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Advances in
MICROBIAL PHYS IOLO GY
This Page Intentionally Left Blank
Advances in
MICROBIAL PHYSIOLOGY edited by
A. H. ROSE School of Biological Sciences Bath University England
D. W. TEMPEST Laboratorium voor Microbiologie Universiteit van Amsterdam Amterdam-C Th-eNetherlands
Volume 15
1977
ACADEMIC PRESS London New York San Francisco A Subsidiary of Harcourt Brace Jouanouich, Publishers
ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road London NWI United States Edition published by ACADEMIC PRESS INC. 111 Fifth Avenue New York, New York 10003
Copyright 0 1 9 7 7 by ACADEMIC PRESS INC. (LONDON) LTD.
All Rights Reserved N o part of this book may be reproduced in any form by photostat, microfilm, or any
other means, without written permission from the publishers
Library ofCongress Catalog Card Number: 67-19850 ISBN: 0-12-027 7 15-8
Printed in Great Britain by William Clowes and Sons Limited London, Colchester and Beccles
Contributors to Volume 15 D. E. ATKINSON, Molecular Institute and Biochemistry Division, Department of Chemistty, University of California, Los Angeles, California 90024, U.S.A. A. T . BULL, Biological Laboratory, University ofKent, Canterbury C T 2 7NJ, England. (Present address :Department of Applied Biology, University of Wales Institute ofStience and Technology, C a r d g CFI 3 N U , Wales) A. G. CHAPMAN, Molecular Biology Institute and Biochemistry Division, Department of Chemistry, University of California, Los Angeles, California 90024, U.S.A. M. CRANDALL, School of Biological Sciences, University of Kentucky, Lexington, Kentucky 40506, U.S.A. I . E. D. DUNDAS, Institutt for Generell Mikrobiologi, Universitetet i Bergen, Bergen, Nonuay R. EGEL, Institut fur Biologie 111 der Universitat Freiburg, 0 - 7 8 0 0 Freiburg, Schanzlestrasse +I I ,Federal Republic of Germuny C. G. ELLIOTT, Botany Department, University of Glasgow, Glasgow, Scotland W. N. KONINGS, De artment ofMicrobiology, Biolo ‘cal Centre, University of Groningen, Kerk Lan 30, Haren, The Netherla s V. L. MACKAY, Waksman Institute of Microbiology, Rutgers University, New Brunswick, NewJersey 08903, U.S.A. A. P. J . TRINCI, Department of Microbiology, Queen Elizabeth College, University of London, London W8 7 A H England
2
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Contents The Physiology and Metabolic Control of Fungal Growth
. .
. . .
A T BULL and A P J TRlNCl
I . Introduction . . . . . . . . . . . . . . . . .‘ I1. Mathematical Modelling of Fungal Growth . . . . . . . . . A. WhyModel? . . . . . . . . . . . . . . . . B . Approaches to Modelling . . . . . . . . . . . . C. The Applicability of Classic Models to Fungal Growth . . . . . I11. Growth of Undifferentiated Mycelia . . . . . . . . . . . A. Regulation of Mycelial Form . . . . . . . . . . . . B . Polarization of Hyphal Growth . . . . . . . . . . . C . Regulation of Branch Initiation . . . . . . . . . . . D. Hyphal Growth Units of Different Strains and Species . . . . . E. Effect of Environment on Hyphal Growth Unit Length . . . . F. RegulationoftheSpacialDistributionofHyphae . . . . . . IV. Colony Growth . . . . . . . . . . . . . . . . . A. Colony Differentiation . . . . . . . . . . . . . B . Mould-Induced Changes in the Substrate . . . . . . . . C. Kinetics of Colony Expansion on Solid Media . . . . . . . D. Colony Expansion as a Parameter of Mould Growth . . . . . E. Comparison of the Colonization of Solid Substrates by Moulds and Unicellular Micro-organisms . . . . . . . . . . V . Fungal Growth in Submerged Liquid Culture.Technical Considerations VI . Kinetics of Fungal Growth in Submerged Liquid Culture . . . . . A. RatesofGrowth . . ,. . . . . . . . . . . . . B . Transient States and Oscillatory Phenomena . . . . . . . C. “Macroregulation” of Growth . . . . . . . . . . . D . Maintained and Starved States . . . . . . . . . . . VII . Transport Controlled Features of Growth . . . . . . . . . A . Transport-Limited Growth . . . . . . . . . . . . B . Transport Regulation . . . . . . . . . . . . . . C. Modulation of Fungal Transport Processes . . . . . . . . VIII . Metabolic Control in Fungi . . . . . . . . . . . . . A. Intermediary Metabolism . . . . . . . . . . . . B . Anaplerotic Metabolism . . . . . . . . . . . . . C. Aspects ofTermina1 Oxidation . . . . . . . . . . . IX. RNA Synthesis and Function: Rate-Limiting Parameters of Growth . . A . Efficiency of Protein Synthesis . . . . . . . . . . . B . Concerning Polyamines . . . . . . . . . . . . . X. Acknowledgements . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . vii
2 3 3 4 7 13 14 14 15 19 20 23 23 23 21 29 33
35 36 40 40 43 46 41 48 48 50 53 51 57 62 61 71 12 14 16 16
viii
CONTENTS
Physiology of Halobacteriaceae
. . .
1 E D DUNDAS
I . Introduction . . . . . . . . . . . . . . . . . I1. Classification of Extreme Halophiles . . . . . . . . . . I11. Intracellular and Extracellular Salt Concentrations . . . . . . A. Intracellular Salts . . . . . . . . . . . . . . . B . Extracellular Salts . . . . . . . . . . . . . . IV. Subcellular Structures . . . . . . . . . . . . . . A. Cell Envelopes . . . . . . . . . . . . . . . B . Ribosomes . . . . . . . . . . . . . . . . C. Vacuoles . . . . . . . . . . . . . . . . . D . Flagella . . . . . . . . . . . . . . . . . V. Halophilic Proteins . . . . . . . . . . . . . . . A. Metabolic Pathways . . . . . . . . . . . . . . B . Halophilic Enzymes . . . . . . . . . . . . . . VI . Lipids in Halobacteriaceae . . . . . . . . . . . . . VII . Electron-Transport Chain . . . . . . . . . . . . . . VIII . Transport Across Membranes . . . . . . . . . . . . IX. Effects of Light . . . . . . . . . . . . . . . . A . Photophosphorylation . . . . . . . . . . . . . B . Effect on Growth and Viability . . . . . . . . . . . X. Nucleic Acids and Their Enzymology . . . . . . . . . . XI . Phage-Host Relationships . . . . . . . . . . . . . XI1. Ecological Considerations on the Existence of Obligate Extreme Halophilism . . . . . . . . . . . . . . . . . XI11. Acknowledgements . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .
85 86 88 88 90 91 91 94 96 99 100 101 102 104 106 107 109 109 110 111 113 114 116 116
Sterols in Fungi: Their Functions in Growth and Reproduction CHARLES G . ELLIOT I. I1. I11. IV. V.
Introduction . . . . . . . . . . . . . . . . . Functions of Sterols: Possible Approaches to the Problems . . . . Sterols in Model Systems . . . . . . . . . . . . . . Subcellular Distribution of Sterols in Fungi. and States of Binding . . Effects of Sterols on Metabolism and Vegetative Growth . . . . . A. e t h i u m and Phytophthora . . . . . . . . . . . . . B . Saccharomyces and Other Fungi . . . . . . . . . . . VI . Effects of Sterols on Asexual Reproduction . . . . . . . . . VII . Sexual Hormones of Achlya . . . . . . . . . . . . . VIII . Effects of Sterols on Sexual Reproduction in Homothallic Species of Pythium and Phytophthora . . . . . . . . . . . . .
121 123 130 135 141 141 144 148 149 152
CONTENTS
ix
IX. Reproduction in Heterothallic Species of Pythium and Phytophthm
. . . . . .
X. Sterols and Sexual Reproduction in Ascomycetes and Basidiomycetes XI . Conclusion . . . . . . . . . . . . . . . . XI1. Acknowledgements . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .
156 162 165 166 166
Active Transport of Solutes in Bacterial Membrane Vesicles
.
WIL N KONINGS
I . Introduction . . . . . . . . . . . . . . . . . I1. Membrane Vesicles . . . . . . . . . . . . . . . A. Isolation Procedures . . . . . . . . . . . . . . B . Physical Properties . . . . . . . . . . . . . . C. Purity of Membrane Preparations . . . . . . . . . D. Functional Properties . . . . . . . . . . . . . . E . Orientation of the Vesicle Membrane . . . . . . . . . F. Localization of D-Lactate Dehydrogenase in Membrane Vesicles from Escherichiu coli . . . . . . . . . . . . . . . . I11. Active Transport Coupled to Electron Transfer Systems . . . . . . A . Coupling to Respiratory Chain . . . . . . . . . . . B . Couplingto AnaerobicElectronTransfer Systems . . . . . . C . Coupling toCyclic ElectronTransferSystems . . . . . . . IV. Energy Coupling to Active Transport . . . . . . . . . . . A. Role ofAdenosine 5'-Triphosphate and the ATPase Complex . . . B . Mechanism of Energy Coupling . . . . . . . . . . . C. Energy-Dependent Binding of Solute to Carrier Proteins . . . . V. Conclusions . . . . . . . . . . . . . . . . . . VI . Acknowledgements . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .
175 177 177 180 183 184 194 200 203 203 217 223 225 225 228 239 243 244 244
Adenine Nucleotide Concentrations and Turnover Rates. Their Correlation with Biological Activity in Bacteria and Yeast
.
.
ASTRID G CHAPMAN and DANIEL E ATKINSON
I . Introduction . . . . . . . . . . . . . . . . . I1. Concentrations and Fluxes ofAdenine Nucleotides in uiuo . . . . . A. Adenine Nucleotide Turnover . . . . . . . . . . . . B . Turnover ofATP . . . . . . . . . . . . . . . C. RegulationofATP Utilizationand Regeneration . . . . . . . D . Sampling of Microbial Cultures for Adenine Nucleotide Determinations E. Changes inAdenineNucleotide Concentrations . . . . . . .
254 256 256 261 268 269 272
X
CONTENTS
I11. Concentration of ATP. Total Adenine Nucleotide Concentration. and . . . . . . . Energy Chargein Relation to Cellular Activities A . Relation between ATP Concentration and Growth Rate . . . . . B . Variations in Adenine Nucleotide Levels during Growth . . . . C. Adenine Nucleotides in Mutant Strains Arrested in Growth . . . . D . Correlation between Kinetics in vitro and Observations in vivo . . . E. Relation between Energy Charge and Total Adenine Nucleotide Concentration . . . . . . . . . . . . . . . . F. Phage Infection . . . . . . . . . . . . . . . . G . Other Nucleotides . . . . . . . . . . . . . . . H . RNASynthesis . . . . . . . . . . . . . . . . I . Protein Synthesis . . . . . . . . . . . . . . . IV. General Discussion . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . .
282 282 285 286 287 289 290 291 293 295 297 300
Physiology of Mating in Three Yeasts
.
.
MARJORIE CRANDALL RICHARD EGEL and VIVIAN L MACKAY
I . Introduction . . . . . . . . . . A. Ecology . . . . . . . . . . . . . . . . . B . General Characteristics C . Lifecycles . . . . . . . . . . I1 . Hamenula Winget . . . . . . . . . A . Mating Type Locus . . . . . . . . B . Haploid Functions . . . . . . . . C. DiploidFunctions . . . . . . . . 111. Schizaracchmomycespobe . . . . . . . A . Mating Type Locus . . . . . . . . B . HaploidFunctions . . . . . . . . C . DiploidFunctions . . . . . . . . IV . Saccharomyces cerevisiae . . . . . . . . A . Mating Type Locus . . . . . . . . B . Haploid Functions . . . . . . . C. DiploidFunctions . . . . . . . . V. Comparative Discussion . . . . . . . A. Steps in Yeast Conjugation Compared . . B . Evolutionary Aspects of Sexual Reproduction C. Comparison with Mammalian Systems . . VI . Acknowledgements . . . . . . . . References . . . . . . . . . . . Author Index . . . . . . . . . . . Subject Index . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
307 309 310 310 313 313 314 327 331 331 336 347 350 350 354 377 384 384 390 391 392 392 399 419
The Physiology and Metabolic Control of Fungal Growth A. T. BULL* and A. P. J. TRlNCl Biological Laboratory, University of Kent, Canterbury CT2 7NJ, England; Department of Microbiology, Queen Elizabeth College, University of London, London W8 7AH. I. Introduction
.
.
.
.
.
11. Mathematical Modelling of Fungal Growth
.
.
. . .
.
. . .
.
. . .
.
. .
.
. . . . . .
A. Why Model? . . . . . . B. Approaches to Modelling . . . . C. The Applicability of Classic Models to Fungal Growth . 111. Growth of Undifferentiated Mycelia . . . . . A. Regulation of Mycelial Form . . . . . . B. Polarization of Hyphal Growth . . . . . . . C. Regulation of Branch Initiation . . . . . . . D. Hyphal Growth Units of Different Strains and Species . . E. Effect of Environment on Hyphal Growth Unit Length . . F. Regulation of the Spacial Distribution of H p h a e . . . 1V. Colony Growth . . . . . . . . . . . A. Colony Differentiation . . . . . . . . . B. Mould-Induced Changes in the Substrate . . . . . C. Kinetics of Colony Expansion on Solid Media . . . D. Colony Expansion as a Parameter of Mould Growth . . E. Comparison of the Colonization of Solid Substrates by Moulds and Unicellular Micro-organisms . . . . . . . V. Fungal Growth in Submerged Liquid Culture. Technical Considerations VI. Kinetics o f Fungal Growth in Submerged Liquid Culture . . A. Rates of Growth . . . . . . . . . . B. Trmisient States and Oscillatory Phenomena . . . . C. "Macroregulatioti" of Growth . . . . . . . D. Maintained arid Starved States . . . . . . . VII. Trailsport Controlled Features of Growth . . . . . A. Traiisport-Liiiiited Growth . . . . . . . . B. Transport Regulation . . . . . . . . . C. Modulatioii of Fungal Transport Processes . . . .
* Present address: Department ol'Applied Biology. Uiiivcrsity ol'W i i h Technology, Cardill; CFI 3NU 1
Illbtittitc
2 3 3 4 7 13 14 14
15 19 20 23 23 23 21 29 33
35 36 40 40 43 46 47 4X *X 50
53
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A. T. BULL AND A. P. J. TRlNCl
VIII. Metabolic Control in Fungi . . . . . . . * A. Intermediary Metabolism . . . . . . . B. Anaplerotic Metabolism . . . . . C. Aspects of Terminal Oxidation . . . . . . . IX. RNA Synthesis and Function: Rate-Limiting Parameters of Growth A. Efficiency of Protein Synthesis . . . . . . B. Concerning Polyamines . . . . . . . . X. Acknowledgements . . . . . . . . . . References . . . . . . . . . . . .
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57 57 62 67 71 12 74 76 76
I. Introduction “The modern era of mould metabolism has only scarcely begun, but signs of an immense advance in the numerous phases of the field are unmistakable. Their importance is recognized, practically and academically’’.Thus commented Jackson Foster in the introduction of his unique contribution to the study of fungal physiology and biochemistry, “Chemical Activities of Fungi” (Foster, 1949). A vast mycological literature had accumulated by the time of Foster’s original persuasion, in 1936, to prepare a critical account of fungal metabolism and, it is worth recalling, several aspects of this subject had already been established during the previous century. Pre-Foster fungal physiology had relied extensively on a classical response-to-stimulus type of approach, an approach unfortunately that generated much conflicting and confusing data. It was Foster’s conspicuous talent and understanding that brought together much of this data in an intelligible form and was instrumental in orientating subsequent researches. I t is appropriate, therefore, by way of a preface to our main discussions, to examine the current status of fungal biochemistry. We would advocate, from an admittedly biased position, that fungi unquestionably are the organisms of choice for the study of numerous aspects of microbial biochemistry: one needs only to ponder their extreme morphological diversity and plasticity, their unrivalled biosynthetic capacities, especially with respect to secondary metabolites, and their utility as model systems in the biochemical analysis of mating behaviour, differentiation, ageing and so on, to find the validity of this assertion. And yet our knowledge of fungal biochemistry remains rather fragmentary, while specific reference to investigation of growth physiology shows that the majority of work has been done with bacteria. Two important reasons for this situation can be proffered that have their bases in: (a) long-standing technical difficulties of growing mycelial fungi; and (b) lack of adequate kinetic analyses of
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
3
their mode of growth. Accordingly, we have devoted a portion of this review to the practicalities of growing filamentous fungi, and have developed at greater length mathematical treatments of mould growth and discussed the applicability of some existent models. We have singled out the growth of surface colonies for particular discussion because, despite the fact that it is a technique used by mycologists ad libitum,it has received little critical analysis; moreover, it has been the subject recently of renewed interest. Strain instability also has plagued the efforts of the fungal physiologist, and this has been an especially acute problem in relation to the Fungi Imperfecti many of which are of major interest in the context of commercial fermentations. Problems of strain selection and strain degeneration can seriously hamper prolonged continuous-flow culture experiments with moulds and care has to be taken to check biological stability under these conditions. The scope of this article is such that we have, of necessity, been selective in our choice of topics to discuss. The selection has been resolved in two ways: by taking those areas of fungal physiology and biochemistry that have been researched in depth, and by attempting to indicate areas that are as yet largely unexplored but seem to us to warrant special attention. In addition we have tried to keep as a theme running throughout the discussion, the modulation of fungal metabolism in response to growth conditions. Palpable absences from this article are references to “secondary” or “shunt” metabolism, at which fungi are most adept, and to the metabolic control of differentiation: in both of these areas, notable advances have been made in recent years (see Smith and Anderson, 1973). But one of our prime objectives in the following pages is to demonstrate to the microbial physiologist and biochemist that the fungi arguably are among the most propitious and alluring of all micro-organisms that he can select for his studies and that von Haller’s eighteenth century portrayal of them as “a mutable and treacherous tribe” is no longer the most apposite of epithets. 11. Mathematical Modelling of Fungal Growth A.
WHY MODEL?
Any mathematical modcl of growth attempts to specify interrelationships between the many components of the system, physical, chemical and biological and, clearly, such components must be
4
A. T. BULL AND A. P. J. TRlNCl
capable of being quantified. The system then is usually described for convenience by a series of differential equations which, depending on the complexity of the-model, may be solved manually or by the aid of computing techniques. A model is likely to be of practical value only if it is mathematically tractable and can be analysed to predict responses to defined environmental conditions, and if it provides a reasonable fit of the experimental data. Topiwala (1973) recently has cautioned that apparently successful models do not conclusively validate basic assumptions made in the model because alternative models may produce similar conclusions. Nevertheless, if a given theory of growth cannot be modelled satisfactorily it is unlikely to be a valid one. What then are the purposes of modelling? First, in formulating models, all descriptions and definitions need to be rigorous, free of ambiguity and be capable of being expressed in mathematical terms. Thus, the microbiologist is obliged to think precisely and systematically about the growth system under study, and for which experimental data can be obtained. Second, models have a conceptual function in helping to focus attention on, and in revealing, fundamental properties of the system. Consequently models enable predictions to be made of the behaviour of the biological system under a limitless range of conditions which may not have been investigated in the laboratory. Indeed computer simulation studies now provide important strategic approaches in both research and process microbiology. An instructive example in the fungal field is provided for the griseofulvin fermentation by Calam et al. ( 197 1). Finally, following on from the latter point, modelling can be a considerable guide in the design of experiments and in the interpretation of experimental data. B . APPROACHES TO MODELLING
Numerous types of mathematical models have been proposed to describe microbial growth and behaviour, and these reflect the particular approaches and objectives prescribed by the modeller. The majority of this research, reported in the chemical-engineering, biochemical and sanitary literature, is frequently overlooked by the microbial physiologist and we feel that it is pertinent to include a brief resume of the main criteria considered in model construction. Although the microbial physiologist most often is concerned with population dynamics, any population of micro-organisms comprises
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
5
individuals whose size, composition and metabolism is distributed over a definable range that itself is dependent on the growth conditions. Thus, models that recognize the differing physiological states of individuals in a population are termed segregated. However, it is more usual to neglect the variations between individuals (thus simplifying the mathematics and avoiding statistical correlations) and instead to treat population dynamics in terms of variations in average properties; models of the latter type are called unsegregated (distributed or non-segregated). In passing we should note that, in models based on a distribution of individuals of different physiological states, the distribution per se may be dependent on the existent environment. These models are referred to as being structured. Again, however, most of the models with which a microbiologist is familiar disregard population structure and more simple unstructured modelling terms of reference are adopted. Unsegregated models also may be endowed with structure if composition changes of the population during the course of cultivation are admitted. For example, biochemical structure was incorporated into an unsegregated growth model by Ramkrishna el al. ( 1966) who divided microbial biomass into G mass (nucleic acids) and D mass (proteins) components. Because the growth rate of individual micro-organisms cannot be predicted with complete certainty, stochastic population, or probabilistic, models have been formulated that take into account the variability of generation times. Once more, due to the “formidable mathematical difficulties” that arise “when one attempts to model only very simple biological phenomena” (Frederickson et al., 19701, stochastic models are usually disregarded in favour of simpler deterministic models. In summary, therefore, most of the commonly used mathematical models of microbial growth are unsegregated, unstructured and deterministic, assumptions that are largely valid when population sizes are large, i.e. the kinetics of microbial growth can be developed on the lines of established chemical reaction kinetics. The classic example of a model of this type is that proposed by Monod ( 1942, 1949) and having its origins in the much neglected researches of M’Kendrick and Pai (191 1). The latter showed: (1) that exponential growth proceeded in batch cultures as long as the “nutriment” supply was unlimited; (2) that the rate of growth was proportional to the number of organisms present (i.e. that growth was autocatalytic); and (3) that they were clearly appreciative of such growth co-efficients as yield and
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A. T. BULL AND A.
