Advances in Microbial Physiology Volume 25

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Advances in

MICROBIAL PHYSIOLOGY

This Page Intentionally Left Blank

Advances in

MICROBIAL PHYSIOLOGY Edited by

A. H. ROSE School of Biological Sciences Bath University, UK

and

D. W. TEMPEST Laboratorium voor Microbiologie Universiteit van Amsterdam The Netherlands

Volume 25 1984

ACADEMIC PRESS (HurcourfBrace Jouunouich Publishers) London Orlando San Diego San Francisco New York Toronto Montreal Sydney Tokyo Sgo Paulo

A C A D E M I C PRESS INC. ( L O N D O N ) L T D . 2 4 2 8 Oval Road London NW1 7DX

US.Edition published by ACADEMIC PRESS INC. (Harcourt Brace Jovanovich, Inc.) Orlando. Florida 32887

Copyright 0 1984 by ACADEMIC PRESS INC. (LONDON) LTD.

AN Rights Reserved

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

British Library Cataloguing in Publication Data

ISBN 0- 12-027725-5 ISSN 0065-291 1

Printed in Great Britain at the Alden Press, Oxford

Contributors Thomas M. Buttke Department of Microbiology, University of Mississippi Medical Center, 2500 N. State Street, Jackson, Mississippi 39216, USA. Iain M. Campbell Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA Leland N. Edmunds, Jr Department of Anatomical Sciences, School of Medicine, Health Sciences Center, State University of New York, Stony Brook, New York 11794, USA Lonnie O’Neal Ingram Department of Microbiology and Cell Science, IFAS, and Department of Immunology and Medical Microbiology, McCarty Hall, University of Florida, Gainesville, Florida 3261 1, USA

D. H. Jennings Botany Department, The University, PO Box 147, Liverpool L69 3BX, England N. Van Uden Laboratory of Microbiology, Gulbenkian Institute of Science, Apartado 14, 2781 Oeiras Codex, Portugal


Quarter and half-way stages on the way to the attainment of a century-be it a life span, a reign or merely a series of book volumes-are often used as an occasion on which to ponder, perhaps celebrate, or just depart temporarily from normal practice. We are taking the opportunity of the publication of this, the 25th volume of Advances in Microbial Physiology, to do something different and present the first ever Preface to appear in this series. Since Volume 1 appeared (in 1967), 111 reviews have been published in Advances. Throughout, our aim has been (and here we speak also for our erstwhile colleagues, John F. Wilkinson and J. Gareth Morris, who shared in editing 14 of these books) to provide in each volume about five articles covering a variety of aspects of microbial physiology. When asking authors to write for us, we have been in the relatively fortunate position of being able to offer them a substantial amount of space-which is where, generally, we score over our competitors-as well as few restrictions on the use of illustrative material (tables and figures). Indeed, having agreed on the particular area of physiology to be reviewed, all we have asked of authors is that they make their articles as broadly based as possible. Feedback from colleagues-be they using Advances for teaching or to feel their way into new areas of research, or both-indicates that comprehensive articles that provide a broad overview are preferred. And discussions with fellow microbial physiologists lead us to conclude that our policy has, on the whole, worked well; moreover Academic Press concur. Hence unless you, our readers, convince us to the contrary, the formula for the next 25 volumes will probably show little change. That does not mean, however, that experiments will not be tried, or that suggestions for improvement in subject coverage or presentation would not be welcome. Times are hard in the publishing business, and the way to survive is to provide information that the readership seeks in a form that they find pleasing. This will be our continuing goal. Finally we would like to thank, most sincerely, all who have contributed to the first 25 volumes of Advances, and also our friends and colleagues at Academic Press who have, throughout, made every effort (and successfullyso) to produce excellent volumes for this series. In this latter regard, our special thanks go to Miss Diana Beaven. A. H. ROSE Bath and Amsterdam January, 1984 D. W. TEMPEST vii


Contents

V

Contributors Preface

vii

Secondary Metabolism and Microbial Physiology IAIN M. CAMPBELL I. Introduction 11. The meaning of the terms secondary metabolism and secondary metabolite 111. Biochemical similarities and differences between primary and secondary metabolism IV. What is meant by the term “physiology”? V. Areas of physiology in which materials of limited taxonomic distribution might be expected to play a role VI. Evidence that some secondary metabolites, distributionally defined, discharge key physiological roles VII. Why have physiological roles not been ascribed to more microbial secondary metabolites? VIII. Epilogue IX. Acknowledgements References

2 2

11 36 39 41 51 56 56 56

Physiology of Circadian Rhythms in Micro-Organisms LELAND N. EDMUNDS, JR I. 11. 111. IV. V. VI.

Introduction Circadian rhythms in protozoa Circadian rhythms in unicellular algae Circadian rhythms in fungi General considerations and conclusions Acknowledgement References ix

61 66 74 115

124 139 139

CONTENTS

X

Polyol Metabolism in Fungi D. H. JENNINGS I. Introduction 11. Enzymes 111. Mastigomycotina IV. Zygomycotina V. Ascomycotina and Deuteromycotina VI. Basidiomycotina VII. Regulation of cytoplasmic pH values in fungi VIII. Comment References

150 150 159 159 160 176 180 185 188

Temperature Profiles of Yeasts N. VAN UDEN I. Introduction 11. The elements of temperature profiles 111. Types of temperature profiles IV. Effects of drugs on the temperature profiles of yeasts V. Targets of temperature effects References

195 196 206 223 239 248

Effects of Alcohols on Micro-Organisms LONNIE O’NEAL INGRAM and THOMAS M. BUTTKE 254 Why study the effects of alcohols on micro-organisms? 255 Effects of alcohols on prokaryotic micro-organisms 270 Effects of alcohols on eukaryotic micro-organisms 280 Effects of alcohols on membrane organization Effects of lipid supplements on alcohol tolerance, growth, 282 survival and fermentation 287 VI. Mechanism of inhibition of fermentation by ethanol 29 1 VII. Conclusions and future directions 295 VIII . Acknowledgements 296 References I. 11. 111. IV. V.

Author Index Subject Index Cumulative Index of Contributors Cumulative Subject Index

305 320 337 340

Secondary Metabolism and Microbial Physiology* IAIN M. CAMPBELL Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

I. Introduction

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11. The meaning of the terms secondary metabolism and secondary

metabolite.

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111. Biochemical similarities and differences between primary and secondary

metabolism . . . . . . . . . . . A. Primary metabolism and the biochemical principles behind it . B. Secondary metabolites and the biochemical principles behind their synthesis . . . . . . . . . . C. What can we con&de from a comparison of the biochemistries of primary and secondary metabolism? . . . . . . IV. What is meant by the term “physiology”? . . . . . V. Areas of physiology in which materials of limited taxonomic distribution might be expected to play a role. . . . . . . VI. Evidence that some secondary metabolites, distributionally defined, . . . . . . . discharge key physiological roles A. Sex hormones . . . . . . . . . . B. Hormones involved in morphogenesis . . . . . . C. Agents that influence spore germination and outgrowth . . D. Metal chelating agents. . . . . . . E. Structural and extracellular protective agents . . . . F. Host-specific toxins . . . . . . . . . VII. Why have physiological roles not been ascribed to more microbial . . . . . . . . secondary metabolites? . VIII. Epilogue . . . . . . . . . . . . IX. Acknowledgments . . . . . . . . . . References. . . . . . . . . . . .

11 11

17 34 36 39 41 41 43 44 47 47 51 51 56 56 56

* Dedicated ta Professor R.A. Raphae1,now of the University of Cambridge, in appreciation of his outstanding stewardshipof the undergraduate,graduate and postgraduateeducation and research missions of the University of Glasgow in the 1950s and 1960s. Many owe him much. ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 25 ISBN 0-12-027725-4

Copyright 01984 by Academic Press, London All rights of reproduction in any form reserved.

2

IAIN M. CAMPBELL

I. Introduction

. . .; this thing of darkness I Acknowledge mine. W. Shakespeare, Tempest, Act V, sc. i. With these words Prospero, the self-assured, soon-to-be reinstated Duke of Milan acknowledges that the half-witted serf Caliban is his kin. The appropriateness of such a quotation as an introduction to this essay is that the latter seeks to establish a kinship that to many seems no less unlikely. For decades, secondary metabolism has been biology’s Caliban, mechanically delivering pharmaceuticals and fine chemicals for its master’s use while conspiratorially endangering that master’s life from time to time through the production of toxins and drugs that can be abused. Throughout these decades the biological Prospero, in the guise of biochemist, geneticist, molecular biologist and ecologist, has largely refrained from acknowledging that microbial secondary metabolism is part of itself, bone of its bone, sinew of its sinew. The notion has arisen that secondary metabolism is a “thing of darkness”, irrational, wanton and capricious, and consequently unworthy of serious consideration or dedicated study. To a significant extent, therefore, serendipity and “black art” govern how we currently produce drugs and fine chemicals and how we seek to control toxin production. The thesis of this essay is that neither this notion nor its immediate consequences are true. The darkling secondary metabolic Caliban is indeed kin to the biological Prospero. Secondary metabolism is a rightful component part of microbial biology, open to and deserving of study as such. The key to proving this thesis is to show that microbial secondary metabolism .is biochemically co-extensive with primary metabolism and that it is physiologically rational. It must be stated at the outset, however, that at this point in time rigorous proof of this thesis is impossible. The experimental data base is too sparse. Considerably more work has to be done at the enzymological, physiological and ecological level before rigour can be approached. What I propose to do is sketch what I believe to be the outlines of that proof and hope that this will stimulate discussion and, more importantly, will suggest to us and others new avenues for productive experimentation that will lead to our eventual complete understanding of the microbial secondary-metabolic phenomenon.

11. The Meaning of the Terms Secondary Metabolism and Secondary Metabolite As already stated, the key to proving my thesis that secondary metabolism is legitimate biology is to show that it is biochemically co-extensive with primary

SECONDARY METABOLISM AND MICROBIAL PHYSIOLOGY

3

metabolism and that it is rational in physiological terms. Such a demonstration requires definitions of primary and secondary metabolism and of physiology that are as fundamental as possible; the definition of biochemistry as the complement of enzyme-catalysed reactions that is required to maintain the life process in all its fullness is sufficiently widely accepted to be used directly as such in this discussion. Approaching a fundamental definition of secondary metabolism as it occurs in bacteria, fungi and the fungal-related protists-the subject area to which this essay is restricted-is my first task. Historically, the terms secondary metabolism and secondary metabolite arose from the practice of segregating the biochemical activity of bacteria, fungi, algae, higher plants and most recently of animal cells (Luckner et al., 1977) into two broad compartments; namely, primary metabolism and secondary metabolism. The practice began in the area of plant biochemistry, having its origin in a lecture given by Kossel in 1891 (Kossel, 1891). The subsequent history of the practice in both plant and microbial systems has been ably chronicled by Mothes (1980). Over the years a variety of criteria were used singly or sometimes in multiples, to attribute products from bacteria, fungi and algae, status as primary or secondary metabolites. Initially, distribution in Nature served the attributive purpose, primary metabolites being defined as natural products that were widely distributed in Nature whereas secondary metabolites were those that were restricted in their distribution. Subsequently, it became evident that products described as primary or secondary on distribution grounds often had additional characteristics in common. Thus, primary metabolites according to the distribution criterion tended: (a) to have relatively simple chemical constitutions; (b) to be produced by biosynthetic pathways that were neither unduly lengthy nor involved; (c) to be turned-over actively through interaction of these biosynthetic pathways with catabolic processes; (d) to be attributed readily a role in the physiological make-up of the producing organism; (e) to be present in that organism throughout its life cycle; and ( f ) to be necessary for the growth of that organism. By contrast, secondary metabolites (distributionally defined) tended: (a) to have complex chemical constitutions; (b) to be produced by biosynthetic pathways that were involved and often lengthy; (c) to be relatively free of turn-over (d) to be difficult to integrate meaningfully into the immediate, general physiology of the producing organism;

4

IAIN M. CAMPBELL

(e) in the case of bacteria and fungi, to be produced under the specific conditions of submerged liquid culture, only after the growth phase of the organism had passed, i.e. in the idiophase, not in the trophophase, according to the nomenclature of Bu’Lock et a f . (1965); and (f) to be unnecessary for growth of the producing organism. Table 1 illustrates these six additional characteristics for a typical primary metabolite, namely palmitic acid (I), for a typical bacterial secondary metabolite, tetracycline (II), and for a typical fungal secondary metabolite griseofulvin (III), all three compounds being attributed primary or secondary metabolic status on distributional grounds (Table 1, row 2). n-CHi(CHJCo COOH

I

Over the years these six additional characteristics came into use as criteria in their own right for making the primary/secondary metabolite attribution in consort with, or in preference to, the original distribution criterion. Mention must also be made of a seventh criterion that has surfaced from time to time. For a variety of reasons-some logical, some accidental (see Campbell, 1983Fmetabolitestermed secondary on distribution grounds tended historically to become the province of study of organic chemists and their ilk, while primary metabolites (distributionally defined) became the province of biochemists and their ilk. This eventuality fostered in some quarters the unfortunate notion that compounds that entered the literature initially through chemical study were intrinsically secondary metabolites whereas those approached initially from the biochemical/biological standpoint were intrinsically primary metabolites. Although secondary metabolism, as it occurs in higher plants, is not of direct relevance to an essay on microbial secondary metabolism, there are good reasons for believing that microbial and plant secondary metabolism are different sides of the same coin. This being the case, it is imperative that those concerned with microbial secondary metabolism keep abreast of definitions and proposed functions that are current in the plant arena and vice versa. In this regard, it is important to note that workers with plant secondary metabolism, historically more conscious of the ecological ramifications of the


5

phenomenon (see, for example, Bell, 1981; Harborne, 1977; Sondheimer and Simeone, 1970; Swain, 1977; Whittaker and Feeny, 1971), have begun in the last decade to characterize plant secondary metabolites in more specific terms, for example those used by Whittaker and Feeny (1971), which classify them as allelochemicals (products of one organism that affect its neighbours), allomones (products that give adaptive advantage to the producer organism) and kairomones (products of one organism that give adaptive advantage to another). In the wake of all this confusion, it is not surprising that many investigators feel the terms “primary” and “secondary” with regard to metabolism/metabolite should be expunged permanently from the scientificvocabulary (see for instance, Jansen, 1978). Terms such as “general/special metabolism/metabolite” have been suggested as replacements (Zahner, 1979). Such a cosmetic change may have merit. Indeed it is interesting to note that Bu’Lock, who first introduced the term “secondary metabolite” into the microbiology vocabulary (Bu’Lock, 1961), referred to primary metabolism initially as “general metabolism”. Replacing “primary metabolism/metabolite” by “general metabolism/metabolite” and replacing “secondary metabolism/metabolite” by “special metabolism/metabolite” would be particularly fitting in terms of the use made of the words “general” and “specific” in the next few paragraphs. In this essay, however, I opt to retain the classic terms primary and secondary for reasons of readers’ familiarity. I would be content, however, if this were my last use of these terms. A more crucial issue that needs to be settled is general agreement on what distinguishes a primary metabolite fundamentally from a secondary metabolite. For reasons that are discussed later, I advocate return to the original distribution-based criterion expressed specifically as follows:

Primary metabolites are products of normal cellular metabolism that are widely distributed in Nature, being found at least in every genus in at least one family; secondary metabolites are products of normal cellular metabolism that are more restricted in their distribution, beingfound in less than every species in a singiefamily. Secondary metabolites may be found sporadically in various species in disparate genera, families, orders, classes, phyla or kingdoms. Some amplification and justification of these definitions are necessary. Firstly, we selected the distribution criterion in the belief that it is more fundamentally significant than any of the six other characteristics listed above. Materials are widely distributed in Nature because they are associated with processes that are themselves widely distributed. It follows that such processes evolved early in time and are successful and general solutions to fairly general biological problems. Returning to Table 1 , palmitic acid is widely distributed because it occurs as a residue in membrane phospholipids

TABLE 1. Criteria used to distinguish primary from secondary metabolites Palmitic acid (I)

Tetracycline (II)

Griseofulvin (III)

Occurrence

In all organisms so far studied.

Streptomyces auireofaciens, Streptomyces Iusitanis, Streptomyces phiaeofaciens, Streptomyces psiunmoticus, Streptomyces sajtamaensis, Streptomyces viridifaciens"

Penicilliurn griseofulvum, Penicillium patulum, (E urticae), Penicillium janczewskii

Number of formal steps in biosynthesis'

Seven repetitions of four basic steps; condensationd, reduction, dehydration, hydrogenation.

Eight repetitions, o f a single basic step (POlYketide formationI),four repetitions of a siingle basic step (polyk:etide aromatization) a nd nine individual reactitons".

Six repetitions of a single basic step (polyketide formation), two repetitions of a single step (polyketide aromatization), three repetitions of a single step (0methylation), and three individual reactions.

Turnover knowne?

Yes-b-oxidation to acetate.

Physiological role known?

Yes-component of triglycerides, phospholipids, sphingolipids, sterol esters; hence role in energy storage and membrane formation; also biosynthetic precursor of longer chain fatty acids.

Synthesized through producer cell's lifetime. . . . . . even during growth?

back

None known in the producing organism.




Yes

No

No

Yes

No

Noj

m

j:

2 T: CI

* 5 P P

See HoSt’alek et al. (1979). See Weinstein (1975). Starting from coenzyme derivatives and excluding steps that associate substrates to enzymes, e.g. coenzyme-A to acyl-carrier protein transfers. See Table 2, example 1 (p. 14). Until recently, secondary metabolite turnover was not an actively studied issue (Barz and Koster, 1981). Some clear-cut instances do occur, e.g. chloramphenicol (Malik and Vining, 1970) and zearalenone (Steele et al., 1976). Whether this is an example of bioconversion (see Section 11, p. 11) or the first instances of a more general occurrence, remains to be seen. Hence my use of the word “known” rather than “occurs” here. ’Although most microbial secondary metabolites are produced after growth in submerged liquid culture, there are some exceptions, e.g.: bacitracin production by Bacillus licheniformis (Haavik, 1975); cephalosporin production by Streptomyces clavuligerus(Aharonowitz and Demain, 1978); chloramphenicol production by a Streptomyces strain (Malik and Vining, 1970); enniatin production by Fusarium sambucinum (Audhya and Russell, 1975); ergot alkaloids by Claviceps paspali (Brar et al., 1968); mycophenolic acid production by Penicillium brevicompacturn (Doerfler et al., 1979), and rifamycin production by Streptomyces mediterranei (Ruczaj et al., 1972).

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8

IAIN M. CAMPBELL

and in triglycerides. Once the processes of palmitic acid synthesis and degradation had evolved and the first palmitic acyl-containing lipid bilayer membranes were formed, the place of palmitic acid (and that of the related fatty acids) in the vast majority of subsequently evolving organisms was secured. It matters not that, in bacteria, the synthetic process is conducted by a loose federation of enzymes whereas in eukaryotes a multi-enzyme complex is involved. It matters not that the fatty acid synthase of Saccharomyces cerevisiae and of Homo sapiens might not be immunologically identical complexes (the active sites of the components of the complex may indeed turn out to be very similar) or that the nuclear code for the enzymes of both complexes are different. The important fact is that all organisms possess the ability to make, and to turn over, palmitic acid. By contrast, materials that are restricted in their distribution are so because the processes with which they are associated are likewise restricted in distribution. These latter processes are restricted because they constitute what can be considered either successful specific solutions to specific biological problems or successful specific solutions to a general biological problem. As will be seen in Section VI1.A (p. 41), sirenin (IV) is produced by cells of the

CH!OH CHI

CH,

IV

female mating type of, inter alia, Allomyces arbuscula to attract cells of the male mating-type to them. This is a specific solution to the specific problem of syngamy in A . arbuscula; it is also a specific solution to the general syngamy problem. If sirenin was widely distributed in Nature, its use as a sex hormone by A . arbuscula would be ineffective. Male cell types of A . arbuscula would receive sirenin-encoded signals from a potentially large number of sources, only one of which would lead to a productive outcome for the water mould. Unlike processes that are found widely distributed in Nature, processes that are of restricted distribution probably evolved late in the phylogeny of the organisms in which the processes are found. Differences in times of evolutionary origins of processes have important biological implications. Indeed, it is because the distribution criterion is so readily projected into functional and evolutionary terms in bacteria, fungi, algae, plant and animal cells that we advocate its use as the primary/secondary metabolite attribution criterion. None of the other characteristics listed above is so immediately fundamental. A second factor in favour of the distribution criterion is that it is easily approached experimentally on a biosphere-wide basis. In having introduced the concept of general and specific solutions to


9

biological problems, it is important to note that nothing was implied about ‘importance’. Secondary metabolites, as already defined, are not intrinsically more or less important than primary metabolites. In biological terms for instance, it is every bit as important for A . arbuscula that cells of opposite mating type meet as that both cells are made of membranes constituted, inter a h , of palmitic acid-containing lipids. My definition of primary and secondary metabolites embodies the terms “widely” and “restricted”, and sets the cut-off between them at the level of “all genera in at least one family”. This is acknowledgedly arbitrary. As our knowledge increases of the distribution of chemical compounds in all species in a genus, in all genera in a family, etc., and in the various subgroups thereof in the biosphere at large, and as our precision in defining taxa improves, this limit may need to be modified. The “all genera in at least one family” limit appears to be quite adequate in dealing with the bacteria and fungi, and will be used in this essay. The impression given by some authors that primary metabolites are ubiquitous, or approach ubiquity, is a misleading one that must be challenged. True, there is a group of chemical compounds of this type. Contained in this group are the following: nucleoside monophosphates found as residues in nucleic acids and their di- and triphospho-counterparts; the 20 amino acid residues found in proteins; glucose and its phosphate esters; the common fatty acids and their esters and amides; thiol esters of acetate, malonate and P-hydroxy-P-methylglutarate; pyridoxal; thiamin, to mention but a few. All of these compounds are clearly primary metabolites and the common processes in which they figure in all cells are ancient, successful, general solutions to global biological problems. However, there are materials such as chlorophyll, cholesterol and acetylcholine that, although not ubiquitous, are clearly general solutions to general biological problems, albeit problems that are not global. The need for such a solution likely emerged only after the complexity of organisms began developing. Chlorophyll is the general solution to the problem of transducing sunlight into chemical energy and is found in those Monera, Protista and Plantae that are phototrophic. Cholesterol (V) is one of several related general solutions to the problem of controlling the fluidity of membranes. Cholesterol is the solution opted for by mammals. The related sterols ergosterol (VI), campesterol (VII), sitosterol (VIII) and stigmasterol (22,23-dehydro-VIII) appear to serve the same general purpose in fungi and plants, respectively. Bacteria have little general commitment to sterol production, and sterols are largely absent from species of the Monera. This, however, does not preclude cholesterol, ergosterol, campesterol, sitosterol or stigmasterol being considered primary metabolites. Acetylcholine (IX) is an even less ubiquitous cell metabolite being found almost exclusively in the Animalia. This was understandable when acetylcholine was recognized as one of several successful general solutions to the general problem of communica-

10

IAIN M. CAMPBELL

V R=H VII R=CHI Vlll R=CH,CH,

VI

CH,COOCH,CH,NCCH,),

IX

tion between nerve cells, a problem that only members of the Animalia have to face. Restriction to one Kingdom does not preclude primary metabolite status for acetylcholine or for any other cell metabolite. Before closing this section on definitions, two further points need to be made. The first concerns the status of polymers. Traditionally in discussions of primary and secondary metabolism, attention has focused principally on non-polymeric cell constituents, with enzymes and nucleic acids being tacitly excluded from the discussion and with biopolymers that are neither enzymes nor nucleic acids being left somewhat in limbo. Apart from the initial startling impact of their inclusion, there are no logical reasons to exclude any type of biopolymer from the primary/secondary metabolite classification. If, as I have argued, the primary/secondary metabolite distinction is essentially one between molecular species that represent general or specific solutions, respectively, to biological problems, polymers are just as likely to be either of such solutions as are their lower molecular-weight brethren. In making the primary/secondary metabolite distinction for enzymes and nucleic acids, however, it would seem essential to pose the question of extent of distribution at the level of the function executed rather than at the particular amino-acid or nucleotide sequences. For exampIe, scanning all species in a family for aspartate carbamoyltransferase activity and finding that activity is present in all species, but in a miscellany of immunologically distinguishable forms, does not mean that the aspartate carbamoyltransferase in each species is a secondary metabolite! The fact that all family members have the activity, irrespective of what protein and DNA sequences give rise to it, makes the activity a primary metabolic one. The second point concerns situations in which the terms secondary metabolism/metabolite should likely not be used. In my opinion, these terms should not be used to describe the metabolic outcome of feeding foreign materials, e.g. naphthalene, cholesterol and reserpine, to micro-organisms. “Bioconversion” is a better word to describe this type of process (see Wang et a/., 1979); the term “biotransformation” should be avoided because of the

SECONDARY METABOLISM A N D MICROBIAL PHYSIOLOGY

11

confusion engendered with genetic transformations. I also believe that caution is necessary in dealing with situations where culture conditions in bacterial or fungal cultures are manipulated, or where the genome in the inoculum has been altered such that large quantities of a material that would normally be considered a primary metabolite are accumulated. Production of citric acid by Aspergillus niger is a good example. A term, first used by Foster (1949) and echoed by Neijssel and Tempest (1979), namely “overflow metabolism”, is an appropriate description for this phenomenon. There is also reason to believe that culture conditions can be developed where a bonaJide secondary metabolite is bioconverted by the same organism that gave rise to it in the first place. For physiological reasons, it will be important to make the experimentally difficult decision regarding where secondary metabolism ends and where bioconversion begins, and label the processes accordingly. It is for this reason that I include the qualifying adjective “normal” in my definition of primary and secondary metabolism. Further discussion of this topic occurs in Section VII (p. 52).

111. Biochemical Similarities and Differences Between Primary and

Secondary Metabolism Before going on to define what is meant by the term “physiology” and seeking to find a place for secondary metabolism within the confines of that meaning, it is appropriate to spend a little time comparing the basic biochemical principles behind primary and secondary metabolism, both distributionally defined. So doing not only establishes that both processes are co-extensiveCaliban is made of the same stuff as Prosper-but it also allows us to make the first faint tracings of secondary metabolism’s possible evolutionary origin from selected components of primary metabolism-Caliban is truly Prospero’s kin. A. PRIMARY METABOLISM A N D THE BIOCHEMICAL PRINCIPLES BEHIND IT

Most textbooks of biochemistry divide primary metabolism into four major areas: lipid metabolism, sugar metabolism-including the tricarboxylic acid (TCA) cycle and associated anapleurotic pathways, amino-acid and protein metabolism, and nucleotide and nucleic acid metabolism. Metabolism of necessary cofactors, such as nicotinamide, pyridoxal and cobalamine, is sometimes added on as a fifth minor metabolic area. Lipid metabolism is primarily the biochemistry of acetic acid. The coenzyme A (CoA) ester, acetyl-CoA, is not only produced during lipid catabolism, but in anabolism it is also used in conjunction with its carboxylation product, malonyl-CoA, to produce fatty acids. When pro-

12

IAIN M. CAMPBELL

cessed through mevalonic acid, acetyl-CoA provides the carbon skeleton of the second major group of primary lipids, the sterols. Sugar metabolism can be seen to focus around the simplest aldotriose, glyceraldehyde, since its monophosphate is the first material to embody both the carbon and the energy entrapped by photosynthesis, it can be converted into glucose which in turn can be polymerized to store that carbon and energy, it is oxidized anaerobically to fermentation products such as lactate and ethanol or aerobically to carbon dioxide and water with release of a proportion of that energy, and it is transformable into a variety of tetroses, pentoses and hexoses which as such, or on further transformation, have a variety of uses in cells. The biochemical reactions that appear in these four processes, constitute sugar metabolism. Amino-acid metabolism is a catabolic and anabolic miscellany in which the 20 amino acids found in proteins plus a few extra are made and turned over. In the anabolic phase, carbon is removed from the pathways of sugar metabolism (widely defined to include the TCA cycle and similar pathways) and is processed to give the amino-acid skeletons which in turn have nitrogen added to them transaminatively. In the catabolic phase, that nitrogen is first removed and the carbon is returned to sugar metabolism and carbon dioxide. Nucleotide metabolism is essentially the construction and degradation of two purine nuclei (adenine and guanine) and three pyrimidine nuclei (uracil, thymine and cytosine), the sugar component being derived separately. From a metabolic standpoint, protein and nucleic acid metabolism is simply the construction and destruction of peptide and phosphodiester bonds, respectively. On initial contact, the biochemistry of primary metabolism appears complex and sophisticated. However, when the many hundreds of reactions that collectively constitute primary metabolism are reviewed individually in chemical terms and with the knowledge that enzyme catalysis inherently ensures stereoselectivity, substrate and product specificity, and the potential for allosteric and/or transcriptional control, it emerges that complexity and sophistication are not the hallmarks of the unit biochemical process. Rather, the common characteristic of these unit processes and the manner in which they are assembled into metabolic pathways appears to be a striving towards simplicity and an avoidance of complexity wherever possible. This fact can be illustrated at several levels. Firstly, over 99% of the reactions occurring in primary metabolism are specific examples of the canonical forms listed in Tables 2 and 3. The former deals with reactions leading to carbon-carbon bond formation, and the latter deals with reactions on preformed carbon skeletons. By and large, cells use the same basic chemical procedures to solve the same basic biochemical problems whether those problems arise in lipid, sugar, amino acid or nucleotide metabolism. Indeed, a basic strategy in the use of cofactors in primary


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II

0

CH,CH+CO,

II

13

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Not a retrocondensation

0

1 p=3 g A * H,O

0

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I

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c=o

* A is the remainder of the thiamin pyrophosphate molecule. FIG. 1. Diagram showing involvement of thiamin in decarboxylation of an a-0x0 acid by a retrocondensation reaction.

metabolism appears to involve converting a reaction that nominally cannot be solved by one of the basic procedures to a situation that is soluble. For instance, the involvement of thiamin in decarboxylations of a-0x0 acids and the removal of carbon atoms C, and CZfrom a 2-ketose (the transketolase reaction) converts both procedures into retrocondensation reactions which are the most commonly used C-C bond-breaking reactions in primary metabolism. Figure 1 illustrates the process for a-0x0 acid decarboxylation. In the interest of completeness, Table 4 contains examples of primary metabolic reactions that do not conform directly to any of the canonical forms found in Tables 2 and 3. Mechanistically, some of the processes listed in Table 4 are understood; others are still under study. It is conceivable that when the mechanistic details of some of the latter are resolved, they will conform to the canonical forms of Tables 2 and 3. Secondly, and with one important and glaring exception (see below), reactions of primary metabolism involve only a small number of atomic centres in the substrate. In the reactions listed in Tables 2 and 3, the chemical change most often involves a single carbon atom (e.g. number 7 in Table 3), two contiguous carbon atoms (e.g. number 17 in Table 3) or a carbon atom contiguous to a heteroatom (e.g. number 1 in Table 3). In a few cases three atomic centres are involved (e.g. number 9 in Table 3). The conformational constraints that an enzyme needs to impose on a substrate to ensure a single product outcome are therefore at a minimum. The glaring exception to this second instance of the basic simplicity in Prospero’s realm is the conversion of squalene oxide (X) into lanosterol (XI) in fungi and mammals, or to cycloartenol (XII) in plants and some protists (Fig. 2). This reaction involves 10 atomic centres, generating in one fell swoop seven (to XI) or eight (to XII) stereochemical centres from an acyclic substrate! How the corresponding

14

IAIN M. CAMPBELL

TABLE 2. Canonical forms of the carbon-carbon bond-forming reactions of primary metabolism 1. Condensation reaction and its reverse, the retrocondensation

H'

X

IJ -c-Y

X

?rH L

7

/ \ II

- -7

I

W

Y-H

m-

c-c/ \ II

V'

VL-c-

II

-'-@ -Y#C.H c

W

W

X=O.N; Y=O,S.C.H. V = H or HOOC. W=O.N.S

Examples: (I) Citric acid (a) formation from oxaloacetate (b) and acetyl-CoA (c) OH

Pt.' H' HO$2CH?C-CO?H

L

7

H+

(b)

I I

HO:C-CHZC-CO?H

HGHI-cosCoA

CH,COSCoA (a)

(C)

(11) The basic polymerization reaction in fatty acid biosynthesis

pr'

CH,-C-S-R

HG-cGHI-cO.SR

I1

0

d 7

H'

I

CH?-CO.SR

7

I

CHI-C0.S

R

COZ (R=CoA or acyl carrier protein)

(111) The decarboxylation stage during C-30 and C-31 demethylation of

sterols

oxidocyclases bind the "floppy" squalene oxide molecule such that the required chair-boat-chair-half chair conformation (XIII) exists exclusivelyis as yet uncertain. This reaction represents a level of organizational sophistication that is quantally distinct from the primary metabolic norm. A third and fourth instance of the underlying simplicity of primary metabolism is found in mechanisms of polymerizations. In such reactions, be they in fatty-acid biosynthesis, polyglycoside formation, polyprenoid pyro-


15

TABLE 2 (cont.) 2. Substitution reaction

H'

H

X=C.O,N; Y=O,N; bonds m a r k e d n may be linked or independent

Examples: (I) Geranyl pyrophosphate (d) formation from isopentenyl (e) and dimethylallyl (f) pyrophosphate CH,

J 4H

CH,

H,C-L-CH-CH,OBO~-OH

c

(el

I

L

7

CHI):C=CH--CH2

(f)

CH,-L=CH-CH20~o~-oH

H+ ( 4

(CHi)ZC=CH-CH:

0-@-0-@-CH

P,O,H;

(11) Conversion of a-aceto-a-hydroxybutyrate(g) to B-hydroxyl-Pmethyl-a-oxovaleric acid (h) during isoleucine biosynthesis COzH I

H+

C02H

I

I

H

phosphate synthesis, protein synthesis, DNA replication or transcription, the following events occur. (1) The eventual chemical form of the added monomer unit is brought into chemical existence before the next monomer is added, e.g. in fatty acid biosynthesis in both prokaryotes and eukaryotes, the 8-ketoacyl unit is successively reduced, dehydrated and further reduced before the next malonyl unit is added. This means that the growing polymer can emerge intact and unprotected from what can be a relatively small and simple polymerization site. This is even so where, in prokaryotic mono-unsaturated de nouo fatty-acid biosynthesis, an a, 8-unsaturated acyl thiol ester is re-arranged to a 8,y-unsaturated system (Moat, 1979). Cells do not need, therefore, to devise

TABLE 3. Canonical forms of the reactions occurring in primary metabolism on preformed carbon skeletons 1. Redox interconversion of alcohols and aldehydes/ketones 2. Redox interconversion of aldehydes and carboxylic acid phosphates 3. Redox interconversion of an a,/?-unsaturated carboxylic ester and the corresponding saturated compound 4. Redox interconversion of a primary or secondary amine into ammonia and an aldehyde or ketone, respectively. 5 . Reduction of an isolated olefinic linkage 6. Oxygen-dependent introduction of an olefinic linkage 7. ATP-dependent phosphorylation" of an alcohol or carboxylic acid 8. ATP-dependentb phosphoadenylation or pyrophosphoadenylationb of an acetal or ketal phosphate 9. Keto-enol and imine-enamine tautomerization 10. Methylation of an alcohol or amine with S-adenosylmethionine 11. Acylation of an alcohol, amine or thiol with an acyl-CoA derivative 12. Interconversion of an olefin and an alcohol 13. Amine-dependent interconversion of an aldehyde or ketone and the corresponding Schiffs base (imine) 14. Allylic rearrangement of an olefin 15. Oxygen-dependent hydroxylation of an aliphatic or aromatic carbon atom 16. Replacement of an acetal or ketal phosphate' with an amino group 17. Oxygen-dependent epoxide formation from an olefin or in an aromatic nucleus 18. AMP-sulphate-dependent sulphate addition to alcohols 19. Oxidative cleavage of a 1,2-dihydroxybenzene derivative through the 1,2- or 2,3-bond 20. Oxidative cleavage of a 1,Cdihydroxybenzene derivative through the 1,2-bond 21. Oxidation of thiols to sulphonates

" Included also are pyrophosphorylation, mono- and diphosphoadenylations Includes also UTP- and CTP-dependent reactions

strategiesto protect a number of highly reactive sites in a nascent polymer and subsequently to remove that protection and process the nascent polymer appropriately for final product formation. (2) Irregular heteropolymers, i.e. polymers in which two or more monomers (A,B) occur in an irregular sequence (AAABABAA), are only formed with the aid of a template orjig. To make tuberculostearic acid (10-methylstearicacid), for instance, cells opt to methylate a preformed fatty acid rather than attempting to incorporate methylmalonate uniquely and consistently into the fourth cycle of acetate/ polymalonate de nova fatty-acid biosynthesis (Moat, 1979). There is one other somewhat more nebulous characteristic of primary metabolism that warrants passing mention. There are very few primary metabolites in which carbon atoms derived from two or more of the major areas of anabolism (lipid, sugar, amino acid, nucleotide) are covalently bound together. Sphingosineis an exception containing fatty-acid and serine carbon in its structure.


17

TABLE 4. Examples of reactions in primary metabolism that do not conform to the canonical form of Tables 2 and 3 1. Cobalamine-dependent interconversion of succinate and methylmalonate 2. Conversion of ribose into 2-deoxyribose 3. “Deformylative” demethylation of the C-32 methyl group in synthesis of, for example, cholesterol. 4. Conversion of two equivalents of farnesyl pyrophosphate into squalene via

presqualene pyrophosphate 5 . Conversion of chorismate into prephenate

6. Conversion of p-hydroxyphenylpyruvateinto homogentisate 7. Insertion of the sulphur atom into desthiobiotin 8. The riboflavin synthetase reaction 9. Prenylation of phenols

B. SECONDARY METABOLITES AND THE BIOCHEMICAL PRINCIPLES BEHIND THEIR SYNTHESIS

Most authors divide the known secondary metabolites into at least three categories according to the perceived source of the carbon from which their skeletons are derived. The three major categories are as follows.

I . Metabolites Derived from Acetate As in primary metabolism, acetate can give rise to two major subgroups. In conjunction with malonate it yields polyketides, a set of compounds in the course of whose biosynthesis a normal poly-/I-diketo aliphatic acid derivative of general structure (XV), can be presumed to occur. The value of n ranges from 3 to 10; metabolites derived from XV (n= 3) are termed triketides, from XV (n=4), tetraketides, and so on. isoprene unit (C,H,)

XVl.@~PO(OH)

Acetate units can also be assembled into mevalonate which, in turn, can give rise to polyprenoid alcohol phosphates of general structure (XVI). The value of m in (XVI)ranges from 1 to 24 but, as far as secondary microbial metabolites are concerned, m ranges only from 2 to 5 . These prenyl pyrophosphates, singly o r as dimers, yield the terpenes. Terpenes are further categorized according to the number of basic isoprene units (C5Hs) they contain; mono-, sesqui-, di-, sester-, tri- and tetraterpenes contain 2 , 3 , 4 , 5 , 6 and 8 isoprene units, respectively.

18

IAIN M. CAMPBELL

1

n

-&7

H

XIV

chair

I

R

'dR CH, CH,

HO

CHI

H

&

HO CH,

CHI

CHI

XI XI1

CH,

FIG. 2. Multicentred reaction leading from squalene oxide (X)to lanosterol (XI)or cycloartenol (XII).

19


2. Metabolites Derived from Amino Acids

Most often, the amino acids in question are those involved in protein synthesis, but on occasion non-protein amino acids are used, e.g. ornithine (XVII), anthranilate (XVIII) and a,y-diaminobutyrate (XIX). Some authors divide amino acid-derived secondary metabolites into two groups, dealing

a:;

NH~(CHZ),CH COZH

I NH,

XVlll

XVll

NH2CHZCH2 CH COZH

I

NH,

XIX

separately with those that retain the amino acid’s a-amino nitrogen atom and those that are derived from non-nitrogenous precursors or degradation products of amino acids.

3. Metabolites Deriuedfrom Components of Sugar Metabolism In this group are placed secondary metabolites that are sugars in their own right (e.g. streptose (XX)) or materials that are derived from modification of any of the pathways of primary sugar metabolism (see Section III.A, p. 12, e.g. itaconic acid (XXI)).

xx

XXI

Following partitioning of the known secondary metabolites into these three groups, it is usually noted that there are some secondary metabolites that derive their carbon atoms from more than a single source. Table 5 cites some examples where carbon atoms originating from more than a single type source (acetate, amino acids, sugars) are fused covalently to provide the structure of a bacterial or fungal secondary metabolite. Instances are also found among secondary metabolites where carbon atoms from more than a single source is linked through an hetero-atom; there are also many cases where carbon atoms from both sections of acetate metabolism (polyketide and terpenoid) are fused covalently and/or by agency of a hetero-atom in a single structure. Divisions of secondary metabolites into groups according to the origin of their skeletal carbon atom has many advantages. For instance, it allows an impression to be gained of the commitment various taxa have to the use of acetate, amino acids and sugars as a fuel for secondary metabolism. Of relevance to this essay are the following facts. (1) Both bacteria and fungi make polyketides, the former yielding predominantly nonapeptides (the

20

IAIN M. CAMPBELL

TABLE 5. Microbial secondary metabolites in which carbon atoms from more than a single source are fused covalently“ Acetate Polyketide Terpenoid Austamides Bassiamin Bovinone Brevianamides Carlosic acid “Crytomycin” Cyclopiazonic acid Cytochalasins Decylcitric acid Echinulin Erythroskyrin Fusaric acid Geldanomycin Hispidin Lysergic acid, etc. Mitomycin Ny bomycin Paxilline Prodigiosin Pseuda n Rifamycin Roquefortine Rubratoxin Sporodesmin Streptovaricin Tenellin Tenuazonic acid

Sugar

Amino Acid X

X

X X X

X

X

X X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X X X

X X

X

X

X X

X X

X

X

X X

X

X

X X

X X

X

X

X

X

X

X

X

a For structural formulae, see Turner (1971), Devon and Scott (1972) and Steyn (1980).

tetracyclines), the latter providing a richer variety of tetraketides through decaketides with benzenoid, benzophenonoid and anthraquinonoid structures predominating. (2) Bacteria have a minimal commitment to terpenoid secondary metabolites; geosmin (XXII), the earthy-smelling factor produced by several species of Streptomyces, 2-methylisoborneol (XXIII) (Bentley and Meganathan, 1981) and the carotenoids are notable exceptions. Fungi are active in sesqui-, di-, sester-, tri- and tetraterpene production. (3) Bacteria and fungi use amino acids extensively with fungi making considerable use of the aromatic amino acids (phenylalanine, tyrosine and tryptophan) in those


XXll

21

XXlll

secondary metabolites derived from a pair of amino acids (Table 6). (4) Bacteria, but not fungi, actively recruit carbon atoms from sugar metabolism for secondary metabolism, principally for producing aminoglycoside antibiotics and saccharide components of macrolides. Division of secondary metabolites into groups according to the source of their structural carbon atoms fails, however, to reveal what could be a more significant and biologically relevant division. It has already been noted that the reaction battery of primary metabolism is restricted (Tables 2 and 3) and that an underlying strategy of primary metabolism appears to be an avoidance of complexity. It is possible to divide known secondary metabolites into three broad groups according to the extent to which they (1) conform in their biosyntheses to these two patterns of primary metabolism, (2) break with the TABLE 6. Fungal secondary metabolites formed, inter aliu, from two amino acids" Aranotins Aspergillic acids Asperphenamate Austamides Brevianamides Cephalosporins Echinulin Gliotoxin H yalodendrins M ycelianamide Penicillins Roquefortine Sirodesmin s Sporidesmin s Tremorgens Verruculogens Verticillins Xanthocillin

2-Phenylalanineb Various combinations of leucine, isoleucine and valine 2-Phenylalanineb Tryptophan plus prolineb Tryptophan plus prolineb Valine plus cysteine Tryptophan plus alanineb Phenylalanine plus serineb Phenylalanine plus serineb Tyrosine plus alanineb Valine plus cysteine Tryptophan plus histidineb Tyrosine plus serineb Tryptophan plus alanineb Tryptophan plus prolineb Tryptophan plus prolineb Tryptophan plus serineb 2-Tyrosineb

a For structural formulae, see Turner (1971), Devon and Scott (1972) and Steyn (1980). Those metabolites containing at least one aromatic amino acid.

22

IAIN M. CAMPBELL

first one, i.e. use new reactions, reactions not commonly found in primary metabolism or (3) break with both patterns, i.e. use new reactions and strategies that do not seem to avoid complexity. This alternative division I shall now develop, realizing that little detailed enzymology has been done on reactions of secondary metabolism. Most of the data available derive from precursor/putative intermediate feeding experiments, and the construction from the data of these experiments of hypothetical biosynthetic pathways. Recent discussion of secondary metabolite biosynthesis will be found in the publications authored or edited by Bell and Charlwood (1980), Conn (1981), Haslam (1979), Herbert (1981), Hutter et al. (1978), Krumphanzel et al. (1982), Mann (1978), Porter and Spurgeon (1981), Rose (1979), Smith and Berry (1976) and Venzina and Singh (1 98 1). 4 . Metabolites Derived Through Use of the Reaction Battery and Basic Operational Principles of Primary Metabolism

The most obvious examples of this type of secondary metabolite are compounds such as polyporenic acid C (XXIV), polyporenic acid A (XXV), viridin (XXVI) and scores of related materials that are clearly derived by applying various reactions listed in Table 3 to lanosterol (XI) in combinations

I

0

I

OH-'

/CO,H CH, HCH,

HO CHI CH,


23

not found in primary metabolism, i.e. combinations not used in the production of sterols, bile acids or steroids. New enzymes must be involved. The substrate specificity of these new enzymes is different from their relatives in primary metabolism but the basic chemistry operating behind both enzyme sets appears to be the same. Other examples of this type of secondary metabolite are given below. (1) Diphenylbenzoquinones such as polyporic acid (XXVII) and related compounds, apparently formed by condensing two equivalents of phenylpyruvic acid (XXVIII). (2) Decylcitrate (XXIX) and itaconate (XXI), formed by

XXVlIl

XXVIl

modifying the input to or fate of citric acid (XXX). (3) Carlosic acid (XXXI) 0

II

-

RCH,CSCoA

+ O=C-COIH I

RCH-COIH

I

HOC-C0,H

I

CHICOIH

R=H

11 I

C-COIH CH,CO,H

CH,-CO:H

XXIX, R=CII,H,, XXX, R = H

XXXlI

CHI

XXI

formed by condensing oxaloacetate (XXXII) with P-oxohexanoate (XXXIII). CO,H

COCHZCH2CHq / CHI

I c=o +

C02H

HO~CCH'

XXXII

I

XXXIll

--

HOmoCOCH?CH2CHi

HOiCCH,

XXXI

It is also appropriate to include in this category materials such as helvolic acid (XXXIV), proposing that their basic skeletons are formed by modifying a squalene oxidocyclase, not to accept a different substrate, but to process the original substrate (squalene oxide) in a slightly different fashion, specifically to stabilize the intermediate cation XIV (Fig. 2, p. 18).

5 . Metabolites Derived Through Use of an Augmented Primary Metabolism Reaction Battery but Still Operating Under the Basic Primary Metabolism Principles When all of the various pathways of secondary metabolism are examined in

IAIN M. CAMPBELL

24

& Y

CH,

CO,H OCOCH,

Squalene

XIV

OCOCH,

0

CHI 0

XXXIV

detail, be those pathways firmly established at the enzymic level or projected from putative intermediate feeding experiments, it is observed that they feature unit processes in addition to those found in primary metabolism (see Tables 2 and 3). Examples of these additional reactions are listed in Table 7. The use of these additional reactions, together with others normally found in primary metabolism, allows a much wider range of secondary metabolites to be formed. Some representative systems are listed below, attention being directed to the key new reaction(s) utilized in their biosynthesis. (1) Acetylenes such as matricaria ester (XXXV) where acetylene bond formation is needed. CH3CH=CH(C=C)2CH=CHCOOCH,

xxxv TABLE 7. Canonical forms of reactions that occur in secondary metabolism but are not found commonly in primary metabolism

1. Conversion of an olefin into an acetylene. 2. Phenol oxidative coupling. 3. S-Adenosylmethionine-dependenta-methylation of a ketone or of a P-diketone. 4. Reduction of a benzyl alcohol to a toluene. 5. Oxidation of an amino group to a hydroxylarnino, nitroso or nitro group. 6 . Oxidation of sulphides to sulphoxides and sulphones. 7. Aromatic halogenation. 8. cr-Halogenation of a ketone or of a fl-diketone. 9. Conversion of a ketone into a lactone. 10. Arginine-dependent transamidations. 11. Peptide-bond formation using a thiol ester derivative of the carboxylic group of the amino acids. 12. Ring expansions of substituted toluenes to tropolones. 13. Conversion of a benzodiazepin derivative into a quinoline. 14. Annulation reactions in penicillin synthesis. 15. Prenylation of indoles.


25

(2) Caldariomycin (XXXVI) where a-chlorination of a P-diketone is needed. (3) Modified diketopiperazines such as aspergillic acid (XXXVII), where hydroxamate formation occurs, and echinulin (XXXVIII) in which indole

I

XXXVI

CHI

bH

XXXVII H

XXXVIll

prenylation takes place. (4) Lysergic acid (XXXIX) and its many derivatives, require indole prenylation and ring C closure. ( 5 ) Aminocyclitol antibiotics NH,

I

I

CO,H

C=NH

CH,

H&

H

?

H

OH

XXXIX

OH H

XL

such as streptomycin (XL), in which a-ketone methylation and transamidation are required. (6) Peptide antibiotics such as gramicidin S (XLI) and depsipeptides such as enniatin B (XLII), which require peptide bond formation using thioesters. (7) Penicillins (e.g. penicillin N (XLIII)) and cephalosporins (e.g. cephalosporin C (XLIV)) which require annelation reactions. It will be noted that the new reactions are, in some cases, mechanistically simple, for example reactions 3 , 4 and 5 in Table 7, whereas some, such as the penicillin annelation reaction, are still under mechanistic study. It will also be

26

IAIN M. CAMPBELL

CH, R L-Leu--D-Phe-L-Pro

I

I

I

L-Om

L-Val

L-Val

L-OITI

I

I

I

L-Pro-i,-Phe-L-Leu

XLI

I

R

I

N-CH-CO-0-CH-CO

I

I

I

i.0

N-CH,

I I 0 I

I I co I

R-CH

CH-R

CO-CH-N-CO-CH-0

I

K

I

CH,

I

R

XLII. R=CH(CH,)? H

H

C02H

XLIII.

R=o-r-Aminoadipoyl

XLlV

noted that the reactions listed in Table 7 are not simply a set of bioconversion processes (see Kieslich, 1978 for an overview of this topic); if this had been so, secondary metabolism would be significantly less interesting.

6. Metabolites Derived Through Use of an Augmented Primary Metabolism Reaction Battery and Without Total Regard for the Basic Operational Principles of Primary Metabolism It was noted in Section 1II.A (p. 12) that in primary metabolism, situations that could produce chemical complexity tended to be avoided. Thus, except for squalene oxidocyclase, reactions were conducted in small theatres with only one, two or three usually contiguous centres of the substrate being involved. Moreover in polymerization reactions, monomers were fully processed before the next monomer was added, an eventuality that precluded protection/deprotection cycles on reactive centres in the growing polymer. If two or more monomers were being used in a consistently irregular fashion, a template or jig was used to direct the synthesis. I argued that these three operating principles, coupled with the limited reaction set, restricted exposure of primary metabolism to unnecessary chemical complications. In secondary metabolism, however, there are two large groups of metabolites that are synthesized in seeming contravention of these three principles. Both groups of metabolites are derived from acetate; they are the polyketides and the non-lanosterol-derived terpenes. I shall deal with the second group first, focusing only on the skeleton-forming reactions; the reactions that eventually decorate that skeleton are all found in Table 3 (p. 16) or Table 7 (p. 24).


27

( a ) What Secondary Metabolism Requires in Making the Non-Lanosterol Derived Terpenes. This is totally a fungal issue since, except for geosmin (XXII), borneol (XXIII) and the carotenoids, bacteria are not active in processing oligoprenyl pyrophosphates other than to larger prenylogues. Fungi synthesize a variety of sesquiterpenes, diterpenes and the majority of Nature’s sesterterpenes (e.g. trichodermin (XLV), helminthosporal (XLVI), illudin M (XLVII), fumagillin (XLVIII) and pebrolide (XLIX)), all sesquiterpenes; pleuromitilin (L), rosenonolactone (LI) and gibberellic acid (LII), all diterpenes; and ophiobolin A (LIII), a sestettetpene.

A

OCOCH,

CH, CH,

XLV

XLVll

XLVI

OCO(CH=CH),CO,H OCOCH,

XLVlll

XLIX

@ CHI

OH

/CHI

OCOCH,OH L

0 LI

0

3 :@

LII

bH Llll

IAIN M. CAMPBELL

28

The key issue as far as this section of the essay is concerned is that these various and varied terpenes are not produced simply by fitting a different substrate into a slightly modified squalene oxidocyclase; each one requires its own specific reaction theatre. More than a single enzyme may be involved. Figs 3 and 4 show formally, for two representatives sesquiterpenes (trichodermin and illudin M) and two representative diterpenes (rosenonolactone and @-OH

( I ) XVI (m=3)

-

y)-o

--

-

Trichodermin (XLV)

+ I

Illudin M (XLVII)

FIG. 3. Necessary transformations carried out at the active sites of the enzymes responsible for synthesis of the carbon skeleton of trichodermin (1) and illudin M (2). It is uncertain how many enzymes are involved; conversions from one carbenium ion into another have to be executed by the same enzyme. “See Cane (1981) for further discussion of the conformational variability of humulene (this molecule).


( I ) XVI. (m=4)

-

29

&l@DO@OH

H + S

&

n

'0 CH,O@O@OH

/

H

H

-(2) XVI (,11=4)

H

Rosenonolactone (LI)

-

*

Gibberellic acid (LII)

CH,O@O@OH

\%-

/

/

FIG. 4. Transformations needed in the active sites of the enzymes responsible for synthesis of the carbon skeletons of rosenonolactone (1) and gibberellic acid (2). It is uncertain how many enzymes are involved; conversions from one carbenium ion into another have to be executed by the same enzyme.

gibberellic acid), the transformations that most likely are essential activities in those reaction theatres. These transformations are diverse and involved, akin to the squalene oxidocyclase in spirit but quite distinct in detail. In making sesqui- and diterpenes, fungi apparently become highly sophisticated. Stereo-

30

IAIN M. CAMPBELL

chemical control must be exquisite and the reactive carbenium ion intermediates must be held stable through several .formal steps. Caliban is venturing far from Prospero’s unloving side; within the bounds of polyketide metabolism that venturing becomes even more bold, as we will now see. ( b ) What Secondary Metabolism Requires in Making Polyketides. Superficially, the production of polyketides is straightforward. Indeed the basic concepts were appreciated by Collie (1907) long before there was any method available for their experimental proof. A tetraketide such as compound XV (n = 4) is formed by condensing an acetyl-CoA molecule “starter” with three equivalents of a malonyl-CoA “extender”. This tetraketide is now allowed to react intramolecularly. The easiest way to cover all possible outcomes is to examine, for reaction potential, each of the possible planar conformations of the tetraketide. Figure 5 shows such an examination. There are 64 theoretical possibilities (22”-2with n = 4 for a tetraketide) of which only 32 need be evaluated since the remaining 32 are their mirror images. Three of these possibilities (A3, A4, C2) can be discarded since they place carbons on top of each other. From the remaining 29 conformations, five products can in principle be formed:

(1) 3,5-Dihydroxyphenylaceticacid (LIV) from conformations A1 and A2 by a condensation reaction (ketone to methyl group). (2) Orsellinic acid (LV) from conformation C 1 by a condensation reaction (ketone to methylene group). (3) Acetylphloroglucinol (LVI) from conformations B2 and D5 by a condensation reaction (acid/ester to methylene group). (4) Tetra-acetic acid lactone (LVII, a 2-pyrone) from conformations A7, B5, C8 and D1 by enolization and lactonization. ( 5 ) The 4-pyrone (LVIII) from conformations C3 and C4 by double enolization and etherification. Although five products are possible in principle, in practice product LV, the product formed by condensing a ketonic carbonyl with a methylene group, is the most prevalent, being the nueleus from which well over half of the fungal tetraketides are formed. Products of type LVI, LVII and, to a lesser extent,

0

C

0

0

qc

0 U C ,

k;,

C

C

0

0

c,

+

-s 5,

Bc

0

5, C

C

c,o

&Y'

5.

C

X

U

i,

Y'

5. 0

i,

c'

0

6

0

1

YE 8 5,

P

32

IAIN M. CAMPBELL

LVIII are found occasionally. Product LIV, the product of coupling a ketonic carbonyl residue to a methyl group, is seldom encountered. Forming a single product from a tetraketide is not logistically simple; the problems are compounded in moving to pentaketides or hexaketides. By leaving the inserted monomer unit (CO-CH2) in an highly reactive state as the polymerization proceeds, a situation that never occurs in primary metabolism, the cell creates a need for some type of active group stabilization/protection. This is patently evident in the case of the bacterial tetracyclines (e.g. 11) where at least half of a nonaketide built from a malonamide starter (LIX) has to be in hand before any aromatization can begin. It has been suggested A

NH, 0

0

0

0

-

Tetracyclines (e.g. 11)

0

LIX

that metal ions play a role in stabilization of polyoxomethylene chains (Bu’Lock, 1967). In view of this suggestion, it is disturbing to note that the 6-methylsalicyclic acid synthase, the only polyketide synthase that has been purified reproducibly to homogeneity, does not appear to have any obvious metal ion requirement (Dimroth et af., 1970). Since, in 6-methylsalicyclicacid biosynthesis, the 50x0 group of the tetraketide is reduced prior to aromatization, the 6-methylsalicylic acid synthase may not, however, be an appropriate system in which to explore the metal-ion stabilization notion. There are two alternative polyketide stabilization possibilities. Firstly, polyketide synthases may have facilities for binding at least 50% of the active sites in a growing polyketide chain. Implicit in this option is the fact that the eventual reaction theatre would be of considerable size, a further divergence from the basic simplicity principles of primary metabolism. Secondly, polyketide synthases may “park” growing polyketide chains in hydrophobic cavities; under anhydrous conditions polyketides might be expected to form internally hydrogen bonded, and hence protected, entities such as compound LX. This latter option has the compelling attribute of simplicity.

LX

The issue of polyketide stabilization, however, does not exist in isolation; the polyketide must eventually be conformed and “deprotected” such that the eventual product can be formed. Since polyketides, unlike polypeptides, lack side chains of varying polarity and size, entropy-driven self-assembly into an


33

appropriate conformation based on secondary and tertiary structural forces is precluded. The desired conformation must therefore be imposed externally. How this is achieved is uncertain. Is the complete polyketide formed intact and then processed subsequently on an appropriate jig; or is the polyketide formed at a fixed point and, by the process of growing, extruded over a series of ketide-binding sites on the enzyme that conform the chain in the desired fashion; or does the synthesis site move over a conformation-dictating jig, laying the chain down as the jig prescribes? Only further work will tell. It is clear, however, that the process is not as logistically simple as fatty-acid biosynthesis was in primary metabolism! Additional levels of seeming complexity exist but need only be touched on in this essay. Firstly, switching to a different starter or to a different extender brings with it no added complexity except when two extendersfigure in the same synthesis. In molecules such as rifamycin (LXI), methymycin (LXII) and tylosin (LXIII), methylmalonate and malonate are used as extenders with methylmalonate being used, respectively, in all but the penultimate, in all but 0

CH, LXII

LXlll

the second, and in all but the third and seventh cycles of the synthesis of these three metabolites. To form irregular polymers consistently in biology, usually requires a template. How is the consistent irregularity achieved in the case of metabolites such as rifamycin, methymycin and tylosin? Do the synthases involved have multicarbon-centred theatres of reactivity that execute polymerization and subsequent polymer processing in a huge multifaceted active

34

IAIN M. CAMPBELL

site? Such an enzyme would outstrip the ribosome in organization complexity. Consider also zearalenone (LXIV). If it is indeed true that zearalenone is - H o p

0

\

0

OH

LXV

LXIV

formed from a polyketide such as compound LXV, what informs the appropriate polyketide synthase to reduce the first carbonyl (counting from the methyl group) to an alcohol, reduce the second to a methylene, leave the third alone, reduce the fourth to a methylene, take the fifth to the olefin level and retain the sixth, seventh and the eighth as the carbonyl groups from which the aromatic ring can be synthesized? Clearly Caliban holds secrets of which Prosper0 knows little. C. WHAT CAN WE CONCLUDE FROM A COMPARISON OF THE BIOCHEMISTRIESOF PRIMARY AND SECONDARY METABOLISM?

From the previous two sections, the clear conclusion can be drawn that primary metabolism and secondary metabolism are co-extensive. Both processes draw carbon from the same sources and, in many cases, use the same chemical compounds to build that carbon into end products. This being so, the first of this essay’s objectives has been realized. Secondary metabolism is, however, no random aping of primary metabolism. In several aspects, secondary metabolism is more biochemically sophisticated than primary metabolism. Indeed, in the areas of polyketide and terpene biosynthesis there is an inherent biochemical complexity present in secondary metabolism that is absent from primary metabolism. Problems of considerable logistical involvement arise in secondary metabolism, and have apparently been solved satisfactorily. The question now inevitably arises: is all of this biochemical effort of any physiological significance? Are secondary metabolites, defined as natural products of limited distribution in Nature, specific solutions to specific biological problems as we have suggested, or are they simply the convoluted chemical doodlings of Caliban’s mad hands? Secondary metabolites are clearly rational in terms of biochemistry. Can their formation be rationalized in physiological terms? To begin answering this question requires a detailed understanding of where such substances might find a physiological role. That is the object of the next section of the essay.


35

Before leaving the biochemical level, however, it seems appropriate to ask how the seeming sophistication and diversity of secondary metabolism arose. Secondary metabolism most likely emerged through mutative modification of areas of primary metabolism. There, is already some tenuous evidence that such is the case. In Penicilliumpatulum, the fatty acid and the 6-methylsalicylic acid synthases are immunologically cross-reactive (Lynen et al., 1978). This suggests that the latter enzyme complex arose from the former by gene duplication (the LY subunit is probably responsible for the condensation and ketone reduction stages; see Stoop and Wakil, 1981), and mutation of the duplicate copy(ies) to yield an enzyme complex that could condense acetate and malonate units to yield polyketides, but could not efficiently reduce the carbonyl functions in those polyketides. Polyketides of various lengths and degrees of reduction may have existed in primitive bacteria and fungi for generations before further mutations led to formation of cyclized systems such as 6-methylsalicylate (LXVI) and subsequent processing of that compound to entities such as patulin (LXVII) and terreic acid (LXVIII), products that conferred a competitive advantage on organisms possessing

LXVI

LXVll

LXVIll

these polyketide-forming and processing capabilities. What those competitive advantages might have been, is considered in the next section of this essay. The origin of the terpenes (from an altered squalene oxidocyclase?) and the various amino acid-derived secondary metabolites (from altered proteases, hence the preference for aromatic amino acids; see Table 6?) is less obvious. Despite this uncertainty, the very fact that the questions can be asked underscores yet again the truth of the statement that secondary metabolism is worthy of the investigative attention of biologists. Caliban is Prospero’s kin; moreover, he appears to be an able and skillful practitioner of that magician’s art. With regard to metabolites of limited distribution (i.e. secondary metabolites) and their evolution, it is interesting to note that Darwin (1859) predicted that competition would be most severe between varieties of the same species or between species in the same genus. A reasonable corollary to this prediction would be that variety-specific or species-specific processes would arise with a significant frequency, the purpose of those processes being to grant a competitive edge to the organism possessing them. Some of these processes would certainly be expected to yield variety- or species-specific metabolites.

36

IAIN M. CAMPBELL

IV. What is Meant by the Term “Physiology”? In most dictionaries, “physiology” is defined as the branch of biology that deals with the normal functionings of organisms, both macro- and microscopic. The contrast is often made to the term “pathology” that deals with the abnormal functionings of organisms. I will have use for this contrast later. For micro-organisms such as the bacteria and fungi, there are three intertwined functions that must be performed, namely growth, adaptation and reproduction. Neither of these three functions, however, is directly approachable in molecular terms, the only terms in which we can expect to find common ground with secondary metabolism. Figure 6 is therefore an attempt to break down the phenomena of growth, adaptation and reproduction into a limited number of processes at the molecular level that can be used in pursuit of this essay’s second objective, namely demonstration that secondary metabolism is physiologically rational. Arbitrarily, Fig. 6 focuses on the flow of nutrients from the organism’s environment into the intracellular compartment. This is an experimentally convenient point of view since, particularly in bacteria and fungi, there is an extensive literature on the nutritional background against which secondary metabolism is conducted (see, e.g. Aharonowitz, 1980; Martin, 1977; Martin and Demain, 1980; Weinberg, 1977). Once in the cell in question, nutrients, whether they be organic or inorganic are processed to serve one (or in a few cases more) of four general functions. They can provide the structural framework of cells or colonies; they can provide the thermodynamic driving force of the life process in both the biochemical and biophysical context; they can discharge operational roles being agents of, or intermediates in, vital processes; they can constitute defense mechanisms of a cell or of a colony. It is convenient to subdivide further the four major functions delineated in Fig. 6 . Again, the subdivisions are abitrary. The structural role can be exercized at the cellular level in the form of cell membranes, subcellular membrane components (save in the bacteria), cell walls and extramural layers (e.g. capsules and mucilages). In those systems in which cells aggregate to form colonies, a structural role in forming the colony can be envisaged. For convenience, the cell-cell recognition aspect of aggregation is considered under the rubric of intracolony communications (see Table 8). The energetic function has three aspects. Cells contain a variety of components that enter directly into biochemical or biophysical processes providing by their entry the thermodynamic driving force for the processes. These components are most commonly anhydrides such as adenosine triphosphate (ATP), or powerful reducing agents such as the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). Intracellular levels of these “high energy” compounds are usually low; hence there exists also a variety of more stable, more abundant cellular factors that can be converted easily into compounds

I

A structural role (1) At the cellular level Membranes Walls Extramural layers (e.g. capsules) (11) At the colony level Intercellular matrix

1

An energy-related role The entities that drive reactions thermodynamically Storage forms of the above Detection/utilization of chemical gradients or radiant energy

I

An operational role ( I ) Conducted intracellularly Information encoding Catalytic Productional Integrational (11) Conducted extracellularly Nutrient acquisition Communications lntracolony lntraspecies lnterspecies

FIG. 6. What an organism has to do to grow, adapt and reproduce.

