysaccharide synthesis (Dedonder and Hassid, 1964). On incubation with UDP-glucose in the presence of Mg2 and Mn2 +,a homopolysaccharide +

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119

containing predominantly @I -,2 linkages was formed. In addition, PI + 3 and PI + 6 linkages were detectable in the product, which closely resembled the polymer formed by intact cells of this bacterium. Sialic acid is another homopolysaccharide which is synthesized by cell-free bacterial preparations viz. of Neisseria meningitidis (Blacklow and Warren, 1962). The enzyme complex was purified from a cell lysate by acetone precipitation and adsorption to alumina gel. After elution from the adsorbent, further purification was achieved by chromatography on DEAE-cellulose. The purest preparation was thought to catalyse the reaction: N-Acetyl-o-mannosamine

+ PEP + N-acetylneuraminic acid + Pi

Like many other preparations accomplishing polysaccharide synthesis, divalent cations were required for activation of the system, the most effective of which was Mn2+ . This mechanism of synthesis was not substantiated by later studies undertaken by Troy et a/. (1975) using E . coli as the source of the sialic acid-synthesizing system. Troy and his colleagues disrupted the bacteria in a pressure cell and found most of the sialyl transferase activity in the supernatant remaining after centrifugation at 48,000 g. Further centrifugation at 229,000 g for 1 hour yielded 15% activity in the pellet and 85% in the supernatant fluid. This system thus appears to be more readily solubilized than many. Both fractions catalysed incorporation of [ ' T I N acetylneuraminic acid from CMP-[ 14C]N-acetylneuraminic acid into lipid soluble and polymeric products. Enzyme preparations depleted of lipid were reactivated by the addition of C, isoprenyl phosphate. Undecaprenyl phosphate (two internal trans double bonds) was more effective than ficaprenyl phosphate (three internal trans double bonds). As bacitracin did not inhibit sialic acid synthesis in vitro, it was concluded that polymer formation did not involve release of polyisoprenyl pyrophosphate. In intact membrane systems, sialic acid synthesis was stimulated by the addition of exogenous lipid (Vijay and Troy, 1975). Such stimulation required incubation at 30°C or (preferably) at 37°C; no stimulation was observed at 20°C. This was considered to result from failure of the lipid phosphate to insert and form a functional complex at the lower temperature (for at 20°C the membrane lipids are relatively immobile).

,

C. CELL-FREE S Y N T H E S I S OF H E T E R O P O L Y S A C C H A R I D E S

As the precursors for heteropolysaccharides are formed intracellularly and many of the enzymes involved in polysaccharide synthesis are membranebound, preparation of cell-free extracts capable of heteropolysaccharide synthesis is more difficult to achieve than the preparation of extracts catalysing the production of dextrans or levans. Some years ago, cell membrane

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I. W. SUTHERLAND

preparations from Streptococcuspneumoniae type 111 were used to synthesize from UDP-glucose and UDP-glucuronic acid a polymer which was then characterized by the use of a specific polysaccharase (Smith et al., 1961). The polysaccharide was composed of a disaccharide repeating unit; consequently, relatively few enzymes are involved in its synthesis. This is probably also true for the exopolysaccharide synthesized by a strain of group A streptococcus, from which a similar membrane fraction was prepared and sedimented by ultracentrifugation at 229,000 g (Stoolmiller and Dorfman, 1969).This preparation, when supplied with a suitable divalent cation, proved capable of synthesizing hyaluronic acid from UDP-glucuronic acid and UDPN-acetylglucosamine. As bacitracin was not inhibitory and UDP, but not UMP, was released during polymer synthesis, this system may differ in certain aspects from that present in cell-free extracts of Enterobacter aerogenes. The disadvantage of using membrane fragments, prepared as described above and in Section V.B, lies in their complexity. Not only do they contain most of the enzymes required for polymer synthesis, but also variable amounts of precursors and lipid carriers. In addition, they may be heavily contaminated with enzymes capable of degrading sugar nucleotides and other precursors. These all affect the results obtained and make their interpretation difficult. A considerable advance was achieved by Troy et al. (1971) in their preparation of lipid-depleted membrane fragments. After ultrasonic disintegration of bacteria and recovery of fragments sedimenting at 30,000 g, acetone, butanol and ether were used to remove much of the lipid present. The resultant material remained stable, although, with time, some loss of transferase activities was noted. Using the solvent-extracted membrane preparation, Troy et al. (1971) observed that addition of exogenous lipid carrier was necessary for restoration of activity. The most active material in restoring enzyme activity was a deacetylated phospholipid fraction. The presence of this phospholipid resulted in transfer of galactosyl 1-phosphate from UDPgalactose to the isoprenoid lipid phosphate in the same manner as in crude membrane preparations. This reaction was inhibited by UMP. A problem associated with the use of membrane systems from which lipid has been extracted, lies in the reconstitution of the various components before attempts at cell-free synthesis can be made. Thus, the formation of exopolysaccharide by membrane preparations from Enterohacter aerogenes type 8 was demonstrated in the presence of divalent cations and the appropriate UDP-monosaccharides (Sutherland and Norval, 1970).The first two enzymes in the biosynthetic sequence, catalysing the transfer of glucose 1-phosphate and galactose respectively, were isolated by extraction with acid butanol (Lomax et al., 1973). Addition of Triton X-100 and 0.lmM dimethylsulphoxide to incubation mixtures was necessary before activity could be detected. Even with such incubation mixtures, no transfer of the second galactosyl residue in the repeat unit was observed.

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121

Reactivation of a specific enzyme involved in precursor formation, C, 5 isoprenoid alcohol phosphokinase from Staphylococcus aureus, has been studied in several laboratories. Sandermann (1974) noted that a variety of lipids could be used to reactivate the apoprotein and that they showed a lack of specificity in respect of their chemical structures. This was confirmed by Gennis and Strominger (1976a, b) who found that lipids which provided a hydrated, loosely packed and highly fluid environment were often the most effective activators. In these lipids, the nature of both the polar and non-polar groups was important. The most effective activator was lysophosphatidylethanolamine. When a series of synthetic lecithins was tested, those showing maximal activity with the phosphokinase were dicapryloyl-L-a-lecithin (C,) and dicaproyl-L-a-lecithin (C, o). When various detergents were similarly tested, only those with short chain and unsaturated chain hydrophobes functioned effectively as activators at 25°C (Gennis and Strominger, 1976b). When it is remembered that these results apply to only one of the membranebound enzymes involved in exopolysaccharide synthesis, the complexity of even reconstituted systems can be realized. The probability of finding conditions under which such systems will function is correspondingly low.

VI. Relationship to other Polysaccharides: Shared Pathways

Shared pathways leading both to exopolysaccharide synthesis and to the production of other polysaccharides have received relatively little attention. This is in part due to the lack of specific methods by which the products of such pathways could be differentiated. The precursors, too, may be shared and their utilization difficult to follow. Thus, UDP-glucose is a common precursor of the succinoglucan and curdlan synthesized by Alcaligen~s,fueculis var. myxogegenes (Harada e f al., 1968); equally, it is required for the formation of both the capsular polysaccharide and colanic acid produced by those strains of E. coli studied by qrskov et al. (1963). I t is also clear that sucrose may be converted into polymers of either levan or dextran type in those bacteria which are capable of forming both types by extracellular processes. Where the substrate does not enter the bacterial cell, there is no requirement for regulated production of common precursors such as sugar nucleotides or isoprenoid lipid-linked intermediates. In a study of the mechanisms of bacterial cell wall synthesis, Anderson et al. ( 1972) examined the biosynthesis of peptidoglycan and teichoic acid. They concluded that sugar nucleotides required for the synthesis of one polymer were inhibitory to the production of the other, whereas isoprenoid lipid molecules were shared between both systems in such a way that the undecaprenol phosphate molecules were returned to the bacterial pool after

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I. W. SUTHERLAND

polymer chains were completed rather than after the formation of a repeating unit and its transfer to elongate another such molecule. Anderson and his colleagues suggested that multienzyme systems involved in the synthesis of the two polymers and sharing precursors such as isoprenoid lipid phosphate, were closely aligned in the membrane. It is probable that similar localization of polysaccharide synthesizing systems may occur in other microbial membranes to permit the sharing of common precursors. This would account for the specialized sites of polysaccharide export described by various authors since their initial discovery by Bayer and Thurow (1977). Problems may also be encountered in interpreting results obtained with cell-free systems. During attempts to obtain synthesis of Enterobacter aerogenes exopolysaccharides by membrane fractions incubated with radioactively labelled UDP-glucose or UDP-galactose, very high levels of incorporation of radioactivity into “polymer” were observed (I. R. Poxton and I. W. Sutherland, unpublished results). Such levels could not be explained on the basis of incorporation of monosaccharides into lipid-linked precursors, and it transpired that much of the radioactivity from the sugar nucleotides was converted into material which could be degraded by amylase, i.e. bacterial glycogen. What is not clear in such systems, is whether the UDP-glucose acts as the immediate precursor (in which case, some mechanism for sharing it between the two pathways would be needed) or whether UDP-glucose is degraded to glucose 1-phosphate and then converted into ADP-glucose. As both ADP-glucose and ADP strongly inhibited “polymer” formation, the latter alternative appears more likely. Presumably in intact bacteria, the enzymes are so organized that substrate moves to the “correct” enzyme system and cannot recycle into glycogen.

VII. Role of Primers and Secretion in Polysaccharide Production A. P R I M E R S O R A C C E P T O R S - A R E

THEY NECESSARY?

A recurring problem in studies of polysaccharide synthesis is the possible involvement of one or more primers (see, e.g. Robbins et al., 1966). Most of the process of synthesis can be explained by the involvement of specific enzymes and precursors such as sugar nucleotides, isoprenoid lipid-linked sugars and oligosaccharides. It is clear, however, that, at least in some systems, these precursors alone are insufficient for polymer synthesis, even in the presence of appropriate enzymes. In the formation of lipopolysaccharide, the primer or acceptor is the “core” to which the “0antigen” or side chains are transferred from the lipid-pyrophosphate-(oligosaccharide), by appropriate enzymes. If the “core” is incomplete, transfer cannot occur. Identifi-

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123

cation of mutant strains bearing lesions of this type in lipopolysaccharide synthesis is relatively simple, as such mutants are altered in their phage sensitivity (Naide et a/., 1965) and the 0 antigen is found as a soluble polysaccharide not sedimentable from phenol extracts by ultracentrifugation (Beckmann et al., 1964). In peptidoglycan synthesis, an acceptor is again involved. In this case, preformed peptidoglycan can be incised by appropriate enzymes, leaving a suitable acceptor to which nascent peptidoglycan can be transferred from isoprenoid lipid-linked intermediates (e.g. Ramey and Ishiguro, 1978). An acceptor has also been postulated in recent studies using a teichoic acid-less mutant of Staphylococcus aureus (Bracha et al., 1978). In this mutant strain, absence of the acceptor containing phosphorylatedN-acetylglucosamine prevents synthesis of the complete acceptor molecule; consequently, ribitol teichoic acid which is ultimately attached to the acceptor is also absent. How does this relate to the situation in exopolysaccharide synthesis? Troy and McCloskey (1979) considered that an acceptor was necessary for sialic acid synthesis in E. coli and that growth at 15°C yielded cells lacking this acceptor. Sialyl transferase was present and functional. The lowered growth temperature uncoupled sialic acid synthesis from production of lipid-linked sialyl derivatives. Addition of exogenous acceptor caused reactivation of the membranes from bacteria cultured at 20°C. In this system, the acceptor was identified as material having sialyl chains with a degree of polymerization of 12. Some of the mutant strains used in studies of exopolysaccharide synthesis are undoubtedly “leaky” and, as a result, any possible acceptor is likely to be present, albeit in small amounts. Consequently, polysaccharide synthesis in such mutants should proceed without any difficulty if an acceptor is needed. In those mutants that are not leaky, it is possible that, as well as failing to form an exopolysaccharide as such, an acceptor is also lacking. A problem not encountered in selecting mutants defective in lipopolysaccharide synthesis, is the recognition and isolation of these mutants. Several studies have been made of acceptor specificity in dextran synthesis. McCabe and Smith (1978) used a solubilized dextran sucrase to demonstrate a requirement for dextran as acceptor. Other polysaccharides did not function as acceptors and low molecular-weight sugars did not compete with dextran in this role. It was suggested that: (i) acceptor of increased efficiency was formed in a slow reaction; (ii) the “improved” acceptor was then rapidly used; and (iii) the rate of dextran synthesis decreased as enzyme was irreversibly activated. Dextransucrase of Leuconostoc mesenteroides formed low molecular-weight dextran, together with a series of oligosaccharides, in the presence of certain sugars as acceptors (Robyt and Walseth, 1978). A further area of uncertainty is the mode of attachment of capsules to the microbial cell surface. Slime-forming (Sl) mutants are frequently found in strains that are normally capsulate (e.g. Enterobacter aerogenes; Wilkinson

124

I. W. SUTHERLAND

et al., 1954). From this, it can be deduced that there is normally a binding process in which, as it is secreted, the polysaccharide is attached to the cell surface. In the S1 mutants, the site of attachment is presumably missing; alternatively, loss of the appropriate ligase enzyme could lead to the same result. As capsulate micro-organisms include Gram-negative and Grampositive bacteria, yeasts and other types of organism, the mode of attachment must differ within these distinct microbial groups. Kozel (1975) suggested that there existed in Cryptococcus neoformans surface receptors for its exopolysaccharide; these were specific in binding the yeast polymers and showed no non-specific binding of other polysaccharides. Equally, the cryptococcal polysaccharide failed to bind to other microbial cells. The polysaccharide receptors on the cells of Cryptococcus neoformans appeared to resist various chemical and enzymic treatments, but were not fully characterized. More information is available on systems from Gram-negative bacteria. A recent report has correlated the presence of a major outer membrane protein with capsular polysaccharide production in E. coli (Paakanen et al., 1979). As the enzymes involved in the synthesis of polysaccharides are thought to reside in the cytoplasmic membrane, such an outer membrane protein could be an attachment site. Further studies on S1 mutants, non-capsulate strains and their parent strains in different bacterial species should help to clarify the involvement of outer membrane proteins in capsular attachment. Nimmich (1969) suggested that the exopolysaccharides of Klebsiella sp. were attached to lipopolysaccharide. The conclusion was based on the identification of small amounts of sugars derived from lipopolysaccharide in the exopolysaccharide preparations. It is much more likely that such traces of lipopolysaccharide are present as contaminating surface material, present even after ultracentrifugation of exopolysaccharide. Such contamination has frequently been observed. There is certainly as yet no evidence for a linkage unit of the type in which a chain of three glycerol phosphate residues links the polyribitol teichoic acid of Staphylococcus aureus to the peptidoglycan present in the walls of these bacteria (Hancock and Baddiley, 1976). Nor is it likely that capsule and lipopolysaccharide are linked in a manner analogous to the attachment of 0-side chains of lipopolysaccharide to the core as described by Nikaido (1969). The elegant electron microscopic studies of Bayer and Thurow (1977) and of Politis and Goodman (1980) in which the exopolysaccharides are seen as strands radiating from the bacterial surface, might indicate the presence of a distinct number of attachment sites. If this is the case, what happens when the attachment sites are filled? Is this the reason for some strains producing soluble slime in addition to capsules? It is difficult to determine how many such attachment sites might exist. Bayer and Thurow (1977) proposed 20G400 “export” sites, but, as these function in export of various macromolecules, they are presumably not identical with attachment sites.

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

125

FIG. 12. Possible modes of attachment of capsule to the cell envelope of a Gramnegative bacterium. Abbreviations: OM. outer membrane; EPS. exopolysaccharide strands.

An alternative possibility is that the attachment only occurs if the polysaccharide molecule is of the “correct” size and polymer molecules of smaller or larger size fail to attach. The latter would then form slime. The exact location of the attachment site has also still to be determined. There are essentially three possibilities as indicated in Fig. 12. The polymer strands may be affixed to the outside of the outer membrane, linked perhaps to a specific protein or lipoprotein, the absence of which would result in S1 mutants. Alternatively, the polymer chains might be linked to some component within the outer membrane. This would not be expected to alter the function of the outer membrane as a permeability barrier such as may happen as a result of various mutations (Coleman and Leive, 1979). A third possibility is the attachment of the polysaccharide to sites on the inner side of the outer membrane or even to the peptidoglycan layer. This appears unlikely, as the periplasmic space should then contain exopolysaccharide and no evidence for this has been reported. Rohr and Troy (1980) in a study of sialic acid biosynthesis in E. coli, failed to find free reducing terminal N acetylneuraminic acid and concluded that this was due to its linkage to an unidentified molecule present in the membrane. Two alternative mechanisms exist for the formation of polysaccharide chains (Fig. 13). In the first of these, monomers from activated precursors (usually nucleoside diphosphate sugars) are added to the non-reducing terminus of the chain. This is typified by glycogen synthesis (Leloir et al., 1959), in which glucose from UDP-glucose is transferred to the non-reducing end of a primer molecule. Recently, two further examples of polymer growth at the non-reducing end of the molecule have been observed in two E. coli lipopolysaccharides, types 08 and 09, in both of which the side chains are polymannose sequences (Flemming and Jann, 1978a, b). In contrast to this,

126

I. W. SUTHERLAND

(b)

4

0-antigen

@

- _ _ _ _n_ P P

I

lipid

P

I P

P

I P

P

I P I

lipid

P

P

I lipid

I

I P

I P

lipid

P

I

P

I

/

P

FIG. 13. Growth at (a) the reducing end of a polysaccharide chain and (b) the nonreducing end of a polysaccharide chain (as illustrated here by the polymerization and chain-elongation mechanism employed in the biosynthesis of the 0-antigen of Sulmonellu newington).

the heteropolysaccharide side chains of Salmonella lipopolysaccharide were formed by the addition of sugars to the reducing end of the acceptor or core molecule (Robbins et al., 1967). In this example, it is the oligosaccharide attached to the isoprenoid lipid pyrophosphate which is transferred. A similar process functions in peptidoglycan biosynthesis, again involving transfer of an oligosaccharide, the disaccharide b-N-acetylmuramyl( 1 -,4)-N-acetylglucosamine, from an isoprenoid lipid pyrophosphate derivative to the reducing terminal of the acceptor (Ward and Perkins, 1973). Robyt and his coworkers (1974) postulated the growth of dextran chains by insertion of glucose (from sucrose) onto the reducing end of the glucan molecule. A further example of a mechanism of this type involved in the synthesis of

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

127

a homopolysaccharide was found in the formation of sialic acid (poly-Nacetylneuraminic acid) by E. coli (Troy, 1979).

B. SECRETION O F P O L Y S A C C H A R I D E S

One of the enigmas of exopolysaccharide production is the presumed final stage of the process, the transfer of the polymer from the site of synthesisthe cytoplasmic membrane-to the normal final location, the exterior of the cell. This problem does not occur in dextran or levan synthesis where the substrate does not enter the cell, but it is, of course, a feature of all heteropolysaccharide synthesis and of bacterial cellulose production. The polymer is presumed to be attached to isoprenoid lipid throughout the biosynthetic process at the cell membrane. As the amount of such lipid is limited and because it also functions in the production of peptidoglycan and lipopolysaccharide, the bacteria must release the exopolysaccharide before the isoprenoid lipid can be returned to the pool in the cytoplasmic membrane. Simultaneously, the hydrophilic exopolysaccharide must pass through the hydrophobic outer membrane. The most likely mechanism involves the adhesion sites (Bayer sites) of the Gram-negative bacterial cell in which the outer membrane and the inner (cytoplasmic) membrane associate with each other. These sites have been postulated to function as export locations for exopolysaccharide synthesis (Bayer and Thurow, 1977). They are also the export channels for lipopolysaccharide and for outer-membrane proteins and thus function in an export capacity in an analogous mode to the import of various substrates through “porins” (Smit and Nikaido, 1978). How the export of the different classes of molecule through the Bayer sites is accomplished remains unclear. Nor is there any real indication as to how the exopolysaccharide, having passed to the outside of the cell and the outer membrane, is then attached to the microbial surface to form a capsule or, alternatively, despatched to the extracellular environment as amorphous slime. In their examination of newly synthesized polysaccharide in capsulate E. coli, Bayer and Thurow (1977) observed the presence of knob-like elements at the termini of many of the polysaccharide filaments. These structures were clearly not composed of lipopolysaccharide, but it was not certain whether they played a role in anchoring the polysaccharide to the cell membrane. The fate of pre-existing polysaccharide was also unclear, i.e. whether “new” polymer displaced the “old” or whether there was, in fact, movement of the site of synthesis. Although various studies have attempted to visualize the synthesis of exopolysaccharide in micro-organisms, with the exception of the findings of Bayer and Thurow (1977) with E. coli, most information has been obtained from studies of homopolysaccharide formation. In particular, examination

128

I. W. SUTHERLAND

of cellulose synthesis in Acetobacter xylinum revealed the formation of knob-like projections on the cell surface (Zaar, 1979). This was followed by growth of microfibrils at the cell surface (Brown et al., 1976). Subsequently, the microfibrils associated to yield a ribbon which extended from the bacterial cell and, under the conditions examined, elongated at a rate of 2 pm min-'. From the ribbons of cellulose, was generated the surface pellicle which is characteristic of liquid media cultures of Acetobacter xylinum. Studies by Zaar (1979) and by Brown et al. (1976) both revealed rows of protuberances arranged linearly on the cell surface and these were considered to be possible sites of cellulose synthesis. These could thus be the external locations of the Bayer fusion sites proposed as export sites for the nascent polysaccharide in the E. colisystem (Bayer and Thurow, 1977). Colvin and Leppard (1 977) noted the presence of irregular features on the surface of rapidly dividing cells of Acetobacter xylinum. These included small polar invaginations and were associated with fine fibrils. In their study of Acetobacter xylinum, Brown et al. (1976) indicated that the row of sites of fibril synthesis was duplicated longitudinally prior to cell division and that these sites were active in polysaccharide synthesis in both putative parent and daughter cells immediately before their separation. Zaar (1977) suggested that each bacterium assembled a single ribbon-like cellulosic structure from a number of microfibrils. This process would differ markedly from the situation in E. coli observed by Bayer and Thurow (1977). In this latter organism, the excreted polysaccharide, although forming microfibrils, did not then become incorporated into a single macromolecule, but rather formed an array of polysaccharide strands forming the bacterial capsule. This may be due to the physical nature of the tertiary structures of E. coli polysaccharides. The situation in Acetobacter may differ from that in enteric and other bacteria, but a word of caution in the interpretation of the structures formed by Acetobacter xylinum was sounded by Colvin et al. (1977). These authors pointed out that there was no unequivocal evidence for linking the nascent ends of cellulose microfibrils to the knob-like structures. On the contrary, they pointed to the direct evidence for export of the polysaccharide to the exterior of the bacterium through large transient pores in the wall, thus envisaging a situation similar to that proposed by Bayer and Thurow (1 977). It would obviously be advantageous to study polysaccharide secretion, capsule and strand formation in a number of micro-organisms in which the chemical nature of the excreted polysaccharide is known and intermolecular interactions of the polysaccharide chains can be postulated as indicated by Morris et al. (1977). Electron microscopy of preparations of the polysaccharide produced by Xanthomonas campestris, the physical properties of which have been widely studied (Morris et al., 1977) and the chemical structure

B I OSY NTH ESIS 0 F M ICRO B IAL E X 0 PO LYSACCHAR ID ES

129

of which is known to be a substituted cellulose (Fig. 2 ) , has been performed by Holzwarth and Prestridge (1977). The physical relationship of the polymer strands to the cells which produce them has not been shown, although the tertiary configuration of this particular polysaccharide has been the subject of numerous studies (see e.g. Morris et al., 1977; Holzwarth, 1976; Moorhouse et al., 1977).

VIII. Modification of Polysaccharides A. C H A N G E S I N P H Y S I C A L P R O P E R T I E S O F P O L Y S A C C H A R I D E S

The current interest in microbial polysaccharides for industrial usage has resulted in increased study of their physical properties. Most attention has been focused on solution viscosity and on gelling capacity and it is clear that considerable variations may be found. Some differences in viscosity may be due to the ability of a single microbial strain to produce several different polymers as is found in glucan and curdlan synthesis by Alcaligenesjaecalis var myxogenes (p. 112). Alternatively, rheological changes may be due to the emergence of variants, particularly during continuous culture. This has been noted in Xanthomonas campestris where small colony variants were found which formed polysaccharide of low pyruvate content (Cadmus et al., 1976). The difference in viscosity between the polysaccharide from the wildtype bacteria and that from the small colony type was considerable. However, such alterations in viscosity are clearly due to the chemical changes observed in the polysaccharide. As such chemical changes are mainly confined to differences in the acyl and ketal groups rather than carbohydrate structures, the appearance of variants of this type may be relatively common, and may have a dramatic effect on solution viscosity. The chemical removal of acetyl groups from the polysaccharide of X . campestris decreased its viscosity by almost 30”/, (Holzwarth and Ogletree, 1979). Alginates of differing physical characteristics have been prepared using carefully controlled culture conditions for Azotobacter vinelandii (Deavin et al., 1977). In this polysaccharide, the changes in physical properties are almost entirely due to the range of D-mannuronic acid to L-guluronic acid contents and this aspect is considered on pp. 134-135. An alternative to chemically changed polymers is also known. A recent observation in our laboratory was that mutant bacterial strains could produce polysaccharides of exceptionally high viscosities though they were identical in chemical composition with the polysaccharide produced by the parent, wild-type bacterium (Sutherland, 1979~).The chemical identity of the deriva-

130

I. W. SUTHERLAND

tive and original polysaccharides in a number of polymer “families” was confirmed by chemical analysis and enzymic hydrolysis. The first such series of viscous mutants was prepared from a strain of E. coli K12 (Fig. 2). Subsequently, similar viscous mutants were prepared from a number of Enferobacter aerogenes serotypes (Fig. 14). These differed from the E. coli in that the original polysaccharide was produced as a discrete capsule. No

(a) Strain A 4 a-Glc A

P

-(3

Strain 8 s

‘14

P

GAL 1 - 3 3 GAL I I PYR 4116

$4

B(3GAL1+3GALI

(b)

3GlcI

-%,3Glc

+

+

ao-Glc A II

P b 3 P P +4Glc1+4Man1~4ManI~

FIG. 14. The structures of Enterobacter aerogenes type polysaccharides (a) in strains A4 and 8S, and (b) in type 30 from which viscous mutants have been derived.

viscous mutants were obtained directly from the capsulate bacteria; an initial mutagenesis to yield slime-forming derivatives (S 1-forms) was required. The progression needed to yield the viscous mutants is indicated in Fig. 15. The apparent absence of chemical change in the polysaccharides indicated that the most likely alteration was in the molecular weight. In linear polymers, a direct relationship exists between molecular weight and viscosity, increased viscosity resulting from increased molecular weight. This might also account for the fact that viscous mutant strains can only be derived from a slimeforming parent, i.e. in capsulate strains, the polysaccharide formed is probably more uniform in size, whereas in slime-forming mutants, this constraint is absent and polymer of increased chain length can be synthesized. This

Wild- type bacteria ( I ipopolysaccharides, capsules, sI ime )

d l

SL mutants

P

E. coll' 5 5 3

( lipopolysaccharides, slime)

Ent cloacae 5920

0 mutants (lipopolysaccharides)

E. coli S61

Viscous mutants

I

15 coli S614 E. coli S617

V V (very (viscous) mutants

90

-

c

a 0

> +

'z 70 0

ln ._ 5

t

50 0 Q Q

a

30

10 0 0

0.1

0.2

0.4

concentration (%, w / v ) (shear 7 0 s - I )

FIG. 15. Production of high viscosity polysaccharides by mutant strains of enterobacteria. (a) Chart illustrating the derivation of various mutant strains, including the SL mutants (e.g. Escherichia coli S53 and Enterobacter cloacae 5920), viscous mutants (e.g. Escherichia coli S61) and very viscous mutants (e.g. Escherichia coli S614 and S6 17. (b) Viscosities of preparations of the polysaccharide colanic acid formed by these mutant strains. The apparent viscosity in each case was measured at 20°C in a Ferranti viscometer at a shear of 70 s - l .

TABLE 8. Molecular weights of some microbial exopolysaccharides Molecular weight

Micro-organism Acetobacter xylinum Xanthomonas campestris Klebsiella pneumoniae Enterobacter aerogenes

Rhizobium japonicum Rhizobium spp. Streptococcus pneumoniae Escherichia coli K87 Pullularia pullulans

Type 1 Type 4 Type 5 Type 6 Type 7 Type 8 Type 9 Type 11 Type 17 Type 21 Type 27 Type 32 Type 44 Type 54 Type 56 Type 57 Type 64 Glucan Type 3

5.67.105 2 .lo6 3.6 .lo6 15 .lo6 2.94. lo6 2.1 .lo5 1.29. lo6 2.5 . l o 6 1.2 . I 0 6 1.13.106 1.2 . l o 6 2 .lo6 9.4 .lo5 4.0 .lo5 9.4 . l o 5 1.2 .lo6 2.6 .lo5 1.2 .lo6 1.7 . l o 5 2.27. lo6 1.7 .lo6 7 .lo4 3 .lo3 2.67.105 2.8 .lo5 1.7 . l o 5

Reference Brown (1962) Dintzis et al. (1970) Milas and Rinaudo (1979) Holzwarth (1 978) Wolf et al. (1978) Churms et al. (1978) Wolf et al. (1978) Dutton et al. (1974) Wolf et al. (1978) Churms et al. (1978) Wolf et al. (1978) Dutton and Folkman (1980a) Churms et al. (1978) Dutton and Folkman (1980b) Churms et al. (1978) Wolf et al. (1978) Churms et al. (1 978) Dudman (1978) Zevenhuizen and Scholten-Koerselman (1979) Koenig and Perrings (1955) Tarcsay et al. (1971) Taguchi et al. (1973)

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

133

approach has now been extended to other bacterial species; it is clearly not restricted to the Enterobacteriaceae. The viscous polysaccharides appear from gel-permeation chromatography to have higher molecular weights than the wild-type material (I. W. Sutherland, unpublished results). Estimation of the molecular weights of exopolysaccharides still presents problems and accurate determination of these values has only been reported for a small number of such polymers. It is clear that the molecular weights of the exopolysaccharides produced by various species of bacteria can differ greatly (Table 8). What has not been determined to any significant extent, is whether there exists any relationship between the conditions under which the bacterium is grown and the molecular weight of the exopolysaccharide(s) it produces. Recent studies on the 0 antigens of Gram-negative bacteria indicate that their substituents and their chain lengths can vary much more than had previously been thought (Goldman and Leive, 1980; Palva and Makela, 1980). Collins ( 1 964) had earlier indicated how the composition of the lipopolysaccharide from Salmonella enteritidis was dependent on the rate at which this organism was grown; rapidly growing bacteria appeared to possess lipopolysaccharide with shorter 0 antigenic chains. McConnell and Wright (1979) demonstrated through phage-inactivation studies, that synthesis of lipopolysaccharide at 20°C yielded many molecules without attached side chains. It seems highly probable that similar variations may be found in exopolysaccharides. Most of the studies reported in Table 8 appear to have been performed on single batches of polymer produced in cultures grown overnight. The effect of growth rate on polymer size has not been appreciated. In recent studies in our laboratory (B. Nisbet and M. Kerr, unpublished work), a considerable range of molecular weights was displayed by a polysaccharide prepared from Enterobacter aerogenes type 30 grown on solid medium. The proportion of material of different molecular weight is shown TABLE 9. Distribution of molecular weights among polysaccharides produced by Enterohacter aerogenes Enterobucter aerogenes type 30 was cultured for four days on solid medium and the molecular weights of the polysaccharides produced were determined using Ultrogel (B. Nisbet. unpublished results) Molecular weight ( x 10-6)

Proportion of total material (%)

3.5

8.7 4.3 30.4 22.8 25.0 8.7

1.8 1.0

0.56 0.32 0.2

134

I. W. SUTHERLAND

in Table 9. Evans et al. (1979) also concluded that the polysaccharide produced by Xanthornonas juglandis in continuous culture at the lowest dilution rate tested (0.03 h - I ) was composed of longer unbranched molecules than that formed at higher dilution rates. Obviously, more studies with different polysaccharides are needed before firm conclusions can be drawn about relationships between the growth conditions or growth rate of producer organisms and the molecular weights of the polysaccharides which they synthesize. Using an in vitro system to study sialic acid synthesis by membranes of E. coli, Rohr and Troy (1980) noted that the polymer chains elongated to approximately 200 residues. After termination, new chain synthesis commenced, but the mode of termination and initiation of the polysaccharide molecules is not yet known.

B. P O S T - P O L Y M E R I Z A T I O N M O D I F I C A T I O N

It is assumed that most exopolysaccharides are secreted into the external environment of the microbial cell in their final form. If they are initially attached to the micro-organism as a capsule, they may eventually become detached, but the polymer remains chemically unchanged. Some polysaccharides may, after excretion, be degraded by polysaccharases secreted by the same micro-organisms which formed them. This is seen in some hyaluronic acid-synthesizing species of bacteria (Faber and Rosendal, 1954) and in alginate-producing strains of Azotobacter vinelandii (Haug and Larsen, 1971). Much less common, is the modification of the polysaccharide without shortening of the chain length after the polymer has been secreted. Such a process has been recognized in eukaryotes e.g. in the biosynthesis of heparin. Iduronic acid in the polymer is formed after polymerization and the enzyme responsible has been partially purified (Malmstrom et al., 1975). The epimerization of poly-D-mannuronic acid by an enzyme from Az. vinelandii was demonstrated by Haug and Larsen (1971). As some of the D-mannuronic acid residues carried 0-acetyl groups (Davidson et al., 1977), they were assumed to be unaffected by the epimerase; only non-acetylated residues could be converted to L-guluronic acid. The degree of specificity displayed by the epimerase remains unclear. Thus it is not known whether mannuronosyl residues adjacent to those which are acetylated are also “protected”. It does appear that L-guluronic acid residues are formed at random along the alginate chain, as fragments containing L-guluronic acid were among the products of Lguluronosyl-specific alginate lyases (Davidson et al., 1976). This was taken to indicate the presence of some adjacent residues in “blocks” of polyguluronic acid. Such sequences of guluronosyl residues can also be isolated by partial acid hydrolysis of the alginate of A z . vinelandii by the method

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

135

of Penman and Sanderson (1972). The major difference between the nature of alginate modification and the introduction of iduronic acid into heparin, lies in the uronic acid sequence; the iduronic acid residues mainly occur singly (Cifonelli and King, 1977). Consecutive units of five to six residues are, however, found in heparin. In alginate, random sequences of both component uronic acids are present.

IX. Acylation Many exopolysaccharides contain acyl groups, the most frequently encountered being 0-acetyl groups and pyruvate ketals; however, succinyl, formyl and other substituents have been identified. In the majority of polymers, the acyl groups appear to occur regularly on the repeating units in the same molar ratio as the monosaccharide constituents. This is typified by a structure such as colanic acid in which one mole of acetate and one mole of pyruvate ketal are present on each hexasaccharide repeating unit (Fig. 2) (Sutherland, 1969). From such results, it was deduced that addition of the acetyl and ketal groups proceeded in a manner similar to that of the monosaccharidesby sequential addition to a presumed lipid intermediate. It was also assumed that failure to add either substituent would lead to the absence of polysaccharide, again by analogy with monosaccharide addition. This view was revised following the isolation and characterization of colanic acid from a mutant of Salmonella typhirnuriurn. In this polysaccharide, 0-acetyl groups were absent, but the normal polysaccharide and ketal structures were present (Garegg et al., 1971a). Acetylation was thus not required for polysaccharide synthesis and failure to acetylate might be due to the absence of the appropriate acetylase enzyme or to the absence of sufficient precursor (presumably acetyl-Coenzyme A). It is also clear that acyl groups may be present in nonstoicheiometrical amounts in some polysaccharides. Unfortunately, purely chemical methods of analysis merely provide an average answer and do not reveal whether acyl groups occur irregularly on one polymer chain or on some chains and not on others.

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

Two polysaccharides have now been studied in sufficient detail for the presence of acyl groups on alternative carbohydrate repeating units to be recognized. In the case of one of these polysaccharides, produced by Enterobacter aerogenes type 54, this was accomplished by sequential degra-

136

I. W. SUTHERLAND

dation with two phage-induced enzymes. The first product obtained was an acetylated octasaccharide. This was isolated and hydrolysed with the second enzyme to yield equal amounts of an acetylated tetrasaccharide and a nonacetylated tetrasaccharide (Fig. 16) (Sutherland, 1967; Sutherland and Wilkinson, 1968). Recently, a similar type of repeating unit in which each alternate hexasaccharide carried a pyruvate ketal was recognized in the Bacteriophage enzyme I

+ FUC-

n[-Glc-,GlcA

G l c +GlcA

7 Fuc] I I

t

Glc

Formyl

Acetyl Bacteriophage

enzyme 2

FIG. 16. The use of bacteriophage-derived enzymes to determine by sequential hydrolysis the structure of a polysaccharide (from Enterobacter aerogenes type 54).

structure of a polysaccharide produced by Enterobacter aerogenes type 70 (Fig. 17) (Dutton and Mackie, 1978). Biosynthesis of these regular structures could still be accomplished by the type ormechanism demonstrated by Troy et al. (1971) provided that enzymes of the correct specificity permitted the addition of the acetyl or ketal groups after the assembly of two repeating units on the lipid intermediate. - 3 ) p o - Gal ( 1 - 2 ) - n -~ Rha ( 1 + 4 ) I \ 3 4 0 0 \I

-Po

- G l c A ( 1 -4)

-n-

L - R h a ( l -2)

- n-

L --ha

(1-2)

-no

Glc ( 1 -

/ C\

CH3

COOH

FIG. 17. The repeating unit of the exopolysaccharide produced by Enterobacter aerogenes type 70. The ketal substituent is located on alternate hexasaccharides (Dutton and Mackie, 1978). B. B A C T E R I A L A L G I N A T E S

In bacterial alginates, there is no repeating unit and the degree of acetylation

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

137

has been found to vary widely (Table 10). As the 0-acetyl groups are associated with the D-mannuronic acid residues (Davidson et a/., 1977), their introduction must occur prior to the post-polymerization epimerization (see p. 134). The process is, therefore, the acetylation of poly-D-mannuronic acid. It is not clear whether the 0-acetyl groups are regularly spaced on the polymer molecule or whether they occur associated only with certain portions of the polysaccharide chains. The absence of suitable enzymes has so far prevented answers being obtained to these questions. Davidson et al. (1977) have suggested, however, that the presence of the 0-acetyl groups is essential to “protect” some of the D-mannuronic acid residues from epimerization. The presence of a high acetyl content would thus ensure a high mannuronic acid content in the final polymer and, in turn, would have a considerable effect on the physical properties of the polymer (Smidsrlad, 1974). No model has so far been produced to account for the 0-acetylation of alginates other than a generalized concept resembling that postulated for the polysaccharides of Enterobacter uerogenes (Fig. 18) (Pindar and Bucke, 1975). Even this may now require some modification, as discussed earlier (p. 101).

C. X A N T H A N - P O L Y S A C C H A R I D E S

OF X A N T H O M O N A S C A M P E S T R I S

Many of the papers dealing with the structure of xanthan show the structure as carrying one mole of pyruvate and one mole of acetate or as each unit carrying acetate and alternate units carrying pyruvate (e.g. Holzwarth, 1976; Morris, 1977; Morris et al., 1977). In the definitive papers postulating this structure, Jansson et al. (1975) proposed that one acetyl group was present on each pentasaccharide repeating unit, whereas only about half were pyruvylated. Similarly, Melton et a f . (1976) presented data which would permit approximately 90% of the repeating units to be acetylated, but only about 33% to carry pyruvate. Indeed, Jansson et al. (1975) realized the probable presence of non-stoicheiometrical amounts of pyruvate in polysaccharides collectively considered to be of the “xanthan type”. This has since been recognized in a number of studies which have shown that the pyruvate content of X . campestris polysaccharides can vary considerably depending on the culture conditions and on the strain used (Tables 4 and 5). Some of these results, together with others in which variable amounts of acyl or ketal groups have been reported for the same or closely related polysaccharides. are presented in Table 10. Examination of a large number of polysaccharides from bacterial isolates now considered to belong to the four recognized Xanthomonas species ( X . campestris, X . albilineans, X . amnopodis and X . fragariu) has shown that the majority closely resembled the previously described xanthan structure in respect of acetyl, pyruvate and monosaccharide content. (In the literature, there are also reports of poly-

TABLE 10. The acyl content of some microbial exopolysaccharides Content (%) of Polymer Polysaccharide from Pseudomonas N C 1B 1 1264 Alginate from Pseudomonas aeruginosa

Carbohydrate

Acetate

Pyruvate

67-80

3.2- 4.0

5.2- 8.8

59-87 50-73

8.5-1 1.0 0 -13.4 2.3-14.7

Xanthan from Xanthomonas campes tr is Succinoglucan from Alcaligenes faecalis Polysaccharide from an Arthrobacter sp.