P. J. TRlNCl
maintenance. Monod related the specific growth rate (p) to the concentration of growth limiting substrate (s) thus: p =pcmax SI(K,+ S), where pmax is the maximum value of p under a given set of growth conditions and K, is the substrate saturation constant ( K , = S at p =pmax/2). Although the Monod expression is formally analogous to the Michaelis-Menten equation relating enzyme reaction velocity to substrate concentration, there are important distinctions between the two models. Thus, the rate constant, p, in the Monod equation is a logarithmic function of S, whereas v in the Michaelis-Menten equation is related linearly to S. Further, whereas the Michaelis-Menten constant, K,, has a mechanistic basis, K , is an empirical constant. From its first proposition, the Monod model was recognized as an oversimplification but it should be remembered that its original objective was in the curve-fitting of experimental data. Two other unsegregated growth models, less widely used than that of Monod, will be mentioned briefly. The first of these, the logistic equation, includes a term to describe the decrease in growth rate as the limiting substrate becomes exhausted and predicts a maximum population or stationary growth phase in batch cultures. The logistic equacion can be variously expressed (cf. Tsuchiya et al. 1966; Hockenhull and MacKenzie, 1968; Maynard-Smith, 1968) one form of which is: x=
xOePt
l-px.(l - P )
where x and xo are microbial biomass concentrations at time t and o, and p is a constant which may be written: = Y(So+ YxJ where Y is the yield factor with respect to the growth-limiting substrate, concentration S,at zero time. Finally we wish to draw attention to a threeconstant growth model (Dabes et al., 1973) which embodies Blackman kinetics (i.e. the concept of a rate limiting step in a biological process). Dabes and his colleagues considered the situation in which two slow enzymic steps were separated by fast reactions, the overall equilibrium constant of which is not large, and their model, unlike the Monod and logistic equations, contain three constants :
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
7
B is the growth-limiting substrate saturation constant, and A is a constant incorporating overall and rate-limiting equilibrium constants. This model has the potential of indicating the proximity of ratelimiting reactions to each other from the magnitude of B relative to Ap,,,; as B approaches zero, the equilibrium constant between the reactions increases. In the following section we examine the applicability of existing models, particularly those referred to above, to describe growth of filamentous fungi. In short, how adequate for fungi are models developed to express the growth of unicellular micro-organisms? C . THE A P P L I C A B I L I T Y O F C L A S S I C MODELS T O FUNGAL GROWTH
At the outset, the extreme morphological plasticity of fungi must be recognized. In liquid culture, a particular species may grow as a diffuse mycelium, in the form of variously sized pellets or other aggregations or, quite frequently, may develop a yeast-like morphology. Diffuse mycelial and pellet growth will be considered here while the growth of surface colonies of fungi will be examined in detail in subsequent parts of this article (p. 29). 1. Distribution $Metabolic Activities in Mycelial Systems
I f we wish to approach fungal growth in terms of unsegregated, unstructural models, an immediate question arises : are metabolic activities distributed evenly among the hyphal cells of a multicellular mycelium? In other words, is the mycelium differentiated biochemically? We will assume for the present that significant morphological differentiation does not occur during unrestricted growth in batch cultures or in steady-state continuous-flow cultures. Unfortunately there have been very few studies of the distribution of activities along fungal hyphae. Fencl et al. (1969) used micro-autoradiography to analyse the distribution of RNA synthesis in Aspergillis niger mycelia growing in batch and multi-stage chemostat cultures. Synthesis of RNA was distributed evenly throughout the mycelium as long as batch cultures were growing exponentially, but differential synthesis occurred in stationary-phase mycelia. This fungus showed diauxic growth on a sucrose-nitrate medium, and unequal RNA and protein synthesis was evident in the mycelia of the second exponential phase (Machek and Fencl, 1973). However, when mycelia from either
8
A. T. BULL AND A. P. J. TRlNCl
the stationary or the second exponential phase were given a nutritional shift-up, the distribution of activity reverted to that typical of the first exponential phase. The results from multi-stage chemostat experiments substantiated these findings. A three-stage chemostat culture was established in which: (1) the dilution rate ( D )in the first stage was 0.04 h-’; (2) fresh medium was fed to the third stage to make D equal to 0.17 h-l; and (3) additions were not made to the second stage, i.e. conditions which were considered to resemble those at the onset of the stationary phase in a batch culture. In all three vessels the mycelium behaved as a homogenous entity, all hyphal cells synthesizing RNA even if at a very low rate as in the case of the stage 2 population. Data from Terui’s laboratory strongly support the findings of the Czech workers. Shinmyo and Terui ( 1970)observed uniform incorporation of 14C-adenineand I4C-guanosine into “pulpy” (i.e. diffuse) mycelia of A . niger growing as hanging drop microcultures, and also noticed that all hyphal cells had a uniform growth potential, i.e. longitudinal growth of apices or formation of branches in subapical cells. Similar observations to the latter have been made on chemostat cultures of A . nidulans (M. E. Bushell and A. T. Bull, unpublished experiments). On the basis of these analyses, therefore, it seems justifiable to treat rate relationships in moulds in terms of the average kinetics discussed above (Section 11, B; p. 4). I t must be stressed that our last conclusion applies on4 to liquid cultures of diffusely growing mycelia. Shinmyo and Terui (1970) pointed out that the distribution of growth activities in the hyphal cells of pellets was highly heterogeneous. Much earlier Camici et al. (1952) had reported that the centres of dense pellets of Penicillium chrysogenum contained mycelium that was frequently dead or autolysing, observations later confirmed in other species by Yanagita and Kogane ( 1963a). Indeed, the latter authors showed that extensive morphological differentiations occurred in mycelia adjacent to the central spaces in pellets while, at a metabolic level, RNA synthesis was strictly limited to the peripheral hyphae of the pellet. Thus, although pelleted growth may be desirable for the formation of certain fungal products, for example ergot alkaloids (Tonolo et al., 1961), citric acid (Clark, 1962) and certain enzymes (a-galactosidase; Kobayashi and Suzuki, 19721, their highly differentiated nature makes them totally unsuitable for most metabolic studies. In passing we should note that, like pellets, surface colonies of fungi
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
9
become differentiated metabolically, and usually morphologically, with increasing age and this process is clearly evident from changing enzyme patterns. Isaac (1964) described a clear differentiation of Rhizoctoniu soluni colonies with respect to the distribution of cellulase
activity; enzyme production was associated with short branched hyphae in old parts of the colony but not with young hyphae produced apically or by subapical branching. A study of phosphorus metabolizing enzymes in Aspergillus niger by Nagasaki ( 1968) illustrates a similar age-dependent differentiation in surface colonies. More recently Skowronski and Gottlieb ( 1970)analysed metabolic changes that occur in the peripheral (actively growing) hyphae from young and old colonies of R. soluni. They made the interesting conclusion that factors responsible for growth limitation in old colonies probably were located in the peripheral hyphae, such hyphae from old colonies having greatly diminished rates of respiration and protein synthesis. Moreover, the deficient protein synthesis appeared to be due to inhibitory factors in the soluble fraction of the hyphae; ribosomes from these hyphae retained the capacity to synthesize protein at close to maximum rates. The plugging of septa1 pores and the consequent prevention of translocation is a further manifestation of differentiation in a fungal colony (Trinci and Collinge, 1974b). We will return to this subject in Section 1V.A (p. 23).
2. Some Examples of Fungal Growth Modelling Only quite recently has it become widely appreciated that filamentous fungi possess the ability to grow exponentially (6.Mandels, 1965), and the perpetuation of the contrary view seems to rest on the fact that these organisms grow by linear apical extension of their hyphae. However, exponential increase in total mycelial length or mass does occur by the generation of new hyphal apices at a rate proportional to the total mycelial length; this condition is realized either by branch formation or by hyphal fragmentation by shearing forces in a stirred culture. In passing it may be noted that very little quantitative data have been published on hyphal fragmentation in stirred fermenters, and information on hyphal-length distribution at different shearing rates is lacking. Recently, however, Japanese workers have made a valuable contribution to the analysis of mycelial strength and have provided a comparative standard against which to assess the intensity of shearing
10
A. T. BULL AND A. P. J. TRlNCl
shock (Tanaka, Takahashi and Ueda, 1975; Tanaka, Mizuguchi and Ueda, 1975). In practice, the Monod model can produce good approximations of fungal gowth, and this is illustrated in Figure l a with reference to the batch cultivation of Geotrichum lactis in a defined glucose-nitrate medium. The departure of the observed data from that predicted is highly suggestive of oxygen limitation occurring when the biomass concentration exceeds about 2 g 1-I. The model also demonstrates the relative unimportance of K , as a determinant of growth under conditions of substrate excess. We noted earlier that the Monod model does not predict a decreasing growth rate as the substrate becomes limiting, and even when the value for K, is very small (Fig. la) a significant deceleration phase may be evident. Consequently, the logistic law, which embodies the principle of a maximum population, may be a more appropriate model to adopt. Constantinides et al. (1970) found that growth of Penicillium was very closely predicted by the logistic equation. Similarly, growth of wild-type Aspergzllus nidulans is fitted much more closely by the logistic than by the Monod equation (Fig. lb). The significant divergence of the Monod plot and the experimental data may be explicable in terms of an over-estimated rate of substrate utilization, or of a substantial channelling of carbon into extracellular product(s). The extensive synthesis of extracellular melanin that begins towards the end of exponential growth (Carter and Bull, 1969; Rowley and Pirt, 1972) is an argument in support of the latter hypothesis. Figure l a also demonstrates the utility of the logistic model; whereas the Monod equation accurately predicts exponential growth, the logistic equation in addition provides an acceptably accurate modelling of the decelerating growth rate phase. The pronounced morphological, as well as biochemical, differentiation which moulds may display necessitates models additional to those discussed so far. Thus, Emerson (1955) argued, on quite reasonable grounds, that the growth rate of a filamentous fungus should be somewhat less than exponential and he predicted that growth would follow a cube root law. Emerson’s own observations with Neurosporu cratsa sustained such a cube root growth model, and other workers have confirmed this fact subsequently. The ensuing confusion over exponential or cube-root expressions of fungal growth has persisted in the literature for many years and was only conclusively resolved by Pirt ( 1966). Pirt deduced that cube root growth was a characteristic of fungi growing as pellets, rather than as diffuse mycelia, and he argued that
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
I
I
I
5
10
I
15 20 Time ( h )
1
I
25
30
11
(a)
Time(h)
(b) FIG. 1. (a).Time-course of growth of a batch culture of Geotrkhum cundidum, from the data of Trinci (1971). Observed values for biomass production are indicated by . Computer simulations based on the Monod model for &value of 9 mg I-' are indicated by . . . ., and for a &value of 24 mg I-' by - - - -,and for the Logistic model by -. Computor simulations (Bushel1 et al., 1976) incorporate the growth constants of Fiddy and Trinci (1975). (b). Time-course of growth of a batch culture of Aspergillus nidulans 224 based on the data of Carter and Bull ( 1969). Observed values for biomass , and the concentration of glucose in the culture by A . production are indicated by . Predictions from the Monod model are indicated by - and from the Logistic model by - - - - (Bushel et ul., 1976).
12
A. T. BULL AND A. P. J. TRlNCl
once a pellet exceeded a critical size growth was restricted to a peripheral zone by constraints on substrate diffusion. Trinci ( 1970), and more recently Huang and Bungay ( 1973), have supplied convincing experimental proof of Pirt’s model and have provided quantitative data on the dimensions of the peripheral growth zone. To our knowledge, Blackman kinetics have not been used to model mould growth, but in their paper Dabes et al. (1973) have made some pertinent observations on yeast respiration and growth. Commenting on the data of Terui and Sugimoto (19691, Dabes and his colleagues argued that, while it was the availability of electrons that determined the maximum rate of respiration, the apparent &value for oxygen “is set by the cytochrome system close to the point of oxygen utilization”. The fit of respiration rates by the three-constant model is claimed to be good support for the idea of two widely separated rate-limiting steps in yeast respiration. These preliminary analyses recommend that more attention should be given to Blackman-type kinetics by the microbial physiologist. All of the models of fungal growth referred to above neglect mycelial differentiation or the effects of cell age. An attempt to model growth of Aspergillus awamori based on the existence of discrete states of mycelial differentiation has been made by Megee et al. (1970). Although they succeeded in modelling many features of mould fermentations on the basis of age-dependent parameters, the differentiation states incorporated into the model were defined with reference to surface colonies, and such an extrapolation may be unwarranted. The concept of a mean cumulative age” introduced by Aiba and Hara (1965) is also relevant in the context of age-related phenomena in filamentous fungi. The mean cumulative age, defined as the cumulative age of all mother and daughter cells of a particular mycelial system divided by the total number of cells in that system, provides a common time scale for comparing batch and continuous-flow cultures. Aiba and Hara (1965) illustrated their hypothesis by reference to the penicillin fermentation, and concluded that mean cumulative age analysis could offer scope for designing continuous processes from observations made on batch culture%. In coklusion, it is essential to recognize that mathematical description of microbial growth is a continuing quest and that refinement of models must be limited by our ability to define physiological changes in precise mathematical terms. Fungal physiologists, and mycologists 66
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
13
in general, usually find the rigorous demands of growth-modelling discouraging and also, perhaps, have been too disposed to seek simple mathematical expressions in the hope of deriving a universal quantitative description of batch growth. Dawson and Phillips (1974) have argued that most growth models have limited utility “if only because simple models have extremely restricted applications and complex models are self-defeating’’ in the sense that critical data needed to test their validity are lacking. At this stage, therefore, the mycologist might adopt mathematical modelling most advantageously as a conceptual aid in the problem of understanding fungal growth. 111. Growth
of Undifferentiated Mycelia
A distinction may be made between differentiated and undifferentiated mycelia (Steele and Trinci, 1975). A fungal spore germinates upon a solid medium to form an undifferentiated mycelium which increases in size and differentiates into a “mature” colony. The differences which distinguish the hyphae of undifferentiated mycelia from those at the margin of mature colonies are listed in Table 1. Most studies of the growth and cytology of moulds have been made upon hyphae at the margin of “mature” colonies. However, the basic features of fungal growth are more likely to be displayed by the hyphae of unTABLE 1. Comparison between the characteristics of hyphae of differentiated and undifferentiated rnycelia Undifferentiated mycelia Hyphae formed during exponential growth on solid media or submerged culture 1. The hyphae of a single mycelium have more or less the same diameter 2. Each hypha has the same maximum extension rate (Emax)
3. Hyphae have relatively short extension zones 4. Hyphae do not usually branch subapically
Differentiated mycelia Hyphae formed at the margin of colonies on solid media The hyphae are usually differentiated into wide “leading” hyphae and narrower branch hyphae “Leading” hyphae have faster rnaximum extension rates than primary and secondary branches “Leading” hyphae have relatively long extension zones “Leading” hyphae often branch subapically
14
A. T. BULL AND A. P. J. TRlNCl
differentiated mycelia since the mechanisms involved in regulating mycelial form may be obscured by the differentiation process involved in colony formation. A.
R E G U L A T I O N O F MYCELIAL FORM
The thallus of a mould, unlike that of a unicellular micro-organism, is well adapted to colonize solid substrates such as plant surfaces, soil
and solidified culture media. An advantage of the filamentous form is that the organism can increase in size indefinitely without altering the ratio between protoplasmic volume and surface area. Thus, exchange of substances between the mycelium and the medium involves transport over only short distances. The fact that hyphae branch at more or less regular intervals ensures that solid substrates are effectively and efficiently covered by the mycelium. At least three mechanisms must be involved in regulating the formation of undifferentiated mycelia: ( l ) Regulation ofhyphal polarity. Hyphal growth is polarized, i.e. extension is confined to the hyphal tip. (2) Regulation of branch initiation. Germ tubes increase in length to form “main” hyphae from which primary branches are produced. In their turn, the primary branches give rise to secondary branches and so on. The predictable form of a mycelium indicates that there is a mechanism which regulates the frequency of branch initiation. (3) Regulation ofthe spatial distribution of hyphae. Hyphae formed by an undifferentiated mycelium tend not to grow in contact with one another. Contact between adjacent hyphae is minimized by an “avoiding” reaction known as autotropism (Robinson, 1973). Thus there is a mechanism which regulates the spatial distribution of the hyphae within a mycelium. B.
POLARIZATION O F HYPHAL GROWTH
The mechanism which initiates and subsequently maintains the polarity of hyphal growth is not known. Growth initially becomes polarized during germ tube formation; fungal spores are often spherical and hence lack any obvious polarity. The onset of polarity may be prevented by manipulating the cultural conditions; for example, at temperatures of 37OC and below, conidia of Aspergillus niger germinate in the normal way and form a mycelium but, on incubation at 44OC, a supra-optimal temperature for this fungus, polarity is in-
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
15
hibited and the conidia increase in volume and dry weight to form giant, coenocytic, spherical “cells” which have very thick walls (Smith and Anderson, 1973). Growth of these structures becomes polarized (the cells germinate forming hyphal outgrowths) when the incubation temperature is lowered to 3 O O C . Mucor rouxii, which also forms a mycelium under normal conditions, grows in a non-polarized, yeastlike manner when it is cultured under anaerobic conditions in the presence of carbon dioxide (Bartnicki-Garcia, 1963). Thus nonpolarized (isotropic) growth may be associated with conditions which are unfavourable far growth. Hyphal polarity is clearly governed by some endogenous regulatory mechanism since the filamentous form is maintained in submerged culture. Further, entire filaments as well as the individual compartments of Geotrichum candidum hyphae grown in batch culture are polarized with respect to branch initiation (Fiddy and Trinci, 1975). Moulds grown in submerged culture are not of course subjected to the environmental gradients associated with colony growth on solid media (Park and Robinson, 1966). The tubular form of a hypha is presumably a consequence of the apical transport and deposition of the vesicles involved in hyphal extension. The vesicle may contain wall precursors and/or enzymes which synthesize and lyse cell-wall polymers (Bartnicki-Garcia, 1973). Hyphal extension (tip growth, branching, spore germination) always appears to be associated with the fusion of vesicles with the existing wall. The mechanism which regulates hyphal polarity presumably operates through its effect on the apical transport and/or deposition of these vesicles. Factors which disrupt normal vesicle transport and/or deposition may thus result in isotropic growth. C . REGULATION OF B R A N C H INITIATION
Growth of undifferentiated mycelia may be studied by following their formation from spores on solid media overlaid with cellophane (Trinci, 1974).The cellophane ensures that the mycelia are formed in a single plane and can thus be photographed in their entirety. Mycelia which have a total hyphal length of only a few millimetres may be regarded as undifferentiated. Certainly, such mycelia lack differentiation into leading hyphae of wide diameter and branch hyphae of narrower diameter (Trinci, 1973a). However, undifferentiated mycelia
16
A. T. BULL AND A. P. J. TRlNCl
may not form a distinct morphology and physiological state of development but simply represent a transient stage in colony formation (Steele and Trinci, 1975). Growth of an undifferentiated mycelium from a spore initially occurs under environmental conditions which remain relatively constant. The physical and chemical characteristics of the medium will only be significantly changed after it has supported a certain amount of biomass production. Therefore the cultural conditions which prevail during the initial stages of mycelial growth on a solid medium will be similar to those present during the early part of the exponential phase of' growth of a batch culture. Certainly the morphology of undifferentiated mycelia produced under these two cultural conditions is very similar (Steele and Trinci, 1975). 1. M aximurn Extension Rates o f Individual Hyphae
of Undgerentiated
Mycelia
The initial rate of extension of a branch of an undifferentiated mycelium is dependent upon its parent hypha. Branch hyphae of Aspergillus nidulans, Geotrichum candidum and Mucor hiemalis attain their maximum extension rates when they are, respectively, about 400, 400 TABLE 2. Mean and maximum extension rates of the hyphae of undifferentiated mycelia grown at 25OC. Mean values are quoted f standard errors of the mean. From Trinci (1974) Species
A.\pergillus nidulans Geolrichum candidum Mucor hiemalis Penicillium chrysogenum Neurospora crassa spco 1
Mean hyphal extension rate ( E , pm h-'1
** 8 * 0.3 21 *I
33 4 48 3 125 k 11
Maximum hyphal extension rate (Em.,, p n h-') 80 120 330
49
and850pm long (Trinci, 1974). These values thus represent the maximum lengths of the peripheral growth zones (Trinci, 1971) of these hyphae. The hyphae of an undifferentiated mycelium thus appear to have a maximum rate of extension which is strain specific (Table 2).
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
2. Mean Extension Rate o f the Hyphae
17
of Undgerentiated Mycelia.
The mean rate of hyphal extension ( E ) of a mycelium may be calculated from the equation:
where H o is the total hyphal length (pm) of the mycelium at zero time, H , the total hyphal length one hour later, Bo the number of hyphal tips at zero time, and B ; the number of hyphal tips one hour later. The mean hyphal extension rate of a mycelium may also be estimated (Steele and Trinci, 1975) from the equation: E=Gp
(2)
where G is the mean length of the hyphal growth unit (Trinci, 1973b) of the mycelium, and p the organism’s specific growth rate. The symbol a has been used to denote specific growth rate in several publications in this field. Since G and p are constants, E must also be a constant. The mean hyphal extension rate (calculated using equation 1) of Geotrichum candidum increased until the mycelium had formed three tips and thereafter remained constant (Trinci, 1974).The low standard deviations calculated for the mean hyphal extension rates of the mycelia of different moulds (Table 2) suggests that E is a specific feature of undifferentiated mycelia. 3. Growth of UndiJerentiated Mycelia from Spores
Undifferentiated mycelia initially increase in total length at an exponential rate (Trinci, 1974); in the case of Mucor hiemalis, exponential growth continued until the mycelium had a total hyphal length in excess of about 15 mm. The exponential phase is followed by a period during which there is a progressive deceleration in growth rate. The duration of the exponential phase is probably influenced by the length of the mould’s hyphal growth unit; that is, deceleration is likely to occur earlier for species which form dense mycelia (e.g. Penicillium chrysogenum) than for species which form sparse mycelia (e.g. Mucor hiemalid. The onset of the deceleration phase is probably correlated with certain adverse changes in the composition of the medium (e.g.
18
A. T. BULL AND A. P. J. TRlNCl
changes in pH value or secondary metabolites or nutrient concentration) and with differentiation of the mycelium (e.g. the formation of narrow branch hyphae and wide “leading” hyphae). During the early part of the stage during which the total hyphal length of the mycelium is increasing exponentially, branches are formed at relatively infrequent intervals. Eventually the number of branches increases exponentially at more or less the same specific growth rate as the total hyphal length of the mycelium (Fig. 2). f
5120
2560 I280 -
--
0 c -4Og 5
320 -
f
160-
80-
20 - 01
0
I 2
4
tip production I
I
I
6
8
10
12
Time ( h )
FIG. 2. Growth of mycelium of Geotrichum candidum on solid medium. The number of hyphal tips (O), total length (0)and length of the hyphal growth unit (0)are plotted as a function of time. The figure is reproduced by permission of Cambridge University Press.