A defensive role ( I ) Conducted intracellularly Foreign agent detoxification Virus replication control Adaptation to physical

stress/insult (11) Conducted extracellularly Protection against physical factors Antagonism to other organisms

50 5

c)

54

! F

2 2 .e

w

4

38

IAIN M. CAMPBELL

TABLE 8. Expansion of the extracellular defense and operations functions that bacteria and fungi need to carry out growth, adaptation and reproduction

I Extracellular ooerations I1 Extracellular defense (i.e. outside the plasma membrane) A. Against physical factors A. Nutrient acquisition (nutrient being Insulation of spores, cysts, or organic, inorganic, simple or sclerotia, against heat/cold. complexly bound) Water-proofing of cysts, sclerotia Extracellular digestive enzymes“, spores and bascidiocarps. e.g. cellulases, peptidases, Shielding of cells, particularly lipases, chitinases. those of reproductive entities Chelating agentsb to assist from ultraviolet radiation. assimilation of inorganic ions Strengthening of basic structural such as Fe3+, Co3+ and Ca2+. features of cell walls, Chemical agents involved in intracellular matrices and invasion of a host by a parasitic cuticles against physical micro-organism, e.g. abrasion appessorium/haustorium functionc. B. Against viruses and predator organisms B. Communications’ Allelopathyj either through direct Intracolony, e.g. sexd or antibiotic action or by spore morphogenetic‘ hormones, germination inhibition. cell-cell recognition/. Protection of spores against Intraspecies, e.g. sex hormonesd, digestion in the intestinal tracts germination inhibitor# of new of vectors/predators. spores in the vicinity of parent Combating the phytoallexisj colony, staling factors, agents of response. hyphal interferenceh. Feeding deterrend against birds, Interspecies, e.g. symbiotic factors slugs, snails, earth worms, (in lichens, root nodules of mites, nematodes and amoebae. leguminous plants and in the rhizosphere in general), vector C. Against “prey” attractants (by colour, visible or Bacterial and fungal toxins ultraviolet; or by odour), territory markers. References: Blain (1975); Byers (1974), Emery (1974), Rosenberg and Young (1974), Zahner (1978); c R (1979); ~ dGooday ~ ~ (1974), Bu’Lock (1976), Turian (1978), Vantk et al. (1981); ‘Newell (1977), Vantk et al. (198l);fManney and Meade (1977); BAllen (1976), Gottlieb (1978), Macko (1981); Ikeduigwu and Webster (1970); ‘Whittaker and Feeny (1971); Newman (1978); jHarborne (1977); kCalam (1979), Harborne (1977); Aaronson (198 1).

’

such as ATP and NADPH. Most often, these cellular factors are biochemical; sometimes they are biophysical. In some cells there may exist a third type of energy-related biochemical, namely an entity that detects and/or utilizes radiant energy or the energy resident in chemical gradients. The mycosporines


39

constitute an example of this type of molecule (see Arpin and Bouillant, I98 I, for an overview). The operations role in cells is a major one involving the genome, the enzyme complement and all cellular components that act as intermediates in biochemical processes. These intermediates range in complexity from mRNA and coenzymes, such as cobalamin, to simple materials such as glucose 6-phosphate, oxosuccinate and Mg2+.Also subsumed under this heading, and often playing a production role simultaneously, are cell components whose intracellular levels regulate metabolic activities of cells and, in so doing, allow for metabolic integration. All of these operational roles outlined are conducted at the intracellular level. However, operations have also an extracellular aspect. Cells may need to secure from their environment necessary supplies of scarce nutrients. They may also have a need to communicate with other cells in the same colony, with cells of the same species but in another colony, or with cells of other species. Although some defense functions could be considered simply as a particularly extreme form of intra- or interspecific communication, it is possibly more appropriate to view defense autonomously. As with the operational role, the defense role may be both intracellular and extracellular. Intracellularly, the principal issues are protection against viral multiplication in cells (Detroy and Warden, 1979),detoxification of noxious substances that have peradventure arrived in the cell, and production of materials that allow cells to withstand intercellular stress produced by such environmental situations as desiccation, excessive heat, cold or osmotic pressure. Extracellularly, the options range’from defense against rival organisms or predators, to pre-emptive attack on a pathogenic organism’s host or “prey”, to protection against such physical factors as heat, cold or radiation.

V. Areas of Physiology in which Materials of Limited Taxonomic Distribution Might be Expected to Play a Role Even the most casual review of Fig. 6 (p. 37) indicates that there are some functions that only materials of limited distribution could fulfill. In general terms, uniformity of molecular strategy and of molecular agency might be expected at the intracellular level. Indeed, evolutionary pressures should tend to produce such uniformity. At the extracellular level, uniformity of strategy may or may not occur, but uniformity of agency is unlikely. This is particularly evident in the area of communication (see the sirenin example in Section 11, p. 8) but is also likely to occur in the areas of nutrient acquisition and defense. Thus, on purely intuitive grounds, it might be predicted that the physiological rationality of secondary metabolism (distributionally defined) would most profitably be sought in areas of extracellular operations and

40

IAIN M. CAMPBELL

defense. Materials of limited distribution may also be found in or around bacteria or fungi as a result of excretion of a detoxified agent or as a result of pathological conditions (mutational, nutritional or cultural) that perturb the natural functionings of the organism. These latter two groups of materials would be considered bioconversion and overflow products respectively. In Table 8, I developed in a little more detail the extracellular operational and defensive options, particular attention being taken to focus attention on specific physiological functions of bacteria and fungi, and functions that might be played by products of these organisms located in the environment outside the cell membrane, i.e. in the cell wall or extramural layers. The table is fairly explicit and requires little commentary. Suffice to note that: (1) in several cases, the biochemical agents that will fulfill the stated roles will be macromolecular, e.g. the extracellular enzymes involved in nutrient acquisition, some agents involved in fungal invasion of a plant, and molecules that promote cell-cell adhesion; (2) not all functions are applicable universally to bacteria and fungi. For instance, feeding deterrence is probably not involved irbacterial consumption by slime moulds (but see Singh, 1942); it could be involved in bird and rodent interaction with sclerotia of Cluviceps spp. on rye grass heads; (3) the listed functions vary from classical ones for which there is solid evidence (e.g. action of sideramines as chelating agents in inorganic nutrition, or sex hormones; see Sections V1.A (p. 41) and V1.D (p. 47), through ones that have an observation basis in micro-organisms (e.g. staling and hyphal interference) or are known to occur in higher plants but have not been demonstrated in micro-organisms (e.g. allopathy or feeding deterrence), to functions that are as yet purely hypothetical (e.g. screening against ultraviolet radiation, protection of spores from a vector’s digestive action, territorial demarcation); (4) some of the unit functions could conceivably be discharged by the same metabolite, e.g. structural strengthening and shielding from ultraviolet radiation; ( 5 ) few of the functions listed will be needed by an organism growing in pure culture in a shake flask or fermentor. It might be expected therefore that, if bacteria and fungi are quick to respond to selective pressures or, if those pressures are intense, the listed functions might be lost on continued or continuous culture in these formats. Key references regarding these unit physiological operations in bacteria, fungi or in higher plants are contained in the footnotes in Table 8. One function that is not included in Table 8 for an obvious reason is the function advanced by Zahner (1978), who suggested that cells may contain metabolites to which evolution has given rise, that are not toxic to cells, but for which the cells have not yet found a use. They are “prefunctional”, as it were. The Zahner notion is going to be difficult to prove or disprove in finite time.


41

VI. Evidence that some Secondary Metabolites, Distributionally Defined, Discharge Key Physiological Roles The objective of the second half of this essay is to establish that secondary metabolites, distributionally defined, discharge legitimate physiological roles in the organism that produces them. In terms of the strategy I have adopted, this amounts to associating bacterial or fungal metabolites that are restricted in their distribution with specific physiological unit functions such as those listed in Table 8. There are several instances where such associations can be made rigorously. These instances are now discussed. A . SEX HORMONES

This topic has been well reviewed (Gooday, 1974, 1978; Bu’Lock, 1976; Turian, 1978; McMorris, 1978; Vantk et al., 1981) and only the salient features will be summarized here. The sesquiterpene sirenin (IV) has already been referred to; it is the sex hormone in the Chytridiomycete genus, Allomyces, a group of barely mycelial fungi that are found in water and damp soil. Sexual reproduction in those members of the genus that engage therein, involves production of separate male and female gametangia on and from the same haploid gametophyte. Following rupture, the male gametangium produces small orange-coloured carotenoid-containing motile zoospores, whereas the female gametangium produces large, unpigmented, less motile zoospores. The female zoospores, although not possessing carotenoids, are not incapable of isoprene synthesis; they produce sirenin and use it as a chemotactic agent with which to attract male gametes. The latter are capable of de-activating sirenin in their immediate neighbourhood. Hence direct chemical-based communication between male and female cells is achieved. A more complex but similarly terpenoid-based communication system is found in the oomycete genus Achlya. The sexual stage in these water moulds involves formation of special sexual structures, namely antheridia (male) and oogonia (female). Growth of the tenticular branches of an antheridium to and then around an oogonium leads to fertilization and formation of a zygote oospore. Production of antheridia and oogonia is controlled by two hormones, namely antheridiol (LXIX) and oogoniol (LXX). The former compound is secreted by the female, potentially oogonium-producing cell type, the latter by the male, potentially antheridium-producing cell types. Both cell types can exist in a single colony (operational homothallism), or one type can predominate in a given colony (functional heterothallism). When male and female cell types are present in the same environment, the oogoniol secreted by the male cells reaches the female cells where, prior to inactivation, it induces formation of oogonia and increased production of antheridiol. In turn, antheridiol diffuses back to the male cells where, prior to inactivation, it

42

IAIN M. CAMPBELL

, , , , , , , , , , , , , ,;OH OH

RO

HO

LXX, Rzacetyl. proplonyl or so-butyryl.

LXIX

induces antheridium formation and increased oogoniol production. The system self-amplifies and leads from two undifferentiated cell types to syngamy and zygote formation. There is a third well-characterizedsex hormone system, namely the trisporic acids of the Mucorales. Trisporic acids are terpenes; however, they appear to be quite broadly distributed in that order, and this may preclude their description as true secondary metabolites. The process depends on the existence of two mating types, a plus cell type that produces the so-called P+ prohormone methyl 4-dihydrotrisporate B (LXXI) and a minus cell type that produces the so-called P- prohormones trisporin B (LXXIIa) and trisporol B (LXXIIb). These prohormones diffuse into the aerial environment and substratum of the producing cell. Cells of the opposite mating type that are in the vicinity absorb the complementary prohormone and convert it/them into trisporic acid B (LXXIII). The presence of trisporic acid B in either plus or

&CH?

C H I COICH, C H ,

CHI &CHI R

0

CHI

0

LXXII: il, R I C H , : b. R=CH-OH

LXXI

0

LXXlll

minus cell types leads to production of aerial zygophores which bend in the direction of the prohormone source. As a result, zygophores of opposite mating type meet, fuse and give rise to a zygospore. A set of hormones in which the side chain ketone is reduced to a secondary alcohol is also effective. They constitute the “C” series of trisporic acids.


43

Mention must also be made of the yeast mating hormones (for an overview, see Crandall et al., 1977; Manney and Meade, 1977). In organisms such as Saccharomyces cereuisiae, the sexual phase of the life cycle involves fusion of two distinct cell types, a-type cells with a-type cells. Cells of the latter type make and excrete one of a set of four oligopeptides (LXXIV a-d). These a-factors appear (Manney and Meade, 1977) to inhibit the next cycle of bud formation in cells of the a-type and to cause a-type cells to secrete a cell-adhesion factor onto their surface. For their part, the a-type cells produce a 600,000 dalton protein into the environment. The factor blocks budding in trp-his-trp-leu-gln-Leu-lys-pro-gly-gln-pro-met-tyr his-trp-leu-gln-leu-ly s-pro-gl y-gln-pro-met-t trp-his-trp-leu-gln-leu-lys-pro-gl y-gln-pro-met his-trp-leu-gln-leu-! ys-pro-gly-gln-pro-met

yr *-tyr *-tyr

LXXIV, a 4 ; met* =met sulphoxide tyr-pro-glu-ile-ser-trp-thr-arg-asn-gly-cys* LXXV; cys* = S-farnesylcys.

glu-his-asp-pro-ser-ala-pro-gly-asn-gly-t yr-cys* * LXXVI; cys** = S-farnesylcys methyl ester.

a-cells and causes them to agglutinate with a-type cells. Following agglutination, zygote formation occurs. The agglutinins are also examples of cell metabolites of limited taxonomic distribution. Sex hormones are known in other yeasts. In Rhodosporidium toruloides, the compound rhodotorucine A (LXXV) is secreted by cells of the A-mating type. Rhodotorucine A induces cells of the a-mating type that encounter it to form a mating tube (Kamiya et af., 1979 and references therein). Tremerogen A-10 (LXXVI) performs the same function in Tremella mesenterica (Sakagami et al., 1979). B. HORMONES INVOLVED IN MORPHOGENESIS

For over a decade, it has been known that a small molecule, produced by wild-type Streptomyces griseus, induces sporulation (and streptomycin production) in some mutants of the same organism that failed to form an aerial mycelium (Khoklov et al., 1973). The proposed structure of this A-factor (LXXVII, Kleiner et a/., 1976) has recently been confirmed (Mori, 1981).

LXXVII

44

IAIN M. CAMPBELL

Table 9 (p. 45) indicates that several other instances of factors inducing formation of aerial mycelium have been reported. The chemical structures of these factors have yet to be elucidated. A slightly more complex situation is found with Gibberella zeae (the perfect form of Fusarium roseurn). Development of perithecia in this fungus appears to be controlled, both positively and negatively, by zearalenone (LXIV); low concentrations of this compound promote perithecium production whereas ten-fold higher concentrations inhibit production (Wolf and Mirocha, 1977, and references therein). Intriguingly, when 100 ascospores from a single perithecium were scored for eventual perithecium formation and zearalenone production, the former correlated inversely with the latter, i.e. low zearalenene producers were phenotypically G . zeae, whereas high producers were phenotypically F. roseum. Mirocha and his colleagues have demonstrated that adenosine 3’,5’-cyclic monophosphate (cyclic AMP) stimulates perithecium production and zearalenone biosynthesis. The argument is made that cyclic AMP stimulates zearalenone production, and that the level of this secondary metabolite determines whether or not perithecia are formed. The best known instance of cyclic AMP being involved in morphogenesis is to be found in the slime moulds (Garrod and Ashworth, 1973; Newell, 1977). Although widely distributed as an intracellular metabolite, its secretion under physiological conditions appears to occur in only a few species of the genera Dictyostelium and Polysphondylium. In the case of the former species but not the latter, cyclic AMP induces ameoboid forms to abandon their solitary existence in the face of nutrient deficiency, and to aggregate to form a migrating slug which in turn yields a fruiting body. As will be seen in Table 9, a compound of as yet undetermined structure appears to perform a similar function in species of Polysphondyliurn. In both organisms, species-specific glycoproteins are produced at the time of aggregation to effect cell-cell association, these are the discoidins and pallidins, respectively. As with the yeast agglutinins above, they are species specific. C. AGENTS THAT INFLUENCE SPORE GERMINATION AND OUTGROWTH

Two types of influence are involved, namely inhibition (retardation) or stimulation of germination and outgrowth. Regarding the former phenomenon, it has long been known that, when dense inocula of fungal or bacterial spores are used in solid or liquid culture, germination is inefficient. This phenomenon, known as self-inhibition or auto-inhibition, has been reviewed extensively (Allen, 1976; Gottlieb, 1978; Macko, 1981). It occurs in a large number of fungi (see Table 1 of Allen, 1976) and in a significant proportion of that number, an extracellular factor is responsible for the inhibition. In several, the structure of the factor has been established. For instance, discadenine (LXXVIII) is the auto-inhibitor of Dictyostelium discoideum (Abe


45

TABLE 9. Some instances where evidence of varying degrees of rigour indicates positive association of microbial secondary metabolism with specific physiological processes. Proposed functions are marked * in the second column. Secondary metabolite Pamamycin (structure unknown) in Streptomyces alboniger Unknown metabolite of Penicillium cyclopium

Physiological process Induces aerial mycelium formation in the producing organism

Induces aerial mycelium formation in the producing organism Diffusible substance of Induces new aerial mycelium Streptomyces spp. formation on aged surface cultures Proteinaceous factor (Factor Causes Streptomyces griseus C) of Streptomyces griseus (strain N52-I) to approach (strain N45-11) sporulation Diffusible gene product of inhibits aerial mycelium the SCPI plasmid present in formation in strains that lack strains of Streptomyces the plasmid coelicolor Heat-stable product of Inhibits spore germination in Drechslera halodes (?) the producing organism Tyrocidin in Bacillus breuis *Counterbalances the effects of gramicidin S in retarding spore outgrowth Protein toxins of the *Control the rapidity of parasporal crystal of Bacillus spore germination in the thuringiensis producing organism Constituent of spores of Inhibits spore germination in Streptomyces the producing organism viridochromogenes Low molecular-weight Increases the efficiency and lipid@)of Stigmatella speed of cell aggregation and aurantiaca allows fruiting bodies to form in the dark Induces amoebae to Low molecular-weight constituents of aggregate into a grex. Polysphondylium violaceum Small molecular-weight, Induces the grex+fruiting acidic volatile metabolite of body conversion Dictyostelium discoideum Diffusible product of Controls sexual development Dictyuchus monosporus in the producing organism Principle of coprophilous *Responsible for the hyphal fungi (e.g. Coprinus interference phenomenon. heptemerus)

Reference McCann and Pogell (1979) Zandem et al. (1982) Dondero and Scotti (1957) Vitalis and Szabo (1969) Chater and Hopwood (1973) Palm and Goos (1980) Ristow el a6. (1975) Stahly er al. (1978) Hirsch and Ensign (1978) Stephens et al. (1982)

Hanna and Cox (1978) Sussrnan et al. (1978) Sherwood (1966) Ikediugwu and Webster (1970)

46

IAIN M. CAMPBELL

TABLE 9 (con?.) Dipicolinic acid and its calcium salt in the Bacillaceae Aminoglycoside antibiotics formed by Streptomyces spp., etc. e.g. streptomycin Monomers of the macromolecular sheaths of aerial hyphae of Streptomyces spp. The macrotetrolides of Streptomyces griseus Pigments of Serratia marcescens and of Chromobacterium uiolaceum.

*Involved in heat resistance of endospores

Murrell (1981)