Succinate

Williams and Wimpenny (1977) Linker and Evans (1976) Evans and Linker (I 973) Piggott (1978)

3.7- 4.7 3.1- 5.2 1 . C 8.2

2.5- 4.7 0.9- 8.5 1G10.6

-

Cadmus et al. (1976) Davidson (1978) I. W. Sutherland (unpublished results)

-

0.3- 1.5

5.4- 5.9 4.9- 6.3

5.8-6.6 0.47.4

81.4

3.7

5.0

5.9

87

Reference

Hisamatsu et al. (l978a) Hisamatsu et al. (1978b) Knutson et al. (1979)

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

139

Carbohydrate s u b s t r a t e

A

Mannose I - p h o s p h a t e

& \1

GDP- mannose pyrophosphorylose

GDP- mannose GDP-rnonnose

dehydrogenase

GDP- mannuronic a c i d

Isaprenaid lipid pyrophosphate mannuronic a c i d

V Isoprenoid lipid pyrophosphate

4

Acetylose

Acetylated polymannuronic a c i d

J,

Epimerose

Alginate

FIG. 18. Possible route of bacterial synthesis of alginate.

saccharides containing galactose or oxodeoxyoctonic acid which are clearly different from “xanthan”, e.g. Fareed and Percival, 1976; Yadomae et a f . , 1978. Whether the bacterial strains are truly species of Xanthomonas is not clear.) As well as the polysaccharides with the same composition as that accepted earlier for “xanthan”, polymers with (i) pyruvate, but negligible acetate; (ii) acetate, but no pyruvate; and (iii) pyruvate, plus approximately two moles of acetate per mole of polymer were identified (I. W. Sutherland, unpublished results). The process of acetylation and pyruvylation of xanthan, therefore, has to account for a wide range of pyruvate contents and also some variation in the acetyl content. The strain whose polysaccharide contains two moles of acetate per mole of polymer is probably exceptional and may well contain a second acetylase capable of adding a second 0-acetyl group to a sugar residue other than the D-mannose adjacent to the cellulose backbone (Fig. 2). The variations in pyruvate content in the polysaccharides from the other strains could be due to either the irregular addition of pyruvate, or the presence of a mixture of pyruvylated and non-pyruvylated strands of polysaccharide. In an attempt to discriminate between these possibilities, an affinity column capable of complexing pyruvylated polysaccharide was prepared and several preparations of 14C-labelled polysaccharide were applied to it. It proved possible to resolve such material into “pyruvate-rich”

1 40

I . W. SUTHERLAND

and “pyruvate-poor” fractions suggesting the possible presence of a mixture of strand types (I. W. Sutherland, unpublished results). This, in turn, would imply that regulatory mechanisms existed which permitted the synthesis of pyruvylated strands under certain conditions of growth and of non-pyruvylated strands under others-a situation resembling that obtaining in the biosynthesis of the colanic acid types with and without acetate. Could this perhaps indicate a dependence on the intracellular level of phosphoenolpyruvate, the presumed precursor of the ketal group? Attempts to obtain cell-free addition of labelled pyruvate from phosphoenolpyruvate to the presumed lipid-linked intermediates or to polymeric material have so far been unsuccessful (C. L. Saunderson, unpublished results).

D. G E N E R A L A S P E C T S O F A C E T Y L A T I O N A N D K E T A L A T I O N

The absence of suitable cell-free systems capable of synthesizing acylated exopolysaccharides has so far prevented direct studies being undertaken of the enzymes responsible for such syntheses. The assumed donor of acetyl groups is acetyl-CoA and the process is probably similar to that found in the acetylation of lipopolysaccharides. Sphaeroplast membranes of Neisseria meningitidis have been used as a source of acetylase for sialic acid synthesis (Vann et al., 1978).This enzyme appeared to transfer acetyl groups from acetylCoA to presynthesized sialic acid, but whether this process is the same as that which occurs in intact cells is uncertain. The addition of pyruvate ketal groups to polysaccharides has also been attempted without success in cell-free systems. There is no evidence to suggest that the addition occurs at the sugar nucleotide stage as in UDP-Nacetylmuramic acid formation (Gunetileke and Anwar, 1968; Wickus and Strominger, 1973). The most likely reaction would be a similar process at the lipid intermediate stage: Lipid-P-P-glycose-glycose+ phosphoenolpyruvate

e

Lipid-P-P-glycose-gl ycose

I1

CH3.C.COO-

Whether such a reaction is a multistage process involving several enzymes and cofactors such as FAD, as demonstrated for UDP-N-acetylmuramic acid production in Staphylococcus epidermidis (Wickus et al., 1973) or a simple reaction involving a single enzyme must await the development of suitable methods. The derivation of non-acetylated polymer from acetylated material was demonstrated with colanic acid-producing bacteria (Garegg et al., 197la). The mutant strain in this instance presumably carried a single mutation resulting in loss of the ability to synthesize the acetylase enzyme. It is possible

BIOSYNTHESIS OF MICROBIAL EXOPOLYSACCHARIDES

141

that similar defects in pyruvylation lead to the production of pyruvate-free polysaccharide in some species of Xunthomonus. However, there has as yet been no report of the derivation of a mutant strain producing polymer of this type from a wild-type strain producing a fully acetylated and pyruvylated polysaccharide. Other acylated and ketalated microbial polysaccharides may resemble the material from Xunthomonus spp. in their variability. In a recent study of polysaccharides from Rhizohium species (M. C. Cadmus and M. E. Slodki, unpublished results), differences were found between the three different species tested. Significant increases in the content of 0-acetyl groups were found in the polysaccharide formed in the early hours of culture growth of R. trifolii, but not of R. phuseoli. As with the polysaccharides produced by Xunthornonus isolates, differences in acetyl and pyruvate contents were observed in polysaccharides formed by different isolates of the Rhizobium species. X. The Future

Several years ago, it seemed that our knowledge of exopolysaccharide synthesis was falling into place with the process appearing closely to resemble the mechanism of formation of peptidoglycan and of some lipopolysaccharides. It is now clear that this optimism was premature and that there are still many unanswered questions. (i) What are the precursors of sugars such as L-guluronic acid and methyluronic acids? (ii) How are the side chains formed of polymers such as xanthan and other polysaccharides with large repeating units? (iii) Are there acceptors for capsular polysaccharides and, if so, what is their chemical nature? (iv) What regulatory mechanisms control (a) addition of side chains in polymers such as xanthan, and (b) the chain lengths of these and other polysaccharides? (v) Are succinyl and acetyl groups added from their CoA derivatives or are there other, as yet unrecognized, acyl donors. (vi) How do the physiological conditions of growth affect the chemical composition of polysaccharides and their physical characteristics? These are only some of the questions to which answers may be forthcoming in the next few years. Fortunately, the current industrial interest in microbial exopolysaccharides has led to studies on many of these polymers from nonenterobacterial sources and this should hopefully extend our knowledge about these macromolecules.

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XI. Acknowledgements The author is indebted to various colleagues for discussions relating to this review and, in particular, to Dr. M. McConnell, Dr. M. Slodki and Dr. M. C. Cadmus for communicating unpublished results. Part of the work in the author’s laboratory was supported by S.R.C. research grant GRA 30049 and by D.O.E. contract OTF 443. REFERENCES

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Robbins, P. W., Bray, D., Dankert, M. and Wright, A. (1967). Science. New York 158, 1542. Robyt, J. F. and Tanaguchi. H. (1976). Archives of Biochemistry and Bioplzysics 174, 129. Robyt, J. F. and Walseth. T. F. (1978). Carbohydrate Research 61, 433. Robyt, J. F.. Kimble. B. K . and Walseth, T. F. (1974). Archives of’Biochernistrj~and Biophysics 165. 634. Rohr, T. E. and Troy, F. A. (1980). Journal qf Biological Chemistry 255, 2332. Sandermann, H. (1974). European Journal of Biochemistry 43, 415. Sandermann, H. (1976). Biochimica et Biophysica Acta 444, 783. Sandermann, H. (1977). Federation of European Biochemical Societies Letters 81.294. Sandermann, H. and Dekker, R. F. H . (1979). Federation of European Biochemical Societies Letters 107, 237. Sandermann, H. and Strominger, J. L. (1972). Journal of Biological Chemistry 247, 5123. Sanders, R. E., Carlson, R. W. and Albersheim, P. (1978). Nature, London 271, 240. Sandford, P. A. (1979). Advances in Carbohydrale Chemistry and Biochemistry 36. 265. Santamaria, F., Reyes, S. F. and Lahoz, R. (1978). Journal of General Microbiology 109, 287. Scales, W. R., Long, L. W. and Edwards, J. R. (1975). Carbohydrate Research 42,325. Scher, M. and Lennarz, W. J. (1969). Journal of Biological Chemistry 244, 2777. Schmidt, G., Mayer, H. and Makela, P. H. (1976). Journal of BacteriologJI 127. 755. Scott, G. J. (1979). Ph.D. Thesis: Nottingham University. Silman, R. W. and Bagley, E. B. (1979). Biotechnology and Bioengineering 21, 173. Silman, R. W. and Rogovin. P. (1970). Biotechnology und Bioengineering 12, 75. Silman, R. W. and Rogovin, P. (1972). Biotechnology and Bioengineering 14, 23. Slodki, M. E., Safranski, M. J., Hensley, D. E. and Babcock, G. E. (1970). Applied Microbiology 19, 10 19. Smidsrod, 0. (1974). Faraduy Discussions of the Chemical Society 57, 263. Smit, J. and Nikaido, H. (1978). Journal of Bacteriology 135, 687. Smith, E. J. (1968). Journal of Biological Chemistry 243, 51 39. Smith, E. E. B., Mills, G. T. and Bernheimer, H. P. (1961). Journal of Biological Chemistry 236, 2 179. Stocker, B. A. D. and Makela, P. H. (1978). Proceedings of the R o j d Society, London Ser B. 202, 5 . Stoolmiller, A. C. and Dorfman, A. (1969). Journal of Biological Chemistry 244, 236. Sutherland, I . W. (1967). Biochemical Journal 104, 278. Sutherland, I. W. (1969). Biochemical Journal 115, 935. Sutherland, I. W. (1971). European Journal of Biochemistry 23, 582. Sutherland, I. W. (1975). Biochemical Societv Transactions 3, 840. Sutherland, I. W. (1977a). In “Extracellular Microbial Polysaccharides” (P. A. Sandford and A. Laskin, eds.) American Chemical Society Symposium 45, pp. 4&57. American Chemical Society. Washington, D.C. Sutherland, I. W. (l977b). Surface Carbohydrates of the Prokaryotic Cell. Academic Press, London, New York and San Francisco. Sutherland, I . W. (1979a). In “Microbial Polysaccharides and Polysaccharidases” (R. C. W. Berkeley, G. W. Gooday and D. C. Ellwood, eds.), pp. 1-34. Academic Press, London, New York and San Francisco. Sutherland, I. W. (1979b). Journal of General Microbiology 111, 21 1. Sutherland, I. W. (1979~).Journal of Applied Biochemistry 1, 60. Sutherland, I. W. and Ellwood, D. C. (1979). In “Microbial Technology-Current

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State and Future Prospects” (A. T. Bull, D. C. Ellwood and C. Ratledge, eds.), Society for General Microbiology Symposium 29, pp. 107-1 50. University Press, Cambridge. Sutherland, I. W. and Norval, M. (1970). Biochemical Journal 120. 567. Sutherland, I. W. and Wilkinson, J. F. (1968). Biochemical Journal 110, 749. Sutherland. I. W. and Williamson. J. (1979). European Journal of Applied Microbiology 6, 233. Taguchi, R., Kikuchi, Y., Sakano, Y. and Kobayashi, T. (1973). Agricultural and Biological Chemistry 37, 1583. Tam. K. T. and Finn, R. K. (1977). In “Extracellular Microbial Polysaccharides” (P. A. Sandford and A. Laskin, eds.), American Chemical Society Symposium 45, pp. 58-80. American Chemical Society, Washington, D.C. Tanaka, A,, Cho, Y., Teranishi, Y., Nabeshima. S. and Fukui, S. (1974). Journal of Fermentation Technology 52, 739. Tarcsay, L., Jann, B. and Jann, K. (1971). European Journal of Biochemistry 23, 505. Tonn, S. J. and Gander, J. E. (1979). Annual Reviews of’Microbiology 33, 169. Troy. F. A. (1979). Annual Reviews of Microbiolog,v 33, 519. Troy, F. A. and McCloskey, M. A. (1979). Journal of Biological Chemistry 254, 7377. Troy, F. A,, Frerman. F. E. and Heath, E. C. (1971). Journal of Biological Chemistry 246. 118. Troy, F. A,, Vijay, I. K . and Tesche, N. (1975). Journal ojBiologica1 Chemistry 250, 156. Umbreit, J. N., Stone, K. J. and Strominger, J. L. (1972). Journal of Bacteriology 112. 1302. Vann, W. F.. Liu, T. Y. and Robbins, J. B. (1978). Journal of Bacteriology 133, 1306. Vijay. I. K. and Troy, F. A. (1975). Journal of Biological Chemistry 250, 164. Ward, J. B. and Perkins, H. (1973). Biochemical Journal 135, 721. Weiner, I. M., Higuchi, T., Osborn, M. J. and Horecker, B. L. (1965) Proceedings o f t h e National Academy of Sciences of the United States of,4mericu 53, 881. Whitfield, C. (1979). Ph.D. Thesis: Edinburgh University, Scotland, UK. Wickus, G. G . and Strominger, J. L. (1973). Journal qf’Bacteriology 113, 287. Wickus, G. G., Rubenstein, P. A., Warth, A. D. and Strominger, J. L. (1973). Journal of Bacteriology 113, 291. Wilkinson. J. F. (1 958). Bacteriological Reviews 22, 46. Wilkinson, J. F., Duguid, J. P. and Edmunds, P. N. (1954). Journal qf General Microbiology 11, 59. Wilkinson, J. F. and Stark, G . H. (1956). Proceedings of the Ro-val Physical Society (Edinburgh) 25, 35. Williams, A. G . and Wimpenny, J. W. T. (1977). Journal qf General Microbiology 102, 13. Willoughby, E., Higashi. Y. and Strominger, J. L. (1972). Journal of Biological Chemistry 247, 5 I 13. Wolf. C.. Elsasser-Beile, V., Stirm, S., Dutton, G. G. S. and Burchard, W. (1978). Biopo1,vmer.y 17, 73 1. Wright, A. (1971). Journal of Bacteriology 105, 927. Wright, A. and Kanegasaki, S. (1971). Physiological Reviews 51, 748. Wright, A. and Tipper, D. J. (1979). In “The Bacteria” (J. R. Sokatch and L. N. Ornston, eds.), vol. 7, pp. 427-485. Academic Press, New York, San Francisco and London. Wright, A,, Dankert, M. and Robbins, P. W. (1965). Proceedings of the National Academy of Sciences of the United States of America 54, 235. Wyss. 0. and Moreland, E. J. (1968). Applied Microbiology 16, 185.

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Yadomae, T., Yamada, H., Miyazaki, T., Omori, T. and Hirota, T. (1978). Carbohydrate Research 60, 129. York, W. S., McNeil, M., Darvill, A. G . and Albersheim, P. (1980). Journal of Bacteriology 142, 243. Yuen, S. (1974). Process Biochemistry 9 , 7. Zaar, K. (1977). Cytobiologie 16, 1 . Zaar, K. (1978). Archives of Microbiology 117, 303. Zaar, K. (1979). Journal of’ Cell Biology 80. 773. Zevenhuizen, L. P. T. M . and Schulten-Koerselman, H. J. (1979). Anronie vun Leeuwenhoek 45, 165.

Note Added in Proof

Although the presence of strains of Xanthomonas campestris synthesizing polysaccharide with little, if any, pyruvate under all conditions tested had been recognized, a mutant has recently been described yielding polymer devoid of pyruvate (Werhau, 1979). Thus, loss of pyruvate resembles loss of acetate (p. 141). Polysaccharide is still produced by the bacteria in good yield, although with apparently very much lower viscosity than the wild-type material (I. Bradshaw, unpublished results). It is possible that this material is of lower molecular weight than the values quoted earlier for xanthan (p. 132). Regulation of alginate production in Pseudomonas aeruginosa has been shown by Fyfe and Govan (1 980) to involve at least one chromosomal marker linked to pro-67, his-5075 and cvs-5605, but it was not possible to determine the number of genes involved. The number of bacterial species capable of synthesizing alginate has been extended following the selection ofcarbenicillinresistant alginate-producing variants of Pseudomona.y”puorescens, Ps. putida and Ps. mendocina (Govan et al., 1981). The product of these strains was an acetylated copolymer similar to that from Ps. aeruginosa and Azotohacter vinelandii. A number of other Pseudomonas species tested failed to form alginate-syqthesizing variants. These included Ps. maltophilia, one strain of which, togetherwith Ps.putidastrains,have been shown to produce “alginases” (Lyle von Riesen, 1980), with specificity for polymannuronic acid portions of the alginate molecule (Sutherland and Keene, 1981). These strains thus resemble A . vinelandii in having the capacity to produce and degrade alginate, although the specificity of the alginate lyases differs. The ability of Agrohacterium tumefaciens to produce polysaccharide fibrils during attachment to plant cells has been demonstrated (Matthysse et a/., 1981). On the basis of staining procedures using calcofluor and enzymic hydrolysis. it was suggested that these fibrils were composed of cellulose. This is of particular interest as A . tumefaciens is one of the group of closely related bacterial species capable of synthesizing both curdlan and succinoglycan (p. 112). If it is also possible that it can form cellulose and perhaps also a low molecular-weight glucan of the type indicated by Zevenhuizen and Schulten-Koerselman (1979) and York et a/. (1980; see main reference

150

I. W. SUTHERLAND

list), complex control mechanisms appear to operate. As yet, there is no other microbial group known to form four distinct exopolysaccharides. REFERENCES

Fyfe, J. A. M. and Govan, J. R. W. (1980). Journal of General Microbiology 119,443. Govan, J. R. W., Fyfe, J. A. M . and Jarman, T. R. (1981). Journal of General Microbiology 125, 217. Lyle von Riesen, V. (1980). Applied and Environmental Microbiology 39, 92. Matthysse, A. G., Holmes, K. V. and Gurlitz, R. H. G. (1981). Journalof Bacteriology 145, 583.

Sutherland, I . W. and Keene, G. A. (1981). Journal of Applied Biochemistry 3, 48. Werhau, W. C. (1979). British Patent 2008 600A

Yeast Cell-Wall Glucans J. H. DUFFUS, CAROLYN LEV1 and D. J. MANNERS Department of Brewing and Biological Sciences, Heriot- Watt Universiiy, Edinburgh EH I I HX, Scotland I. Introduction . . , . . . . . . 11. Structural analysis of yeast glucans . . . . A. General methods . . . . . . . B. Glucans in walls of Sucrhoromycrs rerevisiue . . C . Glucans from other Sacchuromvces species . . D. Glucans in walls of Sc~ii=o.succliurom~ce.~ pnmhe . E. Glucans from Candidu species. . . . . F. Other yeast glucans . . . . . . . 111. Yeast wall glucan synthesis A. Introduction . . . . . . . . B. Studies with inhibitors of glucan synthesis . . C. Glucan synthetases of whole and fractionated cells . D. Glucan synthesis and glucan synthetases in protoplasts IV. Physiological control of glucan content . . . . . . . A. Introduction . B. The cell cycle . . . . . . . . C. Yeast-mycelium interconversion . . . . D. Effects of nutrient limitation . . . . . E. Effects of metabolic inhibitors. . . . . F. Miscellaneous . . . . . . . . V. Acknowledgements . . . . . . . References . . . . . . . . .

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152 155 160 162 162 165 166 166 166 167 169 171 171 171 174 174 176 177 177 178

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I. Introduction Although the chemistry of the yeast cell wall has been the subject of continued investigation in several laboratories, overall progress has been relatively slow. This arises partly from the complex nature of the wall which may contain three, four or even more different polysaccharide components, in addition to protein, phosphorus and lipids, and partly from the relative insolubility

152

J. H. DUFFUS, CAROLYN LEV1 AND D. J . MANNERS

of some of these components which renders purification and structural analysis by conventional methods extremely difficult. Thanks to the sustained work of C. E. Ballou and his coworkers, a substantial amount of information on the structure and metabolism of yeast mannans is now available (for reviews, see Ballou, 1974, 1976). The purpose of this review is to describe the present state of knowledge on the structure and metabolism of the related glucan components of yeast cell walls. Many of the earlier structural studies involved commercial samples of pressed baker’s yeast (i.e. various strains of Succhuromyces cerevisiue) and, although these enabled the main structural features to be identified, they were of limited value from a metabolic point of view. With recent improvements in polysaccharide fractionation and in the methods of structural analysis, it is now possible to use smaller samples of yeast from laboratory-scale cultures as the experimental material, and hence, to relate the changes in cell-wall structure with cell growth. The structural analysis of glucan preparations isolated from various species of yeast will be described in the next section, where all of the constituent monosaccharide residues will be assumed to have the D-configuration. The following section will describe synthesis of glucan from nucleoside diphosphate sugar compounds by a variety of yeast enzyme preparations, and the final section deals with the physiological control of the glucan content of yeast cell walls during various conditions of growth. The literature survey for this review has been selective rather than comprehensive. Much of the early work has been summarized elsewhere, e.g. Phaff (1963, 1971, 1977). MacWilliam (1970), and other current assessments of the literature have been provided by Bacon (1981) and by Fleet and Phaff (1981).

11. Structural Analysis of Yeast Glucans A. G E N E R A L M E T H O D S

1. Prepurution

of Yeust Glucuns

The early work on yeast glucan was carried out using whole pressed cells of baker’s yeast, which were extracted with hot alkali to remove mannan. This was then precipitated as the copper complex after addition of Fehling’s solution. The alkali-extracted cells still contained some glycogen, which was removed by extraction with warm acetic acid, o r by autoclaving with water, and the final residue was regarded as “yeast glucan”. It was characteristically insoluble, a property which prevented examination of its homogeneity by

YEAST CELL-WALL GLUCANS

153

standard methods, and hindered the subsequent structural analysis. The overall composition of the yeast cell wall was usually described (e.g. Northcote, 1962) as glucan (about 30%), mannan (about 30%), protein (about 10-15%), lipid (8-9%) and chitin (1-273, the wall representing about 15% of the dry weight of the cell. The above results do not take account of the presence of about 20% of an alkali-soluble glucan (see Section II.B.2, p. 158), which has only been recognized in more recent years. The true glucan content of many cell walls is therefore about 50%. However, it should be emphasized that all of these analytical results will vary with the species and strains of yeast, its conditions of growth, and with the methods used for the preparation of the cell walls and their fractionation. As an alternative to using pressed cells, the yeast can be disrupted mechanically to give cell-wall preparations which, after being washed with various solvents, are a suitable starting material (e.g. Northcote and Horne, 1952). However, it has recently been realized that the cell walls contain endogenous 8-glucanases, which can partially hydrolyse cell-wall components, causing autolysis. It is therefore essential that these enzymes should be inactivated during the preparation of cell walls. Treatment with Tris buffer or sodium phosphate buffer at pH 8.5 has been recommended (Fleet and Phaff, 1973; Fleet and Manners, 1976). Bacon and his coworkers (1969) reported that glucan is more easily extracted by alkali from isolated cell walls than from intact cells. This difference is discussed in detail by these workers, but the reasons are not yet clearly understood. It is therefore obvious that caution is required in comparing and interpreting the results of alkaline extractions of cell-wall preparations and of intact cells.

2. Analytical Methods We have already noted that the structural analysis of the glucans is hampered by their relative insolubility. As a simple example, total or partial acid hydrolysis by mineral acid is incomplete, without a prior treatment with formic acid. Methylation analysis remains an important technique in polysaccharide studies. With glucans, the proportion of 2, 3, 4, 6-tetra-0-methyl glucose represents the non-reducing terminal residues, whereas the amount of 2, 4, 6- and 2, 3, 4-tri-0-methyl glucose indicates the proportion of (1 + 3)- and (1 -+ 6)-inter-residue linkages. The presence of 2, 4-di-0-methyl glucose, assuming complete methylation, indicates residues linked at C-1 , C-3 and C-6, i.e. branch points, and the amount should be equivalent to that of tetra-0-methyl glucose. However, yeast glucan is notoriously difficult to methylate with the conventional Haworth reagents (dimethyl sulphate

1 54

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

and sodium hydroxide). Bell and Northcote (1950) found 3 1 methylations gave a methoxyl content of 41.7% (theoretical 45.6%), whereas Manners and Patterson (1966) obtained a methoxyl content of 41.5% after 16 treatments. The introduction of improved reagents (e.g. Hakomori, 1964) has, to some extent, facilitated methylation, but doubts about the completeness of the etherification of insoluble glucans still remain. Periodate oxidation provides a useful alternative method of aralysis. The amount of periodate reduced per glucose residue (expressed as molecular proportions, or mol. prop.) is determined by the presence of diol and trio1 groups. The latter arise from (a) terminal glucose residues and (b) ( 1 -6)linked glucose residues. A linear (1 + 3)-/?-glucan should reduce less than 0.1 mol. prop. of periodate, whereas the presence of several ( 1 -6)-linked residues could easily increase this value to 0 4 0 . 6 mol. prop. The amount of formic acid released is also a measure of the relative proportion of nonreducing terminal residues and of (1 + 6)-linked residues. Subsequent analysis of periodate-oxidized glucans involving borohydride reduction and partial acid hydrolysis (i.e. a Smith degradation), or borohydride reduction and total acid hydrolysis, also provides useful information on the location of ( 1 -+ 6)-linked residues within a (1 -+ 3)-P-glucan macromolecule. Readers should consult the series “Methods in Carbohydrate Chemistry” edited by R. L. Whistler and J. N. BeMiller, and published by Academic Press for further details of these methods. A further method of analysis involves selective enzymolysis. Ideally, purified enzymes of known action pattern should be used, so that structural analysis of the end-products gives information on the structure of the enzymically resistant material and also on the nature of the linkages hydrolysed. It should be realized that the specificity of some P-glucanases is controlled not by the linkage which is hydrolysed (b), but by an adjacent linkage (a): (a) (b) - G - G - G Hence, some endo-( 1 -+3)-P-glucanases may hydrolyse (1 -4) or (1 -6)linkages in substrates containing both (1 -3)- and a second type of glucosidic linkage (Perlin and Suzuki, 1962; Cunningham and Manners, 1964). For example, Rombouts and his coworkers (1978) have examined two extracellular ( I -6)-P-glucanases produced by Bacillus circuluns WL- 12, which differ in their ability to lyse yeast cell walls. The non-lytic enzyme hydrolysed ( 1 -6)-P-glucans in an endo fashion, but did not hydrolyse branched (1 -3)-P-glucans containing ( 1 -6)-interchain linkages. By contrast, the lytic enzyme showed both endo-( 1 -6)-p-glucanase activity and activity towards branched (1 3)-P-glucans having (1 -6)-interchain linkages. This indicates an ability of the lytic enzyme to hydrolyse certain ( 1 -3)-linkages in the

-

YEAST CELL-WALL GLUCANS

155

vicinity of a (1 -6)-interchain linkage. In some instances, degradation of a glucan by an unpurified enzyme preparation can be used, provided that deductions are confined to the structure of the products of enzyme action, and do not include the nature and site of the susceptible linkages. Such an experiment provides a means of partial hydrolysis of the P-glucan which is less random than the use of dilute acid. The presence of endogenous j?-glucanases in yeast cell walls has already been mentioned; see for example Abd-el-al and Phaff (1969), Notario et al. (1975) and Villa et al. (1979). Although a detailed discussion of their action falls outside the scope of this review, we should note that their action on substrates in vivo enables some conclusions to be drawn on the molecular structure of the latter. A retrospective and current view of these enzymes has been provided by Phaff (1979).

B . G L U C A N S IN W A L L S O F S A C C H A R O M Y C E S C E R E V I S I A E

1. Alkali-Insoluble Glucans

During the period 1950-1960, differences of opinion developed on the structure of the alkali-insoluble glucan from baker’s yeast. On the basis of a methylation analysis, Bell and Northcote (1950) suggested it had a highly branched structure with relatively short chains of (1 -3)-linked glucose residues interlinked by about 11% of ( 1 -2)-glucosidic linkages. The average chain length, confirmed by periodate oxidation, was nine glucose residues. By contrast, Peat and his coworkers (1958a), using partial acid hydrolysis, concluded that the glucan was linear and contained certain sequences of ( 1 -3)- and (1 -6)-linked j?-glucose residues. The presence of about 10-20% of (1 -6)-linkages was confirmed by tosylation followed by iodination of primary hydroxyl groups (Peat et al., 1958b). In attempts to resolve these differences, Manners and Patterson (1966) carried out further methylation, periodate oxidation and also enzymic degradation studies, and concluded that yeast glucan had a branched structure, containing main chains of (1 -6)-linked P-glucose residues, to which were attached linear side chains of ( 1 -+3)-linked /?-glucose residues. An alternative structure was proposed by Misaki et al. (1968) on the basis of their chemical studies (see Fig. la) which was similar, in some respects, to that proposed by Manners and Patterson (1966). In all of these experiments (19501968), the yeast had been successively extracted with hot alkali, and with acetic acid, and/or autoclaving in water to remove mannan and glycogen respectively, and the residual material was regarded as the “yeast glucan”. A key observation by Bacon and Farmer ( I 968) eventually led to a solution of the problem. These workers showed that yeast glucan prepared as already

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

156

(a)

-~1-~6)-[Gl,-(1-~6)-G-(1-~3)-G-(l-6)-G-(1

+6)-[GIy-

I

I

h

3

j.

6

7 ....G-(l-t3)-G--(l~3)-G.... FIG. 1 . Structure of yeast glucan. (a) Shows the partial structure of yeast glucan as proposed by Misaki er al. (1968). G denotes a /?-D-glucopyranose residue, and x y = 40-50. (b) Shows the partial structure of a segment of yeast /?-(1 -+3)-glucan. a + b + c comprise about 60 glucose residues, although the exact lengths of a, b and c are unknown.

+

described was heterogeneous, and contained an acetic acid-soluble polysaccharide which was shown by partial hydrolysis and infrared spectroscopy to be a (1 +6)-P-glucan. Their suggestion that “yeast glucan” was, in fact, a mixture of a major (1 -+3)-j?-glucan component and a minor (1 +6)-/3-glucan component provided an explanation for many of the earlier experimental results. This significant discovery by Bacon and Farmer (1968) led to a detailed fractionation of bakers yeast, and a chemical characterization of the resulting (1 -+ 3)- and ( 1 + 6)-P-glucan components (Manners et a/, 1973 a, b). The fractionation scheme is outlined in Fig. 2 . The major component (about 85%) was a lightly branched (1 -+ 3)-,!3-glucanof high molecular weight (minimum DP value 1450 f 150, equivalent to a molecular weight of about 240,000) and containing about 3% of (1 -+ 6)-P-glucosidic interchain linkages (Manners et al., 1973a). The minor component (about 15%) had a minimum DP value of 130-140 (equivalent to a molecular weight of about 22,000), and contained a high proportion of (1 -+6)-linkages (65%), together with a smaller number of (1 -+3)-linkages which were present as both interresidue (5%) and interchain (14%) linkages (Manners et af., 1973b). The molecule was highly branched, possessing 16% of non-reducing terminal groups. These analytical results apply only to the glucans from one particular sample of baker’s yeast, and the authors emphasized that glucan preparations from different batches of

YEAST CELL-WALL GLUCANS

157

with 3",, sodium hydroxide at 75 C for 6 hours

Alkali-soluble carbohydrate

Alkali-insoluble carbohydrate

Treated with Fehling's solution

Extracted 27 times with

0.5~ acetic acid at 90 C

glucan glucan (mannan) (mannan)

denatured denatured Iprotein)

1

Soluble Soluble glucans glucans (glycogen and (1 + 6)$D-glucan) I

iodine solution

Iucanase

Precipitated iodinecomplex (glycogen)

Soluble glucan

Treated with z-amy lase

Purified

FIG. 2 . Fractionation of baker's yeast to give purified ( 1 and alkali-soluble glucan.

(1 -,6 ) - 8 - ~ -

3)- and ( 1 -+ 6)-8-~-ghcan.

yeast may differ in the relative proportion of the two components, and that the individual components might vary considerably in degree of branching and other structural parameters. This structural analysis of the major component did not indicate whether single or multiple branching was present, or whether the molecule had a laminated, comb or tree-type structure (or some variant of these) of the kinds

158

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

suggested for the amylopectin component of starch (see Fig. Ib, p. 156). The major ( 1 +3)-/?-glucan is. now generally regarded as providing an insoluble “envelope” which is responsible for the rigidity of the cell wall. The molecular weight of about 240,000 represents a minimum value, and is based on the assumption that each molecule contains one free reducing glucose residue. The validity of this assumption is not yet known, but no other method is available for determining the molecular size of such an insoluble macromolecule. In spite of this uncertainty in the size of the (1 +3)-P-glucan component, it seems clear that numerous individual molecules would be required per cell. The mode of association of the molecules is not known, but the overall degree of branching (about 3%) is so low that some of the linear segments of chains could be held together in triple helix conformations, to give a macromolecular structure with a substantial degree of rigidity and insolubility in water (Rees, 1973). The presence of small but significant amounts of (1 -+6)-/?-glucan in the baker’s yeast cell wall poses questions regarding its biological function, and the control of the relative rates of synthesis of the two types of /?-glucan. Since the proportion of the two types of linkage in the (1 -+6)-/?-glucan are so different from those in the major ( 1 -+3)-/?-glucan, the former is unlikely to be an intermediate in the biosynthesis of the latter. As will be described in more detail later in this section (p. 163), ( 1 -+ 6)-/?-glucan components have been detected in several different yeasts (Manners et al., 1974), so that the questions posed above are of general interest in considering yeast cell-wall glucans as a whole. Since the ( 1 -+6)-/?-glucan is soluble under non-alkaline conditions and has a relatively low molecular weight, it might be considered as a reserve rather than a structural material, although of course, this would duplicate the function normally assigned to glycogen, with which it is closely associated (Evans and Manners, 1971). Alternatively, the glucan could function as a plasticizer or filling material in the relatively rigid envelope constituted by the major ( 1 +3)-/?-glucan, where its function could be to prevent excessive aggregation of linear segments of chains, so that some measure of flexibility is retained in the wall, as would be required for wall expansion during growth (Manners et al., 1974). 2. Alkali-Soluble Glucans

The earlier literature contains several reports on the presence of alkali-soluble glucan in cell-wall preparations of baker’s yeast and Sacch. cerevisiue (Roelofsen, 1953; Kessler and Nickerson, 1959) but its molecular structure was unknown. Fleet and Manners (1976, 1977) accordingly carried out a detailed study of this glucan.

159

YEAST CELL-WALL GLUCANS

The polysaccharide was extracted from cell walls of Sacch. cerevisiar NCYC I 109 and baker’s yeast with cold (4°C) dilute sodium hydroxide under nitrogen for six days. The glucan (GI),which amounted to about 20:4 of the dry weight of the cell wall, precipitated as a gel on neutralization of the alkaline extract (Fleet and Manners, 1976) and the monosaccharide composition is given in Table 1. The GI preparations were insoluble in water, but soluble in 1M sodium hydroxide and dimethyl sulphoxide. Physicochemical studies showed the preparations to be homogeneous. Partial acid hydrolysis showed the presence of both (1 3)- and (1 -+6)-B-glucosidic linkages, and methylation analysis indicated that the latter were present as both interresidue and interchain linkages (Table 1). Electron microscopic examination of the cell walls showed that the alkaline extraction had removed an amorphous surface layer, revealing many bud-scar structures. -+

TABLE 1. Properties of alkali-soluble glucans (GI) from Succhuromyces cerevisiue 1109 and from baker’s yeast Property Composition Carbohydrate Nitrogen Hexosamine Glucose Mannose Molecular size Degree of polymerization Methylation analysis“ 2,3,4,6-tetra2,4,6-tri2,3,4-tri2,4-di “0-methyl

D-glUCitOl

Glucan G , from Succharomyces cerevisiue 1 109

Glucan G I from baker’s yeast

99.0 0.13 0.0 98.5 1.5

98.0 0.26 0.0 97.0 3.0

1330

1810

3.7 84.7 8.3 3.3

4.7 79.3 12.0 4. I

derivatives expressed as mol per 100 mol

In attempts to examine the fine structure of G I , it was subjected to enzymic degradation (Fleet and Manners, 1977). The overall structure that emerged was of a macromolecule with a (1 + 3)-/?-glucan “core” having a low degree of branching (about 2.073, and also containing occasional ( I -+ 6)-linked residues. To this “core” were attached various side chains containing mainly (1 +3)-linked residues, or mainly ( 1 +6)-linked residues, or a mixture of both. Glucan G I always contained a mannan fragment which could be released by the action of an endo-( 1+6)-P-glucanase. This would imply that part of the cell-wall mannan is held in place by some ( 1 -+6)-linkedglucose residues,

160

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

although the nature of this association (i.e. covalent or otherwise) has not yet been established. The biological relationship between the alkali-insoluble glucans and the alkali-soluble glucan has also not been established, but Katohda et al. (1976) have shown that cell walls prepared from large cells of baker’s yeast contained a higher amount of alkali-insoluble glucan, whereas cell walls from small cells contained more alkali-soluble glucan.

C. G L U C A N S F R O M O T H E R S A C C H A R O M Y C E S S P E C I E S

The majority of investigators have examined glucan preparations from Sacch. cerevisiae, and the literature contains only a limited number of references to structural studies on related polysaccharides from other species of yeast. These give the overall impression that they are not greatly different from the glucans of Sacch. cerevisiae, bearing in mind the previous comments on the possible variation in the relative amounts of the three different glucans, and possible variations in molecular size, degree of branching and the relative proportion of (1 -3)- and (1 +6)-fi-glucosidic linkages. Table 2 contains some comparative data on the yields of acetic acidsoluble glucan (B), hot water-soluble material (C) and insoluble glucan (D) from various yeasts, and Table 3 shows the properties of some of these glucan D preparations. The latter consist mainly of ( 1 +3)-P-glucan, with some (1 +6)-glucan in a rather greater amount than was present in baker’s yeast. Related data from Schizosaccharomycespombe (see Section 1I.D)are included for comparative purposes. Partial acid hydrolysates of all the acetic acid extracts B contained gentiosaccharides and maltosaccharides. The latter were removed by treatment of the extracts with a-amylase, prior to partial acid hydrolysis. Fraction B is therefore a mixture of glycogen and a (1 -6)-P-glucan. All of the glucan samples D, on periodate oxidation, reduced more periodate and produced more formic acid than a purified (1 -3)-P-glucan, thus confirming that fraction D was a mixture of a (1 -+ 3)-P-glucan, with small amounts of (1 + 6)-P-glucan. The glucans from Sacch. carlsbergensis are of special interest, since they provided the starting material for one of the first studies on alkali-soluble glucan. Eddy and Woodhead (1968) suspended cell walls in 3% sodium hydroxide solution at 4°C under nitrogen for up to nine days. About 20% of the cell wall dissolved. The soluble polysaccharide was a glucan, with an [a], value of +9 in 3% sodium hydroxide, which was resistant to a- and P-amylases, and it had a molecular weight of about 5. lo5 by sedimentation measurements. This work confirmed earlier observations in the literature (Phaff, 1963) that alkali dissolves some of the glucose residues in the yeast

YEAST CELL-WALL GLUCANS

161

TABLE 2. Fractionation of yeast wall preparations from various yeast species From Manners et a/. (1974) Weight of fractions obtained from 150 g washed organisms

Yeast

Acetic acidsoluble glucan (mg)

Water-soluble material 0%)

Insoluble glucan (g)

460

23

2.80

360 120

61 69

3.60 2.7 I

Saccharomyces jrugilis" (NCYC 100) Saccharomyces ,fermentati (NCYC 161) Schizosuccharomyces pomhe

"This yeast is also known as Klu.vveromyces ,frugilis.