The ratio between the total hyphal length of an undifferentiated mycelium and its number of branches has been called the hyphal growth unit(Caldwel1andTrinci, 1973;Trinci, 1973b).After spore germination, the hyphal growth unit of an undifferentiated mycelium increases in length until the germ tube produces its first branch. At this point the hyphal grown unit is halved. The amplitude of the oscillations in the length of the hyphal growth unit decreases progressively as the undif‘ferentiatedmycelium increases in size. Eventually the hyphal growth unit attains a more or less constant value (Fig. 2). Growth of an un-
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
19
differentiated mycelium may be thought of in terms of the duplication of a hypothetical “growth unit” which consists of a tip and specific length of hypha. The apparently contradictory observations that the biomass of a mould grown in batch culture increases at an exponential rate whilst individual hyphae extent at a linear rate are reconciled by the fact that there is an exponential increase in Up number (Caldwell and Trinci, 1973). Similarly, an individual bacterium may increase in mass at a linear rate whilst the population increases in number exponentially (Kubitschek, 1970).The “hyphal growth unit” of a mycelium is a physiological, but not a morphological, entity. It is simply the mean length of hypha per tip, and it clearly differs qualitatively from the “growth units” (i.e. the cells) of unicellular micro-organisms. The observation that the extension rate of the hyphae of an undifferentiated mycelium of Mucor hiemulis varied from 2 1 to 329 pmlh (Trinci, 1974)suggests that the length of hypha actually associated with each tip probably varies over wide limits ; the observed difference in extension rates presumably reflect differences in the length of hypha associated with each tip (Trinci, 1971). The relationship between hyphal length and tip number (i.e. the hyphal growth unit) may also be investigated by studying populations of undifferentiated mycelia (Caldwell and Trinci, 1973; Trinci, 197313; Trinci and Collinge, 1973; Morrison and Righelato, 1974).The hyphal growth unit of a population of undifferentiated mycelia of Neurospora crussu spco 1 was relatively constant (Trinci, 1973b)suggesting that there is a direct relationship between total hyphal length and tip number. D. H Y P H A L G R O W T H U N I T S O F D I F F E R E N T S T R A I N S A N D S P E C I E S
The hyphal growth unit is strain- (Trinci, 1973a, b ; Morrison and Righelato, 1974) and species-specific (Table 3). The observation that some spreading colonial mutants (spco) of Neurospora crassu have the same specific growth rate as the wild type (Trinci, 1973a) but different hyphal growth unit lengths indicates that these mutations affected the spatial distribution of the organism’s biomass but not its rate of production (Trinci, 1973a).The mean and maximum hyphal extension rates of these strains have presumably also been altered by the mutations. There is a considerable variation amongst the fungi in hyphal growth unit length (Table 3).
A. T. BULL AND A. P. J. TRlNCl
20
TABLE 3. Hyphal growth units ofundifferentiated mycelia of fungi grown at 25OC o n solid defined medium (Trinci, 197 1). The hyphae measured had 3-8 tips. The medium was supplemented with thiamine and biotin for the phycomycetes. Values are quoted k standard errors ot'the mean. ( I . J. Caldwell and A. P. J. Trinci, unpublished results) Hyphal growth unit (G,pm)
Species Cunninghamella sp Rhizopus slolonifer M ucor rammanianus MUCOThiemalis Aclinomucor repens" Aspergillus niger Aspergdlus wenlii Aspergillus gtganteus Penicillium clavtforme Penicillium chyogenum Geolrichum candiduma Cladosporium sp Verlicillium sp Fusarium vaucerium Fusarium avenaceum Trichoderma viride
35 f 9 124 f 31 31 f 10 95 f 22 352 k 97 I? f 14
6 6 k 15 7lf9 104 f 18 48 f 10 110 f 28 59f 1 1 8 2 2 17 682 f 26 620 f 164 160 f 31
"Grown at 3oOC.
E. EFFECT O F E N V I R O N M E N T A L C O N D I T I O N S O N H Y P H A L GROWTH U N I T LENGTH
1. Temperature
Hyphal growth unit length is riot affected by temperature (Trinci, 1973b). This suggests that specific growth rate ( p ) varies directly with mean hyphal extension rate ( E ) (i.e. the ratio Elp is a constant). This certainly appears to be so (Table 4).Presumably the maximum hyphal extension rate (E,,,,J of an undifferentiated mycelium also varies TABLE 4. Specific growth rate and mean hyphal extension rate of undifrerentiated mycelia of NeUrOSPOTa crassa spco I . p was determined in batch culture. From Trinci (1974) Temperature (OC)
25
Mean hyphal extension rate ( E , pn h-')
Specific growth rate (K, h-9
Elp
21 38
0.26 0.45
81 88
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
21
directly with specific growth rate. Thus temperature alters the rate at which the hyphal growth unit is duplicated but not its length. Similarly the mean cell mass of bacterial populations grown in batch culture is not altered by temperature (Schaechter et ul., 1958). 2. Inhibitors
Hyphal growth unit length may or may not be altered by inhibitors of mould growth (Caldwell and Trinci, 1973; Trinci, 1973a, b). The effect of an inhibitor depends upon the nature of’ the inhibition and the concentration employed. When hyphal growth unit length is not altered by an inhibitor, this suggests there is a direct relationship between the effect of the inhibitor on the mould’s specific growth rate and its effect on the mean extension rate of its hyphae (i.e. the ratio E / p is not altered by the inhibitor). Cycloheximide, unlike deoxycholate and triphenyl tin acetate, causes a decrease in hyphal growth unit length. 3. L-Sorbose L-sorbose inhibits the extension rate of Neurosporu crussu hyphae without apparently affecting the mould’s specific growth rate (Trinci and Collinge, 1973). Thus L-sorbose causes a dramatic decrease in hyphal growth unit length, inducing N . CTUSSU to branch profusely. Like the spco mutations of Neurosporu crussu (Trinci, 1973a), the maximum specific growth rate of L-sorbose treated mycelia remains unaltered but the spatial distribution of the mould’s biomass is changed. L-Sorbose, and substances which act like L-sorbose, would be expected to induce moulds to grow in a colonial or semi-colonial manner. 4. Medium Composition
Qualitative changes in medium composition may affect hyphal growth unit length as well as altering specific growth rate (Katz et al., 1972; Morrison and Righelato, 1974). The results of Katz et al. (1972) suggest that there is an inverse relationship between specific growth rate and hyphal growth unit length (Table 5 ) . It would seem that varying the composition of the medium altered the specific growth rate of Aspergillus niduluns without having a corresponding effect on the mean hyphal extension rate of its mycelia (i.e. the ratio, E / p did not remain
A. T. BULL AND A. P. J. TRlNCl
22
TABLE 5. Et'frct of medium composition on hyphal growth unit length Mediuni
Specific growth -rate (p, h-')
Hyphal growth unit length ( G ,pm)
Estimated mean rate of hyphal extension ( E , m h-l)''
(a) A\pergzl/u.\ uidu1an.s at 3OoC (calculated from the data of Katz el al., 1972) Malt extract Defined medium with acetate as the carbon source Defined medium with L-tryptophan as the nitrogen source
0.14
c. 33b c. 7Sb
11.9 10.2
0.11
c. 12Ob
13.2
0.36
(b) Periicillium chrysogenuin T 14 (Morrison and Righelato, 1974) Complex niediuni Defined medium
0.24 0.14
4 3 f 10 60k9
10.3 8.4
Estimated using Equation 2 (p. 1 7 ) . bEstimated from the data of Katz el al., (1972).
a
constant); medium composition appears to have had little effect on mean hyphal extension rate (Table 5 ) . 5 . Conclusions
Under a given set of environmental conditions the total hyphal length of a mycelium, and the number of its tips, increase exponentially at the same specific growth rate (pu).Thus the ratio between total hyphal length and tip number (i.e. the hyphal growth unit) is a constant as is the mean rate of hyphal extension ( E l . The relative constancy of the length of the hyphal growing unit during mycelial development suggests that branch initiation, like the division of unicellular microorganisms, may be regulated by the changes in cytoplasmic volume which accompany growth, i.e. when the mean volume of cytoplasm (length of hypha) per hyphal tip exceeds a critical volume (length) it induces the mycelium to initiate a new branch. Mutations or cultural conditions which cause a deceleration in the mean rate of extension of a mould's hyphae without altering ;he organism's specific growth rate (i.e. altering the ratio, E / p ) will result in
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
23
a decrease in the length of the hyphal growth unit. The spreading colonial mutants of Neurosporu crma are such mutants (Trinci, 1973a,b) and L-sorbose such a factor (Trinci and Collinge, 1973). Mutations or cultural conditions which cause an increase in the organism’s specific growth rate without causing a corresponding acceleration in the mean rate of extension of its hyphae (i.e. again altering the ratio EIp) will also result in a decrease in hyphal growth unit length. Presumably this is what happened when Kau et ul. ( 1972) altered the specific growth rate of Aspergzllus niduluns by changing the composition of the medium (Table 5 ) . Altering the specific growth rate of a mould by temperature changes causes a corresponding change in the mean rate of extension of its hyphae (i.e. temperature does not alter the ratio, Elp) and thus hyphal growth unit length is not affected by temperature. F. REGULATION O F T H E SPATIAL DISTRIBUTION O F HYPHAE
The spatial distribution of the hyphae of undifferentiated mycelia in part results from negative autotropism, i.e. hyphae tend to grow away from each other. Autotropism is thus a mechanism which helps to ensure that solid substrates are effectively and efficiently covered by mycelia. The hyphae at the periphery of a fungal colony grow radially outwards from the centre. This phenomenon has usually been explained in terms of a negative chemotropic response of the hyphae to some unknown factor(s) which accumulate in the environment. However, Robinson (1973) has recently suggested that the phenomenon can be explained in terms of a positive chemotropic response to oxygen. Negative autotropic responses of undifferentiated hyphae have been observed where the responding hypha was up to 30 jm away from the hypha to which it was reacting (A. P. J. Trinci, unpublished observation). Thus, negative autotropism may result from a response to some unknown substances which diffuse from hyphae and accumulate in the environment, or to a gradient in a nutritional factor (including oxygen) which is established in the immediate vicinity of hyphae.
IV. Colony Growth A. COLONY DIFFERENTIATION
A mycelium increases in size and gradually differentiates into a “mature” colony which subsequently extends radially across the substrate at a linear rate. This differentiation probably occurs as a direct
24
A. T. BULL AND A. P. J. TRlNCl
response to the changes induced in the medium by growth of the mould. “Mature” colonies can be divided into at least four morphological zones (Yanagita and Kogane, 1962): (1)the extending zone (which is equal to the peripheral growth zone; Trinci, 197 1 ), made up of the peripheral, sparse network of vegetative hyphae not supporting aerial hyphae; (2) the productive zone, made up of a much denser network of vegetative hyphae supporting aerial hyphae; (3) the fruiting zone, where asexual and/or sexual reproductive structures are formed; and (4)and aged zone, made up of the “aged” and autolysing hyphae at the centre of the colony. Although it is convenient to recognize these zones, there is a continuous differentiation of the colony from its periphery to its centre. 1. The Peripheral Growth Zone The width of the peripheral growth zone of a “mature” colony remains approximately constant as it expands radially across the substrate (Yanagita and Kogane, 1962;Trinci, 1971).The hyphae in the peripheral growth zone, unlike those of young mycelia, are usually differentiated into wide “leading” hyphae and narrower branch hyphae (Butler, 1961; Trinci, 1973a). The “leading” hyphae are oriented radially outwards from the centre of the colony with their apices more or less at the same level, giving a smooth outline to the colony (Butler, 1966). Hyphae at the outer fringe of the peripheral growth zone are thin walled, full of protoplasm (Butler, 1966)and rich in RNA and DNA (Yanagita and Kogane, 1962). The cytoplasm in hyphae more distant from the margin of the colony is vacuolated and the degree of vacuolation increases distally (Park and Robinson, 1967). Intrahyphal hyphae (Lowry and Sussman, 1966 ; Trinci and Righelato, 1970)are rarely if ever formed in the peripheral growth zone, and the septa1 pores of septate hyphae usually remained unplugged (Trinci and Collinge, 1973). Anastomoses (Buller, 1933) are not usually formed between hyphae in the peripheral growth zone. In some moulds, e.g. Neurosporu crassu, formation of a mature colony involves a differentiation process which results in the formation of wide “leading” hyphae having a faster maximum extension rate than the hyphae of the organism’s undifferentiated mycelium (Trinci, 1973b; 1974). This type of differentiation may be lacking in some moulds or not so marked; for example, the maximum extension rate of the leading hyphae of Geotrichum candidum colonies is not very much
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
25
faster than that of hyphae of its undifferentiated mycelium (Trinci, 1974). In most fungi, the hyphae in the peripheral growth zone branch monopodially, i.e. wide, fast growing “leading” hyphae subtend narrower, slower growing branch hyphae. Butler (1961) found that, if the extension rate of the “leading” hyphae of Coprinus dimminutus colonies was taken as loo%, then the extension rates of the primary and secondary branches were 66% and la%, respectively. Primary branches formed by “leading” hyphae at the margin of colonies of Aspergillus niduluns and Geotrichum cundidum had extension rates which were 20% and 30% less than their parent hyphae (Trinci, 1970).Thus, the peripheral growth zone hyphae d o not all have the same maximum extension rate. Butler (1961)found a positive correlation between the extension rate and diameter of “leading” hyphae of colonies of Coprinus disseminutus. There was a similar correlation between the diameter of the leading hyphae of spreading colonial mutant colonies of Neurosporu crassu and their extension rates (Trinci, 1973b). Little consideration appears to have been given by mycologists to the reason why most moulds have a monopodial branching pattern. Leopold (1971) concluded that monopodial branching is the most economical in terms of branch length for the efficient exposure of the leaves of trees to light and drainage of river basins by streams. This suggests that a monopodial branching pattern is probably a very efficient and economical way for a mould to colonize solid substrates, i.e. it ensures efficient cover of the substrate by the mould at the expense of a minimum production of biomass. The leading hyphae of colonies of Allomyces sp. (Emerson, 1955)and Geotrichum cundidum (Trinci, 1970) branch dichotomously as well as laterally, whilst those of Aspergillus niduluns branch sup-apically producing two or more branches per tip (Trinci, 1970). After dichotomous branching in G. cundidum, the extension rate of the branches accelerates until each attains the extension rate of the parent hypha. Dichotomous or sub-apical branching is rarely observed in the undifferentiated mycelia of these same species. Sympodial branching patterns have been observed in Ascobolus immersus colonies where parent hyphal tips are successively overtaken by their branches (Chevaugeon, 1959). The density of the hyphae at the circumference of a mature colony remains more or less constant as it increases in radius. This observation suggests that there is a periodic generation of new leading hrphae
26
A. T. BULL AND A.
P. J. TRlNCl
as the colony increases in diameter. Presumably these new leading hyphae arise as the result of primary branches increasing in diameter and growth rate until they assume the position and characteristics of leading hyphae (Trinci, 1973b). The transformation of a primary branch into a leading hypha probably occurs as a chance event when such a branch happens to extend into a relatively uncolonized part of the substrate at the fringe of the colony. The density of hyphae (Plomley, 1959; Trinci, 197 1) and biomass per unit area (Gillie, 1968) increase from the margin of the colony inwards. In the case of hyphal density, the increase occurs exponentially, suggesting that growth within the peripheral growth zone is rapid. 2. The Productive Zone
Like the peripheral growth zone, the width of the productive zone remains more or less constant. This region consists of a dense mat of vegetative hyphae which, unlike the peripheral growth zone, supports aerial hyphae. Some or all of the aerial hyphae may eventually be associated with reproductive structures. In fungi such as Mucor mucedo, the vegetative aerial hyphae are morphologically distinct from the sporangiophores which will eventually support the sporangia. The differentiation from mycelial to aerial growth is presumably associated with the deceleration in growth rate observed in this region of the colony. The cytoplasm of hyphae in this zone is very vacuolated and well endowed with reserves such as glycogen and lipid (Butler, 1966). The walls of productive zone hyphae are generally thicker than those of hyphae in the peripheral growth zone. Branch hyphae formed within the productive zone become progressively narrower as branching proceeds, and their growth is more meandering and less radially directed (Plomley, 1959). The septa1 pores of septate hyphae are usually plugged (Trinci and Collinge, 1973) and additional septa may be formed (Butler, 1966). Intrahyphal hyphae may be produced in this zone, and anastomoses between hyphae may occur in fungi other than the phycomycetes (Buller, 1933). 3. The Fruiting Zone
The width of the fruiting zone is less constant than the two zones considered previously. The mycelium is very dense and consists of highly vacuolated hyphae, some of which may be autolysing. Intra-
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
27
hyphal hyphae may be present. The mycelium may support asexual and/or sexual reproductive structures, the former usually preceding the latter in their development. The induction of reproductive structures is probably associated with the establishment in the environment of conditions which are relatively unfavourable for growth; for example, exhaustion of a nutrient or accumulation in the medium of inhibitory products. The formation of reproductive structures in at least some species is associated with a turnover of certain cytoplasmic and wall polymers. Exhaustion of the carbon source in the medium induces formation of a-1,s glucanases in Aspergillus nidulans which degrade the a1,3 glucan component of the wall (Zonneveld, 1974). The breakdown products of such wall polymers may supply the carbon and energy required for sporulation. In the fruiting and aged zones of the colony, the nucleic acids which were present in the mycelium may become associated with the reproductive structures (Yanagita and Kogane, 19621, suggesting that there is a turnover of these polymers. 4. The Aged Zone
As a colony increases in radius a progressively larger proportion of it is made up of a central aged zone of indefinite diameter. It is composed largely of autolysing hyphae and reproductive structures. B. M O U L D - I N D U C E D C H A N G E S I N T H E SUBSTRATE
The spatially separated regions of the colony already described reflect temporal changes which occur as the substrate is progressively colonized by the mould. Differentiation of the mould almost certainly reflects, and is induced by, the changes in the substrate which result from mould growth. The physical changes include an increase in the relative humidity above the medium, greater temperature constancy and changes in medium viscosity (Park and Robinson, 1966). However, there is little doubt that the crucial changes in the medium which are correlated with differentiation of the mould are of the chemical type discussed below. 1. Nutrient Concentration
The concentration of nutrients in the environment decreases progressively as colonization proceeds, but whether or not this has a direct effect upon the growth rate of the mould depends upon the original nutrient concentration and the affinity of the mould (K,value)
28
A. T. BULL AND A. P. J. TRlNCl
for the particular nutrient which may eventually limit growth. Moulds, like bacteria, generally have a high affinity for essential nutrients h e . a low K,value); thus, the concentration of the limiting nutrient has to be decreased to a very low level before it causes a lowering in growth rate. For example, the K, (glucose) values of Fusarium aquaeductuum and Geotrichum candidum are 0.3 (Steensland, 1973)and 1.0 mg l-l, respectively (Fiddy and Trinci, 1975). In the case of these fungi, the concentration of glucose in the medium would have to fall below about 45 nig 1-I before growth rate became glucose-limited. Growth of course would not continue for long at such concentrations because the glucose would very quickly become exhausted (cf. Section 11, C,2; p. 9 ) . Nutrient concentrations will obviously have a very significant effect on the maximum biomass of mould per unit area of substrate (i.e. on the yield). I t is a common microbiological practice to compose media so that one particular nutrient, usually the carbon and energy source, becomes exhausted before the rest, and hence determines the final yield. However, it is often difficult to decide which is the limiting nutrient in many of the media conventionally used by mycologists; for example, Vogel’s medium for Neurospora crussa (Vogel, 1956) and Czapek Dox medium (Ainsworth and Bisby, 1961). With these media, it is unlikely that the concentration of the carbon source ultimately limits growth.
2. Oxygen Tension The oxygen tension at the base of a dense mycelial mat probably is decreased to a level at which it limits growth rate. The growth rate of such hyphae will then be limited by the rate of diffusion of oxygen from the air above the colony. A decrease in oxygen tension may lead to the accumulation of secondary metabolites in the medium (e.g. citric acid).
3 . Changes in PH value
The pH value of the substrate may change as a result of the utilization of nutrients during growth (e.g. when the nitrogen source is ammonium sulphate or sodium nitrate) and/or by accumulation of secondary metabolic products (e.g. citric acid). The pH value of the medium is particularly likely to change when it has little buffering capacity.