*Induces cell-wall Barabas and Szabo construction in the (1968) producing organism. Distinguishes the aerial from Kalakoutskii and the vegetative hyphae Agre (1976) *Involved in potassium-ion Kanne and Zahner transport (1976) Protect the producing Singh (1942) organisms from predation by protozoa ~~

~~~

et al., 1976;prior to the work of Abe and his colleagues, the auto-inhibitor was thought to be N,N-dimethylguanosine),quiesone (LXXIX, the isobutyrate of 5-hydroxy-ionone)is the auto-inhibitor of Peronospora tabacina (Leppik et al., 1972), and methyl esters of derivatized cis-cinnamic acids are auto-inhibitors of several rusts, e.g. methyl cis-3,4-dimethoxycinnamate(LXXXa) functions in Uromycesphaseoliand Puccinia helianthi, and methyl cis-3-methoxy-4-hydroxycinnamate (LXXXb) functions in Puccinia graminis (Macko et af., 1971, 1972).

LXXVIlI

LXXIX

LXXX; a. R=CHI b. R=H

Evidence is accumulating that peptide antibiotics might play an auto-inhibitory role in the Bacillaceae. The best studied example is the role of gramicidin S (XLI) in Bacillus breuis (Nandi and Seddon, 1978; Marahiel et al., 1979;Piret and Demain, 1981). Gramicidin S is normally present in spores of B. breuis. If the antibiotic is absent, as the result of mutation or preculturing extraction, the period of time between inoculation of the spores into nutrient medium and their outgrowth is decreased by a factor of five. Other instances of bacterial auto-inhibition are included in Table 9.


47

In fungi, agents that stimulate germination of spores have been encountered and some have been characterized structurally. These stimulating agents emanate from two types of source. The first source is the spores themselves. Examples of such self-stimulating agents are nonanal (LXXXI) and 6-methylhept-5-en-2-one (LXXXII), both constituents of rust uredospores (French

LXXXI

LXXXll

and Weintraub, 1957; Rines et al., 1974). Spore-germination stimulators that are produced by the host in a plant host/predator micro-organism situation are also known (Macko, 1981). D. METAL CHELATING AGENTS

These include sideramines, siderophores and siderochromes (for overview see Byers, 1974; Emery, 1974; Rosenberg and Young, 1974; Zahner, 1978). Bacteria and fungi need to obtain from their environment a range of inorganic cations, principally K + , Mg2+, Ca2+, Fe3+, Cu2+,Zn2+, Mo6+ and Co2+ (Griffin, 1981). Securing supplies of all but the alkali-metal cations can be difficult due to the low solubility products of the corresponding oxides and hydroxides. To combat the insolubility of alkali-earth and transition-metal cations, bacteria and fungi have pressed secondary metabolites into service as agents of chelation. Most of the work currently extant has focused on iron chelation. Table 10 lists representatives of the major iron sideramines. Their chelating properties depend on the presence of carbon- or nitrogen-bound oxygen functions. One presumes that the structural dissimilarity of these compounds is essential to recovery of the iron-laden agent uniquely by the producing organism. If all organisms used the same sideramine, it would be possible for an organism to recover iron in disproportion (negative or positive) to its production of the sideramine nucleus. The extent to which other transition metals and the alkali-earth cations rely on chelation for their importation into cells is uncertain. Chemically, there is no reason for this mechanism not being used generally, i.e. the same strategy would be used but different agents would be featured. As far as the alkali metals are concerned, there is already some evidence to indicate that the so-called ionopherous antibiotics are involved (Kanne and Zahner, 1976). E. STRUCTURAL AND EXTRACELLULAR PROTECTIVE AGENTS

With hormones and chelation agents it is possible to ascertain whether they

48

IAIN M. CAMPBELL

TABLE 10. Some examples of iron-chelation agents produced by bacteria and fungi" Compound

Source

Catechol type: Enterochelin Escherichia coli (= enterobactin) Aerobacter aerogenes Salmonella typhimurium Hydroxamic acid type: Aerobactin Aerobacter aerogenes Disferal ( = ferrioxamine

B) Mycobactins

Aerobacter aerogenes Streptomyces pilosus Several mycobacteria, e.g. Mycobacterium phlei

Ferrichromes

Several Aspergillus spp.

Coprogen

Several Penicillium spp. and Neurospora crassa

Molecular constituents

2,3-Dihydroxybenzoic acid and serine

Lysine, citric acid and acetic acid Cadaverine, aspartic acid and acetic acid Salicylic or 6-methylsalicyclic acid, serine or threonine, lysine and lactic acid or its homologues Glycine or serine, ornithine and acetic acid or B-methylglutaconic acid Ornithine,

3-methyl-5-hydroxypent-2-enoic acid (Eanhydro mevalonic acid) and acetic acid

'For further details and for structures, see Byers (1974), Emery (1974), Rosenberg and Young (1974), Zahner (1978).

are indeed discharging the ascribed function; with structural and protective agents, certainty is less easily established. It is easy to establish whether or not a given material is present on, in, or below a cell wall, or that it is a constituent of a spore coat, cyst exine, or sclerotial rind, hence our reason for including the following cell metabolites in a formal section as opposed to placing them in Table 9. It is more difficult, however, to prove with absolute certainty what role it performs in that location. With this proviso the following metabolites of restricted distribution are introduced.

I . The A l k y l Resorcinols of Azotobacter vinelandii Species of the genus Azotobacter respond to adverse growth conditions by


49

forming cysts (Sadoff, 1975). Inter alia, encystment involves overlaying the cell wall with layers of new or of newly modified material. Cysts are more resistant to radiation and desiccation than are vegetative cells but both are equally susceptible to heat. A major component of the outer layers of cysts of Azotobacter spp. are resorcinols (LXXXIIIa+) and the corresponding 2-pyrones (LXXXIVa, b) (Reusch and Sadoff, 1979,1981). These compounds OH OH

a. b. c. d. e.

R , Z C , , H , ~or C,,H,, R,=R,=R,=R,=H R,=R,=R,=H, R,=galactosyl R?=R,=R,=H. R,=CO?CH, R,=R,=R,=H. R,=OH R,=R,=R,=H. R,=COCH,

LXXXIV. R,=C,,H,, or C,,H,, a. R?=R,=H b. R,, R,=O

are labelled with radioactivity deriving from 3-hydro~y[3-'~C]butyrate and must consequently be considered acetogenic (this somewhat unorthodox precursor was selected since poly-(3-hydroxybutyrate)is a storage form of the carbon used for alkyl resorcinol synthesis). 2. The Amorphous Structural Components of Bacterial and Fungal Cells

Many low molecular-weight secondary metabolites are phenolic and/or contain significant numbers of conjugated double bonds or double bonds disposed in an allylic fashion with respect to each other. In the presence of molecular oxygen and appropriate enzymes, such systems can be polymerized and extensively so. It is not surprising, therefore, that bacteria and fungi contain several high molecular-weight amorphous chemically-refractile polymers. These polymers are very difficult to study; vigorous chemical degradation is the only available access route to structure determination. Likewise, it is difficult to determine with certainty what is their primary role in the producing organism. It is certain, however, that they contribute to the structure of the vegetative cells, spores or sporophores in which they are found, and that their presence therein confers some degree of protection against predator/parasite attack and electromagnetic radiation (Rast et al., 1981). There are two major groups of these polymers. In the mammalian field, melanins are exclusively the products of oxidative polymerization of 5,6-dihydroxyindole (LXXXV), a degradation product of 3,4-dihydroxyphenylalanine (dopa, LXXXVI). In plants, the term has been expanded to

50

IAIN M. CAMPBELL

include all pigments formed by oxidative polymerization of phenols (Thomson, 1976). The same broad definition seems appropriate in the bacterial and fungal fields. Therein, five monomer phenols have been identified as being the basis of melanins (see Bu’Lock, 1967 and Rast et a]., 1981 for reviews): catechol (LXXXVII); dopa (LXXXVI); l&dihydroxynaphthalene (LXXXVII); 1-(4’-glutaminyl)-3,4-dihydroxybenzene(LXXXVIII)); hispidin (LXXXIX); 3-hydroxyanthranilic acid (XC).

;Imco?H NH,

HO

H

HO

LXXXVI

LXXXVII

LXXXV OH

NH-

OH

LXXXVII

LXXXVlll

HO

OH

xc

LXXXIX

Carotenoids have a variety of roles to play in cells, several of which are not directly related to their function in photosynthesis (Krinsky, 1978). One of them appears to be oxidative polymerization to give a highly chemically resistant polymer, sporopollenin (XCI, structure proposed by Brooks and

XCI

Shaw, 1978) that has recently been recognized as a constituent of several fungal spores and bacterial walls (Gooday, 1981).


51

F. HOST-SPECIFIC TOXINS

Bacteria and fungi produce a wide range of materials that are toxic to their fellows, to protists, plants and animals (for overviews, see Ajl et al., 197&72; Andrews and Bulla, 1981; Cuatrecasas, 1977; Thompson, 1979; Wyllie and Morehouse, 1977). Most of these toxins are species-specific, with structures that range from relatively simple molecules, such as the aflatoxins, trichothecins and amatoxins, to the complex toxins produced by Corynebacterium diphtheriae and Clostridium botulinum. For most of these toxins, it remains to be seen if their toxic properties are their prime physiological raison d’ttre. There is one group of toxins, however, for which this issue is already closed, namely the host-specific toxins used by plant pathogens that produce them to kill host tissue (Scheffer, 1976; Yoder, 1980; Durbin, 1981). Typical hostspecific toxins include alternariolide (XCII) (Okuno et al., 1974), a toxin

I

-

NH

I

CO-CH-NH-CO-CH

I

CH 1

1 I

NH

XClll

I

(CH:),-CO-CH-CH,

\ / 0

produced by Alternaria mali, and HC-toxin (XCIII) (Liesch et al., 1982) produced by Helminthosporium catenarium.

VII. Why Have Physiological Roles Not Been Ascribed to More Microbial Secondary Metabolites? In the previous section of this review, several rigorous associations were made between microbial metabolites of limited distribution, i.e. with secondary metabolites, and unit physiological functions. The fact that this is the case supports my thesis that secondary metabolism is physiologically rational, at least in part. Compared with the number of known microbial secondary

52

IAIN M. CAMPBELL

metabolites (many thousands), however, the number of rigorous associations is small. Does this second finding, by its very weight, swamp the fact that some well-defined associations do exist and thereby invalidate my thesis? I do not think so, for the following three reasons. (1) Although not fully elucidated, a number of systems appear to conform to the associative pattern. A representative selection of these systems will be found in Table 9. (2) It is still uncertain how many of the “known” microbial secondary metabolites are “physiologically legitimate”, i.e. for how many do we really need to find rigorous physiological associations? This re-introduces the issue raised in Section I1 (p. 11) concerning the possibility of secondary metabolite bioconversion by the producing organism. Consider the pathway to 6-methylsalicylic acid (LXVI)/patulin (LXVII) in Penicillium patulum. Figure 7 shows what is now known to be the “highway” between 6-methylsalicylic acid and patulin, together with a number of associated “byways” yielding compounds to which secondary metabolite status may be attributed on purely distributional grounds. Are these byways physiologically normal or is their existence an indication that physiologically abnormal, i.e. pathological, conditions were prevailing in the cultures from which they were derived? Are the byways bioconversion pathways of intermediates that accumulated under such pathological conditions? Each of the byway products was obtained from laboratory culture of a single purified strain of P . patulum growing in a defined medium in surface or in submerged culture. Whether these strains were ecologically viable and whether the byways would operate in more ecologically normal circumstances remains to be seen. I suspect that, if the criterion of “production under ecologically normal circumstances” was used to classify known microbial secondary metabolites, the complement of the latter would be significantly lowered. (3) The third reason is in effect a continuation of the second, focussing on the experimental methods used to study bacterial and fungal secondary metabolism. Such studies rely heavily on use of liquid cultures of pure strains. Culturing is mostly of the submerged type, either in shaken flasks or in stirred fermentors. Except for water moulds, submerged liquid cultures are physiologically abnormal for bacteria and fungi. This does not mean to say that this form of culture is not a valid tool under some circumstances. It is so for industrialists desiring a specific drug, toxin or fine chemical, for organic chemists seeking new materials for structural or synthetic work, for bio-organic chemists tracing a biosynthetic pathway and for biochemists interested in the enzymology of the biosynthetic pathway or in the molecular biology of its control. For all of these interests submerged liquid culture of pure strains is both effective and efficient. My criticism of submerged or surface liquid culture (Campbell et al., 1981) should not be taken as an indictment of use of these methods for these purposes. Nor should my criticism be taken as an implied invalidation of the vast body of data relating to control of microbial metabolism that has been culled from

+

LXVI

I

OH

,’ ,’

I

OH I I

o

o

i

H

:

4 0

OH

0

0

LXVIl

FIG. 7. Highways (-+) and byways ( - - - -+) for conversion of 6-methylsalicylic acid (LXVI)into patulin (LXVII) by Penicillium patulum. From Neway and Gaucher (1981).

v,

w

54

IAIN M. CAMPBELL

experiments carried out in submerged culture (see Aharonowitz, 1980; Martin, 1977; Martin and Demain, 1980; Weinberg, 1977). The facts that under such circumstances, for instance, nitrogen depletion of a medium triggers 6-methylsalicyclic acid production by P. patulum (Grootwassink and Gaucher, 1980) or that phosphate depletion triggers candicidin production in Streptomyces griseus (Martin, 1976; Liras et al., 1977) are vitally important and will have to be explained by any eventual model for the intracellular regulation of these syntheses. Only when detailed physiological questions are being asked or when data are being interpreted in physiological terms does my criticism apply. Very, very few of the unit processes listed in Table 8 (p. 38) can be approached meaningfully using submerged liquid culture of pure strains; indeed, as I mentioned with regard to this table in Section V (p. 40),many of the agents of these functions would be expected to be lost in pure culture. Three examples from my own work will illustrate that results from submerged liquid cultures can be misleading in the physiological sense. On solid culture, 6-methylsalicylic acid (LXVI) production by P. patulum does not correlate with the extent of nitrogen depletion as it does unambiguously in submerged liquid culture; rather onset of 6-methylsalicylic acid production correlates with the emergence of aerial mycelia (Peace et al., 1981). We interpret this to mean that 6-methylsalicylic acid is functionally important in a culture of P. patulum that has developed to the point of producing and/or making use of aerial hyphae. In other words, an intra-aerial hyphal nitrogen deficiency is involved in inducing production of salicylic acid in solid culture, whereas nitrogen depletion in submerged liquid culture mimics and/or produces this intrahyphal nitrogen depletion in cells of the aerial hyphal phenotype that form presumptively under what are, for them, the abnormal circumstances of submerged liquid culture. On solid culture (Bird et al., 1981), brevianamides A and B (XCIVa,b) are produced by Penicillium brevicompacrum when conidia begin blebbing off from sterigmata. These photosensitive secondary metabolites accumulate in the penicillus, in the upper regions of the conidiophore and in the 10 or so conidia most proximal to the sterigmatum. Penicilli possessing brevianamides have the ability to rotate on their long axis when illuminated with near-ultraviolet radiation, returning to their original position when illumination is discontinued; penicilli that lack brevianamides do not rotate (Bird, 1981). None of these associations or phenomena would have been apparent in submerged liquid cultures of P. brevicompactum, assuming that steps had been taken to induce the latter to conidiate. (Conidiation is a necessary requirement for brevianamide production, but is a sporadically occurring event in P . brevicompactum submerged liquid cultures unless high concentrations of Ca2+ are present in the medium.) When produced in submerged liquid culture, brevianamides are leached off penicilli by the growth medium. It remains to be seen if results from solid culture hold the key to the physiological role played by the brevianamides. One thing is


55

CH, CH, XClV a.h: diastereoisomers at

XCV

*

I

I

co

co

XCVI

certain; the solid-culture results could not have been deduced from finding the brevianamides free in filtrates from submerged cultures of P . breuicompactum. Penicillium brevicompactum produces two other secondary metabolites in addition to the brevianamides, mycophenolic acid (XCV) and asperphenamate (XCVI). In solid cultures, mycophenolic acid is excreted into the growth medium while asperphenamate is accumulated predominantly in aerial portions of the biomass (Bird and Campbell, 1982). Synthesis of both compounds requires the culture to have developed to the point of producing an aerial mycelium, but development to the point at which conidiation occurs is not required. Such spatial and temporal localization could not be discerned in submerged liquid culture. To explore productively the physiological roles played by metabolites of limited taxonomic distribution, resort needs to be made to culture methods other than that of pure strains in liquid media. Alternatives that warrant investigation include: (1) pure culture on solid substrates such as nutrient agar, purees of fruit, “porridges” of grains, whole fruit, vegetables, plant seedlings, either with or without substrate sterilization; (2) mixed culture in liquid (especially continuous liquid) culture and on solid substrates as already indicated; (3) field examinations, either involving the native population or following seeding with a micro-organism of interest. There is no doubt that such experiments will be demanding technically and that they will require considerable ingenuity in planning and implementation. The challenge that these activities represent is one of the several reasons for being enthusiastic about the future role of secondary metabolism in microbiological research.

56

IAIN M. CAMPBELL

VIII. Epilogue The objective of this essay was to present microbial secondary metabolism as an area of research that is eminently worthy of study by a larger complement of biologists than is currently involved. Secondary metabolism is not a “thing of darkness”, irrational, wanton and capricious. It is biochemically rational, and is beginning to be seen as being rational in physiological terms. To an ever greater extent than primary metabolism, it holds considerable promise for future expansion at both the pure and applied levels. I foresee that much of the value of the “new biology”, including recombinant DNA technology, will be realized in studying and exploiting microbial (and plant) secondary metabolism. The prospects for research and development into microbial secondary metabolism are indeed glorious, provided the necessary human energy and ingenuity are released for application to the problems at hand. Worthy, therefore, is it that the biological Prosper0 acknowledge kinship to the secondary metabolic Caliban and abjure his spirit helper Aerial thus: “Come hither, Spirit. Set Caliban and his companions free.” W. Shakespeare, Tempest, Act V, sc. i

IX. Acknowledgments The author is pleased to acknowledge several stimulating discussions with Drs. R. Bentley and J.W. Bennett on the meaning and implications of microbial secondary metabolism, and the former’s generosity in reviewing the manuscript prior to submission. The assistance of Mss. D. Johnston, S. Wight and R.A. Schulte in preparing the manuscript is gratefully acknowledged. The authors work on fungal secondary metabolism has been supported by U.S. Public Health Service award (GM 25592) and National Science Foundation award (PCM 78-03852). REFERENCES Aaronson, S. (1982). “Chemical Communication of the Microbial Level”, Vols 1 and 2, CRC Press, Boca Raton. Abe, H., Uchiyama, M., Tanaka, Y. and Saito, H. (1976). Tefrahedron Letters 42, 3807. Aharonowitz, Y. (1980). Annual Reviews of Microbiology 34,209. Aharonowitz, Y . and Demain, A.L. (1978). Antimicrobial Agents and Chemotherapy 14, 159. Ajl, S.J.,Ciegler, A., Kadis, S., Montie, T.C. and G. Weinbaum (general eds.) (1970-1972). “Microbial Toxins”. Academic Press, New York. Allen, P. J. (1976). In “Encyclopedia of Plant Physiology” (A. Pirson and M.H. Zimmermann, general eds.), Vol. 4, “Physiological Plant Pathology” (R. Heitefuss and P.H. Williams, eds.), pp. 51-85. Springer-Verlag, Berlin. Andrews, R.E. and Bulla, L.A. (1981). In “Sporulation and Germination” (H.S. Levinson, A.L. Sonenshein and D.J. Tipper, eds.), pp. 57-63. American Society for Microbiology, Washington, DC. Arpin, N. and Bouillant, M.L. (1981). In “The Fungal Spore: Morphogenetic Controls” (G. Turian and H.R. Hohl, eds.), pp. 435-454.Academic Press, London.


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Physiology of Circadian Rhythms in Micro-Organisms LELAND N. EDMUNDS, Jr Department of Anatomical Sciences, School of Medicine, Health Sciences Center, State University ofNew York, Stony Brook, New York, USA

. . . I. Introduction . . . . A. Temporal organization. . . . . . . . B. General properties of circadian rhythms . . . . C. Circadian organization in micro-organisms . . . 11. Circadian rhythms in protozoa . . . . A. Tetrahymena spp.. . . . . . . B. Paramecium spp. . . . . . . . . 111. Circadian rhythms in unicellular algae . . . . . . A. Euglena spp.. . . . . . . . . B. Gonyaulaxspp. . . . . . . . . C. Chlamydomonas spp. . . . D. Acetabularia spp. . . . . . . . . . IV. Circadian rhythms in fungi . . . . . . . . A. Saccharomyces spp. . . . . . B. Neurospora spp. . . . . . . . . . . . . . V. General considerations and conclusions. A. Cell cycle clocks: ultradian, circadian, and infradian interfaces B. Nature of theclock(s) . . . . . . . C. Evolution of circadian rhythmicity D. Multiple cellular oscillators: a unicellular clock shop? . . E. Chronopharmacology and chronotherapy . . . F. Cell cycle clocks in development and ageing . . . . . . . . . . VI. Acknowledgement References . . . . . . . . . . Noteaddedinproof . . . . . . . . .

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I. Introduction A. TEMPORAL ORGANIZATION

The biologist is confronted with a continuously reproducing and evolving set of highly organized living systems. An organism that has thrived by ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 25 ISBN 0-12-027725-4

Copyright 0 1 9 8 4 by Academic Press, London All rights of reproduction in any form reserved.

62

LELAND N. EDMUNDS JR

differential reproductive success is said to be adapted, and its adaptation is reflected in its total organization. This organization is strongly history-dependent, having arisen through the twin processes of natural selection and adaptation. Biological problems, therefore, pivot on the complexities of biological organization (Pittendrigh, 1961). It is almost self-evident that the spatial organization and the functioning of living forms are inextricably intertwined. Of equal importance, however, is the temporal dimension: at the physiological level, for example, not only must the “right” amount of the “right” substance be at the “right” place, but this must also occur at the “right” time (Halberg, 1960). This also holds true for the organism itself, which often must be positioned in time with respect to favourable biotic or physical conditions. Inasmuch as the environment is highly periodic with respect to many of its variables, it would not be surprising (and, indeed, would be essential) for the organism to adapt to these periodicities. That organisms can and do measure astronomical time in some manner (as opposed to the purely “private” time keeping reflected in such variable-period physiological rhythms as heart beat or alpha brain waves) is explicitly demonstrated by four categories comprising quite diverse phenomena occurring throughout the animal and plant kingdoms: (1) persistent rhythms, having daily (circadian), tidal, lunar monthly and yearly (circannual) periods; (2) the Zeitgeduchtnis, or time sense of bees and humans; (3) seasonal photoperiodism, wherein many organisms perform a certain function at a quite specific time of the year by what may well be essentially a daily measurement of the length of the day (or night); and (4) celestial orientation and navigation, in which the sun, moon or stars are used as direction givers, implicating a timing system to compensate for their continuously, but predictably, shifting positions (Bunning, 1973; Palmer et al., 1976; Brady, 1982). All four types of time keeping, or functional biochronometry, have external correlates (generated by the movements of the earth, moon and sun) to which the organism has adapted. Although the last three kinds are commonly found only in higher organisms, and probably are relatively recent, more sophisticated variations on a more ancient evolutionary theme, the first category of persistent rhythms is commonly displayed in most, if not all, eukaryotic (but not prokaryotic) unicells. An understanding of the physiological and biochemical bases of these “simpler” clocks, therefore, may be crucial to the elucidation of the higher level phenomena. The underlying biological clocks which generate the foregoing types of rhythmicity all possess considerably longer periods than those that give rise to ultrafast and rapid chemical reactions, to biochemical rhythmicities of intermediary metabolism (e.g. glycolytic oscillations), and to those rhythms (such as cellular respiration) commonly observed in the epigenetic and genetic time domains (Lloyd et al., 1982). The time domain, therefore, of circadian

63

PHYSIOLOGY OF CIRCADIAN RHYTHMS IN MICRO-ORGANISMS

rhythms-the primary subject matter of this review-lies at the interface (Fig. 1) between the upper border of the genetic domain comprising cell division cycles and those even longer periodicities in the temporal hierarchy of living systems. B. GENERAL PROPERTIES OF CIRCADIAN RHYTHMS

Circadian rhythmicities, having a period of about 1 day, have been documented throughout the plant and animal kingdoms at every level of eukaryotic (but not prokaryotic) organization. Their general characteristics are summarized in Fig. 2. Typically, they can be synchronized, or entrained,

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1 10

64


by imposed diurnal light or temperature cycles to precise 24-hour periods, and can be predictably phase-shifted by single light and temperature signals, yet they are able to “free-run’’ for long time spans as persisting rhythms under conditions held constant with respect to most environmental time cues (Zeifgeber),with a natural period close to, but seldom exactly 24 hours. (Unless otherwise noted, we will always use the term “circadian” in this restricted sense.) Furthermore, the free-running period (z) is remarkably well

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(6)MODULATION OF FREE-RUNNING PERIOD BY LIGHT INTENSITY (Aschoff’s Rules) in diurnal and nocturnal animals

(7) TEMPERATURE-COMPENSATION (temperatureindependent free-running period within the physiological range) (8) GENETIC BASIS (innateness and endogeneity)

FIG. 2. Properties of circadian (sensu stricto) rhythms. A generalized sinusoidal oscillation under entraining (LD) and free-running (dim LL) conditions is shown at the top over a timespan of 7 days. Key: white areas, light intervals; black bars, darkness; hatched areas, continuous dim illumination. Note that the period (T) of the oscillation is longer in dim LL than it is in LD, where it exactly matches that ( r ) of the synchronizing LD cycle. From Edmunds (1975).


65

compensated for changes in the ambient temperature within the physiological range, as might be expected of an accurately functioning clock. Our central thesis is that there is a selectivepremium for temporal adaptation, especially to solar periodicities, and that these adaptive features have been attained by organisms through some sort of timing mechanism: in particular, an endogenous, autonomously oscillatory biological clock which is responsive to, and which can be reset and otherwise modulated by, those environmental periodicities that the organism has encountered throughout its evolutionary history. A number of books and symposia provide comprehensive coverage of circadian rhythms. The following texts afford more general treatment: Sweeney (1969a); Bierhuizen (1972); Bunning (1973); Palmer et al. (1976); Saunders (1976); Brady (1982); Moore-Ede et al. (1982). Major symposia and edited works having similar breadth include: Frisch (1960; Cold Spring Harbor Symposia on Quantitative Biology, volume 25); Aschoff (1965); Menaker (197 1); DeCoursey (1976); Hastings and Schweiger (1976); Naylor and Hartnoll(l979); Suda et al. (1979); Wever (1979); Aschoff (198 1); Follett and Follett (198 1); Aschoff et al. (1982); Edmunds (1984); and the Proceedings

Abbreviations used in this essay LL dim LL DD LD LD: x,y wc:x,y T z

4

4R fA 4 CT

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ZT

continuous illumination free-running continuous dim illumination continuous darkness lightdark cycle light-dark cycle comprising x hours of light and y hours of dark temperature cycle comprising x hours of warm and y hours of cold temperatures (“C) period of an LD cycle or other periodic Zeitgeber (environmental cue) period of a free-running circadian rhythm in constant conditions phase of a rhythm phase marker, or phase reference point change in phase (phase shift) circadian time (where CT 0 indicates the phase point of a free-running rhythm that has been normalized to 24 hours which corresponds to that occurring at the onset of light in a LD: 12,12 reference cycle) phase-response curve [plot of the phase shift of a free-running circadian rhythm engendered by a perturbing light (or other) signal as a function of the circadian time at which it was applied] average generation (doubling) time of a population of cells fundamental quanta1 cell cycle average step-size, or factorial increase in cell concentration (plateau to plateau) after a phased, or synchronized division step environmental (Zeitgeber) time (where ZTO corresponds to the onset of light)

66


of the International Society for Chronobiology (which has met biennially since 1971). Finally, the important monographs by Pavlidis (1973), Goodwin (1976) and Winfree (1980) provide particularly relevant mathematical and theoretical background for some of the considerations raised in this chapter. C. CIRCADIAN ORGANIZATION IN MICRO-ORGANISMS

Circadian organization is not restricted to higher plants and animals, or even to multicellular organisms. Over the past 30 years, overt persisting circadian rhythms have been documented in a number of eukaryotic microorganisms (Table 1) for a wide spectrum of behavioural, physiological and biochemical variables. These periodicities include: mating type reversal, mobility, phototaxis, pattern formation, stimulated bioluminescence, photosynthetic capacity, cell division, chloroplast shape and ultrastructure, susceptibility to noxious agents and treatments, enzymic activities, concentrations of metabolic intermediates, energy charge carriers, biogenic amines and many others. Clearly, virtually all levels of cellular organization display a circadian time structure. Furthermore, in many of these different micro-organisms, several different circadian rhythms have been observed concurrently-in some cases even in isolated, individual cells-with the attendant implication that many or all of the overt rhythms may represent the “hands” of an underlying master pacemaker (see Section V.D, p. 135). These organisms, therefore, constitute attractive systems for the experimental investigation of the finer details of circadian rhythms and the mechanism(s) whereby they are generated. In this general review, it is both undesirable and impossible to consider anywhere close to a majority of the reports of circadian periodicities (now probably numbering in the thousands) in micro-organisms. Rather, only representative rhythms in several of the more intensively studied protozoans and unicellular and lower algae and fungi have been selected for an illustration of the dominant themes and current problems in the field (see Section V, p. 124). Fortunately, there are a number of earlier detailed reviews and comprehensive treatments available that provide discussions of this area. These include Bruce and Pittendrigh (l957), Hastings (1959), Bruce (1965), Ehret and Wille (1970), Vanden Driessche (1970), Sweeney (1972), Ehret (1974), Edmunds (1975, 1982, 1984), Schweiger and Schweiger (1977), Wille (1979), Edmunds and Halberg (1981), Feldman (1982), Feldman and Dunlap (1983). 11. Circadian Rhythms in Protozoa A.

Tetrahymena spp.

1 . Rhythms of Cell Division

One of the most intensively investigated unicellular circadian systems has been


67

Table 1. Some eukaryotic micro-organisms in which persisting circadian rhythms have been demonstrated. Only selected references are cited (to review articles where possible); citations in brackets indicate the sections of this chapter in which more detailed treatment is given. Micro-organism Protozoans Paramecium aurelia Paramecium bursaria Paramecium multimicronucleatum Tetrahymena pyriformis Algae Acetabularia crenulata Acetabularia mediterranea

Amphidinium carteri Asterionella glacialis Biddulphia mobiliensis Ceratium furca Chaetoceros sp. Chlamydomonas sp. Chlamydomonas reinhardi Chlorella pyrenoidosa Cylindrotheca fusiformis Cylindrotheca signata Dunaliella tertiolecta Emliania huxleyi Euglena gracilis Euglena mutabilis Euglena obtusa Gonyaulax polyedra Gonyaulax tamarensis Gymnodinium splendens Hantzschia virgata Hymenomonas carterae Nitzschia tryblionella Olisthodiscus sp. Pavlova lutheri Phaeodactylum tricornutum Prorocentrum micans Pyramimonas sp. Prymnesium parvum Pyrocystis fusiformis Scrippsiella trochoidea Skeletonema costatum

Reference Karakashian (1968) [II.B] Ehret and Wille (1970) [II.B] Barnett (1966) [II.B] Wille (1979) [ L A ] Terborgh and McLeod (1967) [IILD] Schweiger and Schweiger (1977), Vanden Driessche (1980) [III.D] Brand (1982) Brand (1982) Brand (1982) Weiler and Eppley (1979), Adams et al. (1984) Brand (1982) Brand (1982) Bruce (1970), Bruce and Bruce (1981) [III.C] Pirson and Lorenzen (1958), Hesse (1972) Brand (1982) Round and Palmer (1966) [HI-A] Brand (1982) Chisholm and Brand (1981), Brand (1982) Edmunds (1982), Edmunds and Halbert (1981) [III.A] Round and Palmer (1966) [IILA] Round and Palmer (1966) [III.A] Hastings and Krasnow (1 98 I), Sweeney 1981), Wille (1979) [III.B] Brand (1982) [IILB] Hastings and Sweeney (1964) Palmer and Round (1967) Chisholm and Brand (1981) Round and Palmer (1966) Chisholm and Brand (198 1) Brand (1982) Palmer et al. (1964), Brand (1982) Brand (1982) Chisholm and Brand (1981) Brand (1982) Sweeney (1981, 1982) Brand (1982) Brand (1982), 0stgaard and Jensen (1982), 0stgaard et al. (1982)


68

TABLE 1 (continued) Micro-organism Skeletonema menzelii Thalassiosira pseudonana

Fungi Candida utilis Neurospora crassa Saccharomyces ceretlisiae

Reference Brand (1982) Brand (1 982) Wille (1 974) Feldman (1982), Feldman and Dunlap (1983) [IV.B] Edmunds et al. (1979) [IV.A]

the ciliate Tetrahymena pyriformis (reviewed by Ehret and Wille, 1970; Wille, 1979; see Table 2). Earlier studies (Wille and Ehret, 1968a) focused on photo-entrainment of batch cultures of the W strain. Population increase was characterized by a short (1-3 hours) lag period, followed by an exponential growth phase, termed the “ultradian mode” (with typical doubling times of 3 hours at 27”C), and then by a period of extended slow growth, the “infradian” TABLE 2. Some persisting circadian rhythms documented for Tetrahymena pyriformis. Circadian rhythm

Reference

A. Physiological Autotaxis (pattern formation) Cell death Cell division

Wille and Ehret (l968b) Meinert et al. (1975) Wille and Ehret (1968a), Ehret and Wille (1970), Edmunds (1974), Meinert et al. (1975) Oxygen-induction of ultradian growth Ehret et al. (1974, 1977) mode (Pasteur effect) Ehret et al. (1 977) Respiration

B. Biochemical Cyclic AMP ATP DNA, total cellular Glycogen, total cellular RNA, total cellular RNA, “transcriptotypes” Tyrosine aminotransferase

Dobra and Ehret (1977) Dobra and Ehret (1977) Ehret et al. (1974) Dobra and Ehret (l977), Ehret et al. (1977) Ehret et al. (1974) Barnett et al. (1971a,b), Wille et al. (1972) Dobra and Ehret (l977), Ehret et al. (1 977), see Kammerer and Hardeland (1982)


69

growth mode (Szyszko et al., 1968). This ultradian growth exhibited a high degree of temperature dependence; growth curves for cultures in the infradian mode reflected a monotonically decreasing function, in contrast to the so-called stationary phase commonly found in batch cultures of bacteria. Wille and Ehret (1968a) discovered that a sudden increase or decrease in irradiance (such as that afforded by either a DD/LL or a LL/DD transition) could initiate a free-running, circadian (7 19-23 hours) rhythm of cell division in axenic populations of T . pyriformis provided that the switch-up or switch-down occurred at a critical time in the late ultradian growth mode as the cells began to enter the infradian mode. Similar observations were made in continuous cultures achieved by growing cells in an electronically controlled nephelostat, which operated in a manner akin to a chemostat, maintaining a constant cell titre by a programmed dilution with nutrient medium (Wille and Ehret, 1968a): photo-entrainment by an LD:ZO, 14 regimen was consistently observed when the washout rate was infradian (e.g. when it matched a generation time (g) of 40 hours) but was not possible for ultradian growth rates. These studies were extended by Edmunds (1974) in an investigation of the phasing effects of light on cell division in exponentially increasing cultures of T. pyriformis (strains W and GL) grown at relatively low temperatures. Populations maintained on proteose peptone-liver extract in DD or LL (850 lux) at 10 f0.05”Cexhibited values of g of about 20 or 30 hours, respectively. Imposed diurnal LD cycles (e.g. LD: 12,12 or LD: 6,18) induced phased cell division so that doublings of cell number occurred once every 24 hours and were confined primarily to the dark intervals. Finally, long trains of 24-hour oscillations in apparent cell number could be obtained in semicontinuous culture with LD cycles, and the entrained rhythm persisted for at least 6 days with a circadian period (5 23.8-24.4 hours). Thus, once again, cell populations in the infradian (but not ultradian) growth mode were capable of showing circadian periodicities; in this case, however, the infradian state was achieved by lowering the temperature rather than by nutrient depletion. The above findings led Wille and Ehret (1968a) to formulate the “Circadian-Infradian Rule” (Ehret and Wille, 1970), which simply states that when an exponentially growing culture switches from the ultradian (g < 24 hours) to the infradian growth mode (g>24 hours), the cells comprising it are invariably capable of light-synchronizable circadian outputs (not only of cell division). This hypothesis will be discussed again in Section V.A (p. 126). 2. Physiological Rhythms Observed During the Infradian Growth Mode A number of other persisting circadian rhythms (Table 2) have been documented for cultures of Tetrahymena spp. that have reached the slow-growing, infradian growth mode (Ehret and Wille, 1970; Wille, 1979).

70


Thus, dense cultures of this ciliate maintained in a shallow dish exhibited very regular, hexagonally packed cell aggregates. An endogenous circadian rhythm (7 21 hours) in the rate of pattern formation could be initiated by a single DD/LL switch-up which had a specific phase relationship to the similarly initiated cell division rhythm (Wille and Ehret, 1968b). Likewise, a circadian rhythm in respiration (measured continuously as the amount of COZproduced) has been found in cells cultured on the surface of solid enriched proteose-peptone agar plates; maximum values were attained during the latter part of the (subjective) light phase (Dobra and Ehret, 1977). Presumably, an inversely correlated rhythm for the amount of oxygen present in liquid batch cultures might be anticipated. Indeed, a circadian rhythm in the capacity of sudden aeration (by bubbling) to induce cells in the infradian growth phase to return to ultradian growth has been demonstrated (Ehret et al., 1974). Thus, oxygen induction of increased cell division rates is under circadian phase control and appears to represent the Pasteur effect in Tetrahymena spp.; it could be blocked with carbon monoxide, amytal and rotenone, all of which are classical inhibitors of aerobic respiration. Finally, Meinert et al. (1975) have reported what might be construed to be the ultimate in physiological circadian rhythms: chronotypic death. It is well known for this ciliate (as well as for many other micro-organisms) that the attainment of a given cell titre is dependent on the level of dissolved oxygen in the medium. Asynchronous cell populations do not usually exhibit hypoxic death because the titre never exceeds the oxygen support level. In contrast, Meinert et al. (1975) have found that in thermally (WC) entrained cultures [ WC: 7,17 (W= 31 or 29°C; C = 26.5"C)I maintained in constant dim LL, cell death appeared to occur at each switch-up of temperature. This was manifested indirectly by a decrease in cell titre and was observed microscopically as a rupturing of a small percentage of cells in samples taken from the master culture at these phases alone of the WC cycle. These authors speculate that in synchronized cultures overproduction of cells for a given level of oxygen occurs at the time of transition from the infradian to the ultradian growth mode, and cell death then ensues to lower the titre to an appropriate value. If this decrease were to be prevented temporarily, as, for example, by imposition of the WC cycle, then sensitive cells would be induced to die synchronously. In other micro-organisms, however, alternative mechanisms may generate these oscillations in cell number; thus, a circadian rhythm in settling of cells out of the liquid phase of the culture, and attachment to the vessel walls, has been discovered in Euglena spp. (Terry and Edmunds, 1970b; see Section III.A, p. 96).

3. Circadian Rhythmicity in Metabolism and Biochemistry In addition to the many physiological variables discussed in the preceding


71

section, a number of metabolic and biochemical “chronotypes” also exhibit circadian time structure in Tetrahymena spp. (Table 2). Thus, in a photoentrained continuous culture, synchronously dividing during the infradian growth mode (the average generation time was at least 3 days), both total deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis (as measured by [3H]thymidine or [3H]uridine incorporation over a 36-hour time span) displayed circadian rhythmicity (Ehret et af., 1974). Subsequently, the question arose as to whether different RNA species were synthesized at different times of the circadian day, as predicted on the basis of the linear sequential transcription of the “chronon” postulated by Ehret and Trucco (1967) in their model for circadian timekeeping (see Section V.B, p. 129). To test this hypothesis, species of Tetrahymena, as well as Paramecium (see Section ILB, p. 73), were synchronized by a LD: 12,12 cycle in the infradian mode and then pulse-labelled at various time of the day with [3H]thymidine or 32Pto label RNA. Molecular hybridization of DNA-RNA was used to determine the binding capacity of the purified labelled RNA species with single-stranded homologous DNA immobilized on nitrocellulose membrane filters. The experiments were performed with annealing reactions in order to compare the kinetics, saturation and competition behaviour of the various pulse-labelled RNA stocks (Barnett et af.,1971a,b; Wille et al., 1972). The data from these pioneering studies showed clear evidence for the presence of chronotypically characteristic RNA species, or “transcriptotypes”, and thus are consistent with the prediction of the chronon model, which states that temporally differentiated RNA molecules are synthesized during the circadian cell division cycles which are present during the infradian growth mode, during which many other overt circadian rhythms are also observed (but see Section V.B, p. 132). More recently, an entirely different approach has been taken to circadian regulation in T. pyriformis, with primary emphasis being placed on metabolism and energy utilization (Ehret et af., 1977). In highly oxygenated (21% 0 2 ) plate cultures of this ciliate, concentrations of glycogen (a storage product, or energy “sink”) reached maximum values during the ultradian growth phase and then underwent periodic depletion during the ensuing infradian growth. These stepwise decreases were circadian, occurring both during photo-entrainment by LD cycles and during a subsequent free-run. Further, the decrease in glycogen content was in phase with the increase in the production of C02 (see previous subsection). Since other workers had demonstrated that the neurotransmitter substances, epinephrine and serotonin, occur in T. pyriformis (Janakidevi et al., 1966a,b), and that glycogen concentrations could be altered by reserpine, dichloroisoproterenol and aminophylline (Blum, 1967), Dobra and Ehret (1977) investigated the possibility that the regulation of the circadian rhythm of glycogen metabolism in Tetrahymena species might resemble that found in

72


rodent liver, in which glycogen (Haus and Halberg, 1966) and certain enzymes associated with its metabolism, such as tyrosine aminotransferase (Ehret and Potter, 1974), also oscillate with a circadian period. Further, they studied the control of associated pathways for glycogen storage and release, which in the hepatic system involves changes in the intracellular concentrations of cyclic AMP (adenosine 3‘,5‘-monophosphate) through norepinephrine-activated membrane-bound adenylate cyclase. Indeed, circadian rhythms were found in all these components (Dobra and Ehret, 1977): tyrosine aminotransferase, cyclic AMP and adenosine triphosphate (ATP) showed highly significant oscillations (see Table 2). The rhythm of cyclic AMP reached a peak just before the times of greatest glycogen depletion and C02 production; the concentration of ATP underwent maximal increases toward the end of the day-phase, during times of highest glycogen utilization; and tyrosine aminotransferase activity was greatest during the night, preceding the increases in cyclic AMP concentrations and glycogen metabolism. The phase relation of the tyrosine aminotransferase rhythm to that of the adenylate system suggested a regulatory role of biogenic amines in glycogen utilization in Tetrahymena sp. (as for the rat), since this enzyme is known to influence their rate of synthesis when the substrate is limiting. This hypothesis was further supported by the finding that norepinephrine, topically administered to entrained cultures on agar plates, maximally suppressed tyrosine aminotransferase activity at the time when it was known to be synthesized at the highest rate (Ehret et al., 1977). These findings logically led Ehret and coworkers to propose an “energy reserve escapement mechanism” for circadian clocks which intimately associates glycogen metabolism with the Circadian-Infradian Rule alluded to earlier (Ehret and Dobra, 1977; Ehret et al., 1977). In this unifying formulation, a gene-action circadian oscillator, producing phase-specific molecular transcripts and chronotypic enzymes, would ultimately drive overt circadian rhythms, coupling to them through causally interconnected oscillations in glycogenolysis and in biogenic amine metabolism. Glycogen would provide the reliable energy source needed for the clock as well as for infradian intermediary metabolism. This energy would be metered out discontinuously, on a daily basis, by an escapement component comprising either the indoleamine pathway (from tryptophan to serotonin) or the catecholamine pathway (tyrosine to epinephrine). Ehret et al. (1977) provocatively speculate that this exclusively eukaryotic mechanism allows cells to adjust to the “feast or famine” conditions that they encounter: during times of plentiful nutrients, the gene-action clock mechanism programs the ultradian cell division cycle to run as rapidly as possible (temperature-compensation might well be sacrificed for maximal cell proliferation); but under much more commonly encountered restrictive conditions, the eukaryotic cell clock would permit the cell division cycle to be


73

uncoupled and the cell to enter the circadian-infradian growth mode, while all the time continuing to program the circadian cycle of the daily rationing of energy reserve stores and ultimately to generate the multitude of other overt periodicities. In this regard, Kammerer and Hardeland (1982) have recently addressed the question of whether there is any relationship, or common element, between ultradian and circadian oscillator mechanisms and of their separate or joint control of the cell division cycle and other rhythmicities. Cultures of T. pyriformis were synchronized in the ultradian growth mode by a repetitive, 5-hour temperature cycle [WC: 4,l (W=2O"C; C=O"C)]. On transfer to a constant temperature of 20"C, the cells showed a free-running rhythm of tyrosine aminotransferase activity (as well as of cell division) having a period of approximately 5 hours; this rhythmicity persisted even when cell growth was inhibited with cycloheximide. The authors conclude that an independent ultradian oscillator was operating, which, although not identical with the cell division cycle, might control it under normally permissive conditions. The data would seem to suggest that a separate circadian oscillator would control both the cell division cycle and tyrosine aminotransferase activity in the circadian-infradian growth mode, although it is not excluded that the ultradian oscillator might play some role in circadian timekeeping (see Section V.A, p. 124).

B.

Paramecium spp.

One of the first non-green cells shown to possess a circadian clock was the ciliated protozoan Paramecium bursaria, which had been deprived of its symbiotic chlorellae (Ehret, 1951). Mating reactivity between cells of different mating types (syngens) occurs at midday in an LD: 12,12 cycle in populations of non-growing cells. This rhythm was shown to persist for as long as a week in continuous darkness (DD) and could be phase-shifted by brief light pulses. In the visible region, an action spectrum was obtained (Ehret, 1960) for phase shifting (advances) with peaks in the red (650 nm), blue (440 nm) and near ultraviolet (350 nm) regions. Furthermore, Ehret (1959) found that far-ultraviolet radiation also was highly efficient in inducing a phase shift, which could be reversed (photoreactivated) by exposure of the cells to white light. (These and other observations led to the suggestion that nucleic acids were involved in the clock mechanism and in the chronon model for circadian timekeeping (Ehret and Trucco, 1967).) A similar circadian rhythm of mating type reactivity has been demonstrated in P. aurelia (syngen 3) by Karakashian (1965,1968). The free-running period of the rhythm in DD was about 22 hours at 17°C and was relatively temperature-independent. Manifestation of the rhythm seemed to occur only

74


in slow-growing cells (e.g. 56 hours); the system, therefore, adheres to the circadian-infradian rule. One of the most unique cellular circadian rhythms, and one also amenable to genetic analysis, is the rhythm of mating-type reversals, reported first in P . multimicronucleatum (Sonnenborn and Sonnenborn, 1958). Although the mating type of a cell is usually inherited by its asexual progeny, with every cell line normally having a specifically assignable mating type, certain stocks of syngen 2 (mating types I11 and IV) exhibit a fascinating exception to this generalization: the same individual expresses one mating type for part of each day and the complementary type during the remainder. Sonnenborn and Sonnenborn (1958) and Barnett (1959) found that this rhythm could be entrained by LD cycles (e.g. LD: 6,18), and that it persisted for at least three days in DD. Later, in an intensive study of the mating-type reversal rhythm in three separate stocks of P . multimicronucleatum, Barnett (1961, 1965, 1966) found that each stock displayed different, but characteristic, phases in their switch-over times (I11 to IV, and vice versa) in an entraining LD: 8,16 cycle; the rhythm persisted for as long as 6 days in a subsequent D D free-run. The phase of the switch-over time characterizing a given stock was identical in all sublines, as long as the progenitor of that clone derived its macronucleus from one of the segregating macronuclear anlage produced at conjugation. Lastly, she discovered (Barnett, 1961, 1966) that the capability of the stock to cycle depended on the presence of a dominant allele C (cycler); cells homozygous for the recessive allele c (acyclic) did not reverse mating type but were either type I11 or IV as a consequence of macronuclear differentiation. Thus, the nucleus can control not only the phase of a circadian rhythm (as has also been shown in the alga Acetabularia; see Section III.D, p. 112), but also its ability to be expressed in the first place. Finally, photo-entrainable, persisting circadian rhythms of cell division have been reported in P . bursaria (Volm, 1964) and in P . multimicronucleatum (Barnett, 1969; see Table 8). 111. Circadian Rhythms in Unicellular Algae A.

Euglena spp.

The temporal organization of Euglena gracilis Klebs (strain Z ) has been extensively studied in a number of laboratories over the past 25 years (reviewed by Edmunds, 1975, 1982; Edmunds and Halberg, 1981; Wille, 1979), and this micro-organism together with species of Gonyaulax and Tetrahymena have formed a trio that has provided evidence for the so-called “G-E-T Effect”, which is more formally embodied in the Circadian-Infradian Rule formulated by Ehret and Wille (1970) (see Sections II.B, p. 69 and V.A,


75

p. 126). This algal flagellate can be grown on a variety of different completely defined media, either photo-autotrophically in the presence of carbon dioxide, or organotrophically in the light or dark on carbon sources ranging from acetate and ethanol to lactic, glycolic, glutamic and malic acids, and over a wide pH range. This versatility in growth mode, in conjunction with the fact that cell division can easily be synchronized by appropriate 24-hour (and other) lighting schedules (Cook and James, 1960; Edmunds, 1965a) and temperature cycles (Terry and Edmunds, 1970a), has made E. gracilis an important experimental organism for a variety of physiological and biochemical investigations (Buetow, 1968-1982). Many persisting circadian rhythms have been reported, a number of which are given in Table 3. These studies have been aided by the fact that E. gracilis can be maintained in the “stationary” phase of growth (or infradian growth mode) for days or even weeks with little or no net change in cell concentration; circadian output can thus be monitored while divorced from the driving force of the cell division cycle. Likewise, the fact that a number of photosynthetic mutants (or even completely bleached strains devoid of their chloroplast genomes) have been isolated which still exhibit light-entrainable circadian rhythms has effectively eliminated the problem of the dual use of imposed light spans and signals-as an energy source for growth, on the one hand, and as a timing cue for the underlying clock, on the other (Kirschstein, 1969; Jarrett and Edmunds, 1970; Mitchell, 1971; Edmunds et al., 1976). Clearly, then, the Euglena gracilis system provides an excellent case for temporal dzflerentiation: a large number of diverse behavioural, physiological and biochemical activities are partitioned along the 24-hour time axis, thus providing dimensions for both environmental adaptation and for functional integration in time (Edmunds, 1982). This circadian time structure is perhaps best illustrated by an acrophase chart (Fig. 3), which provides a convenient way to indicate the time relationships of periodicities (analysed mathematically by cosinor or other mathematical techniques) both to a synchronizing LD cycle (or other Zeitgeber) and to each other during either entrainment or free-running conditions (Edmunds and Halberg, 1981). In the following subsections, we will survey in greater detail selected classes of circadian rhythms itemized in Table 3 and examine some of the more recent developments in these areas. 1. Circadian Clock Control of the Cell Division Cycle

The relationship between the cell division cycle and circadian oscillators perhaps has been most intensively investigated in E. gracilis Klebs and, consequently, will serve as a basis for subsequent discussion (Edmunds, 1978; Edmunds and Adams, 1981). We will now review the major lines of evidence (Edmunds and Laval-Martin, 1984) that implicate an endogenous, self-sustaining circadian oscillator (see Fig. 2) in the control of the cell division cycle in this unicellular organism (and, by generalization, those in Table 8).

76


TABLE 3. Circadian rhythms in Euglenagracilis Klebs. From Edmunds and Halberg (1981).

Rhythma.h A. Physiological Cell division

Phase marker

StrainC

Onset

Z

dJd

Reference''

CT 11-13

Edmunds (1964,1965a, (180-195") 1975,1978,1982; Edmunds and Funch

(1969a,b);Edmunds and Laval-Martin

(1984)

P4ZUL

Onset

P7ZNgL

Onset

W6ZHL

Onset

W,ZUL

Onset

Y9ZNalL

Onset

CT 10-12 Edmunds (1971,1978); (150-180") Edmunds ef al. (1971, 1974,1976); Jarrett and Edrnunds (1 970) CT 10-12 Mitchell (1971) (1 50-180") CT 10-12 Edmunds (1978); (150-180) Edmunds et nl. (1971) CT l(t12 Mitchell (1971) (1 50-1 8 0 ) CT 10-11 Edmunds ef al. (1976)

(150-165")

Flagellated cells (%) Motility, random (dark) Dunkelbeweglichkeit

Z (1224-5/9, Maximum CT 03 (45") Brinkmann (1966) Gottingen) Schnabel(l968); BrinkZ (12245/9, Minimum CT 18-21 Gott ingen) (270-315") mann (1966,1971); Kreuels and Brinkmann (1979) W,ZHL Minimum CT 12 (180") Kirchstein (1969)

(l2265/25, Gottingen)

pH (external medium)

Z

Photokinesis (photo-activa- Z tion of random motility) Photosynthetic capacity I4CO2 uptake Z, ZR

evolution

Z

Phototaxis (capacity)

Z

0 2

Maximum CT 18-21

(27&3 15")

Maximum CT 0612

K. Brinkmann (personal communication) Brinkmann (1976b)

Walther and Edmunds (1973);Laval-Martin et a/. (1979); Edmunds and Halberg (1981); Edmunds and LavalMartin (1981) Maximum CT 04-06 Walther and Edrnunds (60-90) (1973);Lonergan and Sargent (1978,1979); Lonergan (1983) Maximum CT 04-08 Pohl(l948); Bruce and (60-120") Pittendrigh (1956. 1958); Brinkmann ( 1966);Feldmdn (1967); Feldman and Bruce (1972)

(90-180")


77

TABLE 3 (continued)

Rhythmaqb

StrainC

Settling

Z

Shape

2

Phase marker

4d

Reference'

Maximum CT 2149f Terry and Edmunds (315-135") (1970b) CT 15 (225") Kiefner et al. (1974) Maximum CTO6 (90") Lonergan (1983) elonga- CT 03-09 Brinkmann (1976b) tion ( 4 5 1 35")

z (1224519, Gottingen) Susceptibility ethanol (pulses) trichloroacetic acid Volume

Brinkmann (l976a) Z (1224519, Maximum CT 0 3 4 6 Gottingen) (45-90") Z (1224-5/9, Maximum CT03-06 Brinkmann (1976b) Gottingen) (45-90") 7 Maximum CT 18-21 K. Brinkmann and U. (270-3 15") Kipry (unpublished work)

B. Biochemical Amino-acid incorporation (~~-[3-'~C]phenylalanine) Z Gross metabolic variables8 carotenoids Z

Maximum CT 10-12 (150180") Onset

chlorophyll a

Z

Onset

dry weight

Z

Onset

total protein

Z

Onset

total cellular RNA

Z

Onset

total cellular DNA

Z

Onset

Z

Peak

alanine dehydrogenase

Z

Peak

carbonic anhydrase

Z

Peak

glucose-6-phosphatedehydrogenase

Z

Peak

Enzymic activityh acid phosphatase

Feldman ( 1968)

Z T O (0")

Cook (1961b); Edmunds (1965b) Z T 0 (0") Cook (I961 b); Edmunds (1965b) ZTO (0") Cook (1961b); Edmunds (1965b) ZTO (0") Cook (1961b); Edmunds (1965b) Z T 0 (0") Cook ( 196I b); Edmunds (1965b) ZT08-09 Cook (1961b); Edmunds (120-135") (1964, 1965b) Z T 06-08 Sulzman and Edmunds (90-120") (1972); Edmunds ( 1975) CT O W 8 Sulzman and Edmunds ( 1972, 1973); Edmunds (90-1 20") et al. (1974) ZT OW8 Lonergan and Sargent (90-120") (1978) ZT 06 (90") Sulzman and Edmunds (1972); Edmunds ( 1975)

78


TABLE 3 (continued)

Rhythm",' glutamic dehydrogenase

Strain" 2

Phase marker Peak

4d

Reference''

Z T 06-08

Sulzman and Edmunds (1972); Edmunds (1975) Z T 05-08 Walther and Edmunds (1973); Lonergan and (75-120 ) Sargent (1978) (90-120 )

Z phate dehydrogenase (NADP and NADPHdependent) Z lactic dehydrogenase

Peak

L-serine deaminase

Z

Peak

L-threonine deaminase

Z

Peak

gl yceraldeh yde-3-phos-

Peak

Z T 06 (90 ) Sulzman and Edmunds (1972); Edmunds (1975) Z T 06 (90 ) Sulzman and Edmunds ( 1972); Edmunds (1975) Z T 08 (120 ) Sulzman and Edmunds (1972); Edmunds (1975)

Unless otherwise noted, all rhythms listed have been shown to persist with a circadian period in DD (or LL) and constant temperature following synchronization. All cultures were essentially non-dividing (stationary, or long infradian), except those in which the cell division rhythm itself was monitored. Consult reference for precise culture conditions. The wild-type strain is designated as Z. Unless otherwise noted, it was originally obtained from the American Type Culture Collection (No. 12716) and maintained at the State University of New York at Stony Brook since 1965 or at Princeton University. Also available from the Algal Collection at Indiana University (No. 753) and the Algensammlung Pringsheim at Gottingen (No. 1224-5/9). Other strains are photosynthetic mutants or completely bleached strains incapable of photosynthesis. Phase given in circadian time (CT, hours or degrees after subjective dawn, when the onset of light would have occurred had the synchronizing light cycle been continued). Entraining light cycles were either LD: 12,12 or LD: 10,14 [except in the case of the settling rhythm in which the cultures were synchronized by a 12:12 temperature cycle (1 8/25', LL) before release into LL, and CT 0 denoted the onset of lower temperature]. In those instances where a free-run was not monitored, phase is given in Zeitgeber (synchronizer) time ( Z T 0, onset of light). Only key references are given; no attempt is made to give all citations. fRhythm synchronized by a 12:12 temperature cycle (I8j25"C) in either dividing or non-dividing cultures maintained in LL before release into constant conditions (25°C LL). CTO, onset of lower temperature. g These variables were monitored only in light-synchronized (LD: 14.10) dividing cultures. The phase marker here refers to the point when values start to increase. Although all of the enzymes indicated undergo oscillations in activity in non-dividing cultures in LD: 10,14,only alanine dehydrogenase has been investigated in sufficient detail to demonstrate conclusively that it will persist for long time spans in DD and constant temperature.

*

Circadian System of EupleO VARIABLE CELL DIVISION

z

P , ZUL P,ZNgL WeZHL w, ZUL YgZNalL MOBILITY Molillly, random (dark) Photohlnesls Phototaxls Sellllng MORPHOIBGY Cell volume Cell shape (elongation) Flagellated calla (%) PHOTOSYNTHETIC CAPACITY “ C O ~ incorporatton O2 evolution GROSS METABOLIC PARAMETERS Carotsnolds Chlorophyll a Dry welght Protsln (total) RNA (total) DNA (total) A M I N O A C I D INCORPORATION ENZYMIC ACTIVITY Acld phoephataes Alonlne dehydrogrnase Deoxyribonucleose Glucose-6-phosphate dehydrogenose Glutamlc drhydropenase Glyceraldshyde-3phosphole dehydroqenose Lactic dehydrogrnase Srrlnr deaminass Threonlne deamlnaae SUSCEPTIBILITY Ethanol (puliee) Trlchlaroacetlc a d d (pullel)

OD

60°

120°

180’

ZEITGEBER TIME

240°

300-

360’

(Zr)

FIG. 3. Acrophase chart for Euglena showing the time relations among a number of circadian rhythms documented for this unicellular flagellate (see Table 3). The so-called “external acrophase” (4) relates the peak of the fitted curve to the onset of light in a synchronizing LD cycle, and to “subjective dawn” in DD or LL when the light would have come on had the synchronizer been continued. The bars extending to the left and right of the acrophase points give the 95% confidence interval, when calculated by cosinor analysis. Single points, or points and bars within brackets, indicate subjective estimates of the acrophase from published data without benefit of statistical analysis. From Edmunds and Halberg (1981).

80


a . Entrainability. Photo-autotrophically grown cultures of wild-type E. gracilis (Z strain) can be routinely synchronized, or entrained-a key property of circadian rhythms (see Fig. 2 F b y empirically chosen repetitive, 24-hour LD cycles so that synchrony of cell division is confined entirely to the dark intervals (Cook and James, 1960; Edmunds, 1965a). For example, in LD: 10,14 a batch-cultured population doubles (factorial increase, or ss N 2.0) at each step, the period (7) of the rhythm in the population exactly matching that of the imposed LD cycle, and, by inference, the length of the individual cell division cycles also must average 24 hours (the rate of cell death is insignificant). Long-term synchronous cultures can be obtained using continuous or semi-continuous culture techniques by which the cell titre is maintained at a constant value without reaching limiting conditions (Terry and Edmunds, 1969), as occurs in batch cultures (primarily due to decreased light intensity caused by the mutual shading of the cells). If one decreases the total duration of the light interval (e.g. LD: 8,16) within a 24-hour framework, the amplitude of the cell division rhythm in the population is proportionately decreased (E=1.68), and the average doubling time (g) of the culture is lengthened to about 36 hours (Edmunds and Funch, 1969b). Not every cell divides during each division burst, and the culture is now no longer developmentally synchronous to the extent of a one-to-one mapping between the stages of the cell division cycle in all constituent cells (Edmunds, 1978). Nevertheless, cell divisions, when they do occur, do so during the dark intervals only at intervals of 24 hours, and the culture continues to be synchronized in the sense of event simultaneity (Edmunds, 1978).This rhythmicity observed in LD:8,16 (and in similar LD cycles) stands in sharp contrast to the asynchronous exponential growth curve obtained in LL having a minimum doubling time of 12 to 14 hours (Edmunds, 1965a). Similarly, so-called “skeleton” photoperiods (PPs) comprising the framework of a normal, full-photoperiod cycle (e.g. LD:3,6,3,12) will also entrain the rhythm to a precise 24-hour period; in this case, divisions are confined to the main dark intervals, commencing at their onsets (Edmunds and Funch, 1969b). Entrainment by LD cycles having T # 24.0 hours (e.g. LD:ZO,10) may also occur, but only within certain limits (Edmunds and Funch, 1969b; Ledoigt and Calvayrac, 1979).Additionally, appropriate diurnal temperature cycles (e.g. Z8”C,25”C: Z2,12 or 28”C, 35”C:Z2,12) will synchronize the cell-division rhythm in cultures maintained in LL (Terry and Edmunds, 1970a). Finally, even more conclusive evidence for the role of a basic circadian oscillator in the control of the cell division cycle has been obtained by utilizing photosynthetic mutants (all obligate heterotrophs) of E. gracilis (Edmunds, 1975, 1978). These studies have effectively circumvented the problem of the dual use of imposed light cycles and signals: as an energy source, or “substrate”, for growth, on the one hand, and as a timing cue (Zeitgeber) for

(n


81

the underlying clock, on the other. Thus, the ultraviolet radiation-induced P4ZUL mutant (Jarrett and Edmunds, 1970), although not entrainable to a 24-hour period by diurnal LD cycles when growing in the ultradian mode ( g < 24 hour) at 25”C, where a doubling of the population occurs every 10 hours, could be synchronized by a LD: 10,14 cycle if the temperature were lowered to 19”C,(yielding an exponential curve with g approximating 24 to 26 hours in D D or LL). In this infradian (g > 24 hours) growth mode (Ehret et al., 1977), divisions were set back or delayed 8 to 10 hours at 24-hour intervals. These results have been extended both to the naladixic acid-induced YgZNal L photosynthetic mutant and to the white, heat-bleached W6ZHL strain that totally lacks chloroplasts (Edmunds, 1975; Edmunds et al., 1976), and are consistent with the data of Mitchell (1971) for the pale-green nitrosoguanidine-mutagenized P7ZNgL strain and the white ultravioletbleached W,ZUL mutant. b. Persistence. Although synchronization of the cell division rhythm in E. gracilis by diurnal, full-photoperiod LD cycles is consistent with the notion that a putative circadian clock is entrained by the imposed light regime and, in turn, phases or “gates” cell division to the dark intervals every 24 hours (by acting, perhaps, on one or more key control points of the cell division cycle; Spudich and Sager, 1980), it does not demand it. Light (or darkness) could be acting by directly inhibiting (or promoting) division, and periodic shifts between light and dark would synchronize the culture (Campbell, 1957). A number of other observations, however, render this seemingly straightforward hypothesis untenable. One of the most basic tests for the existence of a circadian rhythm senm stricto, is to determine whether it will continue to freerun for a number of cycles following transfer of the organism to conditions held constant with respect to the major environmental Zeitgeber (light and temperature); characteristically, the period (Z) under such conditions only approximates 24 hours, as might be expected of an imperfect biological clock. Indeed, rhythmic cell division has been found to persist for a number of days (7 = 24.2 hours) in the autotrophically grown Z strain, batch-cultured under dim LL (Edmunds, 1966). This series of experimental results was perhaps the most definitive but was restricted by the low light intensities (800 lux) that had to be used. (The rhythm damped out in more intense LL, and, of course, could not be observed in DD.) Nevertheless, although the division bursts were relatively small (g about 5 days), those cells that did divide did so during their subjective night at the times that they would have done so had the entraining LD cycle previously imposed been continued. We have also observed “free-running’’ circadian rhythms of cell division in a variety of higher-frequency (e.g. LD: 1,3 or LD: 1/3, 1/3) light cycles (Edmunds and Funch, 1969b; Laval-Martin et af., 1979) and even “random”

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FIG. 4. Persistence of the free-running, circadian rhythm of cell division in cultures of Euglena gracilis (Z strain) grown photo-autotrophically at 25°C under two exotic L D regimens, each of which furnished a total duration of 12 hours of illumination (cool-white fluorescent, 7500 lux) and 12 hours of darkness during a 24-hour timespan, but which provided n o 24-hour time cue to the cells. The growth curve (A) a t the top was obtained in a “random” illumination regime, the duration of whose signals varied between 15 and 180minutes according to thedistribution indicated in the inset. That a t the bottom (curve B) was obtained from a culture exposed to a high-frequency LD: I , 1 cycle ( T = 2 hours). The stepsizes (factorial increases in cell titre following division bursts) are given to the left of each step, and periods for the oscillations (hours between the onsets of successive bursts) are encircled to the right. The mean value of the free-running period ( for i) the rhythm obtained in the random L D regimen was 28.5 hours as read from the growth curve, whereas that in LD: 1,l was 27.3 hours. These values corresponded, respectively, to those computed by time series analysis of 28.2 and 26.6 hours (L. Edmunds and D.L. Laval-Martin, unpublished results).

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illumination regimes (Edmunds and Funch, 1969a), although we do not exclude the possibility that such short-period light regimens (which provide no information to the cells with regard to 24 hour periodicities) may modulate the period to some extent (Adams et al., 1984). Those high frequency cycles (Fig. 4B) having symmetrical photo- and scotophases (e.g. LD: 1,l or LD: 3,3), and properly constructed random regimes (Fig. 4A), have proved particularly useful. They afford an amount of light during a 24-hour time span identical to that received in a full-photoperiod LD: 12,12 entraining reference cycle ( T = t = 24 hours) yet elicit free-running circadian rhythms ( T 24 hours). These properties could be restored, however, by the addition of certain sulphur-containing compounds (such as cysteine or methionine) to the medium at the onset of the experiment. If these substances were added at various times to an arrhythmic P4ZULculture in LL (after prior exposure to LD:14,lo), periodic division was likewise induced whose phase was that predicted on the assumption that the underlying clock had been running undisturbed (but unexpressed) throughout the experiment, merely having been uncoupled from division itself until the sulphur compounds were added. This hypothesis could be tested further by simultaneously monitoring another rhythm (such as motility or photosynthetic capacity)-presumably a manifestation of the same oscillator, but whose coupling to the clock would not be affected by these compounds (or lack thereof). c. Initiation. It is a common feature of circadian rhythms that they “damp

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out” or are not observed in continuous bright illumination (in contrast to dim LL or to DD). This also holds true for the cell division rhythm in E. gracilis: in more intense LL (e.g. 3500 or 7500 lux), photo-autotrophic cultures of the wild type (Z strain) at 25°C (Edmunds, 1965a) revert to asynchronous exponential growth (g= 1 1 to 15 hours). A single switch-down in irradiance to 800 lux was sufficient to elicit a free-running circadian rhythm that persisted for at least two or three cycles, with the first C$R being observed about 14 hours after the switch-down (Edmunds, 1966). Two hypotheses could explain these observations. First, either the endogenous oscillators in the individual cells comprising the population were absent or arrested at some specific phase point, and subsequently initiated or released by the transition (primary arrhythmicity) or second, the individual oscillators were all running in bright LL but were out-of-phase with each other (secondary arrhythmicity). The first explanation appears to be more likely (Edmunds, 1966), although this may not be the case for the initiation of rhythmicity (Jarrett and Edmunds, 1970) in arrhythmic cultures of the P4ZUL mutant growing in D D by a single switch-up in irradiance to LL (5000 lux). More recently, we have explored this problem further in cultures of E. gracilis exposed to the higher-frequency cycles discussed in Subsection 1 b (Edmunds and Laval-Martin, 1984).It is not necessary for the cultures to have been exposed to a prior entraining diurnal light cycle in order for rhythmicity to be exhibited: cells maintained in LD: 3,3 or LD: 1,l (Fig. 4, curve B), or in random illumination regimes (Fig. 4, curve A) from the moment of inoculation all were rhythmic. Furthermore, if arrhythmic cultures growing exponentially in LL (7500 lux at 25°C) with a value o fg of 1 1 to 15 hours were placed into any of these cycles, circadian periodicity was quickly induced, with z values varying from 26.6 to 30.6 hours, depending on the sub-strain and regimen used. For example, in some 15 different cultures transferred from LL to LD: 3,3, the C$R peaks of the elicited rhythms were found to occur at intervals after the onset of the first dark pulse of the imposed cycle, given by the expression 19.1 hours CT+n? (where n is an integer and all times have been normalized to 24 hours). Since 4~ is known to occur at about CT 12, with reference to an LD: 12,12 cycle, one can infer that all the oscillators comprising the population were stopped at approximately CT 16.9 in LL and were then set into motion from this phase point by the first transition (or, alternatively, that all the clocks were running in LL and were subsequently reset to CT 16.9 by the dark pulse). This value is somewhat larger than that of CT 12 found as an arrest point for many circadian systems; it may be, however, that the LD:3,3 regime itself may not be without some effect (Adams et al., 1984).

d. Phase-shijtability. Another basic property of circadian rhythms (see Fig. 2) is that their phase can be reset (or sh ifted eb y the lengthening or shortening


85

of the period of one or more oscillations-by single light (or dark) or temperature signals. Indeed, the twin notions of phase and period control are key elements of Pittendrigh’s (1965) theory for the entrainment of circadian clocks by light cycles. It is also characteristic of circadian rhythms that both the sign and magnitude of phase shifts engendered by the light signal are predictably dependent on the subjective circadian time at which the perturbations are applied. Similarly, the phenomenon of phase perturbation is inherent in most, if not all, models for cell cycle oscillators. The response of cells to external influences is often strongly dependent on the time of the cell division cycle at which the agent is imposed (Miyamoto et al., 1973; Zeuthen, 1974; Smith and Mitchison, 1976; Polanshek, 1977; Klevecz et al., 1978, 1980a,b; Edmunds and Laval-Martin, 1984). Despite these investigations, however, a detailed phase-response curve for a circadian mitotic clock has been lacking. Recently, we have managed to fill this void by utilizing photo-autotrophic cultures of E. gracilis free-running in a high-frequency LD: 3,3 cycle (see subsections Ib, Ic) and displaying, under the experimental conditions employed (Edmunds et al., 1982), a stable circadian period of 30.2 hours ( & 1.8 hours for over 156 monitored oscillations). At different times throughout the 30-hour division cycle, 3-hour light perturbations were imposed systematically on free-running cell populations by giving light during one of the intervals when dark would have fallen in the LD: 3,3 regimen. Using the onset of division as the phase reference point, the net steady-state phase advance or delay (kA4) of the rhythm was determined after transients, if any, had subsided (usually in 1 or 2 days) relative to an unperturbed control culture. Both +A 4 and - A 4 were found, with maximum values of approximately 11 to 12 hours being obtained at CT 22 to 23 (the “breakpoint”); little, if any phase shift occurred if the light signal was given between CT 6 and CT 12 (Fig. 5). The phase-shifting curve obtained by plotting new phase (4’) versus old phase (4) was of the type 0 (“strong”) variety. Light perturbations, no matter when imposed, engendered new phases which mapped to a relatively restricted portion (CT 6 to CT 13) of the circadian cycle. Several other interesting features of the phase-response curve for E. gracilis emerge. In the first place, the CT at which delay or maximum advance phase shifts were achieved corresponded, respectively, to the approximate position of the cell division cycle during which division occurred (commencing at about CT 12) and to the first few hours of the GI phase. These times, furthermore, were exactly those when the free-running rhythm of photosynthetic capacity (Laval-Martin et al., 1979; Edmunds and Laval-Martin, 1981) in LD: 3,3 displayed the lowest values; maximum values (amount of COZfixed cell-’ h-I) occurred at the very time (CT 6 to 12) that 3-hour light signals were virtually ineffective in phase shifting the cell division rhythm. This observation demonstrates that, although light is needed as a “substrate” for photosyn-

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LELAND N . EDMUNDS JR 12

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FIG. 5 . Phase-response curve for the action of single 3-hour light signals (0,7500 lux; O, 2800 lux; A, 1500 lux; 0 , 700 lux; A. 300 lux) replacing one of the usual dark intervals on the free-running rhythm of cell division in photo-autotrophic cultures of Euglena gracilis maintained in LD: 3,3 at 25°C. The steady-state phase shift (+Ad), normalized to 24 hours, is plotted as a function of the circadian time (modulo 24) at which the mid-point of the signal was given. The “break-point” inherent in this type of plot, indicated by the dashed line at CT23, would disappear if the curve were drawn in monotonic form with all the phase shifts being treated as delays. If the resultant new C T ( @ = 4 kAr$) following the perturbation were plotted as a function of the C T ( 4 )at which the signal was given, a smooth sinusoidal curve would be obtained, indicating that the phase-response curve is of the type 0 (strong) variety. (Modified from Edmunds ef al., 1982; J.R. Malinowski, D.L. Laval-Martin and L.N. Edmunds, Jr., unpublished work).

thesis and for the progression of the cell division cycle in photo-autotrophically cultured E. gracilis, it serves a quite distinct and separable function in phase shifting and entraining the circadian oscillator(s) hypothesized to underlie the rhythm of cell division, whose rhythmic sensitivity to light is


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reflected in the phase-response curve. Lastly, the phase shifts observed appear to constitute true developmental advances and delays: in the case of the former, a higher cell titre was attained before the same cell concentration was reached in the unperturbed control, whereas the converse held for phase delays. e. Singularity Point. The oscillatory motion of a biological clock can be represented on a plane as a stable trajectory (comprising two first-order differential equations involving two state variables) that closes in on itself, termed a limit cycle (Pavlidis, 1968). Such a system, if disturbed, will always tend to return to an equilibrium configuration. Theoretical studies have further predicted that a circadian oscillator might be rendered arrhythmiccharacterized by a phaseless, motionless state-by a critical pulse of a certain strength and duration given at a specific time (termed the “singularity point”) in the circadian cycle (Winfree, 1970; for an extensive treatment see Winfree, 1980).As Winfree (1980) has pointed out, as the stimulus strength is increased, the transition from Type 1 (weak pulse) to Type 0 (strong pulse) resetting is necessarily discontinuous at one special phase point, the “breakpoint”, corresponding to this unique singularity. This prediction has now been demonstrated for light perturbations in several multicellular circadian systems, and most recently (Taylor et al., 1982a,b) for critical pulses of anisomycin in the dinoflagellate G. polyedra (see Section III.B, p. 107). We have examined the threshold intensity of illumination for phase-shifting the cell division rhythm in E. gracilis by 3-hour light signals (Malinowski et al., 1984). Perturbations having an intensity within the range of 700 to 7500 lux generated the same phase shift when imposed at a given CT. Light signals of lower intensity, however, elicited different responses, some quite dramatic. Thus, a 40 to 400 lux pulse given at CT 0.4(the approximate location of the breakpoint, at about CT 23) induced arrhythmicity, the population reverting to asynchronous exponential growth. The intensity of this annihilating pulse, and the CT at which it was imposed, were found to be quite specific: a 300 lux stimulus given at CT 21.5 merely generated a phase delay of the same magnitude found for 7500 lux signals (Fig. 5 ) . Different degrees of asynchrony were observed as one approached the boundaries (lux, CT) of the critical pulse. The existence of this “critical pulse”, and its corresponding singularity point, not only further supports the hypothesis that a circadian oscillator regulates the cell division cycle in E. gracilis but also suggests (though it does not demand) that the pacemaker may have limit cycle dynamics. Inasmuch as the results were obtained with populations of cells, we cannot deduce the state of the oscillator(s) in individual cells. On the one hand, arrhythmicity in the culture might be the result of a dispersion of the phases of individual cell division cycles (cellular incoherence); on the other hand, the critical pulse may