TABLE 3. Properties of insoluble glucan preparations from various yeast species From Manners et al. (1974)

Periodate oxidation Enzymic degradation

Reduction of periodate (molar proportions)

Trio1 groups

25

0.48

19.5

5.1

26

0.48

18.4

5.4

32

0.27

12.7

7.9

38

0.12

3.2

24

032

22.8

(%I

(%)

Yeast Saccharomyces jragilis" Saccharomyces ,fermentati Saccharomy ces cerevisiae Purified ( 1 + 3)$glucan from Saccharomyces cerevisiae Schizosaccharomyces pomhe

"This yeast is also known as Kluvveromyce.s.fra~ilis

Number of glucose residues divided by the number of trio1 groups

31 4.4

162

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

cell wall, and was the starting point of the detailed analysis of Sacch. cerevisiae described in Section II.B.2 (p. 158). D. G L U C A N S I N W A L L S O F S C H I Z O S A C C H A R O M Y C E S P O M B E

The fission yeast Schiz. pombe differs chemically from budding yeasts in that a-mannan and chitin are absent from their cell walls, and that about onethird of the glucan contains (I -*3)-a-linked glucose residues (Bacon et al., 1968). This finding provides an explanation for the apparent resistance of walls of Schiz. pombe to extensive hydrolysis by (1 -3)- and (1 +6)-flglucanases. Bush et al. (1974) examined the wall structure of Schiz. pombe by a variety of techniques. The major components were galactomannan (9-14%), a-glucan (28%) and fl-glucan. The presence of the last polymer was revealed by digestion of the walls with an exo-( 1 -+3)-fl-glucanase, which gave 42% conversion into reducing sugars, 96% of which was glucose. Gentiobiose (3.5%) and laminaribiose (0.5%) were also produced. The a-glucan had a mainly linear structure with (1 -* 3)-linkages, but there was also some evidence for the presence of about 77{ of ( 1 -+4)-linkages. The b-glucan was heterogeneous and contained three different components (Manners and Meyer, 1977), namely a lightly branched (1 +3)-j?-glucan (R-1) which was insoluble in a!kali and acetic acid, a highly branched (l+6)-bglucan, and an alkali-soluble (1 -,3)-fl-glucan (G-I) which has a different structure from glucan R-I. The properties of two of these glucans are summarized in Tables 4 and 5, together with similar data from Sacch. cerevisiae. In general, the alkali-soluble glucan from Schiz. pombe, has a smaller molecular size, and a higher degree ofbranching than the samples from Sacch. cerevisiae. The ( I -+6)-~-glucans also show significant differences. The alkali-insoluble but acetic acid-soluble and alkali-soluble (G-I) glucans comprise about 2 and 24% of the cell wall, respectively. This study also confirmed the presence of a (1 --.3)-linked a-glucan, for which a D P value of 207 k 20 was reported. Schizosaccharomycespornbe thus contains four distinct glucans, in addition to a galactomannan. The investigation of pathways for biosynthesis of these closely related glucans poses a formidable problem for the biochemist.

E. GLUCANS FROM CANDIDA SPECIES

The cell wall of the pathogenic yeast Candida albicans has been o f special interest, although it is now known that mannans rather than glucans are the major antigenic materials (Yu et al., 1967). A detailed chemical study of alkalisoluble glucan preparations from C. albicans serotypes A and B, and from

163

YEAST CELL-WALL GLUCANS

TABLE 4. A comparison of the properties of some alkali-soluble ( 1 from yeasts From Manners and Meyer (1977)

Schizosucc~iuromyces Succhuromyces pombe cere visiue Fraction G-I NCYC 1109 Baker's yeast

Property Approximate yield

+ 3)-p-glucans

(yo)

[a], in I M sodium

hydroxide Degree of polymerization Methylation analysis" 2,3.4,6-tetra2,4,6-tri2,3,4-tri2,4-di-

24

22

13

+ 20

n.d.

n.d.

809

1330

1810

15.0

3.7 84.7 8.3 3.3

4.7 79.3 12.0 4.1

66.3 4.2 14.6

"Expressed as 0-methyl D-glucitol derivatives, mol per 100 mol. n.d. indicates the value was not determined.

TABLE 5. A comparison of the properties of some alkali-insoluble acetic acid-soluble (1 + 6)-p-glucans from yeasts From Manners and Meyer (1977) Property Approximate yield, from alkaliinsoluble residue (%) [aID in water

Degree of polymerization Methylation analysis" 2,3.4,6-tetra2,4,6-tri2,3.4-tri2.4-di-

Yeast Schizosucchuromyces pombe

Succhuromyces cere visiue

23

13

- 27"

-

32"

186

140

44 5 9 43

16

5 65 14

"Expressed as 0-methyl D-glUCitOl derivatives, mol per 100 mol.

C. purupsilosis was carried out by Bishop et al. (1960) and Yu et al. (1967), and their results are summarized in Table 6. All of the glucan preparations were of relatively low molecular weight, were highly branched, and contained a high proportion of (1 +6)-linked residues. The mono-0-methyl glucose noted in Table 6 is most probably the result of undermethylation, and is not structurally significant. The glucan from C. albicans serotype A differs from the others in having a higher degree of branching and few, if any, ( 1 +3)-

164

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

TABLE 6. Properties of /I-glucans from Candida ulbicans and Cundida parapsifasis From Bishop et al. (1960) and Yu et al. (1967) Property

Candidu albicans Serotype A Serotype B

Candida parapsilosis

D

Degree of polymerization Periodate oxidation Periodate reduced (mol. prop.) Production of formic acid (mol. prop.) Methylation analysis' 2,3,4,6-tetra2,3,4-tri2,4,6-tri2.4-di2,3-di2-mono-

- 35

30

-25'

2"

30 -t 3b

1.60

1.52

1.58

0.72

0.79

0.78

26 48 trace 22

10 61 17 6

-

-

11 63 15 7 5 2

-

30

4

33

* 3b

Determined by hypoiodite oxidation. bDetermined by osmometry of the methylated glucans in chloroform. 'Expressed as 0-methyl o-glucose derivatives, mol per 100 mol.

linked glucose residues. In many respects, these glucans are similar to the alkali-insoluble acetic acid-soluble glucans isolated from Sacch. cerevisiae and certain other yeasts. (see Sections I1.B and ILC, pp. 156 and 163) The above work does ,not, however, represent a complete account of glucans from C. albicans. Firstly, it was carried out before the heterogeneity of yeast glucans had been fully appreciated, so that any alkali-insoluble glucan was not included in the studies. Secondly, the organism shows dimorphism, and exists in blastospore and mycelial forms, the latter being mainly responsible for its pathogenicity (Chattaway et al., 1968). These workers examined cell walls from the mycelial and blastospore forms and separated them into alkali-soluble and alkali-insoluble fractions. The alkali-insoluble fraction of the mycelial form contained three times as much chitin as the blastospore form, but only one-third of the protein. The glucan contents of the two forms were similar (4547%) but differed in fine structure since pglucanase treatment of the mycelium and blastospore released 19 and 39% of glucose, respectively. It is evident that the dimorphism involves many changes in the biosynthetic pathways for glucan, protein and chitin. Candida albicans can be treated with polyene antibiotics, such as amphotericin, but stationary-phase organisms show greater resistance than exponentially grown organisms. This resistance is associated with the cell wall

YEAST CELL-WALL GLUCANS

165

as protoplasts prepared from resistant cultures are sensitive (Gale et al., 1975). However, there are no significant differences in the penetrability of the cell wall to poly(ethy1ene glycols) of different molecular sizes at the end of the growth phase and after prolonged incubation in the stationary phase (Cope, 1980). Cytochemical studies have shown the presence of (1 + 6)-glucan and mannan at the outer surface of the cell walls and, in the case of exponentially growing organisms, there was less staining in the inner regions where ( 1 + 3)glucan and chitin are believed to be more abundant (Cassone et ul., 1979). By contrast, walls from stationary-phase cultures gave a more uniform staining reaction, indicating a greater intermixing of the four polysaccharide components. Treatment of stationary-phase organisms with various hydrolytic enzymes, particularly an endo-( I +3)-/?-glucanase, caused a decrease in the resistance to amphotericin, suggesting that the ( 1 -3)-P-glucan could play a major role in the phenotypic resistance (Gale et al., 1980). Chitinase, trypsin and cr-mannosidase showed similar but less pronounced effects. It is clear that detailed structural studies on all the cell-wall components of C. alhicans are required. Other species of Candida also appear to contain a mixture of glucans in their cell walls but, in general, detailed structural studies have not been reported. One exception is a Canclida sp. which was grown as a possible source of singlecell protein. The cell walls contained both alkali-soluble and alkali-insoluble glucans, composed of varying proportions of (1 +3)- and (1 +6)-/?-glucosidic linkages (Martin, 1982). However, this yeast did not appear to produce a predominantly ( I +6)-linked /?-glucan soluble in acetic acid, but insoluble in alkali, of the type described in Table 3 (p. 161).

F . OTHER YEAST G L U C A N S

The literature contains many reports on cell-wall components of yeasts, but many of these are concerned with the taxonomy, serology or physiology of the organisms, rather than with their detailed molecular structures. I t seems probable that many yeasts will contain one or more glucan components, which will differ in solubility in alkali, and that these will contain a high proportion of (1 +3)-/3-glucosidic linkages. In some instances, (1 -+6)-8glucan components may also be present. For example, with Kfoeckera apiculata, the alkali-insoluble fraction reduced 0.47 mol. prop. of periodate, contained 18.4% of trio1 groups and, on partial acid hydrolysis, yielded gentiosaccharides (Manners et al., 1974). A detailed and systematic study of many of these glucan preparations would be rewarding. There also exist several dimorphic organisms which exist in either mycelial or yeast-like forms, and represent an interesting biological system for the study of biochemical events governing this type of interconversion. For

166

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

example, the pathogenic fungus Paracoccidioides brasiliensis produces a (I-,3)-~-glucan in the yeast form and a mixture of a (I+3)-P-glucan and a galactomannan in the mycelial form (San-Blas and San-Blas, 1977). Biochemically, the interconversion of an cr-glucan and a /3-glucan is unusual, and indeed, remarkable. In addition, both forms contain chitin. The cellwall structure is not stable, but changes according to the environment, especially the temperature at which the organism is grown. Since the cellwall composition appears to correlate with the degree of virulence, it has been suggested (San-Blas and San-Blas, 1977) that the cell-wall polysaccharides, and particularly the ( 1 -+3)-a-glucan, play a role in the active protection of the organism against the defence mechanism of the host. Further detailed chemical and biochemical studies on those organisms that are clinically important is warranted.

111. Yeast Wall Glucan Synthesis A. INTRODUCTION

This section is intended to review the information available on the identification and regulation of the enzymes of yeast cell-wall glucan synthesis. As Farkis (1979) has pointed out, yeast cell-wall glucan synthetases have been very difficult to study. The enzymes responsible for synthesis of (1 -+3)-aand (I+6)-P-linkages are not yet understood in any detail, nor are the mechanisms that determine the proportion of different linkages in wall glucans. However, some of the properties of a (1-+3)-P-glucan synthetase have recently been elucidated (Shematek et af., 1980; Shematek and Cabib, 1980).

B. S T U D I E S W I T H I N H I B I T O R S O F G L U C A N S Y N T H E S I S

Glucose 1-phosphate was implicated as a precursor in the pathway of yeast glucan synthesis by Chung and Nickerson (1954). They found that fluoride inhibition of yeast growth, ascribed to inhibition of phosphoglucomutase and hexose phosphate isomerase, was associated with a decrease in the synthesis of glycogen, mannan and glucan. 2-Deoxyglucose has been used in many studies of synthesis in the yeast wall, although it has multiple effects on cell metabolism (Kratky et a/., 1975). 2-Deoxyglucose appears to cause lysis in growing Schiz. pombe by wall breakage at the sites of wall growth (Megnet, 1965). A hexokinase-deficient mutant was found to be resistant to 2deoxyglucose, implying the necessity of 2-deoxyglucose 6-phosphate synthe-

YEAST CELL-WALL GLUCANS

167

sis for toxicity and for inhibition of wall synthesis (Megnet, 1965). Johnson ( 1 968) confirmed that the sites of wall damage by 2-deoxyglucose in Schiz. pombe, Pichia farinosa and Sacch. cerevisiae were in the regions of wall glucan synthesis. He proposed that wall growth occurred by enzymic breakage of glucan chains, followed by extension of the chains by a glucan synthetase which utilized U D P-glucose. U DP-2-Deoxyglucose, synthesized from 2-deoxyglucose, would prevent chain elongation, but not the initial breakage (Johnson, 1968). However, if (1 3)-/?-glucan chains are extruded through the plasma membrane (see Section III.D, p. 169), as is chitin in Sacch. cerevisiae (Duran et al., 1975), elongation of already externalized chains would require an as yet unidentified glucan synthetase, i.e. not the (1-3)p-glucan synthetase located on the cytoplasmic side of the plasma membrane by Shematek et al. (1980). Biely et a1 (1971) have shown that some 2-deoxyglucose is incorporated into walls of Succh. cerevisiae, which supports the idea that a site of inhibition of wall synthesis by 2-deoxyglucose is at glucan chain elongation (see also Section IV.E, p. 176, in this review). The antibiotic aculeacin A inhibits 14C-glucoseincorporation into the acidinsoluble wall fraction of Sacch. cerevisiae (Mizoguchi et al., 1977). Cell lysis occurs at the growing bud tips (Mizoguchi et al., 1977). The amphophilic antibiotics papulacandin B and echinocandin B (Baguley et al., 1979) specifically inhibit glucose incorporation into glucans synthesized by whole cells and by sphaeroplasts of Sacch. cerevisiae and C . albicans. Synthesis of the alkaliinsoluble glucans by the sphaeroplasts was specifically blocked, whereas synthesis of the alkali-soluble glucans and of mannan was relatively unaffected. It can be inferred from this that alkali-soluble and insoluble glucans are made by different synthetases, or that a secondary factor involved in regulating synthesis of alkali-soluble glucan, possibly the environments of the synthetases in the plasma membrane, is the site of action of these antibiotics.

-

C. G L U C A N S Y N T H E T A S E S O F W H O L E A N D F R A C T I O N A T E D C E L L S

More direct evidence that UDP-glucose was a precursor of wall glucans was obtained by Sentandreu et al. (1 9 7 9 , who fed toluene-treated Sacch. cerevisiae with UDP-[ 14C]glucose.( 1 + 3)-/?-Glucan, as well as some glycogen and chitin, were labelled. Most of the label was recovered in membrane particles of “intracytoplasmic origin”. No lipids were labelled in whole cells, and bacitracin had no effect, indicating a lack of involvement of lipid intermediates. At ImM, A T P inhibited incorporation of label by 50%. Incorporation was stimulated by Mg2+.Sentandreu et al. ( 1 975) proposed that glucan synthesis occurred in cytoplasmic vesicles which fused with the plasmamembrane to externalize the nascent wall glucan.

168

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

Synthesis of B-glucan by a particulate preparation from Sacch. cerevisiae was obtained by B a h t et al. (1976). A membrane fraction incorporated [ ‘‘C]glucose from both UDP-[ ‘‘C]glucose and GDP-[ ‘‘C]glucose into glucans with (1 -+ 3)-8- and (1 -+ 6)-P-linkages. Incorporation from UDPglucose and GDP-glucose was additive. The products from UDP-glucose were more susceptible to digestion by an endo-( 1 -+ 6)-p-glucanase than the products from GDP-glucose. They concluded that the product from UDPglucose was predominantly (1 -+ 3)-P-linked, with some (1 -+ 6)-B-linkages, whereas the product from GDP-glucose was predominantly (1 6)-B-linked and included (1 -+ 3)-B-linkages as side chains. Balint et al. (1976) also suggested that much of the product from GDP-glucose was linked to protein, since it could be solubilized under conditions of B-elimination. Elorza et al. (1976) used a temperature-sensitive mutant of Sacch. cerevisiue blocked in RNA transport into the cytoplasm to study cell-wall synthesis. Mannan synthesis was inhibited more rapidly than glucan synthesis at the restrictive temperature. Cycloheximide also inhibited mannan synthesis, but glucan synthesis continued for at least five hours after application of the inhibitor. Either the mRNA species for synthesis of cell-wall glucan are long lived, as Elorza et al. (1976) concluded, or else the glucan synthetases are long lived and are not constituents of vesicles released into the extracytoplasmic space. Cell-free extracts of mid-exponential phase cells of Sacch. cerevisiae were also used by Lopez-Romero and Ruiz-Herrera (1 977) to demonstrate glucan synthesis from nucleoside diphosphate sugars. UDP-Glucose was the glucosyl donor for glucan synthesis in a particulate “mixed membrane” fraction (0.127 nmol min-’ mg protein-’), and in a fraction containing cell walls (0.49 nmol min- mg protein- l ) . There was no soluble activity. GDP-Glucose was less effective as a glucosyl donor than UDP-glucose (1050 pmol min-’ mg protein-’). The product of the “mixed membrane” fraction was predominantly (1 + 3)-B-linked, but contained 0.6% of (1 -+ 6)j?-linkages and was more soluble in alkali. Lopez-Romero and Ruiz-Herrera (1977) pointed out that the enzymes responsible for the incorporation of GDP-glucose may not be those involved in (1 3)-P-glucan synthesis, but may be the same as those studied by B a h t et al. (1976), since the activity of the glucan synthetase studied by Balint et u1. (1976), with GDP-glucose, was only 0.5-1 pmol min- mg protein- and produced a (1 -+ 6)-B-linked glucan, probably also bound to protein. Further characterization of glucan synthetase activity from the “mixed membrane” fraction (Lopez-Romero and Ruiz-Herrera, 1978), showed that the synthetase had a pH optimum of 6.7, a K , for UDP-glucose of 0.12 mM, and was stimulated by divalent cations. Uridine diphosphate and glucono-b-lactone were inhibitory. There was no apparent requirement for a primer. -+

’

-+

’

’

YEAST CELL-WALL GLUCANS

169

D . G L U C A N S Y N T H E S I S A N D G L U C A N S Y N T H E T A S E S IN P R O T O P L A S T S

One approach to studying synthesis of cell-wall glucan has been to prepare yeast protoplasts or sphaeroplasts by digestion of the cell wall with snail-gut enzyme in the presence of osmoticum (Peberdy, 1979) and follow the course of wall regeneration. Protoplasts of Sacch. cerevisiae form a network of fibrils, without forming a complete wall, when incubated in liquid media; but if embedded in gel, a “fibrillar network” and an “amorphous matrix” are synthesized together, followed by regeneration of a complete wall (NeEas, 1971). Protoplasts from Schiz. pombe form a complete wall in liquid media. A fibrillar network forms and increases in density for 15 hours. This is followed by the appearance of amorphous material and then wall regeneration (NeEas, 1971). Nadsonia elongata regeneration proceeds like that in Schiz. pombe. The nets of Schiz. pombe differ from those of Sacch. cerevisiae, in that microfibrils of ( 1 -+ 3)-fi-glucan are formed, followed by the formation of fibrils of (1 -+ 3)a-glucan (Kreger and Kopecka, 1978). NeEas (1971) suggested that a diffusible factor is required for full wall reversion and that a gel medium, or the very dense fibrillar network formed by Schiz. pombe, serves to retain this unknown substance. Fibrils from Sacch. cqrevisiae are labelled by [3H]glucose (NeEas et al., 1970). P u l s e 4 h a s e experiments have shown that networks are formed by “interposition of new fibrils” all around the protoplast surface (NeEas et al., 1970). The nets are composed of long chain (1+3)-fi-linked glucans arranged in crystalline microfibrils, which are about 40% alkali-soluble (Kreger and Kopecka, 1973, 1975). The lack.of (1 -+6)-fi-linkages makes it appear as though the (1 -+6)-fi-synthetase is inactivated or lost from the protoplast by the procedure used to prepare these structures (Kreger and Kopecka, 1973, 1975). Yeast protoplasts prepared using snail-gut enzyme are leaky to some soluble compounds (Oura et al., 1970). The leakiness and loss of the ( 1 +6)-fi-synthetase may be only part of the damage to the cell surface caused by protoplast formation. The lag period observed before regeneration of cell walls by protoplasts of leaf cells from Antirrhinum mums is a function of the conditions used to prepare protoplasts and is not lengthened by subsequent treatment with wall-dissolving enzymes (Burgess et al., 1978), thus implying that damage to the synthetic machinery during protoplast formation is more critical in delaying regeneration than accumulation of surface polysaccharides. The alkali-insoluble portions of the nets of Sacch. cerevisiae and C . albicans are susceptible to papulacandin B (Baguley et a[., 1979), suggesting that the soluble and insoluble glucans are synthesized by different enzymes or in different environments in the plasma membrane. Synthesis of glucan microfibrils is the most sensitive site of action of 2-

170

J. H. DUFFUS, CAROLYN LEV1 AND D. J. MANNERS

deoxy-2-fluoro-~-glucosefound by Biely et al. (1973b). Shematek et al. (1980) and Shematek and Cabib (1980) carried out a detailed study of glucan synthesis by protoplast membranes of Sacch. cerevisiae. The rates of UDP[ 14C]glucoseincorporation into acid-precipitable polysaccharide were 15 to 35nmolglucose - ’ mgprotein-I (Shemateketal., 1980).Neither ADP-glucose nor GDP-glucose could substitute for UDP-glucose (Shematek et al., 1980) and either ATP or GTP, bovine serum albumin and glycerol are required for activity (Shematek et al., 1980). The product was an unbranched (1 +3)P-glucan and was formed de novo. Lipids were not labelled, dolichol phosphate did not stimulate glucan synthesis and [ 14C]glucosylphosphoryldolichol did not label the glucan. Thus, lipid intermediates are not involved in synthesis of this glucan (Shematek et al., 1980). Glucan synthetase was found to be associated with the concanavalin Astabilized plasmalemma fraction which can be isolated from protoplasts of Sacch. cerevisiae. Pretreatment of lysed, but not of intact, protoplasts with glutaraldehyde inactivated the synthetases. Shematek et al. (1 980) concluded that the (1 +3)-P-glucan synthetase is located on or in the inner surface of the plasma membrane. This does not remove the possibility that synthetase originally on the outside of the plasmalemma is lost in the process of protoplast formation, as is apparently the case with (1 +6)-P-glucan synthetase. The (1 +3)-P-glucan synthetase was subject to regulation; ATP and GTP stimulated activity, but the characteristics of activation by ATP and GTP differed (Shematek and Cabib, 1980). Estimation of the K , values for ATP and GTP were complicated by the presence of phosphatases in the membrane preparation, but appeared to be of the order of 24 ,UM for ATP and 0.16 ,UM for GTP. Activation by ATP was enhanced by time-dependent preincubation with ATP at 30°C but not at O T , and required the presence of a heat-stable, dialysable, “supernatant factor”. Activation by GTP is neither time-, nor temperature-dependent. Magnesium ions are inhibitory, especially to the ATP-activated enzyme, as is EDTA. However, GTP-dependent activation requires EDTA in the initial protoplast lysis, apparently to protect against degradation by phosphatases. On the basis of these results, Shematek and Cabib (1980) have proposed a model whereby, in addition to activation by GTP, the glucan synthetase may be activated by the phosphorylated form of the “supernatant factor” and regulated by its reversible phosphorylation and dephosphorylation. Phosphorylation would require ATP, and dephosphorylation would require endogenous phosphatases and Mg2 . Protection against Mg2 +-dependant phosphate hydrolysis would then be provided by EDTA, but would also inhibit ATP-dependent phosphorylation. Farkis (1 979) has proposed that wall synthesis may be inhibited by the presence of one or more proteinaceous inhibitors. These would be kept in contact with the plasma membrane by an intact cell wall. Loosening of the +

YEAST CELL-WALL GLUCANS

171

wall by endogenous glucanases during normal growth, or by snail-gut enzymes during protoplast formation, would cause loss of the inhibitor(s) and result in activation of polysaccharide synthetases. Acid phosphatase, a low pH-optimum ATPase and a MgZ+-dependent ATPase are found in the cell envelope of Sacch. cerevisiae (Suomalainen and Nurminen, 1973). On the basis of the model of regulation of the ( 1 -+3)-8glucan synthetase described by Shematek and Cabib (1980), at least one of these phosphatases may be a good candidate for a “proteinaceous inhibitor”. However, this could account for only part of the regulation of wall synthesis since, as pointed out by NeEas (1971), complete reversion of yeast protoplasts appears to require a protein- or polysaccharide-retaining network (a gel medium for Sacch. cerevisiue and a dense microfibrillar network for Schiz. pombe).

IV. Physiological Control of Glucan Content A.

INTRODUCTION

In recent years, an increasing proportion of yeast research has been devoted to elucidating the changes that occur in yeast cells during normal growth and division, yeast-mycelial interconversion, and exposure to growth media of different compositions. This research has not only added to our understanding of fundamental biology, but has opened the way to greater control of yeast growth which may enable us to optimize yeast properties of commercial importance. It is possible that studies of yeast glucans in the physiological context have lagged behind other studies and it is hoped that the following review of the current literature will stimulate further progress.

B. T H E C E L L C Y C L E

The first study of bulk glucan changes in relation to the cell cycle appears to be that of Dawson (1967) using Candida utilis in what he called a “continuous phased culture” (Dawson, 1969). It is clear that division was synchronous in this culture but there was no control to establish that the cells were otherwise normal and not subject to the treatment-induced changes that have complicated cell-cycle analysis with synchronous cultures (Mitchison, 1977). However, Dawson (1967, 1969) found that the g1ucan:mannan ratio was constant with a value of 0.5 throughout the cycle in cells using 3% or 5% glucose as the carbon source. In cells using 1% glucose as the carbon

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source, the ratio rose from about 0.3 to about 0.5 during the cycle. Unfortunately only one cycle was studied. Kuenzi and Fiechter (1972) studied changes in the carbohydrate composition of Sacch. cerevisiae during the cell cycle. To achieve synchronization, the yeast was grown in a chemostat and, after six generation times, the medium supply was interrupted. After seven hours, fresh medium was supplied at a dilution rate that ensured complete oxidative growth on glucose. This process induced a cyclic partially synchronized budding. As with Dawson's method, there is no way of knowing what cell abnormalities have been induced by the drastic treatment. In this case, the g1ucan:mannan ratio remains fairly constant, even when budding is taking place, with a ratio of about 0.7 at a dilution rate of 0.05 and about 0.9 at a dilution rate of 0.1. Later, Sierra et al. (1973) investigated the biosynthesis of alkali- and acidinsoluble glucan using synchronized cultures of two strains of Sacch. cerevisiae. The cells of one strain, LK2GI 2, were synchronized by harvesting an asynchronous culture, immersing the cells in ice water to chill them, selectingcells of about the same size from a Ficoll gradient, and re-inoculating them into growth medium. Thus, this strain was subjected to temperature shock, osmotic shock and nutrient depletion, all processes that can modify metabolism dramatically. The other strain, a temperature-sensitive mutant, was transferred from its permissive temperature (25°C) to a restrictive temperature (37°C) for 120 minutes before returning the temperature to 25°C. Again, the inherent assumption that the synchronization of cell division attained must be associated with a normal cell cycle is unlikely to be true. Despite this, Sierra et al. (1973) concluded that both glucan and mannan are synthesized continuously and in an exponential fashion through the whole cell cycle. Recently, Biely (1978) recalculated the results of Sierra et al. (1973) to obtain average rates of glucan and mannan synthesis at 15-minute intervals, and showed that the rates of glucan and mannan synthesis decline during budding. At about the same time, Biely, et al. (1973a) described an electronmicroscope autoradiographic study of cell-wall formation in yeast. These authors found that the pattern and extent of labelling of cell walls of Sacch. cerevisiae with tritium from tritiated glucose varied through the cell cycle. They used exponentially growing asynchronous cultures of the yeast subjected to 15-minute pulses of radioactive glucose. The results, therefore, relate to a cell cycle which has suffered a minimum of distortion. Again it appears that the rate of incorporation of new glucan into a growing bud increases with the size of the bud to a maximum before division begins, when the rate decreases substantially until new bud initiation occurs. Budd (1975) studied incorporation of [U-'4C]glucose, maltose and maltotriose into the polysaccharides of Sacch. cerevisiae in synchronous culture during fermentation of brewer's wort. Synchrony was induced by repeated

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subculture of the yeast cells skimmed from the surface of previous fermentations. This would appear to be a technique for selecting out the smallest cells, i.e. those that have just budded from a parent. If the wort into which they are inoculated is not substantially different from that from which they were isolated, such cells should not be greatly perturbed. On the other hand, any such surface yeast population tends to contain a proportion of abnormal cells, and it is possible that the decrease in oxygen availability accompanying transfer of cells from a surface culture to a totally immersed culture may induce biochemical changes and oscillations not associated with the normal cell cycle. Budd’s (1975) conclusion was that incorporation of 14C into wall glucan was continuous and stable. Hayashibe et a / . (1977) looked at the mode of increase in cell-wall polysaccharides in synchronous cultures of Sacch. cerevisiae prepared using yet another method. Commercial baker’s yeast cells were fractionated into small- and large-sized cells by repeated centrifugation in de-ionized water and the large-sized cells were inoculated into fresh growth medium to give a culture which is highly synchronous with respect to division (Hayashibe and Sando, 1970). Although this is apparently a selection method, establishing synchrony by selecting cells on the point of budding, it is likely that there is a large component of induction brought about by nutrient depletion during the centrifugation, and perhaps also by temperature as the centrifuge temperature is not stated. Thus, abnormal induced fluctations in cell components may occur. The results obtained indicate that the increase in total glucan is almost linear throughout the cell cycle with a decrease in accumulation about the time of cell division. Alkali-soluble glucan accumulates mainly as the buds grow whereas the alkali-insoluble glucan tends to accumulate around the time of cell division and bud initiation. Biely (1978) re-evaluated the results of Biely et a / . (1973a), Sierra et a / . (1973) and Hayashibe et al. (1977) described above, and pointed out that they all agree in showing that the rate of glucan and mannan synthesis is markedly decreased during cell division and budding initiation. He also pointed out that the possibility that it ceases completely cannot be ruled out because of the relatively long sampling intervals used in these studies. In this connection, it may be important to note the work of Maddox and Hough (1971) who observed that bud initiation in Sacch. cerevisiae is accompanied by intensive glucanase and mannanase activity. In conclusion, it may be said that most of the cell-cycle studies have used methods for establishing synchronous cultures that are idiosyncratic in the sense that their use has been restricted to specific laboratories, and hence they lack the background of independent assessment that more widely used techniques would have. This makes it important to repeat and extend these studies with synchronous cultures established by other means. All methods of preparing synchronous cultures have drawbacks, but these can be overcome,

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to some extent, by use of appropriate control cultures. A notable feature of the work described above is the absence of such controls and these must be included in any future investigations.

C. YE A S T-M Y C E L I U M I N TER C 0 N V E R S I O N

Yeast-mycelium interconversion has been reviewed on a number of occasions (Scherr and Weaver, 1953; Romano, 1966; Phaff, 1971) and work covered in these reviews will be described only in so far as it is directly relevant to yeastwall glucans. In 1969, Kanetsuna et al. investigated the yeast and mycelial cell walls of Paracoccidioides brasiliensis. They found that the yeast-wall glucan was almost entirely soluble in alkali ( I M NaOH) but that the mycelial glucan was only 60 to 65% alkali soluble. The alkali-soluble glucan gave a weakly positive response to the periodic acid-Schiff reaction, was not hydrolysed by snail digestive juice and had a-glucosidic linkages. The alkali-insoluble glucan gave a clearly positive response to the periodic acid-Schiff reaction and was hydrolysed by snail digestive juice. Subsequently, it was established that the alkali-soluble glucan from the yeast form of the organism had the properties of a (1 +3)-a-glucan (Kanetsuna and Carbonell, 1970), unlike the equivalent fraction from the mycelial form which proved to be a variable mixture of a- and j-glucans. Electron microscopy showed that the a-glucan occurred on the outer surface of the yeast phase in the form of short fibres (Carbonell et a/., 1970). Synthesis of this glucan can be induced by adding foetal calf serum to the growth medium (San-Blas and Vernet, 1977). Similar studies on Blastornyces derrnatidis showed that the yeast form of this organism had a cell wall composed of 95% a-glucan and 5% P-glucan, whereas the mycelial cell wall was 60% a-glucan and 40% P-glucan (Kanetsuna and Carbonell, 197I). Histoplasma capsulaturn has also been investigated and in this organism the yeast cell walls contain about 47% a-glucans, mainly (1 -,3)-linked, and 31% P-glucan, mainly (1 -3)-linked, whereas the mycelial cell wall has essentially no a-glucan and only 19% P-glucans, again with mainly (1-3)P-linkages (Kanetsuna et al., 1974). Again, electron microscopic studies show the a-glucan to be located on the outer part of the yeast cell wall.

D. E F F E C T S O F N U T R I E N T L I M I T A T I O N

Nutrient limitation has long been known to lead to altered composition of yeast cell walls. This is obvious in batch cultures where exponential-phase cells are much more susceptible to protoplast formation than are stationary-phase cells (Eddy and Williamson, 1957, 1959). Unfortunately, only a few nutrient

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deficiencies have been investigated to any extent, namely deficiencies in inositol, biotin, nitrogen, carbohydrate, phosphate and oxygen. In 1960, Ghosh et ul. studied the effects of inositol deficiency on Sacch. curlsbergensis. They found that daughter buds failed to separate from parent cells and aggregates of up to 50 cells were formed. The cell walls under these conditions contained up to three times as much glucan as walls from cells growing with an adequate inositol supply. Similar results were obtained in experiments with Sacch. cerevisiue (Challinor et al., 1964; Power and Challinor, 1969). When grown in the complete absence of inositol, Sacch. cerevisiae had a much weaker cell wall than when grown in a complete medium, and the wall had a greater percentage of glucan and hexosamine. These results were confirmed by Dominguez et al. (1978) who further demonstrated that the change in wall composition was due to the relative insensitivity of glucan synthesis to inositol deficiency when compared with mannan synthesis. The effects of biotin deficiency on yeast glucan are similar to those of inositol. When Sacch. cerevisiae was grown in biotin-deficient media supplemented with aspartate, the ratio of glucan to mannan increased three-fold (Dunwell et al., 196I). This change is associated with a marked thickening and pronounced multiple layering of the cell walls (Dixon and Rose, 1964). Failure to divide in such media seems to be due to inability to produce a cross septum. The increased ratio of glucan to mannan is not seen in all cells grown in biotin-deficient media (Mizunaga, et al., 1971). At least one strain of Saccharomyces shows a slight decrease in the glucan :mannan ratio when grown in biotin-free medium supplemented with aspartate. Similarly, wall thickening is not always seen. The same strain, which does show wall thickening in a medium containing ammonium ions as the source of nitrogen, shows none in a medium where the nitrogen is supplied as amino acids, even though the glucose: mannan ratio is increased. It was further observed by Mizunaga et al. (1 97 1) that enrichment of glucan occurs in their yeast strain with age of the batch culture, irrespective of the quantity of biotin supplied. Clearly there is a need for such-studies of the effects of nutrient deficiency to be carried out under chemostat conditions. McMurrough and Rose (1967) used continuous culture to study the effects of growth rate and nitrogen and carbohydrate nutrient limitation on the composition and structure of the cell wall of Sacch. cerevisiue. They found that the contents of total glucan and mannan were little affected by growth rate, although the distribution among wall fractions varied. Yeast cells grown at any rate under nitrogen limitation were longer and thinner than those grown at the same rate under carbohydrate limitation, and the electron microscope showed that the walls of these long, thin cells were more porous than normal. In general, there appeared to be a direct correlation between the amount of glucose in the medium and the amount of glucan in the walls. The effects of carbohydrate and nitrogen deficiency on yeast glycogen

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synthesis have also been studied (Becker et al., 1979). Cells of Sacch. carlsbergensis growing in batch culture under either carbohydrate or nitrogen limitation initially deplete their glycogen and this is resynthesized only in later exponential phase. Cells harvested in early logarithmic phase cannot synthesize glycogen, even in glucose-phosphate buffer which supports glycogen

synthesis in stationary-phase cells. Lack of oxygen or addition of ammonia slows down glycogen synthesis in cells grown under carbohydrate or nitrogen limitation. The sensitive control of glycogen metabolism in yeast cells is shown by the rapid commencement of glycogen synthesis within one minute of addition of glucose to a starved cell suspension, and in the equally rapid decrease in glycogen content when glucose is removed from cells that have synthesized glycogen in glucose-phosphate buffer. Another study on yeast glycogen content, this time in Sacch. cerevisiae, showed that the glycogen content of this yeast was higher in anaerobic 1% glucose media than in the same media under aerobic conditions, and that it increased further when the glucose content of the media was increased to 8%. The rest of the yeast glucan behaved similarly but increased only slightly with increasing glucose concentration (Chester and Byrne, 1968). Phosphate limitation affects the glucan content of walls of Sacch. cerevisiae lowering it from 45% to 27% in cells grown in continuous chemostat culture when the medium phosphate concentration is decreased from 3 g-' to 81.6 mg-' at 30°C (Ramsay and Douglas, 1979).

E. E F F E C T S O F M E T A B O L I C I N H I B I T O R S

There are a number of reports of the action of inhibitors on yeast glucan accumulation. Cycloheximide, which inhibits protein synthesis at the ribosome, selectively inhibits synthesis of mannan protein without affecting the formation of glucan microfibrils (Netas et al., 1968; Elorza and Sentandreu, 1969; Elorza et al., 1976). On the other hand, lomofungin, thiolutin and 8hydroxyquinoline at concentrations that inhibit RNA synthesis inhibit both glucan and mannan synthetases (Elorza et al., 1976). This is probably due to their making magnesium and manganese unavailable 'by chelation. Lomofungin was used at a concentration of 50pg ml-. In a subsequent study by Kopecka and Farkgs (1979), lomofungin was found not to inhibit the synthesis of the (1 +3)-/?-glucan network in regenerating protoplasts of Sacch. cerevisiae at a concentration of 20 pg ml-l. 2-Deoxyglucose is probably the inhibitor that affects glucan synthesis most directly. Johnson (1968) reported that it caused lysis of cell walls of Schiz. pombe, Pichia farinosa and Sacch. cerevisiae at sites that co-incided with the regions of growth of their glucan layers. Biely et al. (1971) made similar observations on Sacch. cerevisiae and produced evidence for incorporation of

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2-deoxyglucose into the cell wall. Interestingly, the glucose content of the cell walls from treated cells was unaltered, though the mannose content had fallen by more than a third. Subsequently, Biely et ul. (1974) studied the effects of 2-deoxyglucose on Schiz. pornbe. They observed that formation of the cell plate was more sensitive to the inhibitor than was synthesis of lateral walls. Further, cycloheximide and nalidixic acid could suppress 2-deoxyglucoseinduced lysis suggesting that inhibition of glucan synthesis is not the whole explanation of this phenomenon. Biely et al. (1973b) used the related compound, 2-deoxy-2-fluoro-~-g~ucose, to inhibit synthesis in Sacch. crrevisiae and cause extensive cell lysis. Cells resistant to this compound proved to be defective in the final steps of cell division. Schizosaccharomyces pombe was similarly affected, but was more resistant to the inhibitor than was Sacch. cerevisiae. Three other inhibitors affecting yeast glucan synthesis have recently been reported. 2-Deoxy-~-arabinohexosehas been found to increase the relative amounts of chitin and glucan in the cell wall of Rhodosporidium tordoides (Sipicki and FarkBs, 1979). This is associated with defective cell division and separation of daughter cells, which remain attached to the mother cells giving multicellular aggregates or, growing on yeast extract agar, pseudomycelia.

F. M I S C E L L A N E O U S

Little has been said in this section about yeast glycogen because it is not clear where it is located in the cell. However, Gunja-Smith et al. (1977) have shown that a proportion of the water-insoluble glycogen fraction is associated with a fi-glucan-like polysaccharide which may be a cell-wall component. They also showed that the amounts of yeast glycogen fractions vary during growth in batch culture, and so one may conclude that there are physiological controls which should be further investigated. It might be supposed that changes in cell-wall glucan would accompany yeast conjugation but the evidence from studies of the basidiomycete Tremellu mesenterica seems to indicate that gross changes in wall composition do not occur (Reid and Bartnicki-Garcia, 1976). Finally, the point must be made that many of the physiologically induced changes in yeast-wall glucan probably result from the action of endogenous glucanases. For recent reviews of the role of these enzymes, the reader is referred to the articles by Phaff (1 977, 1979). V. Acknowledgements

The authors are indebted to the Science Research Council for financial

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support (Grant GR/A/7056-4); one of us (D.J.M.) wishes to acknowledge many helpful discussions with J. S. D. Bacon and G. H. Fleet which have contributed significantly to the progress made in this laboratory.