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
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Changes in the pH value of the medium are likely to affect the mould’s growth rate and may also encourage production of certain secondary metabolites. 4. Accumulation
of Secondary
Metabolites
Accumulation of products of secondary metabolism in the medium is likely to lower further the organism’s growth rate, and thus trigger a positive feedback mechanism. Sometimes crystals, or water-soluble pigments, may be deposited in the medium (Raper and Thom, 1949; Nobles, 1948). The changes in the substrate will increase progressively as the fungal biomass per unit area increases during colonization. The chemical changes which occur in one part of the medium will tend to spread throughout the medium by diffusion. However, the radial growth rate of the colonies of most fungi is likely to exceed the rate at which chemicals such as secondary metabolites diffuse through the medium. I t is unlikely, therefore, that secondary metabolites and products of autolysis formed at the centre of the colony will diffuse through the medium at a sufficiently fast rate to attain significant concentrations in the peripheral growth zone of the colony. Hyphae at the margin of a fungal colony continually extend into medium having a composition which is identical, or very similar, to the composition of the original uninoculated medium. C . KINETICS O F COLONY EXPANSION O N SOLID MEDIA
Pirt (1967) pointed out the need to define: (1) the factors which govern the radial growth rate of microbial colonies; (2) the relationship between radial growth rate and mass growth rate; and (3) the factors which cause differentiation within colonies. Some progress has now been made towards these ends. Gillie ( 1968) determined the dry weight of successive one-cm segments of linear colonies of Neurosporu crassu grown on solid medium in growth tubes. Such plots of mould dry weight against distance from the margin of the colony will of course reflect the temporal changes in biomass which occur as any given part of the medium is colonized by the mould. Gillie’s results showed that during the first two days of the growth of N. crussu on a given region of the medium the mould
30
A. T. BULL AND A.
P. J. TRlNCl
biomass increased with time. Subsequently as the mould started to autolyse there was a decrease in biomass per unit area of substrate. The radial expansion of fungal colonies may be divided into four phases (Trinci, 1969):(a)lag, the period between inoculation and germtube emergence; (b) exponential, during which the colony increases in radius at an exponential rate; (c) deceleration, the period between the termination of the exponential phase and the onset of the linear phase; and (d)linear, during which the colony increases in radius at a constant or linear rate. In some fungi there is a deceleration from the linear growth rate as the colony approaches the margin of the Petri dish (see the chapter by Carlile, in Hawker and Linton, 197 1). The exponential and deceleration phases are usually of comparatively short duration, and in Aspergillus nidulans are completed by the time the colony has grown one mm from the edge of the inoculum (Trinci, 1969). Thus for most of its growth a fungal colony expands at a linear rate. The rate of the linear phase is not influenced by the ploidy of the nuclei (Lhoas, 1968). 1. Influence
of Peripheral
Growth Zone Width on Radial Expansion
Pirt (1967) suggested that growth of a microbial colony was restricted to a peripheral annulus and that growth in the centre of the colony eventually stopped due to exhaustion, or near exhaustion, of a particular nutrient. In the case of fungal colonies it has been shown that the peripheral hyphae have a much higher metabolic activity than hyphae of other regions of the colony. The rates of uptake of [3Hlleucine,13H]uridine and [3H]N-acetylglucosamine,for example, are fastest in the peripheral 2 to 3 mm of Trichoderma uiride colonies indicating that the rate of synthesis of RNA, protein and chitin is fastest in this region of the colony (Galun, 1972). Similarly, hyphae of the peripheral 1.5 mm of Aspergillus niger colonies have a faster rate of 32Puptake than other parts of the colony (Table 6). The decrease in the rate of uptake of these various compounds with distance from the colony margin suggests that there is a decrease in growth rate from the periphery to the centre of the colony. Smith ( 1924) was the first to suggest that “although the actual.extension occurs at the tip, there are grounds for believing that the parts of the hypha behind the tip contribute to the activity of the latter”. This hypothesis was subsequently implicitly or explicitly supported by the
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
31
TABLE 6. Uptake of 32P by colonies of Aspergillus n i p . Adapted from Yanagita and Kogane (1963b) Region o f the colony
Peripheral growth zone Productive zone Fruiting zone Aged zone
Width of 3zP uptake 32Puptake expressed as a the region (c.p.m./mgdry wt) per cent of the rate (mm) of uptake in the peripheral zone 1.5 2.0 4.5
3.5
4,800 1,500 300 140
100
31 6 3
~
work of Zalokar (19591, Clutterbuck and Roper (1966), Lhoas (19681, Trinci (1971) and Kau et al. (1972). Trinci ( 197 1) has shown for a number of fungi that the radial growth rate of their colonies (K,) is a function of the width of the peripheral growth zone ( w ) and the organism’s specific growth rate. Thus, K,= wp
(3)
The concept defined by Equation 3 may be considered in terms of unbranched leading hyphae traversing the peripheral growth zone of the colony. The protoplasm in such hyphae increases at, or close to, the mould’s maximum specific growth rate. This hypothetical hypha, and the colony, will only grow at a linear rate if the width of the peripheral growth zone remains constant, i.e. as the peripheral hyphae increase in length by a specific increment at the margin of the colony an equivalent increment of length is removed from the peripheral growth zone of the hypha at its inner margin. However, the hyphae in the peripheral growth zone branch, and thus some of the protoplasm produced by leading hyphae is almost certainly directed to support the initial growth of its branches (Trinci, 1970, 1974). Further, the specific rate of synthesis of protoplasm within the peripheral growth zone is likely to decrease with distance from the margin of the colony. Thus Equation 3 is unlikely to provide more than a first approximation of the rate of expansion of fungal colonies. To summarize, the peripheral growth zone has the following characteristics: (1) In colonies growing at a linear rate the width of the peripheral growth zone remains constant; (2) only growth within the peripheral zone contributes to radial expansion of the colony; regions of the colony distal to the peripheral growth zone increase in biomass (e.g. continue to branch and form a dense mycelium, and also
32
A. T. BULL AND A. P. J. TRlNCl
reproductive structures) but this growth rate does not contribute to radial expansion; and (3) growth within the peripheral growth zone is rapid and occurs at or close to the organism’s maximum specific growth rate for the prevailing conditions. The relationship defined by Equation 3 may be tested by calculating the theoretical radial growth rate of a colony from the width of its peripheral growth zone and the organism’s maximum specific growth rate and comparing this value with the observed colony radial growth rate (Trinci, 1971).
2 . Determination
of Peripheral
Growth Zone Width
The maximum width of the peripheral growth zone may be determined by calculating the minimum length of a hypha which extends at the same rate as the expansion rate of the colony. This may be done by severing peripheral growth zone hyphae and then determining their subsequent growth rate (Ryan et al., 1943; Clutterbuck and Roper, 1966; Lhoas, 1968; Trinci, 1971; Trinci, 1973; Trinci and Collinge, 1973). This method probably overestimates the length of the peripheral growth zone because of the damage which results from cutting. In the case of septate hyphae, the cutting damage appeared to be restricted to three intercalary compartments (Trinci, 197 1). The septa1 pores in the region of the cut may be plugged with Woronin bodies (Reichle and Alexander, 1965; Trinci and Collinge, 1974b)which limit loss of protoplasm. An alternative method of measuring peripheral growth zone length is to determine the length of a branch when it first attains its maximum extension rate (Trinci, 1974). For a number of fungi there is close agreement between the hypothetical colony radial growth rates, calculated from peripheral growth zone and specific growth rate measurements, and observed rates (Trinci, 197 1). The spreading colonial mutants of Neurospora crassa form colonies which are more compact and have a slower rate of expansion than colonies of wild type strains (Garnjobst and Tatum, 1967). Some of these mutants have the same maximum specific growth rate as the wild type but much slower colony radial growth rates. As predicted by Equation 3, there is a linear relationship between the rate of expansion of the colonies of these mutants and the width of their peripheral zones (Trinci, 1973b). In the case of fungi having septate hyphae it is possible that the peripheral growth zone only extends from the tip to the first septum and, in those fungi which have complete septa (i.e. lack pores), e.g.
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
33
Geotrichum candidum, Basidiobolus ranarum and Mucor hiemalis, there is little doubt that the peripheral growth zone is restricted to the apical compartment. The leading hyphae of G. candidum colonies had a mean apical compartment length of 290 k 99 pm compared with an experimentally determined peripheral growth zone width for the colonies of 423 k 129 p.Many fungi, such as Aspergillus nidulans and Neurospora crassa, form septa which initially have unoccluded central pores large enough to allow the translocation of vesicles and organelles such as nuclei (Trinci and Collinge, 1973). It is possible that the peripheral growth zone of these hyphae is limited by the plugging of the septal pores (Trinci and Collinge, 1973). The plugging of septal pores may be initiated by the establishment in the medium of conditions which inhibit growth (see Section IV, B; p. 27). Septa1 plugging may thus be considered as an ageing phenomenon. As mentioned earlier, the establishment in the substrate of conditions which are unfavourable for growth is probably related to the rate of increase in biomass per unit area of substrate. This in turn is probably a function of the organism’s specific growth rate and branching pattern, the rate of increase being fastest for strains with low hyphal growth unit values, i.e. strains which branch profusely. It is possible that there is a direct relationship between hyphal growth unit length and peripheral growth zone width (Trinci, 1973b). Certainly Morrison and Righelato (1974) have also come to this conclusion. D. C O L O N Y E X P A N S I O N A S A P A R A M E T E R O F M O U L D G R O W T H
Colony radial growth rate is only a reliable parameter of growth under conditions where it varies directly with the organism’s specific growth rate (i.e. the ratio KJp is a constant). It follows from Equation 3 that colony radial growth rate will only be directly related to the specific growth rate when the width of the peripheral growth zone of the colony remains constant. 1 . Eflect of Temperature
The width of the peripheral growth zone of a colony remains more or less constant when its rate of expansion is altered by temperature (Trinci, 197 1). Thus, colony radial growth rate may be used to determine the effect of temperature on mould growth (i.e. the ratio KJp is a canstant 1.
34
A. T. BULL AND A. P. J. TRlNCl
2. Effect o f Growth Inhibitors
Inhibitors such as qdoheximide apparently do not alter peripheral growth zone width (Trinci and Gull, 1970) at least at comparatively low concentrations. Under these circumstances, colony radial growth rate is a reliable parameter of growth (K,/c( is a constant). Several workers have shown that colony radial growth rate decreases with the logarithm of the inhibitor concentration in the medium (Trinci and Gull, 1970; Fevre, 1972; Bret, 1972). The basis for this relationship is not known. 3. Effect o f L-Sorbose It has been known for a long time that L-sorbose causes some moulds to grow in a “colonial” form, i.e. form dense colonies which have a lower rate of expansion (Tatum et al., 1959). L-Sorbose caused a
decrease in the hyphal growth unit of Neurosporu crassu (i.e. it branched more profusely) but did not affect the mould’s specific growth (Trinci and Collinge, 1973). The decrease in colony radial growth is correlated with a decrease in the width of the peripheral growth zone which in turn is presumably correlated with the observed increase in branching frequency. Cellobiose may also induce some fungi to grow in a “colonial” form in a similar way (Wilson and Niederpreum, 1967; Wilson, 1970). 4. Nutrient Concentration
The rate of expansion of glucose- and arginine- “limited” fungal colonies increased linearly with the logarithm of the nutrient concentration (Trinci, 1969; Gillie, 1968; Fiddy and Trinci, 1975). Again the basis of this relationship is not known. Most fungal colonies attain their maximum rate of expansion at very low nutrient concentrations (below about 150 mg I-’ for glucose and 80 mg I-’ for arginine). Fiddy and Trinci (1975) have shown that over the “glucose-limited” range there is a direct relationship between the radial growth rate of Geotrichum candidum colonies and the width of their peripheral growth zones. Thus, the deceleration in colony radial growth rate at glucose concentrations below about 100 mg 1-’ could be entirely accounted for by the observed decrease in peripheral growth zone width. Glucose concentration has little effect (Fiddy and Trinci, 1975) or no
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROVVTH
35
effect (Trinci, 1969) on internode length (the mean distance between adjacent primary branches produced by “leading” hyphae). Thus the variation in colony hyphal density with glucose concentration is not related to the frequency of branch initiation, but rather to their subsequent growth. At low glucose concentration branches are initiated, but most of them only grow for a short period of time before the substrate (glucose) in their vicinity becomes exhausted. At low glucose concentrations, therefore, very sparse colonies are formed but they expand at almost the maximum rate. This feature of the growth of fungal colonies clearly has some ecological significance.
5 . Growth of Diflerent Strains and Species Colony radial growth cannot be used as a parameter to compare the specific growth rates of different fungal species (Trinci, 197 1)or strains (Trinci, 1973a). The rapid rate of expansion of the colonies of many phycomycetes is largely due to their having wide peripheral growth zones rather than to their having particularly fast specific growth rates. Colonies of some phycomycetes, e.g. Rhizopus stolonijh, may have wide peripheral growth zones because their hyphae lack septa. In addition the density of hyphae at the margin of such colonies is much less than in the case of colonies of fungi like Penicillium chrysogenum which expand very slowly. E . C O M P A R I S O N O F T H E C O L O N I Z A T I O N OF S O L I D SUBSTRATES BY M O U L D S A N D UNICELLULAR M I C R O - O R G A N I S M S
The filamentous morphology of moulds enables them to colonize solid substrates more efficiently than non-motile, unicellular microorganisms. The polarization of growth within hyphae allows fungi to form colonies which have much wider peripheral growth zones than bacterial colonies (e.g. w equal to about 8.5 mm for Rhizopus stolonijer colonies (Trinci, 197 1) compared with about 90 pm for Escherichia colz colonies (Pirt, 1967)).Thus fungal colonies are able to expand across solid substrate at much faster rates than bacterial colonies although fungi usually have the slower specific growth rates. The wide peripheral growth zones of fungal colonies result in their having radial growth rates which usually exceed the rates of diffusion of chemicals in the medium. Thus secondary metabolites and other products formed at the centre of the colony diffuse through the medium at a slower rate
36
A. T.
BULL AND A. P. J. TRlNCl
than the rate of expansion of the colony, and hence do not affect the growth rate of the peripheral hyphae. Similarly the rate of diffusion of nutrients from uncolonized parts of the medium towards the margin colony is slow compared with the rate of expansion of most fungal colonies. The leading hyphae of fungal colonies are continually growing into uncolonized medium which has approximately the same composition and pH value as the uninoculated medium. However, in the case of bacterial colonies which expand at very slow rates (Pirt, 19671, there will be a tendency for secondary metabolites formed at the centre of the colony to diffuse through the medium and inhibit growth at the periphery of the colony. In addition the concentration of nutrients in the uncolonized region of the medium surrounding the bacterial colony may be significantly lowered or even exhausted because of diffusion towards the colony (Rieck et al., 1973). These effects probably explain the gradual deceleration in the rate of expansion of bacterial colonies with time (Pirt, 1967). The growth of fungal colonies is initiated at much lower nutrient concentrations than bacterial colonies, and fungal colonies attain their maximum rate of expansion at lower nutrient concentrations than bacterial colonies (4 g 1-I for Escherichia coli (Pirt, 1967)compared with 75 mg 1-' for Mucor hiemalis; Trinci, 1969). These latter differences probably resulted from the fact that fungi, unlike bacteria, have a mechanism which regulates biomass density per unit area of substrate according to the concentration of nutrients in the medium. Thus the filamentous habit enables moulds to distribute the biomass which a solid substrate will support to maximum advantage in spreading the colony. Finally the filamentous habit enables fungi, unlike bacteria, to penetrate a solid substrate such as agar-gelled media (Trinci, 1969; Trinci, 1973a); hyphae appear to grow down into the medium at a rate similar to their growth rate across its surface.
V. Fungal Growth in Submerged Liquid Culture. Technical Considerations A variety of operational difficulties accompany the submerged cultivation of fungi. These difficulties are compounded when continuous-flow cultures (the method of choice for analysing fungal growth and metabolism; Bull and Bushell, 1976)are selected, and they
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
37
have undoubtedly inhibited the widespread use of chemostats in fungal research. Basically, the problems are related to the rheological properties of fungal cultures, the propensity of fungi to accrete over fermenter walls and internal surfaces, and to an extremely variable growth morphology. In this section brief consideration is given to the practicalities of fungal fermentations and the solution of common technical problems. A critical assessment of methods for measuring mycelial growth has been made by Calam ( 1969) and, while this topic will not be developed further here, two points may be emphasized. First, that considerable care needs to be taken to obtain representative samples from fungal cultures, especially when small scale laboratory equipment is used, and the position of sampling points, velocity of sample take-off (Solomons, 1972) and culture heterogeneity all exert a pronounced effect on the quality of the sample. Second, it is worth noting that culture absorbance can be used as an accurate growth parameter provided that measurements are restricted to biomass concentrations of less than about 2 g I-' (Trinci, 1972; Solomons, 1975). The relevance of this observation lies in the possibility of continuously monitoring mycelial growth and, thereby, developing turbidostat systems for fungi. As far as we are aware turbidostat culture of moulds has yet to be exploited. The growth form of fungal cultures profoundly affects metabolism; the relationship may be direct and reflect growth of the organism in, say, a yeast-like, mycelial or pelleted form (see p. 14) or, indirect via changes in the rheological properties of the culture. Unlike bacteria, yeasts and fungal pellets, suspensions of diffuse mycelia are nonNewtonian in character; that is their apparent viscosities are a function of the shearing produced by agitation, and the suspension may be heterogeneous with respect to the mass transfer of substrates and products. The apparent viscosity is also dependent on the mould concentration, and Solomons and Weston (19611, among others, have shown conclusively that it may be impossible adequately to aerate high-viscosity cultures in laboratory-scale fermenters. Similarly, the critical dissolved oxygen tensions for fungal cultures become greater (respiration at a submaximum rate) as the culture viscosity increases (Phillips and Johnson, 1961 ; Steel and Maxon, 1966). Unfortunately, even though adequate aeration may be provided, the degree of mixing and distribution of nutrients in a fungal culture may have an adverse effect on growth (Donovick, 1960). These effects can be exacer-
38
A. T. BULL AND A. P. J. TRlNCl
bated in continuous-flow cultures of fungi that are commonly operated at low dilution rates, i.e. < 0.03 h-? At these low rates an increasing proportion- of the energy source is consumed for “maintenance” purposes, a situation that appears to be aggravated by poor mixing. Experimental support for this view was provided by Hansford and Humphrey (1966) who observed that higher fungal yields could be obtained at low dilution rates by use of multifeed distribution and improved mixing. Various other means have been proposed for circumventing growth limitations associated with the rheological characteristics of fungal cultures. Thus, increasing the agitation but not the air flow rate (Brierley and Steel, 19691, lowering the biomass concentration, or establishing pelleted growth (approximation to Newtonian fluid) enhance culture aeration. Some recent work in Moo-Young’s laboratory has revealed that addition of water-soluble polymers to fungal cultures increased both the mass transfer of oxygen to the liquid (Moo-Young et al., 19591, the specific growth rate and other growth parameters (Elmayergi and Moo-Young, 1973). Increased rates of potassium transport induced by higher concentration gradients across the mycelial surface were considered responsible for these effects. For further information on the rheology of mycelial cultures, the reader is directed to the review of Steel (1969) and the morphology model of Roels et af. (1974). Accreted fungal growth creates difficulties in any culture system, and particularly in continuous-flow types. It provides heterogeneous conditions where, for example, anaerobic metabolism may occur in a well aerated fermenter. Bungay et al. ( 1969), using an elegant microprobe technique, showed that respiration could be prevented in microbial accretions of only 150pm thickness. When the thickness of such accretions exceeds the penetration depth of nutrients, detachment from the support surface begins, and subsequent blockage of feed and emuent lines may quickly follow. Accreted growth in chemostats leads additionally to variable culture volumes, the possibility of inadvertent feedback conditions (Solomons, 1972)and an increase the value of D,,,, (Topiwala and Hamer, 197 1). Clearly, each of these effects may vitiate the maintenance of steady-state conditions. The prevention of accreted growth essentially is a biochemical engineering problem, and several fermenter designs and operating conditions have been proposed to alleviate the problem, especially in continuous flow systems. For
THE PHYSIOLOGY AND METABOLIC CONTROL OF FUNGAL GROWTH
39
further information, the interested reader is directed to the papers of Righelato and Pirt (1967), Brunner and Rohr (19721, Bull and Bushel1 (19761, Means et al. (1962) and Dawson (1963). It is sufficient to say here that the conventional stirred-tank type of fermenter can now be adapted easily for continuous culture of fungi, and one such reliable design has been reported by Rowley and Bull (1973). It has been a common experience to find that the critical dilution rate of fungi in chemostat cultures is significantly less than the value of p,,,,,derived from batch cultures grown under similar conditions. Thus, studies with Aspergzllus nidulans, A. niger, Fusarium graminearum and Mucor hiemalis (Carter and Bull, 1969; Fencl and Novak, 1969; Ng et al., 1974; Solomons, 1972; Lynch and Harper, 1974) suggested that steady state dilution rates exceeding about 50%p,,,,, (batch) could not be established. Solomons has suggested (Solomons, 1972 ; Solomons and Scammell, 1974) that such premature washout from chemostats could be due to a growth rate dependency on vitamins, and that the higher growth rates in batch cultures were consistent with a sufficiency of growth factors being present in the spore inoculum. Thus, F. graminearium was found to have a requirement for both biotin and choline. An explanation of this phenomenon, based on the obligatory accumulation of a growth-limiting “intermetabolite”, was favoured by Novak and Fencl(1973).These authors obtained some evidence for the ammonium ion being the critical intracellular metabolite when A. niger was grown in a glucose-nitrate medium; depletion of the ammonium pool occurred when D was approximatrely 4O%pma,(batch),and subsequent nitrite accumulation was considered to cause culture intoxication and washout. However, in our experience, the growth form of the fungus can be the crucial factor in determining Dct.11. Experiments with the hyaline 13 me1 mutant of A. niduluns and the wild-type strain used by Carter and Bull (1969) revealed that D,,,, values within 1% of pm.xCOUld be obtained without any modification of the medium or incubation conditions (M. E. Bushell and A. T. Bull, unpublished results). Similarly the Dorltof both strains could be decreased to about 5 5 4 0 % ,urn.=when chemostat cultures were established from suboptimal spore inocula ( - -i4/[ \
-*2c n
0
8
:.a
2-
FP
I
I
I
I
I
I
NADH Oxidation (nmoler/min/mg protein)
FIG. 16. Relation between reduced nicotinamide adenine dinucleotide (NADH)oxidase activity and NADH-driven amino-acid transport. The initial rate of Lglutamate (0) or L-alanine (A)transport were determined in membrane vesicles from Ban'llus subtills aro D in which the NADH-oxidase activity was reconstitutedto different degrees by addition of different concentrations of menadione. The NADH concentration was 10 mM. Taken from Bisschop and Konings (1976).
results of the experiments shown in Fig. 16 are at variance with this prediction, but are in agreement with the predictions based on an indirectly coupled system. The data given in Fig. 16 supply also information about the efficiency of NADH oxidation in energizing amino-acid transport. For transport of one mole of amino acid, oxidation for 130-250 moles of NADH is needed. This efficiency varies for different amino acids, which indicates a variation in the energy requirement for transport of difTerent amino acids. The same results have been obtained with membrane vesicles from the wild-type B . subtilis W 2 3 (Bisschop et al., 1975c). I t is obvious from these results that only a fraction of the energy supplied by the oxidation of NADH is applied to transport of the amino acid. Similar inefficiencies in energizing amino-acid transport have been observed in membrane vesicles from E . coli (Kaback and Hong, 1973)and Staph. aureus (Short and Kaback, 1974).These observations indicate that more than 99% of the energy generated by electron transfer in the respiratory chain is not, in the membrane vesicles, available for active transport of solutes. This inefficiency can be explained if the membrane vesicles accumulate (orextrude), in ad-
ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 233
dition to the amino acid, other ionic species present in the incubation mixture. However, according to Kaback (197.29, none of the ionic species in the reaction mixture, Mg2; SO:; SO:; PO:; Na+, C1-or K+ (in the absence of valinomycin) is accumulated during D-lactate oxidation by E. coli membrane vesicles. Information about the extrusion of ionic species is not available. In the concept of the chemi-osmotic coupling theory, an explanation for this inefficient use of energy for active transport could be found in a high proton permeability of the membrane vesicles. In other words, the membrane vesicles are leaky for protons. According to the chemi-osmotic coupling theory, inward movement of protons can occur via carrier proteins, or via ATPase. It appears unlikely that leakage of protons takes place via the ATPase complex since the addition of DCCD does not result in a higher efficiency of NADH oxidation in energizing transport. It is postulated that proton translocation from the outer surface of the membrane to the inside, via the carrier protein, occurs only during accumulation of solutes. Active transport of a solute therefore will increase the inward movement of protons (and/or charge) and decrease the proton motive force. This implies that the different transport systems will compete for the available energy, and that active transport of one solute will inhibit the simultaneous accumulation of another solute. This contention is supported by observations made by Schuldiner and Kaback (1975) under conditions of excess supply of energy. Membrane vesicles from E. coli ML 308-225 accumulate, in the presence of D-lactate or ascorbate-PMS, lactose at a much higher rate than proline (V- for lactose is 50 and for proline 1.3 nmoles per mg membrane protein per min) (Kaback and Barnes, 1971; Lombardi et al., 1973).In the presence of 10 mM lactose, the initial rate of proline transport is inhibited by 50%, and of triphenylmethylphosphonium transport (see p. 235) by 40%. Such an inhibitory effect of lactose was not observed in membrane vesicles which lack the lactose transport system. Similar experiments have been performed with membrane vesicles from B. subtilis aro D, incubated under conditions of limited energy supply. Even at low rates of NADH oxidation, transport of one amino acid is not inhibited by the addition of a 50 to 100-fold higher concentration of another amino acid (Bisschopand Konings, 1976).In these membrane vesicles, the energy supply for transport of one amino acid is therefore hardly affected by the simultaneous transport of another amino acid. These results, therefore, do not exclude the possibility that inward
234
WIL N. KONINGS
movement of protons occurs in the membrane vesicles via the carrier proteins, also in the absence of transportable solute. The chemi-osmotic coupling model visualizes the localization of intermediates of the electron-transfer systems partially at the outside and partially at the inside of the cytoplasmic membrane. Recently, observations have been made which indicate a localization of some components of the respiratory chain at the outside of “right-side out” membrane vesicles from B . subtilis (Bisschop et al., 1975b; Konings, 1975). In these membrane vesicles, transport can be energized under anaerobic conditions with NADH in the presence of ferricyanide as an electron acceptor (Fig. 1 7 ) and evidence has been presented that the AEROBIC
-c
10-
ANAEROBIC
5- ( b )
( 0 )
Time (min)
Time (min )
FIG. 1 7 . Effect of ferricyanide on NADH-driven uptake of L-glutamate under aerobic and anaerobic conditions by membrane vesicles from Bacillus subtilis W23. Uptake of Lglutamate was determined in the presence of NADH (10 mM) (01,NADH (10 mM)and or without potassium ferricyanide (10 mM) (A), potassium ferricyanide (10 mM) (O), electron donor or ferricyanide added (w). Taken from Bisschop et al. (1975~).