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have stopped each clock comprising the population (but not the cell division cycles, which continue to run). To distinguish between these two alternatives, a second light perturbation could be given at different times after the first critical pulse; if a unimodal distribution of phases were found, as it was for the Drosophila eclosion system (Winfree, 1970), reinitiation rather than resynchronization would be favoured.

f. Temperature Compensation. A final, and rather remarkable, property of circadian rhythms (Fig. 2) is that their period-but not their amplitude-is only slightly affected by the ambient steady-state temperature over the physiological range (Sweeney and Hastings, 1960). In fact, this isjust what one would anticipate in a functional biological clock measuring astronomical time. In contrast, the duration of the cell division cycle (i.e. g) is commonly thought to be highly dependent on temperature, and, indeed, this is true for E. gracilis also (Terry and Edmunds, 1970a). A recent study (Anderson et al., 1984) of the effects of different constant temperatures, ranging between 16 and 32”C, on the generation time of the wild-type Z strain, and of a 3-(3,4-dichloropheny1)- 1,l -dimethyl urea (DCMU)-resistant line (strain ZR) photo-autotrophically batch-cultured in LD: 3,3, clearly illustrates this dependence (Fig. 6a). Despite this apparent paradox, several lines of evidence suggest that the period of the circadian oscillator hypothesized to underlie rhythmic cell division and to produce the “gating” effect observed in a population of cells is conserved. For example, Klevecz and King (1982) found that the QIOfor cell division of the V79 line of Chinese hamster lung fibroblasts, growing between 34 and 40”C, was between 1.15 and 1.26, thus indicating that the mammalian cell division cycle is temperature-compensated over a span of 6 to 7°C. We have observed previously (Edmunds and Adams, 1981) a similar compensation of the period of the free-running rhythm of cell division in cultures of the P4ZUL photosynthetic mutant of E. gracilis grown in D D over a temperature range of about 7°C (14-21°C). In a more extensive and rigorous comparative study (Anderson et al., 1984) of temperature compensation of the free-running period in the Z and ZR strains of E. gracilis maintained in LD: 3,3 at different steady-state temperatures within the physiological range, we found (Fig. 6b) a Qlo of 1.05 in the former, indicating that it is virtually unaffected by changes in temperature over a 10°C range (22-32°C). The circadian clock was not as well compensated in strain ZR, in which an average value for the QIOof 1.23 was observed over a temperature range of 18-28°C; the spread in the distribution of its period lengths was also greater than for strain Z. Nevertheless, even the circadian oscillator of strain ZR was only modestly affected by temperature compared with typical biochemical rates and other biological processes such as membrane transport. Finally, a critical “permissive” temperature of 22°C

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FIG. 6. (a) Influence of temperature on the average generation time (2) of two different strains [Z, wild type (0);ZR, DCMU-resistant ( O ) ] of Euglena gracilis maintained in LD: 3,3 (7500 lux). Growth curves obtained at each temperature during the exponential growth phase where light intensity had not become limiting (usually between 10,000 and 60,000 cells ml-I) were fitted with straight lines by linear regression (least squares) to obtain their slopes. (b) Temperature-compensation of the period (3 of the free-running circadian rhythm of cell division in two strains [Z, wild type ( 0 ) ;ZR, DCMU-resistant ( O ) ] of E. gracilis cultured in LD: 3,3 (7500 lux). ) the Periods were measured as the time between successive onsets of division ( 4 ~until stationary phase of growth was reached (usually at a cell titre approximating to 100,000 cells ml-I). Error bars denote f 1 standard deviation of the mean. Note that in strain ZR there was some lengthening of the period at lower temperatures. Although growth of the Z strain could be obtained at temperatures less than 22°C (18°C for ZR), rhythmicity disappeared (R.W. Anderson, D.L. Laval-Martin and L.N. Edmunds, unpublished work).

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was found for the Z strain (18°C for strain ZR) and, although slow exponential growth occurred at lower temperatures, synchrony was not seen, This short review has attempted to present a convincing case for the key role of a circadian oscillator(s) in the control of the cell cycle in the algal flagellate E. gracilis, taken to be representative of other eukaryotic micro-organisms (and, perhaps, multicellular systems). The formal properties of circadian clocks--entrainability, persistence, phase shiftability and temperature compensation-were also found to characterize the circadian rhythm of cell division. The recent discovery of a singularity point at which the imposition of a critical light pulse generates an arrhythmic population further suggests that the underlying oscillator may be of the limit cycle type, although it does not demand it. According to our working hypothesis (Edmunds and Adams, 1981; Edmunds and Laval-Martin, 1984), mitosis would not be an essential part of the oscillator but would lie downstream from it: blockage of cell division should not stop the system from oscillating, at least at a subthreshold level. In this sense, then, cell division would be a “hand” of the underlying clock. We have recently tested this hypothesis in two ways (Edmunds and Laval-Martin, 1984). First, vitamin B12deprivation has been shown to completely block the cell division cycle in E. gracilis (BrC et al., 1981). If the division rhythm (free-running in LD: 3,3) was stopped due to low initial concentrations of vitamin B I ~and , if this inhibition subsequently was released by re-addition of vitamin B12 to the medium, the cell division rhythm started up again in phase with an unperturbed control, thereby suggesting that the putative underlying oscillator had continued to run unabatedly throughout the time span during which the cell division cycle had stopped cycling. Second, if a pulse of lactate was given to a free-running culture, thereby temporarily accelerating the cell division cycle (values ofg approached 10 to 12 hours) and overridingcircadian oscillator controls (Jarrett and Edmunds, 1970), the phase of the rhythm when it was finally restored, after the supplemental substrate had been depleted, was in phase with that of an unperturbed control, leading to a similar interpretation. These latter results are consistent with those found previously for the in-phase restoration of rhythmicity in the P4ZUL mutant free-running in LL by the addition of sulphur-containing compounds to the medium (Edmunds et al., 1976; see subsection lb). These problems will be discussed further in Section V.A (p. 127). Another basic question arises from the phase-shifting data reflected in the experimentally derived phase-response curve (see Fig. 5 ) for light signals in E. gracilis. How does a master (circadian) clock generate at the biochemical or molecular level the observed shortenings and lengthenings of individual cell division cycles? The evidence reviewed in earlier sections formally demands that a clock of some sort predictably insert time segments into, or delete them from, the cell division cycle. One way that the cell division cycle might be


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“programmed” would be for a collection of timing loops of different lengths to couple together in various combinations to form a flexible timer, or “cytochron” (Edmunds and Adams, 1981). Temporal loci, or control points, would exist along the cytochron track at which decisions would be made with respect to the addition or deletion of time loops. Based on the view that this time “dilation” or “contraction” has an immediate molecular basis, we have hypothesized (see Section V.B, p. 133) the existence of “chronogenes” (our formal time segments) whose transcription would meter time, and which would be inserted (or deleted) in various numbers into the programmable cytochron by interaction with a circadian oscillator. Thus, the cytochron and the circadian clock are posited to be functionally independent (although not necessarily entirely separate as to mechanism). 2. Circadian Rhythms of Cell Motility Pohl (1948) first demonstrated a daily rhythm of phototactic response in non-dividing (stationary- or infradian-phase) populations of autotrophically grown E. gracilis Klebs. The rhythm showed a maximal response during the day and a minimal response at night. Since then, this and associated motility rhythms in Euglena sp. have been intensively studied. a. Rhythm of Phototaxis. Bruce and Pittendrigh (1956,1958) introduced a high degree of automation into the assay system for this rhythm. This allowed simultaneous recording of the kinetics and degree of aggregation of cells into a narrow, vertical test light beam from a heat-compensated microscope lamp in a number of cultures maintained in transparent Falcon flasks otherwise kept in LD, D D (or other regimen). The pattern thus obtained was termed the phototactic response, which was actually a composite of both general motile and light-oriented behaviours. The response to the test beams during photo-autotrophic growth in synchronously dividing cultures in LD: 14,lO was minimal during the dark phase; the response began to decrease well before the actual L/D transition, as if the cells anticipated its coming (see Edmunds and Halberg, 1981). Inasmuch as the cells all divide (and replicate their flagellae) during periods of darkness (Edmunds, 1965a),one could hypothesize that the decreased response was due to the inability of the cells to swim toward the test beam. This explanation was effectively ruled out by the observations of Bruce and Pittendrigh (1956,1958) and Feldman (1967), which indicated that (a) the rhythm continued to be entrained by LD: 12,12 even in the stationary phase, where little, if any, net increase in cell number occurred and (b) the rhythm subsequently free-ran with a circadian periodicity (ranging from 23.6 to 24.3 hours) for as long as 14 days in D D (except for the intermittent test light, each 2 hours). The free-running period was found to be seemingly independent of temperature,

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or nearly so: over a 15°C range (16.7 to 33.0°C), 7 varied only between 26.2 and 23.2 hours, yielding a Qlo value of 1.01 to 1.10 (Bruce and Pittendrigh, 1956). This lack of dependence of a unicellular clock on temperature was considered to be achieved by virtue of a compensating mechanism within the organism, although component parts of the oscillator might well be temperature-dependent, as suggested by the observation (Bruce, 1960) that a diurnal sinusoidal temperature cycle (18-3 1“C) can modify the phase relation between the rhythm and a simultaneously imposed LD: 12,12 cycle in a manner dependent on the phase angle difference between the two Zeitgeber. Bruce (1960) further investigated the entrainment of the phototaxis rhythm by 24-hour LD cycles having various photofractions (i.e. ratio of light duration to entire cycle length). Entrainment to a precise 24-hour period occurred for photofractions between 1/12 and 516, and the phase of the rhythm depended chiefly on the timing of the L/D transition; for photofractions greater than 5/6 (i.e. cycles having more than 20 hours of light), the rhythm damped out. The system also appears to have relatively wide limits of entrainment, entraining to LD: 3,3, LD: 8,8 and even LD: 24,24 (where T= 6, 16 and 48 hours, respectively). Bruce (1960) noted, however, that what might appear to be entrainment to a short period (for example, 6 hours), might, in fact, be frequency demultiplication by separate subpopulations of cells to 24-hour periods but with differing phase relationships. (Indeed, the rhythm exhibited frequency demultiplication to a 24-hour period in LD: 2,lO and LD: 12,36.) This did not seem likely, however, in view of the fact that the rhythm free-ran in D D with a circadian period following direct entrainment by the high-frequency cycle. Bruce and Pittendrigh (1956, 1957) and Feldman (1967) have demonstrated that the free-running phototaxis rhythm in D D could be reset by light perturbations ranging from 2 to 12 hour duration; as has been found for the majority of circadian rhythms, the sign and magnitude of the steady-state phase shift (A4) thus engendered depended on the phase of the internal oscillation at which the pulse was given. In a phase-response curve obtained for the action of 4-hour light pulses (Feldman, 1967), the maximum delay phase shift was obtained at about CT 16 during the subjective night. Early attempts to affect the period of the phototaxis rhythm yielded negative results: the respiratory inhibitor potassium cyanide, the mitotic inhibitor phenylurethane, the adenine growth factor analogue 2,6-diaminopurine sulphate, the pyrimidine and nucleic acid analogue 2-amino-4-methylpyrimidine, the nucleic acid bases, and the growth factors gibberelic acid and kinetin all were without consistent effect on period and phase (Bruce and Pittendrigh, 1960). Deuterium oxide (2H20), or “heavy water,” which has been shown to reversibly lengthen z in a variety of organisms (Enright, 1971), similarly alters both the period and phase of the phototaxis rhythm in E. gracilis. Thus, a culture adapted to 95% 2H20 increased its period to about 27


93

hours, but returned to a Z of about 24 hours when it was re-adapted to H20 again (Bruce and Pittendrigh, 1960).The problem with such studies, ofcourse, is the fact that the action of 2H20 is quite general and non-specific, and the period-lengthening effects are equally compatible with kinetic, transcriptional and membrane models for circadian oscillators (see Section V.B, p. 129). Indeed, Kreuels and Brinkmann (1979) have found in a comparative study of the effects of 2H20 on the cell-bound circadian oscillator underlying the motility rhythm in E. gracilis (see next subsection), on the cell-free glycolytic oscillator of yeast, and on the Belousov-Zhabontinsky chemical reaction having a known network structure, that although the circadian and glycolytic oscillations were slowed down to an extent depending on the 2H20 concentration, the period of the chemical reaction was either lengthened or shortened, respectively, at high or low catalyst concentrations. They concluded, therefore, that the generalized period-lengthening effect of 2H20 can be explained only by a more complex network approach. More specific (but still not well understood) perturbations have been obtained by altering the nutritional conditions. Feldman and Bruce (1972) reported that the addition of acetate (100 mM) to autotrophic cultures of E. gracilis lengthened the phototaxis rhythm to 27 hours and that 10 mM pulses of acetate administered at different phases of the free-running rhythm induced phase shifts. Pulses of other carbon sources (succinate, lactate, pyruvate), however, caused a temporary cessation of the rhythm and then a resumption with variable phase shifts. They argue that the changes are probably caused by a general metabolic switch (i.e. from autotrophy to mixotrophy) rather than by any specific effect. Finally, and perhaps most specifically, the period of the phototaxis rhythm has been shown to increase (reversibly) by the addition of cycloheximide, an inhibitor of protein synthesis on 80s ribosomes of the cytosol (Feldman, 1967). The effects of this drug appeared to be on the clock itself rather than on some variable controlled by the clock, as confirmed by assaying the position of the light-sensitive oscillation by 4-hour resetting light signals after the addition of cycloheximide in DD (as predicted from the pulse-response curve for light pulses already derived for this unicell). Yet even these results, implicating protein synthesis in circadian clock function, must be interpreted cautiously: cycloheximide may not only have other primary effects besides the inhibition of protein synthesis, but may also produce secondary effects that would modify only indirectly the circadian oscillator (Sargent, 1976).

b. Dark Motility Rhythm (Dunkelbeweglichkeit) . In stationary-phase cultures of E. gracilis (strain 12245/9), a diffusion gradient in the vertical distribution of cells, as indicated by differences in their sedimentation equilibrium, has been observed, which is apparently a function of random cell motility that itself fluctuates with a circadian periodicity in DD for as long as 3 months

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(Brinkmann, 1966, 1971; Schnabel, 1968). This “dark motility” rhythm (Dunkelbeweglichkeit) damps out in LL but may be re-initiated by a single LL/DD transition. The assaying test-light cycle (20 minutes of light every 2 hours) did not entrain the circadian rhythm either directly or by frequency demultiplication (Schnabel, 1968). In contrast to the composite rhythm of the phototaxis response previously described, the random motility rhythm is independent of phototaxis and photokinesis and consequently allows one more easily to distinguish between the influence of light on the circadian oscillator and on the response itself. Schnabel(l968) examined the effects of a wide variety of LD cycles on the motility rhythm in both autotrophic and mixotrophic cultures. Regimes having periods ranging from 16 to 48 hours (L: D ratios of 1 : 1) entrained the rhythm, which subsequently free-ran with a circadian period on release into DD. Under shorter LD cycles, she found that a circadian component was exhibited, as if the rhythm was free-running, ignoring the imposed regimen. A colourless, obligatorily heterotrophic mutant (strain 1224-5/25) also displayed a light-entrainable, circadian rhythm of random motility in DD, just as did the green cultures (Kirschstein, 1969). Finally, Schnabel (1968) determined the phase-response curve for this rhythm using 6-hour light pulses (1000 lux) during the D D free-runs; the phase-response curves of both green and colorless strains were almost identical. The range of entrainment of the motility rhythm by sinusoidal temperature cycles, having driving periods from 4.8 to 55.7 hours, has been explored as well (T. Kreuels and K. Brinkmann, personal communication; see Edmunds, 1982). All cycles used synchronized the overt rhythm, which then reverted to its free-running circadian period on removal of the temperature regime only when the period of the latter had been in the range of approximately 19 to 36 hours. Although passive enforcement occurred with the other temperature cycles (having T=4.8-19 hours, or 36-55.7 hours), the rhythm damped out in D D and constant temperature. Finally, the phase angle difference between the temperature cycle and the synchronized rhythm changed within the range of entrainment (19-36 hours), but was relatively constant in cycles having T > 36 hours. The nutritional mode also plays a role in the effects of constant temperatures on the rhythm of random motility. Brinkmann (1966,1971) has reported that under autotrophic conditions (minimal medium), 7 in D D was more or less independent (23.5k0.3 hours) of temperature in the range between 15 and 35”C, whereas a sudden increase of 5°C or more caused a transitory increase. In contrast, 7 increased with increasing constant temperature in mixotrophic cultures (complex nutrient medium), whereas the phase was not affected by a sudden step-up in temperature. A phase-response curve for single 10°C step-ups, given at different CT times to a free-running autotrophic culture, was also derived (Brinkmann, 1966, 1971) and was


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similar to that for 6-hour light pulses (Schnabel, 1968). The type of response could be switched from one to the other by the addition of either lactate or ethanol to the medium. Brinkmann (1966, 1971) has attributed the different behaviour of the two types of culture, in response to different steady-state temperatures to the participation in the autotrophic strains of two types of reaction-those with a Q l o of 1.0 (photochemical) and those with a Qlo of 2.0 (dark reactionsFwhereas, in mixotrophic cells, the dark reactions would predominate. It now appears (K. Brinkmann, personal communication; see Edmunds, 1982) that the degree of dependence of energy conservation on temperature in turn determines the type of response of the circadian rhythm to different steady-state temperatures. Finally, several other circadian rhythms that have some relevance to the rhythm of random motility have been investigated recently. Brinkmann and coworkers have measured the energy charge and the concentration of glucose 6-phosphate under various sinusoidal temperature cycles within and beyond the limits of entrainment for the motility rhythm. In contrast to glucose 6-phosphate, energy charge never became synchronized, and neither showed rhythmicity in autotrophic cultures during a free-run at a constant 27.5"C, indicating that these variables cannot be responsible for generating the persisting mobility rhythm. Similarly, although the redox state of nicotinamide adenine dinucleotide phosphate (NADP), but not nicotinamide adenine dinucleotide (NAD), was synchronized by a temperature cycle, there was no significant oscillation in the redox state of pyrimidine nucleotides during a free-run. The temperature-enforced NADPH cycle showed a peak coinciding with low glucose 6-phosphate, indicating flux regulation of the pentose-phosphate cycle in response to temperature (K. Brinkmann, personal communication; see Edmunds, 1982). Recently, T. Kreuels and K. Brinkmann (personal communication; Edmunds, 1982)found that the period of the motility rhythm can be lengthened from 23.8 hours to about 25.3 hours by the addition of urea to the culture; indeed, saturation was not obtained (above concentrations of 200 mM, no oscillations were observed, although E. gracilis still could grow). Urea was shown to penetrate the cells but not to be metabolized, suggesting that the period-lengthening effect perhaps was due to structural changes (possibly affecting hydrogen bonds of macromolecules or water structures, or by weakening hydrophobic interactions). Finally, a low-amplitude circadian oscillation of external pH value in weakly buffered cell suspensions has been reported; preliminary evidence suggests that this rhythm is due to the activity of a plasmalemma proton pump and is not generated by the inverse-phase circadian rhythm of photosynthesis (K. Brinkmann, personal communication; see Edmunds, 1982).A similar conclusion was reached by Hoffmans and Brinkmann (1979) for Chlamydomonas reinhardii, whose photosynthetic activity was inhibited by 3-(3,4-dichlorophenyl)-1,l-dimethylurea (DCMU) yet still showed a pH rhythm.

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c. Rhythm of Cell Settling. In addition to the light-oriented rhythm of phototaxis and the dark random-motility rhythm, a circadian rhythm of cell 'settling" has been discovered in E. gracilis, which may occur concurrently with, or in the absence of, cell division (Terry and Edmunds, 1970a,b). This periodicity was reflected during the growth phase by an apparent change in cell concentration in autotrophically batch-cultured populations synchronized in LL by 24-hour temperature cycles (18"C/25"C or 28"C/35"C), although the synchronous division bursts tended to mask the response: small dips were seen in the plateau portion of the growth curves. The settling rhythm maintained the same phase in relation to either entraining temperature cycle (i.e. maxima occurred during the warmer interval) regardless of whether this was the time of maximum cell division (which had a 180" phase difference between the two temperature regimes). In temperature-cycled stationary-phase cultures, the maxima of the settling rhythm also occurred during the warmer phase of the imposed temperature cycle. The rhythm persisted for as long as 9 days in infradian cultures released from the temperature cycle and held at a constant 25°C in LL (Terry and Edmunds, 1970b). Neither experimental artifacts nor cell death (necessarily rhythmic) were responsible for this rhythm: rather, the cells actually tended to settle out of the liquid phase, adhere to the vessel walls, and then subsequently detach themselves and re-enter the medium. This was substantiated by automatically monitoring cell number at two different levels in the magnetically-stirred homogeneous cultures with a dual-sampling system. Attachment could be prevented, and the rhythmic dips in cell number abolished, only by vigorous agitation of a rotary shaker (Terry and Edmunds, 1970b).

3. Rhythms in Photosynthetic Capacity Several studies have reported a rhythm in photosynthetic capacity during the cell division cycle of E. gracilis, although the results were often conflicting (Lovlie and Farfaglio, 1965; Cook, 1966; Codd and Merrett, 1971). Walther and Edmunds (1973) demonstrated a clear diurnal rhythm in the capacity of this unicell to fix C02 (as measured by its ability to incorporate NaHI4C03at saturating light intensities in samples taken from the master culture at different times) in dividing autotrophic populations synchronized by an LD: 10,14 (L= 12,000 lux) at 25°C. Photosynthetic capacity reached a peak value an hour or so before the onset of darkness, at which time cell division ensued. A similar pattern in oxygen evolution was also observed. This daily rhythm also occurred in non-dividing cultures, and could thus be divorced from the cell division cycle. At the time, the rhythm was only weakly persistent under continuous dim LL (750 lux), under which conditions cell division was completely suppressed, the rate of C02 fixation was greatly lowered (making the assay of the rhythm difficult for longer time spans), and, by implication,


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the overall physiology of the cell was disturbed. (Higher intensities of LL caused the rhythm to “damp out”, and D D could hardly be employed in an autotrophic system.) More recently, these problems have been overcome by monitoring the rhythm of photosynthetic capacity and of chlorophyll content in three sub-strains of E. gracilis, synchronized by an LD: 12,12 cycle (L = 7000 lux) during both the exponential and stationary phases of growth, and then released into a LD: 20 minute, 20 minute (L = 7000 lux) regimen (Laval-Martin et a f . ,1979). This particular high-frequency 40-minute cycle was selected to afford an amount of lighting during a 24-hour timespan identical to that received in the full photoperiod, LD: 12,12 cycle. The cell division rhythm is known to persist with a circadian period under these conditions (Edmunds and Funch, 1969a,b; see subsection A.l, p. 81). For all intents and purposes, the high-frequency cycle is perceived by the cells as continuous illumination, at least with regards to rhythmic output. Under these conditions, a non-damped, high-amplitude, persisting rhythm of photosynthetic capacity was observed for some 5-6 days after the entraining 24-hour LD cycle had been discontinued, whose t was estimated to be about 27.5 hours, corresponding closely to that of the cell division rhythm. Similar observations (Edmunds, 1980b; Edmunds and Laval-Martin, 1981) were made in an LD: 3,3 cycle imposed from the moment of inoculation of the culture, which elicited a circadian rhythm ofcell division whose ?, for some 156monitored oscillations, ranged between 27 and 34 hours and averaged 30.2 1.8 hours (Edmunds et al., 1982). Reproducible, high-amplitude, circadian rhythms were also observed in total chlorophyll in both dividing and non-dividing cultures maintained in these high-frequency LD cycles, although there did not appear to be a close correspondence between the chlorophyll rhythm and that of photosynthetic capacity (Edmunds and Laval-Martin, 1981). Indeed, the chlorophyll rhythm sometimes appeared as a bimodal circadian rhythm, or even as an ultradian one having a period of about 13 hours. Likewise, the amplitude of the chlorophyll rhythm was usually considerably less than that for the rhythm in photosynthetic capacity. The fact that the periods, and therefore the phase relations, of the chlorophyll and photosynthetic capacity rhythms varied, not only appears to rule out any simple causality between the two, but also suggests the possibility of desynchronization, or dysphasia, among different rhythms driven by the same clock. It may even suggest the existence of a multicellular oscillator system in a unicellular organism (Section V.D, p. 135) with, perhaps, cell division’s acting as an entraining signal for a hierarchical clockshop in dividing cultures. Lonergan and Sargent (1 978) have also reported a circadian rhythm of 0 2 evolution in E. gracifis (strain Z ) , which persisted for at least 5 days in dim LL and constant temperature but damped out in bright LL. The rhythm could be

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phase shifted by light pulses, and the free-running period length was unchanged over a 10°C span of growth temperature. Although the oxygen rhythm was found in both exponentially dividing cultures and stationaryphase cultures, C02 uptake was clearly rhythmic only in the latter. This may have been due to the sensitivity of the infrared C02 analyser used for the assay. There are at least three non-trivial ways by which the rhythm in photosynthetic capacity could be generated (Edmunds, 1980b, 1982): (a) rhythmicity in total chlorophyll content; (b) rhythmicity in the activity or amount of enzymes regulating electron transport between the photosystems, or mediating the dark reactions, or both; or (c) rhythmicity in the coupling between photochemical events within the two photosynthetic systems and electron flow between them. Although there is an endogenous circadian rhythm in chlorophyll in non-dividing cultures of E. gracilis, perhaps reflecting changes in the functional role of the pigment, it is not sufficient to explain the rhythm in photosynthetic capacity because of the lack of a quantitative relationship between the amplitudes of the two rhythms and the lack of correspondence in their periods and phases (Edmunds and Laval-Martin, 1981). With regard to the second hypothesis (enzymic variations), although a number of photosynthetic dark reactions comprising the Calvin scheme have been examined in E. gracilis, and in several other algal systems (Edmunds, 1980b), both during the cell division cycle and in non-dividing cultures, no enzyme has achieved a consensus as a clear candidate responsible for generating the rhythm in photosynthetic capacity. For example, ribulose 1,5-bisphosphate carboxylase, although sometimes showing fluctuations in activity, does not show sufficient correspondence to satisfy the rates of C02 fixation at all stages investigated (Codd and Merrett, 1971); Walther and Edmunds, 1973). Similarly, although an earlier observation (Walther and Edmunds, 1973) of changes in glyceraldehyde 3-phosphate dehydrogenase suggested a possible mechanism for generating the rhythm in photosynthetic capacity, this does not appear to be the case (Lonergan and Sargent, 1978). Finally, although carbonic anhydrase was found to be rhythmic in photoentrained cultures of E. gracilis in LD: 10,14, with peak activity occurring at the time of the highest rate of 0 2 evolution, the rhythm in enzyme activity disappeared under constant conditions whereas the rhythm in photosynthetic capacity persisted (Lonergan and Sargent, 1978). Lastly, concerning the third hypothesis, although the individual activities of photosystem I (PS I) and photosystem I1 (PS 11)in E. gracilis do not appear to change significantly with time of day (Walther and Edmunds, 1973; Lonergan and Sargent, 1979), the rate of light-induced electron flow through the entire electron chain (water to methyl viologen) was rhythmic both in whole cells and in isolated chloroplasts. The highest rate of flow coincided with the highest rate of 0 2 evolution (Lonergan and Sargent, 1979). Evidence


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consistent with the notion that the co-ordination ofthe two photosystems may be the site of circadian control of photosynthetic capacity rhythms in E. gracilis was obtained from studies of low-temperature fluorescence emission from photosystems I and I1 following preillumination with light wavelengths of, respectively, 710 or 650 nm, whereas there was no indication that changes in total chlorophyll, the ratio of chlorophyll a to b, or the size of the photosynthetic units were responsible (Lonergan and Sargent, 1979). Still further evidence supporting this type of mechanism comes from work with the dinoflagellate Gonyaulax polyedra (Prezelin and Sweeney, 1977; Sweeney et al., 1979) and will be discussed in Section 1II.B (p. 107). 4 . Oscillatory Enzymic Activities

In addition to the numerous gross cellular constituents that have been temporally mapped across the cell division cycle in E. gracilis (Cook, 1961a,b; Edmunds, 1965b) and other biochemical rhythmicities, such as the incorporation of amino acids (Feldman, 1968), the activities of a number of enzymes have been monitored in both synchronously dividing populations and in cultures in the stationary-phase of growth (see Table 2 and Fig. 3). Certain of these enzymes might contribute to some of the overt physiological rhythms concomitantly observed and, indeed, circadian enzymic oscillations can themselves quite validly be considered as indices of an underlying biological pacemaker(s). Although, for technical reasons, not all of the enzymes listed in Table 2 have been assayed rigorously in both dividing and stationary-phase populations under both entraining and free-running regimes, it would seem likely that most would continue to oscillate under LL (or DD) and constant temperature during the exponential growth phase, if one can draw a parallel from the persistence of the circadian rhythm of cell division itself. To attack this problem, and to divorce autogenous enzyme oscillations from those directly generated by the driving force of the cell division cycle (whereby replication of successive genes would lead to an ordered, temporal expression of enzyme activities), light-synchronized photo-organotrophically batch-cultured E. gracilis that had reached the stationary phase of growth have been utilized successfully (Edmunds, 1975, 1982). Relatively large-amplitude oscillations were found in the activities of alanine, lactic and glucose 6-phosphate dehydrogenases, and in L-serine and L-threonine deaminases, with maxima usually occurring during the light intervals (F.M. Sulzman and L.N. Edmunds, unpublished work; see Edmunds, 1982).These rhythmic changes in enzyme activity, therefore, were effectively uncoupled from the cell division cycle. Even more interesting, however, was the finding (Sulzman and Edmunds, 1972, 1973; Edmunds et al., 1974) that the activity of alanine dehydrogenase

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continued to oscillate in these non-dividing (infradian) cultures for at least 14 days in D D (but not in LL), and thus constituted an overt circadian rhythm in itself. The possibility that these oscillations in alanine dehydrogenase activity could be generated by fluctuations in pools of substrates or products, which could change the stability of the enzyme during its extraction and trivially produce the rhythm, was ruled out (Sulzman and Edmunds, 1973). Results from mixing experiments likewise did not suggest the presence of fluctuating pools of effector molecules that could generate the rhythm by altering the activity of the enzyme, nor were there differences in pH optimum, K,,, value, or electrophoretic mobility on polyacrylamide gel of enzyme extracted at different phases of the oscillation. On the other hand, activity determinations of alanine dehydrogenase, extracted from the maximum and minimum points of the free-running rhythm, and partially purified by ammonium sulphate fractionation and polyacrylamide-gel electrophoresis, suggested that periodic de nouo synthesis and degradation may generate the observed variations in its activity (Sulzman and Edmunds, 1973). B.

Gonyaulax spp.

Along with Tetrahymena pyriformis and Euglena gracilis, the marine dinoflagellate Gonyaulax polyedra is one of the most intensively investigated unicellular circadian systems (Wille, 1979; Mergenhagen, 1980; Sweeney, 1981). Of the many documented periodicities (Table 4), two categories of rhythm-bioluminescence and photosynthesis-will be discussed in this section, with particular emphasis being placed on recent advances in these areas.

I . Rhythms in Bioluminescence a. Physiological Characteristics. There are at least two types of rhythmic bioluminescence in Gonyaulax polyedra, the induced flashing rhythm and the spontaneous glow rhythm (Hastings and Sweeney, 1958; Sweeney and Hastings, 1957; see Table 4). The assay in the former case is typically performed by removing an aliquot of culture, mechanically stimulating it by bubbling it with air (or by addition of acid), and measuring the light output (maximum emission at about 475 nm) with a photomultiplier tube. Bioluminescence capacity was greatest in the middle of the dark period in entraining LD cycles, and persisted for many days (7 = 24.4 hours) in dim LL (1200 lux). In more intense LL (3800 lux), Z was 22.8 hours, and at 6800 lux, 7 was decreased to 22.0 hours, and the rhythm gradually disappeared after 4 days. Similarly, in DD, the amplitude of the free-running rhythm (?= 23.G24.4 hours) progressively decreased over several days until it also disappeared (Sweeney and Hastings, 1957; Hastings and Sweeney, 1958).

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TABLE 4. Some persisting circadian rhythms exhibited by Gonyaulax polyedm Circadian rhythm

Reference ~

Bioluminescence, glow

Bioluminescence. stimulated

Cell division K + (intracellular) Luciferase activity Luciferin activity Luciferin-binding protein Photosynthetic capacity

Ultrastructure, chloroplast Ultrastructure, thecal membranes

Sweeney and Hastings (1957, 1958); Hastings and Sweeney (1959); Sweeney et a/. (1959); Hastings (1960); Hastings and Bode (1962); Karakashian and Hastings (1962, 1963); McDaniel et a/. (1974); Taylor and Hastings (1979, 1982); Taylor et a/. (1979, 1982a,b); Dunlap et a / . (1980); Hastings and Krasnow (1981); Njus et a/. (1981); Sulzman et a/. (1982) Hastings and Sweeney (1958, 1959, 1960); Sweeney et al. (1959); Sweeney (1969b, 1974a, 1976b, 1979, 1981); Christianson and Sweeney (1972); Sweeney and Herz (1977); Walz and Sweeney (1979); Hastings and Krasnow (1981) Sweeney and Hastings (1958); Hastings and Sweeney (1964) Sweeney (1 974) Hastings and Bode (1962); McMurry and Hastings (1972b); Dunlap and Hastings (1981); Dunlap et a/. (1981) Bode et a[. (1963); Dunlap et al. (1981) Sulzman et a[. (1978) Hastings et a/. (1961); Sweeney (1960, 1965, 1969b); Prezelin and Sweeney (1977); Prezelin et al. (1977); Govindjee et al. (1979); Sweeney et a/. (1979) Herman and Sweeney (1975) Sweeney (1976a)

The other rhythm of steady, dim, spontaneous glow (Sweeney and Hastings, 1957,1958; Hastings and Sweeney, 1959)reached maximal intensity just at the end of the dark period in LD and, like the flashing rhythm, persisted in dim LL or in DD. Here, the glow rhythm was cleverly monitored by placing small samples of culture in each of the many vials on the turntable of a liquid scintillation counter maintained in a programmed overhead LL or LD regime (to provide energy for photosynthesis). At periodic intervals, the vials were then lowered by a platform elevator into a dark chamber for detecting light emission by the photomultiplier photometer. Not only did this system provide a graphic record of the glow rhythm, but also it made feasible the systematic (and statistical) treatment of the rhythm with a wide variety of chemicals (Hastings, 1960; Hastings and Bode, 1962). More recently, the assay system has reached new heights of sophistication, allowing the simultaneous monitoring of both rhythms (by either a phototube or a fibre-optic light pipe),

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with the mechanical functions and data acquisition both being under software control provided by an Apple I1 microcomputer (Taylor et al., l979,1982a,b). The rhythm of stimulated bioluminescence (luminescence capacity) was found to have wide limits of entrainment: LD cycles ranging from LD: 6,6 to LD:16,16 (i.e. T = 12 to 32 hours) all synchronized the rhythm, which then free-ran with a circadian period upon subsequent release into LL (2000 lux) (Hastings and Sweeney, 1958,1959). The effects of single, 3-hour light (14,000 lux) perturbations, given at different times during a free-run, yielded a standard pulse-response curve. Signals in the late subjective day induced phase delays of as long as 5 hours or more, while perturbations given in the latter part of the subjective night (after peak luminescence response) generated phase advances up to 8-10 hours in magnitude (Hastings and Sweeney, 1958). Maxima in effectivenessfor phase-shifting were found at 475 and 650 nm (Sweeney et al., 1959; Hastings and Sweeney, 1960). Short exposures (2-4 minutes) to ultraviolet radiation also reset the rhythm, yielding only phase advances, whose magnitude depended on the duration of the exposure and the CT time at which they were applied. These effects did not appear to be photoreversible (Sweeney, 1963). The free-running period of both the capacity and glow rhythms (as well as that of cell division; see Table 4) was temperature-(over-) compensated, having a Qloof about 0.85 (Hastings and Sweeney, 1958, 1959). The fact that free-running rhythms of bioluminescence persist for long time spans in populations of G. polyedra implies that either the individual biological clocks that underlie the overt rhythmicity are highly accurate or that the clocks are coupled, in the sense that some sort of chemical (or even photobiological, light-flash) communication, or “cross-talk” exists (see Edmunds, 1971; Section V.D, p. 136). In an attempt to test this hypothesis of cellular interaction, Hastings and Sweeney (1958) mixed equal volumes of two cultures of G. polyedra whose rhythms of luminescence capacity were 5 hours (75”) out of phase, and found no evidence for cross-talk after one cycle under constant conditions. The rhythm continued with the maximum of luminescence in the mixed cultures quite similar to that obtained when the measured luminescence of the separate cultures was summated. Had interaction occurred, one might have expected that a curve would have been obtained representing the resultant of the summated peaks for two separate in-phase cultures, or some other effect. These experiments were extended in more detail, and for several cycles under constant conditions, to the rhythm of glow bioluminescence-a system possessing sharp and reproducible waveforms so that even small changes could be detected (Sulzman et al., 1982). Once again, no cellular interaction was detected: the bioluminescent glow of the mixed cultures matched the algebraic sum of the independent control cultures. Thus, intercellular exchange of temporal information did not play a significant role in maintaining synchrony in circadian rhythms of G. polyedra, implying


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that long-term persistence must arise from the accuracy of the individual cellular clocks. That sufficient accuracy does indeed exist has been demonstrated for the glow rhythm: it is accurate to within 2 minutes a day, and the variance in z among individual cells is about 18 minutes (Njus et al., 1981). The very slow decay of rhythmicity necessarily entailed, in fact, was empirically observed. Finally, the cellular autonomy of the G. polyedra clock(s) has been directly demonstrated by the measurement of the rhythm of both stimulated and glow luminescence in individually isolated cells (Hastings and Krasnow, 1981). Like cell division, they were phased, or gated: some cells emitted, others did not, but those that did emit did so at the “allowed” time (see analogous results for the rhythm in photosynthetic capacity in the following subsection). It is a generalization (Bunning, 1973) that when rhythmic organisms, including G. polyedra (Sweeney and Hastings, 1957), are transferred from bright LL (in which circadian periodicity is lost in most cases) to DD, the rhythms recommence at the phase characteristic of the onset of the night, at CT 12 (Bunning, 1973). The inference is that the clock initially had stopped at CT 12, the rhythms starting up again at this phase point when submitted to DD. As Sweeney (1979) has noted, however, this is not the only interpretation: the underlying oscillator (as opposed to the overt rhythm) could be continuing to run undetected in bright LL and then could be shifted to CT 12 from whatever phase it was in by the LL/DD transition. Indeed, she observed this to be the case for the rhythm of acid-stimulated bioluminescence (as well as of cell division and photosynthetic capacity). Bright light did not cause the circadian clock(s) to stop immediately; some 4 weeks were required under these conditions before all rhythms “damped out”, and when periodicity was restored by a transfer of the cells to D D after different lengths of exposure to LL, the rhythms were always reset to about CT 12-14. Analogously, if cultures of G. polyedra were kept at either 11 or 4”C, the circadian rhythm of glow luminescence was lost, even though the cells were still capable of emitting light at these low temperatures. Rhythmicity was restored, however, when the cultures were returned to a higher temperature of 20”C, with the phase of the re-initiated rhythm apparently being determined by the time of transfer to the permissive temperature (Njus et al., 1977). The re-initiated circadian oscillation started up at CT 12, suggesting to these workers that critical temperatures stopped the clock and held it at this unique phase point (similar to the earlier hypothesis for LL). Further, rhythmicity was found to be lost under combined non-critical low temperatures (i.e. T > 12.5”C)and intensities of LL that otherwise would have been ineffective if individually imposed. Njus et al. (1977) term this lack of persistence at a critical temperature “conditional arrhythmicity”, as opposed to historical arrhythmicity which does not involve the inhibition of an existing rhythm (such as is observed when an organism is raised from the seed under constant

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conditions, and where rhythmicity can be elicited by a single light or temperature perturbation). b. Biochemical Aspects. One time-honored approach toward elucidating the mechanism underlying an overt physiological rhythm, or expression (“hand”) of the clock, is to attempt to thread one’s way back through the biochemical pathways mediating the rhythm-the so-called transducing mechanisms (Sweeney, 1969bFuntil one arrives at their point ofcoupling to the oscillator (Edmunds, 1976). The bioluminescence system of G. polyedra serves as an excellent case history (Dunlap and Hastings, 1981; Dunlap et al., 1981). The reaction responsible for light production involves oxidation of dinoflagellate luciferin (substituted, open-chain tetrapyrrolic structure) by molecular oxygen, catalysed by a specific luciferase enzyme:

Luciferin + 0 2

luciferase

c

Light (A,

475 nm) + products

The absolute level of luciferin (Bode et al., 1963), the binding capacity of its specific binding protein (Sulzman et al., 1978) and the activity of luciferase (Hastings and Bode, 1962) have all been shown to be under circadian clock control. Their activities in extracts made in the middle of the night phase are typically five to ten times greater than those in similar extracts made from day-phase cells, and these cyclic changes continue in cells under constant, free-running conditions. Earlier work (McMurry and Hastings, 1972b) on the cause of rhythmic luciferase activity ruled out simple explanations involving differences in enzyme extractability or extractable inhibitors or activators, leaving open the alternatives of cyclic synthesis and degradation of enzyme (constant specific activity) or cyclic covalent modification of the polypeptide, thus altering its activity (cyclic specific activity). Recently, Dunlap and Hastings (198 1) have purified unproteolysed, higher molecular-weight luciferase from both dayand night-phase cells and compared the two preparations with respect to several physicochemical, enzymic and immunological criteria. A given amount of anti-luciferase inactivated the same amount of luciferase activity in both extracts, indicating that their specific activities were the same, and suggesting that the luciferase was the same polypeptide in day and night preparations but that there were different amounts of the enzyme in each. Thus, the circadian rhythm of luciferase activity could be attributed to circadian clock-modulated synthesis or degradation, or both, of the luciferase polypeptide. Of course, the question now arises (as it always does) as to the next step in the quest for the elusive clock. Another approach toward the biochemical dissection of the clock has been the use of various chemicals and inhibitors (Edmunds, 1976). Earlier work with G. polyedra (and several other unicellular systems) perhaps bore out the old adage “only the uninhibited use inhibitors.” Virtually the entire chemical


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shelf was thrown at the glow luminescence rhythm: EDTA (ethylenediaminetetra-acetate, a chelator), silver nitrate, calcium chloride and ferric chloride (all metabolic poisons); the growth factors, giberellin and kinetin; the mitotic inhibitors, urethane and fluorodeoxyuridine (FUdR); respiratory inhibitors, such as arsenate, cyanide and p-chloromercuribenzoate (PCMB); 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation; and the herbicides DCMU and CMU (chlorophenyl- 1,1-dimethylurea), which specifically inhibit photosynthesis (Hastings, 1960; Hastings and Bode, 1962). In most cases, the compounds were added to vials of the cultures and then washed out later by centrifugation and resuspension of the cells into fresh medium; in this sense, they constituted chemical analogues of the light pulses utilized for phase-shifting the overt glow rhythm. Unfortunately, however, little could be concluded from these studies except that the clock seemed to be remarkably insensitive to perturbations by ordinary agents. In some cases, phase shifts of sorts occurred, whereas in others, the rhythm “damped out.” The problem remained of differentiating between merely affecting the expression of the rhythm (by uncoupling the “hands” of the clock) and actually perturbing the underlying oscillator itself. Nevertheless, one point did emerge: neither photosynthesis nor cell division was needed for clock function inasmuch as the glow rhythm continued to run unabated when these other processes were blocked (on the assumption that one clock controlled all three processes; but see Section V.D, p. 135). As was illustrated by the rhythm of phototaxis in E. gracilis (Section III.A.2, p. 92), heavy water (2H20) reversibly lengthens the free-running period in a variety of organisms (Enright, 1971) and has been formally compared (Pittendrigh et al., 1973) to a diminishing of the apparent temperature (the so-called low-temperature equivalence hypothesis). It was therefore of interest to test this idea using the Gonyaulax glow rhythm, which, as we have seen (Sweeney and Hastings, 1958),exhibits “over-compensation” (i.e. the period is shorter at lower temperatures). Accordingly, McDaniel et al. (1974) assayed glow luminescence in deuterated (6 or 12%) cultures of this dinoflagellate, at either 22 or 16°C in dim LL (1180 lux), following prior entrainment by an LD: 22,12 cycle. The period of the rhythm was lengthened in a dose-dependent manner at either constant temperature. Furthermore, in contrast to the phase angle delay with respect to the LD cycle induced by low temperature and 2H20 in Drosophila spp. (Pittendrigh et al., 1973), the glow rhythm of deuterated cultures of G. polyedra displayed a phase angle advance at the lower temperature, but a phase angle delay at the higher temperature. These results, then, did not support the low-temperature equivalence hypothesis. Attention then turned to the use of somewhat more specific agents, in addition or pulsing experiments, in an attempt to affect either the phase or period of the free-running rhythms of luminescence. Continuous exposure of

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cultures of G. polyedra to moderate concentrations of ethanol (O.l%), which may act at the membrane level, caused a shortening of the free-running period of the glow rhythm (Taylor et al., 1979), contrary to the period-lengthening effects usually reported for other circadian systems (Edmunds, 1980c), including the mobility rhythm of E. grucilis (Brinkmann, 1976a). Ethanol pulses caused phase shifts of both the glow rhythm (Taylor et al., 1979)and of the rhythm of stimulated luminescence (Sweeney, 1974a). In the latter case, the sign and magnitude of the phase shift obtained depended on the CT at which the 4-hour ethanol pulse was applied, the pulse-response curve thus derived closely resembled that for light perturbations in similarly maintained cells. Furthermore, Sweeney ( I 976b) investigated the relationship between alcohol chain length and the corresponding ability to cause phase shifts: methanol was the most effective, and longer-chain alcohols the least (see Sweeney and Herz, 1977). Thus, the notion that alcohols exert their effect on the clock through a non-specific attack on membranes, with alcohols that tend to partition themselves more into the lipid phase of cell membranes being more effective phase shifters, was not supported (see Njus et al., 1976; Section V.B, p. 131). This conclusion also was reached by Brinkmann (1976a) for the mobility rhythm in E. gracilis (Section III.A.2, p. 93), where it appeared that alcohols must be metabolized in order to exert their effects. Indeed, one would anticipate on the basis of Brinkmann's hypothesis that acetaldehyde, an immediate byproduct of ethanol metabolism, should also be able to generate phase shifts. This was empirically confirmed by Taylor and Hastings (1979) for the glow rhythm of G.polyedra,who found that aliphatic aldehydes having a chain length of one to four carbon atoms (formaldehyde, acetaldehyde, proprionaldehyde and butyraldehyde) had a significant phase-shifting effect. Inasmuch as ion transport across membranes has been proposed to be one component of a limit-cycle membrane model for circadian clocks (Njus et al., 1974; Sweeney, 1974b; see Section V.B, p. 131), a number of membrane-active probes have been assayed for their effect on period or phase in the Gonyaulux system (Sweeney and Herz, 1977; Sweeney, 1981). (Indeed, circumstantial evidence, comprising documented circadian rhythms in intracellular K content and membrane ultrastructure (see Table 4), favoured such an involvement of membranes in this micro-organism.) The results from these experiments, however, were not completely consistent with this class of model. Thus, low concentrations of valinomycin (which permits passage of K+), caused reproducible but small phase shifts in the rhythm of stimulated luminescence (Sweeney, 1974a), but other ionophores, such as gramicidin and compound A23 187 (an ionophore for divalent cations), had no effect on phase (Sweeney and Herz, 1977).A potent uncoupler of membrane proton gradients (CCCP, carbonyl cyanide m-chlorophenyl hydrazone) produced rather small phase shifts, and then only if applied during the early subjective day (Sweeney, +


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1976b; Taylor and Hastings, 1979). Solvents such as acetone and dimethyl sulphoxide (DMSO), which might be expected to attack membranes, failed to shift the phase of the stimulated bioluminescence rhythm (Sweeney and Herz, 1977; Sweeney, 1981). Finally, the fact that alcohols that penetrate membranes more easily are not as efficient in phase-shifting as those that do not, as discussed previously, seems inconsistent with a membrane theory for the generation of circadian oscillations. Another avenue of approach toward the clock mechanism in G . polyedra has been the use of inhibitors of macromolecular synthesis. Early work (Karakashian and Hastings, 1963) indicated that the inhibition of DNA synthesis by amethopterin, novobiocin or mitomycin C did not appear to directly affect clock function (glow rhythm). Similarly, the inhibition of DNA-dependent RNA synthesis by actinomycin D (which cannot be removed from the cultures) perhaps only slightly delayed the glow rhythm before it was completely suppressed (Karakashian and Hastings, 1963). Chloramphenicol, which inhibits protein synthesis on 70s ribosomes of chloroplasts and mitochondria, had no effect on the glow rhythm, whereas puromycin (inhibiting protein synthesis on both 70s and 80s ribosomes) generated small delays (Hastings, 1960; Karakashian and Hastings, 1963). On the other hand, quite large (9-12-hour) advance and delay phase shifts were obtained in the rhythm of stimulated luminescence when G . polyedra was pulsed with cycloheximide (Walz and Sweeney, 1979), which inhibits protein synthesis on 80s ribosomes of the cytosol, as it did in both E. gracilis (Feldman, 1967) and Acetabularia sp. (Karakashian and Hastings, 1963)(see Sections III.A.2, p. 93 and III.D, p. 114). Indeed, in G . polyedra, cycloheximide at 0.36 and 3.6 pM exactly mimicked the response to bright light pulses (Walz and Sweeney, 1979). Most recently, Taylor et al. (1982a) have found that 1-hour pulses of anisomycin, streptimidone and cycloheximide at appropriate concentrations caused strong advance or delay phase shifts (of up to 12 hours) in the bioluminescence glow rhythm in G .polyedra. Indeed, even minute-long pulses of anisomycin were sufficient to yield large phase shifts (Taylor and Hastings, 1982); and critical pulses (dose, CT) of this inhibitor can drive the circadian oscillator toward its singularity point, resulting in nearly arrhythmic cultures (Taylor et al., 1982b). These results, therefore, collectively implicate protein synthesis on 80s cytoplasmic ribosomes in the generation of circadian oscillations in G . polyedra and other organisms (Section V.B, p. 132).

2. Rhythms in Photosynthesis In the study of transducing mechanisms from the putative circadian oscillator to observed rhythmicities, photosynthesis is particularly attractive because it comprises a number of relatively well understood processes that can be measured individually, and it was one of the first to be examined in detail in G .

I08


polyedra. Hastings et al. (1961) found that cells exhibited a rhythm of photosynthesis (as measured by incorporation of I4CO2) at the eighth hour of the light interval during a synchronizing LD: 12,12 cycle. Cultures transferred to continuous dim LL (1 100 lux) continued to exhibit only a circadian (f= 26 hour) rhythm of photosynthetic capacity (incorporation measured under 9600-lux illumination in aliquots taken from the master culture). Cultures transferred to bright LL (9600 lux), however, did not show any periodicity. Thus, conditions could be chosen where oscillations would persist although the actual rate of photosynthesis was constant. Using the Cartesian reference diver technique, Sweeney (1960) was able to measure (under saturating conditions) the evolution of 0 2 over a 24-hour time span in single isolated cells of G. polyedra maintained either in LD or in bright LL (8000 to 10,000 lux). The results were similar to those found for populations of cells, thus suggesting that bright LL inhibits rhythmicity in the individual cell rather than merely generating asynchrony among rhythmic cells of the culture. (A similar situation obtains for the loss of circadian rhythmicity of cell division in isolated cells maintained in intense LL (Hastings and Sweeney, 1964).) Further, these observations clearly demonstrate that a bonajide circadian rhythm may occur in a single eukaryotic micro-organism without the necessity of intercellular coupling for its generation or maintenance (Section V.D, p. 136). Attention then turned to the transducing mechanisms involved in the expression of the rhythm of photosynthetic capacity. Earlier efforts concentrated on the dark reactions comprising the Calvin scheme (Sweeney, 1969b, 1972; Bush and Sweeney, 1972), but results were not promising. For example, although ribulose 1,5-bisphosphate carboxylase sometimes showed fluctuations in activity, there was not sufficient correspondence to satisfy the rates of COz fixation at all stages investigated and, indeed, in Acetabularia sp. none of the Calvin cycle enzymes were found to have rhythmic activities (Hellebust et al., 1967; see Section III.D, p. 110). Inasmuch as the enzymes responsible for carbon fixation did not vary over the circadian cycle, experimentation focused on the light reactions of photosynthesis, and results have made it clear that they are indeed regulated by the clock (Sweeney, 1981). Thus, from analysis of the rates of photosynthesis as a function of irradiance, a temporal change in relative quantum yield in dim LL was found, although total chlorophyll, half-saturation constants and the size and number of particles on the thylakoid freeze-fracture faces were constant (PrCzelin and Sweeney, 1977; Prezelin et al.,.1977). More recently, Govindjee et al. (1979) examined fluorescence transients at different times in G. polyedra cells maintained in either LD or LL; no rhythmic changes were found. On the other hand, the intensity of chlorophyll a fluorescence (both initial and peak values) was about twice as high during the day phase of the circadian cycle in LL than during subjective night, and these


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changes were positively correlated with the rhythm of oxygen evolution (Sweeney et al., 1979). This periodicity in fluorescence persisted in the presence of 10 pM dichloromethylurea, which blocks electron flow from photosystem I1 during photosynthesis, indicating that the cause was not due to achange in net electron transport between photosystems I and 11. Although the rhythm of fluorescence could arise from differences in the efficiency of spillover energy from strongly fluorescent photosystem I, such spillover should occur unimpaired at 77K, but this was not the case: the rhythmicity in photosynthetic capacity was abolished at this temperature (Sweeney et al., 1979). The results could indicate that the non-radiative decay of chlorophyll excitation is less during the day than at night, although the reason for such a change remains obscure. Perhaps circadian ion fluxes across the thylakoid membrane generate reversible conformational changes which would couple and uncouple entire photosynthetic units in the light-harvesting pigment-protein complex, and thus induce a circadian rhythmicity in photosynthetic capacity (Prkzelin and Sweeney, 1977; Sweeney, 1981).

c. Chlamydomonas spp. The unicellular green alga, Chlamydomonas reinhardii, like the algal flagellate Euglena gracilis (Section III.A, p. 76), has been well characterized physiologically and biochemically. Furthermore, because it can reproduce sexually, this micro-organism has proved particularly attractive for genetic analysis of cellular behaviour and its circadian regulation (Bruce, 1976; Mergenhagen, 1980; Feldman, 1982). It has been shown that Chlamydomonas reinhardii has a circadian clock which controls the phototactic response of slowly dividing (infradian) or non-dividing cells (Bruce, 1970). This rhythm of phototaxis (photo-accumulation) can be initiated by a sudden, single shift from LL to DD, entrained by LD cycles, and phase-shifted by exposure to short dark perturbations. The free-running period at 22°C is about 24 hours and is temperature-compensated over the range 18 to 28°C. The clock is also manifested by a rhythm in the tendency of cells to stick to a glass surface (Straley and Bruce, 1979), reminiscent of the circadian rhythm in cell settling and adhesion discovered by Terry and Edmunds (1970b) in E. gracilis (Section III.A.2, p. 96). In addition, a circadian rhythm in cell division has been reported (Bruce, 1970), with the possibility existing that the sexual cycle, as well as vegetative growth, also may be under clock control, although recent findings indicate that the mating reactivity of the gametes is not so regulated (Bruce and Bruce, 1981). Subsequent to the discovery of the rhythm of phototaxis, “clock” mutants with both shorter and longer periods than the wild type have been isolated and characterized genetically (Bruce, 1972). For example, two short-period mutants, designated w-c and 90-, displayed periods of about 21-22 hours

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and temperature compensation comparable to the wild type. Similarly, a long-period mutant, Lo-104, had a period of approximately 27 hours. More recently, four long-period mutants, designated per 1,2,3,4,were isolated after nitrosoguanidine mutagenesis, and were analysed in greater detail (Bruce, 1974). These mutants had period lengths ranging from about 26 to 28 hours and unimpaired temperature compensation. Unlike the clock mutants of species of Drosophila and Neurospora (Section IV.B, p. 118), in which the mutations are allelic (Bruce, 1976; Feldman, 1982), the long-period characteristic of the per mutants seems to be unlinked, and to be controlled by several single genes at separate loci. Thus, crosses between single mutants, as well as crosses involving three or four mutant genes, yielded progeny with both parental and recombinant period lengths, including not only normal (wild type) periods, but also extra-long periods (30-33, 33-36 and 3 7 4 0 hours in the double, triple and quadruple mutants, respectively). One of the most provocative findings was that the period-lengthening effect was additive. If one gene lengthened the period by m hours and a second by n hours, then the period of the double mutant was lengthened by m + n hours. Bruce (1974) has interpreted these findings as indicating that the mutations all affect the same rhythmic system rather than independent, autonomously oscillatory systems. It is interesting to note that this additive effect would be a logical consequence of “tape-reading”-type models (Ehret and Trucco, 1967; Edmunds and Adams, 1981) for circadian clocks (see Section V.B, p. 129), in which mutations affecting the period would involve the addition or deletion of tape segments, although the observed lack of clustering of clock mutations at a single locus would not. Finally, Mergenhagen and Hastings (1977) have selected for a number of strains of Chlamydomonas reinhardii having metabolic deficiencies (for example, niacin-requiring, thiamin-requiring, or erythromycin-resistant) which show either an elongation in period (usually by several hours) or an abnormal expression (e.g. arrhythmic behaviour) of the rhythm of phototaxis. Crosses of these mutants with short-period mutants should help clarify whether the observed period deviation is caused by the change in the metabolism for which the strain was selected (Mergenhagen, 1980). D.

Acetabularia spp.

Although there may be those who will balk at the notion of viewing the green alga Acetabularia mediterranea (or A . crenulata) even as a “maxi”-microorganism, it is a unicellular system (at least, until its months-long cell developmental cycle eventually culminates in the formation of a cap), whose large size is one of its strengths, facilitating the monitoring of various functions directly in an individual cell. Inasmuch as A . mediterranea has proved useful to research on circadian clocks (having recently given rise to the


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coupled translation-membrane model (Schweiger and Schweiger, 1977; see Section V.B, p. 129), the salient features of its circadian time structure, and recent advances with this organism, will be briefly reviewed. I . Physiological Rhythmicities A number of persisting circadian rhythms have been documented for A . mediterranea (Table 5). Most of these are associated with the rhythms of

photosynthesis, which have been investigated intensively (see Mergenhagen, 1980). Thus, the number and shape of the chloroplasts, their ultrastructure, their carbohydrate content and RNA synthetic abilities, their Hill reaction capacity, and even their migration rate, have all been shown to be subject to regulation by a circadian clock (see reviews by Vanden Driessche, 1973, 1975 and Broda et al., 1979). Photosynthetic functioning itself has been measured usually in one of two ways: (i) as photosynthetic capacity, reflecting the ability of aliquots of cells to evolve 0 2 or take up C02 under saturating light conditions at different times, TABLE 5 . Some persisting circadian rhythms exhibited by Acetabularia mediterranea Circadian rhythm Cyclic AMP ATP content (chloroplastic) Chloroplast migration Chloroplast number Chloroplast shape Chloroplast ultrastructure Electric potential Hill reaction, activity Photosynthesis, activity

Photosynthesis, capacity

Polysaccharide content, chloroplastic RNA synthesis, chloroplastic

Reference Vanden Driessche et al. (1979) Vanden Driessche (1970) Broda et al. (1979) Vanden Driessche (1973) Vanden Driessche (1966a) Vanden Driessche and Hars (1972a,b) Novak and Sironval(1976); Broda and Schweiger (198 1) Vanden Driessche (1974) Schweiger et al. (1964a,b); Terborgh and McLeod (1967); von Klitzing and Schweiger (1969); Mergenhagen and Schweiger (1973, 1975a,b); Karakashian and Schweiger (1976a,b,c); Schweiger and Schweiger (1977); Vanden Driessche (1979) Sweeney and Haxo (1 96 1); Sweeney et al. (1967); Hellebust et al. (1 967); Terborgh and McLeod (1967); Vanden Driessche (1966a,b; 1967); Vanden Driessche et al. (1970); Sweeney (1972, 1974b) Vanden Driessche et al. (1970) Vanden Driessche (1966b); Vanden Driessche and Bonotto (1969)

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or (ii) as actual photosynthetic activity, wherein photosynthetically produced 0 2 , which is released into the medium, is measured either continuously or over an extended time span under the given culture conditions [which typically are not saturating (e.g. LD or low-intensity LL)]. One of the first reports (Sweeney and Haxo, 1961) of a rhythm of photosynthetic capacity was in populations (10 cells) of A . major, as well as in an individual cell, measured by the Cartesian diver technique. The rhythm was entrained by LD: 12,12(higher activity in the day) in both nucleate and anucleate cells (obtained by simply cutting off the nucleus-containing rhizoid) and persisted in LL for 2 days in the anucleate cells. Similarly, both nucleate and anucleate cells of A . crenuluta could be entrained by 24-hour LD cycles, and their rhythms phase-shifted by the prolongation of the light interval by 12 hours. These earlier results therefore suggested that the nucleus, and, by implication, continuous transcription of the nuclear genome, was not necessary for entrainment or maintenance of persisting circadian rhythms. This conclusion was corroborated in an extension of these studies to A . mediterranea, in which a rhythm of photosynthetic activity was demonstrated in both nucleate and anucleate cells by measuring photosynthetically produced 0 2 by an electrochemical method in LL (2500 lux) at 20°C (Schweiger et al., 1964a).The role of the nucleus in the generation of circadian oscillations was further elucidated by experiments in which rhizoids and nuclei were exchanged (Schweiger et al., 1964b). Two groups of 180” out-of-phase cells maintained in reversed LD: 12,12 cycles were enucleated by removal of their rhizoids; the rhizoids containing the nuclei were then grafted to the anucleate stalks exhibiting rhythms with opposite phase angles from the “donor” plants. The “recipient” cells were observed gradually to gain the phase of the rhizoid donor cells. Similar results were obtained by implanting isolated nuclei into anucleate cells instead of transplanting the entire rhizoid. Once again, it appeared that the nucleus was capable of determining the phase of the rhythm, but was not necessary for its maintenance. Results consistent with this hypothesis were obtained by Vanden Driessche (1967), who was able to induce periodicity in anucleate cells of a strain of A . mediterranea that lacked rhythmicity by grafting on rhizoids from cells that displayed rhythmicity. The development of flow-through techniques for continuous recording of photosynthetic oxygen production, and the use of a highly-sensitive platinum electrode (von Klitzing and Schweiger, 1969; Mergenhagen and Schweiger, 1973), greatly facilitated the measurement and analysis of the photosynthetic activity rhythm in single cells. With this technique the free-running period in nucleated A . mediterranea cells was estimated to be 22.6 f3.2 hours, and was essentially identical to that found for anucleate cells (Mergenhagen and Schweiger, 1973). A Qlo value of 0.8 was calculated (Karakashian and Schweiger, 1976a), indicating an overcompensation similar to that found for


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G . polyedra (Sweeney and Hastings, 1960). It was also possible to phase shift the rhythm of photosynthetic activity using dark pulses to perturb the rhythm that was free-running in LL (Karakashian and Schweiger, 1976a). Finally, by means of the flow-through technique, rhythms in photosynthetic activity were found in a variety of cell fragments. Isolated caps, basal nucleate and anucleate fragments, apical fragments with and without stalks, and small segments taken from the middle of the stalk all displayed circadian periodicity (Mergenhagen and Schweiger, 1975b). Taken at face value, these results would seem to provide dramatic evidence that there is no particular compartment of the cell which is responsible for the expression of the rhythm and no specific site of the “clock.” Lest one think that the only circadian rhythms observed in A . mediterranea are those associated with photosynthesis and chloroplasts, one has merely to note the recent demonstration of a rhythm in electric potential along the longitudinal axis in individual cells (Broda and Schweiger, 1981). The study of this rhythm, initially inspired by the discovery (Novak and Sironval, 1976) of a circadian periodicity of the transcellular current in regenerating enucleated posterior stalk segments, was undertaken to further substantiate the role of membranes in clock function, as called for by the coupled translation-membrane model (Schweiger and Schweiger, 1977; see Section V.B, p. 129). Gradual changes in Z were observed during long (100 days, or more) free-runs in LL. Assuming that this rhythm is shown to exhibit all the other attributes of a full-fledged circadian rhythm (Fig. 2), it would be interesting to monitor it simultaneously, and one or two other periodicities (such as photosynthetic activity and chloroplast migration), over extended time spans to see if mutual dissociation occurs (see Section V.D, p. 136). 2. Biochemical Aspects

Results from experiments involving transplantation and implantation of the nucleus of A . mediterranea, and its role in the determination of the phase of the photosynthesis rhythms, raised the question of whether gene expression was necessary for the generation of circadian rhythms (as called for by the chronon model, for example; see Section V.B, p. 129), and led naturally to the testing of the effects of several inhibitors of transcription and translation. It is only relatively recently that a coherent pattern has begun to emerge. Initial observations (Vanden Driessche, 1966b) showed that the rhythm of photosynthetic capacity, as well as that of chloroplast shape, was abolished in nucleate cells but not in anucleate ones treated with actinomycin D, an irreversible inhibitor of DNA-dependent RNA synthesis (tested only in LD). This result was confirmed with anucleate cells of A . crenulata; furthermore, inhibitors of protein synthesis (puromycin and chloramphenicol) were also ineffective in altering rhythms, despite the fact that they did inhibit

I14


incorporation of labelled precursors into the macromolecules (Sweeney et al., 1967). One was thus confronted with a set of “most unusual paradoxes” (see Sweeney, 1974b): (i) the rhythm continued for months in the absence of a nucleus, yet phase was determined by the transplanted nucleus; (ii) actinomycin D inhibited rhythmicity in intact cells but not in anucleate ones; and (iii) inhibition of RNA synthesis in nucleate cells appeared to affect the rhythm without mediation of protein synthesis. Could differences in the control system for mRNA degradation exist, with short-lived mRNA decaying in nucleate algae but not in anucleate cells? In order to resolve these questions, a re-investigation of the effects of inhibitors was begun using the continuous flow-through system for monitoring the photosynthetic activity rhythm; similar results were obtained with actinomycin D (Mergenhagen and Schweiger, 1975a). Further, in an important experiment Vanden Driessche et al. (1970) demonstrated that rifampicin, an inhibitor of cellular RNA synthesis in Acetabularia sp. (preventing transcription of chloroplastic DNA by competitively inhibiting the binding of RNA polymerase to RNA), was also ineffective in altering rhythmicity in either nucleate or anucleate cells, even though RNA synthesis was inhibited by at least 90%. Thus, daily transcription from either nuclear or cellular DNA did not appear to be required for the functioning of the circadian oscillator. Turning then to translation, Mergenhagen and Schweiger (1975a) found that cycloheximide, an inhibitor of protein synthesis on 80s ribosomes of the cytosol, and puromycin, which attacks both 80 S ribosomes as well as the 70s ribosomes in the cell organelles, reversibly inhibited the expression of the rhythm of photosynthetic activity in both nucleate and anucleate cells. These experiments thus indicated that the observed inhibition was associated with 80s ribosomes, but did not exclude the involvement of 70s ribosomes. [Note the discrepancy with respect to the much earlier results obtained with puromycin on groups of cells (Sweeney et al., 1967).] This question was resolved by using chloramphenicol, which specifically inhibits protein synthesis only on 70s ribosomes. The inhibitor was ineffective in both nucleate and anucleate cells (Mergenhagen and Schweiger, 1975a). The simplest conclusion that could be drawn from these series of experiments was that gene expression at the translational level-specifically on 80s ribosomes-is required for the operation of the circadian oscillator in Acetabularia sp. Either protein synthesis would be part of the clock mechanism itself or a vital short-lived component would require continual resynthesis. [For generalization to other systems, see Vanden Driessche (1975), Mergenhagen (1976), and the Group Report on “The Role of Genes and Their Expression” in Hastings and Schweiger (1976).] This hypothesis was further substantiated by detailed studies of the phase-shifting effects of cycloheximide and puromycin on the circadian rhythm of photosynthetic activity in Acetabularia sp. (Karakashian and


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Schweiger, 1976b). At 20°C 8-hour pulses of both drugs shifted the rhythm when applied between CT 11 and 23 (subjective night) but were ineffective if applied during the subjective day. At 25”C, however, the phases of cycloheximide and puromycin sensitivity to phase-shifting were reversed. These results established a dependence of the rhythm of photosynthetic activity on the synthesis of a protein-perhaps a membrane protein-which would appear at different times of the circadian cycle, depending on the temperature. Indeed, Leong and Schweiger (1978) have reported a periodically appearing membrane protein (polypeptide P39 or polypeptide X) which was synthesized in the presence of cycloheximide and was retained in the chloroplast membrane fraction. Moreover, the timing and amount of synthesis of this polypeptide coincided with the timing and duration, respectively, of the phase of the circadian rhythm of photosynthetic activity sensitive to phase-shifting by pulses of cycloheximide, and depended on temperature in a similar manner. This protein has been purified and found to have a molecular weight of about 39,000 on the basis of analytical polyacrylamide-gel electrophoresis, aminoacid composition and peptide mapping (Leong and Schweiger, 1979). Furthermore, the proportion of intermediate amino-acid groups was high, whereas the proportion of hydrophilic amino-acid groups was well-balanced by that of hydrophobic amino-acid groups, a property characteristic of membrane proteins. The question, of course, is whether polypeptide P39 is a vital part of the clock machinery, as opposed to merely an associated one. If this is determined to be so (perhaps by making antibody against this purified protein), it would constitute one of the “essential” membrane proteins, which, together with the membranes themselves, comprise the two central components of the oscillator system envisaged by Schweiger and Schweiger (1977) in their coupled translation-membrane model for circadian timekeeping (see Section V.B, p. 129).

IV. Circadian Rhythms in Fungi A.

Saccharomyces

SPP.

The interplay among cell-cycle controls, biological clocks and membrane transport in micro-organisms can be quite complex (Edmunds and Cirillo, 1974). Membrane processes may serve as pacemakers in endogenous biological rhythms (Njus e f al., 1974; Sweeney, 1974b); conversely, self-sustaining biological clocks might well underlie oscillations in transport capacity and perhaps modulate the cell division cycle itself (Edmunds, 1978).Inasmuch as the regulation of transport in the common yeast Saccharomyces ceretlisiae has been intensively investigated, and the control of the cell division cycle has yielded to a genetic approach using temperature-sensitive mutants (Hartwell

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et a [ . , 1974; Pringle, 1981). Edmunds et al. (1979) attempted to ascertain whether a circadian clock might generate overt rhythmicities in growth and cell division in this micro-organism as it does in a variety of other unicellular algae and protozoans (Edmunds, 1978, 1984; see Table 8 and Section 1II.A. 1, p. 75) and, if so, whether such long-period oscillations could also be found in the uptake of amino acids in cultures that had attained the infradian (“stationary”) phase of population increase. In addition to the direct inhibitory effects of visible light (cool-white fluorescent, < 3500 lux) on growth rate and amino-acid transport that had been previously reported for yeast (strain Y 185 rho+), grown at a relatively low temperature of 12°C (so that g > 24 hours) in medium containing glucose, yeast carbon base, KH2P04, ammonium ion and proline (Woodward et al., 1978; Edmunds, 1980a), evidence was found for light-entrainable, autonomous, circadian and ultradian oscillators underlying both cell division and transport capacity (Edmunds et al., 1979). Diurnal LD cycles (L N 3000 lux), imposed on yeast cultures previously grown in the dark, phased or synchronized cell number increase to a 24-hour period with bud release being confined primarily to the dark intervals (although not necessarily every cell divided during any given division “burst”). The observed division or budding rhythm free-ran with a circadian period ( N 26 hours) for a number of days in constant darkness (DD), following prior entrainment by LD. Further, a similar light-entrainable circadian rhythm in the uptake of [14C]histidineor [14C]lysineoccurred in non-dividing (or very slowly dividing) cultures synchronized by a 24-hour LD cycle and then released into D D for as long as 10 days. Such oscillations in transport capacity would be anticipated in dividing cultures, if for no other reason than the cell division cycle constitutes a driving force itself. Indeed, Carter and Halvorson (1973) have reported periodic changes in the initial uptake rates of a variety of amino acids at different stages of the cell developmental cycle in synchronous cultures of Succh. cerevisiae fractionated according to cell size and age by zonal centrifugation (although the value o fg for these cultures was usually 1-3 hours). In some experiments (Edmunds et al., 1979), a bimodal (ultradian) periodicity in transport capacity was found in both LD and DD, with secondary peaks, or shoulders, occurring at intervals of about 12 hours, corresponding approximately to subjective “dawn” and “dusk” (although the beginning of the increase in capacity always anticipated the D/L or L/D transitions in the LD cycle). The occurrence of these peaks at intervals of about 11-12 hours is open to several interpretations: (a) a true bimodal circadian rhythm existed; (b) there were two (or more) subpopulations of cells, each of which had a circadian rhythm in amino-acid uptake but which were 180”out-of-phase; (c) the primary and secondary peaks were governed by two different circadian oscillators, keyed to “dawn” and “dusk”; or (d) the rhythm