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Transport of Antibiotics into Bacteria IAN CHOPRA and PETER BALL" Department of Microbiology, Medical School, University of Bristol, Bristol BS8 7 TO, England

1. Introduction . . . . . . . . . . . . , 11. Antibiotics: target sites and uptake . . . . . , . , . 111. Uptake and transport: some definitions . . . . . . . . 1V. Structure of bacterial cell envelopes in relation to antibiotic uptake: an overall . . . . . . . . . . . . . picture . A. Capsules . . . . . . . . . . . . . B. Outer membranes . . , . . . . . . . . C. Periplasmic space D. Peptidoglycans . . . . . . . . . . . . E. Cytoplasmic or inner membranes . . . . . . . . F. Summary . . . . . . . . . . . . . V. Aminoglycosides . . . . . . . . . . . . A. Diffusion of aminoglycosides across the outer membrane . . . , B. Accumulation of aminoglycosides across the cytoplasmic membrane . . VI. Chloramphenicol . . . . . . . . . . . . A. Diffusion of chloramphenicol across the outer membrane . . . . B. Accumulation of chloramphenicol across the cytoplasmic membrane . . VII. D-Cycloserine . . . . . . . . . . . . , A. Diffusion of D-cycloserine across the outer membrane . . . . . , B. Transport of D-cycloserine across the cytoplasmic membrane . VII 1. 3. 4-Dihydroxybutyl- 1-phosphonate . . . . . , . . . . , . . , A. Diffusion of DHBP across the outer membrane . . . . . B. Transport of DHBP across the cytoplasmic membrane IX. Fosfomycin , . . . , . . . . , . . . . . . . A. Diffusion of fosfomycin across the outer membrane , B. Transport of fosfomycin across the cytoplasmic membrane . . . C. Transport of fosfomycin in bacteria other than Esrherirhia coli . . , X. Beta-Lactams . . . . . . . . , . . . . Xi. Norjirimycin . . . . . . . . . . . . . XII. Peptide antibiotics . . . . . . , . . . . . XIII. Showdomycin . . . . . . . , , . . . . A. Diffusion of showdomycin across the outer membrane . . . . * Present address: Pall Biomedical Ltd.. Portsmouth POI 3PD. England

184 185 185

188 188 188

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192 193 194 194 195 200 200 200 20 1 203 203 203 204 204 205 205 208 208 209 212 213 216 217

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B. Transport of showdomycin across the cytoplasmic membrane XIV. Sideromycins . . . . . . . . . . XV. Streptozotocin . . . . . . . . . . A. Diffusion of streptozotocin across the outer membrane . B. Transport of streptozotocin across the cytoplasmic membrane XVI. Tetracyclines . . . . . . . . . . A. Diffusion of tetracyclines across the outer membrane. . B. Transport of tetracyclines across the cytoplasmic membrane XVII. Conclusions . . . . . . . . . . XVIII. Acknowledgements . . . . . . . . . References . . . . . . . . . .

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211 220 222 223 223 225 225 228 233 234 234

Don’t talk to me about permeability-that is the last resort of the biochemist who cannot find a better explanation. Marjory Stephenson, as reported by E. F. Gale (1971)

I. Introduction More than 60 years have elapsed since Ehrlich formulated the concept of antimicrobial chemotherapy, i.e. “the problem of internal disinfection, of destroying living parasites within the infected body” (Himmelweit, 1960). Although Ehrlich’s aim of obtaining “magic bullets” that are innocuous to the host remains an objective of the pharmaceutical industry today, very few therapeutically useful compounds have been obtained de now by laboratory synthesis (Gale et al., 1972). The failure of these compounds probably results from a number of factors, one of which may be the inability of the molecule to penetrate to active sites within the bacterial cell (Gale, 1966). In contrast to man’s failure, micro-organisms have been markedly more effective in the production of antibiotics that penetrate and inhibit bacteria. Therefore an understanding of the factors that lead to the successful uptake of antibiotics could be a step towards the rational design of novel antibacterial agents. Despite its potential importance, the study of antibiotic accumulation has, until recently, been neglected. Thus, during the last decade, only two reviews on the subject have been published (Franklin, 1973; Kadner, 1978). Nevertheless, knowledge in this area has increased considerably during the past few years notably because experimental strategies applied successfully to the study of essential solute accumulation (for reviews see Harold, 1978; Rosen, 1978; Downie et al., 1979; Lanyi, 1979; Dills et al., 1980; West, 1980) have also been used for studies with antibiotics. These recent developments have prompted this review of a topic which may expand considerably within the next decade. Throughout the preparation

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

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of this article, we were constantly reminded of Gale’s (1966) apt comment that “antibiotics are selective agents provided by nature for our enlightenment”.

11. Antibiotics: Target Sites and Uptake

Although some investigators (e.g. Gale as quoted above) regard antibiotics as naturally occurring inhibitors, we consider them as substances produced or derived from living organisms. Furthermore, many of the compounds that will be discussed (Table 1) are used against infectious diseases. Most antibiotics have molecular weights less than 1000 (Table l), but the bacteriocins, bacterial proteinaceous products with bactericidal activity, can also be defined as antibiotics. Although these proteins may prevent infectious diseases (Konisky, 1978) we will not consider them in this review because they are best regarded as toxins (Holland, 1976). Bacteriocins and “true” antibiotics have also been distinguished from each other by other criteria (Hopwood, 1978). Bacteria have antibiotic target sites with different degrees of accessibility to drug molecules entering the cell. By defining the target sites for these inhibitors (Table 1) we can indicate which surface structures the antibiotics must cross to achieve their effects. Thus antibiotics may encounter in turn the following envelope layers. 1. 2. 3. 4. 5.

A capsule An outer membrane A periplasmic space A peptidoglycan layer An inner, or cytoplasmic membrane

In subsequent sections we deal at length with passage of specific antibiotics through envelope regions, particularly the inner and outer membranes. However, before turning to detailed discussions of individual antibiotics, some general aspects of antibiotic uptake are considered.

111. Uptake and Transport: Some Definitions

We regard uptake as the net result of antibiotic interaction with the various envelope regions defined in Section 11. Accumulation of antibiotics can occur by one of the following processes. (a) Passive diffusion-the antibiotic diffuses through the envelope passing

I. CHOPRA AND P. BALL

186

TABLE 1. Selected antibiotics" and their mode of action Antibiotic or group of antibiotics Aminoglycosides e.g. Streptomycin Kanamycin

Molecular weight 580 408

Chloramphenicol

323

D-Cycloserine

102

3, 4-Dihydroxybutyl1-phosphonate

136

Fosfomycin

138

Beta-Lactams e.g. Benzyl penicillin

334

Ampicillin

349

Cephalothin

395

Norjirimycin

179

Peptides e.g. Bacilysin (dipeptide)

270

L - ( N 5 -phosphono) 402 methionine-Ssulphoximinylalanylalanine (tripeptide)

Inhibitory effect

References

Inhibits initiation of protein synthesis and causes miscoding Inhibits initiation of protein synthesis peptidebond formation, and translocation. Also causes miscoding Binds to ribosomes and inhibits peptidyl-transfer reaction Inhibits murein synthesis by inactivating alanine racemase and D-alanine: D-alanine synthetase (cytoplasmic enzymes) Inhibits CDP-diglyceride: sn-glycerol-3-phosphate phosphatidyltransferase and sn-glycerol-3phosphate: N A D oxidoreductase (cytoplasmic enzymes) Inhibits murein synthesis by inactivation of pyruvyl transferase (cytoplasmic enzyme)

Nierhaus and Wittman (1980)

Binding to proteins on outer surface of cytoplasmic membrane leads to altered control of murein hydrolases and loss of murein integrity Inhibits t(- and 8glucosidases (cytoplasmic enzymes)

Tomasz (1979); Georgopapadakou and Liu (1 980); Zimmermann (1980)

Inactivates glucosamine synthetase (cytoplasmic enzyme)b Inactivates glucosamine synthetase (cytoplasmic enzyme)b

Kenig and Abraham (1976); Kenig et a/. (1 976) Diddens et a/. (1976)

Nierhaus and Wittman (1980)

Nierhaus and Wittman (1980) Gale et al. (1 972)

Tyach et a/. (1 976)

Kahan et a/. (1974)

Tanaka (1975)

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

Antibiotic or group of antibiotics Showdomycin

Sideromycins e.g. Albomycin

187

Molecular weight

Inhibitory effect

228

Inactivates nucleoside monophosphate kinase and other cytoplasmic enzymes

Gale et ul. (1972)

1140

Unknown, but probably acts on cytoplasmic target

Bhuyan (1967); Kniisel and Zimmermann (1975); Hartmann ef a/. (1979) Reusser (1971)

Streptozotocin

251

Induces rapid degradation of DNA

Tetracyclines e.g. Tetracycline

444

Minocycline

494

Inhibit binding of aminoacyl-tRNA to the ribosome

References

Barringer et al. (1974); Nierhaus and Wittmann (1980)

a Accumulation of the antibiotics listed here will be considered in detail in the text (see subsequent sections). Some clinically important antibiotics (e.g. macrolides and fusidic acid) have been omitted because there are insufficient data on accumulation to warrant discussion. Apart from the 8-lactams, other antibiotics having targets in the cytoplasmic membrane (e.g. bacitracin, tunicamycin, vancomycin) are not discussed. In Gram-negative bacteria these antibiotics presumably diffuse across the outer membrane, but their passage across this structure has not been examined. After transport the di- and tripeptides are hydrolysed by intracellular peptidases to produce amino acid analogues. It is these amino acids that cause enzyme inhibition.

down a concentration or electrical gradient. For uncharged molecules the intracellular and extracellular concentrations will be equivalent, but cations can produce concentration gradients in response to Donnan equilibria (e.g. as occurs across the outer membrane of Gram-negative bacteria). (b) Facilitated diffusion-the antibiotic forms a complex with a carrier which facilitates diffusion across the membrane. As with passive diffusion, movement down a concentration or electrical gradient can be achieved, but the carrier lends specificity to the uptake of the drug. (c) Active transport via energy-dependent systems-transport across the cytoplasmic membrane mediated by carriers coupled to an energy source. Accumulation against a concentration gradient is achieved.

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IV. Structure of Bacterial Cell Envelopes in Relation to Antibiotic Uptake: an Overall Picture A. C A P S U L E S

In some bacteria, polysaccharide-containing capsules comprise the outer surface of the cell. Various aspects of capsule structure have been reviewed in recent years (Sutherland, 1972, 1979; Powell, 1979; Atkins et al., 1979) to which the reader is referred for detailed information. Capsules provide a negatively charged outer surface to the cell due to numerous free carboxylic acid groups present in the uronic acids and ketals that comprise the capsular polysaccharides (Sutherland, 1972). Capsules may therefore influence the uptake of ionized molecules into the cell, either promoting entry of cationic compounds or retarding anionic species. Since penicillins are relatively strong organic acids (Demarco and Nagarajan, 1972) the latter may explain why encapsulated variants of Pseudomonas aeruginosa are more resistant to penicillins than their non-capsulated counterparts (Govan, 1976).

B. O U T E R M E M B R A N E S

It is well known that the cell envelope of Gram-negative bacteria contains two membranes: the cytoplasmic (or inner) membrane and the outer membrane. The Gram-negative outer membrane contains phospholipid, protein and lipopolysaccharide (LPS). The arrangement and function of these components have been extensively studied in recent years, particularly in Escherichia coli and Salmonella typhimurium (Braun, 1978; Di Rienzo et al., 1978; Inouye, 1979; Nikaido and Nakae, 1979; Osborn and Wu, 1980) and to a lesser extent in other species (Morse et al., 1979; Nikaido and Nakae, 1979). In E. coli and S . typhirnuriurn, the lipid bilayer of the outer membrane is assymetrical with respect to distribution of LPS and phospholipid such that LPS is located in the outer leaflet and phospholipid in the inner leaflet (Fig. 1) (Nikaido and Nakae, 1979; Funahara and Nikaido, 1980). Proteins are located in both halves of the outer membrane with some of them spanning the width of the whole membrane (Fig. 1). The so-called porins (Nikaido and Nakae, 1979) comprise one group of transmembrane proteins. These proteins associate as trimers to produce water-filled pores that traverse the outer membrane. The pores provide relatively non-specific passive diffusion channels for the entry of a large number of hydrophilic solutes including low molecular-weight antibiotics (Nikaido and Nakae, 1979).

Trirners of porin protein ( OmpF, C )

, OmpA

-F 8 nrn

Outer membrane

Lipopol ysaccharide

-d-

r e r I piusiri

8 nrn\ildl

Cytoplasmic membrane

3 1

5:'

Phospholipid

/ Pept i dog I y can

Protein catalysing specific fac i I i t a t e d diffusion

Murein lipoprotei Protein EXTERNAL ENVIRONMENT

(b)

c

,70uter

surface

11

/

4'

Specific carriers

11

Centre

11 - - a l n n e r surface

-c- .

_ _ _ _ _ _

_

_

-

OUTER MEMBRANE - _ _ _ _ _ _ _ _

11

' 'G

Specific carriers

F..

/

PERIPLASM <

11

Outer 'surface

11 11 surface 11

Centre

.+.Inner

_ _ _ _ _ - - _

CYTOPLASMIC MEMBRANE _ _ _ _ _ _ - - _

CYTOPLASM

FIG. 1. Models depicting envelope structure (a) and the routes of entry of antibiotics (b) in Gram-negative bacteria. Cell envelope structure is based on the model of Nikaido and Nakae (1979) and drug entry reproduced by permission from Brown et al. (1979). +---+, Involvement of specific carrier proteins; F=====, passive difpassive diffusion through hydrophilic pores. fusion and partitioning; r , Capsules not indicated.

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I. CHOPRA AND P. BALL

Other outer membrane proteins act as phage and colicin receptors, or are involved in facilitated diffusion of specific solutes (including antibiotics) across the outer membrane, or contribute towards structural integrity. The localization of LPS in the outer leaflet has important consequences for the passive diffusion of antibiotics across the outer membrane because it prevents dissolution of hydrophobic species into non-polar regions (Nikaido and Nakae, 1979). This exclusion does not result simply from the formation of a hydrophilic layer on the outer surface of the membrane contributed by the saccharide side chains of LPS (Nikaido, 1976). The inability of hydrophobic molecules to intercalate in the LPS probably results from the restricted mobility of its fatty acids compared with those in phospholipids (Rottem, 1978). An approximate estimate of the ability of an antibiotic to penetrate the outer membrane can be derived from comparison of the minimum drug concentrations required to inhibit the growth of Gram-negative and Grampositive species (Franklin and Snow, 1975; Chopra and Howe, 1978). High ratios indicate that, for example, the hydrophobic antibiotics rifamycin (ratio lo4) and fusidic acid (ratio 5. lo3) (Chopra and Howe, 1978) are excluded by the outer membrane. Deep rough LPS core mutants of E. coli and S . typhimurium contain phospholipids in the outer leaflet of the outer membrane (Nikaido and Nakae, 1979) and should therefore become more susceptible to hydrophobic antibiotics. In general, this relationship does hold, e.g. for rifamycin the efficacy ratio deep rough mutant: wild-type is below 10 (Nikaido, 1976). Therefore comparison of the susceptibility of wild-type E. coli and a deep rough mutant to an antibiotic can, like the Gram-positive: Gram-negative ratio, indicate whether it is normally excluded by the outer membrane. Not all Gram-negative bacteria have hydrophobic-impermeable outer membranes (Nikaido and Nakae, 1979), but those that do frequently have enteric habitats. This probably reflects evolution of outer membranes to resist the hydrophobic bile acids of the intestinal tract. Although hydrophobic antibiotics do not readily diffuse across the outer membrane of E. coli and S. typhimurium, they can, as mentioned, gain access to the cell interior of deep-rough LPS mutants and presumably also wildtype strains when high external drug concentrations can be maintained. According to Piovant et al. (1978) hydrophobic antibiotics are delivered to the cytoplasmic membrane through envelope regions that connect the inner and outer membranes. The preferential activity of hydrophobic inhibitors of protein synthesis against membrane-bound ribosomes (Hirashima et al., 1973; Piovant et al., 1978) is assumed by Piovant et al. (1978) to reflect the proximity of junction points to the sites of synthesis of envelope polypeptides. In addition to providing a permeability barrier towards hydrophobic compounds the outer membrane, because it participates in the formation of a Donnan equilibrium (Stock et ul., 1977), tends to repel anionic species.

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191

For rapid passive diffusion across the outer membrane an antibiotic would seem to require the following properties: (i) solubility in water; (ii) a molecular weight of less than 600 (to gain access to the transmembrane poressee Nikaido and Nakae, 1979); and (iii) exist as a cation or uncharged species at neutral pH. Clearly, not every antibiotic that is effective against enteric species of bacteria has these properties, and some examples of these exceptions will be discussed in subsequent sections.

C. PERIPLASMIC SPACE

The periplasmic space comprises the aqueous region that lies between the outer membrane and the cytoplasmic (inner) membrane of Gram-negative bacteria. It contains peptidoglycan (see below), lytic enzymes and substratebinding proteins (Heppel, 1971; Rosen and Heppel, 1973; Costerton et al., 1974; Wilson and Smith, 1978). Estimates of the volume of the periplasmic space range from 5% to 40% of the total cell volume (Stock el al., 1977; Nikaido and Nakae, 1979). Antibiotics entering the periplasmic space may become protonated because the pH is below that in the extracellular medium (Stock el al., 1977). Passage of drugs across the periplasmic space may also be influenced by the macromolecules that it contains. Thus, non-specific binding to peptidoglycan (see below) or proteins may occur, or there may be specific interaction with substrate-binding proteins. Involvement of binding proteins in antibiotic transport can be assessed by subjecting bacteria to osmotic shock since such treatment releases these proteins (Rosen and Heppel, 1973). Several plasmidencoded antibiotic-inactivating enzymes are located in the periplasmic space (Davies and Beneviste, 1974; Davies and Smith, 1978). According to Davies and coworkers (Davies and Beneviste, 1974; Davies and Kagan, 1977; Davies and Smith, 1978) further transport of some antibiotics may be abolished because the molecules are altered by the inactivating enzymes. This hypothesis will be discussed more fully in a subsequent section.

D. P E P T I D O G L Y C A N S

All bacteria apart from L-forms, mycoplasmas and archaebacteria are surrounded by peptidoglycan (also called mucopeptide, murein or glycopeptide). Bacterial peptidoglycans have been frequently reviewed (Rogers, 1974; Ghuysen and Shockman, 1973; Braun and Hantke, 1974; Rogers el al., 1978) so that only a brief outline of structure will be provided here. They all consist of pl,4-linked amino sugar (glycan) chains which are joined together by short peptides. The sugar residues are invariably N-acetylglucosamine and N -

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I. CHOPRA AND P. BALL

acetylmuramic acid and the peptides, which are attached to the carboxyl groups of the N-acylmuramyl residues, usually only contain four or five amino acids. The composition of the peptides and the mode of cross-linkage vary between different bacteria. In Gram-positive bacteria the peptidoglycan is organized in multiple layers, but in E. coli and probably other Gramnegative bacteria only one layer occurs. In many Gram-positive bacteria other polymers (e.g. teichoic and teichuronic acids) are covalently linked to the amino sugar chains, but the quantities of these polymers in the wall are variable and depend on the growth conditions. Integrated polymers similar to the teichoic acids do not occur in the peptidoglycans of Gram-negative bacteria. However, a number of outer membrane proteins are peptidoglycan associated, either by covalent linkage (for the lipoprotein) or through ionic interactions (for pgrins) (Nikaido and Nakae, 1979). Synergistic activity towards E. coli between inhibitors of peptidoglycan integrity (lysozyme and penicillin) and other antibiotics have been interpreted by some authors (e.g. Gustafsson et al., 1973) to indicate that the peptidoglycan of E. coli can act as a molecular sieve. However, redistribution of both LPS and outer membrane proteins can occur following treatment of bacteria with lysozyme (Miihlradt and Golecki, 1975; Chopra et al., 1977), so that the conclusions of Gustafsson et al. (1973) must be treated with caution. Indeed, in contrast to the molecular sieving function of the outer membrane porins, bacterial peptidoglycans are probably only able to exclude large molecules ( > 100,000 daltons) (Scherrer and Gerhardt, 1971). Even though estimation of the size of molecules that can penetrate peptidoglycans is subject to considerable error (Cope, 1980), it is unlikely that peptidoglycans prevent permeation of antibiotics because the drug molecules are all relatively small (Table 1). However, peptidoglycan-associated polymers such as teichoic acids are negatively charged and so, like capsules, may affect the accumulation of ionized antibiotics.

E. C Y T O P L A S M I C O R I N N E R M E M B R A N E S

The inner membrane of Gram-negative bacteria broadly corresponds to the cytoplasmic membrane of Gram-positive species. These membranes contain enzyme systems related to electron transfer, active transport of solutes, lipid synthesis and production of cell-wall and envelope polymers. The composition of cytoplasmic membranes and hence their functional properties can be varied according to physiological needs. Extensive reviews dealing with the structure and function of bacterial cytoplasmic membranes have been published (Machtiger and Fox, 1973; Costerton et al., 1974; Salton and Owen, 1976; Salton, 1978), so that detailed discussion of these membranes is not warranted here.

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Water-soluble molecules with molecular weights greater than 100 that have little o r n o lipid solubility cannot passively diffuse across cytoplasmic membranes (Franklin, 1973). Since the majority ofantibiotics are hydrophilic, we may expect their transport across cytoplasmic membranes to be mediated either by facilitated diffusion o r by energy-dependent accumulation systems.

F. S U M M A R Y

Models depicting envelope structure and the routes of entry of antibiotics into Gram-negative a n d Gram-positive bacteria are presented in Figs. 1 and 2.

u

Protein

EXTERNAL ENVIRONMENT

11

~

, ~

-4

~

Specific carriers

y - -

_7Outer surface

It

Centre

11

CYTOPLASMIC M EM BRAN E

- .+Inner surface

11

CYTOPLASM FIG. 2. Models depicting envelope structure (a) and the routes of entry of antibiotics (b) in Gram-positive bacteria. Cell envelope structure is based on the model of Costerton and Cheng (1975) and drug entry on the scheme of Brown et al. (1979). +---+,

Transport by specific carrier proteins; =====, passive diffusion and partitioning. Capsules are not indicated.

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I. CHOPRA AND P. BALL

V. Aminoglycosides

Although some authors (e.g. Tanaka, 1975) consider the aminoglycosides to include antibiotics such as streptozotocin and norjirimycin, we will restrict the use of this term to those antibiotics structurally related to streptomycin (N-methyl-L-glucosoamidostreptosido-streptidine) (Fig. 3), e.g. gentamicin, kanamycin and neomycin. These antibiotics inhibit protein synthesis in bacteria. In the case of streptomycin, the drug primarily binds to protein S12 of the 30s ribosomal subunit (Pestka, 1977) and induces misreading of mRNA (Gale et al., 1972).

NH I1 NHC- NH2

NH

H OH

OH

H

FIG. 3. Structure of streptomycin

Since the target for aminoglycosides is intracellular (Table 1) the drugs must cross both outer and cytoplasmic membranes of bacteria.

A . D I F F U S I O N OF A M I N O G L Y C O S I D E S ACROSS THE OUTER MEMBRANE

In E. coli, aminoglycosides probably cross the outer membrane by passive diffusion through outer membrane porin proteins (Foulds and Chai, 1978). Indeed, the low molecular weight of these compounds (Table 1) is consistent with their entry through pores. Since aminoglycosides are cationic at neutral pH (Holtje, 1978), diffusion of these molecules into the periplasmic space

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

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should be favoured by the Donnan equilibrium across the outer membrane (see Introduction). Indeed, evidence in favour of this view has been obtained (Campbell and Kadner, 1980). In contrast to the situation in E. coli, aminoglycoside accumulation in Pseudomonas aeruginosa may not involve diffusion through outer membrane pores (Hancock et al., 1981). In this case the antibiotics may promote their own uptake across the Pseudomonas outer membrane by interaction with Mg2+ binding sites in the membrane (Hancock et al., 1981).

B. A C C U M U L A T I O N O F A M I N O G L Y C O S I D E S A C R O S S T H E C Y T O P L A S M I C

MEMBRANE

Most measurements of aminoglycoside accumulation have been made using streptomycin (see below). However, unless specifically stated, data obtained with streptomycin can be taken to apply to other aminoglycosides. Early reports on the action of streptomycin indicated that the drug caused alterations in membrane permeability and macromolecular synthesis. Thus, leakage of potassium ions and decreased crypticity of the cytoplasmic enzyme b-galactosidase (EC 3.2.1.23) were detected, as well as inhibition of protein synthesis and alterations in RNA and DNA metabolism (Dubin et al., 1963). However, it was unclear whether these effects resulted directly from interaction of streptomycin with the cytoplasmic membrane or indirectly from inhibition of protein synthesis (Dubin et al., 1963). Both earlier work (Anand et af., 1960; Dubin et al., 1963) and more recent investigations (Bryan and Van den Elzen, 1976, 1977; Holtje, 1978) show that streptomycin uptake is multiphasic. Bryan and Van den Elzen (1977) distinguished three phases of streptomycin uptake; an energy-independent phase (EIP) and two energy-dependent phases (EDPI and EDPII).

1. Energy-Independent Phase

Energy-independent phase (EIP) streptomycin uptake almost certainly represents non-specific binding of the drug to bacteria via anionic groups associated with the cell surface (Bryan and Van den Elzen, 1977). Thus initial binding of streptomycin to bacteria is similar at 0°C and at 37"C, and is reversed by adding cations (Dubin et al., 1963). Although EIP streptomycin uptake might result solely from binding of drug to the cell wall (Dubin et al., 1963), data obtained with E. coli sphaeroplasts indicate that at least part of EIP reflects binding of streptomycin to the cytoplasmic membrane (Bryan and Van den Elzen, 1977).

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I . CHOPRA AND P. BALL

2. Energy-Dependent Phase I

Energy-dependent phase I (EDPI) reflects active accumulation of streptomycin (Bryan and Van den Elzen, 1976, 1977; Bryan et a[., 1980; Campbell and Kadner, 1980). Although the exact mechanism involved is unclear, both streptomycin (Damper and Epstein, 1979) and gentamicin (Gilman and Saunders, 1981) accumulations are driven by the transmembrane electrical potential (A$). Thus, decreased streptomycin uptake occurs in the following cases where A$ is dissipated: (i) by anaerobiosis (Campbell and Kadner, 1980); (ii) when the electron transport chain is blocked by inhibitors (Bryan and Van den Elzen, 1977); (iii) in mutants defective in components of the electron transport chain (Bryan and Van den Elzen, 1977; Campbell and Kadner, 1980); (iv) in bacteria treated with uncouplers (Damper and Epstein, 1979; Gilman and Saunders, 1981); (v) in uncoupled mutants (Bryan and Van den Elzen, 1977). However, alterations in A$ alone cannot explain changes in the level of streptomycin accumulation for the following reasons. (a) Streptococcusfaecalis grown glycolytically and E. coli grown aerobically generate almost identical A$ values. (Bakker, 1978). Under these conditions, S.faecalis is streptomycin resistant and E . coli streptomycin sensitive (Bryan and Van den Elzen, 1977). (b) Anaerobiosis produces almost complete inhibition of streptomycin uptake but only a 10% decrease in proline accumulation (Campbell and Kadner, 1980). Proline accumulation, which is dependent on A$ (Hirata et al., 1974), is presumably driven by A$ generated by ATP hydrolysis during anaerobiosis, but this A$ cannot drive streptomycin uptake. (c) Haem-deficient mutants unable to synthesize functional cytochromes accumulate negligible amounts of streptomycin (Bryan and Van den Elzen, 1977; Campbell and Kadner, 1980). Under identical conditions, proline accumulation is lowered by only 60% (Campbell and Kadner, 1980). Presumably, the residual proline accumulation is driven by A$ derived from ATP hydrolysis, but this A$ is not able to drive streptomycin accumulation. (d) A ubiquinone-deficient mutant accumulates about 50% less streptomycin than does its wild-type parental strain (Bryan and Van den Elzen, 1977). However, serine accumulation [which is driven by A$ (Collins et al., 1976)] is decreased by 90% in the mutant, apparently due to leakage of protons across the membrane (Simoni and Postma, 1975). Indeed, Bryan and coworkers propose that streptomycin uptake involves binding of the drug to quinones in the electron transport chain (Bryan and Van den Elzen, 1977; Bryan et a[., 1980). Nevertheless, certain data are inconsistent with a direct involvement of electron transport chain quinones in streptomycin uptake.

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(a) There is little correlation between quinone content and streptomycin accumulation. Thus, the ubiquinone-deficient mutant studied by Bryan and Van den Elzen (1977) showed only a 50% decrease in streptomycin uptake compared to the parental strain. Conversely, the streptomycin-resistant mutant of Pseudomonas aeruginosa isolated by Bryan et al. (1980) had normal levels of ubiquinone but lower levels of cytochrome c S s 2 and nitrate reduc tase. (b) Ubiquinone-deficient mutants like the one used by Bryan and Van den Elzen (1977) may also be deficient in cytochromes (Bragg, 1979). (c) Haem-deficient mutants accumulate negligible amounts of streptomycin (Bryan and Van den Elzen, 1977; Campbell and Kadner, 1980) but presumably contain normal levels of quinones. Possibly, EDPI streptomycin uptake depends on binding to functional cytochromes. Data supporting this hypothesis are as follows. (a) Anaerobes like clostridia and bacteroides contain negligible amounts of cytochromes (Davis et al., 1973) and are resistant to streptomycin (Bryan and Van den Elzen, 1976, 1977). (b) Facultative anaerobes like E. cofi d o not use cytochromes when grown in the absence of a terminal electron acceptor (Haddock and Jones, 1977). Under these conditions, E . coli accumulates negligible amounts of streptomycin (Campbell and Kadner, 1980). Provision of a terminal electron acceptor such as nitrate to anaerobically grown E. coli restores streptomycin uptake, presumably due to transfer of electrons to nitrate reductase via cytochrome b (Kroger, 1977). If cytochromes are involved in EDPI streptomycin uptake, then the mutant of P. aeruginosa isolated by Bryan et al. (1 980) is presumably streptomycinresistant due to deficiencies in electron transfer by cytochrome css 2 . Similarly, the haem-deficient and ubiquinone-deficient mutants are probably resistant to streptomycin because they contain non-functional cytochromes. The hypothesis that streptomycin uptake depends on functional cytochromes might be tested using S. faecalis. This organism normally lacks cytochromes, but may synthesize them if grown in the presence of haematin (Bryan-Jones and Wittenbury, 1969). If A$ is similar in S. faecalis grown both glycolytically and aerobically in the presence of haematin, it should be possible to correlate streptomycin uptake with the presence of functional cytochromes. Clearly, this depends on the ability of S . faeculis to generate identical A$ values in the presence and absence of haematin. This might not be achieved, since haem-deficient mutants of E. coli apparently generate different A$ values in the presence and absence of functional cytochromes, as shown by differences in proline accumulation (Campbell and Kadner, 1980).

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I. CHOPRA A N D P. BALL

3. Energy-Dependent Phase II (EDPII)

During energy-dependent phase I1 (EDPII), streptomycin is accumulated at a greater rate than during EDPI (Bryan and Van den Elzen, 1977). Holtje (1978) proposed that this increased streptomycin uptake represented induction of a transport system responsible for accumulation of polyamines. However, whereas streptomycin stimulated uptake of the polyamines putrescine and spermidine, these compounds did not stimulate streptomycin accumulation (Holtje, 1978). Also, no evidence for increased synthesis of a putative transport protein was detected in bacteria treated with gentamicin (Ahmad et al., 1980). Therefore, aminoglycosides are probably not accumulated by a polyamine transport system. Rather, increased uptake of polyamines by bacteria exposed to streptomycin probably results from a general stimulation of cation accumulation (Bryan et al., 1980;Campbell and Kadner, 1980). Several experiments show that EDPII is dependent on binding of aminoglycosides to the ribosome. (a) Ribosomal mutants which are streptomycin resistant due to decreased binding of drug do not accumulate streptomycin by EDPII (Dickie et al., 1978; Holtje, 1979). (b) Bacteria carrying plasmids encoding streptomycin adenylyl-transferases accumulate modified drug during EDPII (Dickie et al., 1978; Holtje, 1979). Modified streptomycin does not bind to ribosomes and does not stimulate EDPII streptomycin accumulation (Dickie et al., 1978; Holtje, 1979). In strains carrying plasmids encoding enzymic modification of streptomycin, the rate of modification of the drug during uptake determines the level of binding of antibiotic to ribosomes, and hence the extent of EDPII (Dickie et al., 1978; Holtje, 1979).Thus, the proposal of Davies (Davies and Beneviste, 1974; Davies and Smith, 1978) that modified aminoglycosides are not accumulated by resistant bacteria, and might block a putative transport system involved in EDPII aminoglycoside uptake, is apparently incorrect. The basis of stimulation of EDPII streptomycin uptake by binding of the drug to ribosomes is unclear. EDPII streptomycin uptake is inhibited by N-ethylmaleimide which blocks thiol groups (Fox and Kennedy, 1965) and 2,4-dinitrophenol which uncouples oxidative phosphorylation (Hopfer et al., 1968). Inhibition of streptomycin uptake by N-ethylmaleimide and 2,4dinitrophenol presumably reflects direct blockage of accumulation, rather than indirect disruption of protein synthesis, since both compounds inhibit EDPII streptomycin uptake in bacteria unable to synthesize protein (Hurwitz et al., 1981). Overall, EDPI and EDPII streptomycin uptake result in an intracellular concentration of drug up to 400-fold the extracellular concentration (Bryan

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

199

P

In (b) Sm

out

I

w

U

--- P In

(C) Sm

LSm

out

FIG. 4. Mechanism of streptomycin accumulation (from Bryan and van den Elzen, 1977). (a) Streptomycin (Sm) binds to the cell surface, including the cytoplasmic membrane (cm) (energy-independent phase). (b) Streptomycin (Sm) is accumulated by a process involving respiratory chain components (r) (energy-dependent phase I). (c) Streptomycin binds to polysomes (p) triggering further drug accumulation (energydependent phase 11). Note that although only membrane-bound polysomes are shown for clarity, streptomycin is assumed to stimulate energy-dependent phase I1 uptake by binding to all polysomes.

and Van den Elzen, 1976). However, non-specific binding of drug to ribosomes during EDPII would mean that the intracellular concentration of free drug is lower (Hurwitz et al., 1981). The three phases of streptomycin uptake by bacteria can be summarized as follows (see Fig. 4). (a) Energy-independent binding of drug to the cell surface (EIP) (Bryan and Van den Elzen, 1977). (b) Energy-dependent uptake driven by A$ and involving components of the respiratory chain (EDPI) (Bryan and Van den Elzen, 1977; Bryan et al., 1980; Campbell and Kadner, 1980). (c) Stimulation of energy-dependent streptomycin uptake following binding of drug to ribosomes (EDPII) (Dickie et al., 1978; Holtje, 1979).

200

I. CHOPRA AND P. BALL

VI. Chloramphenicol

Chloramphenicol (D -( - )-threo- 1-p-ni trophenyl-2-dichloroacetamido1,3 propanediol) (Fig. 5 ) is synthesized by Streptomyces venezuelae, and can also be produced synthetically (Glasby, 1976). Chloramphenicol primarily binds to protein L16 of the 50s subunit of the bacterial ribosome, inhibiting peptidyl transferase activity (Harvey and Koch, 1980).

HOCH I HCNHCOCHCt 2 I

CH20H

FIG. 5. Structure of chloramphenicol

Since the target for chloramphenicol is intracellular (Table 1) the drug must penetrate both outer and cytoplasmic membranes of bacteria.

A. D I F F U S I O N O F C H L O R A M P H E N I C O L A C R O S S T H E O U T E R M E M B R A N E

Chloramphenicol apparently crosses the outer membrane by passive diffusion in protein ompF pores (Chopra and Eccles, 1978; Pugsley and Schnaitman, 1978). Thus, mutants deficient in protein ompF are more resistant to chloramphenicol than their parental wild-type strains (Chopra and Eccles, 1978; Pugsley and Schnaitman, 1978). However, chloramphenicol is relatively hydrophobic, and would not be expected to cross the outer membrane via hydrophilic porins (Nikaido, 1976). Possibly, loss of protein ompF leads to loss of other components of the outer membrane essential for entry of chloramphenicol (Nikaido, 1976).

B. A C C U M U L A T I O N O F C H L O R A M P H E N I C O L A C R O S S T H E CYTOPLASMIC MEMBRANE

Certain authors (e.g. Hurwitz and Braun, 1967) propose that chloramphenicol uptake across the cytoplasmic membrane results solely from passive

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

201

diffusion. This contention is supported by data indicating that apparent concentration of chloramphenicol by bacteria probably results from binding of the antibiotic to ribosomes (Harvey and Koch, 1980). Since chloramphenicol is relatively hydrophobic (Nikaido, 1976) passive diffusion of the drug through the hydrocarbon interior of the cytoplasmic membrane is feasible. Irvin and Ingram (1 980) isolated a chloramphenicol-resistant mutant of Pseudornonas aeruginosa which also exhibited decreased amino-acid accumulation. Since amino-acid uptake involves active transport (Anraku, 1978) the data of Irvin and Ingram (1980) could be interpreted in favour of energy-dependent chloramphenicol uptake. However, this mutant may accumulate decreased amounts of amino acids and chloramphenicol due to loss of outer membrane porins (Foulds and Chai, 1978; Pugsley and Schnaitman, 1978). Since Irvin and Ingram (1980) did not confirm that their mutant had a normal outer membrane, their results remain equivocal. In the absence of unequivocal evidence in favour of active accumulation of chloramphenicol, we conclude that the antibiotic probably crosses both outer and cytoplasmic membranes of bacteria by passive diffusion.

VII. D-Cycloserine D-Cycloserine (D-4-amino-3-isoxazolidone)an analogue of D-alanine (Fig. 6) is a broad-spectrum antibiotic synthesized by a number of Streptornyces species. D-Cycloserine blocks mucopeptide biosynthesis by competitive inhibition of D-alanine ligation (Gale et al., 1972). The donor site of Dalanine: D-alanine ligase (EC 6.3.2.4.) is the primary site of action with the acceptor sites of alanine racemase (EC 5.1.1.1 .) and D-alanine: D-alanine ligase as secondary targets (Fig. 7) (Gale et al., 1972). Since the targets for D-cycloserine are intracellular (Fig. 7) the drug must cross both outer and cytoplasmic membranes of bacteria.

FIG. 6. Structure of D-cycloserine (a) and D-alanine (b).

Wa II

I I

I

Membrane

Cytoplasm

I

D-

I

Ala t r a n s p o r t s y s t e m

-_----------------

--

~

I

L-AIo

&(a)

---t----II

I D-AIO

+

D-Ala

To’

D - A I o - D -A10

Undecoprenyl- P --L-AIa -D-GIu-

Cross- linked woll peptidoglycon C ,,

L-

R3-

iso p re no id a I c o h o I UDP-MurNAcL-Ala-D-Glu

Undecoprenyl-P-P

Peptidoglycondisocchoride pentopeptide

F

-

D-GIu

UDP-MurNAc -L-AIo

!-

L-AIo

Fornesyl-P-P

P

UDP-MurNAc NADP@

I

8-Isopentenyl - P - P

I

Undecaprenyl - P - P disaccharide pentopeptide

NADPH

UDP-GIcNAc P-enolpyruvote

UDP-Glc NAc-enolpyruvote

P,

I FIG. 7. Biosynthetic pathway for peptidoglycan, showing sites of action of (a) D-CyClOSerine and (b) fosfomycin. Modified from Ghuysen and Shockman (1973) with permission.

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

203

A. D I F F U S I O N O F D-CYCLOSERINE ACROSS THE OUTER M E M B R A N E

D-Cycloserine is a low molecular-weight hydrophilic molecule. Thus, it probably crosses the outer membrane by passive diffusion in the water-filled porin proteins (see Introduction). Since D-cycloserine is predominantly zwitterionic at neutral pH (Fig. 6) diffusion of the molecule into the periplasmic space is unlikely to be adversely affected by the Donnan equilibrium across the outer membrane (see Introduction).