membrane-impermeable ferricyanide accepts electrons from the terminal part of the respiratory chain, most likely from cytochrome cl. Similar lines of evidence have been presented for an outside localization of anaerobic electron-transfer intermediates in membrane vesicles from anaerobically grown E . coli (Boonstra et al., 1976b). Furthermore, evidence has been obtained for a localization of electron-transfer inter-
ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 235
mediates prior to the coupling site(s) of the respiratory chain at the outside of the membrane, by transport experiments in membrane vesicles from B . subtilis and E . coli with the membrane-impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulphonate (MPS). This electron donor drives transport of amino acids, as well as its lipophilic analogue reduced phenazine methosulphate (PMS) (Konings, 1975; Short and Kaback, 1975). The available evidence from studies in whole cells and membrane vesicles in favour of a chemi-osmotic type of energy coupling has been reviewed by Harold (1972) and Hamilton (1975). In this discussion we will focus our attention only on studies with membrane vesicles. (i) Reeves (197 1) demonstrated that membrane vesicles from E . coli extrude protons during oxidation of D-lactate. (ii) Electron transfer-dependent transport in vesicles from several organisms is severely inhibited by a variety of proton conductors, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), although these agents do not inhibit electron transfer (Barnes and Kaback, 1970, 197 1; Konings and Freese, 1972). Furthermore, a number of mutants of E . coli have been isolated which exhibit pleiotropic transport defects, and vesicles prepared from some of these mutants exhibit increased permeability to protons (Rosen, 1973b; Altendorf et al., 1974). (iii) Dilution of membrane vesicles, which contain internally potassium, into a medium devoid of potassium but containing valinomycin, results in valinomycin-mediated potassium emux and the generation of an electrical potential (Aq) across the membrane, interior negative. Under such conditions, lactose and amino acids are accumulated by the membrane vesicles (Hirata et al., 1973; Lombardi et af., 1974).
(iv) During D-lactate or reduced PMS oxidation, lipophilic cations such as dimethyldibenzylammonium (in the presence of tetraphenylboron) (Hirata et al., 1973; Lombardi et al., 1974; Altendorf et al., 19751,i triphenylmethylphosphonium (Schuldiner and Kaback, 19751, safranine-o (Schuldiner and Kaback, 1975) and rubidium (in the presence of valinomycin) (Lombardi et al., 1973) are accumulated (Fig. 18). There is a quantitative correlation between the steady-state levels of accumulation of the different lipophilic cations (Schuldiner and Kaback, 1975). Furthermore, steady-state levels of lactose and amino
WIL N. KONINGS
236
acid accumulation are directly related to the steady-state level of TPMP-accumulation. 20f
-24 - 18
-
-
+ =
-12
+\.s
a"
z I-
a -
I
h
-
-6
FIG.18. Uptake of triphenylmethylphosphonium(TPMPC)by membrane vesicles from Escherichia coli ML 308-225 in the presence of different electron donors. Triphenylmethylphosphoniumuptake was determined in the presence of sodium ascorbate (20 mM) and phenazine methosulphate(0.1 mM) (01,lithium D-lactate (20 mM) (4,lithium L-lactate (20 mM) (V),sodium succinate (20 mM) (01, NADH (A),or without added electron donor (0). Taken from Schuldiner and Kaback (1976).
Analogous observations have been made with membrane vesicles from anaerobically grown E. coli, during anaerobic electron transfer in the nitrate respiration system and the fumarate reductase system (Boonstra et al., 1976a) and in membrane vesicles from the phototrophic organism Rhodopseudomonas sphaeroides upon light-induced cyclic electron flow (Hellingwerfet al., 1975). (v) Strong support for a chemi-osmotic type of energy coupling comes from transport studies in membrane vesicles from Halobacterium halobium. Upon illumination, the photochemical events which occur in the membrane- bound bacteriorhodopsin result in the extrusion of protons (Bogomolini and Stoeckenius, 1974; Racker and Stoeckenius, 1974; Racker and Hinckle, 19741, and a proton-motive force is generated in the order of 200 mV (Renthal and Lanyi, 1975). Racker
ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 237
and Stoeckenius (1974) observed, in a reconstituted system in which purple membranes from H. halobium and mitochondria1ATPase are incorporated into lipid vesicles, ATP production upon illumination. MacDonald and Lanyi (1975) demonstrated that these vesicles transport leucine in response to light, and presented evidence that the driving force for this transport is the electrical potential. (vi) In agreement with an indirect coupling model, as proposed by the chemi-osmotic theory, is the observation that membrane vesicles from E. coli ML 308-225 contain a large excess of lactose carriers (the product of the y-gene) relative to D-lactate dehydrogenase (Reeves et al., 1973). (vii) Convincing evidence for a chemi-osmotic mechamism of active transport in the vesicle system was supplied by Ramos et al. (1976). It was demonstrated by flow dialysis experiments that membrane vesicles from E. coli generate, in the presence of ascorbate-PMS, a large transmembrane pH-gradient which can reach two pH units at an external pH value of 5.5. Using the distribution of weak acids (Harold and Baarda, 1968), such as acetate, to measure the pH gradient (ApH) and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential across the membrane (A+),the vesicles were shown to generate a proton-motive force (A,&+) of approximately -180 mV at pH 5.5. Membrane vesicles from E. coli accumulate lactose and other substrates to apparent intravesicular concentrations which are one hundred-fold greater, or more, than those of the external medium. In order to sustain concentration gradients of this magnitude, a proton-motive force of at least 120 mV is required. Although these observations lend strong support for a chemiosmotic type of energy coupling in active transport processes, several other observations have been made which are, at this moment, difficult to explain in the framework of this theory (Lombardi et al., 1974). These are: (i) There is no correlation between rates of oxidation of various electron donors, in E. coli, and their relative effects on transport (Barnes and Kaback, 197 1 ; Kaback and Barnes, 197 1). The effectiveness in stimulating transport is much higher for ascorbate-PMS and D-lactate than for NADH or succinate (Table 2). It has been discussed before (p.200)that these observations cannot be explained by a
238
WIL N. KONINGS
specific localization of D-lactate dehydrogenase in the membrane because, in vesicles from mutants which lack D-lactate dehydrogenase, the effectiveness of succinate or NADH as an electron donor reaches similar levels as D-lactate in the wild type. Furthermore, an explanation based on the assumption that part of the membrane vesicles is inverted appears to be unlikely (p. 194). Recently, it was demonstrated that a qualitative relationship exists between the ability of various electron donors to drive transport and their ability to generate both an electrical potential (interior negative) across the membrane (Schuldiner and Kaback, 1975) and a pH-gradient (Ramos et al., 1976). AscorbatePMS and D-lactate produce maximal relative effects for each parameter, while succinate and, especially, NADH produced much weaker efTects. It appears, therefore, than an understanding is required of the role which different electron carriers have in the generation of a pH-gradient, or an electrical potential, in order to explain the different effects of the various electron donors. (ii) Electron-transfer inhibitors, which completely block D-lactate oxidation and D-lactate-dependent transport, have different effects on emux of accumulated substrates. Inhibition at sites of the E. coli respiratory chain distal to the energy-coupling site(s1 (anaerobiosis, amytal, HOQNO and cyanide) results in a rapid efflux of accumulated solutes from vesicles preloaded in the presence of D-lactate, while inhibitors of D-lactate dehydrogenase (oxalate and oxamate) cause little or no efflux from preloaded vesicles (Kaback and Barnes, 197 1 ; Lombardi and Kaback, 1972; Lombardi et al., 1974). Initially these and other observations have led to the postulation of the direct-coupling model (Kaback and Barnes, 1971; Kaback and Hong, 1973). It was postulated that, in E. coli, the carriers with different substrate specificities occupy equivalent sites in the respiratory chain between D-lactate dehydrogenase and cytochrome, b,, and that active transport of a particular sugar or amino acid is associatedwith reduction of the appropriate carrier by D-lactate dehydrogenase. In this model a specific role in the energization of transport is played by that part of the electron respiratory chain, in E. coli, lying between D-lactate dehydrogenase and cytochrome b,. It offered an explanation for the specific effects of D-lactate on transport, and also for the different effects of respiratory chain inhibitors on the emux of accumulated substrate. Inhibition beyond the energy-coupling site maintains the
ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 239
carrier in a reduced state. In such a state, the carrier has a low affinity for the solute and is mobile, and thus it will allow emux to occur. Inhibition of the carriers before the energy-coupling site results in an oxidation of the carrier. In this state the carrier has a high substrate affinity and is immobile, and thus no efflux of accumulated substrate can take place. It is obvious from the previous discussion that a major objection- of this model is that it fails to explain several observations which have been discussed above, such as the uptake of sugars and amino acids upon an imposed ion gradient, electron transfer-driven uptake of lipophilic cations and the action of uncouplers. Furthermore, the different electron donors have similar effects on the transport of lipophilic cations, and respiratory chain inhibitors effect efflux of accumulated lipophilic cations in a similar way to that which has been observed for sugars and amino acids. Transport of lipophilic cations is not carrier-mediated, and an explanation for these observations must therefore be found at the level of the “energized membrane state” and not at the level of the carrier proteins. In order to explain the observations, presented above, in the context of the chemi-osmotic model of energy-coupling, Kaback et al., (1976) suggested that the membrane potential is in equilibrium with the redox state of the respiratory chain at that site between D-lactate dehydrogenase and cytochrome b, which generates the membrane potential. Inhibition of electron flow in a manner which leads to reduction of the energy coupling site results in dissipation of the membrane potential, while inhibition of electron flow in a manner which leads to oxidation of the energy-coupling site does not result in a collapse of the potential. Such an explanation reconciles aspects of the chemi-osmotic model and the direct coupling model. I t emphasizes that the site of the respiratory chain between D-lactate dehydrogenase and cytochrome 6 , plays a special role in generation of the membrane potential. In order to offer a final explanation, more insight appears to be required into the role which various components of the electron- transfer systems have in the translocation of protons and the generation of a membrane potential. C . ENERGY-DEPENDENT BINDING OF SOLUTE TO CARRIER
PROTEINS
Carrier-mediated transport of a solute through the cytoplasmic membrane requires several distinct steps: in one of the initial steps, the
240
WIL N. KONINGS
solute binds to the carrier protein at the outside surface of the membrane; subsequently the carrier-solute complex travels, or rotates, in the membrane in such a way that the solute becomes exposed to the inside surface of the membrane, and finally the solute is released at the inside. Elegant experiments performed by Schuldiner et al. (1975a, b) and Rudnick et al. (1975a, b, c) demonstrated that energy is required for the initial steps of the transport process (i.e. exposure of the carrier to the outer surface of the membrane where it is able to bind the ligand). Photoreactive p-Galactosides
k
Fluorescent /3-Galactosides
i)H
R=
I
FIG. 19. Structural formulae of various dansylgalactosides and azidophenylgalactosides. Taken from Schuldiner et al. (1976).
Schuldiner et a1 (1975a, b) used for these studies the fluorescent pgalactosides shown in Fig. 19. These compounds competitively inhibit lactose transport by membrane vesicles from E. coli ML 308-225, but are not accumulated (Reeves et al., 1973; Schuldiner et al., 1975a, b). When membrane vesicles are incubated with these fluorescent /3galactosides, an increase in fluorescence is observed upon either the addition of D-lactate, the imposition of an electrical potential (interior negative), or dilution-induced carrier-mediated lactose emux. The increase in the fluorescence, induced by D-lactate, is blocked and/or rapidly reversed by addition of /3-galactosides, sulphhydryl reagents, inhibitors of D-lactate oxidation or uncoupling agents. The increase is not observed with a danlysglucoside(Z’-(N-dansyl)aminoethyl 1-thio-
ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 241
p- D-glucopyranoside), nor with membrane vesicles which lack the pgalactoside transport system, indicating that the effects are specific for the galactosyl configuration of the ligand. The affinity of the carrier for substrate is directly related to the length of the alkyl chain between the galactosidic and the dansyl moieties of the dansyl galactosides. The affinity constants of the various dansyl galactosides, as determined by fluorometric titration are in good agreement with their apparent Kt values for lactose transport. Anisotropy of fluorescence measurements with 2-(N-dansyl)aminoethyl-p-D-thiogalactopyranoside(DG,) and 6-(N-dansyl)-aminoethylp-D-thiogalactopyranoside(DG,) demonstrate a dramatic increase in polarization on addition of D-lactate which is reversed by anoxia or addition of lactose (Schuldiner et d., 1975a). These observations indicate that the changes in the fluorescence observed on “energization” of the membrane are the result of binding of the dansyl galactosides rather than binding followed by transfer into the hydrophobic interior of the membrane. The results suggest that the lac carrier protein is inaccessible to the external medium unless energy is provided, and that energy is coupled to one of the initial steps of transport. A similar conclusion was reached in studies with the photoreactive p-galactosides (2-nitro-4azidophenyl-p-D-thiogalactopyranoside (APG,) and 242-nitro-4azidopheny1)aminoethyl-p-D-thiogalactopyranoside(APG,)) (Fig. 19) (Rudnick et al., 1975a, b). Irradiation of these compounds with visible light causes photolysis of the azido group to form a highly reactive nitrene which then reacts covalently with the macromolecule to which the azido-containing ligand is bound. The /?-galactoside APG, inhibits lactose transport in membrane vesicles from E. coli ML 308-225 competitively with an apparent Kt of 75 pM. In contrast to the dansylgalactosides, APG, is actively transported by the membrane vesicles upon the addition of D-lactate, and kinetic studies revealed an apparent K, of 75 pM. Membrane vesicles devoid of lac transport do not accumulate APG, in the presence or absence of D-lactate. When exposed to visible light in the presence of D-lactate, APG, irreversibly inactivates the lac transport system, but this photolytic inactivation does not occur in the absence of D-lactate. Kinetic studies of the inactivation process yield a KD of 7 7 pM. The effects are specificfor the lac transport system, since lactose protects against photolytic inactivation and APG, does not inactivate against amino-acid transport. The p-galacto-
242
WIL N. KONINGS
side APG, behaves similarly with respect to photoinactivation, but this compound is not transported by the vesicles and has a higher affinity for the lac carrier (the Ktfor competitive inhibition of lactose transport and for the KD for photolytic inactivation in the presence of D-lactate are 35 pM). Furthermore, APG,-dependent photolytic inactivation can also be induced by an artificially imposed membrane potential (exterior positive). The studies with dansyl- and azidophenylgalactosides demonstrate the lac protein is accessible to the external medium only when energy is provided. Several possible mechanisms by which energy might lead to exposure or increased affinity of the binding site to the outside surface of the membrane have been considered (Schuldiner et al., 1975a). In view of the evidence presented in favour of a chemi-osmotic type of energy-coupling, it seems attractive to postulate that the lac carrier contains a negative charge and moves in response to a membrane potential to the outside of the membrane where it is able to bind the ligand. Studies on the effects of the sulphhydryl reagent, p-chloromercuribenzenesulphonate (p-CMBS),on APG,-dependent photoinactivation demonstrated that the lac carrier protein contains sulphhydryl groups which are not in the binding site (Rudnick et al., 1975~). Treatment of E . coli membrane vesicles with p-CMBS results in an inhibition of all carrier-mediated aspects of the lactose transport system (Kaback and Barnes, 197 1). However, p-CMBS does not block D-lactate-induced APG,-dependent photo-inactivation; in contrast p-CMBS induces APG, photo-inactivation in the absence of D-lactate. The dissociation constant of APG, for p-CMBS-treated membranes is about 20 p M , a value which is very similar to that determined for D-lactate-induced APG,-dependent photo-inactivation. Rudnick et al. ( 1 9 7 5 ~suggested ) as a possible mechanism thatp-CMBS reacts with a sulphhydryl group of the lac carrier and traps the protein at the outside surface of the membrane. In that position, substrate can bind to the carrier but cannot be translocated. The uncoupler, carbonyl-cyanide m-chlorophenylhydrazone (CCCP), does not inhibit p-CMBS-induced APG,photo-inactivation, and a membrane potential is thus not required for the p-CMBS effect. It is evident that p-CMBS does not block the binding site on the carrier, since APG, binds with high affinity and the p-CMBS-treated carrier protein is protected from APG,-binding by lactose, thiodigalactoside and melibiose. Exposure to the external
ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 243
medium of sulphhydryl groups of the lac carrikr protein appears also to occur upon energization, because inactivation of lactose transport by N-ethylmaleimide is increased two to four-fold by reduced phenazine methosulphate. The results indicate that energization of the membrane leads to an exposure, to the outer surface of the membrane, of a high-affinity binding site and a sulphhydryl group which is not in the binding site. It is suggested that the sulphhydryl group in the lac carrier protein may exist in an ionized form in the hydrophobic milieu of the membrane, and that this functional group in the protein may respond to the membrane potential (Kaback et al., 1976).
V. Conclusions Isolated bacterial cytoplamic membrane vesicles have proved to be an excellent model system for studies of integrated membrane functions. Membrane vesicles, isolated with the lysozyme-EDTA procedure, have the same orientation as the cytoplasmic membrane of intact cells, and these vesicles catalyse a number of membrane-bound functions. Observations made in studies with membrane vesicles demonstrated two types of transport systems : (i) group translocation systems which catalyse vectorial covalent reactions ; and (ii) active transport systems. The active transport systems appear to be the major mechanisms for translocation and accumulation of solutes in bacteria. The energy for active transport can be supplied by electron flow in a number of electron-transfer systems ; respiratory chains with oxygen as terminal electron acceptor; anaerobic electron transfer systems with nitrate or fumarate as terminal electron acceptor, and cyclic electron- transfer systems. Furthermore, light-dependent reactions in bacteriorhodopsin can supply the energy for active transport processes in membrane vesicles from Halobacterium halobium. I t has been demonstrated that energy released by the electrontransfer systems is not coupled to active transport via ATP. It has not yet been thoroughly established whether ATP can serve as the major source of energy for active transport in bacteria grown under glycolytic conditions it might be possible that electron-transfer systems which do not contain cytochromes supply the energy for active transport under these conditions. In view of recent studies, it appears to be beyond dispute that chemiosmotic phenomena are essentially involved in the mechanism of
244
WIL N. KONINGS
energy coupling. Electron flow in the electron-transfer systems results in the generation of a proton-motive force which is the driving force for active transport. Studies with membrane vesicles have demonstrated that the energy is coupled at least to one of the initial steps in the transport process. In order to obtain a complete understanding of the mechanism of active transport, a number of features remain to be elucidated. Among them are : involvement of electron-transfer intermediates in the translocation of protons; the role of the electrical potential, and the pH-gradient, in the energy coupling to active transport of different solutes; the molecular properties of the transport carriers and the mechanism of solute translocation. Attempts are currently in progress which hopefully, in the near future, will supply insight into these and other properties of the active transport systems.
VI. Acknowledgements I would like to express my appreciation to Dr. R. N. Campagne, Mrs. I. Kuipers-Wessels,A. Bisschop, J. Boonstra and P. A. M. Michels for their constructive criticism of the manuscript and their valuable suggestions. Dr. H. R. Kaback and Dr. J. Lanyi kindly supplied manuscripts. prior to publication. I am very grateful to Mrs. M. T. BroensErenstein, Mrs. R. G. Kalsbeek and Mrs. J. W. Schrdder-ter Avest for help in the preparation of this manuscript. The studies performed in the Laboratory of the author were supported by the Netherlands Organization of Pure Scientific Research (ZWO). REFERENCES
Altendorf, K. H. and Staehelin, L. A. (1974). Journal ofEacten'ology 117, 888. Altendorf, K. H., Harold, F. M. and Simoni, R. D. (1974).J o u d o f E i o l o g i c d Chemistry 249, 4587. Altendorf; K., Hirata, H. and Harold, F. M. (1975). Journal ofEiologica1 Chemistry 250, 1405. Ames, G . F. and Lever, J. E. (1970). Proceedings o f t k National Academy ofscience ofthe llnilrd States of America 66, 1096. Anderson, B., Wergel, N., Kundig, W. and Roseman, S. (1971). J o u d ofEiological Chetnislq 246, 7023. Barnes, E. M . (1972). Archives of Biochemistry and Biophysics 152, 795. Barnes, E. M . (1973).Journal of Biological Chemistry 248, 8120.