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was truly ultradian, with a period of about 12 hours. It is difficult to distinguish experimentally among these alternatives. Finally, some preliminary experiments were undertaken to determine the nature of the photoreceptor for these light effects. Previous results indicated that blue light (1.410 nm) was the most effective wavelength of visible radiation for the inhibition of growth and transport in yeast, and that a wide variety of petite mutants either partially or totally lacking cytochromes b or u/a3, or both, show decreased photosensitivity, thus suggesting that these blue-absorbing chromophores are the primary photoreceptors for the inhibitory effects of visible light (Ulaszewski et al., 1979; Edmunds, 1980a). It is provocative that transport in cultures of the Y185 rho- petite mutant, lacking cytochromes a/u3, b and CI,could not be synchronized by LD cycles (Edmunds et al., 1979), a finding that is consistent with, but by no means demands, the hypothesis that cytochromes may constitute photoreceptors for not only the direct inhibitory action of visible light but also the entrainment of biological oscillators by light in this micro-organism. A more rigorous test of the photosensitizing role of the haeme chromophore in light inhibition might be to utilize yeast mutants incapable of synthesizing b-aminolevulinic acid to regulate cytochrome levels and, by hypothesis, the degree of photo-inhibition of growth and transport. Similarly, a more satisfying test of the photoreceptor for the putative biological clock would be the derivation of a high-resolution action spectrum for the phase-shifting effects of single light pulses given at different CT values of the rhythm, free-running in DD in both the rho+ and rho- strains. This would require, however, that the mutants be affected in only one (or very few) genes and that they exhibit some assayable clock function. If, indeed, yeast cytochromes are the primary photoreceptors for the inhibitory and entraining effects of light observed (especially by blue wavelengths), they (together with the flavins) would fall into the larger class of blue-light receptors that are being reported with increasing frequency for a large number of biological phenomena (Senger, 1980). B.

Neurospora spp.

Genetic analysis has proved to be a powerful approach to the elucidation of biochemical pathways, gene organization and gene regulation and, more recently, for the dissection of more complex systems such as bacteriophage assembly, cell division cycles and behavioural responses. There is no reason, a priori, why a similar approach should not be useful in discovering the mechanism(s) that underlie overt circadian rhythmicity. Such a genetic approach has comprised two major avenues of attack. First, clock mutants can be isolated which manifest alterations in the length of the free-running period, in phase, or in sensitivity to various environmental

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Zeitgeber, such as light and temperature. These mutants then can be analysed genetically, physiologically or biochemically, and can be used to localize the clock to specific organs or tissues. Second, biochemical mutants with known metabolic lesions can be isolated, and the effect of such mutations on the functioning of the clock can be determined (Feldman, 1982). In addition to species of Drosophila and Chlamydomonas (see Section III.C, p. 109), a considerable amount of genetic data has been accumulated on the filamentous bread mould, Neurospora crassa, which has proved to be an excellent system for studying circadian rhythms (reviewed by Feldman et al., 1979; Feldman, 1982; Feldman and Dunlap, 1983). Its most intensively examined rhythm is that of conidiation, or asexual spore formation (Sargent et al., 1966). This rhythm can be assayed on agar medium in either “race” tubes or petri dishes, in which the culture produces alternating areas of conidia (or bands) and mycelia; the banding pattern can be measured after an experiment has been completed and recorded with a digitizer interfaced with a microcomputer system (Gardner and Feldman, 1980). Finally, a system for assaying the clock in liquid cultures has been devised (Perlman et al., 1981), involving the transfer of mycelial pieces from liquid medium to race tubes, where the phase of the rhythm of conidial banding reflects the phase in liquid cultures; this important development facilitated the carrying out of many biochemical experiments which previously had not been feasible. The conidial rhythm-on which we will now focus-exhibits three key characteristics of a bonaJide circadian rhythm (see Fig. 2): a circadian period under free-running conditions, temperature-compensation and susceptibility to phase-shifting by light. In addition, circadian rhythms of COZ production (Sargent and Kaltenborn, 1972), DNA and RNA synthesis (Martens and Sargent, 1974), the activities of a number of enzymes (Hochber and Sargent, 1974), and the levels of cofactors (Brody and Harris, 1973; Delmer and Brody, 1975) have been demonstrated in N . crassa.

I . Isolation and Characterization of Clock Mutants At least 20 clock mutants for the conidiation rhythm have been isolated from N . crassa mutagenized with either N-methyl-N-nitro-N-nitrosoguanidine or ultraviolet radiation (Table 6). These mutants, all bearing (not indicated) the mutation bd (band), which expresses a clear pattern of rhythmicity but does not affect the clock itself (Sargent et al., 1966), have either shorter or longer periods than the wild type (where Z= 21.6 hours at 25”C), and more than 12 of these have been rather well characterized both genetically and physiologically (Feldman et al., 1979; Feldman, 1982). A number mapped to a single locus cfrq) on the right arm of linkage group VII and exhibited periods ranging from 16.5 to 29.0 hours. All of thesefrq mutants showed incomplete dominance when tested against the wild-type frq allele and displayed otherwise normal


I I9

TABLE 6. Circadian clock mutants (conidiation rhythm) in Neurospora crassa. From Feldman et al. (1979) and Feldman (1982).

Strain

Period Linkage (hours) Mutagen' group Dominance

Reference

A. Mutants at thefrq (frequency) locus frs- I 16.5 NG VII R Incomplete Feldman and Hoyle (1973) frq-2 19.3 NG VII R Incomplete Feldman and Hoyle (1973) frq-3 24.0 NG VII R Incomplete Feldman and Hoyle (1973) frq-4 19.3 NG VII R Incomplete Feldman and Hoyle (1976) fr9-6 19.2 NG VII R Incomplete Gardner and Feldman (1 980) frq-7 29.0 NG VII R Incomplete Gardner and Feldman ( 1980) frq-8 29.0 NG VII R Incomplete Gardner and Feldman (1980)

B. Mutants at other locib chr (chrono) 23.5 prd-1 (period) 25.8 prd-2 prd-3 prd-4

25.5 25.1 18.0

NG NG UV UV UV

VI L Incomplete Feldman et al. (1979) 111 C Recessive Feldman and Atkinson (1978) V R Recessive Feldman et al. (1979) I C Recessive Feldman et al. (1979) I R Incomplete Feldman et al. (1979)

C. Strains carrying multiple clock mutations fig-3, prd-1 28.5 (28.2)' frq-3, prd-2 frq-3, prd-3 frq-1,prd-4 frq-2, prd-4 frq-I, prd-1 frq-2, prd-1 frq-7,chrgrd-l

Feldman et al. (1979); Gardner and Feldman ( 1980)

28.1 (27.9) 30.6 (27.5) 13.8 (13.5) 16.1 (16.0) 19.3 (20.7) 22.8 (23.5) 38.5 (35.1)

'N G = N-methyl-N'-nitro-N-nitrosoguanidine;UV =ultraviolet radiation. bprd-I.-2,-3, and -4 were formerly frq-5, UV IV-2, UV IV-4 and UV V-7, respectively. Values in parentheses are theoretical additive period lengths (hours).

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growth and development. Provocatively, their periods were not randomly arrayed, but differed from that of the wild type by some integer multiple of 2.5 hours, suggesting the existence of a quanta1 element in the clock mechanism (perhaps the number of copies of thefrq locus) that can be repeated a variable number of times each cycle (see Section V.A, p. 128). Five additional mutants, each at a different locus unlinked to frq or to each other, also have been isolated (Feldman and Atkinson, 1978; Feldman et al., 1979); all showed significantly shorter or longer periods than wild type (Table 6). Finally, in order to determine whether the clock genes act independently or interact with each other, a number of double mutants were constructed (Feldman et al., 1979; Gardner and Feldman, 1980). In nearly every case, the mutations appeared to be not only cumulative (for example, two long-period mutants produced a still longer period than either exhibited individually) but additive as well, suggesting little or no interaction between the paired genes. An analysis of the phase-response curves of the frq mutants has revealed that their periods had not been altered uniformly throughout the circadian cycle, but rather the changes were confined to a 7-hour portion of the wild-type oscillation, corresponding to the early subjective night (Feldman, 1982). This phase was compressed to as little as 2.5 hours in frq-1 and expanded to as long as 14.5 hours infrq-7 andfrqd. Thus, it may be possible to construct a temporal map describing the timing and sequence of the expression of various genes during the circadian cycle. Furthermore, in prd-Z, the amplitude of the pulse-response curve was greatly decreased at 25°C but was almost normal at 20°C. On the other hand, wild-type phase-shifting was severely inhibited at 34”C, suggesting that light-induced phase-shifting in N . crassa may be itself a temperature-dependent phenomenon (Nakashima and Feldman, 1980; Feldman, 1982). Other altered temperature responses as well have been noted in clock mutants. In wild-type N . crassa, the conidiation rhythm is temperature-compensated below 30°C (QIo- l), but above 30°C less well so (QIo= 1.3-1.7) (Sargent et al., 1966;Nakashima and Feldman, 1980). In contrast, whereas the short-periodfrq mutants also are characterized by this pattern of temperature compensation, the longer-period mutants urq-3,-7,-8) are not (Gardner and Feldman, 198l), with the “breakpoint” temperature’s being lowered from 30 to 25°C (frq-3), or even to below 18°C urq-7,-8), where a Q l o value of 1.3 was found over the entire 18-34°C temperature range. These differences in the capacity for temperature compensation displayed by the clock mutants could be interpreted as reflecting changes in the quantity of some gene product that would alter the rate of a metabolic reaction vital to clock timing (Gardner and Feldman, 1981). 2. Altered Clock Properties in Mutants with Biochemical Lesions Although the great majority of auxotrophic and morphological mutants that


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have been isolated in N . crassa do not affect the circadian clock, several mutants with various biochemical lesions have been discovered which exhibit significantly altered clock properties (Table 7). Conversely, certain biochemical mutants have been found in which clock characteristics have not been changed, thereby excluding a particular pathway or sequence as a key part of the oscillator itself (reviewed by Feldman, 1982). For example, one approach has been to attempt to identify the photoreceptor(s) for the clock (see Ninnemann, 1979). Thus, Sargent and Briggs (1967) first showed that the action spectrum for the damping of the conidiation rhythm of N . crassa in LL resembled the absorption spectra of both flavins and carotenoids-typical of the “blue-light photoreceptors” that have been implicated in a multitude of responses in many organisms (Briggs, 1976; Senger, 1980). The carotenoids, however, seemed to be excluded by the fact that LL-induced damping was unaffected in an albino double mutant ( a [ - / , a/-2) in which this pigment was not detected (Sargent and Briggs, 1967). Additional evidence supporting the role of flavins in photoreception has been afforded by the respiratory mutant poky, in which the concentration of non-mitochondria1 cytochrome is decreased: a 50-fold higher threshold intensity for the inhibition of banding in LL was found (Brain et al., 1977a,b; Britz et al., 1977). Finally, Paietta and Sargent (1981) have demonstrated a lowered light sensitivity of the clock for both the damping and phase-shifting responses in two riboflavin auxotrophs, rib-1 and rib-2, grown under conditions where riboflavin was growth-limiting and the concentrations of mycelial flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were decreased. Similarly, because a number of studies had suggested the involvement of cyclic AMP in circadian clock function [see Cummings’ (1975) model and Section V.B., p. 1291, in N . crassa (Feldman, 1975) among other organisms, a genetic approach to testing this hypothesis promised to be fruitful. Several morphological revertants (not exhibiting colonial morphology so that the conidial banding pattern could be expressed) of the mutant crisp- I were utilized (Feldman et al., 1979). Despite the fact that these mutants had been shown to have significantly lower concentrations of adenylate cyclase and cyclic AMP, they entrained normally to an LD:Z2,12 cycle and free-ran with an unaltered period, thus clearly demonstrating that major pools of cyclic AMP are not essential for clock operation. On the other hand, mutations affecting cysteine biosynthesis (Table 7) have been shown to affect the period: cys-X, for example, grew at a slower rate on limiting amounts of sulphur (furnished as methionine) but the clock ran faster, i.e. was significantly shortened (Feldman and Widelitz, 1977). Perhaps even more dramatic was the demonstration that the period could be effectively “titrated” by the addition of unsaturated fatty acids to the medium of the cel mutant of N . crassa, which has a defect in the fatty acid synthetase complex,

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TABLE 7. Biochemical mutants in Neurospora crassa in which clock properties (rhythm of conidiation) have been examined. From Feldman et a/. (1979) and Feldman (1982). Strain

Mutation

Reference

A. Mutations affecting light sensitivity al-I, No detectable carotenoids; damping Sargent and Briggs (1967) of rhythm in constant light al-2(albino) unaltered Poky Respiratory mutant; decrease in Brain et al. (1977a,b); Britz et al. (1977) non-mitochondria1 cytochrome; higher threshold intensity for inhibition of banding in continuous light rib-I; rib-2 Riboflavin auxotrophs; decrease in Paietta and Sargent levels of FAD and FMN in mycelia; (1981) reduction in light-sensitivity of clock for phase-shifting and damping

B. Mutations affecting cyclic AMP Feldman et al. (1979); (see NG 6-3; NG No colonial morphology; decreased 6-1 1 levels of adenylate cyclase and cyclic Feldman, 1975) (revertants AMP; entrainment and free-running of crisp-I ) period unaffected C. Mutations affecting fatty acid synthesis cel Defective fatty acid synthetase Brody and Martins complex; deficient in synthesis of (1979); Mattern and palmitic acid (16:O); addition of Brody (1979); Brody unsaturated or short-chain and Forman (1980); saturated fatty acids lengthened Mattern et al. (1982); period Roeder et a/. (1982) D. Mutations affecting cysteine biosynthesis cys-X; cys-4; Cysteine auxotrophs; shortened period Feldman and Widelitz CYSI2 (1977); Feldman et al. (1979) E. Oligomycin-resistant mutants oli' Resistance to drug oligomycin due to Dieckman and Brody (1980); Brody and mutations in the dicyclohexylcarbodi-imidebinding Forman (1980); Brody protein of mitochondria1 ATPase; (1981) period length shortened F. Cycloheximide-resistantmutants cyh-I; cyh-2 Cycloheximide-resistant 80s ribosomes; clock unaltered, but pulses of cycloheximide could no longer phase-shift rhythm

Nakashima et al. (1981a.b)


I23

and thus a partial requirement for exogenous fatty acids. Brody and Martins (1979) showed that addition of unsaturated fatty acids caused a striking increase in the period of the conidiation rhythm; linoleic acid (cl8:2) was most effective (yielding values of z as long as 40 hours), followed by linolenic acid (C18:3) and oleic acid (C18:l). Although saturated fatty acids, such as palmitic (C16:o)and stearic (C18:0), did not alter the period when added to the medium alone, palmitic acid could reverse the period-lengthening effects of linoleic acid. In contrast, shorter-chain saturated fatty acids (c8to cI3) did lengthen the period of the celmutant, but not of the wild type, from about 21 hours to as long as 40 hours, depending on the supplement (Mattern and Brody, 1979; Mattern et al., 1982). The different responses of the wild type and of cel to exogenous fatty acids could not be attributed to their differential utilization or incorporation by the two strains: their incorporation of [I4C]lauricacid (C12:o) was approximately the same. It is interesting to note that Roeder et al. (1982) demonstrated circadian oscillations in certain of these fatty acids in the growing front of the mycelia: the mol percentages of Cl8:2 and c18:3 fatty-acyl residues (but not of any others or of total lipid content) in the total lipids, and in the phospholipids, oscillated out-of-phase with each other with a period of about 20 hours. It is not yet clear whether these oscillations in fatty acids have a role in the mechanism of the clock, although the possibility that they were merely part of the development rhythmicity itself was eliminated by the demonstration of similar oscillations in the csp-Z strain, which did not express the conidiation rhythm under the growth conditions employed (Roeder et al., 1982). Finally, several drug-resistant mutants of N . crassa have been successfully examined for altered clock properties. Thus, oli' nuclear mutants, resistant to oligomycin, which inhibits mitochondrial adenosine triphosphatase (ATPase), have been found to have periods several hours shorter than wild type at 22"C, and the extent of this shortening is correlated with the degree of oligomycin resistance (Dieckmann and Brody, 1980; Brody, 1981). These results suggest that ATP production, and perhaps mitochondrial function in general, may be important for the function of circadian clocks. Indeed, Brody and Forman (1980) found that if the oli' mutation is introduced into the cel strain, the sensitivity of the latter to the addition of linoleic acid (Brody and Martins, 1979) is eliminated. They speculate that the c18:2 residue alters the clock via its effects on mitochondrial ATPase. Lastly, Nakashima et al. (198 1b) have shown that although the clock is not altered in the mutants cyh-2 and cyh-2, which are resistant to cycloheximide-inhibitionof protein synthesis on 80s ribosomes, neither of these two mutants could be phase-shifted by pulses of this inhibitor. In contrast, the wild type is well known to be sensitive to cycloheximide, and a pulse-response curve has been derived for its action (Nakashima et al., 1981a) which is similar to those found in species of Gonyaulax (Walz and Sweeney, 1979; Dunlap et al., 1980; see Section III.B, p.

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107), Acetabularia (Karakashian and Schweiger, 1976b; see Section III.D, p. 114) and other organisms. This finding demonstrated that the target of cycloheximide is indeed the 80s ribosomes and not some other unknown target.

V. General Considerations and Conclusions

Now that we have completed a survey of the physiology of selected classes of circadian rhythms in micro-organisms, it is perhaps instructive to note some of the more pressing unsolved problems concerning circadian clocks that might be approached using unicellular systems in the hopes of enticing others into the field. Obviously, these topics, embracing entire disciplines in themselves, can be treated only in the most superficial manner, and others necessarily will be slighted. Mea culpa! A. CELL CYCLE CLOCKS: ULTRADIAN, CIRCADIAN AND INFRADIAN INTERFACES

The consideration of mechanisms that control microbial cell cycles often includes the notion of timers and clocks in regulating cell-cycle sequences that culminate in mitosis and cell division (Mitchison, 1974; Klevecz, 1976; Wille, 1979; Edmunds and Adams, 1981;Gilbert, 198 1; Edmunds, 1984). In addition to various dependent pathways, whose blockage would result in the stoppage of all subsequent events of the cell division cycle, there exists the possibility of “independent timers”, which would continue to function even if growth and cell division, for example, were inhibited. Indeed, the cell division cycle is a clock itself in the sense that it measures time-albeit imperfectly-under a given set of environmental conditions. Inasmuch as mitosis is a periodic event of short duration, relative to the total length of the cell division cycle, it is not surprising that various types of autonomous biochemical and macromolecular oscillators have been proposed to underlie this and other ‘‘landmarks’’ comprising the cell division cycle (Edmunds and Adams, 1981; Edmunds, 1984; Shymko et al., 1984). These range from those of the relaxation type, in which a single continuous variable accumulates or declines in the cell, triggering some event in the cell division cycle when it reaches a critical threshold, and the resetting to a baseline value so that the cycle starts again (see, e.g. Sachsenmaier, 1976), to those exhibiting limit cycle dynamics (Pavlidis, 1973; Winfree, 1980). In the latter, a central clock, characterized by a self-sustained oscillation of at least two continuously varying biochemical species, would maintain stable periodic behaviour and co-ordinate the timing of the events comprising the cell division cycle, despite transient perturbations in phase. Although the majority of these putative


125

“clocks” have periods in the neighbourhood of minutes to hours, it is the possibility that they are somehow relevant to circadian oscillators and the regulation of circadian rhythms of cell division that concerns us here. I . Interaction of Circadian Oscillators with the Cell Division Cycle There is abundant evidence that the cell division cycle of many unicellular algae, fungi and protozoans indeed do exhibit persisting circadian rhythms of cell division, or “hatching” (Edmunds, 1975, 1978). A number of these systems are itemized in Table 8, including some quite recent entries (also see Sections II.A, II.B, III.A, III.B, 1II.C and 1V.A). Typically, the division of cells occurs only at a certain phase of the circadian cycle-very often the times (“subjective” nights) in DD or LL corresponding to dark intervals in an environmentally synchronizing LD cycle. This phenomenon, commonly referred to as “gating”, has been particularly well documented in the algal flagellate Euglena gracilis (Edmunds and Laval-Martin, 1984; see Section III.A.l, p. 81), taken to be representative of other eukaryotic micro-organisms (and, perhaps, multicellular systems). Theformal properties of circadian clocks (Fig. 2+entrainability, persistence, phase-shiftability and temperature compensation-were also found to characterize the circadian rhythm of cell division in the organism. The recent discovery of a singularity point at which the imposition of a critical light pulse generates an arrhythmic population further suggests that the underlying oscillator may be of the limit-cycle type, although it does not demand it (Malinowski et al., 1984; Edmunds and Laval-Martin, 1984). According to our working hypotheses (Edmunds and Adams, 1981; Edmunds and Laval-Martin, 1984), mitosis would not be an essential part of the oscillator but would lie downstream from it: blockage of cell division should not stop the system from oscillating, at least at a subthreshold level (see Section III.A.l, p. 90). The question arises as to whether other events comprising the cell division cycle, besides division itself, can be controlled by a circadian oscillator. To approach this question experimentally, one needs an organism having a generation time (g) greater than 1 day, and several clearly distinguishable morphological landmarks. Recently, Sweeney (1 982), working with the large (9.9 mm long) dinoflagellate Pyrocystis fusiformis (Murray), whose cell division cycle has a minimum duration of 5 or 6 days, was able to isolate single cells or dividing pairs in capillary tubes kept in LD or DD and to determine five discrete morphological stages that could be correlated with the classical phases (GI,S, G2, M and C ) of the cell division cycle. Cells changed from one stage to the next only during the night (subjective) phase of the circadian cycle, and cells in all stages displayed a circadian rhythm of bioluminescence. All morphological stages therefore, and not only division, appear to be phased by the circadian oscillator(s). Sweeney (1982) also interpreted these findinp as

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TABLE 8. Some unicellular, eukaryotic micro-organisms for which persisting circadian rhythms of cell division (or “hatching”) have been documented. Updated from Edmunds (1975). Organism Protozoans Paramecium bursaria Paramecium rnultimicronucleaturn Tetrahymena pyriformis Strain W Strain GL

Algae Ceratium .furca

Reference Volm (1964) Barnett (1969) Wille and Ehret (1968a); Edmunds (1974) Edmunds (1974)

P4ZUL mutant P7ZNgL mutant W6ZHL mutant W,ZUL mutant YgZNalL mutant Gonyaulax polyedra Gymnodinium splendens Hymenomonas carterae (Cocco 2) Olisthodiscus sp. Prorocentrum sp. Pyramimonas sp. Pyrocystis fusiformis Murray Skeletonema costatum

Weiler and Eppley (1979); Adams et al. (1984) Bruce (1970); Bruce and Bruce (1981) Pirson and Lorenzen (1958); Hesse (1972) Chisholm and Brand (1981) Edmunds (1 966, 197I); Edmunds and Funch (1969a,b) Jarrett and Edmunds (1970) Mitchell (1971) Edmunds et al. (1 976) Mitchell (1971) Edmunds et al. (1976) Sweeney and Hastings (1958) Hastings and Sweeney (1 964) Chisholm and Brand (1981) Chisholm and Brand (198 1) Chisholm and Brand (1981) Chisholm and Brand (1981) Sweeney (1982) 0stgaard and Jensen (1982)

Fungi Candida utilis Saccharomyces cerevisiae (Y 185 rho+)

Wille (1 974) Edmunds (1980a); Edmunds et al. (1979)

Chlamydomonas reinhardi Chlorella pyrenoidosa Chick (21 1-8b) Emeliania huxleyi (MCH, 45 1B) Euglena gracilis Klebs (Z strain)

being incompatible with a mechanism for circadian oscillations that invokes cycling in G,, the quanta1 cycle which Klevecz (1976) has hypothesized to be appended to the G phase (see subsection A.3).

2. The “Circadian-Infradian Rule” Our survey of the physiology of circadian rhythms in micro-organisms has amply documented the point that in cells not proceeding through their normal


127

cell division cycle (as with cells in the plateau (infradian) stage where little, if any, cell division is occurring [i.e. where the developmental sequence culminating in mitosis has been blocked or arrested)], the circadian clock continues to operate as evidenced by the manifestation of numerous overt circadian rhythms (see, e.g. Table 3 for the E. gracifis system, Section III.A, p. 76). These overt rhythmicities typically can be abolished by changing the environmental conditions so that the overall g of the culture is less than 24 hours (ultradian growth mode); for example, by raising the temperature, increasing the intensity or duration of illumination (photosynthetic systems), or by introducing utilizable carbon sources into an autotrophic system (Jarrett and Edmunds, 1970; Edmunds, 1978, 1981). Presumably, the clock either is operating at a higher frequency, matching that of the fast-cycling cell division cycle (with a lower limit equal to that of the minimum possible g for a species), or alternatively is running with a circadian period but is uncoupled from the cell division cycle, or perhaps is even “stopped” or absent (Ehret et af., 1977; Edmunds, 1978). In any case, circadian rhythms have rarely been observed in cultures of micro-organisms in the ultradian growth mode (Ehret and Wille, 1970). However, there are some recent challenges to this generalization: some phytoplankton species appear to maintain a persisting die1 rhythm under constant conditions, while reproducing at a rate greater than one division a day (Chisholm et af., 1980; Brand, 1982). The mycelial mass of Neurospora crassa sometimes may be doubling at a rate greater than every 24 hours, yet the circadian conidiation rhythm continues to be expressed (J. Feldman, personal communication). We have occasionally observed in photo-autotrophically grown cultures of E. gracilis induced to enter the ultradian growth mode by a pulse of lactate that, although the rhythm of cell division was temporarily suppressed, its phase, when it was finally restored after the supplemental substrate had been depleted, was that of an unperturbed control (J. Rhee, D.L. Laval-Martin, and L. Edmunds, unpublished results; see Section 1II.A.1, p. 90). Ultradian cultures, however, can be phased (if not truly synchronized) by LD cycles whose period is also less than 24 hours; using LD: 6,6 ( T = 12 hours), LeDoigt and Calvayrac (1979) have phased E. gracifis cultures growing on lactate in D D so that a doubling occurred every 12 hours (g = 12 hours). Conversely, as g exceeds 24 hours (e.g. by lowering the temperature or by nutritional limitation), the periods of the basic oscillator, and that of the cell division cycle, seem to start to diverge in the other direction (Ehret et af.,1977; Edmunds, 1978). In the limiting case, where g approaches infinity (as in very slowly dividing, ‘‘stationary’’-phase cultures), low-amplitude division bursts occur at circadian intervals in the population (but cell division cycle is longer than 24 hours), along with numerous other cyclic physiological and biochemical events that are not necessarily related to the cell division cycle.

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This notion of a distinction between g and the circadian cycle in slowly-dividing cell populations has been formalized in the “Circadian-Infradian Rule (Ehret and Wille, 1970; Ehret and Dobra, 1977; Ehret et al., 1977). As g (or CT) tends towards infinity, the circadian cycle (CT) approximates to 24 hours and is almost temperature-independent. Thus, although cell division cycles-like circadian rhythms-are endogenous self-sustaining oscillations, they differ in that the period (z) of the circadian oscillation would not be a function of either the period (g) of the cell division cycle or of the temperature (it arrives at an apparently genetically determined limit value of about 1 day, “circadian”). In contrast, the length of the cell division cycle can take on all values from 24 hours to infinity. (Only in lethal environments would CT exceed circadian values, surely a null case.) But what is happening to the cellular circadian clock during its presumed replication at intervals of less than 24 hours? Ehret et al. (1977) feel that during ultradian growth, cell cycle time (CT) would be a function of g (as well as temperature). That is to say, CT equals g which equals the length of the interdivisional period. According to this scheme, then, the basic iteration period (CT) could take on all values between the minimum g possible under optimal growth conditions for a given cell and approximately 24 hours. More precise answers await further elucidation of biological clock mechanisms.

3. Quanta1 Cell Cycles Klevecz (1976) has raised the notion of a fundamental quanta1 cell cycle (G,), having a period of about 3-4 hours, for cultured mammalian cells in which the generation times are clustered. This model is particularly interesting when applied to the free-running circadian rhythm of cell division in a population of E. gracilis (or other micro-organism) in which the stepsize (amplitude) of the division bursts is less than 2.0 (i.e. where a doubling of cell number does not occur each day; see Section 1II.A.1,p. 81). In this situation, the culture can be described as developmentally asynchronous (Edmunds, 1978), since not all of the phase markers (cytokinesis) of the cells comprising the population show a one-to-one correspondence. Indeed, we presently have no idea to what degree other events in the cell division cycle show a similar registry or lack thereof, much less what the phases are of the cells that are not dividing in a given burst. However, recent findings by Sweeney (1982) with the dinoflagellate Pyrocystis fusiformis indicate that at least four morphological stages preceding division also are gated by the circadian clock (see subsection A.l). From Klevecz’s model, one might assume that individual cells have different g values (a necessary inference from the data on E. gracilis) because the G, subcycle undergoes a varying number of revolutions in different cells, thereby generating the variability in the duration of individual cell division cycles as reflected in varying step size (Edmunds and Adams, 1981).


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If the notion of the G, cellular clock is valid, one might expect to find repeated occurrences of some event in slowly dividing cells as G, undergoes successive revolutions. Indeed, Klevecz (1969, 1976) has observed, in synchronized mammalian cells, oscillations with periods of 3-4 hours in the activity of a number of enzymes that have no obligatory connection with other periodic events (such as DNA synthesis), and has suggested that this might be an expression of the cellular clock. Other markers might also be repetitively expressed. Furthermore, Klevecz and coworkers have found that in synchronous mammalian V79 cells perturbed by serum (Klevecz et al., 1978), heat ,shock (Klevecz et al., 1980b), or ionizing radiation (Klevecz et al., 1980a) at 0.5-hour intervals across their 8.5-hour cell division cycle, display a biphasic 4-hour pulse-response curve comprising both advances and delays in subsequent cell divisions. This evidence supports the notion of an underlying oscillator having a basic period of about 3 4 hours (see Shymko et a[., 1984). Does this mean that the Klevecz fundamental G, cycle is “the clock” in the sense that it underlies and generates circadian oscillations (never unambiguously observed in mammalian cell suspension cultures)? Does this mean that since G, equals 3-4 hours for mammalian cells, then &8(G,) equals 24 hours, or one circadian cycle, in other micro-organisms? If so, then we are dealing with the generation of relatively long 24-hour periods by a type of frequency demultiplication in which 6-8 “ticks” create one “tock” (Edmunds, 1978, 1981). But what, if anything, counts the number of revolutions of G,? Sweeney (1982), however, feels that her findings with Pyrocystis sp. exclude the possibility of such a quanta1 cycle, at least in this dinoflagellate. B. NATURE OF THE CLOCK(S)

In this review of the physiology of circadian rhythms in micro-organisms, it would be inappropriate to attempt to survey the possible molecular basis of circadian rhythms (see the still current proceedings of the Dahlem Workshop on this subject, edited by Hastings and Schweiger, 1976), although several models have been formulated on results obtained with unicellular systems, to which we have frequently alluded in our survey. On the other hand, it is perhaps useful to introduce the potential initiate to this field to some of the extant notions, if for no other reason than to provide reassurance that it is still virgin territory. 1. Classes of Molecular Models for Circadian Clocks

There are several different classes of model for endogenous self-sustaining circadian clocks (Table 9), which are neither mutually exclusive nor jointly exhaustive. They can be grouped into three or four main categories (Edmunds, 1976):(i) strictly molecular models, which rely on the properties of

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TABLE 9. Some molecular models for circadian clocks. Model

Key elements

Periodicity and~ l ~ in the structure ~ the properties of molecules themselves (e.g. alternation between two conformational states of subunits) “Network” Glycolytic Oscillator: by suitable (biochemical selection of allosteric constants feedback loops) and turnover numbers of key enzymes, frequency can be controlled over a large range (in principle, even 24 hours) Cell Energy Metabolism = the clock. Appropriate choice of sink (“deposition effect”) would allow self-oscillatory reactions on a circadian time scale Coupled Oscillators: cross-coupling among high-frequency oscillations in energy metabolism could generate circadian rhythms in energy transduction ~

vitro)

~(in

Transcriptional (“tape-reading”)

Reference Queiroz-Claret l ~ and Queiroz (1981)

~

Hess and Boiteux (1971); Goldbeter and Caplan (1976); Goldbeter and Nicolis (1976); Hess (1976) Das et al. (1 982) Sel’kov (1975, 1979); Reich and Sel’kov (1981) Winfree (1967); Goodwin and Cohen (1 969); Pavlidis and Kauzmann (1969); Wagner and Cumming (1 970); Pavlidis (1971); Vanden Driessche (1973); Wagner (1 976a,b) Cummings (1975)

Cyclic AMP Model: cyclic AMP, ATP, adenylate cyclase and phosphodiesterase are oscillating variables which would exhibit limit-cycle behaviour by allosteric feedback of AMP on adenylate cyclase and phosphodiesterase Heterodyne Endosymbiont Levandowsky (1981) Hypothesis: two prokaryotic colonists of a putatative ancestral eukaryotic cell emitted chemical pulses with slightly different short periods; their coincidence would have yielded a longer, circadian period Chronon Model: sequential Ehret and Trucco (1967); transcription of long DNA Barnett et al. (1971a, polycistron complexes in b); Wille et al. (1972) eukaryotic chromosomes, coupled with time-consuming, rate-limiting, temperature-independent


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TABLE 9 (continued) Model

Key elements

diffusion steps by mRNA to the ribosomes for translation would yield circadian periods Chronogene-Cytochron Model for cell cycle clocks: programmable, sequential transcription of segment of chromosomal DNA without requirement for translation Membrane models Molecule X actively transported into organelles, changing membrane configuration and transport capacity; passive diffusion then occurs until X is evenly redistributed Limit cycle behaviour in which an ion concentration gradient and membrane transport activity are the oscillating variables; slow translational diffusion of membrane proteins; cross-coupling; temperature compensation by changes in membrane lipid saturation Coupled Translation-Membrane Model: assembly, transport and insertion (loading) of essential proteins into membranes Monovalent Ion-Mediated Translational Control Model: intracellular monovalent ion concentration feedback-regulates synthesis and insertion of membrane proteins, resulting in changes in ion concentration

Reference

Edmunds and Adams (1981)

Sweeney ( 1 974b)

Njus et al. (1974, 1976); Njus (1 976)

Schweiger and Schweiger (1977) Burgoyne (1978)

molecules themselves for generating persisting 24-hour rhythms (see the recent and somewhat controversial model of Queiroz-Claret and Queiroz, 1981); (ii) feedback-loop, “network” models for oscillations in energy metabolism (e.g. glycolytic oscillations in yeast or ultradian enzymic oscillations) in which longer periods would be generated (frequency demultiplication) by energy depots (or “sinks”), appropriate allosteric constants and turnover numbers of key enzymes, or by cross-coupling (Pavlidis, 1969,1971)

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among individual oscillators (or even among prokaryotic endosymbionts) within a cell (we will not consider multicellular organization); (iii) transcriptional (“tape-reading”) models, the most prominent of which is the chronon model of Ehret and Trucco (1 967), wherein the transcription of a long polycistronic piece(s) of DNA with associated rate-limiting diffusion steps leading to translation would meter circadian time; and (iv) membrane models (reviewed by Edmunds, 1980c; Engelmann and Schrempf, 1980), in which the transport activity and other properties of various membranes in the cell, intimately related to state transitions in the fluid mosaic membrane itself (which, in turn, affect membrane structure), would in ensemble comprise a stable limit cycle oscillator that ultimately would account for the properties of circadian rhythms. The various models proposed (Table 9) sometimes overlap, for they often incorporate several different notions from each other. This is not at all surprising, since each has strengths and shortcomings for which it attempts to compensate by hybridization. For example, the chronon model (Ehret and Trucco, 1967) combined gene action and cell cycle controls but has fallen into some disrepute with the demonstration that DNA-dependent RNA synthesis does not seem to be important in the clock mechanism of Acetabularia sp., inasmuch as enucleated cells continue to exhibit a rhythm of photosynthesis, and inhibiting extranuclear RNA synthesis with rifampicin does not stop the clock [Section III.D, p. 114; but see Edmunds and Adams (1981) for an updated, variant version]. Similarly, although the “network” models deal with known biochemical oscillations, they have difficulty in accounting for the long period of circadian rhythms and their temperature-compensation. Finally, the earlier membrane models (e.g. that of Njus et al., 1974), though neatly accounting for temperature-compensation, had difficulty in accounting for the circadian period. This deficiency was addressed by Schweiger and Schweiger’s (1977) coupled-translation membrane model, invoking the time-consuming processes of assembly, transport and loading of essential proteins into membranes. Further, it incorporated the recent re-emphasis on the necessary role of protein synthesis in the maintenance (if not generation) of persisting circadian rhythms in species of Acetabularia (Section III.D, p. 114), Gonyaulax (Section III.B, p. 107) and Neurospora (Section IV.B, p. 123). Of course, the search for “essential” proteins may itself be very time-consuming (see Leong and Schweiger, 1979), with no guarantee of immediate success (although the advent of the powerful techniques of recombinant DNA research and gene “cloning” may offer some solace). Lastly, one must note that the search for the elusive clock per se could be doomed to failure if circadian timekeeping is not attributable to any one entity or subset of reactions in a cell; or, at least exceedingly difficult, if “all these aspects of cell chemistry [soluble enzyme kinetics, nuclear message transcription, membranes and the second messenger] are susceptible to circadian


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modification of their regulatory dynamics, and that the clock, volatile as a ghost, lurks now in one room, now in another, in different cell types” (pp. 54-55 in Hastings and Schweiger, 1976). 2. Insertions and Deletions of Time Segments into Cell Cycles: the Cytochron Another basic question arises from the critical problem of variation in the length of individual cell division cycles, particularly in the case of those division cycles modulated by a circadian clock (Edmunds and Adams, 1981; see Section V.A, p. 125), and from the phase-shifting data reflected in the experimentally derived pulse-response curve for light pulses for the cell division rhythm in micro-organisms such as E. gracilis (Edmunds et al., 1982, 1984; see Section III.A.1, p. 84). How are the observed shortenings and lengthenings of individual cell division cycles generated by a master (circadian) clock at the biochemical or molecular level?The evidence reviewed earlier (Section III.A.1, p. 90) formally demands that a clock of some sort predictably insert time segments into, or delete them from, the cell division cycle. One way in which this cycle may be “programmed” would be for a collection of timing loops of different lengths to couple together in various combinations to form a flexible timer, or “cytochron” (Edmunds and Adams, 1981). Temporal loci, or control points, would exist along the cytochron track at which decisions would be made with respect to the addition or deletion of time loops. On the grounds that this time “dilation” or “contraction” has an immediate molecular basis, we have hypothesized (Edmunds and Adams, 1981) the existence of “chronogenes” (our formal time segments) whose transcription would meter time and which would be inserted (or deleted) in varying numbers into the programmable cytochron (which, in turn, would interpret signals from the environment) by interaction with a circadian oscillator. Thus, the cytochron and the circadian clock are posited to be functionally independent (although not necessarily entirely separate as to mechanism). The chronogene would perhaps comprise a small segment of chromosomal DNA involving a hundred or so transcriptional units which would be folded into loops by bridging cross-links (of protein or DNA) at genetically defined loci so as to form a three-dimensional network of anastomosing loops. This entire complex would constitute a giant functional gene capable of metering periods as long as 24 hours by incorporation of 20-40 nucleotides each second, as originally proposed in the chronon model of Ehret and Trucco (1967), but unlike the chronon, not involving a translational step and having a programmable sequence of transcription of the units. The details of this “thought” model await experimental verification (Edmunds and Adams, 198 1).

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C. EVOLUTION OF CIRCADIAN RHYTHMICITY

Whence came circadian clocks? Circadian rhythmicity is part of a continuum of biological oscillations of varying periodicity (see Fig. 1) and was probably derived through the modification of existing cellular oscillations early in the evolution of eukaryotes. In order to answer this question, an arm-chair evolutionary biologist must attempt to understand the selective forces that may have been involved at that time. Pittendrigh (1965, 1966) long ago speculated that the light-dark cycle was the historical selective agent of circadian oscillations and that these periodicities reflected an adaptation of the primitive cell to cope with the deleterious effects of solar radiation on cellular processes such as gene induction and DNA replication. In particular, he reasoned that the strong ultraviolet component of light (not screened at that time by the then underdeveloped ozone layer) would have caused the formation of thymine dimers in the DNA backbone. Thus, an organism that relegated its replicative processes to night-time darkness would have had an advantage; and this, of course, would probably have led to its concentrating its growth and synthetic processes in the day. In this manner, the first circadian clock may have had its genesis. Indeed, supportive arguments for this ingenious hypothesis include the observations that (i) the rhythmic gating of cell division to the dark periods in present-day diurnal LD cycles (see Sections III.A.l, p. 80 and V.A.1, p. 125 would minimize the inhibitory effects of light and (ii) the rhythmic regulation of photosensitive membrane components would minimize the photo-induced loss of membrane function (Epel,.l973; Woodward et al., 1978; Ulaszewski et af., 1979; Edmunds, 1980a). Furthermore, Paietta (1982) recently has proposed that one of the original selective forces involved in evolution of circadian rhythmicity was the joint effect of the LD cycle and the increasing concentration of free oxygen early in eukaryotic evolution. Circadian periodicity would have provided a protective mechanism for minimizing the deleterious effects of diurnally imposed photo-oxidation, on the one hand by prevention of photo-oxidative destruction of sensitive components through excited state quenching by pigments, and, on the other, by restricting the highly photosensitive processes to darkness. As Klevecz (1984) has stressed, however, the search for selective forces is inextricably intertwined with the primitive environment. The evolution of the earth-moon system and the consequent changes in day length through geological time must be taken into account. During pre-biotic evolution the solar day was quite short-probably less than 10 hours, and perhaps only &5 hours-and was punctuated by boiling tides and intense ultraviolet radiation. If a primitive oscillator with the capacity to entrain to sunlight or temperature changes has evolved at that time, it would have had a relatively short free-running period.&e speculates, therefore, that modern circadian rhythms


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5 24 h) have evolved by organismic fusion and coupling of systems with short-period oscillators that first emerged when the earth’s rotation was rapid and the moon was in close proximity. Thus, cellular oscillators as we now see them would represent “vestiges” of a primitive circadian clock having a much shorter period (perhaps even that of the quanta1 cycle, G, (see subsection A.3, p. 128), that he and his coworkers have observed in cultured mammalian cells?).

(z

D. MULTIPLE CELLULAR OSCILLATORS: A UNICELLULAR CLOCKSHOP?

I . Intracellular Clockshops A basic but unanswered question in the field of circadian rhythms concerns the number of clocks that might exist in a single micro-organism. A large part of the difficulty in addressing this question lies in our ignorance of the mechanism of any clock. Probably the preponderance of opinion has it that there is but one central oscillator within the cell and that the numerous overt rhythms observed as circadian outputs are merely different hands of this single driving entity. The observations of McMurry and Hastings (1972a) that four separate rhythms in the dinoflagellate Gonyaulax polyedra (see Section III.B, p. 102) all seem to have the same free-running period with similar temperature coefficients, and that they all respond identically when perturbed by a resetting dark pulse, give rather strong support to this notion. Also, the simultaneous long-term recording of two or more rhythms in Acetabularia sp. do not appear to show any divergence in their respective periods (H.-G. Schweiger and H. Broda, personal communication). On the other hand, some recent work (Laval-Martin et al., 1979; Edmunds and Laval-Martin, 1981) with Euglena gracilis maintained in higher-frequency LD cycles (see Section III.A.3, p. 97) has revealed that the phase relationship between the rhythm of photosynthetic capacity and that of chlorophyll content varied, suggesting the possibility of desynchronization among circadian rhythms in a multi-oscillator unicellular organism. Finally, Dharmananda and Feldman (1979) have reported a spatial distribution of circadian clock phase for the conidiation rhythm in ageing cultures of Neurospora crassa: a gradient of phases was found which was a function of the length of time the clock had been free-running in a given part of the culture. These phase differences within a single interconnected mycelium demonstrated the absence of total internal synchronization between adjacent regions of the hyphae, despite the fact that these regions are replete with cellular junctions (which presumably would permit intercellular communication, yet obviously did not allow effective transfer of phase information).

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2. Intercellular Communication: Coupled Oscillators?

Long-term persisting circadian rhythms in populations of micro-organisms raise another interesting question. Why does not the synchrony decay in the absence of a synchronizing Zeitgeber, in the same manner that cell division synchrony, for example, decays in bacterial and mammalian cell cultures (usually within three to four cycles)? Assuming that no subtle geophysical factors are phasing the population, we are left with two alternatives. First, the free-running period of the oscillator must be exceedingly precise and almost identical with those of other cells or second, some sort of intercellular communication must occur that maintains synchrony within the population, or network, of self-sustaining oscillators (Goodwin, 1963; Edmunds and Funch, 1969b; Pavlidis, 1969, 197 1; Edmunds, 197 1). With regard to the first alternative, much depends on what assumptions are made as to the nature of the variance. Thus, by the “random walk” model (in which “fast-” or “slow-running’’ cells do not necessarily transmit this property to their progeny, or even from cycle to cycle), the rate of dispersion could be quite slow, perhaps requiring several weeks before the peak of some rhythm became so spread out that it was obliterated (“damped out”). Indeed, recent work on the rhythm of glow luminescence in Gonyaulax polyedra (see Section III.B.1, p. 102) suggests that the gradual decay does occur over such an extended timespan because the rhythm is accurate to within 2 minutes a day and the variance in circadian period among individual cells is about I8 minutes (Njus er al., 1981). The alternative hypothesis of intercellular cross-talk has been examined theoretically (Goodwin, 1963; 1976; Winfree, 1967; Pavlidis, 1969) from both a mathematical and a biochemical standpoint. Experimental tests of this hypothesis in micro-organisms, however, have been inconclusive or negative. Thus, Brinkmann (1966) found no evidence for intercellular communication in Euglena gracilis when out-of-phase cultures were mixed and the resultant phase of the mobility rhythm examined. Similarly, mixing experiments with synchronously dividing populations of this flagellate (Edmunds, 197 1) did not support this notion. Finally, mixing experiments for the rhythm of glow bioluminescence\ in Gonyaulax polyedra (Hastings and Sweeney, 1958; Sulzman et al., 1982), as well as experiments with Acetabularia mediterranea cells whose rhythms of photosynthetic oxygen evolution were out-of-phase (Mergenhagen and Schweiger, 1974), and the results for the conidiation rhythm in Neurospora crassa (Dharmananda and Feldman, 1979) discussed in subsection D. 1, all indicated the absence of internal communication and supported the cellular autonomy of circadian clocks. E. CHRONOPHARMACOLOGY AND CHRONOTHERAPY

Although most micro-organisms probably are not the organism of choice for


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these studies, it is perhaps worth noting that circadian and cell cycle clocks can play a profound role in the treatment of disease (see, e.g. Reinberg and Halberg, 1979). In foregoing sections (1II.A. 1, p. 75 and V.A, p. 124) we have documented much of the experimental evidence for viewing the cell cycle as an oscillatory system (Edmunds and Laval-Martin, 1984; Shymko et a/., 1984). In this short synopsis, the role of cell cycle clocks in the pathology and treatment of cancer will be considered. Wille and Scott (1984) have recently discussed cell division cycle-dependent integrated control of cell proliferation and differentiation in normal and in neoplastic mammalian cells. Particularly germane is their exciting speculation that neoplastic transformation might be attributable to defective control of the cell division cycle. They suggest that the two-stage model of carcinogenesis could be explained by specific alterations in the kinetic parameters controlling the dynamics of a non-linear, dynamic biochemical system comprising both a high-amplitude and a low-amplitude stable limit-cycle oscillation “nested” within the mitotic oscillator (Wille, 1979). The importance of chronobiology for basic cancer research and chemotherapy has also been quite recently stressed by Scheving (1984) and by Msller (1984), among others. There is no lack of awareness that the cell division cycle is most significant for cancer treatment, if for no other reason than the fact that studies of synchronized mammalian cells have shown that their sensitivity to a large number of cytostatic drugs (such as cytosine arabinoside), as well as to ionizing radiation, is highly dependent on the stage of the cell division cycle. Indeed, some of these drugs may also lead to a partial synchronization of the cell division cycle and have been so employed to obtain synchronized cell populations both in uitro and in uivo. This strategy can maximize the chance for survival and cure by applying the minimum dose of drug necessary to kill the phased malignant cells at the time of their cell division cycle when they are most susceptible. It is obvious, therefore, that the diurnal (and circadian) rhythmicities observed in cell flux and in the distribution and duration of phases in the cell division cycle (see Edmunds, 1984) must be taken into account in the design of an appropriate chemotherapeutic regimen. But there is another equally important consideration for maximizing the results of radio- and chemotherapy -host tolerance. It is unfortunate that in most of the earlier cancer work there is little mention of the role of rhythmic variations (particular circadian periodicities) in the susceptibility of the whole organism to the toxicity of the drug(s) being utilized for treatment. Both Scheving (1984) and Msller (1984) review the experimental evidence revealing that properly designed protocols (such as sinusoidally varying drug courses during the 24-hour day) can dramatically enhance survival and cure rates by concommitantly maximizing tolerance of the host to the drug through a temporal “shielding” of normal healthy tissues. Thus, “when” to treat must

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assume importance together with the “what” and “where” (Haus el al., 1974; Halberg, 1975). F. CELL CYCLE CLOCKS IN DEVELOPMENT A N D AGEING

It seems somehow most appropriate that in the last section of this chapter we deal briefly with the role of cell cycle clocks in development and in ageing-the a and the R of biological existence. In his state-of-the-art overview, Mitchison (1984) has noted that one line of evidence that supports the existence of “independent-timer’’ periodic controls, which continue to function even if growth or division is blocked or perturbed, is afforded by the development of eggs and early embryos. As he states, these systems are particularly interesting in that “they break many of the normal rules of the cell cycle.” Satoh (1984), for example, has continued on this theme, reviewing the evidence for cell division cycles serving as the basis for timing mechanisms in early embryonic development of animals. He concludes that the developmental clock which determines the time of cellular differentiation is closely associated with the DNA-replication cycle and appears to be independent of the cytoplasmic clock which times early morphogenetic events. Similarly, Belisle et al. (1 984) have given experimental evidence implicating a central timer mechanism in regulation of cell division cycles during development of sea urchin embryos. These authors propose a “sequential interactive network” (SIN) model which couples (by gene action) a semi-autonomous chromosome cycle with an independent metabolic cycle that would regulate the former. At the other end of biological existence, one might consider cell cycle clocks and ageing. Is there a life cycle clock? The viewpoint that ageing can be viewed as the loss of temporal organization has been treated comprehensively by Samis and Capobianco (1978); but what does this mean at the level of the cell? Theories of cellular ageing can be divided into two general categories: the genetic “program” theories, in which ageing would be an event caused by the active expression of specific “ageing genes” or “cell clock longevity genes”, or by the passive exhaustion of vital genetic information; and cumulative error, “wear and tear” theories, in which senescence would result from the progressive damage to organelles or to the accumulating error in cytoplasmic information-carrying molecules (Finch and Hayflick, 1977). Ever since Hayflick and Moorhead’s (1961) report that normal human embryonic fibroblasts, cultured in vitro, were limited in their capacity to replicate (50 & 10 population doublings), there has been considerable controversy as to whether the so-called “phase 111” phenomenon (the period of greatly decreased replicative capacity) is reflective of cellular ageing in viva, and therefore whether the study of cellular senescence in uitro has any relevance to the normal mammalian lifespan. Many investigators feel, nevertheless, that the


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biological changes preceding both phase I11 in vitro as well as mortality in vivo may be at least indirectly responsible for both phenomena. Zorn and Smith (1984) are among this group and have reported recent experiments with reconstructed cells (in which “old” fibroblast nuclei, or karyoplasts, were fused with “young” cytoplasms, or cytoplasts, and vice versa) designed to determine the relative contributions of the nucleus and cytoplasm to the phase 111 phenomenon. Their results, supporting a predominantly nuclear role in cellular senescence, provide evidence that the expression of the phase 111 “clock” is encoded into the genetic material, presumably on one or more chromosomes. If so, the authors note that it should be feasible to transfer this “ticking” clock from an old cell to another younger one that has gone through fewer population doublings (as they did with whole-cell hybrids) and then identify by karyotype analysis those chromosomes responsible for phase 111-i.e. the senescent clock could be localized. Finally, they note the intriguing possibility that cellular ageing and cellular transformation might map on the same chromosome(s) and be related by differential expression of the same genes. See note added in proof, p . 301

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Polyol Metabolism in Fungi D. H. JENNINGS Botany Department, The University, Liverpool, U K

I. Introduction . . . . . . . 11. Enzymes . . . . . . . . A. Glycerol dehydrogenase (NAD+) . . B. Glycerol-3-phosphate dehydrogenase (NAD+) C. Xylitol dehydrogenase (NAD+) . . . D. Mannitol-I-phosphate dehydrogenase. . E. Polyol dehydrogenase (NADP+) . . . . . F. Mannitoldehydrogenase(NAD+) G. Glyceroldehydrogenase (NADP+) . . H. Ribitol-5-phosphate dehydrogenase (NADP+) . I. Mannitoldehydrogenase(NADP+) . J. Glycerophosphate dehydrogenase . . . . . . . K. Glycerol kinase . L. Mannitol kinase . . . . . M. Mannitol-1-phosphatase . . . . . . N. Sorbitol (glucitol) dehydrogenase. . . 0. Glycerol oxidase. . . 111. Mastigomycotina . . . . . . . . . . . . IV. Zygomycotina . A. Absidia glauca , . . . . . V. Ascomycotina and Deuteromycotina . A. Yeasts grown on n-alkanes . . . . B. Ahernaria alternata . . . . C. Utilization of pentose sugars by Candida uti’lis . . . . D. Dendryphiella salina . . . . . . E. Neurospora crassa F. Osmophilic yeasts , . . . G. Pichia (Candida)guilliermondii . . . H. Pyrenochaeta terrestris . . . . I. Saccharomyces cerevisiae . . . . . . J. Sclerotinia sclerotiorum . . . . . . VI. Basidiomycotina A. Agaricus bisporus. . . . . . . . . . B. Schizophyllum commune VII. Regulation of cytoplasmic pH values in fungi . VIII. Comment. . . . . . . . . . . . . . References. ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 25 ISBN 0-12-027725-4

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I. Introduction

Polyols (sugar alcohols) are widely distributed in fungi (Lewis and Smith, 1967; Pfyffer, 1980; Pfyffer and Rast, 1980a,b; Bieleski, 1982). Lewis and Smith (1967) in their seminal review brought to the attention of mycologists and plant biologists the importance of these compounds for fungi and green plants. This review, besides providing a wealth of information about polyol metabolism in fungi, pointed out that polyols could serve the following roles in living organisms: (i) act as carbohydrate reserves, (ii) act as translocatory compounds, (iii) function in osmoregulation and (iv) in coenzyme regulation and storage of reducing power. Since 1967 a wealth of new information has come to hand regarding polyols in fungi, though it has to be admitted that much of this information is fragmentary, with relatively few intensive studies having been undertaken of particular fungi and their polyols. This review attempts to draw together this new information and to integrate it with what was known before 1967. Emphasis has been placed on those fungi for which a relatively coherent body of information about polyol metabolism is available though, where possible, an attempt also has been made to integrate this information into a more general framework.

11. Enzymes

We now know quite a lot about the enzymes concerned with polyol metabolism in fungi. The following sections summarize the major findings, making an attempt to present a general picture of a particular enzyme as examined for a range of fungi. However, as will be seen, in certain circumstances, only one particular fungus has been studied with respect to an enzyme. Hopefully the indication of these cases will encourage the search for and study of the enzyme in other fungi. In many instances, it is not clear as to the degree of purity of the enzyme that was being studied, nor whether the properties described related to its maximum catalytic activity. The fact that most polyols, or their phosphorylated derivatives, can be metabolized by more than one enzyme should sound a note of caution for those investigators who are dealing with relatively unpurified cell extracts. Further, it needs to be remembered that the growth conditions for one fungus may not be the same for another. As will be seen, there are a number of instances of polyols being metabolized by inducible enzymes, and the extent to which such enzymes are induced or repressed may have a bearing on the data obtained, particularly if the extract studied is relatively impure. It has to be stressed that we have no clear idea regarding the exact location of the enzymes being considered. As will be seen, a number of enzymes have

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relatively high K , values for their substrates and a high pH optimum for the oxidation of polyols, which is likely to be a consequence of the buffer more effectively consuming protons at such pH values. It is conceivable that these particular enzymes are membrane-bound. The groups to which the various fungi belong are identified and designated as follows: Mastigomycotina (M), Zygomycotina (Z), Ascomycotina (A), Deuteromycotina (D) and Basidiomycotina (B). A. GLYCEROL DEHYDROGENASE (NAD+)

This enzyme (glycerol :NAD 2-oxidoreductase; EC 1.1.1.6) has been studied in Geotrichum candidum (D) (Itoh, 1982). Glycerol dehydrogenase oxidizes glycerol to dihydroxyacetone. A number of other alcohols, particularly propane- 1,2-diol and butane-2,3-diol, are also oxidized but at somewhat slower rates than that for the oxidation of glycerol. Indeed the highest glycerol dehydrogenase activity was obtained from mycelia grown on propane-1,2-diol (Itoh and Umeda, 1982), and was of the order of eight times that from cells grown on glycerol. The pH optimum for the oxidation of glycerol was found to be 10.5, and the apparent K,,, value for that substrate was 3.7 mM; the pH optimum for the reduction of dihydroxyacetone was 5.5 and the apparentK, for that substrate, 1.2 mM. The enzyme was stimulated (about 60%) by 100 mM calcium chloride, and by 200-500 mM potassium and ammonium chlorides to about the same extent, suggesting that chloride is the effective ion. Phosphate at 100 mM was inhibitory. There are two enzymes in G . candidum with very similar properties and molecular weights (1.35.105 and 1.3.105). +

B. GLYCEROL-3-PHOSPHATE DEHYDROGENASE (NAD')

This enzyme (sn-glycerol-3-phosphate*:NAD+ 2-oxidoreductase; EC 1.1.1.8) has been studied in Succharomyces cereuisiue (A) (Merkell et ul., 1982). Little is known about this enzyme, which is involved in the production of glycerol from glucose in yeast growing under anaerobic conditions (see Section V.1, p. 174). It has been purified 5100-fold and shown to have a pH optimum of between 6.8 and 7.2 for the reduction of dihydroxyacetone phosphate. The equilibrium lies strongly in the direction of glycerol 3-phosphate production. The enzyme is readily inactivated by phosphate and chloride ions. * With respect to the nomenclature of stereoisomers of glycerol, readers should consult the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature on the nomenclature of lipids (Journal of Biological Chemistry (19 ) 242,4845-4849).

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D. H. JENNINGS

c . XYLITOL DEHYDROGENASE (NAD+) (D-XYLULOSE REDUCTASE (NAD+))

This enzyme (xylitol :NAD 2-oxidoreductase (D-xylulose forming); EC 1.1.1.9) has been studied in Candida albicans (D) (Veiga, 1968b; Chakravorty et al., 1962; Horitsu and Tomoeda, 1966), Cephalosporium chrysogenum (Birken and Pisano, 1976), Melampsora lini (B) (mycelium and uredospores) (Clancey and Coffey, 1980), Penicillium chrysogenum (D) (Chiang and Knight, 1959) and Sclerotinia sclerotiorum (A) (Wang and Le Tourneau, 1973). Except for M . h i , the enzyme has been extracted from cultures grown on D-xylose. From the studies on C . utilis and P . chrysogenum, it appears that this enzyme oxidizes D-xylitol to D-xylulose, D-glucitol to D-fructose, D-mannitol to D-fructose and ribitol to D-ribulose, the activities with respect to the first two substrates being about twice those for the second two. In all cases, xylitol was found to be the substrate giving maximal activity. Reduction of carbohydrates has been studied in C . albicans, C . utilis and M . hi;for the last fungus the results are somewhat difficult to interpret because different relative activities have been obtained with uredospores and with mycelium grown on glucose and sucrose. However, there is little doubt that the enzyme from M . lini can reduce D-fructose. The enzyme from C . utilis reduces D-xylulose at a higher rate than other ketoses. At neutral pH values, it is polyol formation that is favoured. The pH optimum for xylitol oxidation by the enzyme isolated from C. utilis was 9.0, for Ce. chrysogenum it was 8.8-10.4 and for S. sclerotiorum, 9.5-10.0. Apparent K , value for the same reaction was 11 mM for the enzyme from C . utilis and 71.5 mM for the Ce. chrysogenum enzyme. The reverse reaction for the latter fungus has an apparent K , for D-fructose of 200 mM. Oxidation of L-arabitol by mycelium of P . chrysogenum grown on L-arabinose may be brought about by a similar enzyme (Chiang and Knight, 1961). L-Xylulose and L-ribulose are produced, with more of the former, suggesting that it may be the major product formed under physiological conditions. +

D. MANNITOL- 1 -PHOSPHATE DEHYDROGENASE (NAD +)

This enzyme (D-mannitol- 1-phosphate :NAD 2-oxidoreductase; EC 1.1.1.17) has been studied in Aspergillus candidus (D) (Strandberg, I969), Aspergillus niger (Kiser and Niehaus, 198l), Pyrenochaeta terrestris (A) (Aitken et al., 1969a), Pyricularia oryzae (D) (Yamada et al., 1961) and Sclerotinia sclerotiorum (A) (Wang and Le Tourneau, 1972). Mannitol- 1-phosphate dehydrogenase does not appear to be present in members of the Basidiomycotina (Hult et al., 1980), though there is a need for critical examination of the procedures used for isolation of the enzyme (see Section V.B, p. 161) and for studies involving a wider range of genera. The +

153


TABLE 1. Mannitol-I-phosphate dehydrogenase; values for apparent K, and pH optima

Fructose 6-phosphate reduction

K, (mM)

Organism

Aspergillus candidus Aspergillus niger Pyricularia oryzae Pyrenochaeta terrestris Sclerotinia sclerotiorum

Optimum pH value

Mannitol 1-phosphate oxidation K, (mM)

Optimum pH value 9.8

0.54

0.3

7.4 7.5

0.038 0.05 0.1

9.5 8.1 9.5- 10.5

enzyme has been isolated from Mucor rouxii (Boonsaeng et a]., 1976) which is surprising, since the fungus does not appear to contain mannitol (Pfyffer and Rast, 1980b). This matter urgently needs to be re-examined; conceivably what was observed relates to the activity of an enzyme whose substrate in vivo is not mannitol 1-phosphate. The enzyme appears to have very high specificity for its substrates, mannitol 1-phosphate and fructose 6-phosphate. In P . terrestris and S . sclerotiorum there was some activity with glucose 6-phosphate 14 and 10% respectively of that with fructose 6-phosphate) and in S. sclerotiorum with glucitol 6-phosphate (6% of activity with mannitol 6-phosphate). The position of the equilibrium of the reaction involving the two substrates, fructose 6-phosphate and mannitol 1-phosphate, lies in the direction of the latter. Table 1 gives data of apparent K , values for the two major carbohydrate substrates, and pH optima for oxidation and reduction of these two substrates. It can be seen that, compared with mannitol dehydrogenase (NADP+)(EC 1.1.1.138; see Section 11.1, p. 156), mannitol-1 -phosphate dehydrogenase has a high affinity for its two substrates. But, like the former enzyme, the latter appears to have optimum activity for oxidation of carbohydrate well into the alkaline region (pH 9-10), while the optimum activity for reduction occurs at less alkaline pH values. In S . sclerotiorum the highest specific activity was found in mycelium that had been grown on D-mannitol, then, in declining order, when grown on glucitol, D-glucose and L-arabinose (Wang and Le Tourneau, 1972). Boonsaeng et al. (1976) have argued that the role of the enzyme is in replenishment of the NAD+ removed during glycolysis. E. POLYOL DEHYDROGENASE (NADP+)

This enzyme (alditol :NADP+ 1-oxidoreductase; EC 1.1.1.21) has been

154

D. H . JENNINGS

studied in Candida albicans (D) (Veiga et al., 1960; Veiga, 1968a), Candida utilis (D) (Scher and Horecker, 1965; Horitsu et al., 1968), Penicillium chrysogenum (D) (Chiang and Knight, 1959)and Pichia quercuum (A) (Suzuki and Onishi, 1975). Polyol dehydrogenase, which is probably better named alditol dehydrogenase (NADP+), has been extracted from cells and mycelium grown on D-xylose. The optimum pH value for reduction of this carbohydrate by the enzyme from Pi. quercuum was 6.3, and for the reverse reaction 10.0-10.5; even so the velocity of this reaction was only 5.8% of the forward reaction at pH 6.3. The enzyme from C . albicans behaves similarly with respect to pH value. The enzyme from the four fungi exhibits broad specificity; thus, one of two similar enzymes from Pi. quercuum was found to act on the following aldoses (with numbers in brackets indicating relative activity) D-xylose (loo), D,L-glyceraldehyde (3 14), D-erythrose (328), D-ribose (89), L-arabinose (1 53) and D-galactose (53). The same broad specificity has been demonstrated for polyols. Relative activity values for the enzyme from C . utilis are as follows: xylitol (loo), galactitol (1 87), L-arabitol (167), glucitol (146), ribitol (57.3, erythritol (40), glycerol (36). The enzyme from C . utilis has a high affinity (apparent K , 0.92 mM) for D-erythrose and a very low affinity for xylitol (apparent K , 530 mM). The affinity for D-xylose was intermediate (apparent K , for the enzyme from C . albicans was 6.7 mM, from C . utilis, 7.7 mM, and from Pi. quercuum, 78 mM). Reduction of L-arabinose by the mycelium of P . chrysogenum grown on the same sugar seems likely to be brought about by a similar enzyme (Chiang and Knight, 1961). L-Arabinose was reduced rapidly to L-arabitol, while D-xylose, L-xylose and D-glucuronic acid were reduced less rapidly. There was no activity with D-glucose or D-mannose. The apparent K , for L-arabinose was 200 mM, and for D-xylose, 120 mM. Concurrent synthesis of glycerol and erythritol in mycelium of Aspergillus niger and Penicillium chrysogenum, when grown in saline media (Adler et al., 1982), suggests that the enzyme may be involved in synthesis of both of these polyols. F. MANNITOL DEHYDROGENASE (NAD+)

This enzyme (D-mannitol :NAD 2-oxidoreductase; EC 1.1.1.67) has been studied in Absidiaglauca (Z) (Ueng et al., 1976;Ueng and McGuinness, 1977), Aspergillus oryzae (D) (Horikoshi et a[., 1965), Chaetomium globosum (A) (Adomako et al., 1972) and Schizophyllum commune (B) (Niederpruem et al., 1965). Mannitol dehydrogenase is able to oxidize not only D-mannitol but also D-arabitol and glucitol at something like l W O % of the rate found with mannitol. Table 2 gives the relevant apparent K , values, and shows that the +

155


TABLE 2. Mannitol dehydrogenase (NADP+); values for apparent K,,, and pH optima Fructose reduction Organism Agaricus bisporus Agaricus campestris Aspergillus candidus Aspergillus oryzae Aspergillus parasiticus Cephalosporium chrysogenum Diplodia viticola Melampsora lini Penicillium chrysogenum

K, (mM)

Optimum pH value

190 1.4

7.9-8.1 -

Mannitol oxidation K,

(mM) 34 0.46

-

-

-

103 -

-

7.0

60 100 71.5

128

-

-

760

Optimum pH value 8.7-8.8 -

9.8 9.0 9.0 9.0-10.0

8.5 -

7.3

pH optimum for reduction of mannitol to fructose is 9.5-9.8, whereas that for the reverse reaction is 7.0-7.3. The enzyme appears to have a low affinity for its two carbohydrate substrates. The kinetics of the enzyme from Absidiu gluucu have been investigated in considerable detail, though the physiological significance of the information obtained has yet to be assessed. A potentially important finding was the fact that mannitol 1-phosphate inhibited the enzyme non-competitively, and probably is significant in terms of regulation of mannitol metabolism. G. GLYCEROL DEHYDROGENASE (NADP+)

This enzyme (glycerol :NADP+ oxidoreductase; EC 1.1.1.72) has been studied in Mucor juuanicus (Z) (Dutler et ul., 1977; Hochuli et ul., 1977), Neurospora crassa (A) (Viswanath-Reddy et ul., 1978)and Rhodotorulu sp.(D) (Sheys and Doughty, 1971a,b; Sheys et al., 1971; Watson et ul., 1969). Glycerol dehydrogenase, unlike polyol dehydrogenase (EC 1.1.1.2l), which is also able to oxidize glycerol and reduce glyceraldehyde in the presence of NADP(H), is relatively substrate-specific acting on glycerol, D-glyceraldehyde, L-glyceraldehyde and dihydroxyacetone. The pH optimum for reduction of D-glyceraldehyde was 6.5 ( N . crussu) and the apparent K m value for the same substrate has been found to be, respectively, 8.0 mM (Mu.juuunicus), 11.5 mM ( N .crussu) and 0.9 mM (Rhodotorulu sp.). The reverse reaction has a pH optimum of 9.C9.5, the apparent K , value for glycerol being 250 m M (A4u.juuunicu.s)and 143 mM ( N . crussa). It has been shown with the enzyme from Rhodororulu sp. that multivalent cations inhibit reduction of aldose and activate oxidation of glycerol. The enzyme from Mu. juuunicus has been

156

D. H.

JENNINGS

-

reported to have a molecular weight of about 1 lo5, with a subunit molecular weight of 2.8. lo4. The molecular weight for the N . crassa enzyme has been estimated to be 1.6.105, with a subunit molecular weight of about 4 . 3 ~ 1 0 ~ . When this enzyme was subjected to isoelectric focussing, there were five protein bands. The enzyme from Rhodotorula sp. has been reported to have a total molecular weight of 6.1 lo4, with two sub-units of molecular weights 3 . 8 ~ 1 0and ~ 2 . 3 ~ 1 0 the ~ ; former has enzyme activity whereas the latter is inactive.

-

H. RIBITOL-5-PHOSPHATE DEHYDROGENASE (NADP+)

This enzyme (~-ribitol-5-phosphate :NADP+ 2-oxidoreductase; EC 1.1.1.137), which converts ribitol 5-phosphate into D-ribulose 5-phosphate, has been extracted from Pichia guilliermondii (A) (Miersch et al., 1980). The apparent K,,, value for ribitol5-phosphate was 0.35 mM, and the pH optimum for oxidation of the carbohydrate was 7.3. The activities with other substrates (relative to ribitol) were glycerol (60%), D-mannitol (53%), D-glucitol (4973, L-arabitol (43%) and myo-inositol (26%). The enzyme is sensitive to ammonium ions. When the cells were made iron-deficient, synthesis of this enzyme was derepressed by a factor of 3-4 compared with non-iron-deficient cells. When glucose, ribitol or 4-aminobutyric acid were provided as carbon sources, enzyme activity was increased relative to those found with carbon sources such as D-ribose, L-alanine and acetate. I . MANNITOL DEHYDROGENASE (NADP+)

This enzyme (D-mannitol :NADP+ 2-oxidoreductase; EC 1.1.1.138) has been studied in Agaricus bisporus (sporocarps) (B) (Ruffner et al., 1978), Agaricus campestris (sporocarps) (B) (Edmondowicz and Wriston, 1963), Aspergillus candidus (D) (Strandberg, 1969), Aspergillus oryzae (D) (conidia) (Horikoshi et al., 1965), Aspergillus parasiticus (D) (Niehaus and Diltz, 1982), Cephalosporium chrysogenum (D) (Birken and Pisano, 1976), Diplodia viticola (A) (Strobe1and Kosuge, 1965), Melampsora h i (B) (mycelium and uredospores) (Clancey and Coffey, 1980) and Penicillium chrysogenum (D) (Boutelje et al., 1983). Mannitol dehydrogenase oxidizes predominantly D-mannitol and NADPH, and reduces D-fructose and NADP+. The enzyme from Agaricus bisporus oxidizes D-glucitol(27% of activity with mannitol); this also is so for the enzymes from Aspergillus oryzae (773, Cephalosporium chrysogenum (16%) and Diplodia viticola (24%). There are indications that D-arabitol can be oxidized, but usually at a very low rate. The enzyme from Melampsora h i , particularly from uredospores, is capable of reducing L-arabinose, D-ribose and D-xylose at relatively high rates. The enzyme from other fungi has not

157


been studied with respect to the range of substrates capable of being oxidized. The activity of the enzyme appears to be dependent on the carbon source used for growth (Strandberg, 1969; Clancey and Coffey, 1980). The enzyme has a high pH optimum (about 9.0; see Table 3) for oxidation of mannitol, except for the enzyme from P . chrysogenum. The reason for this is not clear, but may be a consequence of the buffer used (Strandberg, 1969).The Michaelis constants for the two carbohydrate substrates can be high (Table 3). The oxidative (but not the reductive) capacity of the enzyme with respect to the carbohydrate substrates was stimulated (240%) by 10 mM calcium. Zinc (in the range 1-30 pM) has been shown to be a powerful, apparently competitive, inhibitor of the enzyme from Aspergillus parasiticus. The enzyme from P . chrysogenum is inhibited 30% by 20 pM zinc and 65% by 2 pM cadmium. The following molecular weights have been determined: Aspergillus parasiticus (1.4. lo5,with four equal subunits), Cephalosporium chrysogenum (3. lo5, with apparently 10 subunits) and P . chrysogenum (1.6- lo5, with four equal subunits). J. GLYCEROPHOSPHATE DEHYDROGENASE

This enzyme (~-a-glycerol-3-phosphate:cyclohexane oxidoreductase; EC 1.1.99.5) has been studied in Candida utilis (D) (Halsey, 1982) and Neurospora crassa (A) (Courtwright, 1975a). Glycerophosphate dehydrogenase is located on the inner mitochondria1 membrane and is induced under the same conditions as those found for glycerol kinase (EC 2.2.1.30). There is no activity with NAD+ or NADP+. Flavin adenine dinucleotide (FAD) has been implicated as the co-enzyme; it has been shown to be present in the active enzyme of C . utilis along with iron and copper. The same enzyme has a molecular weight of 5 - lo5. K. GLYCEROL KINASE (GLYCEROKINASE)

This enzyme (ATP :glycerol phosphotransferase; EC 2.7.1.30) has been TABLE 3. Mannitol dehydrogenase(NAD+);values for apparent Km and pH optima

Fructose reduction Km

(mM) Absidia glauca Aspergillus oryzae Chaetomium globosum Schizophyllurn commune

79

Optimum pH values 7.C1.2

-

-

1280

7.3

-

-

Mannitol oxidation Km

(mM) 22 50 240 5.6

Optimum pH values 9.6 9.8 9.5 9.0

158

D. H.

JENNINGS

studied in Candida mycoderma (D) (Bergmeyer et al., 1961) and Neurospora c r a m (A) (Courtwright, 1975a). Glycerol kinase is induced in N . crassa by growth on glycerol. The studies on the enzyme from C. mycoderma were made with cells grown on glycerol. The enzyme from the latter fungus has been shown to phosphorylate dihydroxyacetone. The molecular weight of the enzyme and apparent K , value for glycerol are, respectively, 2.51 los and 6.10-* mM, for that from C . mycoderrna, and 1 . 2 ~ 1 and 0 ~ 5 - lo-' mM for that from N . crassa. The latter enzyme has been shown not to be inhibited by fructose 1,6-bisphosphate, fructose 1-phosphate, fructose 6-phosphate, glucose 1-phosphate or glucose 6-phosphate. L. MANNITOL KINASE

This enzyme (ATP: mannitol-1-phosphotransferase; EC 2.7.1.57) has been detected in crude extracts of Absidiaglauca (Z) (Ueng et al., 1976) (see Section IV.A, p. 160). There is no information about its properties. M. MANNITOL- 1 -PHOSPHATASE