B. T R A N S P O R T O F D - C Y C L O S E R I N E ACROSS T H E C Y T O P L A S M I C

MEMBRANE

D-Cycloserine is accumulated across the cytoplasmic membrane by the D-alanine transport system (Wargel et al., 1971). This system encoded for by gene cycA (Anraku, 1978) is involved in accumulation of malanine, D-glycine and D-serine as well as D-cycloserine (Wargel et al., 1971; Robbins and Oxender, 1973). Thus, mutants in gene cycA are resistant to D-cycloserine and are defective in accumulation of D-glycine and D-alanine (Wargel et al., 1971). The K, values for D-cycloserine uptake (29 FM; calculated from data in Wargel et al., 1971) and D-alanine accumulation (26 PM; Wargel et al., 1971) are almost identical. Presumably, this reflects the structural similarity between D-cycloserine and D-alanine (Fig. 6). Since D-alanine is accumulated by a proton symport (Collins et al., 1976) uptake of D-cycloserine almost certainly involves concomitant proton translocation.

VIII. 3,4-Dihydroxybutyl-l-phosphonate 3,CDihydroxybutyl-1 -phosphonate (DHBP), a synthetic analogue of snglycerol-3-phosphate (Fig. 8), inhibits phosphoglyceride metabolism in both E. coli and Bacillus subtilis (Tyach et al., 1976; Klein et al., 1977). Although the principal target for DHBP is probably CDP-diglyceride: sn-glycerol3-phosphate phosphatidyltransferase (EC 2.7.8.5.) (Tyach et al., 1976) (a)

CH20H I HCOH I CH2CH2 PO$-

(b)

CH20H H~OH I CH 20PO:-

FIG. 8. Structures of 3,4-dihydroxybutyl-l-phosphonate(a) and sn-glycerol-3phosphate (b).

204

I . CHOPRA A N D P. BALL

phosphonic acid derivatives of sn-glycerol-3-phosphate such as DHBP have a variety of intracellular effects (Tang et al., 1979). To reach its target site(s) within the cytoplasm (Table l), DHBP must penetrate the outer and cytoplasmic membranes of bacteria. Since DHBP is accumulated by the sn-glycerol-3-phosphate transport system (Leifer et a[., 1977) it is assumed that accumulation of the drug will be essentially similar to that of fosfomycin (see next section).

A. D I F F U S I O N O F D H B P A C R O S S T H E O U T E R M E M B R A N E

As mentioned above, DHBP is accumulated by the sn-glycerol-3-phosphate transport system. Diffusion of DHBP through the outer membrane presumably involves the glpT protein in a manner similar to fosfomycin (see next section).

B. T R A N S P O R T OF D H B P A C R O S S T H E C Y T O P L A S M I C M E M B R A N E

Accumulation of DHBP involves active transport of the drug across the cytoplasmic membrane by the sn-glycerol-3-phosphate transport system, as described in the next section (see fosfomycin). The affinity of the sn-glycerol3-phosphate transport system for DHBP ( K , 200 p ~ is) about 12-16-fold lower than for sn-glycerol-3-phosphate (Kt 12-16 p ~ (Leifer ) el al., 1977). The low affinity of the transport system for the drug presumably reflects structural differences between DHBP and sn-glycerol-3-phosphate (Fig. 8). DHBP is concentrated about 177-fold by E. coli at extracellular drug concentrations (300 p ~ in) excess of the Kt for the uptake of the antibiotic (Leifer et al., 1977). However, at lower drug concentrations (33 p ~ the ) intracellular concentration of antibiotic is about 700-fold the extracellular concentration (calculated from data in Leifer et al., 1977). Under virtually identical conditions, the concentration gradient for sn-glycerol-3-phosphate is about 1000-fold (Hayashi et al., 1964). Thus, both DHBP and sn-glycerol3-phosphate are accumulated to produce intracellular pools of similar size. Accumulation of DHBP is apparently energy-dependent, since both 2,4dinitrophenol and sodium azide stimulate efflux of the drug from loaded bacteria (Leifer et al., 1977). Presumably, like sn-glycerol-3-phosphate (Boos et al., 1977) DHBP is accumulated by a proton symport.

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

205

IX. Fosfomycin

Fosfomycin (phosphonomycin) (L-cis-1,2-epoxypropylphosphonic acid) (Fig. 9) is a broad-spectrum antibiotic synthesized by Streptomyces,fradiae. Fosfomycin inhibits cell-wall biosynthesis by covalently binding to the enzyme phosphoenolypyruvate: UDP-Glc-NAc-3-enolpyruvyltransferase (b'pyruvyl transferase"; EC 2.5.1.7.) (Fig. 7) at the site normally occupied by phosphoenolpyruvate (Kahan et al., 1974).

(0)

H C ,,

H,

j>O H'

P ' O:-

( b ) HzCOH I HCOH

HztOPOg-

(c)

CHO I HCOH I HOCH I HCOH I HCOH I H2COPO$-

FIG. 9. Structures of fosfomycin (a), sn-glycerol-3-phosphate (b) and glucose 6phosphate (c).

Since the target enzyme for fosfomycin is intracellular (Table 1) the antibiotic must penetrate both the outer and cytoplasmic membranes of bacteria. Fosfomycin is accumulated by both the sn-glycerol-3-phosphate and hexose phosphate transport systems in bacteria, and mutants lacking components of these systems are fosfomycin-resistant (Fig. 10) (Kahan et a/., 1974; Tsuruoka et al., 1978). The involvement of these transport systems in fosfomycin accumulation is described below.

A. D I F F U S I O N O F F O S F O M Y C I N A C R O S S T H E O U T E R M E M B R A N E

1. Probable Involvement o j the sn-Glycerol-3-phosphate Transport (glp T)

Protein In E. coli, the glpT product, apart from mediating uptake of its usual substrate sn-glycerol-3-phosphate, also promotes fosfomycin entry (Kahan et al., 1974; Argast et al., 1977; Silhavy et al., 1978). Fig. 10 shows data which first correlated expression of glpT with susceptibility to fosfomycin and its accumulation by the cell. The glpT structural gene is located in one of several widely spaced operons (Lin, 1976; Miki et al., 1979) that encode products

I. CHOPRA AND P. BALL

lOmM GIu-6-P 4

c 0

U

3

L

0 \

10 m M Glycerol

-

E

No inducer

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

"

70 >700

2

2

; u c C

._ 0

I

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

2 0 >700

:

0 Extrocel lulor spoce

7

Minimum inhibitory concentration ( p ~ )

FIG. 10. Uptake of [3H]fo~fomycinby strains of Escherichia coli after prior exposure to inducers of transport systems. Inocula (60 ml) were grown to A , , , = 0.3 and combined with indicated inducer for an additional 90 minutes. Cultures [strains L-1: glpT+, uhp+ (0) and L-217: glpT-, uhp+ ( O ) ] were chilled and centrifuged, and then resuspended at a 6-fold higher cell density in broth without inducer, which contained 15 p~ [3H]fosfomycin at 6 pCi/pmol. The incubation was conducted at 37 C for 20 minutes, followed by rapid cooling and centrifugation. Cell pellets, which ranged in weight from 80 to 95 mg, were resuspended and counted directly. Dilute samples that represented 10 minute incubated cells of the induced inocula were also plated on nutrient-agar supplemented with fosfomycin to determine the minimum inhibitory concentration (MIC). The extracellular space was determined in separate experiments that employed Blue Dextran 2000 as an indicator of the extracellular volume of the cell pellet, which was found to be 0.4 ml/g. The true intracellular volume is assumed to be 0.42 ml/g. (Reproduced from Kahan et al., 1974 with permission.)

concerned with transport or metabolism of glycerol and sn-glycerol-3phosphate (Table 2). A single repressor (coded by gfpR) regulates the expression of the operons with glycerol or sn-glycerol-3-phosphate as inducers. The role of the gfpT protein in the transport of fosfomycin has been clarified by recent research by Boos and coworkers (Argast et al., 1977; Silhavy et al., 1978). Most probably, this protein acts as a specific porin, producing diffusion channels across the outer membrane through which both sn-glycerol-3-phosphate and fosfomycin enter. Evidence for this contention rests on the following information.

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

207

TABLE 2. Genetic loci and products involved in dissimilation of glycerol and glycerol-3-phosphate in Escherichia coli Map positions are from Bachmann and Low (1980). The symbol] denotes genes that are probably located in the same operon (see Lin, 1976; Miki et al., 1979)

Gene

grpT grpA

1

Map position (minutes)

Gene product or phenotypic trait affected

48

Glycerol 3-phosphate dehydrogenase (anaerobic), EC 1.1.99.5. (Bachman and Low, 1980). 160,000-dalton protein composed of four identical subunits, which have a PI of 4.4. Protein has no binding activity for glycerol phosphate, but my form specific channels through the outer membrane to permit its diffusion (Argast et al., 1977; Silhavy et al., 1978; Nikaido and Nakae, 1979). Glycerol 3-phosphate dehydrogenase (aerobic), EC 1.1.99.5. (Bachman and Low, 1980). Repressor of the glp regulon. Contiguous to glpD (Lin, 1976; Bachman and Low, 1980). Facilitated diffusion of glycerol mediated by an unidentified inner membrane protein which may produce transmembrane pores (Heller et a/., 1980). Glycerol kinase, EC 2.7.1.30. (Bachman and Low, 1980).

48

75

g'pD1

75

dPR

grpF dPK

1

88 88

(a) The protein subunits are acidic (PI 4.4). This is also a feature of well-characterized porins (Nikaido and Nakae, 1979). (b) Part of the glpT protein is exposed on the cell surface. This is also true for transmembrane porins (Nikaido and Nakae, 1979). (c) The glpT protein subunits form stable oligomers in SDS solutions at room temperature, much like porins (Nikaido and Nakae, 1979). (d) The glpT protein is not essential for the energy-dependent active transport of sn-glycerol-3-phosphate in cytoplasmic membrane vesicles (Argast et al., 1977). It may, therefore, function only in the transport of solutes, including fosfomycin, across the outer membrane. Argast et al. (1977) found that the glpT protein was released from whole bacteria by osmotic shock, a procedure known to release periplasmic proteins. However, this observation does not necessarily imply a periplasmic location for the glpT protein since osmotic shock also releases outer membrane porins (Benz et al., 1978; I. Chopra, unpublished observations).

2. Possible Involvement of a Hexose Phosphate Transport Protein

Hexose phosphate accumulation in E. coli, encoded by gene uhpT, is inducible

208

I. CHOPRA AND P. BALL

by glucose 6-phosphate, fructose 6-phosphate and mannose 6-phosphate. Although the transport system is generally considered to lack periplasmic or outer membrane components (Silhavy et al., 1978) uptake by this system is impaired by osmotic shock (Dietz et al., 1968). Thus, diffusion of hexose phosphates across the outer membrane might involve a porin-like protein as just described for sn-glycerol-3-phosphate. Alternatively, uptake of hexose phosphates might involve a periplasmic binding protein.

B. T R A N S P O R T O F F O S F O M Y C I N A C R O S S T H E C Y T O P L A S M I C MEMBRANE

Since phosphorylated compounds probably cannot passively diffuse through the cytoplasmic membrane (Davis, 1958) accumulation of fosfomycin by both the sn-glycerol-3-phosphate and hexose phosphate transport systems is presumed to involve active transport across the cytoplasmic membrane. However, accumulation of fosfomycin by S . typhimurium results in only a lG20-fold concentration gradient (Kahan et al., 1974) and in E. coli only a 4-fold concentration occurs (Fig. 10; Kahan et al., 1974). In contrast, accumulation of sn-glycerol-3-phosphate and glucose 6-phosphate results in concentration gradients of about 1000-fold and 140-fold respectively (Hayashi et al., 1964, and calculated from data in Pogell et a[., 1966). Thus, fosfomycin may enter bacteria by facilitated diffusion rather than by active transport. Both sn-glycerol-3-phosphate (Boos et al., 1977) and hexose phosphate (Essenberg and Kornberg, 1975)are apparently transported across the cytoplasmic membrane by proton symports. We would expect, therefore, that if fosfomycin uptake is active it depends on proton translocation. Unfortunately, no results have been published to confirm this hypothesis.

C. T R A N S P O R T O F F O S F O M Y C I N I N BACTERIA OTHER T H A N E . C O L l

Evidence for accumulation of fosfomycin by components of sn-glycerol-3phosphate transport systems has been obtained in species other than E. coli (Kahan et al., 1974; Lindgren, 1978). In B. subtilis the genetic locus glpT has been ascribed to a system involved in uptake of both fosfomycin and snglycerol-3-phosphate. This locus and the glpT locus in E. coli are unlikely to code for similar products since glpT in E. coli determines synthesis of an outer membrane protein (see previous sections). Presumably, the glpT locus in B. subtilis encodes a cytoplasmic membrane protein involved in transport. The uhpT system is also utilized by fosfomycin to enter bacteria other than E. coli (Kahan et al., 1974).

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

209

X. Beta-Lactams

The p-lactam antibiotics (Fig. 11) have targets which are located in the cytoplasmic membrane (Table 1). Therefore in Gram-negative bacteria these antibiotics must be transported across the outer membrane to exert their inhibitory effects. In early studies the permeability of the outer membrane to p-lactams was expressed as “crypticity”, i.e. the increase in hydrolytic activity of p-lactamase towards a p-lactam following lysis of the bacteria. However, the numerical values obtained depend markedly on the substrate concentration used, so that “crypticity” values give little indication of relative diffusion rates of p-lactams across the outer membrane (Zimmermann and Rosselet, 1977; Nikaido and Nakae, 1979). However, a method based on inactivation of P-lactams by periplasmic p-lactamases is available for reliable determination of diffusion rates (or periplasmic concentrations) in Gramnegative bacteria (Zimmermann and Rosselet, 1977; van Alphen et al., 1978a; Nikaido and Nakae, 1979; Sawai ef al., 1979; Kojo et al., 1980) (see legend to Fig. 12). As with other solutes, the rates of diffusion of p-lactams across the outer membrane depend primarily on the size, hydrophobicity and electrical charge of the molecules (Nikaido and Nakae, 1979). The P-lactams have molecular weights which fall below the exclusion limits for entry through the transmembrane pores and indeed many are sufficiently hydrophilic to diffuse across the outer membrane by this route (Nikaido and Nakae, 1979; van Alphen ef al., 1978a). In E. coli, those p-lactams that diffuse by hydrophilic routes utilize pores formed by the ompF and ompC proteins (van Alphen et al., 1978a). Two groups (Zimmermann and Rosselet, 1977; Sawai et al., 1979) have studied the relationship between diffusion of 8-lactams across the outer membrane and drug hydrophobicity (Fig. 12). Both groups find an inverse correlation between hydrophobicity and the concentration of antibiotic delivered to the periplasmic space. The importance of electrical charge for diffusion of p-lactams across the outer membrane is illustrated by cephalexin. Sawai et al. (1979) found that cephalexin penetrated the outer membrane more readily than cephaloridine or ampicillin even though all compounds have similar partition coefficients (Fig. 12). As noted earlier, the Donnan equilibrium across the outer membrane causes exit of diffusible anions. The rapid diffusion of cephalexin across the outer membrane into the periplasmic space most likely relates to the uncharged form that the antibiotic adopts at neutral pH (Demarco and Nagarajan, 1972).In contrast, both cephaloridine and ampicillin are predominantly monoanionic at neutral pH (Demarco and Nagarajan, 1972), so that their diffusion will be hindered in comparison to cephalexin. It is not immediately obvious why the diffusion rate for ampicillin is markedly less than that for cephaloridine (Fig. 12).

R

1

General formula (with conventional numbering)

Side-chain ( R ) of derivatives Active mainly on Gram-positives

Active mainly on Gram-negatives NH 5CH-CH2-CH2-CH2COO H

Penicillin G (Benzy l penicill in)

Penicillin N (“Cephalosporin N”)

O O . C H 2 Penicillin V (Phenoxyrnethylpeiiicillin)

Ampicillin

Amo xyci Ilin

Methicillin Epicillin

0

CHLOONa

Nafcillin

Carbenicillin

lsoxazolyl penicillins Y

Oxacillin: X=H, Y=H Cloxacillin: X=CI. Y=H Dicloxacillin: X=CI, Y=CI Flucloxacillin: X=CI, Y=F

~

~

~

~

Ticarcillin O - C H = Meci I li nam (an amidinopenicillin) ‘Instead of R-C0.H of side-chain.

FIG. 11. Structure of (a) principal penicillins and (b) principal cephalosporins. Reproduced with permission from Selwyn et al. (1980).

N

FIG. 1 1 -cont.

Dihydro

~-

Available in United Kingdom w i t h side-chains R1

General formula ( w i t h conventional numbering)

Other derivatives w i t h side-chains

81

R2

R2

COOH-CH-(CH2)3-

I

-0-c,

4

NH2

CH3

Cephalosporin C

Cephaloridine

/O

-0-c<

0 - C H -

CH3 H2 Cephalothin

Cephaloglycin

PJaS-CH2Cephapirin

Cephalexin' (>CH-

I

-H

N H2 Cephacetrile

Csphradine N=N,

' N=N'

N-N ,N-CH2-SAS'CH3 Cephazoli n

Cefuroxime

5

(a CeDhamvcinl

'Cefadroxd has an -OH I h y d r o x y l ) a t this position. tCefaclor possesses a -CI (chlorine) instead o f a -CH3 (methyl) group a t the 3-carbon position of cephalexin

I. CHOPRA AND P. BALL 100

(0)

.Cephocetrile

(b)

' C49288

o Ceoholexin

I

0.2

I

i

06

0.4 Par t i t ion coefficient

0.

I

0.2

i

0.4

I

0.6

P a r t i t i o n coefficient

FIG. 12. Hydrophobicity of 8-lactams and their diffusion across the outer membrane of Escherichiu coli. (a) is based on data obtained by Zimmermann and Rosselet (1977) for E. coli 605 (serotype 055). Concentrations in the periplasmic space [SJ* were . is based on data obtained estimated at external concentrations [S,] of 100 p ~ (b) by Sawai et al. (1979) for E. coli ML1410. In this case, concentrations in the periPar- . plasmic space [S,]* were estimated at external concentrations [S,] of 50 l ~ tition coefficientst for all the 8-lactams apart from cephalexin were taken directly from Zimmermann and Rosselet (1977). The partition coefficient for cephalexin (0.14) was estimated by comparison of data in Zimmermann and Rosselet (1977) with that of Sawai et ul. (1979). *The antibiotic concentration in the periplasmic space [S,] ( p ~is) provided by the following relationship (where [s,]is external antibiotic concentration (pM), vi, v d , respectively, are the enzyme activities of a bacterial cell suspension at the substrate concentration of [S,] (WM) before and after disruption and K , is Michaelis constant of the 8-lactamase): [S,] =

(5

vd K m

Km[s11

+ [s,]-

)

(vi/ vd)[s11

t Partition coefficients in isobutanol (2-methylpropan-l-ol)/O~O2Mphosphate buffer (pH 7.4) containing 0.9% (w/v) NaCI.

XI. Norjirimycin Norjirimycin (2:3:4:5:-tetrahydroxy-6-hydroxymethylpiperidine) is a derivative of N-acetyl-D-glucosamine (Fig. 13) synthesized by a variety of Streptomyces species including Streptomyces norjiriensis (Tanaka, 1975). Norjirimycin is a weakly antibacterial antibiotic which inhibits a- and p-glucosidases (Tanaka, 1975). Thus, norjirimycin presumably blocks utiliza-

TRANSPORT OF ANTIBIOTICS INTO BACTERIA ( 0 )

HOCH2 HQH HOoH

(b)

21 3

HOCH2 HH OQHo H

ti

H OH

H HNCOCH3

FIG. 13. Structures of norjirimycin (a) and N-acetyl-D-glucosamine (b).

tion by bacteria of polysaccharides containing a- and P-glucosidic linkages. Since norjirimycin is structurally related to N-acetyl-D-glucosamine (Fig. 13) it is probably accumulated by the same phosphoenolpyruvate: phosphotransferase system (PTS) as streptozotocin (see next section). Indeed, mutants defective in either the enzyme I or HPr components of the PTS (Fig. 13) are resistant to norjirimycin as well as streptozotocin (Ammer et al., 1979).

XII. Peptide Antibiotics Several straight chain peptides have antibacterial activity (see Diddens et al., 1976; 1979 for a bibliography). Since it is now clear that these antibiotics are transported by the systems which usually deliver nutritionally beneficial peptides to the cell (Table I), it is relevant first to consider the general properties of peptide transport. Extensive reviews on the nature of peptide transport in bacteria have been published and the most recent (Payne and Gilvarg, 1978) can be recommended as a basis for understanding transport of the peptide antibiotics. Because of these extensive reviews it is unnecessary in this paper to dwell on peptide transport, so that only a brief summary is provided here. In general, two systems operate for transport: that coded by the locus opt (oligopeptide transport system) and that determined by dpt (dipeptide transport system). As noted by Payne and Gilvarg (1978) it is not known whether the opr system comprises one or several specific, genetically determined components. In E. coli, oligopeptides (which comprise three or more amino-acid residues) are transported virtually exclusively by the opt system, whereas dipeptides can utilize either opt or dpt. The opt and dpt loci are assumed to determine cytoplasmic membrane transport systems that mediate energy-dependent accumulation of the peptides. These transport systems are distinct from amino-acid carriers and deliver intact peptides to the cell interior where they are hydrolysed to free amino acids. The transport systems are sensitive to osmotic shock and have an obligatory requirement for phosphate-bond energy. Nevertheless, there is no direct evidence for the involvement of periplasmic binding proteins in transport. Little is known about transport across

Compound

Structure

Name and transport system

CH3 I O=P-OH

I

1

L-Phosphinothricylalanyl-alanine opt

CH

CH

c‘ II -61 C ‘ -N/H II

+H~N’

0

CH,

I

2

L-(N’-Phosphono)-

CH ‘COO

0

OH

I II

O= S = N - P -OH

I

methionine-(S)sulphoximinyl-alanylalanine opt

y

2

O

0

II I

HO-P-OH

3

Plumbemycin B opt

NH2

I

7H2

C=o

I

CH

II

YH3 CH YH2

CH

‘c-N/H‘c-NN/H‘coo-I1

+H~N’

I1 0

CH I CH

0

CH3

I

o=s =o I

4

L-Methionine-Sdioxidyl-alanyl-alanine opt

CH2

I

y

2

CH + H ~ N ‘c-~ 0 I‘

7H3 CH

&‘c-

11 0

y

3

CH ~H‘COO-

Compound

Name and transport system

Structure

0

II

/ \

H,C I H2C,

5

Bacil ysin opt dpt

HC, I 0

5c'

FH CH + H 3 6C'

CH

-N/H'COO-

II

0

y

6

3

y

3

CH CH + H 3 6 'C-N/H'PO,H

Alaphosphin opt dpt

II

0

H,C-NH

I

1

HO -C-C=O 7

I

(S)-Alanyl-3-[a-(S)chloro-3-(S)-hydroxy-20x0-3-azetidinylmethyl] -(S)-alanine opt dpt

CI -CH CH3 I CH

+H~N'

I y

2

CH

c' - N/H'COOII

0

8

1 -(S)-Hydroxy-2(S,S)valylamidocyclobutane1-acetic acid opt dpt

CH

I

CH

+H,N'

H,C-CH2

'c-tjfr II

I

I

CH-C-CH, I OH

COO-

0 FIG. 14. Peptide antibiotics and transport systems in Escherichia coli. Modified with permission from Diddens et al. (1976, 1979).

21 6

I . CHOPRA AND P. BALL

the outer membrane of Gram-negative bacteria, but hydrophilic peptides with molecular weights less than about 650 are assumed to diffuse through porins (Nikaido and Nakae, 1979). There is clear evidence for transport of peptide antibiotics by the opt and dpt systems in E. coli (Fig. 14). Of particular interest are the recent studies of Diddens et al. ( 1 976, 1979). These authors investigated transport of both tri- and dipeptide antibiotics. Several tripeptide antibiotics (structures 1-3, Fig. 14) were transported by the opt system. Evidence for this contention rests on the following: (i) opt mutants were resistant to the antibiotics; (ii) antagonism tests showed competitive reversal of the antibacterial action by peptides known to be accumulated via the opt system. However, antibiotic action was not compromised by compounds that used different transport systems. Antibiotics 1 and 2 are hydrolysed within the cell to produce compounds that inhibit glutamine synthetase (Diddens et al., 1976, 1979), whereas plumbemycin B (structure 3 in Fig. 14) is cleaved to produce an antagonist of L-threonine (Diddens et al., 1979). Another inhibitor of glutamine synthetase, L-methionine-S-dioxide (methionine sulfone) can be linked to the dipeptide L-alanyl-alanine to produce antibiotic 4 (Fig. 14). Whereas the free L-methionine-S-dioxide was transported by the methionine uptake system, the tripeptide (structure 4, Fig. 14) was transported by the opt system. Although these peptide antibiotics are too toxic for chemotherapy, the synthesis of compound 4 illustrates the principle of producing antibiotics that are capable of entering bacteria via more than one transport system. Dipeptide antibiotics (e.g. compounds 5-8 in Fig. 14) utilize both the opt and dpt systems. Evidence for this was obtained by experiments analogous to those performed with the tripeptide antibiotics. Bacilysin (structure 5 ) is cleaved within the cell to product anticapsin (the C-terminal amino acid of structure 5 ) and it is the latter that inhibits glucosamine synthetase (Kenig and Abraham, 1976; Kenig et al., 1976). The antibacterial activity of compounds 6 8 also probably depends on hydrolysis after transport into the cell. Compounds 6 and 7 produce inhibitors of glutamine synthetase whereas compound 8 may yield a product that interferes with metabolism involving sulphur-containing amino acids (see Diddens et a]., 1979).

XIII. Showdomycin Showdomycin [2-(P-~-ribofuranosyI)maleiimide]which is synthesized by Streptomyces showdoensis (Komatsu and Tanaka, 1972) is a structural analogue of uridine, in which the pyrimidine ring is replaced by a maleiimide moeity (Fig. 15). The antibiotic primarily inhibits nucleotide biosynthesis

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

HO

21 7

OH

FIG. 15. Structures of showdomycin (a) and uridine (b).

by blocking uridine monophosphate kinase (EC 2.7.4.4.) (Gale et at.. 1972). However, showdomycin also inhibits other enzymes, apparently due to reaction between the maleiimide moiety and thiol groups within enzymes (Gale et al., 1972). Since all the target enzymes are intracellular (Table 1 ) the drug must cross both outer and cytoplasmic membranes of bacteria.

A. D I F F U S I O N O F S H O W D O M Y C I N A C R O S S THE O U T E R MEMBRANE

Since nucleotides cross the outer membrane via protein ompF pores (van Alphen et al., 1978b), passage of structurally related compounds including showdomycin probably also involves passive diffusion through these pores.

B. T R A N S P O R T OF S H O W D O M Y C I N ACROSS THE C Y T O P L A S M I C MEMBRANE

Showdomycin is apparently accumulated across the cytoplasmic membrane by one of the transport systems involved in uptake of uridine and other nucleotides (Komatsu and Tanaka, 1972, 1973). However, interpretation of the data of Komatsu and Tanaka (1972, 1973) is complicated by the fact that nucleosides are metabolized either prior to, or immediately after, transport (Hays, 1978) whereas showdomycin is not modified. The use of cytoplasmic membrane vesicles to measure the effect of showdomycin on nucleoside accumulation (Komatsu and Tanaka, 1973) does not overcome this problem since some enzymes involved in nucleoside processing may be membrane-bound (Hays, 1978). However, selection of conditional mutants in enzymes involved in nucleoside processing (Hays, 1978) should permit more meaningful comparisons of showdomycin uptake and nucleoside transport.

SIDEROPHORES

NATURAL SIDEROMYCINS

Fer r i c r o c i n

Ferr ichrorne

Albornycin

6,*

Ferrioxamine 0

Ferrirnycin A,

Sulphanilamido

Sulphanilamido - nicotinic acid

SEMI -SYNTHETIC

f e r r i c r o c i n y l ester

SIDEROMYCINS

Sulphanilamida-

Sulphanilomido - carbonic

carbonyl ferrioxamine B

a c i d ferricracinyl ester

H,N

0

SO,-NH-CO-O-CHz-

- nicotinic

acid ferrioxamine B

H

,

-

N

~

NH

FIG. 16. Siderophores, natural and semi-synthetic sideromycins. *This structure is only tentative (see Hartmann et al., 1979). Structures reproduced with permission from Zahner et al. (1977).

220

I. CHOPRA AND P. BALL

XIV. Sideromycins The sideromycins, e.g. albomycin 6, and ferrimycin A, (Fig. 16), are high molecular-weight antibiotics produced by streptomycetes (Nuesch and Kniisel, 1967). Although these compounds are accumulated within the cytoplasm (and hence have to be transported across outer and cytoplasmic membranes), it is probable that they do not act directly to inhibit growth, but are hydrolysed within the cell to produce smaller growth-inhibitory moieties (Hartmann et al., 1979). Thus the remainder of the sideromycin molecule appears to serve as a vehicle to deliver the antibiotically active pendant group into the cell. The antibacterial spectrum of the sideromycins ranges from those with a broad spectrum (e.g. albomycin) to those with activity against Gram-positive bacteria alone (e.g. ferrimycin) (Niiesch and Kniisel, 1967). The size of these molecules precludes passive diffusion across outer and cytoplasmic membranes and it is not surprising to find that their transport depends on membrane proteins. The proteins utilized (see below) are those normally required for accumulation of siderophores (low molecular-weight iron chelators) to which sideromycins are structurally related (Fig. 16). The variation in antibacterial activity displayed by the sideromycins probably relates to differences in the type of siderophore utilized by a particular organism and hence to the nature of the membrane proteins available for sideromycin accumulation. To understand sideromycin transport it is necessary to consider iron transport itself. Although iron is a relatively abundant element, its availability for microbial growth is limited by its tendency to undergo hydration and precipitation at neutral pH value. To meet their requirement for this essential element, many micro-organisms synthesize low molecular-weight iron chelators (the siderophores) which are accumulated by cells in complex with Fe(II1) ion (Braun et al., 1976; Kadner and Bassford, 1978). Membrane receptors are required for the transport of iron-siderophore complexes into the cell, and some bacteria possess specific transport systems not only for their own siderophores but also for those produced by other micro-organisms (Luckey et al., 1972). The nature of iron transport in E. coli has been examined in detail. In this case, the siderophores include ferric enterochelin (the endogenous chelator), ferric citrate and ferrichrome. E. coli also has a low-affinity iron uptake system which is probably independent of chelators. Details of these systems are provided in several recent reviews on the topic of iron transport in bacteria (Lankford, 1973; Silver, 1978; Kadner and Bassford, 1978). The presence of an effective ferrichrome uptake system in E. coli is surprising since ferrichrome is only produced by certain fungi like

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

221

Ustilago sphaerogena (Braun ef ul., 1976). Nevertheless, albomycin utilizes the ferrichrome transport system in E. coli. Albomycin and ferrichrome transport in E. coli are thought to involve distinct outer and inner membrane components (Kadner and Bassford, 1978). The tonA mutation blocks albomycin and ferrichrome transport and simultaneously confers resistance to colicin M and bacteriophages (p80, T1 and T5 (reviewed by Braun et al., 1976). On the basis of these results, the tonA product was surmised to be in the outer membrane. This was verified by the purification of an 85,000-dalton outer membrane protein which could inactivate both phage T5 and colicin M and reversibly adsorb phages TI and (p80 (Braun et af.,1973; Hancock and Braun, 1976). The manner by which the tonA protein mediates albomycin and ferrichrome transport is not understood. The tonB mutation also results in the inability to transport albomycin and ferrichrome (as well as other iron chelates and vitamin B I Z )(Kadner and Bassford, 1978). The product of this gene has not been conclusively identified [see conflicting data for the molecular weight of the putative tonB product quoted by Postle and Reznikoff (1979) and Plastow and Holland (1979)], but several authors suggest that it is located in the outer membrane where it could receive albomycin and ferrichrome from the tonA protein (Frost and Rosenberg, 1975; Braun et a f . , 1976; Braun, 1978; Kadner and Bassford, 1978). Despite this hypothesis, the tonB product is involved in energy-dependent processes that utilize the proton motive gradient (Kadner and Bassford, 1978) thereby implicating its presence in the cytoplasmic membrane. An alternative theory would therefore propose that the tonB product serves as a transport protein for ferrichrome and albomycin (and other substrates) across the cytoplasmic membrane. Indeed, Wookey and Rosenberg (1978) concluded from experiments with sphaeroplasts of tonB mutants that the tonB product may function at the level of the cytoplasmic membrane. More recently, Weaver and Konisky (1980) challenged Wookey and Rosenberg’s conclusions and themselves indicate an outer membrane location for the tonB product. Weaver and Konisky (1980) also suggest that the tonB product may promote physical association between outer and cytoplasmic membranes which is presumed to be essential for delivery of albomycin and other substrates to the cytoplasmic membrane. Plastow and Holland (1979) concluded that the tonB product is located in the cytoplasmic membrane. Their data may not necessarily contradict the suggestions for an outer membrane location because Plastow and Holland (1979) fractionated inner and outer membrane polypeptides using the detergent Sarkosyl. Location of the presumed tonB product in the Sarkosyl-soluble fraction need not necessarily imply an inner membrane location because some outer membrane proteins can be extracted by Sarkosyl (Chopra and Shales, 1980). Energy-dependent accumulation of sideromycins against concentration

222

I. CHOPRA A N D P. BALL

gradients has also been reported in Gram-positive species such as Staphylococcus aureus and Bacillus subtilis (Kniisel and Zimmermann, 1975). Although this presumably indicates carrier-mediated transport across the cytoplasmic membrane, detailed knowledge of the nature of transport is lacking. Despite uncertainties regarding the precise basis of sideromycin transport their ability to be accumulated by bacteria has encouraged synthesis of semi-synthetic sideromycins (Zahner et al., 1977). A number of sulphonamide derivatives were produced, of which four were tested for antimicrobial activity. These four comprised sulphanilamidonicotinic acid ferricrocinyl ester, sulphanilamidonicotinic acid ferrioxamine B, sulphanilamidocarbonic acid ferricrocinyl ester and sulphanilamido-carbonyl ferrioxamine (Fig. 16). The first two derivatives (with 6-p-aminobenzene-sulphonamidonicotinic acid side chains) were active against Staph. aureus, but not B. subtilis or E. coli. The siderophores ferricrocin and ferrioxamine B (Fig. 16) competitively reversed the anti-staphylococcal activity of these two semi-synthetic sideromycins, thereby implying that the derivatives utilize normal siderophore transport functions. The action of these semi-synthetic sideromycins could also be prevented by p-aminobenzoic acid, the specific antagonist of sulphonamides. This indicates that the derivatives act in the cell as sulphonamides. The third and fourth compounds which have sulphonamide groups directly linked to the siderophore (Fig. 16) had no antimicrobial properties. Their inactivity might reflect failure to split the transport moiety from the reactive pendant group within the cell.

XV. Streptozotocin Streptozotocin (2-deoxy-2-(3-methyl-3-nitrosoureido)-~-glucopyranose) an N-methyl-N-nitrosourea derivative of N-acetyl-D-glucosamine (Fig. 17) is a broad-spectrum antibiotic produced by Streptomyces achromogenes (Reusser, 1971). The drug also exhibits antitumour, mutagenic and diabetogenic properties (Reusser, 1971). (a)

(b)

HOCH, HQH HO

NO I

H HN-CO-N-CH3

HOCH2 HO HQH H HNCOCH3

(C)

HOCHZ HO HQH H NH2

FIG. 17. Structure of streptozotocin (a), N-acetyl-D-glucosarnine (b) and glucosarnine (c).

D-

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

223

Streptozotocin induces rapid DNA degradation in Bacillus subtilis by causing breakage of DNA strands (Reusser, 1971). This may result from interactions between the drug and cytosine residues in the DNA (Reusser, 1971). To reach its target site within the cytoplasm (Table 1) streptozotocin must penetrate both outer and cytoplasmic membranes of bacteria.

A . DIFFUSION O F STREPTOZOTOCIN ACROSS THE OUTER MEMBRANE

Since streptozotocin is a low molecular-weight (Table 1) hydrophilic molecule (Ammer et al., 1979) it probably crosses the outer membrane by passive diffusion in the water-filled porin proteins (see Introduction). Streptozotocin is predominantly uncharged or weakly anionic at neutral pH values, therefore diffusion of the drug across the outer membrane is unlikely to be adversely affected by the Donnan equilibrium (see Introduction).

B. T R A N S P O R T O F S T R E P T O Z O T O C I N A C R O S S T H E C Y T O P L A S M I C MEMBRANE

Streptozotocin is accumulated across the cytoplasmic membrane by the phosphoenolpyruvate: phosphotransferase system (PTS) (Saier, 1977) (Fig. 18) normally responsible for N-acetyl-D-glucosamine uptake (Ammer et al., 1979). This transport system is encoded by gene nagE (Lengeler, 1980) which specifies the membrane-bound enzyme I1 complex responsible for N-acetylD-glucosamine uptake (enzyme IINag) (White, 1968; Lengeler, 1980). Thus, mutants defective in either the enzyme I or the HPr components of the PTS

In

I

lout

FIG. 18. Probable organization of the phosphoenolypyruvate: phosphotransferase system (PTS) involved in accumulation of N-acetyl-D-glucosarnifie, D-glucosamine, streptozotocin and norjirimycin. The substrate (S) is transported by enzyme I1 (11) which is located in the cytoplasmic membrane (Saier, 1977). The substrate is phosphorylated by enzyme I11 (111) or directly by the heat-stable protein (HPr) (Saier. 1977). HPr is phosphorylated by enzyme 1, which is phosphorylated at the expense of phosphoenolpyruvate (PEP).

I. CHOPRA AND P. BALL

224

(Fig. 18) (Ammer et al., 1979; Lengeler, 1980) or in enzyme IINag(Lengeler, 1980) (Fig. 18) are streptozotocin-resistant. Streptozotocin uptake is inhibited by N-acetyl-D-glucosamine, and, to a lesser extent, by D-glucose (Ammer et al., 1979). However, neither D-glucosamine, nor the non-PTS sugar lactose, inhibited streptozotocin accumulation (Ammer et al., 1979). Since D-glucosamine (Fig. 17) is a substrate for enzyme IINag (Lengeler, 1980) it should competitively inhibit streptozotocin uptake. The explanation for this lack of inhibition of streptozotocin uptake by D-glucosamine is unknown. Streptozotocin is phosphorylated during uptake (Ammer et al., 1979) as would be expected for a PTS substrate. Since mutants deficient in enzyme I or the HPr components of the PTS are streptozotocin-resistant (Ammer et al., 1979) phosphorylation of the drug almost certainly involves active group translocation (Saier, 1977) rather than exchange group translocation (Saier, 1977). The steady-state intracellular concentrations of both streptozotocin and N-acetyl-D-glucosaminewere about 70-fold greater than the extracellular concentrations of the two compounds (Ammer et al., 1979). Thus, both drug and normal substrate are accumulated to a similar extent by enzyme IINag. Both uptake of streptozotocin and accumulation of N-acetyl-Dglucosamine were lowered byp-chloromecuribenzoate, which inhibits enzyme I of the PTS (Ammer et al., 1979). However, whereas the respiratory chain inhibitor sodium cyanide (Pudeck and Bragg, 1975) and the uncoupler of the transmembrane protonmotive force (AjiH+)2,4-dinitrophenol (Hopfer et al., 1968) inhibited N-acetyl-D-glucosamine uptake by about SO%, they had no effect on streptozotocin accumulation (Ammer et al., 1979). Since reduction of AjiH by anaerobiosis and uncouplers stimulates accumulation of a methylglucoside by enzyme IIG" of the PTS (Saier and Moczydlowski, 1978), uptake of both N-acetyl-D-glucosamine and streptozotocin by enzyme IINag should be insensitive to both sodium cyanide and dinitrophenol. Possibly, reduction of N-acetyl-D-glucosamine uptake by sodium cyanide and dinitrophenol results from the presence of two transport systems for N-acetylD-glucosamine, one of which is part of the PTS and one of which is dependent on AjiH+. If so, streptozotocin would be accumulated by the former system, but not by the latter. Alternatively, inhibition of N-acetylD-glucosamine uptake by sodium cyanide and dinitrophenol under the conditions used by Ammer et al. (1979) might result from secondary effects of these compounds not associated with membrane de-energization. ~

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

225

XVI. Tetracyclines The tetracyclines (Fig. 19) are a group of broad-spectrum antibiotics most of which are synthesized by various Streptomyces species and some of which are produced semi-synthetically (Mitscher, 1978). Tetracyclines inhibit protein synthesis by blocking aminoacyl-tRNA binding to the bacterial ribosome (Gale et al., 1972). Since their target is intracellular (Table 1) the tetracyclines must cross both outer and cytoplasmic membranes of bacteria. Some aspects of tetracycline transport were considered in a previous review (Chopra and Howe, 1978) but, since then, further progress has been made in this area.