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Barnes, E. M. (1974). Federation Proceedings. Federation of American Societies f o r Expm’menlal Biology 33, 1475. Barnes, E. M. and Kaback, H. R. (1970). Proceedings ofthe National Academy ofscience of the United States of America 66, 1190. Barnes, E. M. and Kaback, H. R. (1971). J o u m l o f B i o l o g i d Chemistry 246, 5518. Berger, E. A. (1973). Proceedings of the National Academy of Science ofthe United States of Amprica 70, 1514. Berger, E. A. and Heppel, L. A. (1972). Journal ofBiologica1 Chemistry 247, 7684. Berger. E. A. and Heppel, L. A. (1974). J o u m l ofBiological Chemistry 249, 7747. Bhattacharyya, P. (1975). Journal of Bacteriology 12S, 123. Bhattacharyya, P., Epstein, W. and Silver, S. (197 1). Proceedings ofthe National Academy of Science of the United States of America 68, 1488. Bisschop, A., Doddema, H. and Konings, W. N. (1975a).JournalofBactcriology 124,613. Bisschop, A., Boonstra, J., Sips, H. J. and Konings, W. N. (1975b). Federation of European Biochemical SocietiCs Letters 60, 1 1. Bisschop. A., Jong de, L., Lima Costa, M. E. and Konings, W . N. (1975~). J o u m l of Bacteriology 121, 807. Bisschop, A. and Konings, W. N. (1976). Manuscript in preparation. Bogomolini, R. A. and Stoeckenius, W. (1974).Journal ofSupramolecu1lr Structure 2, 775. Boonstra, J., Huttunen, M. T. Konings, W. N. and Kaback, H. R. (1975a).Jounal of Biological Chemistry 250, 6792. Boonstra, J., Gutnick, D. L. and Kaback, H.R. (1975b).JournalofBacteriology 124, 1248. Boonstra. J., Kaback, H. R. and Konings, W. N. (1976a). Manuscript in preparation. Boonstra, J., Sips, H. J, and Konings, W. N. (1976b). In press. Boos, W. (1974). Annual Review of Biochemistry 4S, 123. Boos, W. (1975). In “Current Topics in Membrane and Transport”, (A. Kleinzeller and F. Bronner, eds.), Vol. 5, pp. 51-136. Academic Press, NewYork. Bronner, F., Nash, W. E. and Golub, E. E. (1975). In “Spores VI”, (P.Gerhardt, H. L. Sadofrand R. N. Costillow, eds.), pp. 356461. American Society for Microbiology, Washington, D.C. Butlin, J. D. (1973). Ph.D. Thesis: Australian University, Canberra City, Australia. Cirillo, V. P. (1961). Annual Review ofMicrobiology 15, 197. Cirillo, V. P. (1973). International Association of Microbiological Societies 1, Abstract, p. 57. Cohen-Bazire, G. (1963). In “Bacterial Photosynthesis”, (H. G a t , A. San Pietro and L. P. Vernon, eds.), pp. 89-110. The Antioch Press, Ohio. Cohen, G . N. and Monod, J. (1957). Bacteriological Reviews 21, 169. Cole, J. A. and Wimpenny, J. W. T. (1968). Biochim’ca et Biophysica Acta 162, 39. Cox, G. B.. Newton, N. A,, Gibson, F., Snoswell, A. M. and Hamilton, J. A. (1970). Biochemical Journal 117, 55 1. Cox, G. S., Thomas, E., Kaback, H. R. and Weissbach, H. (1973). Archives ofBiochemistry and Biophysics 158, 667. Devor, K. A.. Schairer, H. K., Renz, D. and Overath, P. (1974). European Journal of Biochemistry 45. 45 1. Dietz, G. W. (1972). Journal ofBiological Chemistry 247, 4561. Dutton, P. L., Petty, K. M., Bronner, H. S. and Morse, S. D. (1975). Biochimka et Biophysica Acta 387, 536. Enoch, H. G . and Lester, R. L. (1974). Biochemical and Biophysical Research Communications 61, 1234. Farrand, S . K. and Taber, H. W. (1973). Journal ofBactm’ology 115, 1021. Fein, J. E. and MacLeod, R. A. (1975). Journal ofBacteriology 124. 1177.
246
WIL N. KONINGS
Fournier, R. E., McKillen, M. N., Pardee, A. B. and Willecke, K. (1972). Journal of Biological Chemistry 247, 5587. Fox, C. F., Carter, J. R. and Kennedy, E. P. (1967). Proceedings ofthe National Academy of Science of the United States America 57, 698. Frerman, F. E. (1973). Archives of Biochemistry and Biophysics 159, 444. Futai, M. (1973). Biochemistry, Tokyo 12, 2468. Futail, M. (1974a). Journal ofMembrane Biology 15, 15. Futai, M. (1974b). Biochemistry, Tokyo 13, 2327. Ganesan, A. K. and Rotman, B. (1966). J o u m l of Molecular Biology 16, 42. Ghei, 0. K. and Kay, W. W. (1972). Federation ofEuropean Biochemical Societies Letters 20, 137.
Gibson, K. D. (1965). Journal of Bacteriology 90, 1059. Gordon, A. S., Lombardi, F. J. and Kaback, H. R. (1972). Proceedings ofthe National Academy o f Science of the United States of America 69, 358. Groen, A., Konings, W. N. and Harder, W. (1976). Manuscript in preparation. Guymon, L. F. and Eagon, R. G. (1974). Journal ofBacteriology 117, 1261. Halpern, Y. S. (1974). Annual Review of Genetics 8, 103. Halpern, Y. S. and Even-Shoshan, A. (1967). Journal of Bacteriology 93, 1009. Hamilton, W. A. (1975). Advances in Microbial Physiology 12, 1. Hampton. M. L. and Freese, E. (1974). J o u m l ofBacten’ology 118, 497. Hare, J . F., Olden, K. and Kennedy, E. P. (1974). Proceedings ofthe National Academy o f Science o f the United States o f America 71, 4843. Harold, F. M. (1970). Advances in Microbial Physiology 4, 45. Harold, F. M. (1972). Bacteriological Reviews 36, 172. Harold, F. M. and Baarda, J. R. (1968). J o u m l ofBacteriology 95, 816. Harris, P. and Kornberg, H. L. (1972). Proceedings ofthe Royal Society B 182, 159. Hellingwerf, K. J., Michels, P. A. M., Dorpema, J. W. and Konings, W. N.(1975). European Journal o f Biochemistry 55, 397. Hengstenberg, W. ( 1973). Intenzational Association o f Microbiological Societies 1, Abstract, p. 53. Heppel, L. A. (1967). Science, New York 156, 1451. Hertzberg, E. L. and Hinkle, P. C. (1974). Biochemical and Biophysical Research Cmmunicalions 58, 178. Hirata, H., Asano, A. and Brodie, A. F. (197 1). Biochemical and Biophysical Research Comminications 44, 368. Hirata, H., Altendorf, K. H. and Harold, F. M. (1973). Proceedings of the National Academy o f Science of the United States of America 70, 1804. Hochstadt-Ozer, J . (1972). Journal o f Biological Chemistry 247, 2419. Hochstadt-Ozer, J. and Rader, R. L. (197s). International Association o f Microbiological Societies 2, Abstract, p. 80. Hochstadt-Ozer, J . and Stadtman, E. R. (197 la).JournalofBiological Chemisty 246,5294. Hochstadt-Ozer, J. and Stadtman, E. R. (1971b). Journal of Biological Chemistry 246, 5304.
Hochstadt-Ozer, J. and Stadtman, E. R. (197 lc).Journal ofBiological Chemistry 246.53 12. Holden, J. T. (1962). In “Amino Acid Pool”, (J. T. Holden, ed.), American Elsevier, New York, 566-594. Holt, S. C. and Man-, A. G. (1965). Jounal ofBacteriology 89, 1402. Hong, J. S. and Kaback, H. R. (1972). Proceedings ofthe National Academy ofscience ofthe United Slates of America 69, 3336. Horecker, B . L., Thomas, J. and Monod, J. (1960).Journal ofBiologica1 Chemistry 235, 1580.
ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 247
Joenje, H., Konings, W. N. and Venema, G. (1974).Jouml ofBactenohgy 119 (31, 784. Joen,je, H . , Konings, W. N. and Venema, G. (1975).J o u d o f B a c t e ~ o l o g y121 (3). 771. Johnson; C. L., Cha, Y. A. and Stern, J. R. (1975).Journal ofBacteriology 121, 682. Kaback, H. R. (1968).Journal of Biological Chemistry 243, 37 11. Kaback, H. R. (1969a). In “Molecular Basis of Membrane Function”, (D. C. Testeson, ed.), pp. 421-444, Prentice Hall, inc., Englewood Cliffs. N.J. Kaback, H. R. (1969b). Proceedings ofthe National Academy ofscience ofthe United States of America 63, 724. Kaback, H. R. (1970a). In “Current Topics in Membranes and Transport”, (A. Kleinzeller, and F. Bronner, eds.), Vol. 1, pp. 35-99. Academic Press, New York. Kaback, H. R. (1970b). Annual Review of Biochemistry 39, 561. Kaback, H. R. (1971). In “Methods in Enzymology”. (W. B. Jakoby, ed.), Vol. 22, pp. 99-120. Academic Press, New York. Kaback, H. R. (1972). Biochimica et Biophysica Acta 265, 367. Kaback, H . R. (1974). Science, New York 186, 882. Kaback, H . R. and Barnes, E. M. (1971).Journal ofBiologica1 Chemistry 246, 5523. Kaback, H. R. and Deuel, T. F. (1969). Archives of Biochemistry and Biophysics 132, 118. Kaback, H. R. and Hong, J. S. (1973). Critical Reviews in Microbiology 2, 333. Kaback, H. R. and Kostellow, A. B. (1968). J o u m l of Biological Chemistry 243, 1384. Kaback, H . R. and Milner, L. S. (1970). Proceedings ofthe National Academy ofscience ofthe United States of America 66, 1008. Kaback, H. R. and Stadtman, E. R. (1966).Proceedings ofthe National Academy ofscience of /he United States of America 55, 920. Kaback, H. R., Rudnick, G., Schuldiner, S.,Short, S. A. and Stroobant, P. (1976).In “The Structural Basis of Active Transport”, (Y. Hatefi and L. qjavardi-Ohaniance, eds.), pp. 107-128, Academic Press, New York. Kaczorowski, G., Shaw, L., Fuentes, M. and Walsh, C. (1975). Journal of Biological Chemistty 250, 2855. Kepes, A. (1970). In “Current Topics in Membranes and Transport”, (A. Kleinzeller, and F. Bronner, eds.), Vol. 1, pp. 101-134. Academic Press, New York. Kenvar, G. K., Gordon, A. S. and Kaback, H. R. (1972).Journal OfBiological Chemistry 247, 291. Klein. W. L. and Boyer, P. D. (1972).Journal of Biological Chemistry 247, 7257. Kohn, L. and Kaback, H. R. (1973).Journal ofBiological Chemistry 248, 7012. Komatsu, Y. and Tanaka, K. (1973). Biochimica et Biophysica Acta 311, 496. Konings, W. N. (1975). Archives of Biochemistry and Biophysics 167, 570. Konings, W. N. and Bisschop, A. (1973). International Association of Microbiological Societies. 1, abstract, p. 78. Konings, W. N. and Boonstra, J. (1976). In “Current Topics in Membranes and Transport’’, (A. Kleinzeller and F. Bronner, eds.), Vol. 9, in press. Academic Press, New York. Konings, W. N. and Kaback, H. R. (1973).Proceedings of the National Academy ofbcience of [he United States of America 70, 3876. Konings, W. N. and Freese, E. ( 197 1). Federation of European Biochemical Societies Letters 14, 65. Konings, W. N. and Freese, E. (1972).Journal of Biological Chemistry 247, 2408. Konings, W. N., Barnes, E. M. and Kaback, H. R. (1971).Journal ofBiologicalChemistry 246, 5857. Konings, W. N., Bisschop, A. and Daatselaar, M. C. C. (197-2).Federation ofEuropean Biochemical Societies Letters 24, 260.
248
WIL N. KONINGS
Konings, W. N., Bisschop, A., Veenhuis, M. and Vermeulen, C. A. (1973). Journal 01 Bnrlenohgy 116, 1456. Konings, W. N., Boonstra, J., Vries, W. de (1975). Journal ofBacteriology 122, 245. Kornbrrg, H. L. (1972). In “Molecular Basis o f Biological Transport”, (J. F. Woessner and F. Huyings, eds.), pp. 157-181. Academic Press, New York. Kundig, W. (1976). I n “The Enzymes o f Biological Membranes” (A. N. Martonosi, ed.), Vol. 3, in press. Plenum Publishing Corporation, New York. Kundig, W., Ghosh, S . and Roseman, S. (1964). Proceedings ofthe National Academy of Sriencc of /he United States of America 52, 1967. Lagarde. A. E. and Stoeber, F. R. (1974). European Journal of Biochemistry 43, 197. Lester. R. L. and DeMoss, J. A. (1971). Journal of Bacteriology 105, 1006. Levinson, S. L. and Krulwich, T. A. (1974). Archives ofBiochemistry and Biophysics 160, 445.
Lin, E. C. C. (1971). I n “Structure and Function of Biological Membranes”, (L.J. Rothfield. ed.), pp. 285-341. Academic Press, New York. Lo. T. C. Y., Ravman. M. K. and Sanwal, B. D. (1974). Canadian Journal ofsiochemistry 52, 854.
Lombardi, F . J . and Kaback, H. R. (1972). Journal of Biological Chemistry 247, 7844. Lonibardi, F. J., Reeves, J. P. and Kaback, H. R. (1973). Journal of Biological Chemistry 248. 3551.
Lombardi, F . J.. Reeves, J. P., Short, S. A. and Kaback, H. R. (1974). Annals ofthe New k‘ork Arademy of Sciences 227, 312. Lowrv, 0 . H., Carter, J.. Ward, J. B. and Glaser, L. (1971).JournalofBiological Chemistry 246, 651 1.
MacDonald, R. E . and Lanyi, J. K. (1975). Biochemistry, New York 14, 2882. MacLeod, R. A., Thurman, R., Rogers, H. J. (1973). Journal ofBacteriology 113, 329. Matin, A. and Konings, W. N. (1973). European Journal of Biochemistry 34, 58. Matin, A., Konings, W. N., Kuenen, J. G. and Emmens, M. (1974). Journal ofGenera1 Microbiology 83, 3 1 1. Miki, K. and Lin, E. C. C. (1973). Journal ofBacteriology 114, 767. Miki, K., Sekuzu, I . and Okunuki, K. (1967). Annual Reports ofscientific WorkJ, Faculty o f Scienre. Osaka University 15, 33. Mitchell, P. (1966). Bzologtcal Reviews 41, 445. Mitchell, P. (1970). Symposium ofthe Society for General Microbiology 20, 121. Mitchell, P. (1973). Bioenergetics 4, 63. Murakawa, S., Izaki, K. and Takahashi, H. (197 1). Agricultural and Biological Chemistry 35, 1992.
Murakawa, S. Izaki, K., Takahashi, H. (1973). Ap’cultural and Biological Chemistry 37, 1905.
Oelze, J. and Drews, G. (1972). Biochimica et Biophysica Acta 265, 209. Oesterhelt, D. and Hess, B. (1973). European Journal of Biochemistry 37, 316. Oesterhelt, D. and Stoeckenius, W. (1973). Proceedings ofthe National Academy of Science o f /he United States of America 70, 2853. Or, A., Kanner, B. I. and Gutnick, D. L. (1973). Federation of European Biochemical Societies Letters 35, 2 17. Oxender, D. L. (1972). Annual Review ofBiochemistry 41, 777. Pardee, A. B. (1968). Science, New York 162, 632. Parnes, J. R. and Boos, W. (1973). Journal of Biological Chemistry 248, 4429. Patel, L., Schuldiner, S . and Kaback, H. R. (1975). Proceedings ofthe National Academy o f Science of the United Staks of America, 72, 3387
ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 249
Padasova, E. and Harold, F. M. (1969). Journal ofBactenology 98, 198. Pressman, B. C., Harris, E. J.. Jagger, W. S. and Johnson, T. H. (1967).Proceedings of the National Academy of Science ofthe United States of America 58, 1949. Prezioso, G., Hong, J. S., Kenvar, G. K. and Kaback, H. R. (1973). Archives of Biochemistry and Biophysics 154, 575. Racker, E. and Hinckle, P. E. (1974).Journal ofMembrane Biology 17, 181. Racker, E. and Stoeckenius, W. (1974).J o u m l of Biological Chemistry 249, 662. Ramos, S., Schuldiner, S. and Kaback, H. R. (1976). Proceedings ofthe National Academy o f Science ofthe United States ofAmerica, 73, 1892. Rayman, M. K., Lo,T. C. Y. and Sanwal, B. D. (1972).Journd ofBiologica1 Chemistry 247, 6332. Reeves, J . P. ( 197 1). Biochemical and Biophysical Research Communications 45, 93 1. Reeves, J . P.. Schechter, E., Weil, R. and Kaback, H. R. (1973).Proceedings ofthe National Academy of Science ofthe United States of America 70, 2722. Renthal, R. and Lanyi, J. K. (1976).Biochemistry, New York, 15, 2136: Romano, A. H., Eberhard, S. J., Dingle, S. L. and McDowell, T. D. (1970).Journal of Bacteriology 104, 808. Roseman, S . (1972). In “Metabolic Pathways”, (L.E. Hokin, ed.), Vol. 4, pp. 41-67. Academic Press, New York. Rosen, B. P. (197 1).Journal of Biological Chemistry 246, 3653. Rosen, B. P. (1973a).Journal ofBiologica1 Chemistry 248, 1211. Rosen, B. P. (1973b). Biochemical and Biophysical Research Communications 53, 1289. Rosen, B. P. and Adler, L. W. (1975). Biochimica et Biophysica Acta 387, 23. Rosen, B. P. and McClees, J. S. (1974). Proceedings ofthe National Academy ofbcience ofthe United States of America 71, 5042. Rosenberg, H., Cox, G . B., Butlin, J. D. and Gutowski, S. J. (1975). BiochemicalJoumal 146, 417. Rudnick, G., Weil, R. and Kaback, H. R. (1975a).Journal ofBiological Chemistry 250, 1371. Rudnick, G., Weil, R. and Kaback, H. R. (1975b). Federation Proceedings. Federation o f American Societies of Experimental Biology 34, 49 1-abstract 1525. Rudnick, G., Kaback, H. R. and Weil, R. ( 1 9 7 5 ~ )Journal . of Biological Chemistry 250, 6847. Ruiz-Herrera, J . and DeMoss, J. A. (1969).Journal ofBacteriology 99, 720. Schairer, H. U. and Haddock, B. A. (1972). Biochemical and Biophysical Research C m municntions 48, 544. Schuldiner, S. and Kaback, H. R. (1975). Biochemictry, New Yorh 14, 5451. Schuldiner, S., Kenvar, G . K., Weil, R. and Kaback, H. R. (1975a).Journal ofBiologica1 Chemistry 250, 1361. Schuldiner, S., Kung, H., Kaback, H. R. and Weil, R. (1975b).Journal ofBiologica1 Chemistry 250, 3679. Schuldiner, S., Rudnick, G., Weil, R., Kaback, H. R. (1976). Trends in Biochemical Sciences 1, 41. Shemyakin, M.M., Aldanova, N. A., Vinopradova, E. J., Yu, F. M. (1963). Tetrahedron Le/ter.c 28, 1921. Shemyakin, M. M., Ovichinnikov, Yu. A., Ivanov, V. T., Antonov, V., Vinogradova, E. J., Shkrob, A. M., Malenkow, G. G., Eustanov, A. V., Laine, I. A., Melnik, E. J. and Rvabova, I . D. (1969).Journal of Membrane Biology 1, 402. Short, S. A. and Kaback, H. R. (1974).Journal ofBiological Chemistry 249, 4275. Short, S. A. and Kaback, H. R. (1975). Journal ofBiological Chemistry 250, 4291.
250
WIL N. KONINGS
Short, S. A., White, D. C. and Kaback, H. R. (1972a).Journal ofBiologica1 Chemistry 247, 298. Short, S. A., White, D. C. and Kaback, H. R. (1972b).Journal ofBiologica1 Chemistry 247, 7452. Short, S. A., Kaback, H. R., Kaczorowski, G . , Fisher, J., Walsh, C. T. and Silverstein, S. C. ( 1974a). Proceedings of the National Academy ofscience of the United States of America 71, 5032. Short, S. A., Kaback, H. R. and Kohn, L. D. (1974b). Proceedings ofthe National Academy of Scieurr of the United States of America 71, 1461. Simoni, R. D. and Postma, P. W. (1975). Annual Review ofBiochemistry 44, 523. Simoni, R. D. and Shallenberger, M. K. (1972). Proceedings of the National Academy of Scirnces OjIhr United States of America 69, 2663. Singh, A. P. and Bragg, P. D. (1975). Biochimica et Biophysica Acta 396, 229. Smith, L. (1961).In “The Bacteria”, (I. C. Gunsalus and R. Y.Stanier, eds.), Vol. 2, pp. 365-396. Academic Press, New York. Sprott, G. D. and MacLeod, R. A. (1972). Biochemical and Biophysical Research Communicalions 47, 838. Sprott, G. D . and MacLeod, R. A. (1974).Journal ofBacteriology 117, 1043. Stinnett, J. D., Guymon, L. F. and Eagon, R. G. (1973). Biochemical and Biophysical Rr.\rnrch Communications 52, 284. Stoeckenius, W. and Lozier, R. ( 1974). Journal of Supramolecular Structures 2, 769. Tanaka, S., Lerner, S. A. and Lin, E. C. C. (1967).Journal ofBacteriology 93, 642. Tanigurhi, S. and Itagaki, E. (1960). Biochimica et Biophysica Acta 44, 263. Thomas, E. I., Weissbach, H. and Kaback, H. R. (1972). Archives ofBiochemistry and Biophysics 150, 797. Thomas, E. L., Weissbach, H. and Kaback, H. R. (1973). Archives of Biochemistry and Bin/ihy.ric.$157, 327. Tosteson, D . C., Andreoli, T. E., Tiefenberg, M. and Coole, P. (1968).JoumlofCeneral P/!~~!,’.iiolop 51, 3735. Tsuchiya, T. and Rosen, B. P. (1975a). Biochemical and Biophysical Research Communications 63, 832. Tsuchiva, T. and Rosen, B. P. (1975b).Journal ofBiologica1 Chemistry 250, 7687. Tsuchiva. T. and Rosen, B. P. (1976). Biochemical and Biophysical Research Communication 68, 497. Turtle, A. L . and Gest, H. (1959).Proceedings ofthe National Academy ofscience ofthe United Sln1e.r nf Amrricn 45, 1261. Van Thienen. G . and Postma, P. W. (1973). Biochimica et Biophysica Ada 323, 429. Walsh, C. T. and Kaback, H. R. (1974). Annals of the New York Academy of Sciences 235, 5 19. Walsh, C. T., Schonbrunn, A., Lockridge, O., Massey, V. and Abeles, R. H. ( 1972a). Journal of Biological Chemistry 247, 6004. Walsh, C. T., Abeles, R. H. and Kaback, H. R. (1972b).Journal ofBiologica1 Chemistry 247, 7858. Watanabe, N. and Po, L. (1974). Biochimica et Biophysica Ada 345, 419. Weiner. J . H. (1974).Journal of Membrane Biology 15, 1. Weiner, J . H. and Heppel, L. A. (1971).Journal of Biological Chemistry 246, 6933. Weissbach, H., Thomas, E. and Kaback, H. R. (1971). Archives of Biochemistry and Bioiphwics 147, 249. Willerkr, K. and Lange, R. (1974).Journal of Bacteriology 117, 373. Willecke, K. and Pardee, A. B. (1971).Journalof Biological Chemistry 246, 1032.