This enzyme (D-mannitol- 1-phosphate phosphohydrolase; EC 3.1.2.22) has been studied in Aspergillus candidus (D) (Strandberg, 1969), Penicilliurn notatum (D) (Boonsaeng et al., 1976), Pyrenochaeta terrestris (A) (Aitken et al., 1969a), Pyricularia oryzae (D) (Yamada et al., 1961) and Sclerotinia sclerotiorurn (A) (Wang and Le Tournaeu, 1972). The activity of this enzyme is stimulated by magnesium ions and inhibited by nickel and zinc, among a number of other cations. Manganous ions have been reported to be both inhibitory and stimulatory, as also was found for the enzyme from Pyricularia oryzae. The same enzyme, and that from Pyrenochaeta terrestris, only shows activity with mannitol 1-phosphate and has an apparent K , value of 3 mM. The exact specificity of the enzyme from the other fungi is not clear because of the impure nature of the preparations. N. SORBITOL (GLUCITOL) DEHYDROGENASE (NAD +)

This enzyme has been isolated from Aspergillus niger (D) grown on glucitol (Desai et al., 1969). There is said to be an absolute specificity for glucitol, and for the product of the reaction (fructose). However, no investigation was made regarding the reactivity of L-iditol; reduction of the latter polyol would indicate that the enzyme should be called L-iditol dehydrogenase (EC 1.1.1.14). The enzyme has an apparent K , value for glucitol and fructose of 0.098 mM and 0.66 mM, respectively, the pH optimum for the oxidation of the former being 9.0.


159

0.GLYCEROL OXIDASE

This enzyme has been studied in Aspergillus japonicus (D) (Uwajima et al., 1980; Uwajima and Terada, 1980). It catalyses the oxidation of glycerol without the mediation of NAD(P)+: Glycerol

+0 2 -+ glyceraldehyde + H202

The enzyme is synthesized when the fungus is grown on glycerol. Species of Aspergillus, Neurospora and Penicillium also form this enzyme when grown under similar conditions, but it has not been found to be present in Mucor racemosus, Rhizopus japonicus, Rhizopus oryzae, Candida lypolytica, Pichia haplophyla or Coprinus cummatus. The enzyme has a molecular weight of 4 - lo5and a pH optimum with glycerol as substrate of 7.0. There was found to be 50% activity with dihydroxyacetone. The apparent K,,, value for glycerol was 10 mM. Zinc, magnesium, nickel, cobalt and manganous ions activate the enzyme; sodium azide and hydroxylamine are inhibitory.

111. Mastigomycotina

The oomycetes are said by Pfyffer and Rast (1980b) not to contain polyols. However, Luard (1982~)reported trace amounts of arabitol in Phytophthora cinnamoni. More significant was her finding that substantial amounts of arabitol and mannitol were synthesized by Pythium debaryanum in an external medium of - 1.5 MPa osmotic potential. This fungus was not included in the survey by Pfyffer and Rast (1980b) and in any case neither polyol was produced when the external osmotic potential was close to zero.

IV. Zygomycotina Examination by Pfyffer and Rast (1980a,b) showed that only glycerol, and occasionally ribitol (in Actinomucor elegans, Mortierella rammaniana, Mucor and Zygorrhincus moelferi), were the major polyols present. Nevertheless, there can also be traces of arabitol and mannitol. Curtis et al. (1980) have found ribitol to be present in five Circinella sp., although the polyol was not found in Circinella naumorii. As will be seen below, Absidia glauca can metabolize D-mannitol when the fungus is induced to grow on this polyol as the sole carbon source. The mechanism of formation of either glycerol or ribitol in any of the members of the Zygomycotina has not yet been investigated.

160

D. H. JENNINGS

A.

Absidia glauca

On the basis of studies using cell-free extracts and radioactive substrates, such that the products of the reaction could be identified, Ueng et ul. (1976) and Ueng and McGuinness (1977) showed that the following conversions could take place when the fungus is induced to grow on mannitol. (i) Fructose could be reduced to mannitol utilizing NADH, (ii) fructose could be phosphorylated in the C-6 position via the action of hexokinase, (iii) fructose 6-phosphate could be reduced to mannitol 1-phosphate utilizing NADH, and (iv) mannitol could be phosphorylated in the C-1 position by the action of a kinase. There was no evidence of mannitol-1 -phosphatase. Oxidation of mannitol to fructose by mannitol dehydrogenase (NAD+) (EC 1.1.1.67) was inhibited by mannitol 1-phosphate. The relationship of this inhibition to the steady-state kinetic properties of the enzyme have been described by Ueng and McGuinness (1977).

V. Ascomycotina and Deuteromycotina It is expedient to consider together information obtained from both these groups of fungi, because there are extremely strong similarities between them with respect to their polyol metabolism. There are, of course, good mycological arguments for considering the majority of imperfect fungi as being related to members of the Ascomycotina (Hawker, 1966). Adomako et al. (1971a) have pointed out that, with respect to the metabolism of hexoses by resting mycelium, ascomycete and basidiomycete fungi appear to fall into three principal categories based on the quantitative relationship between polyols, non-reducing disaccharides and glycogen. The first group is characterized by a considerable synthesis of glycogen and trehalose. The second contains many fungi in which polyols, notably mannitol, are synthesized rapidly from hexoses, while in the third group mannitol, trehalose and glycogen are produced in comparable quantities. In this latter group, mannitol is preferentially synthesized from fructose while the others are synthesized from glucose. Obaton (1929,1930) first described this in a strain of Aspergillus niger. Though it is not clear whether a particular metabolic pattern is species-specific, there is plenty of evidence that a particular genus can show different patterns. Thus, while Succhuromyces cereuisiae produces trehalose and glycogen (Trevelyan and Harrison, 1956), other species produce a range of polyols. This has been demonstrated by Peterson et al. (1958), and the particular case of Succh. rouxii is considered further in the section on osmophilic yeasts. For Succh. cerevisiae, there is no evidence that, apart from glycerol, polyols are produced by this yeast. Certainly it cannot grow on higher polyols such as glucitol and mannitol (Barnett, 1968).

POLYOL METABOLISM I N FUNGI

161

It is probable that allocation of fungi to one of the above three groups may not be based on absolute differences in the metabolic machinery but on the consequences of quantitative differences in rates of the various enzyme-catalysed reactions. Thus Chaetomium globosum appears qualitatively like members of the third group, yet is quantitatively much better allocated in the first group (Adomako et al., 1971a,b, 1972). Many of the species considered in this section, and also the lichenized fungi, clearly belong to the second group. However, there is little doubt that Sclerotinia sclerotiorum belongs to the third group (Wang and Le Tourneau, 197I). I shall argue that a number of other fungi considered in association with Alternaria alternata also belong to this third group.

A. YEASTS GROWN ON N-ALKANES

Studies by Hattori and Suzuki (1974a,b,c) have shown that certain Candida sp., which can grow on n-alkanes (containing a mixture of C I to~ CISchains), can produce polyols in the medium when the pH value is kept below 4.0. Candida zeylanoides KY6 166 produces meso-erythritol but, if the phosphate concentration of the medium is maintained at between 0.3 and 1.7 mM, mannitol and not the tetritol is produced. Candida tropicalis KY6224 produces arabitol, when grown under the same conditions as those in which C. zeylanoides produces erythritol. It is not known whether these polyols are produced in media with pH values above 4.0, but are not released into the medium. It is certainly the case for Dendryphiella salina that, in media below pH 4.5, mannitol and arabitol, which are normally retained in the mycelium, are released into the medium (Holligan and Jennings, 1972b).

B.

Alternaria alternata

When fat was being produced by this fungus, it was possible to show by the use of [U-'4C]glucose that the NADPH required for its synthesis could be produced by oxidation of mannitol (Hult and Gatenbeck, 1978). There is no doubt that mannitol is simultaneously formed and utilized, though the calculations leading to the above conclusion, using the published data, are not completely clear. The following enzymes have been found in cell extracts (Hult and Gatenbeck, 1979; Huh et al., 1980): (i) glucose-6-phosphate dehydrogenase (EC 2.7.1.1; responsible for the production of NADPH), (ii) hexokinase (EC 1.1.1.49), (iii) mannitol-1-phosphate dehydrogenase (EC 1.1.1.17), (iv) mannitol-1-phosphatase (EC 3.1.2.22) and (v) mannitol dehydrogenase (NADP+) (EC 1.1.1.138). No mannitol dehydrogenase (NAD+) (EC 1.1.1.67) appears to be present. Thus there is evidence for a cycle (Fig. 1) from fructose 6-phosphate to

162

D. H. JENNINGS

NADPH

ATP

4

I

Fructose 6-phosphate

NADH\

Mannitol

Mannitol I-phosphate

NAD'

FIG. 1 . The mannitol cycle proposed by Huh and Gatenbeck (1978).

mannitol 1-phosphate, to mannitol and finally to fructose. One turn of the cycle gives the following overall reaction: NADH + NADP+ + ATP

+ NAD+

+ NADPH +ADP + PI

The formation of free fructose means that there will be competition between glucose and fructose for hexokinase. Investigations of the flux through mannitol dehydrogenase and mannitol- 1-phosphatase have indicated that the level of the flux is such that the sites of regulation of glycolysis by the withdrawal of fructose 6-phosphate may be monitored by NADH availability. The importance of the mannitol cycle for NADPH production is supported by a calculation which shows that mannitol oxidation alone can meet the total need of NADPH for fat synthesis. An alternariol-producing strain of Alternaria alternata has a lower rate of mannitol oxidation, and therefore a lower rate of fat synthesis than the non-producing strain which could imply that a shortage of NADPH in the mycelium might explain why the polyketide alternariol is produced. On the other hand, the low rate of mannitol oxidation in the producing strain could be an adaptation to a low rate of fat synthesis (Hult and Gatenbeck, 1978). On the basis of studies using crude extracts, Hult et al. (1980) concluded that the following fungi also possess the cycle: Aspergillus niger, Botrytis cinerea, Cladosporium cladosporoides, Penicillium frequentans, Penicillium islandicum, Thermomyces lanuginosus and Trichothecium roseum. All are fungi imperfecti, though the converse does not hold, since the cycle does not appear to be present in Candida utilis. Though, from the studies of Hult et al. (1980), the cycle does not appear to be present Pyricularia oryzae (i.e. since mannitol- 1-phosphate dehydrogenase activity always was low), Yamade et al.


163

(1961) demonstrated a high specific activity for the enzyme in this same fungus. The presence of all the enzymes needed for the cycle have been found in Aspergillus candidus (Strandberg, 1969), Microsporum gypseum (Leighton et al., 1970) and Sclerotinia sclerotiorum (Wang and Le Tourneau, 1972). Polyol metabolism by the latter fungus is dealt with in detail in Section V.J. (p. 175). There it is suggested that the relevant investigations indicate that there are two different biosynthetic pathways to mannitol. Further, very little activity of mannitol dehydrogenase (NADP+) was found in that fungus. Hult et al. (1980) pointed out that the cycle also may be present in the ascomycetes Ceratocystis multiarmulata, Gibberella zeae and Neurospora crassa. Mannitol- 1-phosphate dehydrogenase was not present, but it was argued that the situation may be similar to that in Aspergillus nidulans where the specific activity of mannitol- I-phosphate dehydrogenase is regulated by the nitrogen source in the medium (Hankinson and Cove, 1975). The concept of the NADPH-regenerating mannitol cycle will be discussed later.

c. UTILIZATIONOF PENTOSE SUGARS

BY

Candida utilis

This fungus is well known for its capacity to utilize pentoses. Early studies showed that resting cells grown on xylose reduced this sugar to xylitol with the mediation of NADPH (Veiga et al., 1960)and that the pentitol was oxidized to xylulose with the mediation of NAD (Chakravorty et al., 1962). Further details of the dehydrogenase (EC 1.1.1.21) that catalyses the first step are given elsewhere (see Section II.E, p. 153). It is possible that there is an alternative pathway for conversion of xylose into xylulose. Although Chakravorty et al. (1962) indicated that D-xylose ketol-isomerase (EC 5.3.1.5) was absent from yeasts grown on D-xylose, Tomoyeda and Horitsu (1 964) showed the enzyme to be present. Horitsu et al. (1 970) subsequently demonstrated the presence of L-arabinose ketol-isomerase (EC 5.3.1.4). These two isomerases convert D-xylose and L-arabinose into D-XylUlOSe and L-ribulose respectively. These enzymic steps are similar to the first stage in the metabolism of pentoses by bacteria (Horecker, 1962). Thus, in C . utilis, there appear to be two different initial steps in the utilization of pentose (Fig. 2), either by isomerization to the pentulose or by a reduction to the alditol, utilizing NADPH, followed by oxidation to the pentulose, utilizing NAP+. This latter pathway of pentose metabolism (through the polyol) appears to be widespread in fungi (Chiang and Knight, 1960; Lewis and Smith, 1967; Barnett, 1968). Failure to demonstrate the presence of these isomerases in fungi may be related to their need for Mn2+,or less frequently other bivalent cations, which strongly activate the isomerase (Tomoyeda and Horitsu, 1964; Horitsu et al., 1970) plus the fact that, if the same holds for

D. H. JENNINGS

I64 4

- -

-

I

o-Xylose

Xylitol

1

L-Arabinose 4

i)-Xylulose

3

2

1

1.-Arabitol

?

__c

I

-Ribdose

i)-Xylulose 5-phosphate

- I 8

-

t>-Ribulose 5-nhosnhate

I

Glucose

Glyccraldehyde 3-phosphale


FIG. 2. Some of the pathways of carbohydrate metabolism in Cundidu utilis are shown. 1, polyol dehydrogenase (EC 1.1. I .21); 2, xylitol dehydrogenase (NAD+)(EC 1.1.1.9); 3, D-xylulose kinase (EC 2.1.1.1.17) (Chakravorty e f u/., 1962);4, xylose isomerase (EC 5.3.1.5); 5, L-arabinose keto-isomerase (EC 5.3.1.3); 6,glucose-bphosphate dehydrogenase (EC 1.1.1.47) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) (Osmond and apRees, 1969; Domagk et al., 1973); 7, ribose-5-phosphate ketol isomerase (EC 5.3.1.6) (Horitsu and Tomoeda, 1966); 8, see Barnett (1976) for some possible pathways for the subsequent metabolism of L-arabitol and L-ribulose; this review also contains an excellent summary of the metabolism of pentose sugars by yeasts, as well as the metabolism of other carbohydrates, including polyols.

fungi as well as bacteria, these enzymes are inhibited by polyols at relatively low concentrations (approximately 5 mM). One possible explanation for the presence of two pathways for the initial dissimilation of pentoses may lie in their transport across the plasma membrane. If the process of transport were to be a proton-linked symport, as is the case in a number of fungi (Eddy, 1982), then pentitol formation could be seen as a sink for the protons. This idea will be argued further in Section VII (p. 180). Essentially, pentitol formation can be considered as a means by which the pH value of cytoplasm is prevented from becoming more acid. Conceivably, if conditions within the fungus are such that metabolism is tending to make the cytoplasm more alkaline, then the conversion of pentose into the pentulose, as a result of the isomerase, might be the more likely possibility, though the reaction itself would do little to alter the cytoplasmic pH value. The possibility that the isomerase might be switched off, as the pentitol concentration rises, suggests how acidification might switch off the pathway to pentulose. Irrespective of these ideas, it is interesting to note that, for the yeast Pachysolen tannophilus, mutants have been isolated that are deficient in xylitol dehydrogenase (Maleszka et al., 1983). Growth was poor on xylitol, but the mutants grew readily on D-xylulose which was converted into ethanol. D-Xylose isomerase could not be detected in the fungus. Further, although


165

there is a need for caution over the assay conditions, as indicated above, recent studies by Gong et al. (1983) have failed to demonstrate D-xylose isomerase in many yeasts. However, it should be noted that the enzyme has been shown to be present in a yeast other than C . utilis, namely Rhodotorulagracilis (Hofer et al., 1971);the enzyme was produced after growth on D-xylose. Cells grown on the pentose cannot break down xylitol, even if it is accumulated in the cells. Gong et al. (1983) have provided a most valuable survey of the ability of yeasts to convert pentoses, the information from which could form the basis of valuable comparative enzymic and metabolic studies. D.

Dendryphiella salina

There is a considerable amount of information available on the relationship between the concentrations of the various polyols in the mycelium of this marine hyphomycete grown under different cultural conditions (Holligan, 1971; Holligan and Jennings, 1972a,b,c,d, 1973). The major polyols present are arabitol, erythritol, glycerol and mannitol. The hexitol was found to accumulate in the mycelium when it was grown in the presence of the following carbon sources: glucose, fructose, sucrose, trehalose, myo-inositol, mannitol, succinate, acetate and ethanol. Very little was produced when the fungus was grown on arabitol. In non-growing mycelium, mannitol was the only polyol produced in the presence of glucose. Arabitol only began to be produced by the mycelium when it entered the logarithmic phase of growth. In media containing glucose, fructose and sucrose, arabitol accumulation generally exceeded that of mannitol. By contrast, relatively small amounts were formed from acetate and succinate, and very little, if any, from glucitol, trehalose and myo-inositol. The mannito1:arabitol ratio was found to alter with changes in the concentration of glucose in the medium. At low concentrations there was more arabitol than mannitol; as the glucose concentration was increased, the balance of products changed in favour of mannitol. Owing to insufficient data, the conditions favouring glycerol production have not yet been properly defined. The polyol appears to be lost from the mycelium much more readily than arabitol and mannitol, and the amount of glycerol lost often has not been determined. Nevertheless, the available evidence indicates that glycerol accumulates only in growing mycelium. Variation of nitrogen source, in media containing glucose, has a significant effect on accumulation of arabitol, but not of mannitol (Holligan and Jennings, 1972b). Most arabitol appears to be produced in the presence of nitrate, and least with tryptone; intermediate amounts were formed when ammonium or glutamate was the nitrogen source. The level of arabitol attained also depended on the rate of utilization of the carbon source supplied; the faster the rate, the more was arabitol produced.

166

D. H. JENNINGS

Studies with [1-I4C]-and [6-'4C]glucose indicate that the activity of the pentose phosphate pathway was greatest with nitrate as the source of nitrogen (Holligan and Jennings, 1972~).Support for this conclusion comes from labelling patterns in mannitol which indicated that it was synthesized partly by a direct route from the glucose entering the hyphae, and partly from hexose phosphate derived from the pentose phosphate pathway. The proportion of mannitol synthesized by this pathway has been estimated to be greater in the presence of nitrate (37% after a 6-hour period) than in the presence of ammonium (26%) and glutamate (19%). The greater activity of this pathway with nitrate is almost certainly due to an increased requirement for NADPH needed to bring the level of reduction of nitrogen to that of an amino group. This also suggests that production of mannitol requires this reduced coenzyme. Activities of extracted enzymes indicate that there are two possible pathways for the synthesis of mannitol (Holligan and Jennings, 1972b; see Fig. 3). A similar pattern of labelling in the arabitol present in mycelia grown with nitrate was obtained with all the following specificallylabelled carbon sources: [ 1-'4C]glycerol, [ 1-I4C]-,[2-I4C]-or [6-'4C]glucose(Lowe and Jennings, 1975). Examination of the pattern indicated that synthesis of arabitol occurred via the non-oxidative part of the pentose phosphate pathway, as well as via the oxidative part. The final s t ep r e d u c t i o n of pentose to arabitol-involved reduction of xylulose. This conclusion comes from the pattern of labelling obtained with [ l-'4C]ribose and [2-'4C]glucose, and the ability of cell-free extracts to bring about the conversion of xylulose into arabitol (Holligan and Jennings, 1972b). Thus, there are two routes for arabitol synthesis-that via the oxidative part of the pentose phosphate pathway and that via the non-oxidative segment (Fig. 4).

Glucose

NADPH

Glucitol

Overall: Glucose+NAD

Glucose Fructose

ATP

NADPH

+

- NAD+

Fructose

+2NADPH

Glucose 6-phosphate

NAI)PH

-

Mannitol P~lrthn~rr~~ I

Mannitol+ NADH + 2 N A D P '


-

Mannitol

Overall: Glucose+ATP+NADPH

-

P a l k n ~ 2~ l ~ ~

Mannitol+ADP+P,+NADP+

FIG. 3. The putative pathways of mannitol synthesis from glucose in Dendryphiella salina.

Glucose

ATP

Glucose 6-phosphatc

Overall: Glucose+ATP+NADP.+

Glucose

ATP

H,O

Glucose 6-phosphate

-

2NADP+

Nori-osiclit/ir~e.si~gt?iiw/

Pentose phosphate

- -

-

NA I)( P It I

Arabitol

O.\-idu/iw seggri i c i i I Arabitol+ADP+P,+NADPH +CO,


I

Xylulose 5-phosphate

Overall: Glucose+If A T P + N A D H

Pentose

ATP

Fructose 1, 6-bisphoaphale

1

Glyceraldehyde 3-phosphate

-

Ardbitol

Arabito1-t I + A D P + N A D +

FIG. 4. The pathways for arabitol synthesis from glucose in Dendryphiella salina (Lowe and Jennings, 1975).

168

D. H. JENNINGS

There are two ways in which there can be both net synthesis ofmannitol and regeneration of reduced coenzyme. First, there is the coupled synthesis of mannitol via pathway 1 (Fig. 3) and arabitol (Fig. 4), and second, there is the synthesis of mannitol via pathway 2 (Fig. 3), the NADPH being regenerated via the pentose phosphate pathway. The pentose phosphate formed as a consequence of the production of NADPH can be reconverted into glucose and thence to mannitol by the non-oxidative segment of the pentose phosphate pathway. The latter pathway seems to be the more likely route to mannitol in non-growing mycelia metabolizing glucose, and in growing cells presented with high concentrations of glucose. On the other hand, coupled synthesis of mannitol and arabitol is the probable route in cells growing in the presence of lower concentrations of metabolizable carbohydrate, under which conditions, as indicated above, arabitol accounted for a significant fraction of the polyol produced. However, as noted above, data for radioactive incorporation with specifically labelled sugars indicates the importance of the non-oxidative segment of the pentose phosphate pathway as the route to arabitol. Whatever the route, one presumes that NADH will be regenerated either by mannitol breakdown or via glycolysis; the extent to which this NADH is available for the production of arabitol from xylulose will depend on the competition between these particular metabolic steps and oxidation of NADH by mitochondria (Watson, 1976). Support for the idea that there is a competition between xylulose and mitochondria for NADH comes from studies by Metcalf and Jennings (1982) and D.H. Jennings and J.D. Thornton (unpublished work) on the effect of the non-metabolized sugars 3-O-methylglucose and L-sorbose on the metabolism of Dendryphiella salina. It can be argued that these compounds act in a similar manner to uncouplers. There is evidence that L-sorbose acts in this way in N . crassa (Crocker and Tatum, 1968) and that non-metabolizable sugars act in a similar manner to uncouplers when inducing rhythmic growth in Podospora anserina (Lysek and Jennings, 1982). In keeping with the idea that 3-O-methylglucose acts like an uncoupling agent, Metcalf and Jennings (1982) found that the sugar increased the flux of carbon from polyols to organic acids. The extent to which the polyols are replaced depends on the breakdown of polysaccharide. With 3-O-methylglucose there was a net fall in the concentration of polyol because the sugar prevented glycogen breakdown (McDermott and Jennings, 1976). This was not so for L-sorbose; with this sugar, there was breakdown of arabitol but net synthesis of mannitol (D.H. Jennings and J.D. Thornton, unpublished observation). The breakdown of arabitol is presumed to be brought about by the increased rate of oxidation of NADH accompanying an increased glycolytic flux. It can be argued that increased net synthesis of the hexitol is provoked by influx of protons accompanying uptake of L-sorbose.


169

When Dendryphiella salina is grown in media salinized with either NaCI, MgCl2 or Na2S04, glycerol becomes increasingly the major soluble carbohydrate, with decreasing osmotic potential of the external medium (J.M. Wethered, E. Metcalf and D.H. Jennings, unpublished observation). When, on the other hand, a non-growing mycelium (provided with a source of glucose) was exposed to a range of concentrations of NaCI, it was mannitol and arabitol that increased with decreasing medium osmotic potential (Jennings, 1973). Since mycelia grown in media in which the osmotic potential was changed by non-ionic solutes (e.g. inositol) also d o not produce glycerol, it would seem that synthesis of the compound requires that the mycelium be growing in a suitable ionic environment. The features of that environment are not clear, but conceivably it should allow an appropriately high flux of ions into the mycelium. Irrespective of whether the external osmotic potential is changed ionically or by the use of an organic solute, total soluble carbohydrate concentration within the mycelium, after a similar period of growth, was the same. Further, total carbohydrate concentration increased with the osmotic potential of the growth medium. Within that total concentration, values for individual carbohydrates could vary very considerably between various treatments. A somewhat analogous situation holds when non-growing mycelia are presented with the non-metabolized sugar 3-0-methylglucose. The compound was actively accumulated, but total soluble carbohydrate concentration was adjusted such that overall it remained constant (Jennings and Austin, 1973). The adjustment occurred predominantly via the breakdown of mannitol, but there was some breakdown of arabitol. A major locus for the effect of 3-0-methylglucose seemingly is the inhibition of glycogen breakdown such that net synthesis occurs (McDermott and Jennings, 1976). When, as described earlier, only very slowly metabolized L-sorbose was presented to non-growing mycelia, arabitol breakdown occurred just as for 3-O-methylglucose, but there was a significant rise in the concentration of mannitol (D.H. Jennings and J.D. Thornton, unpublished observations). This argues against an effect of 3-0-methylglucose through turgor regulation, and suggests that adjustment of the concentration of soluble carbohydrate, which occurs when the sugar enters the mycelium, is metabolically regulated. These matters relating to control of soluble carbohydrate concentration within mycelia of De. salina require further investigation. Nevertheless, the fact that the mycelium can regulate the concentration of soluble carbohydrates, as indicated above, suggests that we must consider the synthesis and breakdown of individual polyols as part of a system of metabolic reactions involving all the polyols within the mycelium which act in concert. The evidence from De. salina is part of the case that I wish to make that polyols can provide a fungus with a physiological buffering system (see Section VIII, p. 185).

170

D. H. JENNINGS

E. Neurospora crassa

Although there is apparently little information available regarding the hexitol or pentitol metabolism in this fungus, there are some important details known about glycerol metabolism. Figure 5 indicates the possible pathways for the metabolism of glycerol in fungi generally. When grown on minimal medium with glycerol as the sole carbon source, N . crassa grows at rates comparable to those measured with fermentable carbon sources. In liquid medium, vegetative growth occurs but is limited by the rapid onset of extensive conidial production (Courtwright, 1975a). Studies on glycerol metabolism have been made using mutants that do not produce conidia, or with the wild-type organisms using media in which acetate was a supplementary carbon source. In the absence of glycerol, and when glucose was the external carbon source, it would seem, from the specific activities of a range of enzymes that have been investigated, that the most likely pathway for glycerol assimilation would be by its conversion, via ,glycerol dehydrogenase (NADP+) (EC 1.1.1.72) into glyceraldehyde, and then into 3-phosphoglycerate by means of either an NAD+-linked aldehyde dehydrogenase (EC 1.2.1.3) or glyceraldehyde kinase (Tom et al., 1978). The presence of glyceraldehyde kinase (albeit at one-fifth of the specific activity of aldehyde dehydrogenase) may mean that dissimilation of glycerol can lead to D-glyceraldehyde 3-phosphate. When mycelia were grown in the presence of glycerol, the syntheses of glycerol kinase (EC 2.7.1.30) and glycerol-3-phosphate dehydrogenase (EC Glycerol 3-phosphate

/Dih/!oxyacetone

Glycerol

-

5&

p

h

o

k

Fructose I. 6-bisphosphate

Dihydroxyacetone

Glyceraldehyde

i9

Glycerate

-

10

6

Glyceraldehyde 3-phosphate

I

3-phosphoglycerate

FIG. 5. Possible pathways for glycerol metabolism in fungi. 1, glycerol kinase (EC 2.7.1.30); 2, glycerol dehydrogenase (NAD+) (EC 1.1.1.6);3, glycerol dehydrogenase (NADP+)(EC 1.1.1.72);4, glycerol-3-phosphate dehydrogenase (NAD+)(EC 1.1.1.8) (FAD-linked) (EC 1.1.99.5); 5, dihydroxyacetone kinase (May et al., 1982); 6, D-glyceraldehyde kinase (Tom et al., 1978);7, triose phosphate isomerase (EC 5.3. I . 1); 8, fructose-1,6-bisphosphatealdolase (EC 4.1.2.13);9, aldehyde dehydrogenase (EC 1.2.1.3);10, glycerate kinase (EC 2.7.1.31); 11, glycerol oxidase (Uwajima and Terada, 1980).


171

1.1.99.5) were induced (North, 1973, 1974; Courtwright 1975a,b; ViswanathReddy et al., 1977). The former enzyme is present in the cytosol and the latter on the inner face of the mitochondria1 membrane, and is thought to be a flavoprotein. The pathway of glycerol breakdown using these induced enzymes would be via glycerol 3-phosphate and dihydroxyacetone phosphate, though it is unclear how events in the cytosol are related to those in the mitochondria. Glycerol-3-phosphate dehydrogenase can be detected at low specificactivity in wild-type strains (Viswanath-Reddy et al., 1977) but there appears to be no evidence for an NAD+-linked oxidative activity leading to dihydroxyacetone. Further information about the genetic characterization of the inducible glycerol dissimilatory pathway has been presented by Denor and Courtwright (1978, 1982). Growth of Candida utilis on glycerol appears to involve similar metabolic pathways to those described above (Gancedo e t a / . , 1968). On the other hand, there is good evidence that, in Schizosaccharomyces pombe strain NCYC 132 there is a specific dihydroxyacetone kinase present which catalyses the second step in the pathway for glycerol utilization (May et al., 1982) in which the first step is the oxidation of glycerol involving NAD+ (May and Sloan, 1981). A similar kinase appears to be present in N . crassa (Tom et al., 1978). F. OSMOPHILIC YEASTS

von Richter (1912) gave this term to those yeasts that can grow well in an environment of low osmotic potential. Since 1912, a large number of yeasts have been discovered which can grow in the presence of high concentrations of either sugars or salts. Jennings (1983a) has pointed out that such yeasts are only part of a considerable spectrum of fungi which can grow under conditions of low osmotic potential classified variously as osmophilic (Onishi, 1957), osmotolerant (Anand and Brown, 1968), osmotophilic (van der Walt, 1970), osmotrophic (Sand, 1973), xerophilic (Pitt, 1975) and xerotolerant (Brown, 1976). Although higher marine fungi are not necessarily so able to tolerate equally low osmotic potentials (Jones and Jennings, 1964, 1965), this ecological grouping appears to behave similarly with respect to polyol levels in the mycelium as a function of osmotic potential of the external medium (Jennings, 1983a). It is now clear from numerous studies (Onishi, 1963; Brown, 1976, 1978; Troke, 1976; Adler et al., 1978, 1982; Adler and Gustafsson, 1980; Metcalf, 1980; Luard, 1982a) that a wide variety of fungi, not necessarily those belonging to the above groups (e.g. Chaetomium globosum and Saccharomyces cerevisiae), produce an increased amount of glycerol in response to a decrease in the osmotic potential of the medium. Work by Onishi and his colleagues, reviewed by Onishi (1963), has suggested that glycerol synthesis (on a dry weight basis) is stimulated by sodium or

172

D. H. JENNINGS

potassium chloride. However, from studies on Dendryphiella salina, this may not be a specific effect on glycerol synthesis itself, but a consequence of a larger mycelial osmotic volume such that the concentration of glycerol synthesized is the same for the same external water potential (J.M. Wethered and D. H. Jennings, unpublished observation). Not only do high concentrations of NaCl stimulate glycerol production, but there is a change of pattern from that for media in which little NaCl is present. Thus, when Saccharomyces rouxii was grown in the presence of 3.0 M NaCl, in a medium containing 0.5 M glucose, 70% of the total polyol formed was glycerol while, in the presence of 1.7 mM NaCl and 1.6 M glucose, the value was SO%, with a high proportion of the remainder being arabitol (Onishi, 1960a). For Pichia miso, grown in media of similar composition, in the presence of the high concentration of NaCl, Onishi (1960b) found that only glycerol was produced whereas, in the presence of the much lower salt concentration, the yeast produced glycerol, D-arabitol and erythritol. Adler and Gustafsson (1980) have shown that arabitol can be the major polyol present in cells of the marine yeast Debaryomyces hansenii which had been growing in the presence of 2.7 M NaCl but that was only when the cells had entered the stationary phase. The increased synthesis of arabitol was associated with a rapid drop in the concentration of glycerol in the cells. The amount of polyol produced by Sacch. rouxii was dependent upon the oxygen tension of the medium (Spencer et al., 1957; Spencer and Shu, 1957). As the oxygen tension was increased from a low value, the ratio of polyol to ethanol increased. Onishi (1960a) demonstrated for the same yeast that, in the presence of 3.0 M NaCl in a medium containing 0.5 M glucose (compared with a medium containing 1.7 mM NaCl and 1.6 M glucose), while the quantity of polyol synthesized per unit of glucose consumed increased, the rate of production of ethanol was decreased by a factor of eight. Though there is a need for care in discussing this topic, since data for comparable experimental conditions are needed (Fiechter et al., 198 I), the effect of salt on the fermentation of osmophilic yeasts seems similar to the Pasteur effect in which fermentation is inhibited by the presence of oxygen. This seems particularly so when comparisons are made between (i) the growth of Sacch. cerevisiae on galactose under anaerobic and aerobic conditions (Fiechter et al., 1981), and (ii) the growth of Sacch. rouxii in the absence and presence of high concentrations of NaCl (Onishi, 1960a). Spencer and Spencer (1978) believe that glycerol synthesis in Sacch. rouxii occurs by a pathway similar to that in Sacch. cerevisiae (see Section V.1, p. 174). They point out that we need to know how the reactions of the Embden-Meyerhof pathway are regulated such that ethanol formation is suppressed and more NADH is diverted to the reduction of dihydroxyacetone phosphate to glycerol phosphate. The labelling pattern in glycerol, and in ethanol, in the osmophilic yeast Torulopsis magnoliae, obtained after feeding with [1-I4C]-and [2-'4C]glucose,was found by Spencer et al. (1956) to be as


173

expected if both compounds (glycerol and ethanol) were synthesized by the same pathways as in Sacch. cerevisiae. Although this conclusion is not unlikely, it would be gratifying to have further experiments performed, particularly some concerned with the enzymology of the pathways. Spencer et al. (1956) also examined the pattern of labelling in arabitol in T. magnoliae, using glucose specifically labelled as above. The results obtained, which also included data for incorporation of 14Cinto carbon dioxide, were consistent with the hypothesis that glucose was being dissimilated not only via the Embden-Meyerhof pathway but via phosphogluconate oxidation accompanied by transketolase-mediated transformations, and that these reactions were important in arabitol formation. The labelling patterns were consistent with the formation of arabitol directly from D-ribulose 5-phosphate by sequential reduction and dephosphorylation. Ingram and Wood (1965) found that, when fed to Saccharomyces rouxii, ~ - [ 6 - ' ~ C ] g h . ~ oyielded se ~ -[5 -~ ~ C]arab ito andl ~-[5-'~C]ribose in the ribonucleic acid fraction, which is consistent with the pathways postulated by Spencer et al. (1956). Blakley and Spencer (1962) showed that, although Sacch. rouxii was able to oxidize both D-ribulose and D-xylulose, growth on the latter pentulose was much better and produced significant amounts of arabitol (only trace amounts could be detected after growth on D-ribulose). Feeding of ~-[5-~~C]xylulose resulted in the production of arabitol with two-thirds of the radioactivity in C-1 . This is consistent with production of arabitol from D-xylulose emanating from the pentose phosphate pathway. The presence in the cells of a xylulose reductase, requiring NADH and with high specificity for D-xylulose, was demonstrated.

G.

Pichia (Candida) guilliermondii

This yeast is flavinogenic, overproducing riboflavin. When the yeast was grown on D-glucose, ribitol and D-arabitol were produced which were first released into the medium but subsequently taken up and metabolized in the course of growth (Miersch and Reinbothe, 1974; Salewski et al., 1976; Miersch, 1977). The labelling pattern in the polyols isolated after exposure of the cells to [1-I4C]-,[6J4C]-, [1-3H]-,[3-3H]-,[4JH]- or [6-3H]glucosehas been determined (Salewski et al., 1976). The distribution of label in arabitol and ribitol was close to that expected if these two pentitols were formed from D-ribulose, or its phosphate ester, produced via the pentose phosphate pathway. Synthesis from D-ribose seems unlikely. As one might anticipate, there is a randomization of label via the aldolase and transaldolase-transketolase reactions. Subsequent studies (Miersch et al., 1980) indicated that ribitol was produced from ribitol 5-phosphate formed by the action of ribitol-5phosphate dehydrogenase (EC 1.1.1.137) on ribulose 5-phosphate, and

I74

D. H. JENNINGS

requiring NADPH. A ribitol dehydrogenase was not detectable; apparently no attempt was made to detect the presence of ribitol-Sphosphatase. H.

Pyrenochaeta terrestris

This fungus has been shown by Wright and Le Tourneau (1965) to utilize a wide range of carbohydrates when tested in a mineral salts medium with nitrate as the nitrogen source. The pH value of the medium rose during growth in the presence of sucrose. Mannitol was the only polyol accumulated inside the mycelium for most of the carbohydrates that were tested, the exceptions being glucitol, with L-sorbose and with glucitol as carbon sources, and L-arabitol, erythritol and glycerol with the same polyols in the medium. Interestingly, D-xylose and D-ribose supported good growth, with slower growth in the presence of L-arabinose; yet the equivalent polyol was not found inside the mycelium. Instead, ribose was found together with the individual pentose used as the carbon source. Mannitol gave as fast a growth rate as glucose, and the polyol was utilized within the mycelium when it was transferred to a mineral salts medium without carbon source (Wright and Le Tourneau, 1966). The enzymic evidence (Aitken et al., 1969a,b) indicates that fructose 6-phosphate is reduced to D-mannitol 1-phosphate, using NADH, and mannitol is produced by the action of mannitol-1-phosphatase (EC 3.1.2.22). There is no evidence of the enzyme steps to fructose 6-phosphate from the various carbon sources that will support growth, of which D-mannose is a particularly intriguing example. Wright and Le Tourneau (1965) showed that this carbohydrate supported the fastest rate of growth. I.

Saccharomyces cerevisiae

Glycerol is the only polyol produced by this yeast. Gancedo et al. (1968) showed that cell extracts contained NADH-dependent enzymic activity, reducing triose to a-glycerophosphate. Potassium ions, added at a concentration of 0.1 M, and magnesium ions, at 10 mM, caused a disappearance of activity. This does not seem to be a specific ion effect. The properties of the enzyme involved-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8Fare described elsewhere (see Section II.B, p. 151). The presence of a-glycerophosphatase was confirmed, indicating the hydrolysis of a-glycerophosphate as the final step in the production of glycerol. During growth of the yeast under anaerobic conditions, only about 10% of the glucose consumed was converted into biomass (Fiechter et al., 1981). The remainder appeared as ethanol, glycerol and pyruvate which were lost into the medium. Glycerol was released only under anaerobic conditions. According to Oura (1977), glycerol synthesis is a means of regenerating NAD+. Thus he was unable to demonstrate glycerol excretion in all instances where there were


175

other ways of removing the excess NADH generated (e.g. presence of reducible compounds such as acetaldehyde and 0x0 acids, and even small amounts of oxygen). Sprague and Cronan (1977) have isolated mutants of Succh. cerevisiue which are defective in their ability to utilize glycerol. Two types were obtained-one deficient in glycerol kinase (EC 2.7.1.30) and the other in sn-glycerol-3-phosphate dehydrogenase (EC 1.1.99.5). This indicates that glycerol is utilized in a manner similar to that in Neurosporu crussa and Cundidu utilis (see Section V.E, p. 170). It appears that some form ofcatabolite repression is the principal factor controlling the activity of the above two enzymes in Succh. cerevisiue. There is little induction above non-repressed enzyme levels when cells are grown in the presence of glycerol. In contrast, in C . utilis there was a 7-fold increase (Gancedo et ul., 1968) and, in N . crussa, a ICL15-fold increase in induced activity. The control mechanisms involved in Sacch. cerevisiue must be different from those in the other two fungi. J.

Sclero t inia sclero tior urn

This fungus is well-known for producing sclerotia during growth of mycelium in stationary culture; they are not formed in shaken cultures. There have been several studies of the soluble carbohydrate content of sclerotia, mycelia and culture media when cells were grown under a variety of nutrient regimes (Le Tourneau, 1966; Cooke, 1969; Wang and Le Tourneau, 1971). Highest weights of mycelia and sclerotia appeared to be formed in media that contained raffinose, sucrose, maltose, lactose, D-mannose, D-glucose, D-fructose or L-arabinose. Regardless of the carbon source, mannitol usually was found to be present in culture filtrates, while trehalose, mannitol and usually small quantities of glucose and fructose were present in mycelia and sclerotia, irrespective of the carbon source. The other soluble carbohydrates present depended on the carbon source. With glucose, fructose, sucrose and trehalose, no other soluble carbohydrates were present in detectable amounts. With other sugars, the free sugar was present, as well as the appropriate polyol (e.g. L-arabinose and arabitol, D-xylose and xylitol, D-ribose and ribitol; Wang and Le Tourneau, 1971). From studies on cell-free extracts, it would seem that mannitol is synthesized by reduction of D-fructose 6-phosphate involving NADH, with D-mannitol I -phosphate being converted into mannitol by mannitol-I-phosphatase (EC 3.1.2.22) (Wang and Le Tourneau, 1972). There was only very limited mannitol dehydrogenase (NADP+) (EC 1.1.1.138) activity. With respect to pentitol metabolism, both pentitol: NADP+ and NAD+ dehydrogenases were present in cell-free extracts of mycelia and sclerotia grown on D-xylose (Wang and Le Tourneau, 1973). NADPH, but not NADH, was oxidized in the presence of ~-xylose,L-arabinose, D-ribose and possibly D-arabinose. Reaction products of D-xylose and L-arabinose

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were xylitol and arabitol, respectively. Extracts of mycelia and sclerotia grown on a medium containing L-arabinose or D-ribose behaved similarly, with highest activity shown towards the sugar that had served as the carbon source. Dialysed cell-free extracts of sclerotia, grown on D-xylose medium, reduced NAD+ in the presence of xylitol, L-arabitol, ribitol and possibly D-arabitol. There was no reaction with NADP+. Mycelial preparations were not as active as those from sclerotia. Similar results were obtained with preparations of mycelia and sclerotia grown on media containing L-arabinose and D-ribose. Irrespective of the carbon source, the greatest rate of NAD+ reduction was shown with xylitol as substrate. Neither pentitol dehydrogenase (NADP+) nor pentitol dehydrogenase (NAD+) activity could be detected in mycelia or sclerotia grown on glucose media. The enzymic basis for the metabolism of pentose sugars was as would be expected from studies on Candida utilis (see Section V.C, p. 163).

VI. Basidiomycotina

Our knowledge of the metabolism of polyols in the Basidiomycotina is singularly limited. It is only for Agaricus bisporus and Schizophyllum commune that any detailed information (see below) concerning the metabolism of polyols is available. Nevertheless, there are some intriguing items of information which indicate that polyol metabolism within this group merits very considerable study. Threitol (L-erythritol) has been reported to occur in Amillaria mellea (Birkinshaw et al., 1948) and in mycelial cultures of Claviceps purpurea (Vining and Taber, 1964). For the latter fungus, the presence of the polyol depended on the culture medium. Indeed, under the same conditions that threitol was produced, there were greater amounts of erythritol (mesoerythritol). Neither this polyol nor threitol has been reported in sclerotia of the same fungus (Cooke and Mitchell, 1969, 1970; Corbett et al., 1975). More recent studies (H.I. Granlund, D.H. Jennings and W. Thompson, unpublished observations) suggest that it is erythritol, not threitol, which is present in Amillaria mellea (mycelium and rhizomorphs grown on a medium containing malt and yeast extract), reaching around 50% of the total soluble carbohydrate content on a molar basis. The metabolism of erythritol by Schizopyllum commune is considered below. Brownlee and Jennings (1 98 1) have demonstrated that trehalose is the sugar translocated through the mycelium of Serpula lacrimans, the disaccharide being converted into arabitol in the younger regions. Two points need to be made regarding this observation. First, trehalose also has been demonstrated to be (or postulated to be) a major constituent of carbon translocated into the basidiocarps of Agaricus bisporus (Hammond and Nichols, 1976) and


177

Flummulinu uelutipes (Kitamoto and Gruen, 1976), and in the mycorrhizal fungus of Dactylorchis purpurellu (Smith, 1967). In the larger algae such as Macrocystis sp., mannitol appears to be the major carbon compound translocated (Schmitz, 1981); it is this fact which has led to a view that polyols might be involved in translocation within fungi (Lewis and Smith, 1967). Jennings (1983b) has pointed out that it may be significant that the disaccharide trehalose is the form in which carbon appears to be translocated in fungi. When the disaccharide is converted into arabitol, as in Serpula lacrimam, or into mannitol, as in Agaricus bisporus, the osmotic potential of a solution due to the presence of the soluble carbohydrates is approximately doubled. Thus, at the end of the translocation pathway, trehalose could be metabolized such that one glucose moiety was utilized in respiration (and in the provision of carbon skeletons for structural material) and the other moiety contributed to the maintenance of the osmotic potential. Second, the data of Brownlee and Jennings (198 I), and of other studies on Serpula lucrimans, indicate that the carbohydrate metabolism of this fungus changes significantly as it grows and differentiates. This indicates in a striking manner what may happen in a wide range of fungi, and thus should urge the investigator to pay careful attention to the growth phase of the fungus under study. A.

Agaricus bisporus

Mannitol contributes up to 40% of the dry weight of fruit bodies (Rast, 1965). The accumulation of the hexitol in these bodies during their growth was not accompanied by an increase in mycelial mannitol (Hammond and Nichols, 1976). Trehalose, the other major soluble carbohydrate in the sporophore, decreased throughout its growth, and a parallel decrease was observed in the mycelium along with decreases in glucose and sucrose. Mannitol is synthesized in cell-free extracts by the NADPH-dependent reduction of free fructose (Diitsch and Rast, 1969). Synthesis by the reduction of glucose to glucitol, with epimerization to mannitol, does not seem to occur. The purified enzyme is, however, capable of oxidizing glucitol (Ruffner et al., 1978; see Section 11.1, p. 156). The postulated synthesis of mannitol via the reduction of fructose, utilizing NADPH, is supported by experiments involving the 'H and 14Cincorporation into mannitol from [1-I4C]-,[6-14C]-,[3,4-I4C]-and [ lJH]-, [3-'H]-, [6-'H]glucose (Diitsch and Rast, 1972). There was a minimum randomization of I4C between carbon atoms 1,6 and 3,4 which indicated that thecarbon skeleton of the hexitol came almost direct from glucose. With 'H, the specific activity of mannitol was greatest in the order [3-)H] > [ 1-)H]> [6-3H], as would be anticipated if 'H entered mannitol not only via the carbon skeleton but also through NADP 'H produced by the first and third steps of the pentose-phos-

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phate pathway. Likewise the label of 3Hwith C-1, C-6 and C-2 and C-2 to C-5, from the three specifically labelled 3H-glucose molecules, is in keeping with this mechanism of incorporation of 3H. Hammond (1977) showed that the C-1 :C-6 ratio, determined from the activity of I4CO2produced in the first 8 hours after presentation of [1-I4C]-or [6-14C]glucose,was greater for pileus slices than for vegetative mycelium. Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity was greater in the sporophore than in the vegetative mycelium, while the reverse was true for glucose phosphate isomerase (EC 5.3.1.9). There was no significant difference between mannitol dehydrogenase activity in sporophore and mycelium. Thus there is a strong body of evidence that, in uiuo, the reductive step leading to the production of mannitol involves NADPH produced by the pentose-phosphate pathway. Further support that that step involves the reduction of fructose comes from the studies of Hammond and Nichols (1977) who fed [U-14C]glucoseand [U-14C]fructoseto growing sporophore tissue. Mannitol became slowly labelled with I4Cfrom fructose, but only weakly with I4C from glucose. With glucose, proportionately more label entered trehalose than with fructose. With [U-'4C]fructose 6-phosphate there was a lower entry of label into mannitol than with labelled-fructose. B.

Schizophyllum commune

Niederpruem and Hunt (1967) have described the occurrence and changes of polyols during the morphogenesis of Schizophyllum commune. Dikaryotic fruit bodies and basidiospores contain both mannitol and arabitol. Before spores start to elongate, arabitol disappears; there is also a drop in the content of mannitol. These changes require the presence of both carbon and nitrogen sources, and are inhibited by the protein-synthesis inhibitors, cycloheximide and p-fluoro-DL-phenylalanine (Aitken and Niederpruem, 1970). Once hyphal elongation commences, the mannitol concentration starts to increase, returning within 24 hours to that found initially within the spores. The arabitol content remains low for 5-7 days and cannot be increased by high concentrations of glucose (Cotter and Niederpruem, 1971). When acetate is the sole carbon source, there is an increase in the concentration of both arabitol and mannitol. Studies with [1-14C]-, [2-I4C]-,[3,4-I4C]-and [6-'4C]glucose indicated that, during the first 4 hours of germination, there was likely to be increased activity of the pentose-phosphate pathway, which was more important than glycolysis in the breakdown of glucose during this period. Subsequently glycolysis appeared to be the more important pathway (Aitken and Niederpruem, 1973). This contrasts with the situation in Dendryphiella salina, where increased activity of the pentose phosphate pathway was associated with the synthesis of arabitol (see Section V.D, p. 165).


I79

Studies on the activities of various enzymes during basidiospore germination (Speth and Niederpruem, 1976) indicated that, at 4 hours after germination on medium containing glucose and asparagine, there was an increased reductase activity involving NAD+ and D-ribulose and D-fructose, as well as an increased dehydrogenase activity involving NADH. There was much less change in D-xylulose reductase and D-mannitol dehydrogenase activities (both involving NAD' and NADH, respectively). Reduction of xylulose was shown to produce xylitol, and reduction of ribulose, arabitol. After 4 hours, all enzyme activities declined such that, at 24 hours, they were at a level similar to that extant before germination commenced. When the mycelium was transferred to a medium containing acetate and ammonium phosphate (conditions that stimulate production of isocitrate lyase), there was an increase in all three reductases, and in both dehydrogenase activities, by far the greatest being in the D-xylulose reductase activity. The lack of any xylitol production under these conditions is intriguing. Presumably synthesis of mannitol and arabitol from acetate occurs by gluconeogenesis (Holligan and Jennings, 1973). Glucose, added together with acetate, suppressed arabitol formation and, under these conditions, isocitrate lyase activity was not stimulated. There was, however, significant arabitol synthesis with maltose, trehalose and cellobiose, and without any stimulation of isocitrate lyase activity (Cotter and Niederpruem, 1971). Although the various changes in enzyme activity described above have been thought to explain the observed changes in polyol concentrations (Speth and Niederpruem, 1976), knowledge gained from studies of other fungi, particularly of Dendryphiella salina, suggests that much more information is needed before the factors controlling the synthesis and catabolism of arabitol and mannitol in Schizophyllum commune can be said to be elucidated. Erythritol can act as the sole carbon source for the germination of basidiospores of Schizophyllum commune (Niederpruem and Denne, 1966), though for germination to take place, the period of exposure to the polyol is much longer than with glucose. Following growth in erythritol and glycerol, an NAD+-dependent erythritol dehydrogenase could be detected within the mycelium (Isenberg and Niederpruem, 1967).The acquisition of the ability to metabolize erythritol was inhibited by cycloheximide (Braun and Niederpruem, 1969). The enzyme was absent from mycelia grown on other carbon sources. Interestingly, in the presence of NAD+, rnycelial extracts are capable of oxidizing D-mannitol, glucitol, ribitol, xylitol and D- and L-arabitol, irrespective of the carbon source used for growth. However, again irrespective of the carbon source used for growth, an erythrose reductase (coupled to NADPH) was present which had a pH optimum of 7.0 and an apparent K,,, value for erythrose of 5 mM (Braun and Niederpruem, 1969). Some further details of the ability of mycelial extracts to oxidize a range of polyols is given

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by Niederpruem et al. (1965) who also demonstrated the presence of a xylitol dehydrogenase involving NADP+ .

VII. Regulation of Cytoplasmic pH Values in Fungi If fungi are like other organisms, one must anticipate that the internal pH is under regulation. There is experimental support for this contention from studies on three fungi, Neurospora crassa (Sanders et al., 1981; Sanders and Slayman, 1982), Rhodotorula gracilis (Hofer and Misra, 1978; Kotyk, 1980) and Saccharomyces cerevisiae, for which there are a number of observations reported, using a variety of methods, all of which have been reviewed by Borst-Pauwels (1981). Essentially, all the studies have shown that as the external pH value is changed there are very much smaller changes in the internal pH value. Thus, for Sacch. cereoisiae, as the external pH value was raised from 4.0 to 9.0, the internal pH value increased at the most by 2.0 units, from 5.9 to 7.9 (Kotyk, 1963). The actual change may be somewhat smaller than this, since there is evidence that these values were obtained with a method (dye-distribution) which probably gave erroneous results at high pH values (Borst-Pauwels, 1981). It is for this reason that the results for Rh. gracilis, where the internal pH change over the same range of external pH values was much greater, probably do not indicate the true situation within the cell. Measurement of the pH value of the cell homogenate obtained after disrupting a thick suspension of cells, or the 3'P-nuclear magnetic resonance chemical shift of intracellular orthophosphate within cells of Sacch. cerevisiae, indicated changes somewhat less than those mentioned above. Similar results have been obtained for N . crassa. All these results by themselves, although indicating that the cytoplasmic pH is regulated, d o not necessarily mean that there is direct metabolic involvement. The buffer capacity of the cell contents can be sufficient to restrict the pH changes to the recorded values (Sanders and Slayman, 1982).The evidence that there is direct involvement of metabolism comes from studies on N . crassa, in which changes in cytoplasmic pH were determined for various metabolic states. The results of these investigations are dealt with below. Though investigations with other fungi are clearly necessary, one should not be surprised (if the results of studies on animal cells are an example) to find that metabolism directly regulates cytoplasmic pH. While proteins may buffer the cell over the short term, they will not be able to cope with the prolonged production of hydrogen ions by oxidative metabolism. Until recently the view, first outlined by Raven and Smith ( 1 973), has been gaining ground that the proton pump at the plasmalemma plays a major role in the control of the cytoplasmic pH value in plant cells. The essential process is the pumping of protons from the cytoplasm across the plasma membrane


181

into the external medium, the process involving utilization of free energy of hydrolysis of ATP. Smith and Raven (1 979) have developed their ideas further to take into account data that have come forward since 1973. It has been argued (Raven and Smith, 1976) that the overriding importance of the pump in controlling the pH through excretion of protons into the external medium, means that those parts of a multicellular plant away from the external medium, must incorporate hydrogen ions into some compound which can be considered a sink for them. The compound is then either moved into the vacuole or to those cells in contact with the external medium. In these latter cells, the compound can be broken down and the hydrogen ions thereby released pumped into the external medium. The presence of a proton pump in the plasma membrane of N . crussa and Succh. cerevisiue is well documented, and there is little doubt that it is an ATPase (Eddy, 1982). The properties of the proton-translocating ATPase of fungal plasma membranes have been reviewed by Goffeau and Slayman (1981). It is well known that suspensions of fungal cells or mycelia readily acidify the external medium, and that the proton pump must play a major role in this process of acidification. This was demonstrated most dramatically by Conway and OMalley (1946) who showed that resting cells of Succh. cerevisiue, whilst fermenting glucose, could generate 20 mM hydrogen ions in the external medium. It was the further studies of Conway and his colleagues (see Jennings, 1963) which laid the foundation for our present understanding of proton excretion by fungi. The extent of acidification of the medium by a fungus will depend on the buffering effect of the following (see Sigler et ul., 1981b): (i) water with its content of dissolved COz, (ii) CO2 extruded from cells during glycolysis and respiration, plus bicarbonate formed by hydration of the gas at pH values above 4, (iii) acidic metabolites (succinic, malic, lactic and acetic acids in Succh. cerevisiue) extruded into the medium, the acids referred to having pKa values ranging from 3.5 to 5.2, (iv) polyelectrolytes on the cell surface, and (v) active membrane processes (extrusion of OH- or absorption of H + ) tending to maintain a constant external pH value. The process of acidification of the medium by Succh. cerevisiue has been examined in detail by Sigler et uf. (1981a,b). An important finding was that there is a fairly good correlation between acidification and respiration with glycolytic substrates such as glucose, sucrose and galactose (after induction), but there was almost complete absence of acidification with substrates utilized below the level of the glyceraldehyde 3-phosphate-+ 1,3-diphosphoglycerate reaction. This was particularly true for ethanol, acetate and lactate; the other compounds studied, particularly Krebs cycle acids, had only a very small effect on respiration (less than 8% of that of glucose). Glucose-induced acidification was stimulated by univalent cations in the sequence

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K + > Rb+ 9 Li+==Cs+==Na+,in keeping with the view that hydrogen ion excretion via the pump was the major contribution to medium acidification. Some of the other characteristics of it will be considered later. A word of caution is required at this stage. We need to be careful in assuming that what is observed with a non-growing culture holds also for one in which the fungus is growing. Certainly, when the growth of mycelium of Gibberella fujikuroi was studied using a very large volume of medium, the pH value of the medium remained constant (Borrow et al., 1964). This might be due to the buffering capacity of the medium, but equally net extrusion of hydrogen ions, or indeed of hydroxyl ions, may only represent that situation when the organism is not growing. I have referred to hydroxyl ions here, because growth can be associated with an increase in pH value. This is the case for Dendryphiella salina when assimilating nitrate (Holligan and Jennings, 1972b). Sanders and Slayman (1 982) have examined the role of the proton pump in controlling the internal pH value of N . crassa. They examined the response of internal pH value to changes in the pH value of the medium, to inhibition of the proton pump by vanadate and to inhibition of respiration by cyanide. Changes brought about by the first treatment were in line with those findings for Sacch. cerevisiae, and could be explained in terms of the buffering capacity of the cellular contents. Vanadate had no substantial effect. Sodium cyanide at 1 mM caused a drop, although not immediate, in internal pH value from 7.0 to 6.3. Vanadate also had no effect on this drop, confirming the view that the pump does not necessarily regulate the internal pH value. If this were not so, there would be a greater drop when the pump was inhibited. Nevertheless, there is evidence, from the time course of the effect of vanadate alone on pump activity and respiration, that the inhibitor, by switching off the pump, signals a decrease in oxidative activity which is the source of hydrogen ions in the hyphae. Sanders and Slayman (1982) indicate that the signal is a changed ATP:ADP ratio which exerts a negative feedback on oxidative metabolism (i.e. the pump, by not hydrolysing ATP, decreases the availability of ADP which in turn limits the rate of glycolysis). There is already strong evidence for this type of control from studies on beech mycorrhizal roots where the basidiomycete fungal sheath contributes a major proportion of respiratory activity (Jennings, 1963). The small effect of external pH on internal pH values has been shown to be due to the low rate of proton leakage across the plasma membrane. Application of weak acids and bases, whose undissociated molecules are lipid soluble, can alter theinternal pH value, though not to anymarkeddegree,partlyas the result of the buffering capacity of the cell contents, but also as a result of increased pump activity. This increased activity is accompanied by increased conductance to another ion, most probably an organic acid anion, such as to allow anet movement of protons into the medium (Sanders et al., 198 1).


183

While the above information indicates that, under certain circumstances, the pump can help to control cytoplasmic pH value, it is the view of Sanders and Slayman (1982) that metabolism plays the dominant role. In particular, they cite their observation that there is recovery in cytoplasmic pH value after cyanide treatment which occurs at the same rate whether or not the pump is working normally. Thus there is not only production of hydrogen ions by metabolism, but also their consumption. The hydrogen ions produced by metabolism are hypothesized to be associated with the synthesis of organic acids. The metabolic basis of hydrogen ion consumption was not alluded to. Berry (1981) has pointed out that the numerous redox reactions occurring within the living cell are sinks or sources for protons. We can consider polyols as providing such sinks or sources of protons through their reductive synthesis from, or their oxidation to, other carbohydrates. In this manner, polyols may help in the regulation of cytoplasmic pH value. The information for Dendryphiella salina (see Section V.D, p. 165) indicates some of the metabolic complexities which need to be taken into account in establishing how polyol metabolism might be involved in pH regulation. Dendryphiella salina is a mould that can be cultured as mycelium dispersed in liquid media, and even in its natural habitat seems likely to be in a liquid medium of significant magnitude to receive and buffer secreted hydrogen or hydroxyl ions. Consequently it is simplistic to over-emphasize the role of polyol synthesis in pH regulation when proton excretion and organic acid synthesis and breakdown are also part of the armoury of the fungus for the removal of hydrogen or hydroxyl ions from the cytoplasm. Nevertheless, there is some indication that mannitol, synthesized by reactions involving NADPH, might act as a sink for protons. The NADPH can be generated by the pentose phosphate pathway, and any pentose phosphate produced also converted into mannitol. It needs to be remembered, however, that the necessary regeneration of ATP by oxidative processes will generate protons. Nevertheless, the presence of a similar pathway for the synthesis of mannitol in the sporophore of Agaricus bisporus, and the particular problems of regulating pH value within hyphae distant from the external medium, as will be the case in the fruit body, suggests that hexitol formation could function as a sink for protons. There is a further point to be considered with respect to mannitol synthesis in A . bisporus, namely the source of hexose. Available evidence (Hammond and Nichols, 1976;Jennings, 1983)indicates that trehalose is the carbohydrate translocated into the fruit body. If this disaccharide is taken intact into those hyphae intact, where the mannitol is synthesized, and the transport across the membrane only requires one proton, then, after hydrolysis of the trehalose to give two molecules of glucose, the subsequent synthesis of the hexitol could be a sink for protons not only entering via the co-transport system, but for those produced by metabolism.

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D. H. JENNINGS

In the situation where there is a readily available sugar, it would seem that polyol synthesis, concomitant with transport of the sugar, could act as a sink for protons. This has been presumed to be so for glucose uptake into De. salina and A . bisporus. It is difficult to rationalize the metabolism of pentoses by C . utilis (and a wide variety of other fungi) via the equivalent pentitol if it did not act as a sink for protons co-transported with the pentose. However, in the absence of adequate information about the generation of NADPH for pentitol production in C. utilis and other fungi, it has to be admitted that, at present, this idea is no more than a possibility. In spite of the inadequacy of the data available, it is nevertheless worth stating in explicit form the hypothesis alluded to. This is that polyol synthesis, acting as a sink for protons, will be involved in the regulation of cytoplasmic pH values and, in particular, in the removal of those hydrogen ions generated by the movement of protons across the plasma membrane from the external medium. By this means a proton gradient can be maintained across the membrane with minimum mediation of the proton pump. This will not necessarily mean that the pump will not be working. There will be some hydrogen ion production within the cell as the result of a need to regenerate ATP. In any case, the outside of the plasma membrane (even just in the wall) will need to be kept more acid than the cytoplasm to maintain the proton gradient. This can be done by the pump, but it will be working only at a low level of activity. A system operating as just described avoids the need for either cation uptake and organic acid production, or excretion of organic acids without net cation uptake. Thus polyols acting as sinks for protons entering in association with sugar transport into the hyphae are particularly relevant for those situations where sugar transport has to be maintained in the absence of a readily available mechanism for removing protons excreted into the local environment, or where the concentrations of cations available for entry into the hyphae may be limiting. I have indicated that the growing sporophore of A . bisporus may be a representative locale, but the same comments are likely to apply to carbohydrate movement between the phycobiont and mycobiont in a lichen (Smith, 1974, 1975, 1978), and possibly to biotrophic symbionts, particularly those producing extensive mycelium on the surface of an aerial organ of the partner. Of course, it is axiomatic that if carbohydrate entry is not qccurring, polyol synthesis or breakdown may be another element in a range responsible for buffering the cytoplasmic pH value. Two final points need to be made. First, many lower fungi are apparently devoid of polyols. These organisms require investigation with respect to the mechanism of internal pH regulation. Second, the sporophores of Coprinus sp. do not produce polyols (Moore et al., 1979). This also needs investigation, though it seems likely that the metabolism of the sporophore is geared not to removal of hydrogen ions, but to neutralization of nitrogen bases consequent on the habitat of the species.


185

VIII. Comment It is surprising how relatively little we know about the metabolism of polyols in fungi in spite of their ubiquity among the higher genera and their occurrence among the lower genera. It is regrettable that the study of polyol metabolism in many fungi has been confined to only a few aspects. There are few fungi which have been studied with any intensity, as is shown by the various sections in this review dealing with information for specific fungi. Even for these, the emphasis has been on either the elucidation of those enzymes present, or on changes in polyol content under certain external conditions. As a consequence, we often have only a rudimentary idea about how polyol metabolism is controlled in a particular fungus. There may be a number of pathways by which a particular polyol may be synthesized or broken down. This is evidenced by the metabolism of glycerol in N . c r a m (see Section V.E, p. 170) and in Succh. cereuisiue (see Section V. 1 , p. 174), and the several possible enzymes involved in the metabolism of mannitol which have been discovered. The information regarding glycerol indicates that part of the diversity in enzyme complement for the metabolism of this polyol relates to when it is an exogenous substrate for growth. The factors governing the presence of a particular enzyme are not clear. As already indicated in Section V.B (p. 161), the presence of several enzymes in a filamentous fungus may represent temporal changes in mycelial metabolism, as the colony grows, such that a particular polyol is metabolized by a different set of enzymes in the older mycelium from that in the younger. There might also be spatial separation within a cell or hyphal compartment. Indeed we do not know whether polyols are sequestered in different hyphal compartments. This matter requires urgent consideration as, indeed, does the matter of location of the various enzymes. The presence of a membrane-bound D-sorbitol dehydrogenase in Gluconobucter suboxydans (Shinagawa et al., 1982)should make us aware of a similar possibility for the membrane-location of polyol metabolizing enzymes in fungi. While there is evidence that certain members of the Deuteromycotina may contain a similar complement of enzymes relating to the metabolism of mannitol 1-phosphate (Hult et ul., 1980; see Section V.B, p. 161), it appears that relevant enzymes are not present in C. utilis, nor in De. salinu (Holligan and Jennings, 1972b). This suggests that we need to be careful over generalizing, even from studies of what at first sight seem to be a representative group of fungi. Lewis and Smith (1967) suggested that polyols could perform the following functions in plants: (1) carbohydrate storage, (2) translocation of combined carbon, (3) osmoregulation, and (4) coenzyme regulation and storage. The information from studies on long-distance translocation of carbon through mycelium of members of the Basidiomycotina (see Section V.A, p. 176) does

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not support the idea that polyols are the form in which carbon is translocated. Nor is there much firm evidence that polyols are involved in carbohydrate storage in the sense that we can point to well-defined instances in which functional mycelium, subjected to a decreased supply of external carbohydrate, metabolizes internal polyols. Studies on De. salina (Holligan and Jennings, 1972a) indicate that a drop in the level of mannitol within the mycelium under such conditions may be one of the concurrent events of autolysis. The best authenticated instance of a polyol acting as a storage product is that of mannitol in the harvested sporophore of Agaricus bisporus (Hammond and Nichols, 1975; Hammond, 1978). The decrease in mannitol levels could account for about one-half of the post-harvest production of COZ over a 4-day period. There is evidence for a decline in the activity of the pentose phosphate pathway, presumably lowering the NADPH :NADP+ ratio and thus allowing the reversal of mannitol synthesis, even though there is a decrease in mannitol dehydrogenase (NADP+) concentrations during the post-harvest period (see Section VI.A, p. 177). There are now good indications (see glycerol production in Sacch. cerevisiae; Section V.1, p. 174 and polyol metabolism in De. salina, Section V.D, p. 165) that polyols are involved in regulating the concentrations of NAD(H) and NADP(H) in fungi. But very much more work is needed to confirm this, and then with an intensity of the approach used for the study of nicotinamide nucleotide metabolism in animal cells (Krebs, 1973). The situation with respect to the metabolism of polyols may be particularly complex because they may be involved in maintaining pH value as well as redox balance. It is conceivable, and indeed likely, that where phosphorylated derivatives of polyols are present they are involved in the regulation of ATP concentrations within the cytoplasm. For this reason, we need information on the extent to which the different reactions involving polyols are close to or distant from equilibrium, such that we can identify those reactions that are regulatory. The problems of making such investigations on plants have been discussed (apRees, 1974, 1977). There is also a need for information concerning the extent of the possible interactions between trehalose metabolism which requires ATP (Blumenthal, 1976)and mannitol metabolism when the polyol is synthesized via mannitol- 1-phosphate dehydrogenase (EC 1.1.1.17). Jeffery and Jornvall (1983) have proposed a bypass for the first stages of glycolysis (from glucose to triose) in animal cells involving the reduction of glucose to sorbitol (glucitol) and the subsequent conversion into fructose. fructose 1-phosphate and triose (Fig. 6). The bypass involves oxidation of NADPH so that the bypass and the pentose phosphate pathway act in concert. The bypass may produce glycerol. The data for De. salina (see Section V.D, p. 165), and studies on Puccinia graminis (Maclean and Scott, 1976; Manners et al., 1982), indicate the possible presence of this pathway in fungi.

187

POLYOL METABOLISM IN FUNGI NADPH A Y Iurse,

N

Sorbitol

I

Glucose 6-phosphate

i

Pentose phosphate pathway Fructose 6-phosphate

NAD+=lI

NADH NADH Fructose

t

Fructose I-phosphate \

1

\ Dihydroxyacetone phosphate-

+

Glyceraldehyde NAD(P)H NAD(P)+

=1

Glycerol

I

Glycolysis

FIG. 6. The sorbitol (glucitol) bypass as it might apply to fungal metabolism.

In toto, it is clear there is a need for a detailed examination of the regulation of sugar metabolism in those fungi that produce polyols. There is no doubt from studies on De. safina, osmophilic yeasts and other fungi (see Sections V.D, p. 165 and V.F, p. 171) that, compared with controls, polyol concentrations are increased in mycelium and cells after growth in environments of decreased osmotic potentials. Glycerol is frequently a major component of these increased polyol concentrations, but not necessarily so (e.g. in Geotrichum candidum no glycerol is synthesized when the fungus is grown in the presence of increased concentrations of NaCl in the external medium, though there is an increase in the amount of arabitol; da Costa and Niederpruem, 1982;J.M. Wethered and D.H. Jennings, unpublished observation). Further, though polyols increase in concentration in response to the external osmotic potential, where measured, as in Chrysosporium fastidium and Penicillium chrysogenum (Luard, 1982a), and in De. salina (J.M. Wethered and D.H. Jennings, unpublished observation), the total contribution may only be as high as 30% in Ch.fastidium and P . chrysogenum grown in medium of - 10 MPa osmotic potential, produced with glucose, and as low as 12% in De. salina grown in medium of 0.8 osmol kg-' water produced by

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magnesium chloride. Brown (1976) has pointed out the value of polyols in helping to maintain a suitable environment for enzyme activity within cytoplasm, in the face of a potentially unfavourable external osmotic potential. Whatever the mechanism by which polyols exert their protective effect, it is clear that one may have to take into account the fact that they have to exert this effect in the presence of relatively high concentrations of ions. If we are to ascribe a role for polyols in terms of a beneficial effect on enzyme activity, then there must be studies on the relevant enzymes in a solute millieu corresponding to that in the cytosol. To date, such studies have not been made. Indeed it is possible that increases in polyol content within a fungus in response to a decreased medium osmotic potential may not only be due to the need to protect enzymes but be a consequence of changed metabolism brought about by the changed external environment. Certainly, to date, there have been no studies on fungal solute composition in which the osmotic potential of medium has been decreased with a non-penetrating solute. Such studies need to be made before one can obtain a proper view of the role of polyols in fungal growth in media of low osmotic potential. Finally, a word of caution about the use of the term osmoregulation. Strictly we should use the term turgor regulation (Cram, 1976). Further, though Lewis and Smith (1967) ascribe a role for polyols in osmoregulation (i.e. that their concentrations in the cytoplasm respond to changes in external osmotic potential), it is only Luard (1982b) who has attempted to discover whether or not polyols have this role. It has been said that, in this author’s view, the data which she obtained are rather ambiguous, but it is clear from them that further studies would be very worthwhile. Although there might be doubts about the osmoregulatory role of polyols in fungi, there is no doubt that, in general, we can think of these compounds as acting as ‘physiological buffering agents’ in that they can assist in the maintenance of the cytoplasm in the appropriate redox state at the required pH value, and probably help to maintain a suitable millieu for enzyme activity. The information from studies on De. salina indicate that no one polyol is necessarily and particularly involved, but that if several polyols are present they are synthesized and catabolized in concert. The fact that their total concentration may not change has important implications for the regulation of the cytoplasmic environment, because such regulation can take place without involvement of ions which may be either absorbed or expelled, or metabolically produced. However, more data are required concerning polyol metabolism in fungi before this concept of ‘physiological buffering agents’ can be convincingly supported. REFERENCES Adler, L. and Gustafsson, L. (1980). Archives of Microbiology 124, 123. Adler, L., Gustafsson, L. and Norkrans, B. (1978). In “Energetics and Structure of Halophilic


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Veiga, L.A. (1968b). Journal of General and Applied Microbiology 14, 79. Veiga, L.A., Bacila, M. and Horecker, B.L. (1960). Biochemical and Biophysical Research Communications 2,440. Vining, L.C. and Taber, W.A. (1964). Canadian Journal of Microbiology 10,647. Viswanath-Reddy, M., Bennett, S.N. and Howe, H.B., Jr. (1977). MolecularandGeneralGenelics 153. 29. Viswanath-Reddy, M., Pyle, J.E. and Howe, H.B., Jr. (1978). Journal of General Microbiology 107., 289. von Richter, A. (1912). Mycologisches Cenrralblart 1, 67. Wang, S.-Y., C. and Le Tourneau, D. (1971). Archiu f i r Mikrobiologie 80, 219. Wang, S.-Y., C. and Le Tourneau, D. (1972). Archiu f i r Mikrobiologie 81, 91. Wang, S.-Y., C. and Le Tourneau, D. (1973). Archiu f i r Mikrobiologie 93, 87. Watson, K. (1976). In “The Filamentous Fungi,” Vol. 2 “Biosynthesis and Metabolism” (J.E. Smith and D.E. Berry, eds.), pp. 92-102. Edward Arnold, London. Watson, J.A., Hayashi, J.A., Schuytema, E. and Doughty, C.C (1969). Journal of Bacteriology loo, 110. Wright, J.R. and Le Tourneau, D. (1965). Phycologia Plantarum 18, 1044. Wright, J.R. and Le Tourneau, D. (1966). Phycologia Planlarum 19, 702. Yamada, H., Okamoto. K., Kodama, K., Noguchi, F. and Tanaka, S. (1961). Journal of Biochemistry 49, 404.


Temperature Profiles of Yeasts N. VAN UDEN Laboratory of Microbiology, Gulbenkian Institute of Science, 2781 Oeiras Codex, Portugal

I. Introduction . . . . . . . 11. The elements of temperature profiles . . . A. Cardinal temperatures . . . . . B. Specific rates of growth and thermal death . C. Activation parameters . . . . . . . . 111. Types of temperature profiles A. Associative profiles. . . . . . B. Dissociative profiles . . . . . IV. Effects of drugs on the temperature profiles of yeasts A. Ethanol and other alkanols . . . . B. Other drugs . . . . . . . . . V. Targets of temperature effects . A. Basic aspects . . . . . . . B. Thermodynamiccompensation . . . C. Membranes and mitochondria . References . . . . . . . .

.

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

.

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

.