A. D I F F U S I O N O F T E T R A C Y C L I N E S A C R O S S T H E O U T E R MEMBRANE

Passage of tetracyclines across the outer membrane appears to occur predominantly by passive diffusion with no evidence for involvement of facilitated diffusion systems (Chopra and Howe, 1978). As discussed elsewhere in this review, two factors in particular affect the passive diffusion of molecules across the outer membrane: hydrophobicity and electrical charge. These factors are relevant to the tetracyclines because this group R' R2

R3 R 4

\

HO #@

Antibiotic Tetracycline Oxytetracycline Chlortetracycline Demethylchlortetracycline Methacycline Doxycycline Minocycline

OH

N(CH312

CONH2

\

0

OH

R' H H CI CI H H -N(CH3)2

0

R2 CH, CH, CH, H =CH2 CH, H

R3 OH OH OH OH H H

R4 H OH H H OH OH H

FIG. 19. Structure of tetracycline and some of its analogues. From Colaizzi and Klink ( 1969).

Copyright 1969, Journal of Pharmaceutical Sciences, American Pharmaceutical Association. Washington. D.C. All rights reserved. Reproduced with permission.

I. CHOPRA AND P. BALL

226

TABLE 3. Partition coefficients of some of the tetracyclines Those in chloroform/water were based on data quoted by Barza et a/. (1979) and those in octanol/phosphate buffer were from Colaizzi and Klink (1966) Partition coefficients Chloroform/water Octanol/phosphate buffer

Antibiotic Minocycline Doxycycline Tetracycline Oxytetracycline

30 0.48 0.09 0.007

1.48 0.92 0.05 0.09

FIG. 20. Dissociation schemes for (a) tetracycline hydrochloride, (b) oxytetracycline hydrochloride and (c) rninocycline hydrochloride. Based on Chopra and Howe (1978) and data of Barringer et al. ( 1 974).

of antibiotics comprises members which have different partition coefficients and which produce various ionic species (Table 3; Fig. 20).

1. Effect of Hydrophobicity on Passive Diffusion

Those tetracyclines that are used clinically (Fig. 19) have different partition coefficients, with being minocycline the least water soluble (Table 3). Indeed, since minocycline is much more hydrophobic than tetracycline it is surprising that comparable external concentrations of these two antibiotics achieve the

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

227

TABLE 4. Susceptibility of Escherichia coli and Bacillus subtilis to tetracyclines Concentrations of antibiotic (pg!ml f I standard deviation) required to cause a 50% decrease Organism Escherichia coli K-I2 JC3272 Bacillus subtilis NCIB10106

in growth rate: Tetracycline Minocycline

0.29 & 0.02" 0.35 f 0.0Ib

0.42 & 0.08" 0.025 f 0.005'

References: "from Shales rt al. (1980). bfrom Eccles et al. (1981). '1. Chopra. unpublished observations

same degree of growth inhibition in E. coli (Table 4). Although it is not yet possible to determine diffusion rates for tetracyclines across the outer membrane, comparison of minocycline susceptibility data for E. coli and B. subtilis (Table 4) implies that the outer membrane does constitute a penetration barrier towards minocycline. Thus the E. coli: B. subtilis inhibitory concentration ratio was 16.8 for minocycline, but only 0.8 for tetracycline. Furthermore, the ratio for tetracycline implies that it can readily cross the E. coli outer membrane. Since the growth of wild-type E. coli is impaired to the same extent at equivalent external concentrations of minocycline and tetracycline, then minocycline itself is likely to be a more active inhibitor of protein synthesis than tetracycline. This is supported by studies on the activities in vitro of tetracycline and minocycline (Tritton, 1977). The mutation in E. coli that leads to the synthesis of deep-rough lipopolysaccharide also results in increased sensitivity to minocycline (Ball et al., 1977). As noted in Section IV.B, this also indicates exclusion of minocycline by the outer membrane of wild-type E. coli. In contrast, tetracycline itself is both sufficiently hydrophilic and small enough to diffuse through pores formed by the ompF porin (Chopra and Eccles, 1978). As discussed in Section IV.B, Piovant et al. (1978) proposed that when hydrophobic antibiotics enter E. coli they d o so by diffusion through regions that connect the cytoplasmic and outer membranes. The preferential activity of hydrophobic inhibitors of protein synthesis against membrane-bound ribosomes (Hirashima et al., 1973; Piovant et al., 1978) is assumed by Piovant et al. (1978) to reflect the proximity ofjunction points to the sites of synthesis of envelope polypeptides. Diffusion of minocycline through apolar outer membrane regions could therefore locate the antibiotic at sites favourable for entry across the cytoplasmic membrane via junction points. Recently, Chopra and Shales (1981) tested this model for minocycline uptake by determining its activity against the synthesis of cytoplasmic and envelope proteins,

228

I . CHOPRA AND P. BALL

but found no evidence for preferential entry of minocycline through junction regions. 2. Effect of Charge on Diffusion of Tetracyclines

The tetracyclines are acids which can ionize in aqueous solution (Fig. 20; Table 5). At neutral pH the forms TH,, TH- and qH,, cpH- predominate for tetracycline and oxytetracyline (see Chopra and Howe, 1978), whereas for minocycline MH, is the major species (Barringer et al., 1974).The Donnan equilibrium across the outer membrane will not hinder diffusion of the uncharged species, and indeed the forms TH,, q H z and MH, probably enter Gram-negative bacteria (see Chopra and Howe, 1978). However, diffusion of the anionic species across the outer membrane is likely to be severely hindered. Binding of divalent cations by the TH- and q H - anions will lead to the formation of cationic chelates (Fig. 20) that should be able to cross the outer membrane in response to the Donnan equilibrium. Nevertheless, the sensitivity of a number of bacteria to tetracycline is decreased in the presence of increasing concentrations of magnesium (see Chopra and Howe, 1978). This effect may be due to the reduced solubility of tetracycline-metal chelates compared to the free antibiotics (Barringer et al., 1974). B. T R A N S P O R T O F T E T R A C Y C L I N E S ACROSS T H E C Y T O P L A S M I C MEMBRANE

Detailed studies have only been conducted on accumulation of tetracycline and chlortetracycline. Nevertheless, we refer to “tetracycline” accumulation throughout most of this Section. This is taken to include not only tetracycline and chlortetracycline, but also the majority of other tetracyclines, whose

TABLE 5. Ionization constants ( K , , K , ’ , K 2 , K 3 ) of tetracycline, oxytetracycline and minocycline hydrochlorides and stability constants K, of complexes with magnesium Data from Barringer et al. (1974) and Chopra and Howe (1978) Antibiotic

PK I

PK;

PK2

PK3

PKS

Tetracycline Oxytetracycline Minocycline

3.3 3.3 2.8

-

1.7 7.3 7.8

9.7 9.1 9.5

4.16-4.29 3.80-3.96 n.d.

-

5.0

n.d. indicates not determined, but minocycline does form magnesium chelates (Barringer et al., 1974).

TRANSPORT OF ANTIBIOTICS INTO BACTERIA

229

transport across the cytoplasmic membrane is probably identical with that of the other two drugs. Indeed, both chlortetracycline (Weckesser and Magnuson, 1979) and minocycline (Samra et al., 1979; P. R. Ball, unpublished observations) competitively inhibit tetracycline accumulation. Tetracycline accumulation has been measured using [ 7-3H]tetracycline (e.g. Dockter and Magnuson, 1975; Weckesser and Magnuson, 1976, 1979; McMurray and Levy, 1978; Fayolle et al., :980) and by spectrofluorimetry (Dockter and Magnuson, 1974, 1975; Weckesser and Magnuson. 1976, 1979; Dockter et al., 1978; Smith et al., 1981). Both methods are imperfect, because: (i) [ 7-3H]tetracycline decomposes by radioautolysis, and the degradation products readily bind to bacteria (Levy et al., 1977); (ii) spectrofluorimetry measures fluorescence enhancement resulting from entry of tetracycline into the bacteria cell (Dockter and Magnuson, 1974) and correlation of fluorescence with total accumulation may not always be possible because of variation of the quantum yield of accumulated tetracycline. Data demonstrating correlation between fluorescence and uptake of tetracycline (Dockter and Magnuson, 1975; Weckesser and Magnuson, 1976; Ball et al., 1980) indicate that quantum yield changes are normally inconsequential. Nevertheless, correlation between uptake and fluorescence should be established for each individual system studied. Tetracyclines are actively accumulated by bacteria (Franklin, 1973; Dockter and Magnuson, 1974; Weckesser and Magnuson, 1976, 1979; McMurray and Levy, 1978; Fayolle et al., 1980; Smith et al., 1981). This almost certainly represents transport of the drug across the cytoplasmic membrane, a contention supported by the following data. (a) Tetracycline accumulation is saturable (Dockter and Magnuson, 1974; Weckesser and Magnuson, 1976; Smith et al., 1981). These data indicate that the drug binds to a membrane-located carrier protein during uptake. The affinity of the putative carrier for tetracycline is apparently low, since the K, for drug uptake is higher than values quoted for amino-acid transport (Table 6). (b) Tetracycline uptake is sensitive to membrane fluidity (Dockter and Magnuson, 1974, 1975; Dockter et al., 1978). Thus, Arrhenius plots of I n initial rate of uptake versus absolute temperature show inflections, corresponding to melting of membrane lipid domains surrounding the carrier (Dockter and Magnuson, 1975; Dockter et al., 1978). (c) Tetracycline accumulation is energy-dependent (Franklin, 1973; Dockter and Magnuson, 1974; Weckesser and Magnuson, 1976, 1979; McMurray and Levy, 1978; Fayolle et al., 1980; Smith et al., 1981). (d) Tetracycline accumulation produces intracellular concentration gradients of about 500-fold in E. coli (P. R. Ball, unpublished observations). Three proposals have been made for the carrier involved in tetracycline accumulation by bacteria.

TABLE 6. K, values for tetracycline accumulation by Strapliylococcus aureus, Rhodopseudomonas sphaeroides, Escherichiu coli and Bacillus .subtilis. K , values for the amino acids are included for comparison KI(PM) Bacterium Staphylococcus uureus Rhodopseudomonas sphueroides Escherichia coli Bacillus subtilis

Measured using spectrofluorimetry I07 f 2W/254 300'/400 87 f 8 190 28

Amino acid Threonine Phenylalanine Lysine Chlortetracycline. Tetracycline. ' K, = 300 P M determined in osmotically shocked bacteria. a

32b

Klol M) 0.39 2 0.5

Measured using [7-3H]tetracycline

Reference

N.D. 540 N.D. N.D.

Dockter and Magnuson (1974) Weckesser and Magnuson (1976) Smith et al. (1981) S. J . Eccles (unpublished work)

Anraku ( 1 978)

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231

(i) Tetracycline is accumulated as a drug-ion chelate by magnesium transport systems (Franklin, 1973). However, mutants of E. cofi K12 defective in magnesium transport were as sensitive to the drug as their parental wildtype strain (Ball et al., 1977). Thus, tetracycline accumulation is unlikely to depend on functional magnesium transport per se. (ii) Tetracycline is accumulated by a dicarboxylate transport system (Plakunov, 1973). However, this proposal by Plakunov (1973) that tetracycline could be accumulated by dicarboxylate transport systems because of structural analogy with oxaloacetic acid is probably invalid, since oxaloacetic acid is not a substrate for the dicarboxylate transport system in E.coli (Kay and Kornberg, 1971). Furthermore, expression of this system is inducible (Lo, 1977), whereas tetracycline accumulation involves a system expressed constitutively. Preliminary experiments with mutants defective in the dicarboxylate transport system obtained from T. C. Y. Lo (see Lo, 1977) indicate that tetracycline accumulation does not involve this system (P. R. Ball, unpublished observations). (iii) Tetracycline accumulation could involve a glutamate transport system because there is weak structural analogy between the drug and glutamate (Mitscher, 1978). However, no results exist to support a role for amino-acid transport systems in tetracycline accumulation. As previously mentioned, tetracycline accumulation is energy-dependent. The mechanism of energy coupling in E. coli has been established using inhibitors and uncouplers, as well as in mutants defective in energy coupling. The findings (Smith et af., 1981) are as follows. (a) The uncouplers carbonylcyanidechlorophenylhydrazone and 2, 4-dinitrophenol (Hopfer et af., 1968) inhibit tetracycline accumulation at low concentrations (Smith et af., 1981). Inhibition of tetracycline uptake by these uncouplers apparently reflects dissipation of the transmembrane protonmotive force (AFH+) p e r se since both inhibitors block drug uptake immediately after their addition to bacteria (Smith et al., 1981). (b) Potassium cyanide, an inhibitor of terminal electron transfer by the respiratory chain (Pudeck and Bragg, 1979, inhibits tetracycline accumulation at concentrations of potassium cyanide that inhibit respiration (Smith et af., 1981). Since APH- is generated by respiration in aerobically grown E. cofi (Rosen and Kashket, 1978) inhibition of tetracycline accumulation by potassium cyanide presumably reflects reduction in AILH+(Smith et al., 1981). (c) Dicyclohexylcarbodiimide an inhibitor of the bacterial membranebound C a 2 + M g 2 +- ATPase (BF,Fo) (Harold et af., 1969) did not inhibit tetracycline accumulation (Smith et a/., I98 1). Under identical conditions, dicyclohexylcarbodiimide inhibited leucine uptake, which depends on ATP hydrolysis (Rosen and Kashket, 1978) but not proline accumulation, which is driven by the transmembrane electrical potential (A$ ) component of

232

I. CHOPRA AND P. BALL

AFH+(Hirata e t a / . , 1974; Smith et a/., 1981). Thus, tetracycline accumulation apparently does not depend on ATP hydrolysis. (d) An uncB mutant and its parental wild-type strain (Bragg and Hou, 1977) accumulated similar amounts of tetracycline (Smith et al., 1981). Proline accumulation, which is driven by the A$ component of A j i H . (Hirata et al., 1974) was also similar in the uncB mutant and its parent (Smith et al., 1981).These data support the contention (see point (c) above) that tetracycline accumulation does not depend on ATP hydrolysis (Smith et af., 1981). Tetracycline accumulation measured by fluorescence shows an apparent pH optimum between 6 and 7 (Table 7). Uptake of the drug could therefore depend on the transmembrane pH gradient (ApH) component of A & - . However, using an alternative method to study tetracycline accumulation we have recently found little difference in tetracycline accumulated by E. coli within the pH range 5.5-8.0 (Smith et al., 1981). These results are therefore inconsistent with a transport system dependent solely on the ApH component of A,&& (Rosen and Kashket, 1978). Since valinomycin and nigericin specifically collapse A$ and ApH respectively (Ramos et al., 1976) measurement of the relative contributions of A$ and ApH to tetracycline uptake should be possible. TABLE 7. Apparent pH optima for tetracycline accumulation measured by fluorescence in various bacteria Bacterium Staphylococcus aureus Rhodopseudomonas sphaeroides Escherichia coli K 12 Bacillus subtilis

pH optimum

Reference

6.0 7.0

Dockter and Magnuson (1974) Weckesser and Magnuson (1979) Smith et a/. (1981) P. R. Ball (unpublished work)

6.5 6.5

Since the ionizing pK of tetracycline is 7.7 (Table 5) the drug is probably accumulated as a neutral species (Fig. 20). However, since the interior of the bacterial cell is normally alkaline (Ramos et a/., 1976) accumulated tetracycline molecules are probably present as anions (Fig. 20). To maintain electroneutrality, the cell must accumulate two positive charges, i.e. two monovalent cations or one divalent cation. By analogy with other organic anions (Ramos et al., 1976) this might involve concomitant translocation of two protons per tetracycline molecule. However, no results are available for the proton stoicheiometry of tetracycline accumulation.

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XVII. Conclusions Accumulation of antibiotics by bacteria has, in the past, appeared to be a paradox because it was believed that few drugs could use transport systems normally involved in uptake of essential solutes. Thus Franklin (1973) comments that “there are exceptional cases where an antibiotic sufficiently resembles a normal nutrient of the cell so as to be absorbed by the nutrient transport system”, and Kadner (1978) states that “there is no evidence at present that antibiotics enter the cells by means of usual transport systems”. We are no longer faced with a paradox because many, if not all, hydrophilic antibiotics with intracellular targets will probably prove to be actively accumulated by solute transport systems. Several examples of antibiotics which behave in this manner have been described here and the situation is not only confined to antibiotics, because various heavy metal ions are also accumulated by systems that usually function to transport essential ions (Webb, 1970; Nelson and Kennedy, 1971, 1972; Park et al., 1976; Weiss et al., 1978; Willsky and Malamy, 1980). In the introduction to this article we made the claim that studies on antibiotic transport could be of chemotherapeutic value. Although the subject of antibiotic transport into bacteria is still in its infancy, we believe that encouraging developments have been made. Kahan et al. (1974) provide one indication of the value of studies on antibiotic transport. These authors demonstrated potentiation of fosfomycin activity in infected mice by coadministration of glucose 6-phosphate. This resulted in induction of uhpT in the bacteria within the animal and increased transport of drug into the micro-organisms. Another promising approach involves transport of antibiotics linked to natural substrates, so that the transport system is misused by the adduct. Examples of this strategy include the peptide derivatives (Section XII) and the semi-synthetic sideromycins (Section XIV). As noted by Diddens et af. (1976) the synthesis of such adducts could be beneficial for the following reasons: (i) poorly accumulated antibiotics might be actively transported as drug-nutrient complexes; (ii) addition of adducts sharing a common antibiotic, but different linking molecules, might allow simultaneous entry of the drug on different transport systems; (iii) plasmid-mediated antibiotic resistance involving decreased antibiotic accumulation (e.g. tetracyclineBall et al., 1980; Shales et al., 1980) might be overcome by administration of adducts. Although some adducts may hnve chemotherapeutic potential, other hybrid molecules may prove ineffective because they will become too bulky for transport. This may be particularly critical for Gram-negative bacteria

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where the exclusion limit for passive diffusion through outer membrane porins is usually a b o u t 600 daltons (Nikaido and Nakae, 1979).

XVIII. Acknowledgements W e thank Stuart Shales for invaluable help with artwork, Betty Fry for photographic assistance and Sheila Travers for secretarial assistance. W e also thank several colleagues for the provision of d a t a before publication. M u c h of our original research work has been supported by project grants (G975/ lOOC, G978/67/SB, G978/834/S and G979/642/SB) from the Medical Research Council t o I.C. REFERENCES

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Rosen, B. P. and Kashket, E. R. (1978). In “Bacterial Transport” (B. P. Rosen, ed.), p. 559. Marcel Dekker, New York and Basel. Rottem, S. (1978). Federation of European Biochemical Societies Letters 95, 121. Saier, M. H. (1977). Bacteriological Reviews 41, 856. Saier, M. H. and Moczydlowski, E. G. (1978). In “Bacterial Transport” (B. P. Rosen, ed.), p. 104. Marcel Dekker, New York and Basel. Salton, M. R. J. (1978). Symposium of the Society for General Microbiology 28, 201. Salton, M. R. J. and Owen, P. (1976). Annual Review of Microbiology, 30,451. Samra, Z., Krausz-Steinmetz, J. and Sompolinsky, D. (1979). Microbios 21, 7. Sawai, T., Matsuba, K., Tamura, A. and Yamagishi, S. (1979).Journal of Antibiotics32, 59. Scherrer, R. and Gerhardt, P. (1971). Journal of Bacteriology 107, 718. Selwyn, S., Lacey, R. W. and Bakhtiar, M. (1980). “The Beta-Lactam Antibiotics” (S. Selwyn, ed.), Hodder and Stoughton, London. Shales, S. W., Chopra, I. and Ball, P. R. (1980). Journal of General Microbiology 121, 221. Silhavy, T. J., Ferenci, T. and Boos, W. (1978). In “Bacterial Transport” (B. P. Rosen, ed.), p. 127. Marcel Dekker, New York and Basel. Silver, S. (1978). In “Bacterial Transport” (B. P. Rosen, ed.), p. 221. Marcel Dekker, New York, Basel. Simoni, R. D. and Postma, P. W. (1975). Annual Review of Biochemistry 43, 523. Smith, M. C. M., Ball, P. R. and Chopra, I. (1981). JournalofBacteriology, submitted. Stock, J. B., Rauch, B. and Roseman, S. (1977). Journal of Biological Chemistry 252, 7850. Sutherland, I. W. (1972). Advances in Microbial Physiology 8, 143. Sutherland, I. W. (1979). In “Microbial Polysaccharides and Polysaccharases” (R. C. W. Berkeley, G. W. Gooday and D. C. Ellwood, eds.), p. 1. Academic Press, London. Tanaka, T. (1975). In “Action of Antimicrobial and Antitumour Agents” (J. W. Corcoran and F. E. Hahn, eds.), p. 340. Springer-Verlag, Heidelberg, New York. Tang, C. T., Engel, R. and Tropp, B. E. (1979). Biochimica et Biophysica Acta 572, 472. Tomasz, A. (1979). Annual Review of Microbiology 33, 113. Tritton, T. R. (1977). Biochemistry 16, 4133. Tsuruoka, T., Miyata, A. and Yamada, Y. (1978). Journal of Antibiotics 31, 192. Tyach, R. J., Engel, R. and Tropp, B. E. (1976). Journal qf Biological Chemistry 251, 6717. Van Alphen, W., Van Boxtel, R., Van Selm, N. and Lugtenberg, B. (l978a). Federation of European Microbiological Societies Microbiology Letters 3, 103. Van Alphen, W., Van Selm, N. and Lugtenberg, B. (1978b). Molecular and General Genetics 159, 75. Wargel, R. J., Shadur, C. A. and Neuhaus, F. C. (1971). Journal of Bacteriology 105. 1028. Weaver, C. A. and Konisky, J. (1980). Journal of Bacteriology 143, 1513. Webb, M. (1970). Biochimica et Biophysica Acta 222, 428. Weckesser, J. and Magnuson, J. A. (1976). Journal of Supramolecular Structure 4, 515.

Weckesser, J. and Magnuson, J. A. (1979). Journal of Bacteriology 138, 678. Weiss, A. A., Silver, S. and Kinscherf, T. G. (1978). Antimicrobial Agents and Chemotherapy 14, 856. West, I. C. (1980). Biochimica et Biophysica Acta 604, 91. White, R. J. (1968). Biochemical Journal 106, 847.

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Willsky, G. R. and Malamy, M. H. (1980). Journal of Bacteriology 144, 366. Wilson, D. B. and Smith, J. B. (1978). In “Bacterial Transport” (B. P. Rosen, ed.), p. 495. Marcel Dekker, New York and Basel. Wookey, P. and Rosenberg, H. (1978). Journal of Bacteriology 133, 661. Zahner, H., Diddens, H., Keller-Schierlein, W. and Nageli, H-U. (1977). Journal of Antibiotics 30,5. Zimmermann, W. (1980). Antimicrobial Agents and Chemotherapy 18, 94. Zimmermann, W. and Rosselet, A. (1977). Antimicrobial Agents and Chemotherapy 12, 368.

Note Added in Proof Since the main part of this article was prepared, many additional relevant publications have come to our notice. A review on aminoglycoside transport has been published by Hancock (1981), and this author has also discussed antibiotic transport across the outer membrane of Pseudomonas aeruginosa (Benz and Hancock, 1981). Further evidence that some b-lactams diffuse across the Gram-negative outer membrane via porins has been obtained by Harder et af.(198 1). Ringrose (1980) has reviewed aspects of peptide antibiotic transport, and further experimental studies in this area have been reported by Perry (1981). Additional studies on ferrichrome transport (relevant to sideroymycin accumulation) in E. cofi have also been published (Schneider et af., 1981; Wookey et af., 1981). Further experiments on the transport of tetracycline into E. cofi have also been reported McMurry et a f .(1981), which suggest that the proton-motive force drives the active transport of tetracycline. Work from this laboratory on the composition of outer membranes from E. cofihas also recently been completed (Shales and Chopra, 1982).Our results show that it may be necessary to re-consider some aspects of the theory of Nikaido and colleagues regarding the molecular basis by which hydrophobic antibiotics are prevented from crossing the Gram-negative outer membrane. REFERENCES

Benz, R. and Hancock, R. E. W. (1981). Biochimica et Biophysica Acta 646, 298. Hancock, R. E. W. (1981). Journal of Antimicrobial Therapy 8, 249. Harder, K. J., Nikaido, H. and Matsuhashi, M. (1981). Antimicrobial Agents and Chemotherapy 20, 549. McMurry, L., Cullinane, J. C., Petrucci, R. E. and Levy, S . B. (1981). Antimicrobial Agents and Chemotherapy 20, 307. Ringrose, P. S . (1980). In “Micro-organisms and Nitrogen Sources” (J. W. Payne, ed.), p. 641. John Wiley and Sons, London. Perry, D. (1981). Journal of General Microbiology 123, 145. Schneider, R., Hartmann, A. and Braun, V. (1981). Federation of European Microbiological Societies Microbiology Letters 11, 115. Shales, S . W. and Chopra, I. (1982). Journal of Antimicrobial Chemotherapy (in press). Wookey, P. J., Hussein, S. and Braun, V. (1981). Journalof Bacteriology 146, 1158.

Author Index A

B

Abd-El-Al., A. T., 15, 178 Abe, J., 112, 138, 144 Abe, N., 160, 173, 179 Abraham, E. P., 186,216,237 Abrahams, A,, 231, 236 Ahmad, F., 175, 179 Ahmad, M. H., 198,234 Aida, K., 175, 180 Albersheim. P., 1 1 I , 114, 147, 149 Alcorn, M. E., 50, 51, 52, 65,69 Allen, K. E., I I , 12, 69 Alsbach, E. J. J., 14, 76 Altendorf, K., 196, 204, 208, 232, 234, 23 7 Amemura,A., 82, 112, 121, 138,142,144 Ames, B. N., 220, 238 Ammer, J., 213, 223, 224, 234 Anand, N., 195,234 Anderson, R. F., 88,91, 142 Anderson, R. G., 121, 142 Andreesen, M., 91, 95, 142 Andrews, K. J., 205, 207, 238 Ankel, H., 106, 146 Anraku, Y . . 201, 203, 208, 230, 234. 235 Anwar, R. A., 140,144 Apperson, A., 184, 235 Argast, M., 205, 206,207,234 Armitage, K., 195, 234 Armstrong, W. McD., 16, 69 Arnett, A. T., 115, 142 Arnott, S., 129, 146 Asensio, J., 14, 76 Atkins, E. D. T., 188,234 Austin, M., 91, 145 Austrian, R., 109, 142 Avigad, G., 55,69 Avni, H., 105, 110, 11 I , 142, 144 Azuma, I., 174, 179

Baarda, J. R., 231,236 Babcock,G. E., 88,91, 132, 143, 147 Bachmann, B. J . , 207,234 Backen, K., 31, 33, 71 Bacon, J . S. D., 152, 153, 155, 156. 162, 178 Baddiley, J., 121, 124, 142, 144 Bagley, E. B., 92. 147 Baguley, B. C.. 167, 169, 178 Baine, H., 86, 142 Baird, J . K., 116. 118, 142 Bakhtiar, M., 212, 239 Bakker, E. P., 196,234 Balint, S., 168, 178 Ball, P. R., 192, 227, 229, 230. 231, 232, 233,234,235,236,239 Ballou, C. E., 152, 178 Barlow, A. J. E., 164. 178 Barnett, J . A,, 3, 46, 47, 53. 69, 77 Baron, C., 231,236 Barringer, W. C . , 187, 226, 228. 234 Bartnicki-Garcia, S., 177, 181 Barts, P. W. J. A., 15. 16, 69 Barza, M., 226, 234 Bassford, P. J., 220, 221, 237 Bauer, S., 166, 167, 168, 170. 172, 173, 176, 177, 178, 180 Bayer, M., 122, 124, 127, 128, 142 Beary, M . E., 16, 70 Bechet, J., 23, 72 Becker, J. M., 41, 74 Becker, J . U . , 176, 178 Beckmann, I., 123,142 Bell, D. J.. 154, 155, 178 Belsky, M. M., 61, 69 Beneviste, R. E., 191, 198, 235 Benitez, T., 155, 180 Benz, R., 207,234 Berg, P. E., 105, 110, 1 1 I , 142. 144 Berge, A. M. A., Sen, 53, 69 Berkeley, R. C. W., 105, I 18, 143

242

AUTHOR INDEX

Bernheimer, H. P., 109, 120, 142, 147 Bhuyan, B. K., 187,234 Biely,P., 166, 167, 170, 172, 173, 176, 177, 178, 180

Bigger, L. C., 31, 70 Bishop,C.T., 162, 163, 164, 178, 181 Bjorndal, H., 156, 180 Blacklow, R. S., 119, 142 Blank, F., 162, 163, 164, 178, 181 Blasco, F., 1 I , 12, 61,69, 76 Bock, A., 198,234 Boehler-Kohler, B. A,, 207, 234 Boehm, C., 12, 14,40,45, 71 Boller, J., 27, 71 Boller, T., 18, 30,69, 71 Bonsall, V. E., 169, 178 Booij, H. L., 43, 78 Boos, W., 204, 205, 206, 207, 208, 234, 239

Booth, I. R., 65, 66,69 Bordes, A. M., 20, 21,23, 24, 78 Borer, R., 175, 179 Borst-Pauwels, G. W. F. H., 13, 14, 15, 16, 17, 35, 60, 61, 69, 70, 72, 75, 76, 78

Boutry, M., 13, 14, 16, 17, 36, 69, 71 Bower, B., 167, 179 Bowman, B. J., 1 I , 12,69 Braatz, J. A,, 166, 167, 170, 181 Bracha, R., 123, 142 Brady, T. G., 12, 70 Bragg, P. D., 197,224,23 I , 232,234,235, 238

Brandriss, M . C., 21, 23, 69 Braun, C. B., 198, 199,200,237 Braun, V., 187, 188, 191, 220, 221, 234, 235, 236

Bray, D., 126, 147 Brennenstuhl, M., 213, 223, 224, 234 Bretscher, A. P., 84, 142 Brey, R. N., 12, 14,69 Britton, H. G., 39, 69 Brocklehurst, R., 34,35,43,48,49,50,53, 54, 5 5 6 9 , 71

Brooker, B. E., 97, 142 Brown, A. M., 132,142 Brown, M. R. W., 189, 193,235 Brown, R. B., 226,234 Brown, R. M., 128, 142 Bryan, L. E., 195, 196, 197, 198, 199, 235

Bryan-Jones, D. G., 197,235

Buchanan, C. E., 110, 1 1 1 , 142, 145 Bucke, C. W., 137, 146 Budd, J. A., 172, 173, 178 Burchard, W., 132, 148 Burckhardt, G., 32,69 Burdett, I. D. J., 191, 239 Burger, M., 54,69 Burgess, J., 169, 178 Burton, K. A., 87,91, 129, 138, 142 Bush, D. A., 162, 178 Bussey, H., 26, 41, 69 Button, D. K., 61,69 Byrne, M . J., 176, 178

C Cabib,E., 166, 167, 170, 171, 17Y, 181 Cacciapuoti, A. F., 188, 238 Cadmus, M . C., 87,88,91, 129, 138, 142 Campbell, B. D., 195, 196, 197, 198, 199, 235

Carbonell, L. M., 174, 178, 179 Carlsson, J., 106, 142 Carlson, R. W., 11I , 147 Carminatt, H., 125, 145 Carr, G., 14, 18, 24, 26, 33, 37, 38, 48, 77

Carton, E., 12, 14, 70 Cassidy, P. J., 186, 205, 206, 208, 233, 23 7

Cassone, A,, 165, 178 Catley, B. J., 88, 89, 142 Caulfield, M . P., 105, 118, 143 Chai, T-J., 194, 201, 236 Challinor, S. W., 175, 178, 181 Chambert, R., 118, 146 Chan, P. Y., 23, 70 Charalampous, F., 175, 179 Charles, M., 87, 143 Chattaway, F. W., 164, 178 Chene, L., 114, 128, 143 Cheng, K.J., 191, 192, 193, 235 Cherniak, R., 86, 142 Chester, V. E., 176, 178 Cheung, L., 89, 143 Chevallier, M. R., 23, 24, 29, 39, 41, 65, 66, 70, 73, 74, 75

Childs, G., 190, 227, 237 Childs, R. E., 67, 72 Chluzdinski. A. M., 117, 143

AUTHOR INDEX

Cho, Y., 85, 148 Chojnacki, T., 107, 145 Chopra, I., 190, 192, 200, 221, 225, 226, 227,228,229,230,23I , 232,233,234, 235,236,239 Christensen, H. N., 4, 76 Christensen, M. S., 40, 42, 44, 45, 70, 74 Christenson, J. G., 107, 143 Chung, C. W., 166,178 Churms, S. C., 132,143, 144 Cifonelli, J. A,, 135, 143 Cirillo, V. P., 17, 18, 24,40,42,44,45,46, 70, 73, 74, 78 Clark, W. H., 89, 143 Cluskey, J . E., 91, 145 Cockburn, M., 34, 35, 60, 61, 70 Cohen, G. N., 20, 29, 72, 73 Colaizzi, J. L., 225, 226, 235 Coleman, W. G., 125, 143 Collins, F. M., 133, 143 Collins, S. H., 196, 203, 235 Conway, E. J., 3, 12, 14, 15, 16, 31, 70 Colvin, J. R., 100, 114, 128, 143 Cooney, C. L., 106, 116, 146 Cooper, F. P., 162, 163, 164, 181 Cooper, T. G., 23, 25, 27, 28, 29, 38, 65, 70, 77, 78 Cope, J . E., 165, 178, 192, 235 Corpe, W. A., 86, 143 Cossins, E. A,, 23, 70 Costerton, J . W., 191, 192, 193, 235 Couperwhite, I., 88, 143 Cowie, D. B., 18, 30, 70, 72 Cox, G. B., 184,236 Crabeel, M . , 21, 22, 23, 24, 26, 70, 72 Crane, R. K., 7, 68, 70 Creanor, J., 53, 75 Cressnell, R. C., 61, 75 Cuesta Casada, M. C., 33, 75 Cummins, J. E., 22, 24, 25, 70 Cundliffe, E., 184, 186, 187, 194,201,217, 225,236 Cunningham, W. L., 154, 178 Cuppoletti, J., 5, 6, 25, 60, 70 Curran, P. F., 58, 77

D Dame, J. B., 12, 70 Damper, P. D., 196,235

243

Dankert, M., 99, 100, 101, 122, 126, 144, 146, 147 Daoust, V., 114, 143 Darte, C., 21, 23, 70 Darvill, A. G., 114, 149 Davidson, I. W., 90,94,95, 134, 137, 138, 143 Davidson, R., 123, 142 Davies, B. D., 195, 234, 236 Davies, D. D., 20, 76 Davies, J. E., 191, 198, 235 Davies, R., 31, 70 Davis, B. D., 197, 208, 235 Davis, J . B., 85, 146 Davis, R. H., 18, 77 Dawson, E. C., 44, 78 Dawson, P. S. S. 171, 179 Deak, T., 35, 52, 70 Deavin, L., 86, 87, 90, 129, 143, 145 De Bongioanni, L. C., 33, 36, 75 Decker, M., 57, 58, 70, 71 Dedonder, R., 118, 143 Dekker, R. F. H., 100, 114, 147 De La Fuente, G., 42, 44, 45, 46, 53, 70, 72, 77 Delhez, J., 13, 70 Demarco, P. V., 188, 209, 235 Derks, W. J. G., 16, 70 De Robichon-Szulmajster, H., 20, 21, 23, 24, 3 1, 77, 78 Diamond, J. M., 3, 70 Dickie, P., 198, 199, 235 Dicks, J . W., 86, 143 Diddens, H., 186,213,215,216, 222, 233, 235,240 Dieterle, R., 207, 234 Dietz, G. W., 208, 235 Dills, S. S., 184, 235 Din, G. A., 60, 74 Dintzis, F. R., 132, 143 Di Rienzo, J., 188, 235 Dixon, B., 175, 179 Docherty, A., 227,236 Dockter, M . E., 229,232, 235, 236 Dogerloh, M., 213, 215, 216, 235 Dominguez, A., 175, 179 Dorando, F. C., 68, 70 Dorfman, A., 103, 120,147 Douglas, L. J., 176, 181 Downey, M., 3, 12, 70 Downie, J. A,, 184, 236 Dubbelman, T. M. A. R., 46, 70

244

AUTHOR INDEX

Dubin, D. T., 195, 236 Dubois, E., 21, 70 Dudman, W. F., 86,99, 132, 143 Dufour, J. P., 13, 14, 36, 70, 71 Duggan, F., 14, 31, 70 Duguid, J. P., 89, 123, 143, 148 Dulbecco, R., 197, 235 Dunker, S. S., 61,69 Dunlop, P., 15, 22, 23, 76 Dunlop, R. C., 19, 23, 71 Dunn, E., 21, 75 Dunwell, J. L., 175, 179 Duran, A,, 167, 179 Diirr, M., 18, 27, 30,69, 71, 78 Dutton, G. G. S., 132, 136, 142, 144, 148

F Faber, V., 134, 144 Fareed, V. S., 139, 144 Farkis, V., 166, 168, 170, 176, 177, 178, 179, 181 Farmer,V.C., 153, 155, 156, 162, I78 Fayolle, F., 229, 236 Fenzl, F., 57, 71 Ferenci, T., 205, 206, 207, 208, 239 Fiechter, A., 172, I80 Fiedler, F., 101, 144 Fiedler, H-P., 187, 220, 236 Finn, R. K., 85, 87, 148 Fisher, B. E., 91, 145 Fleet,G. H., 152, 153, 154, 158, 159, 179, 181

E Earnshaw, P., 34, 35, 48,60, 61, 70, 71 Eaton, N. R., 48, 54, 73, 78 Eccles, S. J., 200, 227, 231, 234,235, 236 Eddy, A. A., 7, 9, 12, 14, 15, 18, 21, 23, 24, 26, 28, 31, 32, 33, 34, 35, 36, 37, 38, 43, 47, 48, 49, 50, 54, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 69, 70, 71, 72, 75, 76, 77, 160, 174, 179 Edmunds, P. N., 123, 148 Edwards, J. R., 97, 147 Edwards, T. E., 155, 180 Eilers-Konig, C., 176, 178 Elander, B., 106, 142 Elloway, H. F., 188, 234 Ellwood, D. C., 83, 84, 86, 91, 92, 93,95, 96, 116, 118, 134, 142, 144, 147 Elorza, M. V., 167, 168, 176, 179, 181 Elsasser-Beile, V., 132, 148 Elsen, H. N., 197,235 Engel, R., 186, 203, 204, 237, 239 Enns, L. H., 12, 76 Epstein, W., 196, 235 Essenberg, R. C., 208,236 Evans, C. G. T., 91, 92, 93, 95, 96, 134, 144 Evans, H. L., 19, 23, 71 Evans, L. G., 138, 145 Evans, J. M., 155, 180 Evans, L. R., 138, 144 Evans, R. B., 158, 179 Even, H. L., 15, 22,23, 76

Flemming, H. C., 102, 125, 144 Flores-Carreon, E., 47, 71 Folkes, J. P., 31, 70 Folkman, T. E., 132, 143 Forbes, E., 21, 75 Foret, M., 35, 40, 71, 75 Foulds, J., 194, 201, 236 Foury, F., 13, 14, 16, 17, 36, 40, 69, 71 Fox, C. F., 192, 198,236,238 Franklin, T. J., 184, 190, 193, 229, 231, 233,236 Fransson, L-A,, 134, 145 Freese, E., 61, 78 Frerman, F. E., 99, 100, 113, 120, 136, 148 Fritsch, A., 118, 143 Frost, G. E., 221,236 Frumkin, S., 208, 238 Fuhrmann, G-F., 12, 13, 14, 16, 17, 40, 41, 45, 71 Fukui, L., 117, 144 Fukui, S., 85, 148 Fukui, Y., 117, 144 Funahara, Y . , 188,236 Fyfe, J., 11 1, 144

b

Gale, E. F., 31, 70, 165, 178, 179, 184, 185, 186, 187, 194,201,217,225,236 Gamble, C., 226, 234 Gander, J. E., 84, 148 Garcia, R. C., 100, 101, 144