ACTIVE TRANSPORT OF SOLUTES IN BACTERIAL MEMBRANE VESICLES 251
Willecke, K., Cries, E. M. and Oehr, P. (1973).Journal ofBiologica1 Chemistry 248, 807. Wimpenny, J. W. T. and Cole, J. A. (1967). Biochimica et Biophysica Acta 148, 233. Wolfson, E. B. and Krulwich, T. A. (1974). Proceedings ofthe National Academy ofScience of (he United States of America 71, 1739. Wolfson, E. B., Sobel, M . E., Blanco, R. and Krulwich, T. A. (1974). Archives of Biochemistq and Biophysics 160, 440. Wu, H . C. P. (1967). Journal ofMolecular Biology 24, 213. Yamamoto, T. H., Mevel-Ninio, M . and Valentine, R. C. (1973). Biochimica et Biophysica Acta 314, 267.
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Aden ine Nucleot ide Concentrations and Turnover Rates. Their Correlation with Biological Activity in Bacteria and Yeast ASTRID G. CHAPMAN and DANIEL E. ATKINSON Molecular Biology Institute and Biochemistry Division, Department of Chemistry, University of California, Los Angeles, California 90024, U.SA. I. Introduction . . . . . . . . . . . . 11. Concentrations and Fluxes of Adenine Nucleotides in uivo . . . A. Adenine Nucleotide Turnover . . . . . . . . B. Turnover of ATP . . . . . . . . . . C. Regulation of ATP Utilization and Regeneration . . . . D. Sampling of Microbial Cultures for Adenine Nucleotide Determinations . . . . E. Changes in Adenine Nucleotide Concentrations 111. Concentration of ATP, Total Adenine Nucleotide Concentration, and . . . . . Energy Charge in Relation to Cellular Activities A. Relation between ATP Concentration and Growth Rate . . . . B. Variations in Adenine Nucleotide Levels during Growth C. Adenine Nucleotides in Mutant Strains Arrested in Growth D. Correlation between Kinetics in vitro and Observations in uiuo E. Relation between Energy Charge and Total Adenine Nucleotide . . . . . . . . . . . Concentration F. Phage Infection . . . . . . . . . . . G. Other Nucleotides . . . . . . . . . . H. RNA Synthesis . . . . . . . . . . . I. Protein Synthesis . . . . . . . . . . . IV. General Discussion . . . . . . . . . . . References . . . . . . . . . . . .
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I. Introduction
Because the adenine -nucleotides are involved in every metabolic sequence, and in most metabolic energy transductions (both chemical and mechanical), and are also intermediates in the synthesis of nucleic acids, a discussion of the roles of these compounds could include nearly all aspects of the genetics, metabolism, physiology, and locomotion of microbes, as well as much of the work reported on properties of enzymes from micro-organisms studied in vitro. Such a comprehensive treatment would, however, not only exceed the competencies of the reviewers, but also greatly exceed the available space; thus drastic and largely arbitrary limitations of the areas to be covered have been necessary. This review therefore deals mainly with reports on the concentrations of ATP, ADP, and AMP, and on the ratios of these concentrations found in intact microbial cells, and with changes in these concentrations (or concentration ratios) that occur under various conditions. Further we shall review the correlations, both stoicheiometric and regulatory, that have been reported between the concentrations of the different adenine nucleotides and the occurrence or rates of integrated functions such as growth or production of storage compounds. Even within these areas, only a sampling was possible; other equally important papers had to be omitted, with no derogatory implications. Although much of the conceptual framework within which the results will be considered was derived initially from work on enzymes in vitro, experiments of that type will not be described here. Some of the earlier results have been reviewed elsewhere (Atkinson, 1969). Moreover the whole area of cyclic AMP effects has been excluded (for a recent review of cyclic AMP in prokaryotic organisms, see Rickenberg, 1974). The roles of the adenine nucleotides may be conveniently classified under three headings. First, they are intermediates in the biosynthesis of nucleic acids and histidine, and also of nucleotide cofactors such as NAD, NADP, FAD, and coenzyme A. Second, they constitute an energy-transducing system that stoicheiometrically couples all metabolic processes and thus plays the central role in stoicheiometric correlation of metabolism. Third, they kinetically regulate the activities of a large number of enzyme reactions and probably of all metabolic sequences. In serving as intermediates, the adenylates d o not differ in principle from dozens of other compounds, but the second and third
ADENINE NUCLEOTIDE CONCENTRATlONS AND TURNOVER RATES
255
functional categories are unique. Metabolism is organized around the adenine nucleotides, and these compounds must be taken into account in the study of nearly every aspect of functional biology. Processes in which the adenine nucleotides participate stoicheiometrically (categories 1 and 2 in the above classification) are summarized in Fig. 1. Interconversions between the different adenine nucleotide species that occur in the course of energy transduction and stoicheiometric coupling between metabolic sequences (category 2) are indicated within the circle. These include the large number of reactions where ATP serves as a phosphate donor; those in which AMP is the immediate or ultimate product; the reformation of ATP from ADP by oxidative phosphorylation or by substrate-level phosphorylation; and the interconversion of ATP, ADP and AMP through the adenylate kinase reaction. The adenyl cyclase-mediated formation of CAMP from ATP, and the subsequent hydrolysis of CAMP to AMP, are also indicated in the figure, although these reactions will not be discussed in this review. All these reactions involve the removal and replacement of the y- and P-phosphate groups of ATP, and they do not affect the sum of the concentrations of the different adenine nucleotide species. Conversion of ATP to ADP or AMP, and its regeneration, will be referred to as ATP turnover. NAD FAD CoASH etc. k
Histidine, Protein
,dADP, DNA
Adenine
Adenosine
De KO
biosynthesis--,-IMP
\$.
FIG. 1. Reactions involved in adenine nucleotide formation, utilization and interconversion. Adapted from an unpublished figure by Jean. S . Swedes.
256
A. G. CHAPMAN AND D. E. ATKINSON
Reactions of category 1, in which the adenylates serve as biosynthetic intermediates, are indicated by arrows extending from or entering the adenine nucleotide circle. These reactions include the synthesis and degradation of AMP and the net utilization of adenine nucleotides in biosynthesis. The adenylate utilization and resynthesis represented by these reactions will be referred to as turnover of the adenine nucleotide pool, as distinguished from ATP turnover as already defined. 11. Concentrations and Fluxes of Adenine Nucleotides in vivo A. ADENINE NUCLEOTIDE TURNOVER
The rate of utilization of adenine nucleotides for the biosynthesis of macromolecules can be estimated from the cellular composition and the generation time of the organism. Such calculations are shown in Table 1 for Escherichia coli and Salmonella typhimurium grown in different media so as to allow a ten-fold range of growth rates to be obtained. Although the figures are compiled from different papers, and the growth rates at which total adenine nucleotides were measured differed slightly from those for the other determinations, as indicated in the footnotes, the calculated turnover rates should be approximately correct. The amount of adenylate and deoxyadenylate residues in RNA and DNA, and of histidine in'protein (ATP contributes a carbon and a nitrogen atom to the biosynthesis of histidine), is calculated from the macromolecular compostion of the cell according to the assumptions listed in the legend to the table. At rapid growth rates, net RNA synthesis represents as much as 75% of the adenylate consumption. Even at slow growth rates, where the cellular RNA content is lower, about 50% of the adenylate utilization is accounted for by RNA synthesis. The net rate of adenylate incorporation per minute into polymers is shown in the second column from the right. Figures for the two organisms are very similar. As would be expected, slower growth rates are accompanied by lower rates of net adenylate utilization. The last column shows that at very rapid growth rates the adenine nucleotide pool is replenished approximately every 40 seconds, in contrast to a turnover time of four to six minutes in slowly growing cells. The rate of adenylate utilization decreases somewhat more sharply than the growth rate because of the lower RNA content of the slowly growing cells. The difference between the rates of decrease is not great, how-
TABLE 1. Turnover of the adenine nucleotide pool' Organism
Salmonella typhimurium'
Doubling time (mid
Net adenine Sum of adenine incorporation nucleotides ( p o l e s / g into polymers DNA RNA Protein DNA RNA Protein Total dry wt) (pmoleslminlg dry wt.)
D 0 Turnover of rn adenine nucleotide pool (seconds) rn
$ z C
25 50 300
35 310 37.. 220 37 180 40 120
670 740 780 830
28 29 29 32
233 165 135 90
55 61 64 68
316 255 228 190
8.0' 3.7 6.0 4.0
12.64 5.10 2.28 0.63
38 44 158 38 1
24 40 110
26 23 32
670 750 800
21 18 25
231 172 122
55 61 66
307 251 213
9.5f 8.3 6.8
12.79 6.28 1.94
45 79 210
100
Escherichiacoli'
v o l e s adenine residuedg dry wt!
mg/g dry wt.
307 229 163
'The following assumptions were used for the calculations: the average molecular weights of nucleotide monophosphate residues in RNA, the deoxynucleotide monophosphate residues in DNA, and the amino-acid residues in protein are equal to 333, 3 1 7 and 122, respectively. Adenylates constitute 25% of the RNA bases in E. coli (Spahr and Tissieres, 1959). and it is assumed that DNA contains 25% dmxyadrnylatr rrsidurs. Histiditir cotistimtes 1% of the amino-acid residues in protein from E. coli (Roberts el nl., 1955). The net utilization of adenine nucleotidcs for thr li)rniatioti ol' lrrr histidine and pyridine nucleotides has been ignored in these calculations. "denylate residues in RNA, deoxy-adenylate residues in DNA, and histidine residues in protein. 'The composition of Sal. lyphimutium is from Maalw and Kjeldgaard ( 1966). 'The composition of E. coli is from Forchhammer and Lindahl(l97 I). 'The concentrations of total free adenine nucleotides in Sal. typhimurium at doubling times 30, 50, 105, and 240 min are from Smith and Maalee (1964). f The cellular concentrations of the adenine nucleotides at the growth rates observed were not determined by Forchhammrr i i i i t l 1.incliilil (10711. Instead, values obtained from other laboratories at similar growth rates were substituted as follows: At a doubling time of 36 min, from Dietzler tf 01. (1974~). The units are converted from jnnoleslg protein by assuming protein equal t o 67% of dry wright; iit ii doubling tinic ,4fi tiiiti. IIotti S\vc.tlrs ef 01. (1975).The units are converted fr.om pnoles/g protein by assuming protein equal to 75%of dry weight; at a doubling time of 120 min, lrom Lowry ef al. (1971).
Prn
2 #
0 0
z Gz -I n
30 c)
-I
C
3
z
2rn n
2 v)
hl
cn -J
258
A. G. CHAPMAN AND D. E. ATKINSON
ever. I t can be calculated from Table 1 that the adenine nucleotide pool turns over 30 to 50 times per generation; thus it appears that the turnover time of the adenylate pool in enteric bacteria is about 2 to 3% of the generation time. 1. Balance between Adenine Nucleotide Synthesis and Utilization
In growing cells, in the absence of exogenous nucleosides or free bases, biosynthesis de nouo must account for rLetpurine nucleotide formation (sequence 1, Fig. 1). There is also a constant influx of AMP into the adenylate pool from breakdown of unstable RNA (reaction 2, Fig. 1). The flow of ATP into mRNA is rapid, but because of the short lifetime of mRNA, only a small part of this flow, proportional to the increase in cell mass and the steady-state concentration of mRNA, represents net utilization of adenylates. For the purposes of this discussion, mRNA synthesis may be considered as an indirect conversion of ATP to AMP. The regulation of purine nucleotide biosynthesis and the maintenance of adenylate and guanylate levels have been discussed by Blakey and Vitols (1968), Stadtman (19701, Burton (19701, and Henderson and Paterson (1973). The pathway is subject to feedback control by purine nucleotides through their effects on phosphoribosyl pyrophosphate (PRPP)synthase (Atkinson and Fall, 1967; Switzer, 1967; Switzer and Sogin, 1973), and on the first committed enzyme of the sequence, PRPP amidotransferase (Nierlich and Magasanik, 1965; Rowe and Wyngaarden, 1968; Wyngaarden, 1972 (a review)). Under some conditions, nucleosides may be formed in the degradation of RNA or encountered in the environment. In such cases the “scavenger” reactions, whereby AMP or IMP is formed frcm the reaction of PRPP with adenine or hypoxanthine respectively, probably provide important routes for replenishment of the intracellular adenine nucleotide pool (reaction 3, Fig. 1). The reactions are catalysed by adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase, both of which are present in micro-organisms (Henderson and Paterson, .1973; Krenitsky et al., 1970; Hochstadt-Ozer and Stadtman, 197 1 ; Sin and Finch, 1972). Competition for a common, limiting supply of PRPP between the “scavenger” reactions and PRPP amidotransferase appears to be part of the mechanism whereby exogenous nucleotides, nucleosides, or free purine bases inhibit biosynthesis de nouo, since the “scavenger” enzymes have much greater affinity for PRPP than do the enzymes in the pathways by which nucleotides are
ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES
259
synthesized de n o w (Bagnara and Finch, 1973, 1974). Since the synthesis of purines is metabolically expensive in terms of ATP equivalents, replacement through the “scavenger” reactions represents a significant saving in energy and intermediates. Adenosine kinase appears to be missing from bacteria (Hoffmeyer and Neuhard, 1971; Yagil and Beacham, 1975)and so is not indicated in Fig. 1. Ribonucleic acid synthesis (sequence 4, Fig. 1) accounts for the major and most variable net utilization of adenine nucleotides and will be treated separately in a later section. The reduction of ADP or, in some bacteria, ATP (O’Donovan and Neuhard, 1970) to deoxyribonucleotides initiates the utilization of adenylates for DNA synthesis (sequence 5, Fig. 1). According to Table 1, DNA synthesis represents a minor ( 10 to 15%) but relatively constant adenylate consumption per cell, although the rate of adenylate utilization by this process probably fluctuates considerably during the cellular growth cycle. In exponentially growing cultures of E. coli, DNA synthesis can account for the entire turnover of the dATP pool (Neuhard and Thomassen, 197 1). The N- 1 and C-2 atoms of the purine ring of ATP are incorporated into the imidazole ring of histidine, and the remainder of the ATP molecule, 5’-phosphoribosyl-5-amino-4-imidazole carboxamide, can be re-utilized as an intermediate in purine nucleotide biosynthesis, entering the path of synthesis de novo two reactions prior to IMP formation. Biosynthesis of histidine and its subsequent utilization in protein formation (sequence 6,Fig. 1) accounts for 18 to 35% of adenylate consumption in E. coli and sul. typhimurium (Table 1). The total pyridine nucleotide content of a number of microorganisms is typically around 3 to 10 p o l e s per gram dry weight (Schon, 197 1; Brody, 1972; Wimpenny and Firth, 1972).Therefore, unless there is a very rapid flux through the pyridine nucleotide pool, which seems highly unlikely, biosyntheses of these compounds and of other cofactors containing adenylic acid contribute only negligibly to net adenylate utilization (sequence 7, Fig. 1). Lundquist and Olivera (1971) have estimated that in E. coli growing with a doubling time of 40 min there are 550 molecules of pyridine nucleotides synthesized per second per cell, or approximately 0.09 ,umoles/min/g dry weight (assuming a typical cellular mass at this growth rate of 59 x lo-” g; Franzen and Binkley, 196 1).This is equal to about 1.5% of the adenylates utilized for synthesis of macromolecules in E. coli at this growth rate according to Table 1.
260
A. G. CHAPMAN AND D. E. ATKINSON
2. Adenine Nucleotide Catabolism The conversion of AMP to IMP, adenosine or adenine (reactions 8 , 9 and 10 in Fig. 11, and subsequent conversion of these compounds to yield hypoxanthine, xanthine or inosine, is often referred to as adenylate catabolism since there is no obvious biosynthetic role for this process and since the end-products appear to accumulate or to be excreted from the cells. However, in some micro-organisms these degradation products might be converted back to purine nucleotides and might fulfil a transient biosynthetic need. Regulation of purine ribonucleotide catabolism, with emphasis on mammalian systems, has been reviewed by Fox (1974).Adenylate deaminase, which catalyses the conversion of AMP to IMP, appears to be missing from microorganisms (Zielke and Suelter, 1970; Schramm and Leung, 19731, in contrast to its obiquitous presence in mammalian cells. However, a non- specific enzyme has been isolated from Desuyouibrio desuljkicans and other organisms that can catalyse deamination of all of the adenine nucleotides (Yates, 1969). The enzyme catalysing removal of the phosphate group, 5’-nucleotidase, has been isolated from a large number of bacteria and yeasts (for review, see Drummond and Yamamoto, 1970). The enzyme has a very broad specificity, but has high affinity for AMP. A specific protein inhibitor isolated from E . coli is reported to prevent the action of the enzyme on AMP and ATP (Dvorak, 19681, and might be related to regulation of the rates of adenine nucleotide degradation. Adenosine monophosphate nucleosidase, which catalyses the cleavage of AMP to adenine and ribose 5-phosphate, has been observed in Azotobacter uinlandii, E . coli and Pseudomonas diminuta (Schramm and Leung, 1973; Schramm and Lazorik, 1975). The enzyme appears to be largely inactive during normal growth, but is activated under conditions of low adenylate energy charge and low phosphate concentration. Very little is known about the substrate specificity in uzuo, the physiological role, or the extent of these catabolic reactions in the intact cell. If degradation of AMP is appreciable in uiuo, the rates of adenine nucleotide turnover estimated in Table 1 would have to be revised upward. However, based on the lack of accumulation of adenylate degradation products during active growth, and by analogy with ascites tumor cells where adenylate catabolism accounts for a loss
ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES
261
of less than 3% from the adenine nucleotide pool per hour (Snyder and Henderson, 1973; Crabtree and Henderson, 197 11, it is assumed that little or no adenine nucleotide catabolism occurs normally during active growth, and that the enzymes involved remain largely inhibited in the cell except under circumstances that will be discussed later. 8 . TURNOVER OF
ATP
Since the rate of ATP formation is equal to the rate of ATP utilization at steady state, the rate of turnover of the y-phosphate group of ATP might in principle be estimated by measuring either process if the other could be abolished. For instance, initial rates of ATP utilization have been estimated by determining the decrease in ATP concentration following cessation of ATP generation caused by removing oxygen from obligate aerobes or light from photosynthetic bacteria, or after addition of metabolic inhibitors. In estimating the rate of ATP utilization by this approach, one must assume that no ATP is generated by any alternative path, and that the rate of utilization of ATP continues at an undiminished rate, at least initially, in the absence of ATP regeneration and in the face of a decrease in the concentration of ATP, and probably of an increase in that of ADP. Neither of these assumptions is inherently plausible, and the second is inconsistent with what is known about metabolic regulation. The probability that either assumption is even approximately valid decreases rapidly with time. A linear decrease in the concentration ofATP, suggesting an unaltered rate ofATP utilization lasting for several seconds, has however been reported by Knowles and Smith (1970) and MioviC and Gibson (1973). Even if constant, however, the rate need not be the same as that when energy is available, and these authors, as well as Slayman (19731, have emphasized that this approach can produce only a minimal estimate of the rate of ATP utilization. The reverse approach, where the rate of ATP production is estimated from the rate of recovery of the ATP concentration following, for instance, the removal of inhibitor, has been used less often, and estimates based on it are especially doubtful. The extent of simultaneous ATP utilization is unknown; it cannot be assumed that the rate of ATP regeneration during recovery from a state of energy depletion is representative of steady-state ATP production, and the concentrations of the adenine nucleotides are necessarily abnormal. If the total adenine nucleotide concentration has decreased, as
262
A. G. CHAPMAN AND D. E. ATKINSON
frequently happens during energy depletion, the rate of ATP regeneration might be limited by the low concentration of ADP, and thus may not reflect the rate- of phosphorylation of ADP under normal circumstances. Other approaches to evaluating the rate of ATP production or utilization include: calculation of the ATP yield to be expected from the amount of growth substrate oxidized or fermented; estimation of the rate of ATP formation from the rate of respiration of organisms growing aerobically; and estimation of the ATP cost of biosynthesis and growth. The first of these approaches, the estimate of microbial ATP yields from substrate utilization, requires a knowledge of the metabolic pathways involved and quantitation of the rates of substrate degradation and product formation. A very large number of such investigations have been conducted with the somewhat different objective of determining molar growth yields, YATP . This parameter was introduced by Bauchop and Elsden (1960) who proposed that there is a relatively constant yield of anaerobically grown bacterial cells (about 10.5 g of dry weight) per mole of ATP produced from substrate utilization (see reviews by Forrest, 1969; Stouthamer, 1969; Payne, 1970; Forrest and Walker, 197 1 ; Stouthamer and Bettenhaussen, 1973; Penning de Vries et al., 1974; and Rogers and Stewart, 1974). Combined with the growth rates of the cells, this information can be used to calculate the rate of ATP formation, as has been done by Stouthamer and Bettenhaussen (1973). These authors, and others, have also challenged the universality of a molar growth yield of 10.5. They have argued convincingly that the value of YATP is affected by specific growth rates, the maintenance requirement of the cell, and the resulting cell composition and extent of formation of storage material. Temperature changes may also affect the molar growth yield (Coultate and Sundaram, 1975). Jain (1972) reported different apparent cell yields of Saccharomyces cerevisiae during different stages of the cell cycle. Calculation of the rate of ATP production from the rate of respiration is dependent on reliable estimates of P/O ratios in bacteria. In contrast to the commonly observed and accepted P/O ratio of 3 for mammalian systems, the stoicheiometry of bacterial oxidative phosphorylation is a subject of considerable dispute (see discussions in the growth yield reviews cited above; also Gibson and Cox, 1973; Van der Beck and Stouthamer, 1973; Hempfling, 1970). Membrane fractions from micro-organisms normally exhibit very low P/O ratios, while
ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES
263
calculations of P/O ratios in vivo based on estimated ATP yields vary from 0.5 to 3. The value of the molar growth yield parameter, YATP, determined anerobically, is often used to estimate these aerobic ATP yields. But since the apparent value of YATp may vary with growth rate, this method entails uncertainties (Stouthamer and Bettenhaussen, 1973). Estimates of the ATP cost of biosynthesis and cell growth presuppose not only knowledge of cellular composition and of the complete set of biosynthetic pathways and polymerization reactions (and the cost of each of these processes in terms of number of ATP molecules or ATP equivalents utilized), but also an appraisal of the contribution to the total energy requirement of such cellular activities as transport, maintenance of concentration gradients, motility, and turnover of cell components. Very little information on the magnitude of the ATP demand by these latter activities is available. Therefore, such an approach cannot lead to a useful estimate of total ATP production and consumption. It may be interesting, however, to compare the calculated requirement for biosynthesis with estimates of total growth requirement obtained by using one of the other approaches as a basis for estimating the relative magnitudes of the ATP requirements for biosynthesis and for other cell functions. Atkinson (1971a, b), Forrest and Walker (1971) and Penning de Vries et al. (1974) have calculated the metabolic costs of several intermediates and of different polymerization reactions. Table 2 shows that all of these different approaches have been utilized by different authors in order to estimate rates of ATP hydrolysis and regeneration. In order to distinguish between methodological uncertainty and legitimate variations in rate of ATP turnover, it would have been interesting to see all of these different methods applied to one organism growing under one set of conditions. At present, interpretation of the tabulated results remains speculative. Three of the lowest sets of values (those for Azotobacter vinelandii, Neurospora crassa, and Proteus mirabilis)were observed in stationary-phase cells, and reflect the expected low rate of turnover in the absence of growth. Species difference is probably also a factor. The estimates based on ATP yields from respiration and substrate utilization give the highest estimated rates of ATP turnover in Table 2, while estimates based on the initial linear rate of decrease of the ATP concentrations following cessation of ATP regeneration give the lowest values. These low values probably
TABLE 2. Turnover of ATP hl
Species
Doubling time (mid
Approach
ATP
(pmoles/ g dry wt)
Rate of A T P Use or Synthesis
A T P turn- Reference
P
over time (sec)
(pnioles/niin/ g d1-v wrt)
Escherichia coli
44 78 1 I4
Aerobacter ( Klebsiella) aerogenes Klebsiella aerogenes Saccharomyces cerevisiae Neurospora crassa Proteus mirabilis Chromutiurn sp. Azotobacter vinelandii
62 208 300 97 416 C
E
300 C
6. I 6.5 4.5
10
8b Sb 6.4'
8.0 6.2'
6
Yield from respiration and glycolysis, assuming P / O = 3 From YATp, corrected for maintenance energy Yield from respiration,
assuming P / O = 3 Yield from respiration, assumingP/O = I A T P depletion following cyanide addition Yield from respiration, assuming P/O = 2 A T P increase on aeration A T P decrease on darkening Cost of synthesis A T P decrease on oxygen exhaustion A T P increase on aeration
"An ATP concentration of 6 pmoleslg dry weight is assumed. 'The average level of ATP in growing yeast, according to Weibel el al. (1974). 'Not growing. 'Converted from mmoles/kg of cell water by the factor: intracellular w a t d d r y wt = 2.54, from the same publication. An apparent typographical error caused a discrepancy of 60 between ATP turnover rates given in the text and in the summary. The value tabulated is believed to be correct.