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

.

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

.

195 196 196 199 202 206

210 22 1 223

223 234

239 239 240 244 248

I. Introduction When an Arrhenius plot is prepared of the specific growth rates of a yeast for temperatures that range from suboptimal to supermaximal values and an Arrhenius plot of the specific rates of thermal death is superimposed on the Arrhenius plot of growth, one obtains by definition the temperature profile of this yeast. Temperature profiles of the specific rates of growth and thermal death may be enriched with curves displaying the temperature dependence of other significant parameters, such as growth yield, specific rates of production of extracellular enzymes and excretion of metabolic products. Temperature profiles of growth and death provide at least three types of information. (1) The position of the cardinal temperatures of the strain within the range of biological temperatures. (2) The dependence of the specific rates ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 25 ISBN 0-12-027725-4

Copyright 01984 by Academic Press, London All rights of reproduction in any form reserved.

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N. VAN UDEN

of growth and death on the temperature, i.e. their activation parameters. (3) The presence or the absence of linkage between growth and thermal death in the superoptimal temperature range. From an analysis of the temperature profiles, general predictions may be made regarding the relation between the target sites connected with the upper temperature limit for growth (Tma, sites) and the target sites connected with thermal death (thermal death sites). Although temperature profiles are an expression of the temperature dependence of growth and death in batch culture, once established they permit predictions regarding the temperature dependence of growth and death in open systems of industrial or ecological interest. Metabolites that accumulate in the medium and cells and added drugs of industrial, medical or general scientific interest may profoundly change the temperature profiles of yeasts. Analysis of such modified profiles may throw light on the nature and localization of the receptor sites and, depending on the case, allow predictions with respect to the temperature dependence of yeast performance in industrial fermentations, the temperature relations of the effects of preservatives on yeasts in food, wine and other beverages, and the effects of fever or deliberate hyperthermia on drug action in yeast infections of man and animals (Roberts, 1979). Finally the study of temperature profiles of yeasts, taken as models of eukaryotic cells, may give useful hints on possible effects of temperature, alone or combined with drug action or radiation, on growth and death of human cells and cell systems including malignant ones (Streffer, 1978).

II. The Elements of Temperature Profiles A. CARDINAL TEMPERATURES

A temperature profile covers, by definition, a temperature range that includes the maximum temperature for growth (T,,,,,), the optimum temperature for growth (Top)and, ideally, also the minimum temperature for growth (Tmi"). While the temperature range of microbial growth in general extends from several degrees below the freezing point of water to a few degrees below its boiling point at normal pressure (Precht et al., 1973; Ingraham, 1973; Ingraham and Stokes, 1959; Stokes, 1962; Farrell and Rose 1967a,b; Brock, 1967; Larkin and Stokes, 1968; Brock and Freeze, 1969; Bott and Brock, 1969; Babel et al., 1972), the temperature ranges of individual cell strains do not normally comprise more than 40 to 50°C and are often much narrower. This is also the case with the yeasts (Fig. 1). Depending on whether the T,,, value is well above 50°C, between about 25°C and 50"C, or below 25"C, micro-organisms are conventionally and roughly subdivided into three temperature groups. These are referred to as

197

TEMPERATURE PROFILES OF YEASTS

S d i izo.urrc.cliriro t ti !jce.v oct 0s)J o rll ,v Picli iti t t I e t t i / I rti t i ( i efucic.t Klrr ! l w r otti!lces.frtrgili.s

I

0

I

10

I

1

-1

20

30

40

SO

FIG. 1. Examples of temperature ranges for growth of several species of yeasts. From Phaff et al. (1978).

thermophilic, mesophilic and psychrophilic micro-organisms, respectively. Nearly all known yeasts are mesophilic, a few are psychrophilic while thermophilic yeasts, as defined above, have so far not been detected. Yeasts such as Cyniclomyces guttulatus, Saccharomyces telluster (Candida bovina) , Candida sloofii and Torulopsispintolopesii, which are able to grow only within a narrow range of temperatures with 20-30°C as the lower limit and 4245°C as the upper limit, are sometimes referred to as “thermophilic” yeasts (Watson et al., 1980) but are more appropriately called “psychrophobic” yeasts (do Carmo-Sousa, 1969). Widely scattered references indicate that many yeasts are able to grow at 37°C and some at 45°C (Guilliermond, 1920; Wickerham, 1951; van Uden, 1963; Bridge-Cooke, 1965; Lodder, 1970; Phaff et a/., 1978). Stokes (1 97 I), surveying the literature, compiled cardinal temperatures of 40 yeast strains belonging to 31 species. Interest in the biology of the Antarctic and other cold habitats, which reached a peak in the 1960s, led to the description of a number of psychrophilic yeasts with Tmax values around 20°C (Sinclair and Stokes, 1965; di Menna, 1966; Fell et al., 1969). At the other extreme, the highest T,,, value for a yeast so far reported is 49-50°C for a strain of Hansenula polymorpha (van Uden et al., 1968). Wickerham (1951) in his monograph on the genus Hansenula introduced the ability to grow at 37°C as a test in yeast identification. This test was adopted in the taxonomic treatment of a number of yeast genera (Lodder, 1970). Van Uden and Farinha (1958) and Van Uden and do Carmo-Sousa (1959) found that the T,,, value was fixed within narrow limits on the species level in a number of yeast species. The value for Tmaxas a character in yeast identification was later adopted by several authors. As a consequence, the T,,, ranges of a large number of yeast species belonging to the following genera

198

N. VAN UDEN

have become available: Lipomyces (Slooff, 1970a); Metschnikowiu (Miller and van Uden, 1970); Nematospora (do Carmo-Sousa, 1970a); Schizossucharomyces (Slooff, 1970b); Candida (van Uden and Buckley, 1970); Oosporidium (do Carmo-Sousa, 1970b); Torulopsis (van Uden and Vidal-Leiria, 1970); Trichosporon (do Carmo-Sousa, 1970c) and Trigonopsis (Slooff, 1970c). Vidal-Leiria et al. (1979) determined the T,,, values of 594 yeast strains belonging to 1 12 species of the genera Candida, Torulopsis, Hansenula, Pichia, Metschnikowia and Leucosporidium. Less than 2% of the strains were psychrophilic having T,,, values below 24°C. More than 98% consisted of mesophilic strains with T,,, values ranging from 26 to 48"C, with the highest frequency in the 34-38°C range. No thermophilic strains were encountered (Fig. 2). The intraspecific variation of the T,,, value in 41 species of which more than five strains were studied did not exceed a range of 5°C in 78% of the species, the highest frequency pertaining to the 3°C range (Fig. 3). When the T,,, values of a collection of yeast strains supposedly belonging to the same species cover a wide range of temperatures, with subgroups of strains clustering around distinct T,,, values, these subgroups may represent distinct species. Thus Meyer et al. (1975) retained the name Candida sake' only for those of the strains included in this species by van Uden and Buckley (1970) that had their T,,, values around 30"C, and they excluded strain C . maltosa from C .sake' restoring it to species level based on its Tmax value around 40°C as well as on other characters. Similarly, Walsh and Martin (1977)

48

12

22 24

.rl! 26

28 30

32 :

LI I6 48

T,,, value P C )

FIG. 2. Distribution of the maximum temperature for growth (TmaX) among 594 yeast strains. From Vidal-Leiria et al. (1979).


199

12

n E

21

3

4

6

7

Ronge of T,,,

8

volues ("C)

FIG. 3. Distribution of the range ofvariation in the maximum temperature for growth (Tmax)among 41 species of yeast of which six or more strains were studied. From Vidal-Leiria et al. (1979).

re-introduced the name Saccharomyces carlsbergensis for strains classified in Sacch. uvarum, as defined by van der Walt (1970), that had their T,,,,, values around 3 3 T , retaining the latter name only for strains with T,,, values around 39°C. B. SPECIFIC RATES OF GROWTH AND THERMAL DEATH

1. Growth When a yeast population is growing in a stirred liquid medium of suitable composition with aeration (if needed) at a constant suitable temperature and when a linear measure of the population density (turbidity, cell numbers or viable counts) is plotted on semilogarithmic paper against time, a number of sequential growth phases, characteristic for micro-organisms in general (Monod, 1942), become evident. Normally more than 90% of growth is represented in such a plot by a single straight line (above the Topvalue in some cases, as we shall see, two sequential straight lines with different slopes may characterize this phase). This linear relationship may be expressed as follows: In X , = In Xo+k,t

(1) where X , and XO are population densities at time t and time zero (on the straight line), respectively, and the constant k, is the so-called specific growth

200

N. VAN UDEN

rate. By taking antilogs on both sides of equation (l), it is seen why this growth phase is correctly called “exponential” (rather than “logarithmic”): X , = X , ekgr Differentiation of equation (2) leads to:

dX dt

-=

kgX

(2)

(3)

which, on re-arrangement, gives the definition of the specific growth rate:

k g-

dX 1 dt X

(4)

Exponential growth is “balanced” (Campbell, 1957; Painter and Marr, 1968) when all of the constituents of the biomass increase with the same specific rate:

where XI,X 2 . . . X,, are concentrations in the culture of biomass and biomass constituents (e.g. cells, protein or nucleic acids). When different methods for measuring biomass (e.g. turbidity or viable counts) lead to markedly different estimates of the specific growth rate, growth is likely to be unbalanced. The distinction between balanced and unbalanced growth is critical in determinating the T,,, value, which by definition is the temperature above which sustained and balanced exponential growth is impossible. At a given constant temperature at which sustained, balanced exponential growth is possible (i.e. between Tminand T,,,), the specific growth rate depends on a number of environmental factors such as the chemical composition of the growth medium (particularly the nature and concentration of the carbon source), pH value, water activity, oxygen tension, the presence of growth inhibitors (e.g. residual detergent on the glassware) and concentration of excreted metabolites (e.g. ethanol) in addition to other factors. If one wishes to study the temperature dependence of the specific growth rate of a given yeast with the objective of establishing its temperature profile, these variables should be under control. Even so, there is some evidence that unknown factors outside the control of the operator, possibly connected with meteorological conditions and solar activity (Bortels, 195l), may cause considerable variation of the specific growth rate of yeasts under otherwise controlled conditions (Stanley, 1964; Martinez-Peinado and van Uden, 1977). 2. Thermal Death When a suspension of micro-organisms is exposed to a high enough


20 1

temperature, thermal death will occur. The semilogarithmic survival plot may be entirely linear or the linear part may be preceded by a shoulder, indicating a number of target sites greater than unity or the operation of repair mechanisms. When the population contains a subpopulation with greater heat resistance, the plot may display a "tail". Formal treatments of such plots were presented by Johnson et al. (1 954), Wood (1 956) and Moats (1971). The type of semilogarithmic survival plot commonly encountered in yeasts is shown in Fig. 4. The linear part of the semilogarithmic survival plot may be expressed by the following equation: In N1 = In No-kdt

(6)

where N1and NOare the numbers of viable cells after time t and time zero (on the straight line), respectively, and the constant kd is the so-called specific death rate: kd=

dN 1 dt N

(7)

By taking antilogarithms on both sides ofequation (6), it becomes evident that

50C 30C 20c

--ae

1oc

P ._ C 3

W

5

0 .> r

0 L

W

5 z

1c

e

c Time (min)

FIG. 4. Semilogarithmic survival plots of Saccharomyces cereuisiae exposed to various temperatures (A, 52°C; 0, 50°C; Q48"C). From van Uden et a/. (1968).

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N. VAN UDEN

the linear part of the semilogarithmic survival plot represents exponential death: Nt = NOe-kd' (8) The specific death rate for a given strain at a given temperature is a constant only under defined and controlled conditions. It may vary depending on the composition, pH value (also the chemical nature of the buffer; Cerny, 1980), the water activity of the suspending medium, the presence of death-enhancing drugs (including residual detergents on the glassware) and, most importantly, the physiological state of the cell population. If one wishes to study the influence of a single independent variable (such as the temperature or the concentration of a death-enhancing drug) on the kinetics of thermal death of a yeast strain, preparation of the cell populations should be carefully standardized. The composition and pH value of the growth medium, the method of stirring or shaking, the supply of oxygen (where needed) and the temperature of incubation should be the same from one experiment to the next. Furthermore, the cells to be used as inoculum in death experiments should be harvested at a defined point on the growth curve, for example in the mid-exponential phase of growth. The heat resistance of exponentially growing populations of Sacch. cereuisiue increased as the growth temperature was increased (Fintan Walton and Pringle, 1980). One should keep in mind that changes in growth temperature are accompanied by changes in specific growth rate. The separate effects of these two variables can be studied independently by using the chemostat at constant temperature and varying the dilution rate, or at constant dilution rate and varying the temperature (Hunter and Rose, 1972). Stationary-phase yeast cells are more heat resistant than exponentially growing cells (Schenberg-Frascino and Moustacchi, 1972; Parry et al., 1976). Use has been made of this fact for developing a procedure for enrichment of yeast mutants (Fintan Walton et ul., 1979). C. ACTIVATION PARAMETERS

When specific thermal death rates of a yeast are determined for a number of temperatures with the use of cell populations grown under carefully controlled conditions, typically, a plot of the logarithms of the specific rates against the reciprocals of the corresponding absolute temperatures is linear. Within temperature limits, the same is true for many other biological rates (Johnson et al., 1954). Thus, the specific thermal death rate behaves with respect to the temperature in the same way as a chemical rate constant as expressed by the Arrhenius equation; for detailed treatments of this subject, see textbooks on physical chemistry. Consequently we may write: kd = A e(-E/RT) (9)


203

and E l In kd = ln A - - RT

where E is the “energy of activation”, R the gas constant and A an empirical parameter. A totally explicit form of equation (9) is based on the theory of absolute reaction rates (Eyring, 1935; Stearn, 1949; Johnson et al., 1954):

where k g is Boltzmann’s constant, h Planck’s constant, AS# the entropy of activation and AH’ the enthalpy of activation. Its logarithmic form is, for all practical purposes of experimental biology (i.e. over the temperature ranges normally used in biological experiments), a linear equation. However, for calculation of AS# and AH‘ from experimental data the following truly linear form should be used: kd kg AS’ In - = In -+--T h R

AH’ R

1 T

-

I shall refer to plots of In kd/T against 1/T as “modified Arrhenius plots”. In the following paragraphs an attempt will be made, for the benefit of the biological reader, to discuss the significance of equation (1 1) and the limits of its applicability to biological rates. Consider a simple chemical reaction: AeB

(13)

The first basic idea of the theory of absolute reaction rates is that the transformation of A into B passes through an activated form A # which, in most cases, has a greater free energy of formation than either A or B . The second basic idea is that A and A # are virtually at equilibrium with each other, so that: A‘ A

-=

Kf,

and that classical equilibrium thermodynamics may be applied to KG, the equilibrium constant of activation:

+-

Keg

( - A c + / R ) (IIT)

(15)

and consequently: #

Keg

-

( A S f / R )e ( - A H # / R )

(16)

where AG’, AS’ and AH‘ are the free energy, the entropy and the enthalpy of

204

N. VAN UDEN

activation respectively (for a highly readable introduction into chemical thermodynamics, see Bent, 1965). The third basic idea of the theory is that the decay of activated form A + into product B is governed by a universal rate constant: dB dt

dA+ - kBT dr - h [A+]

According to classical chemical kinetics we also have:

-dB _ - k [A1 dt where k is the rate constant of the forward reaction. From equations (17) and (18) it follows that: kBT A + k =--h A

By substituting equations (14) and (16) in equation (19), equation (11) is readily obtained. The term A H + , the enthalpy of activation, is the difference (under standard conditions) in heat content between one mole of A + and one mole of A . Similarly A S + , the entropy of activation, is the difference in entropy content (a measure of disorder and randomness) between one mole of A + and one mole of A . When estimates of A H + and AS+ of thermal death are obtained from modified Arrhenius plots, the question arises as to what physical significance one may attribute to these values. If it is assumed that the specific thermal death rate is equal to the specific thermal inactivation rate of a thermosensitive target, A H + and A S + , if taken literally, would represent the difference in heat and entropy content between one mole of the activation target (a membrane protein for example) and one mole of the non-activated form (under standard conditions). Reservations must be made, even for the case of this most simplest of model, with respect to A S + . This thermodynamic quantity is contained in the value of the vertical intercept of the modified Arrhenius plot and may be calculated by the use of equation (1 2): AS#

=

kg R (Intercept - In -) h

(20)

However, since the universal rate constant kBT/h may need corrections from case to case by use of the appropriate “transmission coefficient” (Eyring, 1935; Stearn, 1949; Johnson et al., 1954), values for A S + calculated by the use of equation (20) may be only rough approximations. In most other cases of biological rates, it is not legitimate to estimate A S # values from modified Arrhenius plots. Consider, as a pertinent example, a yeast cell population in balanced exponential growth. The growth rate is


205

related to the consumption of nutrient S through the appropriate yield coefficient y (Monod, 1942): dX dt

- --

Y

dS (-5)

By dividing both sides of the equation by the instant population density Xand applying equation (4), we obtain: k, = y ks

(22)

where ks, the specific rate of transfer into the biomass of nutrient S, is defined as:

The specific transfer rate ks is dependent on the capacity and affinity of transport systems and enzymes, and the concentrations of substrates, products, effectors and inhibitors of a complex multistep reaction system that leads from the extracellular nutrient to the final biomass and other end-products. Each of the enzymes and transport proteins involved in the system has a certain weight with respect to the value of ks as expressed by the so-called sensitivity coefficient (Kacser and Burns, 1968). For the present purpose, it is sufficient to consider the extremely simplified case in which the first transport step wholly controls the overall rate, i.e. its sensitivity coefficient is unity (van Uden, 1971) and behaves as a true “master reaction”. Under this condition and assuming that nutrient S is used at concentrations that saturate the transport step across the cytoplasmic membrane, equation (22) transforms into:

k,

y k-r ET

(24) where ET is the total concentration per unit biomass of the carrier protein that transports the nutrient, and kT is the rate constant of transport by the loaded carrier. The form of equation (12) applicable to this extremely simplified case would be: k, ke AS+ AH+ 1 In - = In y + In ET+ In -+--h R R T T where the activation parameters refer to the transport step. Since estimates of ET are normally not available, a value for AS# cannot be estimated in the simple case presented, much less in realistic cases. What is usually done in these cases (Johnson et al., 1954) is to lump the known and unknown constants together in an empirical constant and write: =

206

N. VAN UDEN

When a straight line is obtained, AH# may be calculated from the slope and referred to as the enthalpy of activation. It may be seen that equation (26) is similar to equation (lo), the classical Arrhenius equation. The relation between the “energy of activation” calculated from a classical Arrhenius plot as expressed by equation (10) with the enthalpy of activation calculated from the modified Arrhenius plot is obtained by differentiating equation (1 1) which reveals that: E

=

AH#+RT

giving a difference of about 2.5 kJ (600 calories) at biological temperatures. In the author’s experience the use of activation thermodynamics in the analysis of Arrhenius plots of thermal death rates and of other biological rates is sometimes not well received by biologists, either because the equations are felt to be too formidable or because it is thought that the application of thermodynamics to living systems is being overdone. The basic experimental facts which are contained in a modified Arrhenius plot are the value of the slope and the value of the vertical intercept. Rejecting activation thermodynamics as a legitimate tool for processing these data should not lead to rejection of the data themselves, and thus of the information they may provide. Indeed, if so wished, the slopes and the intercepts may be used directly in the appropriate equation rather than the thermodynamic quantities calculated from them. For example, equation (39) given in Section IV (p. 229), which states that the entropy of activation of thermal death of Succh. cereuisiue in the presence of an alkanol is a linear function of alkanol concentration, might be rewritten to express directly the experimental results. This would reveal that the vertical intercept of the modified Arrhenius plot of thermal death of Succh. cereuisiue in the presence of an alkanol is a linear function of the concentration of the alkanol. This primary treatment would still lead to the verifiable prediction that, under isothermic conditions, the specific death rate is an exponential function of the alkanol concentration. However, it would be at a loss to provide a plausible explanation of why the slope of the linear relations between intercepts and alkanol concentrations are correlated with the lipid-buffer partition coefficients of the various alkanols. Though it should be kept in mind that the theory of absolute reaction rates contains speculative elements and that its applicability to biosystems is an open question, its use in the analysis of biological Arrhenius plots is potentially fruitful as it may lead to fresh theories open to experimental verification. 111. Types of Temperature Profiles

Up to the optimum temperature for growth, the specific growth rate of yeasts and other micro-organisms is an approximate Arrhenius function of the

207


temperature. Ratkowsky et af. (1982) contested this, and reported to have observed in many bacteria and some yeasts a linear relationship between the square root of the specific growth rate and the growth temperature in the suboptimal temperature range. Though we have been unable to confirm their findings when applying their equation to our own yeast data, the matter warrants further study. At temperatures above To,,, in most instances the value for k, declines sharply till T,,, is reached (Fig. 5). Establishment of an optimum temperature for growth and the sharp decline of k, values in the superoptimal temperature range require the simultaneous occurrence of at least two opposite processes, namely a constructive one (i.e. biosynthesis leading to growth) with a relatively low enthalpy of activation and a destructive one with a relatively high enthalpy of activation. Growth-rate equations have been proposed on the basis of different concepts of the destructive process. Hinshelwood (1946) derived an equation based on the assumption that the destructive process is irreversible, whereas an equation proposed by Johnson et al. (1954) implies that the specific growth rate in the superoptimal temperature range is limited by the concentration of the native (i.e. catalytically active) form of a key

c

/

7

0

40-

: -

.;”

._

0

w

I

30 20:

c

0

E’

c

t ._

z

4-

d

I I

3-

I I

I I

’‘47 I m 6 4 1 1

310

1

1

1

I

I

315

I

I

I

I

I

‘3:’ :5 I

I

1

I

I

30 Temperature (‘C) l

I

I

1

I

I

I

1

1

1

I

25

20

I

~I l I I I

I I I I

320 325 330 335 340 105.Reciprocal of the absolute temperoture

I 15

1 1 345

FIG 5 . Relative rate of multiplication (G) of Escherichiu coli growing in a simple medium as a function of temperature. The maximum rate is arbitrarily taken as 100. The points are data from experiments, and the solid line is the curve calculated in accordance with the following equation and constants. G = c Tec-AHo#/Rv + 1+ e(-AHo/Rn where c = 0.3612e24.w, A@ = 150,000, A H + = 15,000 and AS‘ = 476.46. From Johnson et ul. (1954).

208

N.

VAN UDEN

protein which is in thermodynamic equilibrium with its reversibly denatured form. Under the assumption that the sensitivity coefficient (Kacser and Burns, 1968) is sufficiently near to unity to provide it with dominant weight in the overall growth reaction, I shall refer to this protein as the T,,, site. At equilibrium we have: ED EN

-=

Keq

where EN and EDare the concentrations at equilibrium of the native and the reversibly denatured form of the T,,, site. From equation (28) it follows that:

where E is the sum of EN and ED.Following Johnson et al. (1954) the specific growth rate may now be expressed as: E k' k, = C'1 +Keq where k' is the rate constant of the step catalysed by the T,,, site (an enzymic reaction or a transport step) and C' is a proportionality coefficient. When k' and Keq are written as their respective temperature function and some of the constants are lumped together with C' of equation (30), one ends up with the equation originally proposed by Johnson et al. (1954): C T e(-AH#/R) (I/T) (31) kg = 1 + e (ASoIR) e ( - A H ~ / R T ) In this equation, AS" and AH0 are the standard entropy and the standard enthalpy of reaction (28), whereas A H Z is the enthalpy of activation of growth. For reasons explained on p. 205, ASz of growth cannot be determined and is therefore best lumped together with the other known and unknown constants in coefficient C. The equation fits some experimental results rather well (Fig. 5 ) , both in the suboptimal and the superoptimal range of temperatures. At relatively low temperatures, the slope of the Arrhenius plot of growth and thus the activation enthalpy are often found to increase. Readers interested in this aspect of temperature profiles may consult Ingraham (1973). Arrhenius plots of growth of yeasts may present peculiarities which depend on the relations with the respective Arrhenius plots of thermal death. The Arrhenius plots of thermal death of a number of mesophilic yeasts studied by van Uden et al. (1968) and by van Uden and Vidal-Leiria (1976) formed a positional sequence which followed the same order as the numerical sequence of the respective values of the maximum temperatures for growth. As can be seen in Fig. 6, the sequence related to T,,, value was no longer maintained by

209


c

100

a?

c

e

:200

" ._ 5

la? Q n

.

10-

6-

0

z1 2-

1

I

300

31 0

lo5 * Reciprocal I

60

I

50

I

56

I

1

I

305

54

I

52

I

50

I 325

I

31 5

320

of absolute temperature I

40

I

1

46

44

Temperature

I

42

I

40

I

30

l

36

l

34

(OC)

FIG 6. Arrhenius plots of the specific rates of thermal death of eight mesophilic and two psychrophilic yeasts. The mesophilic yeasts were A, Hansenula polymorpha (T,,, 4849°C); B, Candida albicans (T,,, 4546°C); C, Saccharomyces cerevisiae (T,,, 4142°C); D, Candida utilis (Tmax 4142°C); E, Torulopsis candida (T,,, 37-38°C); F, Torulopsis haemulonii (T- 37-38°C); G, Candida marina (T,,, 32-33°C); H , Torulopsisfujisanensis (T,,, 27-28°C). The psychrophilic yeasts were I, Candida nivalis (Tmax 22-23°C); J, Candida frigida (T,, 22-23°C). From van Uden et al. (1968).

sections of the plots extrapolated to much higher or much lower temperatures. The positioning of the plots was a consequence of an interplay between AH# and A S # of death in such a way that the plots, when extrapolated to the respective T,,, values, indicated values for the specific thermal death rate high enough to be measurable should they exist. This behaviour of the plots was formally expressed as follows (van Uden et al., 1968):

cY -

AHZ Tmax

+n

AS*

where Cyis a constant shared by the yeast strains and n is the number of degrees above the respective T,, value at which the constant applied. In the case of mesophilic yeasts, n was a small number between 1 and 4°C the constant having a value of from 301 to 330 J (72 to 79 cal) mol-l degree-'. Values of n for two psychrophilic yeasts (Fig. 6) were much higher (15-1 7"C), an expression of the fact the extrapolated Arrhenius plots indicated theoretical values for the specific death rate at Tma, values that were far below

210

N. VAN UDEN

measurable values. The results obtained by van Uden et al. (1968) suggested that there might be at least two distinct types of temperature profiles in yeasts, namely profiles in which there is some form of biologically significant association between the Arrhenius plots of growth and thermal death and other profiles in which growth and thermal death are dissociated. A. ASSOCIATIVE PROFILES

I . Batch Culture Wild and industrial strains of Sacch. cerevisiae (van Uden and MadeiraLopes, 1970; van Uden and Duarte, 1981; Loureiro and van Uden, 1982), but not necessarily genetic strains (Madeira-Lopes and van Uden, 1979), display typical associative profiles. The principal characteristics of such profiles are summarized in Table 1 and in Fig. 7. When a population of Sacch. cerevisiae growing at a suboptimal growth temperature is transferred to a liquid stirred medium incubated at a temperature between Topand T,,, and, when the logarithms of absorbance and viable cell counts are plotted against time, curves are obtained of the types depicted in Figs 8 and 9. In both cases, after a lag phase of longer or shorter duration (during which unbalanced growth may take place; Shaw, 1967) an initial period of exponential growth occurs during which the specific rate of mass growth (measured as absorbance) is equal to the specific rate of increase of the viable population (measured as viable cell counts). The duration of this first period and the respective specific growth rate decrease with increasing temperature. The first period of exponential growth is followed by a second period of exponential change during which exponential growth concurs with exponential thermal death. The true specific growth rate of the viable population during the second period is equal to k,, the specific growth rate of the first period. The apparent or net specific growth rate of the viable population during the second period is equal to the difference between k, of the first period and kd, the specific thermal death rate of the second period. the so-called final maximum temperature for growth, Between Topand Tmaxp k, is greater than kd and net exponential increase of the viable population takes place. The duration of the second exponential period between To, and Tmaxr is therefore unlimited as long as the culture medium is appropriate. The specific thermal death rate increases with the temperature whereas k, decreases. At Tmaxf k, equals kd and no net change of the viable population takes place during the second period (Krouwel and Braber, 1979). Between Tmaxf and T m q , the so-called initial maximum temperature for growth, the true specific growth rate of the viable population is smaller than its specific thermal death rate. Consequently, net exponential death prevails, which must lead to the eventual extinction of the viable population. Above Tmaxi, a first period


TABLE 1. Characteristics of exponential growth and death as a function of temperature in yeasts with associative temperature profiles Process temperaturea

Characteristics of exponential periodsh

T < Top One exponential period (Fig. 7 curve A) Duration endless as long as the medium is appropriate:

dN 1 - k, dt N

--

Value for k, increases with increasing temperature. Top< T < Tmaxf Two exponential periods (Fig. 7, curve B) First period: duration decreases with increasing temperature:

dN 1 _ - - k, dt N Value for k g decreases with increasing temperature. Second period: duration endless as long as the medium is appropriate:

dN 1 dt N

-_

=

k,-kd

kg > kd Value fork, decreases while that for kd increases with increasing temperature.

Tmaxr< T < T,,,, Two exponential periods (Fig. 7, curve C) First period: duration decreases with increasing temperature: dN 1 - k, dt N Value for k , decreases with increasing temperature. Second period: population eventually becomes extinct: dN 1 - kg-kd dt N

kg < kd Value for k , decreases while that for kd increases with increasing temperature.

21 1

212

N. VAN UDEN

TABLE 1. (continued) Process temperaturea

Characteristics of exponential periodsh

Tmaxi .

- 103

.Z

-fl c

-102

2u 0

ii -10

0.001

0

5

10

15

20

25

5

30

Time (h)

Time ( h )

FIG. 38. Exponential growth, death and petite mutation in a strain of the yeast Succhuromyces cereuisiue. pg,specific growth rate; pd, specific death rate; p,,,', apparent specific mutation rate; pm, true specific mutation rate. (a) At 38°C without ethanol (reproducedwith permission from Sim6es-Mendes et al., 1978). (b) At 36.2"Cwith 5% (v/v) ethanol (from Cabega-Silva et ul., 1982).

247


o - ~.

1

(a)

1 o-d

---

10-z

'In

E

x 10-E u

10.~ rn

x

D

= i 1

o-~

10-6

10.'

4

1

1

1

1

40 38 36 L

320

1

1

l

34 32 30 28 Temperature ("C) I

325

I

330

l

26 I

335

lo5 -Reciprocal of absolute temperature ( K-')

FIG. 39. Temperature profiles of specific growth rates and specific petite mutation rates in a strain of Saccharomyces cereuisiae grown (a) without ethanol (reproduced with permission from Simdes-Mendes et al., 1978) and (b) with 5% (v/v) ethanol. From CabeGa-Silva et al. (1982). Abbreviations: ps, specific growth rate during the first exponential period (0); pg-pd, net specific growth rate during the second exponential period ( 0 ) ;pd, specific death rate; pm. specific petite mutation rate (0).

lower temperatures without disrupting it (van Uden and Duarte, 1981; Loureiro and van Uden, 1982) and to enhance petite mutation (Zakharov and Bandas, 1979; Bandas and Zakharov, 1980), had a temperature profile of mutagenic action which coincided with the supraoptimal range of temperature corresponding to the ethanol concentration used (Figs 38b and 39b; Cabeqa-Silva et al., 1982). The correlations observed suggest that, in Sacch. cereuisiae, the ethanoland temperature-sensitive petite mutation sites are located in the same membrane as the ethanol and temperature-sensitive T, sites and thermal death sites, namely the inner mitochondria1 membrane (Cabeqa-Silva et al., 1982). Should this hypothesis be confirmed, one should of course not exclude

248

N. VAN UDEN

a role for the nuclear genome of Sacch. cerevisiae in determining the temperature profile of this yeast and its sensitivity to ethanol and other drugs, since a large part of the mitochondria1 composition is under nuclear control. Furthermore, the mechanisms that underly the temperature profile of Sacch. cerevisiae and their sensitivity to drugs are not necessarily the same in all other yeasts or in eukaryotic cells in general.

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Effects of Alcohols on Micro-Organisms LONNIE O’NEAL INGRAM Department of Microbiology and Cell Science, IFAS, and Department of Immunology and Medical Microbiology, University of Florida, Gainesville, Florida, USA

and THOMAS M. BUTTKE Department of Microbiology. University of Mississippi Medical Center, Jackson, Mississippi, USA

I. Why study the effects of alcohols on micro-organisms? . . XI. Effects of alcohols on prokaryotic micro-organisms . . . . A. Growthandsurvival . . . . . . . B. Cellmorphology . . . . . . . . . . . . . . . C. Peptidoglycan synthesis D. Macromolecular synthesis . . . . . E. Biosynthesis of outer membrane and secreted proteins . . . F. Fatty acid composition and biosynthesis . . . . . . . . . G. Phospholipid composition and biosynthesis . H. Membrane-bound enzymes and transport systems . . I. Membrane leakage . . . . , 111. Effects of alcohols on eukaryotic micro-organisms . . . . A. Growth and morphogenesis . . . . . . B. Transport systems . . . . . . . C. Membrane leakage . . . . . . . D. Lipid composition and biosynthesis . . . . . . . . E. Thermal tolerance in yeasts . F. Ethanol-induced leakage and excretion in yeasts . . . . . . . . IV. Effects of alcohols on membrane organization . V. Effects of lipid supplements on alcohol tolerance, growth, survival and fermentation . . . . . . . . . . . A. Escherichia coli . . . . . . B. Saccharomyces cerevisiae . . . . . . . VI. Mechanism of inhibition of fermentation by ethanol . A. Saccharomyces cerevisiae: The “staling” effect . . . , B. Zymomonasmobilis . . . . . . . . . ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 25 ISBN 0-12-027725-4

254 255 255 257 257 260 261 262 266 268 269 270 270 272 274 274 277 278 280 282 282 284 287 287 290

Copyright 0 1 9 8 4 hv Academic Press, London All rights of reproduction in any form reserved.

2 54

LONNIE O’NEAL INGRAM AND THOMAS M. BUTTKE

VII. Conclusions and future directions A. Alcohols: A biophysical problem B. Membranes are involved! .

C. Futuredirections. VIII. Acknowledgements . References. .

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291 291 292 295 295 296

I. Why Study the Effects of Alcohols on Micro-organisms? Alcohols are ubiquitous small molecules which are produced both chemically and as products of microbial fermentation. Accumulation of alcohols in the microbial environment represents a form of environmental stress, analogous to extremes in pH value and temperature. By understanding the effects of alcohols on microbial systems and the biochemical changes that allow cells to adapt to alcohols, we hope to gain new insights into the regulation of cellular activities and the relationships between cellular structure and function. Biomedical interests in alcohol research stems from its prominence as our single most used and most abused drug. The mechanism by which ethanol depresses central nervous system functions is not understood (Janoff and Miller, 1982). Microbial research forms the backbone of our knowledge of intermediary metabolism, genetics and gene regulation in Man as well as other eukaryotic organisms. Bacteria, yeasts, various moulds and protozoa have been used to study the effects of alcohols. These latter simple eukaryotes may be of particular value due to their similarity to animal cells in having well differentiated organelles and membranes containing sterols. Recent studies have linked alcohol tolerance in animal cells with increased membrane sterol content (Chin and Goldstein, 1977; Chin et al., 1978).The ease with which the sterol composition of unicellular eukaryotes can be modified should serve to make these organisms useful model systems for studying the relationship between alcohol tolerance and membrane sterol content. Investigations of the effects of alcohols on microbes may provide important clues to mechanisms for alcohol tolerance, dependence and intoxication in Man. The political instability of many of the nations supplying petroleum and our relatively recent realization that petroleum reserves are not infinite has provided additional impetus for alcohol research. Both Brazil and India utilize ethanol from microbial fermentations as a principal energy source and as a feedstock for chemical industries. In Europe, the United States, Canada and other countries, ethanol is now used as an octane enhancer in premium lead-free gasoline replacing up to 10% of the petroleum. Further research into the mechanisms of action of ethanol on alcohol-producing micro-organisms could lead to substantial decreases in the cost of microbial alcohol production. It has long been recognized that industrial ethanol fermentations terminate prematurely, despite an unlimiting supply of substrates. Early studies

EFFECTS OF ALCOHOLS ON MICRO-ORGANISMS

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demonstrated that this inhibition of fermentation was due to a “staling effect”, which refers to accumulation of toxic products resulting from the fermenting organism’s own metabolism. Premature inhibition of fermentation by end products limits the final concentration of alcohol which can be achieved (Gray, 1941). Ethanol is now well established as the primary end product responsible for inhibiting fermentation. Thus, it is not surprising that numerous workers have, for at least 50 years, sought to define the mechanisms by which ethanol inhibits the growth of yeast and causes staling. Recent studies have provided much valuable information regarding the effects of ethanol and other membrane-active agents on prokaryotic and eukaryotic micro-organisms. Alcohol research has shown that, in many cases, fermentation can be prolonged by supplementing specific lipid molecules. This result has already found practical application in industrial alcohol production. In this review, we will summarize research on the action of ethanol and other alcohols on micro-organisms, and attempt to identify some of the important mechanisms of action. For additional information regarding biomedical research with ethanol, the recent reviews by Michaelis and Michaelis (1983) and Janoff and Miller (1982) will serve as an excellent starting point. The review by Seeman (1972) provides an excellent summary of the older literature on the pharmacological effects of ethanol.

11. Effects of Alcohols on Prokaryotic Micro-organisms A. GROWTH AND SURVIVAL

Alcohols have been employed for many years both as a disinfectant and as a preservative. Concentrations of ethanol above 15% result in immediate inactivation of most vegetative organisms, with spores being considerably more resistant (Dagley et al., 1950; Hugo, 1967; Harold, 1970). Low concentrations of ethanol also render bacteria more sensitive to inactivation by ionizing radiation (Jacobs, 1981) and by lipophilic acids (Corner, 1981). Most bacteria exhibit a dose-dependent inhibition of growth over the range from 1% to 10% ethanol, and few grow at concentrations above 10%. Growth of Escherichia coli is inhibited at ethanol concentrations (v/v) above 6% (Ingram, 1976) while that of Zymomonas mobilis, an organism capable of producing high concentrations of ethanol (Rogers et al., 1982; Stokes et al., 1982), can grow in concentrations up to 8% (Fig. 1). Organisms of the genus Lactobacillus are exceptional in that many of these grow in higher alcohol concentrations. Lactobacillus heterohiochii and Lactobacillus homohiochii are the most resistant micro-organisms known (Demain et al., 1960). These two organisms cause spoilage in sake, and have been reported to grow in over 20% ethanol (Kitahara et al., 1957; Uchida and Mogi, 1973; Uchida, 1975a,b).

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FIG. 1. Inhibition of growth by ethanol in three bacteria. Inocula were grown at 30°C in complex medium lacking ethanol. These were diluted approximately 100-fold into fresh media containing a range of ethanol concentrations. Growth was measured as absorbance at 550 nm. Values plotted were measured after 48 hours and did not change appreciably during subsequent incubation. Escherichia coli (0) is seen to be the most ethanol-sensitive organism and Lactobacillus heterohiochii ( 0 )is the most resistant an obligately fermentative organism that produces organism. Zymomonas mobilis (O), ethanol, is intermediate in its ability to grow in the presence of ethanol (L.O. Ingram, unpublished observations).

Growth of L. heterohiochii is actually stimulated by low concentrations of ethanol. The toxicity of alcohols is directly related to their chain length and hydrophobicity (Harold, 1970; Hugo, 1967). Longer-chain alcohols, up to a chain length of around 10 carbon atoms, are much more potent inhibitors than are the shorter-chain alcohols. Longer fatty alcohols (over 10 carbon atoms in length) are relatively ineffective as inhibitors. For organisms such as E. coli, the growth toxicity roughly doubles with each additional aliphatic carbon atom up to octanol. Cyclic alcohols, such as benzyl alcohol and phenethyl alcohol, have also been shown to inhibit growth, and the extent of their toxicity was consistent with the hydrophobicity of these alcohols (Lang and Rye, 1972; Wilson et al., 1981). The presence of short-chain alcohols such as ethanol dramatically lowers the thermal tolerance of Zymomonas mobifis (Benschoter et al., 1983; Dombek and Ingram, 1983) as shown in Fig. 2. Conversely, elevated temperatures dramatically reduce the ethanol-tolerance of these organisms. Like growth, isolated membranes from 2.mobilis are more sensitive to disorganization by ethanol at elevated temperatures (Dombek and Ingram, 1983). Thermophilic


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organisms such as Clostridium acerobutylicum (Linden and Moreira, 1982; Ulmer et al., 1982) and Cl. thermocellum (Herrero and Gomez, 1980) are also particularly alcohol sensitive at elevated growth temperatures. Janoff and Miller (1982) proposed that this increase in alcohol-sensitivity at elevated temperatures may be due to an increase in the amount of alcohol that partitions into the membrane. An understanding of the mechanism by which these two stress factors interact should be instructive in determining the basis for inhibition of growth by alcohols. Inhibition of growth by alcohol is substantially increased by glucose concentrations (w/v) above 10% (Carey and Ingram, 1983). Recent studies by Dombek and Ingram (1983) have shown that alcohol-induced fluidization of isolated membranes from Z . mobilis is more extensive in the presence of high concentrations of glucose, implying a membrane-mediated effect.

B . CELL MORPHOLOGY

Fried and Novick (1973) have reported that growth of E. coli in the presence of ethanol resulted in interference with cell division, and production of elongated cells which divided on removal of ethanol. Other studies have reported the production of similarly filamentous cells of E. coli during growth in the presence of hexanol (Ingram, 1981). Fried and Novick (1973) isolated ethanol-resistant mutants that did not exhibit this pleomorphy, and growth of one group of these was actually stimulated by low concentrations of ethanol. These effects appeared to result from non-specific actions of ethanol since other alcohols and dimethyl sulphoxide gave similar results. Sturgeon et al. (1975) have shown that addition of ethanol and other short-chain alcohols (C, and shorter) have the opposite effect on a group of cold-sensitive cell-division mutants of the blue-green bacterium Agmenellum quadruplicatum. Addition of ethanol to filamentous cells of A . quadruplicatum stimulated a co-ordinate burst of cell division. These effects in E. cofi and in A . quadruplicatum were thought to result from alcohol-induced alterations of membrane function. The sporulation process in Bacillus subtilis is also inhibited by short-chain alcohols (Bohin and Lubochinsky, 1982). A single-gene mutation was found to confer a decrease in ethanol sensitivity and to cause pleotropic changes in membrane composition.

C. PEPTIDOGLYCAN SYNTHESIS

Synthesis of bacterial peptidoglycan is a multistep process which involves synthesis of complex precursors within the cytoplasm, transport of these precursors through the plasma membrane by the translocase and their assembly to form the bag-shaped macromolecule which prevents osmotic lysis

LONNIE O'NEAL INGRAM AND THOMAS M. BUTTKE

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2.0 I

1.6

-

1.2

L c in

x

E

e

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0.8

0 Ethanol concentration

(%)

FIG. 2. Interdependence of temperature and ethanol concentration on growth of Zyrnornonas rnobilis strain CP4. An exponentially growing culture was diluted into media containing a range of ethanol concentrations and these were incubated at various temperatures. Growth was measured at Asso. (a) Effect of incubation temperatures ( 0 , 20°C; 0,30°C; 0,40"C) on ethanol tolerance. (b) Effect of ethanol 2%, v/v; 0 , 5%, v/v; 0 , 7%, v/v; m, control) on thermal tolerance. concentrations (0, Increasing growth temperatures severely limited alcohol tolerance in Zymornonas rnobilis. Conversely, increasing ethanol concentrations severely limited thermal tolerance in this bacterium. From Benschoter et al. (1983) and Dombek and Ingram (1983).

in bacteria. Studies in vitro using membrane fragments of Staphylococcus aureus (Lee et al., 1980) have shown that butanol stimulates the activity of the lipid phospho-N-acetylmuramylpentapeptide translocase. Studies in uivo with A . quadruplicatum (Dickens and Ingram, 1976) have shown that addition of ethanol to filamentous cells of a mutant results in stimulation of peptidoglycan synthesis during cell division. Again, the basis of this stimulation of peptidoglycan synthesis was thought to involve interactions of alcohol with the cell membrane.

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Short-chain alcohols (C, to Cq) cause an unusual growth-dependent cell lysis in some strains of E. cofiwhich also results from effects on peptidoglycan assembly (Ingram and Vreeland, 1980). Cultures lyse precipitously after 3 4 hours growth in the presence of modest concentrations of ethanol (2-5%,v/v). Longer-chain alcohols such as hexanol do not cause similar lysis despite their effectivenessas growth inhibitors. The basis of this lysis has been investigated in some detail (Ingram, 1981). These modest concentrations of ethanol lower the extent of peptidoglycan cross-linking in the growing cell wall, analogous to the action of the antibiotic penicillin (Blumberg and Strominger, 1974). Peptidoglycan continued to become progressively weakened during growth in the presence of ethanol. Cells became swollen and lysed due to the large osmotic pressure difference across the plasma membrane. Mutants have been isolated which are resistant to this growth-dependent lysis by ethanol and the peptidoglycan cross-linking in these mutants was not affected by ethanol.

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The mechanism by which ethanol affects cross-linking reactions is unusual in that it appears to involve the colligative properties of the growth medium rather than a direct action of alcohol on a target enzyme (Ingram, 1981). The cross-linking enzyme is thought to be attached to the outer surface of the cell’s plasma membrane, and to face outward toward the nascent peptidoglycan substrate (Lee et al., 1980). Both ethanol and chaeotropic salts such as sodium thiocyanate and sodium perchlorate weaken hydrophobic interactions (Hansan and Rosen, 1977). Sub-lytic concentrations of ethanol and chaeotropic salts were additive in their ability to cause growth-dependent lysis. Antichaeotropic salts, such as sodium chloride or potassium sulphate, strengthen hydrophobic interactions and prevented ethanol-induced lysis during growth. If added after an initial period of growth in the presence of ethanol, however, neither antichaeotropic salts nor removal of ethanol by washing prevented lysis during subsequent growth. The hypothesis was presented that ethanol and other chaeotropic agents may prevent development of functional associations between newly synthesized cross-linking enzymes and the cell membrane. Growth of bacteria in the presence of sub-lytic concentrations of antibiotics, which inhibit peptidoglycan biosynthesis, frequently affects cellular division, resulting in the production of filaments and other pleomorphic cells (Blumberg and Strominger, 1974). It is likely that the pleomorphic cells formed during growth with aliphatic alcohols also result from alcoholinduced alterations in peptidoglycan synthesis and assembly.

D. MACROMOLECULAR SYNTHESIS

Phenethyl alcohol (phenethan-2-01) has been investigated in some detail as a bacterial inhibitor of macromolecular synthesis. Gram-negative bacteria, such as E. coli, are more sensitive to this compound than are Gram-positive organisms, and this alcohol has been recommended for use in distinguishing these groups of bacteria (Tilley and Shaffer, 1926; Lilley and Brewer, 1953). Berrah and Konetzka (1962) examined the effects of phenethyl alcohol on biosynthesis of RNA, DNA and protein in E. coli. In this study, a concentration of 0.25% (v/v) appeared to be a potent, selective and reversible inhibitor of DNA synthesis. As with other inhibitors of DNA synthesis, cell division was also blocked with production of filamentous cells in the absence of genome duplication. High concentrations of phenethyl alcohol have been shown to disrupt the association between replicating DNA and the cell membrane (Masker and Eberle, 1972). Further studies revealed that phenethyl alcohol did not cause an immediate inhibition of DNA synthesis but acted to inhibit initiation of a subsequent round of DNA replication (Treick and Konetzka, 1964; Lark and Lark, 1966). Other studies investigat-


26 1

ing the effects of phenethyl alcohol on induction of alkaline phosphatase (Rosenkranz et al., 1964, 1965) and of P-galactosidase (Provost and Moses, 1966) hypothesized that RNA synthesis is the most sensitive target. Analyses of phenethyl alcohol-resistant mutants revealed that, in many mutants, this resistance resulted from mutations in genes encoding proteins involved in DNA replication (Wada and Takashi, 1974). Phenethyl alcohol also affects synthesis of membrane proteins and membrane lipids. These investigations are discussed in Section 1I.E. (p. 261). In contrast to phenethyl alcohol, short-chain aliphatic alcohols, such as ethanol, cause a retardation in the rates of RNA, DNA and protein synthesis in experiments in vivo with E. coli (Ingram, 1977b, and unpublished results) and do not act as specific inhibitors of these processes. Addition of methanol, ethanol or propanol caused a decrease in growth (the bulk of which is protein) accompanied by corresponding decreases in the rate of RNA accumulation (Mitchell and Lucas-Lenard, 1980). Guanosine tetraphosphate, a regulatory metabolite which couples RNA synthesis and protein synthesis (Haseltine et al., 1972), increased on addition of these alcohols, consistent with the co-ordinate decrease in the rates of RNA and protein biosynthesis. In vitro, alcohols, dimethyl sulphoxide and other organic solvents have been reported to stimulate synthesis of DNA by polymerase I11 over twofold (Heinze and Carl, 1975). This unusual property has proven useful to distinguish DNA polymerase 111 from other polymerase enzymes (Kornberg and Gefter, 1972; Gefter, 1974). Although the mechanism by which this stimulation occurs is unclear, it is thought to involve the membrane association of this enzyme (Heinze and Carl, 1975). Although DNA polymerase I11 is affected by aliphatic alcohols, these effects do not lead to an increase in mutagenicity (Osztovics et al., 1981). Acetaldehyde, a metabolite of ethanol, does, however, cause an increase in mutation rate (Igali and Gazso, 1980). Allylic alcohols, such as butenol and propenol (allyl alcohol), are also mutagenic in the Ames’ test with Salmonella typhimurium (Lutz et al., 1982). Mutants of E. coli have been isolated which are resistant to allyl alcohol. These were deficient in alcohol dehydrogenase, preventing the conversion of allyl alcohol into its aldehyde (Lorowitz and Clark, 1982). E. BIOSYNTHESIS OF OUTER MEMBRANE AND SECRETED PROTEINS

In E. coli, biosynthesis of several outer-membrane proteins and secreted proteins is inhibited by concentrations of phenethyl alcohol equivalent to those that inhibit DNA synthesis (Tribhuwan et a[., 1970; Tribhuwan and Pradhan, 1977; Pages et al., 1978; Halegoua and Inouye, 1979; Lazdunski et al., 1979; Pugsley et al., 1980). These proteins are normally synthesized on membrane-bound polysomes, transported through the plasma membrane

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with processing by signal peptidase and either secreted into the periplasmic space or inserted into the outer membrane (DiRienzo et af., 1978). Phenethyl alcohol blocks the processing and release of alkaline phosphatase into the periplasmic space (Pages and Lazdunsky, 1981). In addition, phenethyl alcohol prevents processing of three of the major outer-membrane proteins and causes their accumulation as precursor forms in the cytoplasmic membrane (Halegoua and Inouye, 1979; Pugsley el al., 1980). Similar effects have been shown to occur as a result of changes in membrane lipid composition and after addition of anaesthetics (DiRienzo et af., 1978; DiRienzo and Inouye, 1979) suggesting that these effects of phenethyl alcohol may result from interactions of phenethyl alcohol with the plasma membrane. Recent studies by Daniels et a f .(1 98 1) have demonstrated a requirement for membrane potential during the processing and secretion of proteins into the periplasmic space. In these experiments, both carbonylcyanide rn-chlorophenylhydrozone (a proton ionophore) and phenethyl alcohol blocked the processing and secretion of b-lactamase and leucine-binding protein into the periplasmic space. Early studies by Silver and Wendt (1967) showed that similar concentrations of phenethyl alcohol cause an increase in the rate of leakage of potassium ions. Thus, disruption of the proton gradient could be the basis for the preferential sensitivity of secreted and outer-membrane proteins to phenethyl alcohol. F. FATTY-ACID COMPOSITION AND BIOSYNTHESIS

Escherichia cofi has been used extensively as a model system to investigate the effects of alcohols on membrane composition and function during the last several years. The fatty-acyl composition of E. cofi is relatively simple and consists of palmitic acid ( C I ~ : Oas) the principal saturated residues and palmitoleic (Cl6:l)and vaccenic (C18:I)as the major unsaturated residues. The growth of E. cofi in the presence of alcohols caused major shifts in fatty-acyl composition (Ingram, 1976; Sullivan el af.,1979; Zemlyanukhina et a f . ,1981). These changes began immediately and were immediately reversible on removal of alcohol as shown for ethanol in Fig. 3. Changes in fatty-acyl composition occurred as a result of dilution during lipid synthesis and did not result from deacylation and reacylation of existing lipids. Alcohols can be divided into three groups based on chain length and the type of change in fatty-acyl residue induced (Ingram, 1976; Sullivan et a]., 1979; Zemlyanukhina et af.,1981). Short-chain alcohols up to butanol caused cells to synthesize increased concentrations of vaccenyl residues with corresponding decreases in the proportion of palmityl residues (Ingram, 1976). This change is identical with those changes induced by a shift-down in growth temperature (Marr and Ingraham, 1962). Longer-chain alcohols, from pentanol to octanol, caused cells to synthesize lipids with a higher

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1 0 0

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90

0

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120

Time (min)

FIG. 3. Effects ofethanol on the fatty-acyl composition ofEscherichiu colistrain CSH 2. (a) Ethanol (4%, v/v) was added to exponentially growing cells at zero time and samples were removed for fatty-acyl analyses during subsequent growth. (b) Cultures were grown for 3 hours in the presence of ethanol (4% v/v). These were diluted 10-fold into fresh medium lacking ethanol at zero time. Samples were removed for fatty-acid analysis during subsequent growth. In both (a) and (b), 0 represents proportion of palmitic acid residues (Cleo), and 0 , represent vaccenic (Cla:l)-acid and palmitoleic (Cls:l)-acid residues, respectively. Addition of ethanol caused an immediate shift in fatty-acyl composition with an increase in the proportion of vaccenic-acid and a decline in palmitic-acid residues during growth. Dilution of ethanol resulted in an equally rapid reversal of the ethanol-induced changes. Replotted from Ingram (1976).

proportion of palmityl and a decrease in vaccenyl residues (Ingram, 1976; Sullivan et al., 1979) analogous to the changes observed following an increase in growth temperature (Marr and Ingraham, 1962). Butanol caused changes similar to those with ethanol in some strains of E. coli (Ingram, 1976) and like those of pentanol in other strains (Sullivan et al., 1979). The longer-chain fatty alcohols, such as nonanol and decanol, had no effect on fatty-acyl composition and these fatty alcohols did not retard the growth rate of the organism (Sullivan et al., 1979). A variety of other organic solvents have been examined for their effects on fatty-acyI composition (Ingram, 1977a; Nunn, 1975). The relative potency of alcohols up to octanol and of the other solvents was directly related to their hydrophobicity, indicating a hydrophobic site of action. In all cases, the small relatively polar solvents, such as ethylene glycol and acetone, induced changes in fatty-acyl composition similar to that caused by the short-chain alcohols,

264


whereas the more hydrophobic compounds, such as amyl acetate and analine, induced changes like those caused by hexanol, illustrating the lack of strict structural specificity for these effects. The mechanisms by which alcohols alter fatty-acyl composition have been investigated in some detail in E. coli. The fatty-acyl composition of the plasma membrane in E. coli is determined both by biosynthesis of fatty acids and by the selectivity of the acyl transferase enzymes which assemble these residues to form phospholipids (Raetz, 1978). Ethanol-induced changes in fatty-acyl composition could be prevented by supplementing cultures with exogenous palmitic acid to increase the supply of this residue (Buttke and Ingram, 1978). Supplementing cultures with unsaturated fatty acids ( C I ~ or : I C I ~ :did I ) not prevent the ethanol-induced decrease in palmityl residues. These results suggested that ethanol was altering the fatty acid composition by diminishing the synthesis or supply of palmitic acid rather than by affecting the specificity of the acyl transferase enzymes that catalyse incorporation of fatty acids into phospholipids. This was confirmed in vivo using mutants that accumulate free fatty acids in the absence of phospholipid synthesis, and using temperatureconditional fatty acid-synthesis mutants which were fed both saturated and unsaturated fatty acids (Buttke and Ingram, 1978). Subsequent experiments in vitro (Buttke and Ingram, 1980; Ingram, 1982) showed that the ratio of unsaturated to saturated fatty acyl products from the soluble enzymes of fatty acid synthesis increased both with increasing concentrations of ethanol and with a shift down in incubation temperature. These changes occurred both in crude and in purified preparations that were substantially free from membrane, indicating that the effect of ethanol on fatty acid synthesis in E. coli is not dependent on the effects of ethanol on the cell membrane. Further studies have revealed a surprising mechanism of action of ethanol which involves the colligative properties of the solvent. A decrease in incubation temperature, addition of ethanol and addition of chaeotropic salts all weaken hydrophobic interactions and all cause similar and additive effects, increasing the proportion of unsaturated fatty acids synthesized (Ingram, 1982). This appears to be an important part of the mechanism for ethanol-induced and temperature-induced changes in the fatty-acyl composition of E. coli. Analogous types of experiments have been performed in vivo and in vitro to determine the mechanism of hexanol-induced changes in fatty-acyl composition (L.O. Ingram, unpublished observations). Hexanol is a much more hydrophobic compound and caused changes in the fatty-acyl composition of E. coli which were opposite to those induced by ethanol (Ingram, 1976). The hexanol-induced increase in the proportion of palmitic acid appears to result from a change in the specificity of the acyl transferase enzymes embedded in the plasma membrane. Temperature changes have been shown to alter both the selectivity of the


265

acyl transferase system (Sinensky, 1971, 1974) and the composition of acyl products of fatty acid synthesis (Ingram, 1982). Investigations with long- and short-chain alcohols have allowed us to dissect this regulatory process into two parts, and to establish that a change in either part is sufficient to cause shifts in membrane fatty-acyl composition. The effects of alcohols on the fatty-acyl composition of other bacteria have also been examined. An increase in the ratio of unsaturated to saturated fatty-acyl residues was reported in the phospholipids of Mycobacterium smegmatis following growth at 27"C, analogous to a shift to a lower growth temperature (Taneja and Khuller, 1980). However, ethanol caused the opposite change during growth at 37°C. No data were presented for specific fatty-acyl residues. Zymomonas mobilis, an obligately ethanol-producing bacterium, does not change its membrane fatty-acyl composition in response to added ethanol (Carey and Ingram, 1983). However, the phospholipids of this organism are particularly rich in vaccenic acid which accounts for over 60% of the total fatty-acyl residues (Carey and Ingram, 1983;Ohta et al., 1981; Tornabene et al., 1982). Gram-positive bacteria have also been investigated. Rigomier et al. (1980) have shown that, during growth in the presence of ethanol, an increase in the proportion of C I 6and C I 8residues occurs with corresponding decreases in the amounts of Cl5 and CI, residues. In addition, there was a decrease in the proportion of branched-chain fatty acids. Kates et al. (1962) have reported very different changes in Bacillus cereus. Both ethanol and propanol caused increases in the proportion of C I Sbranched-chain fatty-acyl residues with a decrease in the proportion of branched-chain C16 and C17 fatty-acyl residues in total lipid extracts. Very few normal chain residues were present in this micro-organism when it was grown in the presence or absence of ethanol. Herrero et al. (1982) have shown a variety of changes in the proportions of fatty-acyl residues in the total lipids of Clostridium thermocellum, a cellulytic organism that grows and produces ethanol at 60°C. Ethanol caused a decrease in the proportion of normal saturated fatty-acyl residues and an increase in branched-chain and unsaturated residues. This was accompanied by an increase in C12 and C13 residues. An alcohol-resistant mutant was isolated and exhibited similar trends. The effects of the changes in fatty-acyl residues on membrane composition reported in B. subtilis, B. cereus and Cl. thermocellum are difficult to interpret, due to the mixed analyses of phospholipids (membrane components) and the sizable neutral-lipid fraction which may also be pesent. Neutral lipids (di- and triacylglycerides) are storage lipids and may obscure significant changes in the composition of the plasma membrane. The hiochii bacteria, Lactobacillus homohiochii and L. heterohiochii are the most alcohol-resistant organisms known (Uchida and Mogi, 1973) and are commonly found as spoilage organisms in saki (Kitahara et al., 1957).

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Vaccenic acid and its cyclopropane residue represent over 60% of the total residues in L. homohiochii with a ratio of unsaturated to saturated residue of 3.0 (Uchida, 1975b). This ratio increases dramatically to 40 during growth in the presence of 10% (v/v) ethanol. All alcohols tested (C, to C,) caused a similar change in the fatty-acyl composition of L. homohiochii (Uchida, 1975b) in contrast to their effects on E. coli (Ingram, 1976). The potency of alcohols for inducing changes in fatty-acyl composition in L. homohiochii correlated well with their triolein/water partition coefficients indicating a hydrophobic site of action (Uchida, 1975b). Although data were not given for individual residues, vaccenyl residues were the dominant ones in this organism. It is likely that the increases in unsaturation reflect increases in this residue and its cyclopropane derivative as shown by a comparison of strains grown in complex medium and in sakt (Uchida and Mogi, 1973). Lactobacillus heterohiochii has a unique fatty-acyl residue composition characteristic of this species with over 20% of its total cellular fatty-acyl residues being O length (Uchida, 1974). In this organism, monoenoic acyl chains over C ~ in addition of ethanol caused a substantial increase in the proportion of unsaturated fatty-acyl residues (especially vaccenyl) at the expense of saturated residues (Uchida, 1975a), with a slight increase in the average chain length of the residues.

G. PHOSPHOLIPID COMPOSITION AND BIOSYNTHESIS

Although phenethyl alcohol has been used as an inhibitor of initiation of DNA synthesis in E. coli, as already described, this action may result from the effects of phenethyl alcohol on the phospholipid composition of the plasma membrane (Barbu et al., 1970). Unlike DNA synthesis, phospholipid synthesis was immediately affected by addition of phenethyl alcohol to growing cultures (Barbu et al., 1970; Nunn and Tropp, 1972; Nunn, 1975). Cells grown in the presence of phenethyl alcohol contained lower levels of phosphatidylethanolamine with increases primarily in cardiolipin (Nunn, 1975).These effects resulted from a preferential inhibition of phosphatidylethanolamine synthesis and a stimulation in the rate of conversion of phosphatidylglycerol into cardiolipin (Nunn, 1975). There was a net inhibition of phospholipid synthesis resulting in a decrease in cellular phospholipid, and this was not relieved by addition of exogenous fatty acids. Acylation of the lysophosphatidic acid, phosphatidylserine synthetase and phosphatidylglycerol synthetase all appeared resistant to phenethyl alcohol (Nunn et al., 1977). The initial acylation of sn-glycerol 3-phosphate was sensitive to phenethyl alcohol and was proposed as the primary target leading to the decrease in cellular phospholipid (Nunn et al., 1977). Other aliphatic alcohols caused similar effects on synthesis of phospholipids


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in E. coli (Ingram, 1977b). Cells grown in the presence of aliphatic alcohols contained an elevated proportion of acidic phospholipids (phosphatidylglycerol together with cardiolipin). Alcohols of chain lengths CI to Cs caused a dose-dependent inhibition of phospholipid synthesis resulting from a preferential inhibition of phosphatidylethanolamine biosynthesis. Phosphatidylserine synthetase has been proposed as the site of alcohol inhibition in E. coli (Ingram, 1977b).Cells (Ingram, 1977b)and plasma membranes (Dombek and Ingram, 1984) from cultures grown in the presence of ethanol were deficient in phospholipid and exhibited low phospholipid :protein ratios. Alcohols did not affect the rate of phospholipid turnover. Alcohol-induced changes in fatty-acyl composition were uniformly reflected in each major class of phospholipid (Ingram, 1977b). In the presence of ethanol, cells synthesized increased levels of phospholipid species with unsaturated fatty-acyl residues at both positions, whereas hexanol stimulated synthesis of phospholipids containing one saturated and one unsaturated residue (Berger et af., 1980). The distribution of different molecular species of phospholipids following growth in the presence of alcohols was the same as that observed at altered growth temperatures (Berger et af., 1980). Clark and Beard (1979) have reported the isolation of alcohol-resistant mutants of E. coli. These mutants exhibited an overproduction of acidic phospholipids, consistent with the alcohol-induced increase in acidic lipids being important for alcohol tolerance. The effects of alcohols on phospholipid composition have also been examined in other bacteria. Growth of M . smegmatis (Taneja and Khuller, 1980) in the presence of ethanol resulted in a decrease in the amount of phospholipid (mg of phospholipid (g dry cell weight)-'), a decrease in the amount of phosphatidylethanolamine, and an increase in the proportion of phosphatidylinositol mannosides (acidic lipids). Added ethanol had little effect on the phospholipid composition of Z . mobilis, causing a small decrease in phosphatidylethanolamine and phosphatidylglycerol with increases in cardiolipin and phosphatidylcholine (Carey and Ingram, 1983). Exogenous ethanol did, however, cause a substantial decrease in the membrane phospholipid: protein ratio of Z . mobifis (Carey and Ingram, 1983). Ethanol also caused a substantial decrease in the amount of phospholipid in B. subtilis and an increase in the proportion of phosphatidylethanolamine (Bohin and Lubochinsky, 1982; Rigomier et af., 1980). In contrast, addition of propanol has been reported to increase the phosphatide content of B. cereus and to decrease the neutral-lipid fraction (Kates et a f . , 1962). In C f .acetobutylicum, the proportion of phosphatidylethanolamine was decreased with an increase in phosphatidylglycerol in cells grown with ethanol (L. Vollherbst-Schneck, L. Thompson, M. Krajci, J.A. Sands and B.S. Montenecourt, unpublished observations).

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H. MEMBRANE-BOUND ENZYMES A N D TRANSPORT SYSTEMS

The effects of ethanol on a variety of active membrane functions have been examined in E. coli (Eaton et al., 1982; Fried and Novick, 1973; Ingram et al., 1980a; Sullivan et al., 1974). Addition of 0.67 M ethanol did not cause a significant inhibition of the Na+, K+-dependent ATPase, NADH oxidase or D-lactate oxidase (Eaton et al., 1982). High concentrations of ethanol (1.7 M) were required to cause a 20% inhibition of these enzymes with ATPase being the most resistant enzyme examined (Eaton et al., 1982; Ingram et al., 1980a). In contrast, succinate dehydrogenase was more sensitive, showing 20% inhibition with 0.67 M ethanol and 50% inhibition with 1.7 M ethanol. Transport systems were uniformly more sensitive to inhibition by ethanol. The lactose permease system exhibited a dose-dependent inhibition with increasing concentrations of ethanol (Fried and Novick, 1973; Ingram et al., 1980a). Uptake of glutamate, proline, leucine and the lactose permease were inhibited 1&300/, by 0.67 M ethanol and 60-80% by 1.7 M ethanol (Eaton et al., 1982). Inhibition of both the membrane-bound enzymes and transport systems was substantially relieved after removal of alcohol by washing. Changes in fatty-acyl composition caused small changes in the sensitivities of these enzymes and transport systems to ethanol but no clear trends were apparent (Eaton et al., 1982). Longer-chain alcohols (C, to C,) have been shown to stimulate activity of the lactose permease system in E. coli at assay temperatures below the membrane phase transition (Sullivan et al., 1974). The basis of this stimulation was thought to be membrane fluidization by these longer-chain alcohols. Arrhenius plots of the activity of the lactose permease exhibited a discontinuity, the position of which was related to membrane composition (Sullivan et al., 1974). These longer-chain alcohols decreased the temperature at which this discontinuity occurred. The discontinuity was shown to result from changes in the apparent K,,, value of the lactose permease. The effects of ethanol have also been examined on Arrhenius plots of the activity of lactose permease and ATPase in E. coli (Ingram et al., 1980a). In contrast to the longer-chain alcohols, ethanol caused an increase in the break temperature. The reason for this ethanol-induced increase in break temperature is unknown. A series of experiments were performed in E. coli to examine the sensitivity of the lactose permease during growth in the presence of ethanol (Ingram et al., 1980a). The specific activity of the lactose permease returned to the control level during growth in the presence of ethanol. Growth was required and the time necessary for this recovery increased with alcohol concentration and decreased with growth rate of the organism. The time frame for this apparent adaptation was consistent with ethanol-induced changes in membrane composition playing a major role in recovery (Ingram et al., 1980a).


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Linden and Moreira (1982) have examined the effects of alcohols on the ATPase of CI. acetobutylicum. As with other micro-organisms, the relative potency of alcohols was directly related to their chain length and hydrophobicity. Cells grown previously in the presence of butanol were somewhat more resistant to inhibition by butanol suggesting that these cells adapt during growth. This adaptation may be related to alcohol-induced changes in membrane composition (L. Vollherbst-Schneck, L. Thompson, M. Krajci, J.A. Sands and B.S. Montenecourt, unpublished observations). The addition of phenethyl alcohol has been reported to inhibit reversibly the uptake of a-aminoisobutyric acid in a marine pseudomonad (Thompson and DeVoe, 1972). This was shown to be caused by dissipation of the potassium gradient by phenethyl alcohol, required for a-aminobutyric acid uptake. A similar phenethyl alcohol-induced leakage of potassium has been reported for E. coli although the Na+, K+-dependent ATPase was reported to be substantially resistant to this alcohol (Silver and Wendt, 1967). Active transport of lactose and amino acids in E. coli requires ion gradients as an energy source. Pate1 et al. (1975) have shown that chaeotropic salts, such as sodium thiocyanate and sodium perchlorate, inhibit lactose transport by increasing proton leakage across the membrane. Hansan and Rosen (1977) have shown that this increased permeability to protons is due to release of the catalytic subunit of ATPase. Alcohols are also chaeotropic agents and increase leakage (Eaton et al., 1982; Ingram, 1981). This increase in leakage may be the basis of the increased sensitivity of transport systems (as compared to membrane-bound enzymes) to ethanol.

I. MEMBRANE LEAKAGE

Disruption of the cellular permeability barrier is thought to be the basis of bacterial killing by high concentrations of alcohols (Harold, 1970; Hugo, 1967). High concentrations of alcohols solubilize lipids and denature proteins leading to membrane destruction. Lower concentrations of alcohols have been shown to increase the rates of leakage of small molecules in model membrane systems and in native membranes (Pang et al., 1979). The potency of aliphatic alcohols for leakage is directly related to their hydrophobicity implying a hydrophobic site of action. Increased leakage of ions and metabolites may be responsible for the decreased rate of growth in the presence of many alcohols. Phenethyl alcohol has been shown to facilitate the rapid leakage of protons and of potassium ions from E. coli at concentrations that cause partial inhibition of growth (Daniels et al., 1981; Silver and Wendt, 1967). Mutants of E. coli have been isolated which were resistant to phenethyl alcohol, and these were shown to be less permeable to other small molecules such as azide (Yura and Wada, 1967). Ethanol, butanol and hexanol have

270


been reported to increase efflux of protons and potassium in Pseudomonas aeruginosa (Bernheim, 1978). Ethanol has been shown to increase the rate of leakage of nucleotides in E. cofi and this leakage has been correlated with cell killing (Eaton et al., 1982). Nucleotides are relatively large highly charged molecules and small ions such as protons and potassium would be expected to be even more susceptible to leakage. Conditions that decreased the rate of nucleotide leakage (increase in vaccenic acid content, addition of magnesium) also increased survival in E. coli (Eaton et al., 1982).

111. Effects of Alcohols on Eukaryotic Micro-organisms A. GROWTH AND MORPHOGENESIS

Among eukaryotes, Saccharomyces spp. appear to be the most alcohol-resistant organism. These organisms are able to grow in concentrations of 8-12% (v/v), to survive exposure to concentrations of up to 15% (v/v), and to ferment glucose to produce ethanol up to concentrations of around 12% (v/v) for normal fermentations and up to 20% (v/v) during sakC fermentations (Rose, 1980, 1983). All strains of Saccharomyces spp. are not equally tolerant, implying that alcohol resistance is determined in part by genetic composition (Gray, 1941). In a subsequent study, Gray (1948) correlated alcohol tolerance with cell fat content, and was among the first to suggest a relationship between alcohol tolerance and lipid composition. Details of this remarkable ethanol tolerance and its relationship to membrane lipids will be dealt with in Sections V (p. 282) and VI (p. 287) of this article. Alcohol-sensitive and alcohol-resistant mutants of Sacch. cerevisiae have been described (Williamson et al., 1980; Sugden and Oliver, 1983).Williamson et al. (1980) isolated mutants which were resistant to allyl alcohol. These mutants were shown to be defective in alcohol dehydrogenases, preventing reduction of allyl alcohol to the toxic compound, acrolein. The alcohol-sensitive strain was originally described as being deficient in vacuolar hydrolases and the basis for its increased alcohol sensitivity is not known (Sugden and Oliver, 1983). Wilkie and Maroudas (1969) have shown that phenethyl alcohol inhibits the growth of Succh. cerevisiae by inducing a cytoplasmic respiratory deficiency. These authors attributed the deficiency partly to the induction of petite mutations, and partly to a direct inhibition of respiration. Inhibition of respiration was proposed as resulting from increased mitochondria1 permeability. Turian et al. (1972) have examined the effects of phenethyl alcohol on mycelial growth and differentiation of Neurospora crassa. Addition of 0.08%


27 1

phenethyl alcohol repressed conidial differentiation without preventing mycelial growth. Conversely, when N . crassa was grown as conidia, higher concentrations of phenethyl alcohol (0.16-0.32%, v/v) blocked the formation of mycelial filaments with a resultant accumulation of budding elements (Turian et al., 1972). Phenethyl alcohol was similarly shown to block reversibly dimorphism in Candida albicans (Lingappa et al., 1969), Mucor rouxii, Rhizopus arrhizus and Zygorhynchus moelleri (Terenzi and Storck, 1969a,b). The effects of phenethyl alcohol on growth and dimorphism depended on the nature and concentration of the carbon and energy sources provided. In the presence of 5% (w/v) glucose, mannose, fructose or galactose, phenethyl alcohol induced M . rouxii to grow with a yeast-like morphology (Terenzi and Storck, 1969a). However, when lower concentrations (1-273 of the same sugars were present, this alcohol did not affect dimorphism. Inhibition of respiration by phenethyl alcohol in Sacch. sakP was similarly dependent on the concentration of glucose provided (Yamashiro et al., 1971). The studies of Terenzi and Storck (1969a) also demonstrated that, when M . rouxii was grown in the presence of either xylose, maltose, sucrose, glucosamine or amino acids as the carbon source, phenethyl alcohol (even at 5%, v/v) did not prevent formation of mycelia. This apparent dependency of phenethyl alcohol effects on carbon source may result from differences in cellular energy metabolism. Terenzi and Storck (1969b) observed a stimulation of aerobic fermentation by phenethyl alcohol in Mucor rouxii and Rhizopus arrhizus, leading to an enhanced production of ethanol (Terenzi and Storck, 1969a). Turian et al. (1972) reported a similar effect of phenethyl alcohol on ethanol production by N . crassa. Whether the effects of phenethyl alcohol on growth and dimorphism are structurally specific or result from its action as a membrane-active agent is not known. Lester (1965) compared the growth-inhibitory effects of a variety of substituted aromatic compounds, and found that the interposition of one or two carbon atoms between the benzene ring and the hydroxyl group of benzyl alcohol, to yield phenethyl alcohol and phenylpropanol respectively, led to increased inhibitory activity. Interestingly, phenylpropanol also induced a yeast-like morphology of M . rouxii (Terenzi and Storck, 1969a,b). However, an unexplored possibility is that the effects of phenethyl alcohol on growth and dimorphism may depend on the concomitant presence of intracellular or extracellular ethanol. The diverse and complex effects of phenethyl alcohol on morphogenesis in various fungi may provide useful tools for further studies to delineate the role of cell membranes in morphogenesis. The effects of ethanol and phenethyl alcohol on growth and morphology have also been examined in Tetrahymena pyriformis, a unicellular ciliated protozoan (Nandini-Kishore et al., 1977; Nozawa et al., 1979). Both alcohols inhibited growth in a dose-dependent fashion, with the higher doses of ethanol