AUTHOR INDEX

Gardner, D., 14, 15, 35,36, 37, 38,43,48, 49, 50, 54, 55, 60, 61, 64, 65, 66, 67, 69, 71 Gardner, P. E., 163, 164, 178 Garegg, P. J., 99, 135, 140, 144 Gasdorf, H., 91, 142 Gayda, R. C., 105, 110, 111, 142, 144 Geck, P., 7, 72 Gennis, R. B., 121, 144 Georgopapadakou, N. H., 186,236 Gerhardt, P., 192,239 Germaine, G. R., 117, 143 Ghosh, A,, 175, 179 Ghuysen, J . M., 191, 202, 236 Gibb, L. E., 32, 71, 76 Gibbons, R. J., 97, 144 Gibson, F., 184,236 Gil, F., 174, 178, 179 Gilbert, P., 189, 193, 235 Gill, C. O., 46, 47, 71 Gilman, S., 196, 236 Gilvarg, C., 213, 238 Ginsberg, H. S., 197,235 Ginsburg, V., 98, 144 Gits, J. J., 23, 71 Glasby, J. S., 200, 236 Glaser, L., 101, 144 Goffeau, A,, 13, 14, 16, 17, 36,40,69, 70, 71 Gohring, W., 186, 213, 215, 216, 233,235 Goldemberg, S. H., 125, 145 Goldman, R. C., 107, 133, 144 Goldstein, S., 61, 69 Golecki, J. R., 192, 238 Goodman, R. N., 124,146 Gorski, M., 23, 28, 38, 77 Gotschlich, E. C., 105, 124, 146 Govan, J. R. W., 1 1 I , 144, 188, 236 Gradman, D., 7, 9, 10, 71, 75 Grant, W. D., 98, 110, 144 Greener, A,, 50, 73 Grenson, M., 21, 22, 23, 24, 26, 70, 71, 72, 73, 75 Griffin, C. C., 50, 51, 52, 65,69 Groseclose, R., 39, 68, 72 Gross, S. K., 107, 143 Griineberg, A., 35, 58, 59, 63, 72 Gruner, J., 167, 169, 178 Guggenheim, B., 106, 117, 144 Gunetileke, K. G., 140, 144 Gunja-Smith, Z., 177, 179 Gustafsson, P., 192, 236

245

H Haass, D., 57, 58, 62, 65, 71. 73 Hacking, C., 14, 15, 35, 36, 37, 38, 60. 61, 64, 65. 66, 67, 69, 71, 72 Haddock, B. A , , 197,236 Hagler, A. N., 30, 72 Hakomori, S., 154, 179 Halvorson, H. O., 18, 20, 48, 53, 54, 55, 65, 72, 75 Hamilton, W. A., 7, 65, 66, 69, 72, 196, 203,235 Hammerman, M. R., 32, 77 Hancock, I., 124,144 Hancock, R., 195,236 Hancock, R. E. W., 195, 220, 221, 235. 236 Hansen, U-P., 7, 9, 10, 56, 66, 71, 72, 77 Hantke, K., 191, 220, 221, 234, 235 Harada,T., 82,85,112, 121,138,142,144 Harold, F. M., 7, 72, 184, 196, 231, 232, 236,237 Harris, G., 48, 72 Hartig-Beecken, I., 204, 208, 234 Hartman, K., 220, 221,235 Hartmann, A., 187, 220, 236 Harvey, R. J., 200, 201, 236 Hasenclever, H. F., 162, 163, 164, 181 Haskovec, C., 46, 72 Hassid, W. Z . , 1 1 8, 143 Hauer, R., 50, 51, 72 Haug, A,, 83, 88, 98, 99, 134, 144 Hawthorne, D. C., 53, 72 Hayano, K., 167,180 Hayashi, S., 204, 208, 236 Hayashibe, M., 160, 173, 179 Haynes, W. C., 91,145 Hays, J. B., 217, 237 Heath, E. C., 99, 100, 113, 120, 136, 148 Hedenstrom, M., von, 53, 73 Heinz, E., 6, 7, 68, 72 Hejmova, L., 54, 69 Heller, K. B., 207, 237 Hemming, F. W., 106, 144 Hennaut, C., 21,23, 72 Henney, H. R., 89,143 Hensley, D. E., 88, 91, 147 Heppel, L. A., 191, 208, 235, 237, 238 Heredia, C. F., 42, 45, 72 Higashi, Y., 107, 144, 148 Higuchi, T., 99, 148 Himmelweit, F., 184, 237

246

AUTHOR INDEX

Hirashima, A,, 190, 227, 237 Hirata, H., 196, 232, 237 Hirota, T., 139, 14Y Hisamatsu, M., 82, 112, 138, 142, 144 Hoeberichts, J. A,, 14, 15, 16, 6Y, 72 Hofer, M., 35, 47, 50, 51, 52, 65, 72, 73, 74

Hoffman, E. K., 61, 75 Holland, I. B., 185, 221, 237, 238 Holloway, B. W., 196, 197, 198, 199,234 Holtje, J-V., 194, 195, 198, 199, 237 Holman, G . D., 5, 72 Holme, T., 135, 140, 144 Holmes, M. R., 164, 178 Holzer, H., 45, 48, 56, 72 Holzman, M., 42, 77 Holzwarth, G., 129, 132, 137, 145 Hook, M., 134, 145 Hopfer, U., 39, 68, 72 Hopfer, V., 198, 224, 231, 237 Hopwood, D. A,, 185,237 Horak, J., 41, 46, 72, 75 Horecker, B. L., 99, 148 Horisberger, M., 162. I 7 8 Horman, I., 162, 178 Horne, R. W., 153, 180 Hou, C., 21, 22, 23, 72, 232, 235 Hou, C. T., 85, 145 Hough, J. S., 173, 180 Housset, P., 22, 25, 72 Howe, T. G . B., 190, 192,225,226,228, 235 Huang, S., 115, 145

Huber-Walchli, V., 27. 28, 72 Hulsebos, T. J. M., 14, 72 Hunter, D. R., 25, 72 Hurwitz, C., 198, 199, 200, 237 Hussey, H., 121, 142

J Jackson, R. W., 91, 142 Janczura, E., 107, 145 Janda, S., 50, 51, 53, 73 Jann, B., 112, 121, 132, 146, 148 Jann, K., 101, 102, 112, 121, 125, 132, 144, 145, 146, 148

Jansson, P. E., 82, 113, 137, 145 Jarman, T. R., 84, 86, 87, 89, 90, 91, 93, 129, 143, 145, 146

Jarvis, A. W., 196, 203, 235 Jaspers, C., 20, 75 Jaspers, H. T. A., 43,46,47, 54, 55, 73 Jauniaux, J-C., 18, 73 Jaynes, P. K., 56, 73 Jeanes, A., 91, 129, 138, 142, 145 Johnson, A. M., 165, 179 Johnson, B. F., 167, 176, 179 Johnson, J., 155, 156, 180 Johnson, J. G., 102, 114, 145 Joiris, C. R., 23, 73 Jones, C. W., 197,236 Jones, D., 153, 162, 178 Jones, G . E., 19, 75 Jones, M., 17, 73 Jorgensen, C. B., 61, 75 Joyeux, Y., 118, 143 Jozon, E., 118, 143 Jund, R., 23, 24, 29, 39, 65, 66, 70, 73, 74

Jung,G., 186,213,215,216,233,235

K Kaback, H. R., 232, 238 Kadner, R. J., 184, 195, 196, 197, 198, 199,220,221,233,235,237

I Indge, K. J., 12, 17, 21, 24, 26, 28, 30, 31, 33, 37, 67, 71, 72

lngram, J., 165, 17Y Ingram, J. M., 191, 192, 201, 235, 237 Inkson, C., 23, 35, 47, 49, 77 Inouye, M., 188, 190, 227, 235, 237 lrvin, J. E., 201, 237 Isaac, D. H., 188,234 Ishiguro, E. E., 123, 146 Iwashima, A., 16, 73

Kadomtseva, V. M., 17. 30, 75 Kagan, S. A., 191,235 Kahan, F. M., 186, 205, 206, 208, 233, 23 7

Kahan, J. S., 186, 205, 206, 208, 233, 237 Kaiser, D., 84, 142 Kamamura, S., 112, 121, 144 Kanegasaki, S., 100, 102, 145, 148 Kanetsuna, F., 174, 178, 179 Kang, K. S., 84, 145 Kang, S., 110, 145 Karst, F., 24, 73

AUTHOR INDEX

Kashket, E. R., 231, 232, 239 Katohda, S., 160, 179 Kay, W. W., 231,237 Keenan, M. H. J., 22, 23, 73 Keller-Schierlein, W., 222, 240 Kelley, D. P., 17, 75 Kenig, M., 186, 216, 237 Kenne, L., 82, 137, 145 Kennedy, E. P., 198, 233,236,238 Kennedy, L. D., 99, 145 Kepes, A., 29, 73 Kerridge, D., 165, 178, 179 Kessler, G., 158, 179 Khan, N. A,, 48, 50, 54, 73, 78 Kholodenko, V. P., 17, 30, 41, 75 Kikuchi, Y., 104, 132, 148 Kimblei, B. K., 126, 147 King, J. A,, 135, 143 Kinghorn, J. R., 21, 75 Kinne, R., 32, 69 Kinscherf, J. G., 233, 239 Kirkwood, S., 155, 156, 180 Klaassen, A,, 15, 16, 69 Klein, D. A,, 203, 237 Kleinzeller, A,, 46, 54, 69, 73 Klemperer, R. M. M., 189, 193, 235 Kligerman, A,, 95, 145 Klink, P. R., 225, 226,235 Kloepfer, H. G., 106, 146 Kloppel, I., 52, 73 Kniisel, F., 187, 220, 222, 237, 238 Knutson,C. A,, 87,91, 129, 138, 142, 145 Kobayashi, H., 97, 146 Kobayashi, M., 105, 145 Kobayashi, T., 104, 132, 148 Koch, A. L., 200, 201, 236 Koch, J. P., 204, 208, 236 Koenig, V. L., 132, 145 Koh, T. Y., 165, 179 Kojo, H., 209,237 Komatsu, Y., 216, 217,237 Komor, B., 58, 62, 65, 73 Komor, E., 35, 39, 48, 57, 58, 59, 62, 65, 66, 68, 72, 73, 77, 78 Koningsberger, V. V., 54, 55, 65, 66, 74 Konisky, J., 185, 221, 237, 239 Kopecka, M., 169, 176, 179, 180 Kopmann, H. J., 101, 145 Kornberg, H. L., 3, 46, 69, 76, 208, 231, 236,237 Kotyk, A., 24, 41, 42, 45, 46, 47, 50. 51, 52, 54, 55, 63, 68, 70, 72, 73, 75

247

Kovarik, J., 167, 170, 172, 173. 176, 177, 178 Kozel, T. R., 124, 145 Kraas, H., 186, 213, 215, 216, 233, 235 Kratky, Z., 166, 167, 176, 177, 178, 180 Krausz-Steinmetz, J., 229, 239 Kreger, D. R., 169, 180 Kreisle, R. A,, 13, 36, 76 Kroger, A,, 197, 237 Kroon, R. A., 54, 55,65, 66, 74 Kropp, H., 186,205,206,208,233,237 Kuenzl, M. T., 172, 180 Kulaev, I. S., 17, 30, 75 Kunst, F., 118, 143 KUO,S-C., 42, 44, 45, 46, 73, 74 Kuraishi, H., 175, 180 Kuramitsu, H. K., 117, 145 Kuypers, G. A. J., 35, 60, 76 Kuznar, J., 21, 23, 24, 74

L Lacroute, F., 23, 24, 29, 39, 65, 66, 70, 73 Lacey, R. W., 212,239 Lagoda, A. A,, 87,88,91,142 Lahoz, R., 88, 147 Lampen, J. O., 30, 53, 74, 78 Lankford, C. E., 220, 237 Lanyi, J. K., 7, 74, 184, 237 Larimore, F. L., 15, 21, 22, 23, 76 Larimore, F. S., 13, 36, 41, 74, 76 Larsen, B., 83, 88, 98, 99, 134, 144 Laskin, A. I., 85, 145 Lawford, G. R., 95, 145 Lawford, H. G., 95, 145 Lawson, C. J., 86, 87, 90, 129, 134, 137, 143 Lazdunski, C., 190,227,238 Leal, J. A., 88, 145 Lee, H. C., 115, 145 LeFevre, P. G., 5, 74 Lehle, L., 106, 107, 145, 146 Lehninger, A. L., 198, 224, 231, 237 Leifer, Z., 204, 237 Leisinger, T., 18, 78 Leive, L., 125, 133, 143, 144 Leloir, L. F., 125, 145 Lengeler, J., 223, 224, 237 Lennarz, W. J., 104, 147 Lepesant, J. G., 118,143

248

AUTHOR INDEX

Lepesant-Kejzlarova, J., 118, 143 Leppard, G. G., 128, 143 Levy, J. S., 15, 21, 76 Levy, S. B., 229, 237 Lewis, M. J., 19, 30, 72, 74 Lichko, L. P., 17, 30, 75 Lichstein, H. C., 26, 28, 75 Lieberman, M. M., 110, 11 1,145 Lin, E. C. C., 204,205,207,208,236,238 Lindahl, U., 134, 145 Lindberg, B., 82, 99, 113, 135, 137, 140, 144,145, 156,180 Lindgren, V., 208,238 Lindsay, R. J., 196, 203,235 Linker, A,, 138, 144, 145 Linstead, P. J., 169, 178 Liu, F. Y., 186, 236 Liu, T. Y., 140, 148 Ljunggren, H., 113, 137, 145 Lo, T. C. Y., 231,238 Lomax, J. A., 109, 120, 145, I46 Long, L. W., 91, 147 Long, R. A,, 66, 74 Long, W. S., 7, 9, 10, 71, 77 Longyear, V. M. C., 116, 118,142 Lopez-Romero, E., 168, 180 Losson, R., 39, 74 Lostau, C. M., 168, 176, 179 Low, K. B., 207,234 Lu, C. Y-H., 7, 10, 77 Luckey, M., 220,238 Ludwig, I1 J. R., 30, 74 Lugtenberg, B., 209, 217, 239, Lusk, J. E., 233,238 Lysko, P. G.. 188,238

M McCabe, M. M., 123, 145 McCallum, M. F., 88, 143 McCloskey, M. A., 103, 123, 148 McClure, F. T., 18, 30, 70 McConnell, M., 113, 133, 145 McCready, R. G. L., 60, 74 McDonough, J. P., 56, 73 Machtiger, N. A., 192,238 Mackie, G., 110, 145 Mackie, K. L., 136, 143 McLaughlin, C. S., 30, 74 McMurray, L., 229, 237, 238 McMurrough, I., 175, 180 McNeely, W. H., 84, 145

McNeil, M., 114, 149 MacQuillan, A. M., 47, 76 MacRobbie, E., 7, 74 MacWilliam, I. C., 152, 180 Maddox, I. S., 173, 180 Magada-Schwencke, N., 21, 23, 24, 74 Magasanik, B., 21, 23,69 Magnuson, J. A., 229,230,232,235,236, 239 Mahler, H. R., 56, 73 Maity, B. R., 208, 238 Makela, P. H., 105, 110, 123, 124, 133, 146, I47 Malamy, M. H., 233, 240 Malmstrom, A,, 134, 145 Maloney, P. C., 65, 74 Malpartida, F., 13, 74 Mankowski, T., 107, 145 Manners, D. J., 153, 154, 155, 156, 158, 159, 161, 162, 163, 165,178,179,180, 181

Markovitz, A,, 105, 109, 110, 11 I , 142, 144,145, 146 Marks, C., 61, 78 Martin, D., 165, 180 Martin, W. G., 66, 74 Marzluf, G. A., 60, 74 Masson, A. J., 156, 158, 161, 165, 180 Matile, P., 12, 74 Matsuba, K., 209, 212, 239 Matsuda, K., 105, 145 Matsui, M., 160, 173, 179 Maxwell, W. A,, 45, 74 Mayer, H., 110, 147 Mayer, R. M., 115, 142, 145 Megnet, R., 166, 167, 180 Meister, A., 19, 74 Melling, J., 105, 118, 143 Melton, L. D., 137, 146 Melvin, E. H., 91, 145 Menna, M., 61,69 Meredith, S. A,, 42, 45, 46, 74 Merkel, G. J., 41, 74 Merrifield, E. H., 132, 143 Metzler, R., 45, 74 Meyer, G. M., 13, 36, 76 Meyer, M. T., 162, 163, 180 Mian, F. A,, 89, 90, 91, 146 Michaljanicova, D., 42, 45, 54, 55, 73 Midgley, M., 61, 74 Miki, K., 205, 207, 238 Milas, M., 132, 146

249

AUTHOR INDEX

Mills, G. T., 109, 120, 142, 147 Mindt, L., 137, 146 Mirelman, D., 123, 142 Misaki, A,, 112, 121, 144, 155, 156, 180 Misra, P. C., 35, 50, 51, 65, 72, 74 Mitchell, P., 2, 4, 7, 33, 39, 50, 74 Mitchison, J. M., 22, 24, 25, 53, 70, 75, 171, 180 Mitscher, L. A,, 225, 231, 238 Miyata, A., 205, 239 Miyazaki, T., 139, 149 Mizano, K., 167, 180 Mizoguchi, J., 167, 180 Mizunaga, T., 175, 180 Moczydlowski, E. G., 224,23Y Montville, T. J., 106, 116, 146 Moor, M., 12, 74 Moorhouse, R., 129, 146 Mooz, E. D., 20, 75 Moreland, E. J., 85, 148 Moreno, R. E., 174, 179 Morgan, H. E., 5, 75 Moriyama, T., 117, 144 Morris, E. R., 128, 129, 137, 146 Morrison, C. E., 26, 28, 75 Morse, M. L., 61,69 Morse, S. A., 188, 238 Mousset, M., 23, 72 Muhlradt, P. F., 192, 238 Muhlenthaler, K.. 12, 74 Mummert, H., 7, 75 Murer, H., 32. 6Y Murphy, J. T., 38, 75

N Nabeshima, S., 85, 148 Nagarajan, R., 188,209.235 Nageli, H-ll., 222, 240 Nagy, M., 22, 25, 30, 31, 71, 72, 75 Naide, Y., 123, 146 Naider, F., 41, 74 Nakae, T., 188, 189, 190, 191, 192, 207 209,2 16,234,238

Nakamura, K., 188, 235 Nash, R. A., 187,226, 228, 234 NeEas, O., 169, 171, 176, 180 Neilands, J . B., 220, 238 Nelson, D. L., 233, 238 Neuhaus, F. C., 203, 239 Neville, M. M., 55, 56, 65, 75

Newbrun, E., 106, 117, 144 Nicas, T. I . , 195, 196, 197, 198, 199, 235,236

Nickerson, W. J., 158, 166, 178, 179 Nierhaus, K. H., 186, 187,238 Nikaido, H., 102, 123, 124, 127, 146, 147. 188, 189, 190, 191, 192,200,201.207, 209,216,234,236,238 Nikaido, K., 102, 146 Nimmich, W., 124, 146 Nishida, M., 209, 237 Nordstrom, K., 192,236 Normark, S., 192, 236 Norris, P. R., 17. 75 Northcote, D. H., 153, 154, 155, 178, 180 Norval, M., 99, 108, 113. 120, 146, 148 Nose, Y.. 16, 73 Notario, V., 155, 165, 179, 180, 181 Nowacki, J. A., 31, 33, 36. 71 Niiesch, J., 220, 238 Nurminen, T., 169, 171, 180, I81 Nygaard, M., 97, 144

0 Ogletree, J . , 129, 145 Okada, H., 48, 53, 54, 55, 65, 75, 78 Okorokov, L. A,, 17, 30, 75 Olavarria, J. M., 125, 145 Oliver, S. G., 30, 74 Omori, T., 139, 149 Onigman, P., 229,237 Onn, T., 99, 135. 140, 144 Opekarova, M., 41, 75 Orskov, F., 112, 121, 146 Orskov, I., 112, 121, 146 Osborn, M. J., 99, 146, 148, 188,238 Oshima, U., 53, 78 Oura, E., 169, 180 Owen, P., 192, 239 Oxender, D. L., 203,238 Ozawa, Y., 97, 146

P Paakanen, J., 105, 124, 146 Pages, J. M., 190. 227,238 Pdlamarczyk, G., 107, 146

250

AUTHOR INDEX

Pall, M . L., 56, 78 Palva, E. T., 133, 146 Panos, C., 107, 108, 146 Park, M . H., 233,238 Parlebas, N., 41, 75 Pascal, M., 118, 143 Patel, R. N., 85, 145 Pateman, J. A., 21, 75 Patil, N. B., 177, 179 Patterson, J . C., 154, 155, 156, 158, 161, 165,180 Pauling, K. D., 19, 75 Payne, J. W., 213,238 Peat, S., 155, 180 Peloerdy, J. F., 169, 180 Pefia, A,, 13, 14, 16, 17, 75 Penman, A,, 135, 146 Penninckx, M., 20, 75 Pepper, E. A,, 105, 118, 143 Percival, E., 139, 144 Perkins, H., 126, 148 Perlin, A. S., 154, 180 Peberdy, J . F., 169, 180 Perrings, J. D., 132, 145 Perry, M., 114, 143 Pestka, S., 194, 238 Peters, P. H. J., 13, 75 Petit-Glatron, M . F., 118, 146 Petrotta-Simpson, T. F., 23, 42, 75 Phaff, H. J., 30, 74, 152, 153, 154, 155, 160, 174, 177,178, 179, 181 Philo, R., 34, 35, 71 Philo, R. D., 32, 48, 75 Pickard, M . A,, 198, 199, 235 Pickering, W. R., 24, 75 Pierce, J . S., 17, 73 Piggott, N. H., 90, 138, 146 Pindar, D. F., 137, 146 Piovant, M., 190, 227, 238 Pittsley, J. E., 87, 91, 129, 138, 142, 145 Plakunov, U. K., 231,238 Plastow, G. S., 221, 238 Pogell, B. M., 208, 238 Polak, A,, 22, 23, 75 Politis, D. J., 124, 146 Pollack, J. R., 220, 238 Ponec, M., 24, 73 Postle, K., 221, 238 Postma, P. W., 196, 239 Powell, D. A., 188, 238 Power, D. M., 175, 178, 181 Poxton, I. R., 107, 109, 120, 145, 146

Pragnell, M . J . , 17, 73 Prestridge, E. B., 129, 145 Privitera, G., 229, 236 Pudeck, M. R., 224,231,238 Pugsley, A. P., 200, 201, 238

R Radjai, M. K., 87, 143 Raffle, V. J., 195, 236 Rainbow, C., 19, 74 Raizada, M . K., 106, 146 Ramey, W. D., 123, 146 Ramos, E. H., 33, 36, 75 Ramos, S., 232, 238 Ramsay, A. M., 176, 181 Rankin, J. C., 91, 145 Rapaport, G., 118,143 Rasmussen, L., 61, 75 Ratledge, C., 46, 47, 71 Rauch, B., 190, 191, 239 Raven, J. A,, 7, 11, 14, 57, 75, 77 Raymond, R. L., 85, 146 Rechenmacher, A,, 198,234 Recondo, E., 100, 101, 144 Rees, D. A., 128, 129, 137, 146, 158, 181 Rees, T. A. V., 61, 75 Regen, D. M., 5, 75 Reich, J., 169, I80 Reichert, U., 35, 40, 71, 75 Reid, I. D., 177, 181 Reid, M . , 32, 76 Reiner, M., 42, 77 Reusch, V. M., 107, 108, 146 Reusser, F., 187, 222, 223, 238 Reyes, E., 47, 71 Reyes, S. F., 88, 147 Reynolds, P. E., 184, 186, 187, 194, 201, 217,225,236 Reznikoff, W., 221, 238 Richardson, C. L., 128,142 Richmond, M. H., 184, 186, 187, 194, 20 I , 2 17,225,236 Riemersma, J . C., 14, 76 Riggs, T. R., 4, 76 Righelato, R. C., 86, 87, 89, 90, 91, 129, 143,145, 146 Rikova, L., 24, 73 Rinaudo, M., 132,146 Rist, C. E., 91, 145

AUTHOR INDEX

Robbins, J. B., 140, 148 Robbins, J. C., 203, 238 Robbins, P. W., 99, 101, 107, 122, 126, 143,146,147,148

Robins, R. J., 20, 76 Robyt, J. F., 115, 123, 126, 147 Rodriguez, J., 174, 179 Rodriguez-Navarro, A., 14, 15, 76 Roelofsen, P. A., 158, 181 Rogers, H. J., 191, 238 Rogovin, P., 91, 92, 147 Rogovin, S. P., 129, 138, 142 Rohr, T. E., 125, 134, 147 Romano, A. H., 42, 45, 46, 74, 76, 174, 181

Rombouts, F. M., 154, 181 Rommele, G., 167, 169, 178 Roomans, G. M., 15, 16, 17, 35, 60, 61, 76, 78

Roon, R. J., 13, 15, 19, 21, 22, 23, 36, 41, 71, 74, 76

Rosano, C. L., 198, 199,237 Rose, A. H., 22, 23, 73, 179, 180 Roseman,S., 55,56,65, 75, 190, 191,239 Rosen, B. P., 12, 14, 69, 184, 191, 231, 232, 238, 239

Rosenberg, H., 221,236,240 Rosenberg, T., 42, 76 Rosendal, K., 134, 144 Rosselet, A,, 209, 212, 240 Rothstein, A., 12, 15, 16, 17, 42, 43, 45, 69, 71, 76, 78

Rottem, S., 190, 239 Royt, P. W., 47, 76 Rubenstein, P. A., 140, 148 Ruiz-Herrera, J., 47, 71, 168, 180 Ruperez, P., 88, 145 Ryan, H., 12, 14, 15, 70, 76 Ryan, J. P., 12, 14, 15, 76 Rytka, J., 19, 21, 23, 76

S Sachs, G., 12, 76 Sacktor, B., 32, 76, 77 Safranski, M. J., 88, 91, 147 Saier, M. H., 184, 223, 224, 235, 239 Saito, H., 112, 121, 144 Saito, T., 167, 180 Sakano, Y ., 104, 132, 148 Salton, M. R. J., 192,239

251

Samra, Z., 229,239 San-Blas, F., 166, 181 San-Blas, G., 166, 174,181

Sancho, E. D., 14, 15, 76 Sandermann, H., 101, 108, 109, 114, 121, 147

Sanders, R. E., 1 1 I , 147 Sanderson, G. R., 135, 137, 146 Sandford, P. A,, 84, 147 Sando, N., 173, 179 Sano, K., 82, 138,144 Santamaria, F., 88, 147 Sartirana, M. L., 53, 75 Sasak, W., 107, 145 Saunders, R. M., 229, 237 Saunders, V. A., 196,236 Sawai, T., 209, 212,23Y Scales, W. R., 97, 147 Scaletti, J. V., 155, 156, 180 Scarborough, G. A., 1 I , 12, 55, 56, 65, 70. 76. 77 Schachtele; C. F., 117, 143 Schachter, H., 5, 76 Schaller, K., 221, 234 Scher, M., 104, 147 Scherr, G. H., 174, 181 Scherrer, R., 192, 239 Schindler, P., 213, 223, 224, 234 Schlegel, H. G., 91, 95, 142 Schmidt, G., 110, 147 Schmidt, M. R., 184, 235 Schmidt, R., 40, 71, 75 Schnaitman, C. A,, 200,201,238 Schneider, E. G., 32,76, 77 Schneider, H., 66, 74 Schneider, R. P., 55, 56, 77 Schuldiner, S., 232, 238 Schulten-Koerselman, H. J., 132, 149 Schultz, S. G., 58, 77 Schultz, W., 187, 226, 228, 234 Schumacher, G., 205,206,207,234 Schutzbach, J . S., 106, 146 Schwab, W. G. W., 39,48, 57, 73, 77 Schwencke, J., 21, 23, 24, 30, 31, 71, 74, 77 Scott, G. J., 101, 147 Seaston, A,, 14, 15, 18, 21, 23, 24, 26, 28, 33, 35, 36, 37, 38, 47, 48, 49, 60, 61, 64, 65, 66, 67, 71, 72, 77 Sebald, M., 229,236 Segel, I. H., 5, 6, 25, 60, 68, 70, 72, 77 Selwyn, S., 21 1 , 239

252

AUTHOR INDEX

Sentandreu, R., 167, 168, 172, 173, 175, 176, 179, 181 Serrano, R., 13, 35, 44, 46, 48, 74, 77 Shadur, C. A., 203, 239 Shales, S. W., 221, 227, 229. 233, 234, 235,236,239 Shanks, C., 226,234 Shapiro, S., 208, 238 Shematek, E. M . , 166, 167, 170, 171, 181 Shigi, Y., 209, 237 Shockman, G. D., 191,202,236 Sierra, J. M., 172, 173, 181 Siewert, G., 107, 144 Sieger, G. M., 187, 226, 228, 234 Silhavy, T. J., 205,206,207,208,238,239 Silman, R. W., 91, 92, 147 Silver, S., 220, 233, 239 Simoni, R. D., 196, 239 Sims, A. P., 47, 77 Sinskey, A. J., 106, 116, 146 Sipicki, M., 177, 181 Sire, J., 20, 21, 23, 24, 78 Sison, Y., 175, 179 Slayman, C. L., 7, 8, 9, 10, 35, 56, 66, 71, 72, 77, 78 Slayman, C. W., 8, 9, 11, 12, 35, 56, 66, 69, 77 Slocombe, S., 86, 87,90, 129, 143, 145 Slodki, M. E., 88,91, 147 Sly, W., 20, 21, 23, 24, 78 Smidsrsd, O., 83, 137, 144, 147 Smit, J., 127, I47 Smith, D. I., 191, 198, 235 Smith, E. E., 123, 145, 177, 179 Smith, E. E. B., 109, 120, 142, 147 Smith, E. J., 98, 147 Smith, F., 155, 156, I80 Smith, F. A,, 7, 1 I , 77 Smith, J. B., 191, 240 Smith, M. C. M., 229,230,231,232,239 Snow, G. A., 190,236 Sobotka, H., 42, 77 Sols, A., 42, 45, 46, 53, 70, 72, 77 Sompolinsky, D., 229,239 Sooka, J., 169, 180 Sorenson, E. N., 12, 14,69 Sowden, L. C., 114, 128, 143 Spence, K . D., 23, 38,42, 75 Spoerl, E., 45, 74 Stange, G., 32,69 Stark, G. H., 86, 89, 148

Steinmetz, M., 118, 146 Stephanopoulos, D., 30, 74 Stephen, A. M., 132, 143, 144 Stirm, S., 132, 148 Stock, J. B., 190, 191, 239 Stocker, B. A. D., 110, 123, 142, 146, 147 Stone, K . J., 107, 148 Stoolmiller, A . C . , 103, 120, 147 Stoppani, A. 0. M., 33, 36, 75 Strominger, J . L., 107, 109, 121, 140, 144, 147,148 Stroobant, P., 1 I , 77 Struzinsky, R., 63, 68, 73 Subbiah, T., 123, 142 Subramanian, K . N., 18, 77 Sumrada, R., 23, 25, 28, 29, 38, 65, 70, 77 Suomalainen, H., 169, 171, 180, 181 Surdin, Y., 20, 21, 23, 24, 78 Suskind, S. R., 55, 56, 65, 75 Sutherland, 1. W., 81, 83, 84, 86, 95, 98, 99, 106, 107, 108, 109, 110, 113, 114, 120, 129, 134, 135, 136, 137, 143, 144, 145, 146, 147, 148, 188.239 Sutton, D. D., 53, 78 Suzuki, H., 97, 146 Suzuki, S., 154, 180 Svoboda, A,, 176,180 Syrett, P. J., 61, 75

T Taguchi, R., 104, 132, 148 Takai, M., 114, 128, 143 Talmadge, J . E., 23, 42, 75 Tam, K . T., 85,87, 148 Tamura, A., 209, 212, 239 Tanaka, A., 85, 148 Tanaka, K., 216,217,237 Tanaka,T., 186, 194,212,217,237,239 Tang, C. T., 204,239 Tanaguchi, H., 115, 147 Tanner, W., 35, 48, 57, 58, 62, 65, 66, 68, 70, 71, 73, 78, 106, 107, 145, 146 Tarcsay, L., 132, 148 Tate, S., 19, 74 Tatum, E. L., 9, 77 Tauchova, R., 50,51, 73 Taylor, E. S., 24, 78 Taylor, J . F., 153, 178

253

AUTHOR INDEX

Tempest, D. W., 86, 143 Teranishi, Y., 85, 148 Terui, G., 53, 78 Tesche, N., 102, 1 19, 148 Theuvenet, A. P. R., 14, 15, 16, 17, 35, 40, 45, 60, 71, 76, 78 Thines, D., 13, 70 Thorn, D., 128, 129, 137,146 Thompson, C. C., 48, 72 Thompson, T. E., 198, 224, 231,237 Thurow, H., 122, 124, 127, 128,142 Tipper, D. J . , 101, 103, 107, 112, 148 Titovsky, V. T., 17,30, 7.5 Tobin, R., 132, 143 Tomasz, A,, 186, 239 Tongue, R. J., 175, 178 Tonn, S. J., 84, 148 Trevillyan, J. M., 56, 78 Tritton, T. R., 227, 239 Tropp, B. E., 186, 203, 204, 237, 239 Troy, F. A., 84, 99, 100, 102, 103, 113, 119, 120, 123, 125, 127, 134, 136,147, 148 Trumble, W. R., 229,236 Tsuruoka, T., 205, 239 Tuchiya, H. M., 91, 14.5 Turoscy, V., 23, 25, 29, 38, 77, 78 Turvey, J. R., 155, 180 Tyach, R. J., 186,203,239

Van Steveninck, J., 42, 43, 44, 45. 46. 47. 54, 55, 70, 73, 78 Van Wezenbeck, P. M . G. F., 14, 72 Varenne, S., 190, 227, 238 Vernet, D., 174, 181 Vijay, I. K., 102, 119, 148 Villa, T. G., 155, 180. 181 Villanueva, J. R., 155, 167, 168. 1 72, 173, 175, 176, 179, 180, 181 Vohman, H. J., 176, 178 Vorisek, J., 41, 78

W

Wakabayashi, Y., 16, 73 Walker, L. M., 4, 76 Walkinshaw, M. D., 129, 146 Walseth, T. F., 123, 126, 147 Ward, J . B., 126, 148, 191,239 Wargel, R. J., 203, 239 Waring, M. J., 184, 186, 187, 194, 201, 217, 225,236 Warncke, J., 7, 9, 10, 56, 71, 78 Warren, L., 119, 142 Warth, A. D., 140, 148 Watson, G., 31, 33, 71 Watson, T. G., 17, 78 Wayman, F., 165,179 Wayne, R., 220,238 Weaver, C. A., 221, 239 U Weaver, R. H., 174, 181 Webb, M., 233, 239 Uemura, T., 175, 180 Webley, D. M., 162, 178 Ullrich, K. J., 7, 78 Weckesser, J., 229, 230, 232, 239 Umbarger, H. E., 26,41,69 Wehrli, E., 12, 71 Umbreit, J . N., 107, 148 Wehrli, W., 167, 169, 178 Urech, K., 30, 71 Weiner, I . M., 99, 146, 148 Urrestarazu, L. A., 18, 73 Weinstein, L., 226, 234 Weiss, A. A., 233, 239 Weiss, R. L., 18, 77 v Welsh, E. J., 128, 129, 137. 146 Wermundsen, I. E., 109, 142 Van Alphen, W., 209, 217, 239 West, I. C . , 7, 32, 62, 78, 184, 239 Van Boxtel, R., 209, 239 Whelan, W. J., 155, 180 Vandamme, E., 186,216,237 White, R. J., 223, 239 Van Den Berg, T. P. R., 16, 17, 76 Whiteman, P., 61, 78 Van den Elzen, H. M., 195, 196, 197,198, Whitfield, C . , 95, 113, 148 199,235 Wiame, J. M., 18, 20, 21, 23, 70, 72, 73, Vann, W. F., 140,148 75 Van Selm, N., 209,217,239 Wickus, G. G., 140, 148

254

AUTHOR INDEX

Wiemken, A,, 18, 27, 28, 30, 69, 71, 72, 78 Wigglesworth, L., 20, 75 Wilbrandt, W., 42, 76 Wiley, W. R., 55, 56, 77 Wilham, C. A,, 91, 145 Wilkinson, J. F., 84, 86, 89, 98, 110, 123, 136, 143, 144, 148 Wilkinson, R. G., 123, 146 Williams, A. G., 86, 89, 138, 148 Williams, R. J. P., 69, 78 Williams, T., 95, I45 Williamson, D. H., 174, 179 Williamson, J., 81, 95. 148 Willison, J. H. M., 128, 142 Willoughby, E., 107, 148 Willsky, G. R., 13, 78, 233, 240 Wilson, D. B., 102, 110, 114, 145, 191, 240 Wilson, T. H., 65, 74, 207, 237 Wimpenny, J. W. T., 86,89, 138, 148 Winter, W. T., 129, 146 Wipf, B., 18, 78 Wittenbury, R. H., 197, 235 Wittman, H. G., 186, 187, 238 Wolf, C., 132, 148 Wolff, H., 221, 234 Wong, B. B., 233,238 Wong, J. T.. 68, 78 Wood, W. B., 197,235 Woodhead, J. S., 160, 179 Woods, R. A,, 24, 75 Woodward, J. R., 17, 18, 24, 78 Wookey, P., 221,240 Wright, A,, 99, 100. 101. 102, 104, 107,

112, 113, 122, 126, 133, 145, 146, 147, 148 Wright, E. M., 3, 70 Wu, H. C. P., 188,238 Wursch, P., 162, 178 Wyss, O., 85, 148

Y Yadomae, T., 139, 149 Yamada, H., 139, 149 Yamada, K., 97, 146 Yamada, Y., 205,239 Yamagishi, S., 209, 212, 239 Yamamoto, L. T., 11 I , 144 Yeo, R. G., 91, 92, 93, 95, 96, 134, 144 York, W. S., 114, 149 Yoshimura, T., 85, 144 Yu, R. J., 162, 163, 164, 181 Yuen, S., 88, 149

Z Zaar, K., 89, 128, 149 Zacharski, C. A,, 23, 27, 28, 29, 77, 78 Zahner, H., 186, 213, 215, 216, 222, 223, 224,233,234,235,240 Zehnbauer, B., I 1 1,142 Zevenhuizen, L. P. T. M., 132, 149 Zimmermann, W., 186,187,209,212,222, 237,240 Zimmermann, F. K., 48, 54, 73, 78

Subject Index A

structure, 215

-, L-methionine-S-dioxidylananyl-strucAcetobacter xylinum ture, 214 cellulose synthesis, secretion, 128 L-phosphinothricylalamyl-, structure, exopolysaccharide production by, li214 pids intermediate in, 100 -, ~-(N~-phosphono)methionine-S-sulpolysaccharide production by, culture phoximinyl-alanylmedium, 86 mode of action, 186 water-soluble glucan production by, structure, 214 1 I4 Alaphosphin, structure, 21 5 Acetylation, microbial exopolysaccha- Albomycin, mode of action, 187 rides, 14CL-141 Albomycin 6,, transport into bacteria, Achromobacter georgiopolitanum, exo220 polysaccharides from, 98 Alcaligenes faecalis var. myxogenes Achromobacter mucosum, exopolysacglucan and curdlan synthesis, 129 charide production, substrates, 97 polysaccharide production, I 12 Aculeacin A, inhibition of glucan synsuccinoglucan and curdlan synthesis, thesis by, 167 shared pathways, 121 Acylation Alginates microbial exopolysaccharides, 135acetylation, 136-1 37 141 biosynthesis, 83 polysaccharides, on alternate repeating L-guluronic acid in, 98 units, 135-136 production, 90 Adenine by Azotobacter vinelandii, 87 uptake, by Schizsaccharomyces pombe, varying physical properties, 22 129 by yeasts, purine pool and, 25 by Pseudomonas aeruginosa, 89 Adenosine, vacuolar transport systems in mechanism, 139 yeasts, 31 Allantoates, transport in yeast, gradient Adenosine triphosphatase, solute transcoupling and, 38 port and, 2 Allantoin ADP-glucose as substrate for exopolyinflux kinetics by yeasts, 29 saccharide production, 98 transport in yeast, gradient coupling Aeration, polysaccharide production by and, 38 Rhizobium meliloti and, 86 absorption by yeasts, genetically disAerobacter levanicum, levan synthesis by, tinct pathways, 2&22 1I8 concentration by yeasts, 37 Agrobacterium spp., polysaccharide bioin yeast vacuolar compartments, 27 synthesis in, 82 influx from yeasts, proton gradient hyAgrobacterium tumefaciens, Bl,2-linked pothesis and, 38 glucans from, 1 I5 Myxococcus xanthus growth on, 84 Alanine, S-alanyl-3[a-S-chloro-3-S-hy- retention by yeasts, 24 droxy-2-oxo-azetidinylmethyl]-, transport in yeast, 1 7 4 2