1895 1740 2040 1100 800 500
0.19 0.22 0. I3
.f
0.3" 0.4" 1.2
g
o
h
%
66 7 167 67 158
0.7 2.9 6 2.4
i
z
z
j
I13
105 50'
4.6 7.5
k 1
79' 2 12
4.6 180 30
m
.Converted from nmoles/mg protein by the factor: protein dry wt. from the same publication. 'Helms et d.(1972). 'Stouthamer and Bettenhaussen ( 1973). *Harrison and Maitra (1969). 'Rogers and Stewart (1974). 'Slayman (1973). %an der Beek and Stouthamer (1973). 'MioviCand Gibson(l971). Knnwlm and Smith [ 1870).
?
n I
P
m
x3z m
0
z
= 60% of
ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES
265
reflect regulation of ATP utilization under ,these conditions. The ATPlADP concentration ratio and the adenylate energy charge (defined on page 268) are strongly stabilized in vivo, and as a consequence of regulatory interactions the decrease in ATP concentration comes to a halt well before total depletion ofATP (Knowlesand Smith, 1970; Miovid and Gibson, 197 1 ; Slayman, 1973). Most of the errors to be expected will cause the estimated turnover times to be too long. Thus, despite the uncertainties of the estimates, it seems likely that in growing bacteria the entire ATP pool is utilized and regenerated in a second or less, in contrast to the much slower turnover of the total adenine nucleotide pool shown in Table 1. 1. ATP Turnover at Dgerent Growth Rates. Maintenance Requirement
Holms et ul. (1972) found no significant variation in the rate of ATP regeneration in E . coli cultured at five different growth rates (doubling times ranging from 114 to 44 mid, whereas Stouthamer and Bettenhaussen ( 1973) reported a linear relationship between the rate of ATP regeneration (720 to 1200 pmoleslminlg dry weight) and rate of growth (doubling time 360 to 53 min) in Klebsiellu (Aerobacter) aerogenes. Such a linear relationship was also observed in cultures of Sacch. cerevisiae (doubling time 786 to 97 min; ATP regeneration 62 to 667 pmoleslminlg dry weight) and Candida parupsilosis (doubling time 1050 to 208 min; ATP regeneration 50 to 266 pmoleslminlg dry weight) by Rogers and Stewart (1974). The implication of a constant molar growth yield, of course, is that the rate of ATP regeneration is proportional to the growth rate. This assumption presumably cannot be precisely correct; the degree of error involved will depend on the magnitude of the maintenance energy requirement of the cell. Gunsalus and Shuster ( 196 1) calculated that the expected maximum yield of cells per mole of ATP is 33 g dry weight. The implication of molar growth yield of about 10 g is that two-thirds of the ATP produced is consumed by processes other than biosynthesis. Stouthamer and Bettenhaussen ( 19731, by extrapolating to conditions of no growth (dilution rate = 01, estimated a rate of ATP production equal to 645 pmoleslminlg dry weight, which they termed the maintenance coefficient. According to this value, net biosynthesis and formation of macromolecular cell constituents account for 10 to 50% of the ATP utilization, depending on the growth rate. Other recent estimates of maintenance coeficients include
?
n TABLE 3. Steady state. values of ATP concentration, adenine nucleotide pool and energy charge in exponentially growing microorganisms Organism
Conditions
Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Enterobacter aerogenes Salmonella typhimurium Klebsiella aerogenes Hydrogenomonus eutropha Rhodospirillum rubrum Clostridium kluyueri Acetobacter aceti Bacilllus subtilis
Different carbon sources Different carbon sources
2.9-5.8 2.9-8.5 -
Different carbon sources
Different carbon sources PO, above 0.2 mm H g Light and dark aerobic Different carbon sources
-u
pmoles/g dry wt ATP
10.8* 55-73' 5.0' 6.7' 2.0-5.3 6.1-6.5 5.7 3.0-4.3' 4.5-7.0 7.6 0.4
2D
Sum of adenine nucleotides 3.6-6.8 3.8- 1 0.9 10' 12.8* 4.2-9.Oc.* 9.5' 7 .3'
-
3.7-8.0 8.6-9.1 8.1 4.7-8 .O' 8.3-1 1.2 8.0
-
Energy charge value
Reference
z
D
z D
z 0
P 0.9 1-0.94 0.75-0.85 0.80 0.90 0.94 0.74 0.90 0.80-0.95 0.46-0.73 0.8 1-0.85 0.82 0.54-0.80 0.76-0.79 0.87 0.70
m Frauen and Binkley ( 196 1 ) L o w r y e t a / . (1971) Chapmanetal. (1971) E z Mathews (1972) v) Bagnara and Finch (1973;1974) Dietzler et a/. (1974~) Swedes et al. (1975) Wiebe and Bancroft (1975) Smith and Maalse (1964) Harrison and Maitra (1969) Bowien and Schlegel(1972) Schon (1969) Decker and Pfitzer (1972) Bachi and Ettlinger (1973) Hutchison and Hanson ( 1974)
3
Azotobacter vinlandii Peptococcus prevotii Myxococcus xanthus Chromatium Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Neurospora uassa
Different light intensities Steady-state condition in second half of growth phase Different carbon sources Steady-state conditions in late exponential phase
'Units are converted from pmoles/absorbance unit to jmoleslg dry weight by assuming an A.sonmof 1.0 corresponds to 0.317 mg dry weighdrnl for E . coli (Franzen and Binkley, 1961). or an Amurn of 1.0 corresponds to 0.189 mg dry weighthl for yeast (Talwalkar and Lester, 1973), or using any other dry weight information given in the publication cited. *Units are converted kom pmoles/g wet weight or estimated mM concentration to pmoleslg dry weight on the assumption that dry weight equals 25% of wet weight. For E . coli dry weight is equal to 22 to 27% of wet weight, whereas the average value for a large number of bacteria is 20% (Luria, 1960).
-
-
5.4" 0.85 2.9-3. lC 6. 1" 7.6-8.5
7.9" 1.06 4.0-4.1" 6.9" 11.5-12.3
0.84 0.82 0.86 0.8 1-0.85 0.88 0.78-0.86
Liao and Atkinson (197 1) Montague and Dawes (1974) Hanson and Dworkin (1974) Miovik and Gibson (1973) Talwalkar and Lester (1973) Weibel el al. (1974)
6.5
8.0-9.6* 8.9
0.80-0.90 0.82
Ball and Atkinson (1975) Slayman(l973)
? I
rn
zz rn
z
C
0, rn
0
2
=Units are converted from pmoleslg protein to pnoles/g dry weight rn U on the assumption that protein represents 67% of the dry weight, as C) reported for E. cob (Forchhammer and Lindahl, 1971) and Sal. lyphimurium (Maaloe and Kjeldgaard, 1966) at fast growth rates. At slower C) rn growth rates the protein content increases to 80% of dry weight. z "The AMP concentration was assumed by the authors to equal 0.2.5 of the ADP concentration.
2
30 z v)
D
z 0 -I
268
A. G. CHAPMAN AND
D. E. ATKINSON
315 pmoleslminlg dry weight (Hempfling and Mainzer, 1975), and 680 pmoleslminlg dry weight for E . coli and 40 pmoleslminlg dry weight for Klebsiellapneumoniae (Brice et al., 1974) and 80 ,umoles/min/g dry weight for Bacillus megaterium (Downsand Jones, 1974). These values were con-
verted from the reports of moles/h/g cells by assuming dry weight equal to 25% of wet weight. Rogers and Stewart (1974) estimated lower values for maintenance energy of Sacch. cerevisiae and C . parapsilosis (3.5 to 31 pmoleslminlg dry weight) and reported that the values differed under different growth conditions. The relationship between ATP turnover and bacterial growth rate would therefore depend on the magnitude of the maintenance requirement and on the growth rates. At slow growth rates where maintenance processes apparently account for a very sizable fraction of the ATP utilization, the observed variation of total ATP requirement with changes in growth rate would be less pronounced than at higher growth rates. The amount of growth substrate needed to maintain bacteria viable but with no net growth has been a subject of earlier reviews (Marr et al., 1963, Mallette, 1963; Dawes and Ribbons, 1964). C . REGULATION OF
ATP
UTILIZATION A N D REGENERATION
1. The Adenylate Energy Charge
The turnover time of ATP in growing bacteria is short (one second or less) and the reactions in which ATP is hydrolysed or regenerated are numerous, yet the ATP concentration is kept at a relatively stable value in the cell. This can only be accomplished if ATP-utilizing and ATP-regenerating processes are kept in balance through precise and fast-acting regulation of their rates. The adenylate energy charge, (ATP + 1/2 ADPMATP + ADP + AMP), is a linear measure of the amount of metabolic energy stored in the adenine nucleotide pool. The responses to variations in the energy charge of enzymes catalysing certain ATP-utilizing or ATPregenerating reactions and the roles of these responses in stabilizing the cellular energy charge have been discussed previously (Atkinson, 1968, 1969, 1971a, b, 1972). The predictions, from enzyme response patterns observed in uitro, that the energy charge in viuo must be stabilized in the range 0.8 to 0.95 during growth and normal metabolism have been verified in nearly all organisms and tissues examined
ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES
269
(see Chapman et al., 1971 for a compilation). The ATP and total adenine nucleotide concentrations observed in a number of microorganisms during active growth are shown in Table 3, along with the calculated energy charge values. With the exception of the low value for Myxococcus xanthus, the total adenine nucleotide levels observed during growth are all within an approximate three-fold range (about 4 to 12 ,umoles/g dry weight). The large majority of the energy charge values fall within the predicted range, 0.8 to 0.95. It is quite remarkable that this degree of stabilization is maintained in the cell, when it is considered that during energy imbalance the entire ATP pool could be depleted in less than a second in the absence of any form of adenylate control. D. SAMPLING OF MICROBIAL CULTURES FOR ADENINE NUCLEOTIDE DETERMINATION
Many of the reported estimates of adenine nucleotide concentrations in intact cells published before 1960 lead to calculated energy charge values below 0.8, while the great majority of those published since about 1960 correspond to energy charge values of 0.8 or above (Chapman et al., 197 1). The low values from many earlier papers, and a few more recent ones, probably result from insufficient attention being paid to speed of sampling and to avoidance of ATP degradation in the sample. Indeed, it is our present belief that values around 0.8 published in, or derived from, a number of recent papers, including one of our own (Chapman et al., 19711, are slightly low because of an unrecognized loss of ATP during handling, and that the true value of the energy charge is probably near 0.9 in most, if not all, normally metabolizing cells. A brief comment on sampling requirements when adenine nucleotides are to be determined seems appropriate. Since no feasible sampling technique is more rapid than the rate of ATP turnover, estimations of adenine nucleotide concentrations can be valid only if these concentrations remain unchanged during sampling and processing. Thus enzyme activity must be abolished before the environment of the cells in the samples comes to differ significantly from that in the bulk culture as a result, for example, of depletion of substrate or especially of oxygen in the sample. It is also necessary that the inactivating agent act rapidly, so that all enzymes are destroyed essentially
270
A. G. CHAPMAN AND D.
E. ATKINSON
simultaneously. It is evident that if several enzymes that catalyse reactions in which ATP is used remained active, even for very short periods, after enzymes catalysing ATP regeneration were inactivated, the concentrations of ATP, ADP, and AMP observed would not reflect those in the intact cell, but would correspond to an energy charge value lower than the true value. Enzymes are usually inactivated and the cells destroyed (promoting extraction of the nucleotides) by addition of perchloric acid either directly to the cell suspension, as removed from the culture, or after filtration or centrifugation. The effect of varying the time between sampling and inactivation will of course depend on the species and the conditions (aerobic or anaerobic and high or low cell density, and others). Sampling requiring more than six seconds was found to cause a decrease in the ATP concentration in Neurospora c~assa(Slayman, 1973). A 3 to 30 second delay in the sampling of E. coli does not usually affect the ATP level. The oxygen tension in samples was not substantially lowered until 40 secor,ds at the cell densities employed by Holms et al. (1972).Decreases in ATP levels after a one-second delay have been reported in Sacch. cereuisiae (Weibel et al., 1974). Filtration at 4 O C decreases ATP values in Sacch. cereuisiae more than 50% (Weibel et al., 1974). However, the same ATP level was observed following direct sampling or filtration of Myxococcus xanthus (Hanson and Dworkin, 19741, and E . coli (Lowry et al., 1971), where high energy charge values have been observed even after a 10min delay imposed by filtration (Franzen and Binkley, 1961). Freshly filtered E . coli cells exhibited a lowered energy charge following resuspension, however, and two to five minutes was required for this parameter to return to control values (Chapman et al., 197 1). Cole et al. (1967) showed that centrifugation caused a 50% decrease in the ATP level in E . coli, and washing the cells caused a 90% decrease. This has been confirmed by Lowry et al. (1971). Lundin and Thore (1975) showed that centrifugation decreased the amount of adenine nucleotides extracted by 10 to 50% in five different species of bacteria ( E . coli, Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, and Klebsiella pneumonia) and led to energy charge values in the range of 0.2 to 0.5. The ATP content of Halobacterium halobium is also sharply lowered by centrifugation (Danon and Stoeckenius, 19 74). Prolonged centrifugation of Nitrobacter winogradskyi leads to a decrease in the ATP level. The concentrations of ATP and the energy charge values were very low,
ADENINE NUCLEOTIDE CONCENTRATIONS AND TURNOVER RATES
271
compared to those observed in other growing bacteria, even after attempts to inhibit ATP hydrolysis during centrifugation by addition of glutaraldehyde (Eigener, 1975; Eigener and Bock, 1975). Knowles and Smith (1970) reported that centrifugation and washing did not affect the ATP level in Azotobacter, but this treatment was followed by resuspension and aeration for two minutes, which probably allowed time for recovery of the ATP levels, as has been shown by Strange et al. ( 1963) with suspensions of Aerobacter aerogenes. Centrifugation and resuspension under strictly anaerobic conditions led to lower nucleotide levels than direct sampling of cultures of the anaerobic organism Clostridium Kluyveri (Decker and Pfitzer, 19 72). Centrifugation of Bacillus lichenformis (Leitzmann and Bernlohr, 1965) and Bacillus subtilis (Chow and Takahashi, 1972) that were in the exponential growth phase resulted in very low energy charge values, whereas Sacch. cereuisiae (Ball and Atkinson, 1975) and Polytomu uvella (Mangat, 1971) maintained high energy charge values after centrifugation. Higher energy charge values were observed in E. coli (Swedes et al., 1975) and in Bacillus brevis (Davison and Fynn, 1974) if acidprecipitable material was removed by centrifugation before neutralization of the perchloric acid extract. Failure to carry out this procedure resulted in a moderate decrease in the observed energy charge in E. coli, and a large change in B . brevis, presumably due to re-activation of ATP-destroying enzymes after neutralization. Addition of ethylenediamine tetra-acetic acid (EDTA) during extraction of several bacterial species with various acids and solvents likewise inhibited ATP hydrolysis (Lundin and Thore, 19 7 5 ). Freezing Sacch. cereuisiae before acid extraction was found to lower the observed ATP value by 60% (Weibel et al., 1974). Low energy charge values in Sacch. cereuisiae treated in a similar fashion have also been observed by Akbar et al. (1974) and Polakis and Bartley (1966). During certain phases of growth, a portion of the adenine nucleotides, largely AMP, have occasionally been shown to be excreted into the growth medium by E . coli (Chapman et al., 1971; Moses and Sharp, 19721, Chromatium sp. (MioviC and Gibson, 19731, Acetobacter aceti (Bachi and Ettlinger, 1973) and Myxococcus xanthus (Hanson and Dworkin, 1974). In such cases the adenine nucleotide determinations on cell suspensions must be corrected for nucleotides in the medium in order to estimate intracellular nucleotide concentrations.
272
A. G. CHAPMAN AND D. E. ATKINSON E. CHANGES IN ADENINE NUCLEOTIDE CONCENTRATIONS
Typical reports of changes in the concentrations of the adenine nucleotides in response -to changes in environmental conditions, or relation of these concentrations to biological capacities or functions, such as viability, glycogen synthesis, or sporulation and germination, are tabulated in this section. 1. Depletion $Energy Source
Reports of changes in energy charge or concentrations of the nucleotides on depletion of the carbon and energy source are presented in Table 4. Results obtained with E . coli in several laboratories are essentially in agreement in that all measurements of ATP concentration, or of the total adenylate pool, show a sharp drop when the substrate is exhausted. In marked contrast, the value of the energy charge changes much less drastically. Both of these changes are rapidly reversed if the energy source is restored. Although consistent for E . coli, this pattern does not hold for all micro-organisms. In two obligate aerobes, B . subtilis and Acetobacter aceti, depletion of the energy source was reported to cause a large decrease in the concentration of ATP and in the energy charge, with a much smaller change in the total adenylate pool. In baker’s yeast, growing under various conditions, the energy charge may either be maintained at relatively high values or fall sharply on substrate exhaustion, but changes in the size of the adenylate pool are small. Saccharomyces cerevisiae does not ordinarily synthesize hnctional mitochondria in the presence of glucose, and it appears that the ability to maintain a high value of the energy charge during aerobic starvation may depend on the presence of functional mitochondria (Ball and Atkinson, 1975).
2. Re-addition o f Substrate Table 5 summarizes the effects of the re-addition of substrate to starving cells of various microbial species. A rapid increase in the energy charge to normal values is seen in both yeast and E . coli. In Peptococcus prevotii, which catabolizes the ribose moiety of nucleotides during starvation, and thus decreases the adenylate pool to very low levels, the total adenylate concentration as well as the energy charge returns fairly rapidly to values typical of growing cells. Addition of an oxidizable
TABLE 4. Effects of depletion of energy source Species
Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Escherichia coli Peptococcus preuotii Bacillus subtilis Acetobacter aceti Saccharomyces cereuisiae Saccharomyces cereuisiae Saccharomyces cereuisiae Saccharomyces cerevisiae
Conditions
Glucose depletion Glucose depletion Resuspended minus glucose Glycerol depletion Glucose depletion Resuspended minus glucose Serine depletion Glucose depletion Acetate depletion Glucose depletion Glucose depletion Glucose depletion Ethanol depletion Glucose depletion Resuspended minus glucose
(
.Chapman et d.(197 1 ) . bSwedesetal. (1975). cHolms et d.(1972). 'Lowly et 01. ( 197 1 ). 'Coleelal. (1967). JMontague and Dawes (1974). 'Hutchinson and Hanson (1974).
Energy
(aerobic) (aerobic)
ATP
+
+ (0.8 -0.7)
+
+
+
(0.9 -0.8)
(aerobic) (aerobic) (aerobic) (aerobic) (anaerobic) (anaerobic) (aerobic) (aerobic) (aerobic) (aerobic) (aerobic) (aerobic) (anaerobic)
N o change + (0.8- 0.7) N o change + (0.85- 0.7) + (0.8 0.5)
(aerobic)
+
Sum ot'adenine nucleotide concentrations
+
(0.8 -0.7)
+
+ +
-
(0.85 0.6) (0.7 - 0 . 3 ) (0.87- 0.1)
+ 45%
+
-50% -55%
90%
>75% >90%
-
Littlechange +-75% N o change N o change N o change i -15%
+ -25%
(0.9 -0.2)
rn rn
Oxygenuptaker
+
> 0
zZ
-50%
+-75%
+ +
Reference
-50%
4 -90%
+ (0.84 --c 0.78)
Comments
During 10 h During 10 h Catabolizes purines
b
rn
2
b
2
c
d
o
f g h
z
i
0
J
W
k k k
> c +
2 d z
z
C W
"0