256

SUBJECT INDEX

energy coupling during, 3 1-37 metabolism and, 18-19 Amino acid-binding proteins, yeastmembrane vesicles and, 4&42 Amino acid permease in yeasts, 21 Aminoglycosides accumulation across cytoplasmic membranes, 195-1 99 diffusion across outer membranes, 194-195 mode of action, 186 transport into bacteria, 194-199 Amino-uronic acids in exopolysaccharide from Achromobacter georgiopolitanum, 98 Ampicillin, mode of action, 186 Analysis, yeast glucans, 152-1 66 Antibiotics target sites, 185 transport into bacteria, 183-234 uptake, 185 Antirrhinum mazus, protoplasts, glucan synthesis in, 169 D-Arabinohexose, 2-deoxy-, effect on glucan synthesis in Rhodosporidium toruloides cell wall, 177 Arginine absorption, by Saccharomyces cerevisiae, 22 by yeasts, 21 in Saccharomyces cerevisiae, 18 in yeast vacuolar compartments, 28 uptake by yeasts, inhibition by histidine, 30 Arginine permease in yeasts, regulation, 26 D-Asparaghe, assimilation in yeasts, 19 Aspartates in Saccharomyces cerevisiae, 17 Aspergillus nidulans amino acid absorption in, 21 exopolysaccharide production by, 88 nutrient transport in, 2 ATP, ion transport in Neurospora crassa and, 9-1 1 ATPase as electrogenic proton pump in Neurospora crassa, 11 from vanadate-sensitive plasma membranes, phosphorylation, 13 Aureobasidium spp., pullulan biosynthesis in, 82

A ureobasidium pullulans exopolysaccharide production by, 88 pullulan synthesis by, 88, 89, 104 Azotobacter vinelandii alginate production by, 90 post-polymerisation modification, 134 varying physical properties, 129 exopolysaccharide production, 87 lipid precursors and, 101 polysaccharide production by, culture medium, 86

B Bacillus circulans, ( I --* 6)-/l-glucanases from, in enzymolysis of yeast glucans, 154 Bacillus subtilis levan synthesis by, 118 levansucrase in, regulation, 105 phosphomycin transport into, 208 tetracycline accumulation by, 230 pH and, 232 Bacilysin mode of action, 186 structure, 21 5 Bacteria, transport of antibiotics into, 183-234 Bacteriocins, 185 Bactoprenol in bacteria, 106 Batch culture, growth phase, exopolysaccharide production and, 89 Bayer sites, 127 Beijerinckia indica, exopolysaccharide production by, 99 Binary capsulation in Streptococcuspneumoniae, 109 Biotin, deficiency, effect on glucan synthesis by yeast cell walls, 175 Blastomyces dermatidis, yeast-mycelium interconversion, glucan synthesis and, 174 Budding, glucan and mannan synthesis and, 173 I-Butanephosphonic acid, 3,4-dihydroxydiffusion across outer membranes, 204 mode of action, 186 transport into bacteria, 203-204

SUBJECT INDEX

C Calcium ions alginate synthesis by Azotobacter vinelandii and, 87 polysaccharide production by Enterobacter uerogenes and, 86 transport, in Neurospora crassa, 8 in Saccharomyces cerevisiae, I6 Canamycin, transport into bacteria, 194 Candida spp., monosaccharide transport in, 47 Candida albicans cell walls, glucans, structural analysis, 162 /?-glucans, structural analysis, 164 protoplasts, glucan synthesis in, 169 purine transport in, 22 Candida parapsilosis cell walls, glucans, structural analysis, 163 /?-glucans, structural analysis, 164 Candida utilis amino acids in, 18 glucan content, cell cycle and, 171 phosphate symport systems, 60 vacuolar compartments, solute transport and, 27 Capsules, structure, antibiotic uptake and, 188 Carbohydrates deficiency, effect on glucan synthesis by yeast cell walls, 175 steady state, 61-69 transport in yeasts, 42-55 Carrier models, solute transport, eukaryotic micro-organisms, 5 Cell cycle, glucan content and, 171-174 Cell division, glucan and mannan synthesis and, 173 Cell envelopes, bacterial, structure, antibiotic uptake and, 188-193 Cell walls Candida albicans, glucans from, structural analysis, 162 Saccharomyces cerevisiae, glucans, structural analysis, 155 Schizosaccharomyces pombe, glucans, 162 yeasts, glucans, synthesis, 166171 Cell wall glucans, yeast, 151-177 Cell wall polymers, microbial production,

257

sugar nucleotides as substrates. 97 Cellulose, bacterial, biosynthesis. 82 Cephalexin. diffusion across outer membranes, 209 Cephalothin, mode of action, 186 Cerulenin, levansucrase control in Bacillus subtilis, 105 Chloramphenicol accumulation across cytoplasmic membranes 200-20 1 diffmion across outer membranes. 200 mode of action, 186 transport into bacteria, 20&201 Chlorella spp. carbohydrate symptoms, 62 nutrient transport in, 2 Chlorella vulgaris active hexose-uptake system, 57-59 6-deoxyglucose accumulation in, 66 hexose uptake, respiration and, 63 3-0-methylglucose in, 62 Chromobacterium violaceum, polysaccharide production by, culture medium and, 86 Citrulline in Saccharomyces cererisiae. 18 Clostridiirm perfringens, exopolysaccharide production, 86 Cobalt ions, transport in Saccharomyces cerevisiae. 16 Colanic acid digestion by fucosidases, 1 14 microbial production, 107 production by Escherichia coli. 102, 110, 112 structure, 135 Concentration of amino acids by yeast, 37 Continuous culture, nutrient limitations in, exopolysaccharide production and, 89-95 Cryptococcus laurentii. exopolysaccharide production regulation in. 106 Cryptococcus laurentii var. flavescens. exoploysaccharide production by, 88 Cryptococcus neoformans. exopolysaccharide production, surface receptors in, 124 Culture medium, exopolysaccharide production by micro-organisms and, 8488

258

SUBJECT INDEX

Curd1an production by Alcaligenes,faecalis var. myxogenes, 112, 129 shared pathways, 121 Cyclobutane- 1 -acetic acid, 1-S-hydroxy2-S,S-valylamido-, structure, 21 5 Cycloheximides, effect on yeast cell glucan synthesis, 176

D D-Cycloserine diffusion across outer membranes, 203 mode of action, 186 site of action in biosynthetic pathways for peptidoglycans, 202 transport across cytoplasmic membranes, 203 transport into bacteria, 201-203 Cytoplasmic membranes aminoglycoside accumulation across, 195- 199 chloramphenicol accumulation across, 200-20 1 D-cycloserine transport across, 203 3.4,-dihydroxybutyl-l-phosphonate transport across, 204 phosphomycin transport across, 208 showdomycin transport across, 21 7219 streptozotocin transport across, 223224 structure, antibiotic uptake and, 192193 tetracycline transport across, 228-232 Cytosine, accumulation, pump-leak models, 66 Cytosine permease, 41 Dental caries, mutan in, 116 Deoxy sugars in exopolysaccharide from A chromobacter georgiopolitanum, 98 Dextran sucrases in Leuconostoc mesenteroides, regulation, 105 Dextrans biosynthesis by micro-organisms, 80 microbial synthesis, acceptors in, 123 from sucrose, 97 production by Leuconostoc mesenteroides, 95 synthesis by cell-free preparation, 1 15118 Dio-9, effect on plasma membrane, 13

Disaccharides, proton symport with, in yeasts, 47-50 Dolichol phosphate, glycosylation control by, 106

E Echinocandin B, inhibition of glucan synthesis by, 167 Electrogenesis, ion transport in Neurospora crassa and, 9-1 1 Energetics, solute transport, in eukaryotic micro-organisms, 2-7 Energy coupling in amino-acid transport in yeast, 31-37 Energy-dependent phase I, streptomycin accumulation in, 196-197 Energy-dependent phase 11, streptomycin accumulation in, 198-199 Energy-independent phase, aminoglycoside accumulation across cytoplasmic membrane, 195 Enterobacter aerogenes cell-free extracts, heteropolysaccharides production by, 120 exopolysaccharides, biosynthesis, 80 production, growth phase and, 89 lipids as substrates 99 isoprenoid alcohol kinase, 109 lipopolysaccharide production by, 108 membrane preparations from, exopolysaccharide synthesis, 120, 122 monosaccharide transfer to lipids in, 100

polysaccharides from, 133 acylation on alternate repeating units in, 135 production, culture medium, 84, 86 varying physical properties, 130 structure, 136 xanthan production by, 113 Enterobacter cloacae, high-viscosity polysaccharide production, 130 Enzymes plasma-membrane proton pump and, 11-12 regulation, exopolysaccharide synthesis, 109-1 11 yeast plasma-membrane proton pump and, 12-17 Enzymolysis in analysis of yeast glucans, I54

259

SUBJECT INDEX

Escherichia coli capsular polysaccharide production, outer membrane proteins and, 124 colanic acid production by, 102 exopolysaccharides, biosynthesis, 80 P-galactoside permease in, 29 8-galactoside transport in, 62 high-viscosity polysaccharide production, 130 lactose transport in, 66 methylthio-a-galactoside accumulation in, 66 outer membranes, aminoglycoside diffusion across, 194 structure, antibiotics uptake and, 188 peptide antibiotics and transport systems, 214215 phosphomycin uptake by, 206 poly-N-acetylneuraminic acid production by, 102 polysaccharide excretion, 128 polysaccharide production, changes in, 112 culture medium, 84 sialic acid synthesis by, 127 attachment site, 125 sialic acid production by, 102 tetracycline accumulation by, 230 pH and, 232 Eukaryotic micro-organisms, solute transport in, 1-69 Exopolysaccharides acylation, 135 microbial, biosynthesis, 79-141 molecular weights, 132 physiological aspects of production, 84-95 production, sugar nucleotides as substrates, 97 molecular weights, 133 post-polymerisation modification, 134135 shared pathways, 121 synthesis by Enterobacter aerogenes membrane fractions, 122

F Feedback control, solute uptake in yeasts, 25-27

Ferrichrome transport system in Escherichia coli, 221 Ferrimycin A,, transport into bacteria, 220 Fluorides as inhibitors of yeast cell-wall glucans synthesis, 166 Fosfomycin, mode of action, 186 Fructan, preparation from Streptococcus mutans, 118

G Galactose proton symport in yeasts, respiration and, 50 transport in Saccharomyces cerevisiae, phosphorylation, 42 in yeasts, phosphorylation and, 44, 45 Galactose, 2-deoxy-, transport in yeasts, phosphorylation and, 44 P-Galactoside, methylthio-, steady-state accululation in Escherichia coli, 66 b-Galactoside permease in Escherichia coli, 29 /3-Galactosides, transport in Escherichia coli, 62 GDP-mannose pyrophosphorylase, synthesis, derepression, 1 11 GDP-mannuronic acid as substrate for microbial exopolysaccharide production, 97 Genetic regulation, exopolysaccharide synthesis, 109-1 11 Genetics, solute transport and, in eukaryotic micro-organisms, 2 Gentamycin, transport into bacteria, 194 (1 3)b-Glucan in Saccharomyces cerevisiae, molecular weight, 158 yeast glucan component from Saccharomyces cerevisiae, 156 (1 +6)-/?-Glucan in Saccharomyces cerevisiae, biological functions, 158 yeast glucan component from Saccharomyces cerevisiae, 156 Glucan synthetases, 167-1 68 in protoplasts, 169

-

260

SUBJECT INDEX

Glucans alkali-insoluble, in cell walls of Saccharomyces cerevisiue, structural analysis, 155-1 58 alkali-soluble, from Sacchuromyces cerevisiae, 158-160 from Candidu spp. structural analysis, 162-165 in cell walls of Saccharomyces cerevisiue, structural analysis. 155-160 in cell walls of Schizosuccharomyces pombe, 162 synthesis, by Alcaligenes faecalis var. myxogenes, 129 by particulate preparations from Saccharomyces cerevisiue, 168 in protoplasts, 169-171 water-soluble, production by Acetobucter xylinum, 114 yeast, cell walls, 151-177 physiological control, 171-177 synthesis, 166-171 inhibitors, 166-167 methylation, 153 preparation, 152-1 53 structural analysis, 152-166 Glucose active uptake system of Chlorella vulgaris, 57 proton symport in yeasts, 54 transport systemsin Neurosporu crassu, 55-56 transport in Saccharomyces cerevisiae, phosphorylation, 42 -, 1-deoxy-, active uptake system of Chlorella vulgaris, 57, 59 -, 2-deoxyas inhibitors of yeast cell wall glucans synthesis, 166 effect on yeast cell glucan synthesis, 176 transport, in Sacchuromyces cerevisiae, 46 in yeasts, phosphorylation and, 44 -, 6-deoxyaccumulation in Chlorella vulgaris, 66 active uptake system of Chlorella vulgaris, 57, 59 -, 2-deoxy-2-fluoroeffect on glucan synthesis in Succharomyces cerevisiue cell walls, 177 effect on synthesis of glucan micro-

fibrils, 169-170 3-0-methylactive uptake system of Chlorella vulgaris, 57 electrogenic symport in Neurospora crassa, 64 steady state in Chlorella vulgaris, 62 Glucoside, a-methylproton symport in yeasts, 54 transport into yeast cells, 55 -, a-phenyl-, proton symport in yeasts, -,

54

-, a-thioethylaccumulation, pump-leak models, 66 proton symport in yeasts, 54 Glucosides, hydrolysis in yeasts, 53 Glutamates in Saccharomyces cerevisiae, 17 y-Glutamyl cycle in yeasts, 19-20 Glutathione, transport in yeasts, 19-20 sn-Glycerol 3-phosphate transport protein in phosphomycin diffusion across outer membrane, 205-207 Glycine concentration by yeasts, 37 in yeast vacuolar compartments, 28 uptake by yeasts, feedback control, 26 Glycogen microbial synthesis, mechanism, 125 in yeast, 177 Glycosylation, control by dolichol phosphate, 106 Gradient coupling purine transport in yeast and, 38-40 pyrimidine in yeast and, 38-40 Growth phase, in batch culture, exopolysaccharide production and, 89 Guanine, uptake by Schizsaccharomyces pornbe, 22 Guanosine, vacuolar transport systems in yeasts, 31 L-Guluronic acid in bacterial alginates, 98

H Hunsenula cupsulatu, exopolysaccharide production by, 88 Hansenulu holstii, exopolysaccharide production by, 88 Heparin, biosynthesis, 134

SUBJECT INDEX

Heteropolysaccharides biosynthesis by micro-organisms, 80 cell-free synthesis, 119-121 extracellular bacterial, lipids as substrates, 99 microbial production, sucrose as substrate, 95 Hexose, active uptake system of Chlorella vulgaris, 57-59 Hexose phosphate transport protein in phosphomycin diffusion across outer membranes, 207-208 Hexoses, methyl, from Beijerinckia indica, 99 Histidine permeases, feedback inhibition, 26 Histoplasma capsulatum, yeast-mycelium interconversion, glucan synthesis and, 174 Homopolysaccharides cell-free synthesis, 118-1 19 production by Salmonella spp., lipid precursors, 102 Homopolysaccharide synthetases, regulation, 105-106 Hyaluronic acid cell-free synthesis, 120 formation by Streptococcus spp., 103 Hydrocarbons microbial polysaccharide production from, 85 Myxococcus xanthus growth on, 84 Hydrolysis, cc-glucosides, in yeast, 53 Hydrophobicity, effect on passive diffusion of tetracyclines, 226228 Hypoxanthine, vacuolar transport systems in yeasts, 31

I Indo-( 1+3)-p-glucanases in enzymolysis of yeast glucans, 154 Inner membranes - - - See Cytoplasmic membranes Inosine, vacuolar transport systems in yeasts, 31 Inositol deficiency, effect on Saccharomyces carlsbergensis glucan synthesis, 175 Ion transport in Neurospora crassa, 7-12 Intracellular compartments eukaryotic

261

micro-organisms, solute transport and, 24-25 Iron complexes, siderophores, transport into bacteria, 220 Isoprenoid alcohol kinase in exopolysaccharide synthesis, 108 Isoprenoid alcohol phosphokinase from Staphylococcus aureus, 12 1

K Kanamycin, mode of action, 186 Ketalation, microbial exopolysaccharides, 140-141 Kinetics, solute transport, in eukaryotic micro-organisms, 2-7 Klehsiella spp., exopolysaccharide production, lipopolysaccharide and, 124 Kloeckera apiculata, glucans, structural analysis, 165 Kluyver effect, 46 Kluyveromyces lactis, glucose uptake in, 47

L p-Lactams mode of action, 186 transport into bacteria, 209-21 2 Lactose, transport in Escherichia coli, 33, 66 Leucine, uptake in Saccharomyces sp., inhibition, 26 Leuconostoc mesenteroides cell-free preparations, polysaccharide synthesis by, 115 dextran production by, 95 dextran sucrase synthesis, acceptors in, 123 regulation, 105 exopolysaccharide production by, substrates, 97 Levan sucrases from Bacillus subtilis, 1 18 regulation, 105 Levans microbial synthesis, 118 from sucrose, 97 synthesis by cell-free preparation, 1 15118

262

SUBJECT INDEX

Lipids as substrates for microbial exopolysaccharide production, 99-1 04 isoprenoid, microbial exopolysaccharide production regulation by, 106109 Lipopolysaccharides microbial production, primers in, 122 sugar nucleotides as substrates, 97 Lomofungin, effect on yeast cell glucan synthesis, 176 Lysine, absorption by yeasts, 21 Lysine permease in yeasts, regulation, 26

Magnesium ions exopolysaccharide production and, 89 by Xanthomonas campestris, and, 95 polysaccharide production by Enterobacter uerogenes and, 86 transport in Saccharomyces cerevisiae, 16 Maltose permease in yeasts, genetic basis, 47-48 Mammalian cells, nutrient transport in, 9 L

Manganese ions, transport in Saccharomyces cerevisiae, 16 Mannans synthesis, by Micrococcus lysodeikticus, 104 by Saccharomyces cerevisiae, 168 Membrane permeability, nutrient transport and, 2 Metabolic inhibitors, yeast cell-wall glucans and, 176-1 77 Metabolism, amino-acid transport and, 18-19 Methionine, uptake by yeasts, feedback control, 26 -, S-adenosyltransport in yeast, gradient coupling and, 38 in yeast vacuoles, 30 Methylamine, concentration by Saccharomyces cerevisiae, 15 Methylation, yeast glucans, 153 a-Methylglucoside permeases, Saccharomvces SDD.. 53-55 L

A

I

Microbial surface, polysaccharide production control and, 104105 Micrococcus lysodeikticus lipopolysaccharide production, 107 mannan production by, 104 Minocycline, mode of action, 187 Moenomycin as inhibitor of C,, isoprenoid alcohol kinase, 108 Moniliniu fructigens, exopolysaccharide production by, 88 Monosaccharides proton symport with, in yeasts, 4750 transport in yeasts, phosphorylation and, 42-46 Mutans formation by Streptococcus mutans, dental caries and, 116 microbial synthesis from sucrose, 97 synthesis by cell-free preparation, 1 15118

Myxococcus xanthus, culture medium, 84

Nudsonia elongata, protoplasts, glucan synthesis in, 169 Neisser ia men ingit idis cell-free preparations, sialic acid preparation by, 119 spharoplast membranes, acetylase for sialic acid synthesis from, 140 Neomycin, transport into bacteria, 194 Neurospora spp., carbohydrate symports, 62 Neurosporu crassa glucose-transport systems, 55-56 ion transport in, 7-12 3-0-methylglucose electrogenic symport, 64 nutrient transport in, 2 sulphate transport in, 60 Nickel ions sugar fermentation and, 45 transport in Saccharomyces cerevisiae, 17 Nitella sp., electrogenic proton pump in, 7 Ni trollen deficiency. effect on glucan synthesis by yeast cell walls, 175

-

SUBJECT INDEX

effect on exopolysaccharide production, 86 exopolysaccharide production by Xanthornonas juglandis and, 92 Norjirimycin mode of action, 186 transport into bacteria, 194, 212-213 Nutrient limitation, effect on yeast cellwall glucan synthesis, 174-176 Nutrients in continuous culture, exopolysaccharide production and, 89-95

0 Ornithine in Saccharornyces cerevisiae, 17, 18 Outer membranes aminoglycoside diffusion across, 194195 chloramphenicol diffusion across, 200 D-cycloserine diffusion across, 203 3,4-dihydroxybutyl-I -phosphonate diffusion across, 204 phosphomycin diffusion across, 205208 showdomycin diffusion across, 2 17 streptozotocin diffusion across, 223 structure, antibiotic uptake and, 188191 tetracycline diffusion across, 225-228 Oxygen deficiency, glucan synthesis by yeast cell walls and, 176

P Papulacandin B, inhibition of glucan synthesis by, 167 Paracoccidioides brasiliensis glucans, structural analysis, 165 yeast-mycelium interconversion, glucan synthesis and, 174 Passive diffusion, tetracyclines, hydrophobicity and, 226228 Penicillin, benzyl-, mode of action, 186 Penicillium notatum, sulphate symport systems, 60 Peptide antibiotics, transport into bacteria, 2 13-21 6 Peptides, mode of action, 186 Peptidoglycans

263

biosynthetic pathway, 202 in Staphylococcus aureus, polyribitol teichoic acid linked to, 124 microbial synthesis, 121 acceptors in, 123 structure, antibiotic uptake and, 191 Periodate oxidation, yeast glucans, 154 Periplasmic space, antibiotic uptake and, 191 Phenylalanine, transport in Saccharomyces cerevisiae, 18 Phosphates, deficiency, glucan synthesis by yeast cell walls and, 176 Phosphate symport in yeasts, 6&61 Phosphomycin diffusion across outer membranes, 205-208 site of action in biosynthetic pathways for peptidoglycans, 202 transport, across cytoplasmic membranes, 208 into bacteria, 205-208 Phosphorus, fungal exopolysaccharide production and, 88 Phosphorylation a-methylglucoside transport into yeast and, 55 monosaccharide transport in yeast and, 4 2 4 6 Physarurn falviconum, exopolysaccharide production, growth phase and, 89 Physarurn polycephalum, exopolysaccharide production, growth phase and, 89 Pichia farinosa cell walls, glucan synthesis, effect of 2-deoxyglucose on, 176 glucan synthesis, 2-deoxyglucose inhibition of, 167 Plasma-membrane vesicles,yeasts, carbohydrate transport in, phosphorylation and, 45 Plumbemycin B, structure, 214 Poly-N-acetylmuraminic acid, production by Escherichia coli, 102 Poly-D-mannuronic acid, epimerization in Azotobacter vinelandii, 134 Polyols proton symport in yeasts, 47-50 respiration and, 52 Polyribitol phosphate, production by Staphylococcus aureus, 101

264

SUBJECT INDEX

Polyribitol teichoic acid, linked to peptidoglycan of Staphylococcus aureus, 124 Polysaccharides microbial production, changes in, 1 12115

control, 104-1 15 microbial secretion, 127-129 modification, 129-135 physical property changes, 129-1 34 shared pathways, 121-122 synthesis by cell-free preparation, 1 15121 Porins, 188 Potassium ions amino-acid transport in yeast and, 31 exopolysaccharide production by Xanthomonas campestris, and, 95 polysaccharide production by Enterobacter aerogenes and, 86 transport in Saccharomyces cerevisiae, 12-17 Primary active transport, solutes, eukaryotic micro-organisms, 4 Primers in microbial polysaccharide production, 122-129 Proline permease in yeasts, 21 Proton gradient hypothesis, amino-acid influx from yeasts and, 38 Proton pump electrogenic, in Neurospora crassa, ATPase as, 11 ion transport in Neurospora crassa and, 7 in yeast plasma membrane, enzymic basis, 12-17 plasma-membrane, Neurospora crassa, enzymic basis, 11-12 Proton symport in Rhodotorula gracilis, respiration and, 50-53 in yeasts, with carbohydrates, 47-50 Protons amino-acid transport in yeast and, 31 as cosubstrate for amino-acid permeases, 33 transport in Saccharomyces cerevisiae, 12-17 Protoplasts, glucan synthetases in, 169171 Pseudomonas spp., exopolysaccharide

production, growth phase and, 89 Pseudomonas aeruginosa alginate production, 89, 90 regulation, 11 1 exopolysaccharide production by, 90 growth phase and, 89 outer membranes, aminoglycoside diffusion through, 195 Pullulan bacterial, biosynthesis, 82 preparation by Aureobasidium pullulans, 88, 89, 104 Purine adenine uptake by yeasts and, 25 transport in yeasts, 22-24 gradient coupling and, 3 8 4 0 Pyrimidine transport in yeasts, 22-24 gradient coupling and, 38-40

Q Quinoline, 8-hydroxy-, effect on yeast cell glucan synthesis, 176

R Regulation, microbial polysaccharide production, 104-1 1 1 Respiration in Rhodotorula gracilis, proton symport and, 5&53 Rhizobium spp., exopolysaccharides, 99 Rhizobium japonicum, cell-free extracts, homopolysaccharide production by, 1 I8 Rhizobium leguminosarum exopolysaccharides from, 1 15 polysaccharide production, regulation, 111 Rhizobium meliloti, polysaccharide production by, culture medium, 86 Rhizobium phaseoli, exopolysaccharides from, 115 Rhizobium trifolii, exopolysaccharide production, 1 13, 1 15 Rhodopseudomonas spheroides tetracycline accumulation by, 230 pH and, 232 Rhodosporidium toruloides - - - See Rhodotorula gracilis

SUBJEC T INDEX

Rhodotorula spp., nutrient transport in, L

Rhodotorula gracilis cell walls, glucan synthesis, effect of 2-deoxy-2-arabinohexose on, 177 glucose uptake in, 47 a-glucoside hydrolysis in, 53 monosaccharide transport in, 47 proton symport in, respiration and, 50-53

S Saccharomyces spp. a-methylglucoside permeases, 53-55 nutrient transport in, 2 phosphate sympdrt systems, 60 Saccharomyces carlsbergensis glucan synthesis, inositol deficiency and, 175 nutrient limitation and, 176 structural analysis, 160 melibiose hydrolysis in, 53 proton symport with carbohydrates in, 47 Saccharomyces cerevisiae alkali-soluble glucans from, structural analysis, 159 amino-acid absorption, genetically distinct pathways, 20-22 amino acids in, 17 cell walls, glucans, structural analysis, 155 glucan synthesis, effect of 2-deoxyglucose on, 176 glucan content, cell cycle and, 172 glucan synthesis, 2-deoxyglucose inhibition of, 167 inositol deficiency and, 175 monosaccharide transport in, phosphorylation and, 42 particulate preparation, p-glucans synthesis by, 168 potassium ion transport in, 12-17 proton transport in, 12-17 protoplasts, glucan synthesis in, 169, 170 purine transport in, 22 sodium ion transport in, 12-17 sucrose hydrolysis in, 53

265

sulphate symport systems, 60 uracil transport in, 39 vacuolar compartments, solute transport and, 27 Saccharomyces fragilis 2-deoxyglucose uptake by, 47 proton symport with lactose in, 47 Saccharomyces uvarum - - See Saccharomyces carlsbergensis Salmonella spp., homopolysaccharide production by, lipid precursors, 102 Salmonella anatum, lipopolysaccharide production by, 113 Salmonella enteritidis, lipopolysaccharides, composition, 133 Salmonella newington, 0-antigen, biosynthesis, 126 Salmonella typhimurium colanic acid from, 135 exopolysaccharide production by, 99 lipopolysaccharide production, 106 outer membranes, structure, antibiotics uptake and, 188 phosphomycin accumulation by, 208 Schizosaccharomyces pombe cell walls, glucans, structural analysis, 162 glucan synthesis, effect of 2-deoxyglucose on, 176 glucans from structural analysis, 160 synthesis, 2-deoxyglucose inhibition of, 167 a-glucoside hydrolysis in, 53 protoplasts, glucan synthesis in, 169 Secondary active transport, solutes, eukaryotic micro-organisms, 4 Secretions in microbial polysaccharide production, 122-129 Shock excretion, amino acids, from yeasts, 29-30 Showdomycin diffusion across outer membranes, 2 17 mode of action, 187 transport, across cytoplasmic membranes, 2 17-2 19 into bacteria, 2 1 6 219 Sialic acid biosynthesis, in Escherichia coli, attachment site, 125 in micro-organisms, 82 production by Escherichia coli, 102 ~

266

SUBJECT INDEX

synthesis, acetylase for, from Neisseria meningitidis sphaeroplast membranes, 140 by cell-free preparations from Neisseria meningitidis, 119 by Escherichia coli, 127, 134 acceptors in, 123 Sideromycins mode of action, 187 structure, 218-219 transport in bacteria, 220-222 Siderophores, structure, 218-219 ‘Slip’ models, solute accumulation, 6668 Sodium ions phosphate symport systems and, 61 transport in Saccharomyces cerevisiae, 12-17 Solute accumulation, steady state, 61-69 Solute transport in eukaryotic micro-organisms, 1-69 reversibility, 24-25 in yeasts, pyrimidine, gradient coupling and, 38-40 purines, in yeast, gradient coupling and, 38-40 Solute uptake, feedback control, yeasts, 25-27 Sophorose from Acetobacter xylinum polysaccharides, 114 L-Sorbose, proton symport in yeasts, 54 Sporobolomyces spp., exopolysaccharide production by, 88 Staphylococcus aureus exopolysaccharide synthesis, acceptors in, 123 isoprenoid alcohol kinase, 109 isoprenoid alcohol phosphokinase from, 121 polyribitol phosphate production by, 101 polyribitol teichoic acid linked to peptidoglycan in, 124 tetracycline accumulation by, 230 pH and, 232 Staphylococcus epidermidis, UDP-Nacetylmuramic acid production in, 140 Streptococcus spp., hyaluronic acid production by, 103 Streptococcus faecalis, free isoprenoid alcohol in, 107

Streptococcus mutans dextran sucrases in, regulation, 106 dextran sucrase preparation from, 117 fructan preparation from, 1 18 mutan production by, dental caries and, 116 Streptococcus pneumoniae cell-free preparations, heteropolysaccharides preparation from, 120 exopolysaccharides, biosynthesis, 80 polysaccharide production, genetic regulation, 109 Streptococcus pyogenes C s s lipid phosphate degradation in, 107 lipopolysaccharide production by, 108 Streptococcus sanguis dextran sucrases in, regulation, 106 dextran synthesis by, 115 Streptomycesfradiae, phosphomycin synthesis by, 205 Streptomyces norjiriensis, norjirimycin synthesis by, 212 Streptomyces sho wdoensis, showdom ycin synthesis by, 21 6 Streptomyces venezuelae, chloramphenicol synthesis by, 200 Streptomycin accumulation, mechanism, 199 mode of action, 186 structure, 194 Streptozotocin diffusion across outer membranes, 223 mode of action, 187 transport, across cytoplasmic membranes 223-224 into bacteria, 194, 222-224 Strontium ions, transport in Saccharomyces cerevisiae, 16 Substrates, microbial exopolysaccharide production and, 95-104 Succinoglucan production by Alcaligenes faecalis var. myxogenes, 112 shared pathways, 121 Sucrose as substrate for microbial heteropolysaccharide production, 95 proton symport in yeasts, 54 Sugar nucleotides as substrates for microbial exopolysaccharide production, 97-99

267

SUBJECT INDEX

Sulphate symports in yeasts, 60-61 Sulphur, exopolysaccharide production by Xanthomonas juglandis and, 92 Symport mechanisms, conclusions, 6869

T Target sites, antibiotics, 185 Teichoic acid microbial synthesis, 121 sugar nucleotides as substrates, 97 Tetracyclines diffusion across outer membranes, 225-228 diffusion into bacteria, effect of charge on, 228 mode of action, 187 transport across cytoplasmic membranes, 228-232 transport into bacteria, 225-232 Thiamin, concentration by Saccharomyces cerevisiae, 16 Thiolutin, effect on yeast cell glucan synthesis, 176 Torulopsis dattila, D-fucose absorption by, 47 Transinactivation in feedback control of solute uptake by yeasts, 25-27 Transinhibition in feedback control of solute uptake by yeasts, 25-27 Transport antibiotics, definition, 185-1 87 into bacteria, 183-234 Trehalose, proton symport in yeasts, 54 Tremella mesenterica, cell walls, glucan synthesis, 177 Tyrosine, transport and metabolism in yeasts, 19

UDP-galacturonic acid as substrate for microbial exopolysaccharide production, 97 UDP-glucose as substrate for exopolysaccharide production, 97 common precursor of succinoglucan and curdlan, 121 UDP-glucose pyrophosphorylase, synthesis, derepression, 11 1 Uptake antibiotics, 185 definition, 185-187 Uracil, transport in Saccharomyes cerevisiae, 39 Uracil permeases, regulation in yeasts, 26 Urea influx kinetics by yeasts, 29 transport in yeast, gradient coupling and, 38 Uridine permeases, regulation in yeasts, 26 Uronic acids, methyl, in Rhizobium polysaccharides, 99

v Vacuolar compartments in yeasts, role in solute transport, 27-29 Vacuolar transport systems, amino acids, in yeasts, 20-31 Valine, transport and metabolism in yeasts, 19 Viscosity, polysaccharides, 129

W Whey, microbial exopolysaccharide production and, 87

U

X

UDP-N-acetylmuramic acid, production in Staphylococcus epidermidis, 140 UDP-galactose as substrate for exopolysaccharide production, 97 UDP-galactose-4-epimerase, colanic acid production in Escherichia coli and, 110

Xanthan production by Enterobacter aerogenes, 113 structure, 137-140 Xanthomonas albilineans, polysaccharides from, structure, 137 Xanthomonas axonopodis, polysaccharides from, structure, 137

268

SUBJECT INDEX

Xanthomonas campestris exopolysaccharide production, 86, 90, 92, 94, 113 lipid precursors, 101 nutrient limitation in continuous culture and, 92 polysaccharide, production, 128 modification, 129 structure, 137-140 Xanthomonas fragaria, polysaccharides from, structure, 137 Xanthomonas juglandis exopolysaccharide production, nutrient limitation in continuous culture, 92 polysaccharides production, 134 nutrients and, 96

Yeast-mycelium interconversion, cell wall glucans and, 174 Yeast permeases, 23 Yeast plasma membrane, proton pump, enzymic basis, 12-1 7 Yeasts amino acids, concentration by, 37 transport in, 17-42 carbohydrates, symports, 62 transport in, 42-55 cell-wall glucans, 15 1-1 77 synthesis, 166171 glucans, structural analysis, 152-1 66 proton symport with carbohydrates in, 47-50 purine transport in, gradient coupling and, 38-40 pyrimidine transport in, gradient coupling and, 3 8 4 0 sulphate symport systems in, 60

Y D-Xylose, proton symport in yeasts, respiration and, 50-51 Yeast membrane vesicles, amino-acid binding proteins and, 4&42

z Zinc ions, transport in Saccharomyces cerevisiae, 16

Note added in proof Mechanisms of solute transport in selected eukaryotic micro-organisms A. A. EDDY

Two recent studies with purified enzyme preparations have strengthened the evidence that the plasma membrane adenosine triphosphatase (ATPase) of both Schizosaccharomyces pombe (Amory et al., 1980) and Saccharomyces cerevisiae (Malpartida and Serrano, 1981a) forms an acylphosphate intermediate. The purified ATPase from Sacch. cerevisiae was reconstituted in liposomes. In the presence of ATP, these quenched the fluorescence of an acridine dye, in a manner indicating that the enzyme functioned as a proton pump that tended to acidify the interior of the vesicles (Malpartida and Serrano, 1981b). The balance of somewhat conflicting evidence favours the view that proton pumping in these preparations was electrogenic (Malpartida and Serrano, 1981b, c). Significant progress has been made with the problem of characterizing membrane proteins that appear to be involved in uptake of phosphate by a certain strain of Candida tropicalis. This yeast possesses two phosphate-transport systems. One of these is enhanced dyring phosphate starvation and exhibits a relatively high affinity for phosphate corresponding to an apparent K , value of about 5 phi. There is preliminary evidence that this system is a proton symport. The other constitutive system exhibits an apparent K , value of about 1 mM (Blasco et al., 1976). When the yeast preparations were subjected to a controlled osmotic shock, protein components were released which bound phosphate in assays based on a Sephadex filtration technique. Evidence for the presence of two main protein components was obtained, one of which exhibited a high affinity for phosphate and the other a lower affinity. These components, which lack alkaline phosphatase activity, appear to be involved in phosphate transport (Jeanjean and Fournier, 1979; Jeanjean et al., 1981). Thus, the fractionated materials slightly stimulated phosphate uptake into the hypotonically shocked yeast cells. Furthermore, specific antisera which partially inhibited phosphate binding to the protein fractions, also partially inhibited phosphate uptake by the intact yeast (Jeanjean et al., 1981). Woodward and Kornberg (1980) identified at least three proteins associated with plasma membrane preparations from a strain of Sacch. cerevisiae carrying the general amino-acid permease and which are absent from a mutant yeast strain lacking this permease. A further component of the permease which bound the tryptophan analogue 6-N-chloroacetyl ornithine appeared to be located in the periplasmic space. While it was not released during a controlled osmotic shock, it was released during conversion of cells into sphaeroplasts. Woodward and Kornberg (1980) suggest that the general amino-acid permease is a multiprotein complex which may contain proteins with apparent molecular weights of 53,000,45,000and 30,000, respectively, together with the loosely bound component of about 14,000 apparent molecular weight. Biosynthesis of a protein of about 31,000 molecular weight occurred during reactivation of the general amino-acid permease following an earlier period of exposure of the yeast to valine which inactivated the permease (transinhbition). Correspondingly, a similar protein component was missing from the plasma membrane of yeast preparations that had been subjected to transinhibition by valine (Woodward and Kornberg, 1981). These observations provide a fresh insight into the possible molecular basis of the control of the activity of the general amino-acid permease of yeast. The mechanism of uptake of basic amino acids by vesicles derived from the yeast

vacuole has been greatly clarified with the demonstration that a MgZ+-ATPaseis involved in the process. Ohsumi and Anraku (1981) propose: (1) that the ATPase is electrogenic and acidifies the interior of the vacuoles, producing a proton gradient of the order of 0.17V; and (2) arginine is concentrated therein by means of an H H+/arginine antiport. Some evidence for uptake of tyrosine and isoleucine was also obtained, but not of glycine, proline or glutamate. These observations add weight to the view that both the plasma membrane and the vacuolar membrane of yeast carry amino-acid pumps. Rodriguez-Navarro et al. (1981) have recently put forward the novel suggestion that efflux of Li+ from yeast, and possibly that of K + and N a + , may be based on an antiport with protons whereby the net process is electrogenic, more than one proton being absorbed with each Lic expelled. REFERENCES

Amory, A,, Foury, F. and Goffeau, A, (1980). Journal of Biological Chemistry 255, 9353. Blasco, F., Ducet, G . and Azoulay, E. (1976). Biochimie 58, 351. Jeanjean, R. and Fournier, N. (1979). Federation of European Biochemical Societies Letters 105, 163. Jeanjean, R., Attia, A,, Jarry, T. and Colle, A. (1981). Federation of European Biochemical Societies Letters 125, 69. Malpartida, F. and Serrano, R. (1981a). European Journal of Biochemistry 116, 413. Malpartida, F. and Serrano, R. (198 1b). Federation of European Biochemical Societies Letters 131, 351. Malpartida, F. and Serrano, R. (1981~).Journal of Biological Chemistry 256, 4175. Ohsumi, Y. and Anraku, Y. (1981). Journal of Biological Chemistry 256, 2079. Rodriguez-Navarro, A., Sancho, E. D. and Perez-Lloveres, C . (1981). Biochimica et Biophysica Acta 640, 352. Woodward, J. R. and Kornberg, H. L. (1980). Biochemical Journal 192, 659. Woodward, J. R. and Kornberg, H. L. (1981). Biochemical Journal 196, 531.