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M.BUTTKE

causing cells of T.pyriformis to appear shrunken and move more slowly than control cells (Nandini-Kishore et al., 1977). Growth of T . pyriformis was inhibited by concentrations of phenethyl alcohol as low as 5.5 mM (0.07%, v/v), which is in the range of phenethyl alcohol concentrations that inhibited growth of yeasts and moulds. By contrast, the molar concentration of ethanol needed to inhibit growth was nearly 80-fold higher, which reflects the difference in the octanol-water partition coefficients for the two alcohols (Leo et al., 1971). The relationship between toxicity and lipid solubility for ethanol and phenethyl alcohol in T. pyriformis implies that the two compounds are acting on plasma membranes. B. TRANSPORT SYSTEMS

Alcohols have been shown to inhibit transport systems in eukaryotic micro-organisms. In N . crassa, Lester (1965) examined the effects of phenethyl alcohol on uptake of glucose and amino acids. At concentrations that inhibited growth and macromolecular synthesis, this alcohol inhibited glucose incorporation by 45% and amino acid uptake by 75-95% (Lester, 1965). Phenethyl alcohol has also been shown to inhibit the uptake of adenine and aspartic acid in Sacch. sake' (Yamashiro et al., 1971). Several more recent studies have examined the effects of ethanol and other alcohols on solute accumulation by Sacch. cereuisiae. Using D-xylose as a non-metabolizable analogue, Lea0 and van Uden (1982b) studied the effects of ethanol and several other alkanols on glucose transport. Their results showed that none of the alcohols tested affected the affinity of the transport system for D-xylose (as judged by the K m value), but the alcohols did lower the rate ( V m a x value) of sugar uptake. Further, with increasing concentrations of the alcohols, there was a corresponding decrease in the V,,, value, allowing Lei0 and van Uden (1982b) to calculate a characteristic inhibition constant (k)using the equation: u

=

S V:,, e c k x K,+S

where S is the concentration of D-xylose, K , is the Michaelis constant, v is the rate of initial uptake, Vkaxis the maximum uptake of D-xylose in the absence of alcohol, and x is the alcohol concentration (LeSio and van Uden, 1982b). For each of the alcohols tested, a k value was determined. As shown in Fig. 4, the inhibitory constants of the alcohols could be correlated with their lipid-buffer partition coefficients. The authors also reported a relationship between increases in the molecular size of alcohols and extent of inhibition. LeSio and van Uden (1982b) concluded that alcohols inhibited sugar uptake in Sacch. cerevisiae by changing the lipid environment of the plasma membrane.

EFFECTS OF ALCOHOLS O N MICRO-ORGANISMS

0

-

0.9

273

Ethanol propo on-2-01

'5

E

a

Propanol

0.3

0 0

Eutonol

0.6

1.2 1.8 Partition coefficient

2.4

FIG. 4. Inhibition by alcohols of D-XylOSe uptake in Saccharomyces cerevisiae. The concentration of alcohols which inhibited D-xylose uptake by 50% is plotted against the lipid-buffer partition coefficient for each alcohol. These results illustrate a direct relationship between alcohol potency for inhibition of sugar uptake and hydrophobicity of the alcohol.

The degree of this inhibition was dependent on the lipid solubility and the molecular volume of the alcohol tested. In a similar study, Loureiro-Dias and Peinado (1982) examined the effects of several alcohols on maltose transport. Once again, alcohols were observed to lower the rate of sugar uptake without affecting the affinity of the transport systems for the substrate. The degree of inhibition for each alcohol was determined by its lipid-buffer partition coefficient. These results were in good agreement with the findings of Le2o and van Uden (1982b) and Loureiro-Dias and Peinado (1 982) and provide evidence consistent with the membrane as the site of action. Further support for the importance of alcohol effects on membranes comes from a study by Thomas and Rose (1979). Saccharyomyces cerevisiae was grown anaerobically to inhibit sterol and unsaturated fatty acid biosynthesis (see p. 284) and supplemented with ergosterol and either oleic acid or linoleic acid. Yeast cells enriched with either of the two unsaturated residues were then exposed to ethanol, and the effects of the alcohol on growth and sugar or amino-acid uptake were determined. With unsaturated fatty acids, ethanol addition led to an immediate decrease in rates of growth and nutrient transport. The degree of this inhibition was dependent on the nature of the unsaturated fatty-acyl residue. Yeast cells grown with linoleic acid were more resistant to ethanol inhibition than cells supplemented with oleyl residues. Assuming that changing the nature of the unsaturated supplement causes changes only in membrane lipids and not membrane proteins, then the results

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LONNIE O’NEAL INGRAM A N D THOMAS M. BUTTKE

of Thomas and Rose (1979) indicate a relationship between inhibition of growth and nutrient transport by alcohol and membrane-lipid composition.

C. MEMBRANE LEAKAGE

Alcohols are membrane-active compounds and their addition has been shown to increase membrane permeability to ions and small metabolites in many types of cells. However, few studies have addressed this point in eukaryotic micro-organisms. In N . crassa, phenethyl alcohol has been shown to promote leakage of a non-metabolized amino acid, y-aminoisobutyric acid, from cells that had previously been loaded with this compound. Hayashida and Ohta (1978) demonstrated that ethanol promotes leakage of molecules from Sacch. saki. Concentrations of ethanol which inhibited growth of this organism caused leakage both of enzyme proteases and of small ultraviolet-absorbing molecules. Despite this paucity of studies with eukaryotic micro-organisms, studies with model membranes (Pang et al., 1979), with animal cell membranes (Michaelis and Michaelis, 1983) and with bacteria (see p. 269) provide overwhelming evidence that alcohols promote membrane leakage. It is likely this leakage in yeast and other eukaryotic micro-organisms exhibits similar characteristics, with the potency of the alcohols increasing both with chain length and hydrophobicity and small ions such as protons and potassium being most susceptible to leakage. An alcohol-induced collapse of the permeability barrier in yeast would be expected to have broad ramifications. In recent years it has become apparent that the energy used to transport nutrients actively into cells is derived from the coupling of proton currents and ion fluxes. The excellent review by Eddy (1982) summarizes the critical role of transmembrane gradients as a major energy source for transport systems in selected eukaryotic micro-organisms including Saccharomyces spp. This is further illustrated by the ability of uncoupling agents such as dinitrophenol to inhibit amino-acid uptake in Sacch. cereuisiae (Keenan and Rose, 1979). Thus alcohol-induced ion leakage would be expected to interfere with nutrient accumulation and, ultimately, to inhibit cell growth.

D . LIPID COMPOSITION AND BIOSYNTHESIS

In previous sections of this review, the lipid composition of cellular membranes was discussed as being of critical importance for alcohol tolerance. Since Saccharomyces spp. can survive in concentrations of alcohol as high as 10-12%, this organism might also be expected to have an unusual lipid composition. In terms of their lipid composition, Saccharomyces spp. are intermediate between bacteria and animal cells. Like most prokaryotes, they


275

cannot synthesize polyunsaturated fatty acids and thus contain only monounsaturated and saturated fatty-acyl esters in their phospholipids. However, like animal cells, Saccharomyces spp. contain large proportions of phosphatidylcholine and membrane sterols, which are largely absent from prokaryotes. Nevertheless, the lipid composition of Saccharomyces spp. is unique in that this organism synthesizes ergosterol rather than cholesterol, and the phospholipids contain very high proportions (7040%) of unsaturated fatty-acyl residues. As will be discussed later, the presence of ergosterol and the high unsaturated fatty-acyl content in yeast membranes may be important for alcohol tolerance. Yamashiro et al. (1967) reported that sake yeast grown anaerobically in the presence of ethanol were more resistant to growth inhibition by ethanol than were cells that had not previously been exposed to ethanol. Such a differential in alcohol sensitivities implies that, on exposure to ethanol, yeast cells can adapt in some way to compensate for the detrimental actions of ethanol. Recent studies by Beavan et al. (1982) suggest that Sacch. cerevisiae adapts to ethanol during growth by altering its membrane fatty-acyl composition. Yeast cells were grown in the presence of increasing concentrations of ethanol and subsequently analysed for phospholipid fatty-acyl compositions. Analogous to the results obtained by Ingram (1976) in E. coli., Beavan et al. (1982) reported that yeast grown in the presence of ethanol displayed a dose-dependent increase in the content of mono-unsaturated fatty acids (primarily oleic ) by a decrease in saturated residues. These results acid, C I ~ : ,accompanied suggest that Sacch. cerevisiae can alter its membrane lipid composition as an adaptive response to ethanol. As a free-living protozoan, T. pyriformis is unlikely to encounter very high concentrations of ethanol or other anaesthetics in its environment. Nevertheless, when grown in the presence of ethanol, phenethyl alcohol or methoxyflurane, T. pyriformis displayed significant changes in its membrane lipid composition. These changes have been interpreted as being adaptive, ameliorating the effects of the membrane-active agent (Nandini-Kishore et al., 1977,1979; Nozawa et al., 1979). Addition ofmethoxyflurane to cultures of T. pyriformis caused the organism to incorporate increased amounts of [I4C]acetate into palmitic and myristic acids, with correspondingly less radioactivity being associated with palmitolyl and polyunsaturated fatty-acyl residues. The shift in fatty-acyl composition towards a more saturated content was similar to the type of fatty-acyl changes observed when T. pyriformis was grown at an elevated growth temperature (Martin et al., 1976). Both high temperatures and methoxyflurane are known to decrease membrane order. The authors proposed that the changes in fatty-acyl composition in methoxyflurane-treated cells were compensatory for the presence of the drug (Nandini-Kishore et al., 1977). In a subsequent study, Nandini-Kishore et al. (1979) reported that ethanol

276


could also induce changes in the membrane fatty-acyl composition of T. pyriformis. Despite the fact that ethanol, like methoxyflurane, seemed to increase membrane fluidity, the alcohol-induced fatty acid changes were quite dissimilar from those reported previously. Instead of increased levels of saturated fatty-acyl residues (Nandini-Kishore et al., 1977), ethanol-grown cells displayed an increase in linoleyl residues, with a concomitant decrease in C16 mono- and diunsaturated residues (Nandini-Kishore et al., 1979). To complicate matters further, Nozawa et al. (1979) reported that phenethyl alcohol-induced changes in the fatty-acyl composition of T. pyriformis were distinct from those resulting from addition of ethanol or methoxyflurane. Specifically, phenethyl alcohol led to elevated levels of linoleyl and linolenyl residues which were offset by decreased proportions of myristyl and palmitoleyl residues. It should be noted that phenethyl alcohol was also expected to increase membrane fluidity (Nozawa et al., 1979). Thus we are faced with a dilemma. Three agents that have similar effects on bilayer order bring about distinct changes in fatty-acyl composition. There are at least two possible explanations for this discrepancy. Firstly, although each drug appears to have a similar effect on membrane fluidity, this crude measurement of membrane order may not be the significant action of ethanol. Alternatively, some of the changes induced by these agents may be counterproductive for tolerance to the alcohol or anaesthetic. Growth of T.pyriformis in the presence of ethanol and of phenethyl alcohol also led to major changes in cellular phospholipid composition (NandiniKishore et al., 1979; Nozawa et al., 1979). Again, the two alcohols elicited different effects. Ethanol increased the content of phosphatidylethanolamine while decreasing that of the 2-aminoethylphospholipid (Nandini-Kishore et al., 1979). With phenethyl alcohol, there was an increase in the content of phosphatidylcholine and a decrease in phosphatidylethanolamine and 2-aminoethylphospholipid (Nozawa et al., 1979). Unfortunately, changes in phospholipid head groups are even more difficult to interpret than alterations in fatty-acyl composition, so the significance of the alcohol effects on polar lipid composition is obscure. Additional experiments are needed to evaluate the adaptive value of alcohol-induced changes in fatty-acyl and phospholipid compositions in T . pyriformis. Tetrahymena pyriformis is unique in that it does not synthesize sterols, although it does produce a pentacyclic triterpene, tetrahymenol, which probably performs the same functions as membrane sterols (Mallory et al., 1963). However, when sterols are included in the culture medium, synthesis of tetrahymenol by T .pyriformis is inhibited and exogenous sterols are used for membrane biogenesis (Mallory and Conner, 1970). In this fashion, a variety of sterols were shown to be effective in supporting the growth of T. pyriformis (Mallory and Conner, 1970). Considering the importance of sterols for alcohol tolerance in yeasts (Thomas et al., 1978) and their link to alcohol

277


tolerance in humans (Michaelis and Michaelis, 1982), this organism seems particularly attractive for further study.

E. THERMAL TOLERANCE IN YEASTS

In 1941, Gray reported that the sensitivity of Sacch. cereuisiae to inhibition by ethanol was increased at elevated temperatures. van Uden’s laboratory has recently reported a very interesting group of experiments which demonstrate that the converse of this is also true. In the presence of ethanol, the sensitivity of yeasts to thermal inactivation is increased. In studies with Sacch. cereuisiae, van Uden and da Cruz Duarte (1981) found that, as the concentration of extracellular ethanol was increased, the maximum temperature at which the yeast could grow was lowered (Fig. 5 ) . On the basis of their studies, the authors suggested that the sites which determine maximum growth temperatures are located in the cell membrane, and that ethanol affected these membrane sites to make them more temperature sensitive (van Uden and da Cruz Duarte, 1981). Additional support for this paradigm was provided by the studies of Lea0 and van Uden (1982a) in which several normal alkanols were compared for their effects on temperature-induced death. With increasing chain length, the

45r

30

0

1

2

3 4 5 6 7 Ethanol concentration (%, w/v)

0

9

FIG. 5 . Effect of ethanol on the maximum growth temperatures of Saccharomyces cerevisiae. From van Uden and Cruz Duarte (1981).

278


alkanols became more effective in altering thermal death in a non-specific fashion which was solely dependent on their lipid solubility. These results lead Lei0 and van Uden (1982a) to extend their membrane target hypothesis and to propose that alcohols alter the lipid vicinity of the thermal death sites so that the same amount of heat leads to a greater amount of disorder. Presumably, as the concentration of alcohol within the lipid vicinities increased there would be a greater amount of disorder associated with the target site. For a given alcohol, the concentration at the target site would be determined by its partition coefficient, and hence the basis for the relationship between thermal-death point and alcohol chain length. Although several aspects of the model remain to be proven, the results of van Uden and his co-workers, coupled with the findings of Gray and Sova (1956), provide strong evidence for the involvement of cell membranes in the growth inhibition of yeast by alcohols. It is worth noting that all of the alcohols tested by Le5o and van Uden have positive temperature coefficients which means that higher temperatures will enhance their partitioning into membranes (Janoff and Miller, 1982). Thus, the enhanced potency of alcohols at elevated growth temperatures may result not only from increased membrane disorder, but also from a higher concentration of alcohol within the membrane. In this regard, future studies with anaesthetics that have negative temperature coefficients, and therefore become concentrated within the membrane at lower temperatures, would be of particular interest.

F. ETHANOL-INDUCED LEAKAGE AND EXCRETION IN YEASTS

Thus far we have primarily been concerned with the effects of extracellular ethanol on cells as if they are resting. However, the fermentation industry is primarily interested in the alcohol tolerance of yeast under conditions where they are rapidly producing alcohol. This situation is somewhat different in that the alcohol is being introduced on the intracellular versus the extracellular side of the plasma membrane. In fact, several workers have recently proposed that high concentrations of intracellular ethanol are responsible for the premature inhibition of fermentation (Beavan et al., 1982; Nagodawithana and Steinkraus, 1976; Navarro and Durand, 1978). Supportive evidence for this theory initially came from studies with yeast undergoing “rapid fermentation” (Nagodawithana and Steinkraus, 1976). At 30°C, the initial stages of fermentation proceeded extremely rapidly but later slowed down due to alcohol toxicity. Toxicity could be avoided, in part, either by lowering the incubation temperature to 15°C or by lowering the number of cells in the culture. Nagodawithana and Steinkraus (1976) suggested that the fermentation rate was sufficiently slower at 15°C to allow ethanol efflux to keep pace


279

with ethanol production. They also proposed that by lowering the number of cells in the culture, the extracellular ethanol concentrations would be lower and the greater differential between intracellular and extracellular ethanol concentration would favour a more rapid efflux. In this fashion, low temperatures and low cell numbers would prevent accumulation of toxic concentrations of ethanol intracellularly. Note that the model proposed by Nagodawithana and Steinkraus (1976) implies that the plasma membrane of yeast poses a formidable diffusion barrier for ethanol. This concept was later supported by the studies of Navarro and Durand (1978) and by Panchal and Stewart (1980, 1981a,b). Thomas and Rose (1979) measured the internal ethanol concentration of yeast cells grown anaerobically in the presence of oleic or linoleic acids. Yeast cells enriched in oleyl residues contained roughly twice as much ethanol as linoleic acid-grown cells, leading Thomas and Rose (1979) to propose that the latter fatty acid was more effective in promoting extracellular efflux of ethanol. A subsequent study by Beavan et al. (1982) reported that intracellular ethanol concentrations reached values of 1-3 M. During fermentation, the intracellular ethanol concentration was always higher than the extracellular concentration, implying the existence of a downhill gradient for ethanol efflux. According to Rose and his co-workers, enriching cells with linoleyl residues increases membrane permeability so that ethanol can diffuse out of yeast cells at a faster rate (Thomas and Rose, 1979; Beavan et al., 1982). Intracellular ethanol concentrations may not climb as high in linolenyl residue-supplemented cells due to increased leakage, and this may be responsible for the enhanced survival. The experimental measurement of intracellular concentrations of ethanol is technically very difficult due to complications of metabolism and the ease with which ethanol can be washed from cells. Although the notion that intracellular ethanol is responsible for inhibiting yeast growth and that lipids promote ethanol efflux are appealing, there are at least two factors which must be addressed. The first concerns the established relationship between growth temperature and alcohol toxicity. The experiments of Navarro and Durand (1978) clearly demonstrate that the rate of ethanol production by yeast increases with elevated temperatures. However, the studies of Kleinans et al. (1 979) show that, at higher temperatures, yeast plasma membrane permeability also increases. Therefore, one would expect that as the fermentation rate increases so would the rate of ethanol efflux. At elevated temperatures, it is unlikely that the plasma membrane would not be freely permeable to ethanol. The second complication is that changes in fatty-acyl composition, which have been reported to protect cells during active growth and fermentation (i.e. increased unsaturation), have also been shown to protect resting cells from extracellular ethanol. If the major purpose of the lipid changes is to facilitate enhanced diffusion of ethanol out of the cell during fermentation, then it is

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LONNIE O’NEAL INGRAM A N D THOMAS M. BUTTKE

difficult to imagine how this increase in permeability would also protect resting cells against extracellular ethanol.

IV. Effects of Alcohols on Membrane Organization A variety of physical methods have been used to investigate the effects of ethanol and other alcohols on the organization of model and biological membranes. These are summarized in excellent reviews by Michaelis and Michaelis (1982) and Janoff and Miller (1982). All studies agree that the addition of relevant concentrations of alcohols (chain lengths CI to CS) increases the freedom of motion within the membrane (membrane fluidity), lowers the phase-transition temperatures of model membranes and decreases membrane order. As with the biological effects of alcohols, the relative potency correlates well with alcohol hydrophobicity, increasing with longer chain length. These effects on membrane composition result from a complex combination of physical effects both directly on the membrane and on the membrane environment. Although long- (pentanol to nonyl alcohol) and short- (methanol to butanol) chain alcohols both cause an increase in membrane fluidity, these groups frequently have different effects on biological systems. Long- and short-chain alcohols induce opposite changes in the fatty-acyl composition of E. coli (Ingram, 1976) and exhibit different actions on bacterial peptidoglycan synthesis (Dickens and Ingram, 1976; Ingram, 1981), on the widths of phase transitions in model membranes (Jain and Wu, 1977) and on the stability of non-bilayer conformations within membranes (Hornby and Cullis, 1981). These differences in effects may result from fundamentally different modes of action between long- and short-chain alcohols despite the superficial appearance of a continuous relationship between hydrophobicity and alcohol potency. Alcohols have two basic functional groups, namely, a hydroxyl function and a hydrocarbon tail. The effects of short-chain alcohols such as ethanol would be expected to be dominated by their polar function. In longer-chain alcohols, such as hexanol, the hydrophobic nature of the hydrocarbon tail would be expected to dominate its actions. The addition of short-chain alcohols such as ethanol has a variety of biophysical effects. Ethanol partitions very poorly into hydrophobic environments. Relatively high concentrations of ethanol (e.g. 0.67 M) cause a smaller increase in membrane fluidity than a 2°C increase in temperature (Ingram et al., 1980b). To cause appreciable effects on biological systems, relatively high concentrations are required and have profound effects on the colligative properties of the aqueous environment surrounding the membrane. Addition of ethanol results in an apparent increase in pH value (Jukes and Schmidt, 1934)due to replacement of water by an essentially aprotic solvent, a decrease


28 1

in the strength of hydrophobic interactions (Yaacobi and Ben-Naim, 1974), an increase in the strength of coulombic interactions (Franks and Ives, 1966) with a decrease in the tendency for molecules to ionize, and in the availability of an additional bulk-phase molecule which is capable of participating in hydrogen bonding and competing with water. In addition, a small proportion of the ethanol partitions into the membrane and directly disturbs packing. On the basis of measurements of the partitioning of ethanol into membranes (Conrad and Singer, 1981; Rottenberg et al., 1981; Seeman et af.,1971), the intramembrane concentration would be expected to be no more than one-tenth that in the aqueous phase. Hydrophobic interactions are the principal driving force for the biological self-assembly of membranes and both electrostatic and hydrophobic interactions are involved in maintaining the spatial organization of membrane components. Addition of ethanol would be expected to cause profound changes in this complex organization. Unfortunately, the simplicity of the tools currently available do not allow us to probe many of the details of form and function within membranes and primarily report bulk changes reflecting averages of the changes exhibited by different membrane components. Indeed, it is highly unlikely that the simplistic measurements of alcohol-induced increases in membrane fluidity are an important physiological change. However, this change does provide direct physical evidence that some change in organization has occurred. Using ethanol as an example, let us consider the hypothetical ways in which the permeability properties of a membrane could be affected. The hydrophobic interior of the membrane serves as the primary permeability barrier of the cell preventing free exchange of polar molecules. Intercalation of ethanol into this hydrophobic interior would tend to increase the polarity of this region, weakening this barrier. The decreased strength of hydrophobic associations would also tend to decrease the extent of van der Waal’s interactions among acyl chains and hydrophobic protein surfaces, further weakening the permeability barrier. The increased strength of coulombic interactions would tend to decrease the extent of ionization, but would also increase the strength of charge repulsion between the polar head groups. This could result in an increase in the surface area occupied by each phospholipid molecule and a decrease in membrane thickness, again increasing membrane permeability. The positioning of proteins within the membrane also represents a complex balance of charge interactions and hydrophobic associations. Analogous reasoning would provide a multitude of possible effects on the orientation and function of membrane-bound and cytoplasmic proteins. Although the physical principles of ethanol action would be similar for all proteins, the specific consequences for any given protein would depend on the specific properties and structure of that protein. The effects of longer-chain alcohols, such as hexanol, would be expected to

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be dominated by their hydrocarbon portion. This alcohol partitions effectively into membranes and causes substantial increases in freedom of motion (Jain and Wu, 1977). Its impact on the polarity of the hydrophobic interior would be far less than ethanol due to its more hydrophobic nature. However, the fluidizing effect of this alcohol would disrupt short-range interactions and also facilitate increased leakage. The effects of this alcohol on the colligative properties of the aqueous environment would be less than that of ethanol owing to, in large part, its use at much lower concentrations. Recent studies in our laboratory have addressed the localization of alcohol within the membrane (Dombek and Ingram, 1984). We have used a series of anthroyloxy fatty-acyl derivatives in which the fluorophore is attached at different positions along a stearic or palmitic acid backbone (Thulborn and Sawyer, 1978). These molecules insert into the membrane localizing the reporting function at different depths within the bilayer (Podo and Blasie, 1977; Thulborn and Sawyer, 1978). As expected, the probes located near the surface at C-2 were the least mobile and mobility was increased with increasing probe depth. Although the addition of ethanol caused a dosedependent increase in the motion of all probes, the probes near the surface were preferentially affected. Following addition of hexanol, the probes located more deeply within the membrane were also disturbed. On the basis of these results, we proposed that the hydroxyl function'of both ethanol and hexanol serve to orient and preferentially localize these alcohols near the membrane surface probably by virtue of their participation in hydrogen bonding (Fig. 6). These results are consistent with the proposed location of phenethyl alcohol in the membrane based on nuclear magnetic resonance studies (Metcalfe, 1970). However, these do not agree with electron spin resonance studies using nitroxide probes located at different depths (Michaelis and Michaelis, 1983). The reasons for the disagreement between electron spin resonance and fluorescence results is not immediately apparent, although these methods measure somewhat different aspects of membrane organization.

V. Effects of Lipid Supplements on Alcohol Tolerance, Growth, Survival and Fermentation A.

Escherichia coli

The fatty-acyl composition of E. coli can easily be manipulated using lipid mutants and fatty-acid supplements. This approach has been used to examine the effects of membrane composition on growth and survival in the presence of ethanol (Ingram et al., 1980b). As described in earlier sections of this review, there is a general tendency for cells that have been grown in the


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FIG. 6. Proposed model for the interaction of ethanol (a) and hexanol (b) with membranes. In this model, the hydroxyl functions of both ethanol and hexanol are shown as hydrogen bonding (- - - -) with ester linkages of fatty-acyl residues and with water molecules that have intruded into the membrane, two polar functions likely to serve as hydrogen-bond acceptors. Although this diagram depicts hydrogen bonding only to fatty-acyl residues in the C-1 position, analogous interactions should be equally possible with residues in the C-2 position. In biological membranes, carbohydrates and proteins also offer many additional sites for hydrogen bonding. Hydrogen bonding to polar components in the membrane surface would be expected to localize preferentially hydroxyl groups in this region. From Dombek and Ingram (1983).

presence of ethanol or organisms that inherently exhibit increased ethanol resistance to contain high proportions of c18:1fatty-acyl residues. Mutant strains of E. cofi which are unable to synthesize vaccenic acid (C18:l)contain membranes composed of nearly equal proportions of palmityl and palmitoleyl residues. These mutants were hypersensitive to growth inhibition and death by ethanol in comparison to wild-type organisms (Ingram et af., 1980b). Exogenously added fatty acids were readily incorporated into these organisms, and addition of CIS:,fatty acids to the growth medium resulted in an increase in the growth rate of the mutant in the presence of ethanol and a restoration of survival to that of the wild-type strain. No other fatty acids were beneficial although addition of palmitic acid (opposite to the change in fatty-acyl composition induced by ethanol) rendered the cells even more sensitive to killing by ethanol (Ingram et al., 1980b).

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Unpublished studies from our laboratory have examined the structural requirements of the fatty acids for increased alcohol tolerance. Saturated fatty acids over 16 carbon atoms in length and mono-unsaturated fatty acids over 18 carbon atoms in length were not incorporated very efficiently and could not be evaluated. Of the C18:,fatty acids examined (L.O. Ingram, unpublished observations), the two trans unsaturated fatty acids, elaidic and trans-vaccenic, were the most effective followed by cis-vaccenic (normally occuring in E. coli) and then oleic acid. Petroselenic acid was ineffective. From these studies, we concluded that increases in C18:1fatty-acyl residues (analogous to the changes in fatty-acyl composition induced by ethanol) were beneficial for the growth and survival of E. coli in the presence of ethanol. Since trans-unsaturated fatty acids were even more effective than cis-unsaturated fatty acids, the increase in chain length rather than the increase in membrane fluidity appears to be of more benefit to E. coli. B.

Saccharomyces cerevisiae

Sake production involves a complex fermentation of rice with both yeast and Aspergiffus oryzae over a period of months at a low temperature. This fermentation proceeds to much higher concentrations of ethanol than do fermentations of wines and beers. In 1974, Hayashida et al. reported that sake yeasts could produce up to 20% ethanol when grown in a chemically defined medium supplemented with a “high concentration alcohol-producing factor”, derived from A. oryzae. This factor, a proteolipid isolated from the envelope of A. oryzae, was subsequently shown to be composed primarily of phosphatidylcholine and protein, with a small amount of steryl ester (Hayashida and Ohta, 1978). Hayashida et a f . (1974, 1975) found that the component of proteolipid that was required for alcohol tolerance was phosphatidylcholine. Purified phosphatidylcholine was also effective when solubilized with either albumin or methylcellulose. A comparison of phosphatidylcholines with different fatty-acyl compositions showed that egg-yolk lecithin promoted production of high concentrations of ethanol, but dipalmitoylphosphatidylcholine was ineffective. Thus, unsaturated fatty acids seemed to be required for promoting alcohol tolerance. Indeed, analyses of the phospholipid acyl-chain composition of sake yeasts grown in the presence of A. oryzae proteolipid (Hayashida et al., 1974; Hayashida and Ohta, 1978, 1980) showed that the supplemented cells contained high proportions of linoleyl residues, the major residue of the proteolipid phosphatidylcholine (Hayashida et a f . , 1976). Similarly, sake yeast grown in the presence of egg-yolk phosphatidylcholine were enriched in oleyl residues, the major unsaturated residue present in the egg-yolk phosphatidylcholine (Hayashida and Ohta, 1980). By comparison, saki yeast grown in the absence of either proteolipid or phosphatidylcholine contained low proportions of


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unsaturated fatty-acyl residues and were enriched with medium-chain saturated fatty-acyl residues (Hayashida et a[., 1974; Hayashida and Ohta, 1978, 1980). In further studies, Hayashida and his coworkers reported that proteolipid not only promoted yeast growth (and alcohol production) but also affected the “durability” of the yeast cells (Hayashida et al., 1975; Hayashida and Ohta, 1980). Sake yeast cells were grown in the presence or absence of proteolipid followed by incubation in a buffer containing 20% (v/v) ethanol for 48 hours at several temperatures (Hayashida et af., 1975; Hayashida and Ohta, 1980). These “dipped” cells were then compared for their fermentative capacities as measured by evolution of carbon dioxide. The results of these studies clearly showed that cells incubated in the presence of proteolipid were more “durable” since they were able to undergo fermentation even after a 48-hour exposure to 20%(v/v) ethanol. By contrast, cells grown in the absence of proteolipid showed a marked decrease in their ability to evolve carbon dioxide. Additional studies have shown that supplementation of sake yeasts with proteolipid also enhanced sphaeroplast stability and lowered membrane leakage (Hayashida and Ohta, 1978). The most recent studies (Hayashida and Ohta, 1978, 1980; Ohta and Hayashida, 1983) have focused on the role of ergosterol, which is also present in the proteolipid. According to their reports, addition of unsaturated fatty acids appears to be responsible for enhancing the growth and fermentation activities of the sake yeasts, whereas ergosterol is reported to confer alcohol-endurability. Thus Hayashida and his coworkers have convincingly shown that unsaturated fatty acids and ergosterol play an important role in alcohol tolerance. Since the initial studies of Hayashida et al. (1974), numerous investigators have examined the effects of adding various lipids to fermenting yeast cultures (Lafon-Lafourcade et al., 1977, 1979; Larue et a[., 1978; Panchal and Stewart, 1981a,b; Traverso-Rueda and Kunkee, 1982; Ohta and Hayashida, 1983). Addition of exogenous lipid supplements enhanced alcohol production, thereby supporting the findings of Hayashida and his coworkers. The biochemical basis for the increased alcohol tolerance of yeast grown in the presence of unsaturated fatty acids and sterols probably lies in the oxygen-dependent enzyme systems for their biosynthesis (Bloomfield and Bloch, 1960). In a completely anaerobic environment, yeast have an absolute requirement for exogenous supplements of unsaturated fatty acids and sterols (Andreasen and Stier, 1954a,b). To some extent, Sacch. cerevisiae can substitute medium-length saturated fatty acids for unsaturated residues (Meyer and Bloch, 1963), but a low concentration of unsaturated fatty acid seems to be essential for normal phospholipid metabolism (Buttke et al., 1982; Buttke and Pyle, 1982). Similarly, much of the organism’s sterol requirement can be satisfied by a variety of sterols, but there is also an absolute requirement for a small amount of ergosterol (Rodriguez et al., 1982; Ramgopal and

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Bloch, 1983; Pinto et al., 1983) which may also be needed for phospholipid metabolism (Ramgopal and Bloch, 1983). Rose and his coworkers have investigated the structural requirements of the lipid supplements for alcohol tolerance in Sacch. cerevisiae by exploiting the anaerobically-induced requirement of this yeast for exogenous sterol and unsaturated fatty acid. Thomas et al. (1978) were able to modify the membrane composition at will and to compare a variety of lipids for their ability to promote alcohol tolerance. Of particular interest were their findings that yeast cells enriched with linoleyl residues remained viable after exposure to I M ethanol to a greater extent than did cells enriched with oleyl residues. In the same study, Thomas et al. (1978) examined the effect of fatty-acyl chain length on ethanol resistance. In a comparison of mono-unsaturated fatty acids, enrichment with the unsaturated fatty acid, palmitoleic acid (CI6:l) residue, was more effective than either oleyl (CI~:.) or cetolyl (C2k.) residues. Linoleic acid, a polyunsaturated fatty acid (C18:2),was even more effective as a medium supplement. The 0°C melting point of palmitoleic acid is about 12°C lower than that of oleic acid, and 32°C lower than that of cetoleic acid and, since linoleic acid (melting point of -5OC) is even more effective than palmitoleic acid, these results tend to suggest that the degree of fluidity is of more consequence than chain length. However, certain other changes in fatty-acyl composition also occurred. A subsequent study by Thomas and Rose (1979) reported that uptake of glucose and amino acids was less affected by ethanol in cells of Sacch. cereuisiae enriched with linoleyl residues rather than oleyl residues. Thus, the results of Hayashida and his coworkers and the findings of Rose and his colleagues implicate membrane fatty-acyl residues as being important determinants of alcohol resistance. The structural requirements of sterols for alcohol tolerance were also investigated (Thomas et al., 1978). Saccharomyces cerevisiae grown anaerobically was supplemented with a variety of sterols which led to a substantial modification in membrane sterol composition. The cells were then exposed to 1 M ethanol and cell survival was determined with time. Cells enriched with ergosterol were significantly more resistant to the toxic effects of ethanol than cells containing the predominant animal sterol cholesterol (Thomas et af., 1978). Similar results were subsequently obtained with a mutant of Sacch. cereuisiae defective in ergosterol biosynthesis (Buttke et al., 1980). Ergosterol is the sterol normally synthesized by yeast in the presence of oxygen. On the basis of these two studies, it is tempting to speculate that synthesis of ergosterol by yeast is not merely fortuitous but rather the result of evolutionary pressure to survive in the presence of high concentrations of ethanol. A great deal of work remains to be done to determine the molecular organization of the membrane and how changes in this composition facilitate increased alcohol tolerance.


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VI. Mechanism of Inhibition of Fermentation by Ethanol A.

Saccharomyces cerevisiue:

THE “STALING” EFFECT

Early attempts to determine the basis of “staling” focused on the potential roles of pH value (due to production of organic acids), oxygen deprivation (resulting from the evolution of carbon dioxide) and accumulation of ethanol, the primary non-gaseous end-product of glycolysis (Brown, 1905; Richards, 1928; Rahn, 1929). Brown (1905) examined the effects of volatile and non-volatile non-gaseous fermentation products on yeast cell reproduction, and the results showed that the volatile products, consisting primarily of ethanol, were more inhibitory towards growth than the non-volatile products. Nevertheless, further studies by Brown led him to conclude that carbon dioxide and not ethanol was responsible for inhibition of yeast growth during fermentation. Later studies by Richards (1928) and Rahn (1929), however, clearly showed that ethanol was the by-product most responsible for inhibition of growth. Roughly 12 years later, Gray (1941) confirmed the growth-inhibiting properties of ethanol and demonstrated that different strains of Succh. cerevisiue varied considerably in their sensitivity to ethanol. Since ethanol is a direct by-product of the Embden-Meyerhof glycolytic pathway, it was not unreasonable that some of the earlier workers should suggest that ethanol retards yeast growth through a specific feedback inhibition. To examine this possibility, Troyer (1955) measured growth of several strains of Sacch. cerevisiue in the presence of methanol. His results showed that methanol also inhibited yeast growth, but it seemed to be less inhibitory than ethanol since higher concentrations of the former alcohol were necessary to inhibit growth. Thus Troyer (1955) concluded that “the action of ethanol in inhibiting glucose utilization may be associated with its properties as a member of a general class of substances, rather than with its role as a yeast metabolite”. The concept that alcohol inhibition was due to its chemical properties has been widely documented for many alcohol effects on microorganisms and higher forms. Subsequent studies by Gray and Sova (1956) measured the alcohol tolerance values for 14 alcohols differing in size (number ofcarbon atoms) and polarity (number of hydroxyl groups). These studies used glucose consumption as a measure of alcohol tolerance. Of particular interest were the results they obtained when they compared a series of normal alkanols ranging from C, to Cs. The results of this study clearly showed that the concentration of alcohol needed to inhibit glucose utilization by 50”:, was inversely proportional to the size of the alcohol. Thus, the alcohol tolerance value for ethanol was 1.305 M, whereas for pentan-1-01 it was 28 mM. Gray and Sova (1956) also examined the effects of the positions of the hydroxyl group on alcohol tolerance (e.g. butan- 1-01 was compared with butan-2-01 which in turn was

288

LONNIE O’NEAL INGRAM AND THOMAS M . BUTTKE

compared with tertiary-butanol), as well as the effects of including additional hydroxyl groups (e.g. propanol compared with propane-I ,Zdiol and glycerol). In general, they found that moving the hydroxyl group from the terminal carbon atom to a more interior position lowered the alcohol tolerance value, as did addition of a second or third hydroxyl group. However, the concentrations of polyhydric alcohols needed to inhibit glucose utilization were more than 10-fold higher than the concentration of the corresponding primary alcohol. This large discrepancy in alcohol tolerance values led Gray and Sova (1956) to conclude that the inhibitory actions of polyhydric alcohols resulted from their actions as plasmolysing agents, whereas the inhibitory effects of monohydric alcohols could be related to their potency as an anaesthetic. Indeed, if the logarithm of the alcohol tolerance values obtained by Gray and Sova (1956) for monohydric alcohols are plotted against the logarithm of the phospholipid-buffer partition coefficients for the same alcohols, there is a surprisingly good correlation between inhibition of glucose utilization and lipid solubility (Fig. 7). This correlation with lipid solubility and potency provided substantial evidence that a hydrophobic site, such as the cell membrane, is the target for alcohol-inhibition of fermentation.

-5

1Methanol

0

W

0 c

0 .c

8 a

0

Ethanol

1.01

--

0

Propan-2-01

-

.Tertiary

-

0

butonol

Propan-1-01

Butan-1-01 0.1 .

I

I

I

I

I

9

FIG. 7. In ibition by alcohols of fermentation in Saccharomyces cerevisiae. A.Jc..c concentrations causjng equivalent levels of inhibition o f fermentation (glucose utilization) are from the data of Gray and Sova (1956), and are plotted as a function of the membrane (dirnyristoylphosphatidylcholine liposomes) : water partition coefficients for each alcohol. The partition coefficients used were determined by Katz and Diamond (1974). There is an excellent correlation between hydrophobicity (partition coefficients) and the potency of these various alcohols as inhibitors of fermentation.


289

Once it had been firmly established that accumulation of ethanol was responsible for the premature termination of yeast fermentations, there was considerable interest in determining the basis for the ethanol inhibition. Possibilities included feedback inhibition of alcohol dehydrogenase, specific inhibition of one or more enzymes of the glycolytic pathway, non-specific effects on cytosolic or membrane-bound enzymes and perturbation of other membrane functions. Since ethanol is an end product of the Embden-Meyerhof pathway, it seemed reasonable that ethanol might inhibit one or more of the enzymes on this pathway. To examine this possibility, several investigators determined the effects of ethanol on various glycolytic enzymes in vitro (Augustin et al., 1965; Llorente and Sols, 1969; Nagodawithana et al., 1977; Navarro, 1980; Millar et al., 1982). In general, most of the earlier studies reported that hexokinase was inhibited by lower concentrations of alcohol than other glycolytic enzymes. Since this enzyme acts at the beginning of the glycolytic pathway, it appeared to be a logical site for end-product inhibition. However, the recent in vitro studies of Millar et al. (1982) showed that hexokinase was less sensitive to ethanol inhibition than pyruvate decarboxylase, phosphoglycerate kinase and several other glycolytic enzymes. From their results Millar et al. (1982) concluded that a general inhibition of glycolytic enzymes by ethanol may slow metabolism overall and lead to a decline in fermentation rate. The glycolytic enzymes were surprisingly resistant to denaturation by ethanol with 12-25% ethanol causing only a 10% loss in activity (Millar et al., 1982). Enzyme activity was somewhat more sensitive. At concentrations above 12% (v/v), ethanol significantly inhibited several glycolytic enzymes including glyceraldehyde phosphate dehydrogenase, but the physiological importance of such high concentrations of ethanol is suspect since, in vivo, extracellular ethanol concentrations as low as 1% (v/v) can significantly decrease the rate of ethanol production (Moulin et al., 1980). However, accumulation of high concentrations of intracellular ethanol during fermentation, which are augmented by extracellular accumulation, would provide one means of inhibiting fermentation. If inhibition of glycolytic enzymes by ethanol is responsible for the inhibitory effects of the alcohol on growth and fermentation, then, as pointed out by Rose and his coworkers (Beavan et al., 1982), the enzymes from alcohol-resistant organisms should be less susceptible to inhibition by ethanol than the same enzymes from alcohol-sensitive organisms. The early studies of Llorente and Sols (1 969) addressed this question by comparing the ethanol sensitivities of glycolytic enzymes from the alcohol-sensitive Kloeckera apiculata to those of the alcohol-resistant Saccharomyces evilormis. The authors reported that the enzymes of K . apiculata were denatured by concentrations of alcohol which did not affect the enzymes of Sacch. evilormis. However, since the denaturing effects of ethanol were determined by

290


incubating intact yeast cells with ethanol before assaying activity of the enzymes (Llorente and Sols, 1969), the possibility that the resistant yeast was simply less permeable to ethanol cannot be ruled out. Thus, additional studies need to be performed with cell-free enzyme extracts isolated from a variety of micro-organisms which have different values of alcohol tolerance. In addition, it would be of interest to compare the effects of ethanol with that of other alcohols to determine if the inhibitory effects of the former are due to its role as a metabolic end-product, or merely a reflection of its chemical properties as proposed by Gray and Sova (1956). Until such experiments are performed, it cannot be stated with certainty that ethanol inhibits growth and fermentation by acting on glycolytic enzymes. It should be noted that several studies have reported that fermentation continues even after growth has ceased (Navarro and Durand, 1978; Panchal and Stewart, 1981a,b). These findings demonstrate that retardation of growth is not simply due to an inhibition of glycolysis, and argue for at least one other target site for this action of ethanol. There are many problems with the hypothesis that glycolytic enzymes are the primary target for ethanol inhibition of growth, survival or fermentation in yeast. The evidence of Gray and Sova (1956) clearly indicates that a hydrophobic site is involved. It is certainly true that hydrophobic regions of enzymes could be the sites involved. However, the relative insensitivity of many enzymes and transport systems make this less attractive. More importantly, the notion that the glycolytic enzymes serve as the primary sites of inhibition is not readily compatible with the vast literature which shows that Sacch. cerevisiae, as well as bacteria, adapt to ethanol following changes in membrane composition. Further, a dominant role for glycolytic enzymes is inconsistent with the observations of structural changes in the cell membranes of micro-organisms which have evolved to become tolerant to high concentrations of ethanol. In some instances, these organisms exhibit an extreme of adaptive trends found in less tolerant organisms. After this tirade, we are sadly embarrassed that we cannot provide the reader with a final answer, but we are confident that it will involve the structural arrangement of the cell membrane and will be forthcoming in the decade ahead. B.

Zymomonas mobilis

Zymomonas mobilis is an obligately fermentative bacterium which produces extracellular ethanol concentrations equivalent to that of Sacch. cerevisiae (Rose, 1983). Unlike this yeast, Z . mobilis utilizes the Entner-Doudoroff pathway for glycolysis with the generation of a single molecule of ATP for each glucose molecule consumed (Swings and DeLey, 1977). Zymomonas mobilis has been reported to have a number of advantages over yeast for industrial production of alcohol which include greater efficiency of conver-


29 1

sion, higher thermal tolerance, higher osmotolerance and the ability to achieve more rapid rates of conversion (Rogers et al., 1982; Lawford et al., 1983). The recent renewed interest in fuel alcohol has resulted in several investigations into the mechanism of alcohol inhibition of fermentation in this organism. Fermentation slows dramatically as ethanol accumulates to concentrations above 9% (w/v) (Doelle et al., 1983). Millar et al. (1982) examined the effects of ethanol on glucokinase and the enzymes of the Entner-Doudoroff pathway in extracts of Z . mobilis. Glucokinase was reported to be unaffected by 15% (w/v) ethanol and the unique enzymes of the Entner-Doudoroff pathway were only slightly affected over the range from 0-1 5% (w/v) ethanol. Although data were not presented, the inhibitory effects on other glycolytic enzymes were reported to be similar to that in Sacch. cerevisiae. In Sacch. cerevisiae, phosphoglycerokinase and pyruvate decarboxylase were the most sensitive enzymes exhibiting approximately 50% inhibition in the presence of 10% (w/v) ethanol. These enzymes were discussed as potential sites of inhibition for declining rates of alcohol production during the course of fermentation in 2. mobilis. Laudrin and Goma (1982) investigated the intracellular concentrations of alcohol in 2. mobilis, and found these to be somewhat higher than that of the medium during maximal rates of fermentation. This accumulation could be partly responsible for the depression of fermentation rates. Initial studies from Doelle’s laboratory suggested that accumulation of carbon dioxide was a major factor involved in inhibition of fermentation in 2. mobilis (Doelle et al., 1982). More recent studies (Burrill et al., 1983),however, have clearly shown that accumulation of ethanol is the primary culprit responsible for the decline in fermentation rates. These authors hypothesized that membrane integrity rather than metabolism limits the final concentration of ethanol. Recent studies in our laboratory have examined the inhibition of glucose utilization by ethanol in Z . mobilis (Osman and Ingram, 1983). Over the range I-lO% (v/v), ethanol dose-dependently inhibited glucose utilization in 2. mobilis. This inhibition was not due to a shift in affinity for glucose ( K , 2 mM, unaffected by 5% ethanol). However, ethanol significantly decreased the VmaX value (25% decrease by 5% ethanol). Studies are now underway to define conditions to prevent this inhibition by ethanol. VII. Conclusions and Future Directions A. ALCOHOLS: A BIOPHYSICAL PROBLEM

The basic actions of alcohols on both eukaryotic and prokaryotic organisms share the same general principles. These effects appear to be dominated by the

292


physicochemical properties of alcohols rather than involving specific receptors. All hydrophobic and electrostatic interactions in the cytosolic and envelope components of cells can potentially be affected. These include membranes, conformations of enzymes and macromolecules, activity coefficients of metabolites, permitivity, ionization potentials, pK values of functional groups and pH value. It is indeed remarkable that the potency of alcohols for many biological effects can be correlated so well with the relative ability of alcohols to be partitioned into hydrophobic solvents, consistent with a hydrophobic site of action. Let us not be unduly misled by this correlation, however. This correlation by no means proves that all alcohols have the same mode of action nor does it prove that the cell membrane is the prime target. Further, all effects of alcohols need not share the same target. It is likely that many “targets” will be discovered for alcohol-related phenomena with new “targets” becoming relevant at different concentrations of alcohol. An extreme example of this would be to consider the inhibition of a transport process at modest concentrations of ethanol compared with complete disruption of the membrane barrier also “inhibiting transport” at high concentrations of ethanol. From a chemical consideration, one would expect the short hydrocarbon tail of methanol and ethanol to be of little consequence while that of hexanol imparts a significant hydrophobic character. Ethanol is very polar and partitions poorly into the membrane (Fig. 8). In contrast, the hydrocarbon tail of hexanol favours its concentration within the membrane. There are ample examples of differential effects of these alcohols on biological systems which, when examined in detail, reveal unrelated mechanisms of action. The differences in the chemical properties of the these alcohols are undoubtedly responsible. B. MEMBRANES ARE INVOLVED!

The most convincing evidence that the plasma membrane is involved in alcohol tolerance comes from studies involving the experimental manipulation of membrane composition. Addition of specific lipids which are incorporated into membranes has been shown to result in production of higher concentrations of alcohol during fermentation (Section V.B, p. 284). This effect appears specific for particular types of lipid molecules adding further evidence for a causal relationship. Addition of specific lipids and their incorporation into the plasma membrane has been shown to affect directly both growth and survival in the presence of alcohol (Thomas et al., 1978; Ingram et a[., 1980b). Additional evidence is provided by the evolution of micro-organisms that exhibit increased resistance to alcohol as an environmental stress. The membranes of these organisms such as the hiochii bacteria (Uchida, 1975 a,


293

L FIG. 8. Model showing interactions of hexanol and ethanol with a membrane. Hexanol is much more hydrophobic than ethanol and is depicted as partitioning favourably into the lipid portion of the membrane with lower equilibrium concentrations of this alcohol in the aqueous milieu. In contrast, ethanol is very polar in nature and is depicted as partitioning unfavourably into the membrane. Higher concentrations of ethanol are shown in the aqueous milieu than in the membrane. The polar hydroxyl function of both alcohols is visualized as being preferentially localized near the membrane surface by virtue of participation in hydrogen bonding with polar membrane functions. Although alcohols appear freely permeable to most membranes, hydrogen bonding with polar constituents and unfavourable partitioning into non-polar solvents would tend to maintain higher concentrations of short-chain alcohols near the membrane surface than in the membrane interior.

b), Z . mobilis (Carey and Ingram, 1983), and fermentatively grown yeast (Beavan et ul., 1982) share the characteristic of having predominantly mono-unsaturated fatty-acyl residues in their polar lipids with low proportions of saturated residues. In all cases, the dominant unsaturated fatty-acyl residue is a cis C18:1 oleyl (yeast) or vaccenyl (bacteria) residue, the longest residues synthesized by these organism. The synthesis of ergosterol, a unique and non-condensing sterol, may also represent an evolutionary trait facilitating alcohol tolerance in Succh. cerevisiue. The bacterium, L. heterohiochii, appears to be the exception which proves the rule and synthesizes even longer mono-unsaturated fatty acids which are incorporated into membrane lipids in high proportions (Uchida, 1975a). , C22:1, C24:1and CXI)are These long-chain mono-unsaturated residues (C~O:, not found in appreciable quantities elsewhere in living organisms. Among other effects, these longer-chain residues would tend to increase the thickness

294


of the cell membrane and the hydrophobicity of its interior by increasing the number of methylene groups capable of van der Waal’s and other short-range interactions. This would tend to compensate for the decrease in hydrophobicity of the membrane core caused by growth of this organism in up to 22% (v/v) ethanol. Organisms adapted to growth in environmental extremes such as temperature, salinity or pH value frequently become dependent on these extremes for growth. In an analogous fashion, L. heterohiochii requires ethanol for optimal growth (Demain et al., 1961). The third major line of evidence that membranes are involved in alcohol tolerance is a series of correlations between two different organisms. During growth in the presence of ethanol, Sacch. cerevisiae and E. coli synthesize lipids that are preferentially enriched in C18:1 fatty-acyl residues and compensated for by a decrease in palmityl residues (Beavan et al., 1982; Ingram, 1976). This is particularly remarkable when we consider that the pathways for synthesis of cl8:l fatty-acyl residues are entirely different enzymically and undoubtedly involve completely different types of enzymes as control sites (Fulco, 1974). Escherichia coli synthesizes unsaturated fatty-acyl residues by inserting a cis double bond during the middle stages of fatty acid elongation using soluble enzymes without an oxygen requirement. In Sacch. cereuisiae, a fully saturated fatty-acyl residue (CI~:O) is first synthesized on a large multi-enzyme synthetase protein and released (Stoops et al., 1978). Cis double bonds are subsequently inserted into this fatty-acyl residue by an oxygen-dependent desaturase enzyme with elongation to form CIS:^, oleyl residue (Bloomfield and Bloch, 1960). The common change in fatty-acyl composition induced by ethanol in E. coli and Sacch. cerevisiae may represent an example of parallel evolution between a eukaryote and a prokaryote. It is curious that E. coli and Sacch. cerevisiae both exhibit ethanol-inducible changes in their fatty-acyl compositions while the fatty-acyl composition of the obligately ethanol-producing bacterium Z . mobilis fails to alter its fatty-acyl composition in response to various ethanol concentrations in the medium (Carey and Ingram, 1983). This lack of responsiveness in Z . mobilis may reflect a retrograde evolutionary adaptation. Both E. coli and Sacch. cerevisiae are capable of shifting between oxidative and fermentative metabolism depending on environmental conditions, and both produce ethanol as a fermentation product (Chesbro et al., 1979; Rose, 1980). These shifts in metabolism involve the co-ordinate regulation of many enzyme activities and, in yeasts, the control or mitochondria1 biosynthesis. Zymomonas mobilis possesses the Entner-Doudoroff pathway characteristic of the obligately oxidative pseudomonad-type bacteria and parts of a cytochrome system, but has lost the ability to shift to an oxidative metabolism (Swings and DeLey, 1977). This organism produces very high proportions of c18:I fatty-acyl residues which represents the extreme of the ethanol-inducible trends in E. coli and Sacch. cereuisiae. Along with loss of ability to grow


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oxidatively, Z . mobilis may have also lost the requirement and ability to shift lipid compositions. Thus Z . mobilis may be obligately adapted, in terms of fatty-acyl composition, to compete and survive the accumulation of its own waste product, namely ethanol. Other membrane changes are also induced by ethanol in bacteria. These include an increase in the proportion of acidic phospholipids and a decrease in the phospholipid :protein content of membranes. These may also be adaptive. Mutants of B. subtilis have been isolated which are resistant and sporulate in the presence of ethanol (Bohin and Lubochinsky, 1982). These mutants contain elevated concentrations of acidic phospholipids compared with the parent organism. Similarly, ethanol-resistant mutants of E. coli have been isolated and these also contain an increase in the proportion of acidic lipids (Clark and Beard, 1979). The decrease in the phospholipid :protein ratio in the membrane may also be beneficial. Proteins serve as barriers to the passive diffusion of ions and molecules through membranes except for specific transport enzymes. In order to diffuse across the membrane, molecules must first dissolve in the lipid portion of the membrane. Thus a decrease in the amount of phospholipid in the membrane would decrease the available surface area for passive diffusion. C. FUTURE DIRECTIONS

What avenues of research should prove most successful? To understand the mechanisms of action of ethanol, we need to continue to unravel the details of membrane organization and the intimate relationships between membranes and cytosolic metabolism. For biomedical interests, micro-organisms should provide an important tool to investigate the significance of sterols for alcohol tolerance and to learn new fundamental mechanisms of cellular regulation. For commercial alcohol production, the benefits of adding lipids to fermentations to enhance productivity are already being realized in some areas. The alcohol-resistant hiochii bacteria probably set an upper limit for the functioning of metabolism in the presence of ethanol at somewhat over 20% (v/v). In order to meet or exceed this limit, considerable support from industry is needed to carry out basic research on the biochemical mechanism of alcohol inhibition of fermentation and its relationship to temperature. Once this biochemical mechanism is established, it may be possible to chemically or genetically engineer organisms with superior fermentations rates, superior thermal tolerance and higher final alcohol concentrations.

VIII. Acknowledgements This work was supported in part by grants from the National Science

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Foundation (LOI, PCM-8204928), the Florida Alcohol Research Center (LOI, NIAAA AA 05793), the National Institute of Health (TMB, HL 28468), and the Florida Agricultural Experiment Station (Journal Series No. 4968). LO1 is the recipient of a National Institute of Alcohol Abuse and Alcoholism research career development award (KO 00036).

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Physiology of Circadian Rhythms in Micro-organisms LELAND N. EDMUNDS, Jr Note Added in Proof

A number of important articles have appeared since the initial literature survey for this review was completed. Sweeney (1983) speculates on the possible role of phosphorylation as a general oscillator control mechanism in her discussion of models and hypotheses for circadian time-keeping in eukaryotic cells. On the rationale that both the direct transitory activation and inhibition of a putative element of a circadian clock should each yield a phase shift in an overt rhythm generated by the underlying oscillator, K. Goto, D.L. Laval-Martin and L.N. Edmunds, Jr. (1984, unpublished work) have derived phase response curves for short perturbations of NAD+, NADP+, p-nitrophenylphosphate (a competitive inhibitor of NADP phosphatase), W7 and chlorpromazine (inhibitors of Ca2+-calmodulin),chlortetracycline (a membrane permeable chelator of Ca2+) and sodium acetate, nitrogen and dinitrophenol (enhancers of mitochondrial Ca2+ uptake or efflux) on the free-running circadian rhythm of cell division in Euglena sp. (see Section III.A.1, p. 75). The results suggest that NAD+, the mitochondrial Ca2+-transport system, Ca2+-calmodulin, NAD kinase and NADP phosphatase in ensemble might constitute a self-sustained circadian oscillator loop. A circadian rhythm in total RNA content (acridine orange-fluorescence) has been discovered in stationary-phase cultures of Gonyaulax sp. (see Section III.B, p. 100); and in-phase rhythm in ribosomal RNA synthesis (32P incorporation) was also found, with new species RNA (shown by gel electrophoresis) appearing at CT 18 and disappearing 3 to 4 hours later (Waltz et al., 1983). These findings suggest the role of transcription in the expression of circadian rhythms and possibly in the mechanism of the primary oscillator itself. In this same micro-organism, the circadian rhythm in luciferase activity, which is partly responsible for rhythmic bioluminescence (see Section 1II.B.1, p. 104), in turn has been shown to be attributable to a corresponding circadian change in the concentration of immunologically reactive luciferase protein; thus, protein turnover may be a common means of oscillator confrol over biochemical pathways (Johnson, et al., 1984). Similarly, changes in photosystem I1 (PS 11) have been shown to account for the circadian rhythm in photosynthesis in Gonyaulax sp. (see Section III.B.2, p. 107);electron flow ( 0 2 30 1

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uptake in cell-free extracts from late log cultures in LL for 3 days) through both PS I and PS I1 and through PS I1 alone was rhythmic, but flow through PS I alone, including or excluding the plastoquinone pool, was not (Samuelsson et af.,1983). Mergenhagen (1984) has genetically characterized a short-period clock mutant for the circadian rhythm of phototaxis in Chlamydomonas sp. (see Section III.C, p. 109). A diurnal rhythm of fatty acid content has been found in Acetabularia sp. (see Section III.D, p. 110) with a peak about 9 hours after the onset of light in LD: 12, 12; circadian rhythmicity in oleic acid content in entire alga and in palmitic acid content in chloroplasts persisted for 3 days in LL (Jerebzoff and Vanden Driessche 1983).Nakashima (1982a,b) has reported that diethylstilbestrol, a membrane ATPase inhibitor, not only generated phase shifts but also prevented light-induced phase shifting of the conidiation rhythm of Neurospora crassa (see Section IV.B, p. 117) at pH values above 6, suggesting that membrane-bound ATPase might be a clock protein; the relative effects of diethylstilbestrol and several of its analogues on plasma-membrane ATPase, however, did not parallel their phase-shifting activities, nor did their relative activities as inhibitors of mitochondrial ATPase. In this same fungus, Nakashima (1983) also examined the effects of phenylmethanol, 2-phenylethanol and 3-phenylpropan- 1-01 (all presumably affecting membrane permeability) on both the conidiation rhythm and the fatty acid composition of the phospholipids; only 2-phenylethanol altered the period (but not temperature compensation of tau or the amplitude of light-induced phase shifts), shortening it in proportion to its concentration (above 1 mM), whereas the ratio of linolenic to linoleic acid decreased in proportion to the concentrations of all three chemicals in all phospholipids examined, as did the proportion of phosphatidic acid and diphosphatidylglycerol to total phospholipids. Thus, the period-shortening effects of 2-phenylethanol could not be explained by changes in phospholipid fatty acid composition (see Section IV.B, p. 123). A phase-response curve for the effectsof pulses of the CaZaionophore A23 187 on the conidiation rhythm of N . crassa in divalent cation-free medium has also been obtained (contrast with Gonyaufaxsp.; seep. 106);phase-shifting was completely inhibited by the addition of CaC12 (but not by MgC12) (Nakashima, 1984).Thus, the presumed raising of internal cellular Ca2+levels did not affect the clock, though lowering it did. The addition of antimycin A inhibited respiration and lowered ATP content by 85 to 90%, and, if given with A23 187, caused phase delays at CT 10 which were not reversed by CaC12. These results caused Nakashima (1984) to conclude that rhythmic changes in mitochondrial Ca2+content, even if they do occur, are not a main component of the clock. Unpublished results from experiments by D.O. Woodward (as cited in Feldman and Dunlap, 1983, pp. 343-344) in which heterokaryons of N . crassa were created in D D in “Y”-shaped race tubes between two strains previously entained by LD to different phases unexpectedly suggest that a “phasing substance” may exist in

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the mycelium and that a significant part of the clock mechanism may be compartmentalized (see Section V.D.2, p. 136). Finally, recent and provocative results from the STS 9 flight of Space Shuttle Columbia revealed that although the period of the conidiation rhythm of this micro-organism in DD was the same as that of earth-based controls, there was a marked reduction in amplitude of the rhythmicity and apparent arrhythmicity in some culture race tubes; the data were insufficient, however, to determine if the circadian timing mechanism itself was affected or only its expression (Sulzman et af., 1984). References Jerebzoff, S. and Vanden Driessche, T. (1983). Comptes Rendus des Shances de I’dcademie des Sciences, Shries III-Sciences de la vie 2%, 3 19. Johnson, C.H., Roeber, J.F. and Hastings, J.F. (1984). Science 233, 1428. Mergenhagen, D. (1984). European Journal of Cell Biology 33, 13. Nakashima, H. (1983). Plant and Cell Physiology 24, 1121. Nakashima, H. (1984). Plant Physiology 74,268. Samuelson, G., Sweeney, B.M., Matlick, H.A. and Prezelin, B.B. (1983). Plant Physiology 73, 329. Sulzman, F.M., Ellman, D., Fuller, C.A., Moore-Ede, M.C. and Wassmer, G. (1984). Science (in press). Sweeney, B.M. (1983). In “Progress in Phycological Research” (F.E. Round and D.J. Chapman, eds.), Vol. 2, pp. 189-226. Walz, B., Walz, A. and Sweeney, B.M. (1983). Journal of Comparatiue Physiology 151, 207.


Author Index

s.,

A

Abe, H., 46, 56 Abe, K., 43, 58 Abranches, P., 197, 201, 208, 209, 210, 241,244,251 Adachi, O., 185, 192 Adams, K.J., 67, 75, 83, 84, 88, 90, 91, 110, 124, 125, 126, 128, 131, 132, 133, 139, 141 Adler, L., 154, 171, 172, 188, 189 Adomako, D., 154, 160, 161, 189 Agre, N.S., 46, 58 Aharonowitz, Y., 7, 36, 54, 56 Ahearn, D.G., 198, 250 Aiba, S., 223, 248 Aisaka, K., 159, 192 Aitken, W.B., 152, 158, 174, 178, 189 Ajl, S.J., 51, 56 Akita, H., 159, 192 Aldrich, H.A., 257, 299 Algar, E., 289, 291, 298 Allen, P.J., 38, 44, 46, 56, 58 Amerine, M., 227, 250 Ameyama, M., 185, I92 Anacleto, J., 234, 235, 236, 237, 248 Anand, J.C., 171, I89 Anderson, E., 225,248 Anderson, M.S., 51, 58 Anderson, R.E., 198,250 Anderson, R.W., 88, 139 Andreasen, A.A., 285,296 Andreeva, E.A., 243,248 Andrews, R.E., 51,56 Anisova, L.N., 43, 58 Antal, A., 261, 298 Antipa, G.A., 68, 70,71,72,81, 127, 128, 142, 144 apRees, T., 164, 186, 189, 191 Apter, R.I., 68, 116, 117, 126, I42 Arnold, W.J., 155, 192 Aronson, A.I., 45,60

Arpin, 39, 56 Arslanian, M.J., 294, 299 Arthur, H., 197,245,248,251 Aschoff, J., 65, 139 Ashworth, J.M., 44, 58 Astrachan, L., 101, 108, 143 Atkinson, C.A., 119, 120, 142 Audhya, T.K., 7,57 Augustin, H.W., 289,296 Austin, S., 169, 191 Awad, E.S., 294,299

B Babel, W., 196, 248 Bacila, M., 152, 154, 163, 164, 189, 193 Bajpai, P., 226, 250 Balasheva, T.A., 43, 58 Ball, A., 207, 250 Bandas, E.L., 247,248,251 Banks, B.E., 241,248 Barabas, G., 46, 5 7 Barbu, E., 266,296 Bard, M., 279,297 Barnes, E., 255, 294,296 Barnes, J.H., 68, 70, 71, 142 Barnes, R., 240, 241, 244,248 Barnett, A., 67, 68, 71, 74, 126, 130, 139, 148 Barnett, J.A., 160, 163, 164, I89 Bartman, C.D., 7, 52, 54, 57, 59 Barz, W., 7, 57 Baty, D., 261, 298 Bauer, K., 45,59 Bazua, C.D., 225,248 Bean, H.S., 232, 248 Beard, J.P., 267, 295,296 Beavan, M.J., 225, 233, 248, 250, 275, 278,279, 289, 293, 294,296 Belisle, B.W., 138, 139 Bell, E.A., 5 , 22,57

305

306

AUTHOR INDEX

Ben-Naim, A., 281,300 Bennett, S.N., 171, 193 Bennetzen, 270, 300 Benschoter, J.A.S., 256, 258, 296 Bent, H.A., 204,248 Bentley, R., 20, 57 Berger, B., 267, 296 Bergmeyer, Hrll., 158, 189 Bernheim, F., 270,296 Berrah, G., 260, 296 Berry, D.R., 22, 60 Berry, M.N., 183, 189 Betz, A., 165, 190 Bieleski, R.L., 150, 189 Bierhuizen, J.F., 65, 139 Bird, B.A., 52, 54, 55, 57 Biemann, K., 271, 298 Birken, S., 152, 156, 189 Birkinshaw, J.H., 176, 189 Bittner, AS., 265, 300 Blackwood, A.C., 173, 192 Blain, J.A., 38,57 Blakey, M., 197,251 Blakley, E.R., 173, 189 Blasie, J.K., 282, 299 Bloch, K.. 285, 286, 294, 296, 298, 299 Block, R., 261,297 Bloomfield, D.K., 285, 294, 296 Blum, J.J., 71, 140 Blumauerova, M., 7, 38, 41, 58, 60 Blumberg, P.M., 259, 260, 296 Blumenthal, H.J., 186, 189 Bode, V.C., 101, 104, 105, 140, 143 Boguslawski, G., 243,251 Bohin, J.P., 257, 265,267, 295,296,299 Bohm, H., 3,58 Boiteux, A., 130, 143 Bole, D.G., 262, 269, 296 Bonotto, S., 11I , 114, 147 Boonsaeng, V., 153, 158, 189 Borrow, A., 182,189 Borst-Pauwels, G.W.F.H., 180, 189 Bortels, H., 200, 248 Bott, T.L., 196,248 Bottomley, W., 46, 58 Bouillant, M.L., 39, 56 Boutelje, J., 156, 189 Bowler, K., 241, 249 Boze, H., 289, 298 Braber, L., 210,215,249 Brachet, J., 11I , 114, 147

Brady, J., 62, 65, 140 Brain, R.D., 121, 122, 140 Brand, L.E., 67, 68, 126, 127, 140 Brar, S.S., 7, 57 Braun, M.L., 179, 189 Bre, M.H., 90, 140 Brechot, P., 285, 297 Brewer, J.H., 260, 298 Bridge-Cooke, W., 197, 248 Briggs, W.R., 118, 120, 121, 122,140,146 Brinkmann, K., 76, 77, 93, 94, 95, 106, 136, 140, 143, 144 Britz, S.J., 121, 122, 140 Brock, T.D., 196,248 Broda, H., 87, 101, 102, 107, 111, 113, 140, 147 Brody, S., 118, 122, 123, 140, 141, 144, I46 Brooks, J., 50, 57 Brown, A.D., 171, 188, 189 Brown, A.J., 287,296 Brown, F.A., Jr., 62, 65, 145 Brown, R.E., 198,250 Brown, S., 182, 189 Brown, S.W., 225,248 Brownlee, C., 176, 177, 189 Bruce, N.C., 67, 109, 126, 140 Bruce, V.G., 66, 67, 76, 91, 92, 109, 110, 126, 140, 142, 146 Buckley, H., 198, 199, 251 Buetow, D.E., 75, 140 Bulder, C.J.E.A., 242, 245, 248 Bulla, L.A., 45, 51, 56, 60 Bullock, J.G., 243, 248 Bu’Lock, J.D., 4, 5, 32, 38, 41, 50, 57 Biinning, E., 62, 65, 103, 140 Burakova, A.A., 243,249 Burgoyne, R.D., 131, 140 Burns, J.A., 205, 208,249 Burns, R.O., 243,248 Burrill, H., 291, 296 Bush, K., 108, 140 Busse, H.G., 130, 141 Butler, R.D., 184, 191 Buttke, T.M., 264,268,285,286,296,297 Byers, B.R., 38, 47, 48, 57 Byrd, E.W., 138, 139

C Calam, C.T., 38, 57

307

AUTHOR INDEX

CabeCa-Silva, C., 197,201,208,209,210, 225,241, 243,244,246, 247,248,251 Caillaud, J.M., 226, 249 Cairns, W.L., 111, 147 Caldarola, P.C., 105, 145 Calvayrac, R., 80, 127, 144 Campbell, A., 81, 140, 200, 248 Campbell, I.M., 4, 7, 52, 54, 55, 57, 59 Campbell, L.L., 239,259 Cane, D.E., 28,57 Cannell, E., 226, 250 Caplan, S.R., 130, 143 Capobianco, S., 138, 146 Carey, V.C., 257, 265,267,282,293,294, 296 Carl, P.L., 261,297 Carmo-Sousa, L. do, 197, 198,248,251 Carr, H.S., 261, 299 Carter, B., 202, 249 Carter, B.L.A., 116, 140 Carty, C.E., 267,296 Cerf, O., 244, 248 Cerny, G., 202,248 Chaitiumvong, S., 284, 297 Chakravorty, M., 152, 163, 164, 189 Chang, T.L., 269, 274,299 Charley, R., 291, 298 Charlwood, B.V., 22,57 Charpentier, C., 225, 248, 275, 278, 279, 289,293, 294,296 Chater, K.F., 45,57 Cheng, P.-J., 266, 298 Chesbro, W., 294,296 Chiang, C., 152, 154, 163, 189 Chilla, R., 164, 190 Chin, J.H., 254, 296 Chisholm, S.W., 67, 126, 127, 140 Christianson, R., 101, 141 Christophersen, J., 196, 250 Chuang, L., 76, 83,142 Ciegler, A., 51,56 Cirillo,V.P., 115, 116, 117, 134, 141, 147, 148 Clancey, F.G., 152, 156, 157, 189 Clark, D.P., 261,267,295,296,298 Claypool, T.A., 165,190 Codd, G.A., 96,98,141 Coakley, W.T., 243, 248 Coffey, M.D., 152, 156, 157, 189 Cohen, M.H., 130,143 Collie, J.N., 30, 57

Conant, N.F., 243,248 Conn, E.E., 22,57 Conner, R.L., 276,298 Conrad, M.J., 281, 296 Conrard, D.J., 261, 262, 299 Conway, E.J., 181, 189 Cook, J.R., 75, 77, 80, 96, 99, 141 Cooke, R.C., 159, 175, 176, 189, 190 Cooney, C.L., 10,60 Corbett, K., 176, 189 Cordes, E.H., 262, 263,299 Corner, T.R., 255,296 Cosbey, E.S., 105, 145 Cotter, D.A., 178, 179, 189 Courtwright, J.B., 157, 158, 170, 171, 189, 190 Cove, D.J., 163, 190 Cox, E.C., 45,523 Cram, W.J., 188, 189 Crandall, M., 43, 57 Crocker, B., 168, 189 Cronan, J.E., 175, 192 Cuatrecasas, P., 51, 57 Cudlin, J., 38, 41, 60 Culatti, J., 116, 143 Cullis, P.R., 280, 297 Cumming, B.G., 130, 148 Cummings, F.W., 121, 141 Curtis, F.C., 159, 190 Cutaia, A.J., 225, 250

D Daan, S., 65, 139 Daasch, L.W., 47, 59 Dabbagh, R., 243,248 Da Costa, M.S., 187, 190 Dagley, S., 255, 296 Dally, E.L., 255, 299 Damjanovics, V., 241, 248 Danders, A.W., 46,59 Daniels, C.J., 262, 269, 296 Darwin, C., 3 5 5 7 Das, J., 130, 141 Daum, G., 245,248 Daurelles, J., 226, 249 Davies, P.J., 202, 250 Dawes, E.A., 255, 296 Day, A., 225,248 Decedue, C., 243,251 DeCoursey, P., 65, 141

308

AUTHOR INDEX

De Ley, J., 290, 294, 299 Delmer, D.P., 118, 141 Demain, A.L., 7, 10,36,46,54,56,59,60, 255,294,296 Denison, R.A., 118, 119, 120, 121, 122, 142 Denne, D.W., 179,191 Denor, P.F., 171, 190 De Sa, R., 101, 104, 140 Desai, B.M., 158, 190 Detroy, R.W., 39, 57 Deutsch, R., 266, 298 Deville-Duc, T., 226, 249 DeVoe, I.W., 269,299 Devon, T.K., 20,21,57 Devouge, C., 226,249 Dewey, V.C., 71, 143 Dharmananda, S., 135, 136,141 Diamond, J.M., 288, 297 Dickens, B.F., 258, 268, 280,296,297 Dickerson, A.G., 176, 189 Dieckmann, C.L., 122, 123, 141 Diltz, R.P., Jr., 156, 191 Dimroth, P., 32,57 Dingmann, D.W., 45,60 DiRienzo, J.M., 262,296 Dobra,K.W.,68,70,71,72, 128,141,142 Doege, K.J., 11 1, 147 Doelle, H.W., 291, 296 Doerfler, D.L., 7, 52, 54, 57, 59 Doering, K.M., 164, 190 Domagk, G.F., 164, 190 Dombek, K.M., 256, 257, 258, 267, 282, 283,296 Dondero, N.C., 45,57 Doughty, C.C., 155, 192, 193 Duarte, H., Cruz da, 210, 213, 215, 225, 226, 232,247,251,277,300 Dunhill, P., 10, 60 Dunlap, J.C., 66, 68, 87, 101, 102, 104, 107, 118, 123, 141, 142, 147 Durand, G., 225,250,278,279,290,298 Durbin, R.D., 51, 57 Dutler, H., 155, 190 Dutsch, G., 177, 190 Duvnjak, I., 226, 248

E Eaton, L.C., 268,269,270,280, 282,283, 292,296,297

Eberle, H., 260, 298 Eddy, A.A., 164, 181, 190, 274,297 Eder, E., 261,298 Edmundowicz, J.M., 156, 190 Edmunds, L.N., Jr., 62,65,66,67,68,69, 70,74,75,76,77,78,79,80,81,83,84,

85,86,87,88,90,91,94,95,96,97,98, 99, 100, 102, 104, 106, 109, 110, 115, 116, 117, 124, 125, 126, 127, 128, 129, 131, 132, 133, 134, 135, 136, 137, 139, 141, 142, 143, 144, 145, 146, 147, 148 Edwards, S.W., 62, 63, 144 Egel, R., 43, 57 Ehret, C.F., 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 110, 126, 127, 128, 130, 132, 133, 139, 141, 142,144, 147, 148 Eifert, R., 294, 296 Eider, W.J., 69, 147 El Ferjani, E., 90, 140 Elhiti, M.M.Y., 184, 191 Elizondo, E., 241, 249 Elkins, D., 272,298 Emery, T., 38, 47, 48, 57 Engelmann, W., 77, 132, 142, 144 Engeser, H., 35, 58 Enright, J.T., 92, 105, 142 Ensign, G.C., 45, 58 Epel, B.L., 134, 142 Eppley, R.W., 67, 126, 148 Esser, A.F., 244, 249 Evans, P.R., 241,249 Evans, T., 294,296 Evans, W.E., 202, 243,249,250 Eveleigh, D.E., 255, 299 Evison, L.M., 242, 249 Eyring, H., 201, 202, 203, 204, 205, 207, 208,239,249 F Farfaglio, G., 96, 144 Farinha, M., 197, 251 Farrell, J., 196, 249 Feeny, P.F., 5, 38, 60 Fein, J., 291, 298 Feldman, J.F., 66, 68, 76, 77, 91, 92, 93, 99, 107, 109, 110, 118, 119, 120, 121, 122, 123, 135, 136, 141, 142, 143, 145 Fell, J.W., 197, 249 Feng, D.D., 284,285,297 Fiechter, A., 172, 174, 190

309

AUTHOR INDEX

Finch, C.E., 138, 142 Finn, R.K., 223,249 Fintan Walton, E., 202, 244, 249 Follet, D.E., 65, 142 Follett, B.K., 65, 142 Forman, L.R., 122, 123, 140, 144 Foster, J.W., 1 1 , 58 Franks, F., 281,297 Freeberg, J., 121, 122, 140 Freeze, H., 196,248 French, R.C., 47, 58, 59 Fried, V.A., 257, 268, 297 Friedrick, J., 35, 58 Frisch, L., 65, 142 Fuhrmann, G.F., 172, 174,190 Fujino, M., 43, 59 Fukui, S., 43, 58, 271, 272, 275, 300 Fulco, A.J., 294, 297 Fuller, C.A., 65, 145 Funch, R.R., 76, 80, 8 1 , 83, 97, 126, 136, 141

G Galzy, P., 226, 249, 289, 298 Gancedo, C., 171, 174, 175, 190 Gancedo, J.M., 171, 174, 175, 190 Gardner, G.F., 118, 119, 120, 121, 122, 142, 143

Garrod, D., 44, 58 Gatenbeck, S., 152, 156, 161, 162, 163, 185, 189, 190

Gaucher, G.M., 53, 54, 58, 59 Gazso, L., 261,297 Gefter, M.L., 261, 297 Gerber, N.N., 58 Ghose, T.K., 225,249 Gilbert, D.A., 124, 143 Gilbert, W., 261, 297 Glatz, H., 245, 248 Goffeau, A., 181, 190 Goldbeter, A., 130, 143 Goldstein, D.B., 254, 296 Goma, G., 225,251,291,298 Gomez, R.F., 257, 265,297 Gong, C.-S., 165, 190 Gooch,V.D., 101,102,103,104,106,131, 136, 145, 146, 147

Good, D., 291,298 Gooday, G.W., 38,41, 50,58 Goodwin, B.C., 66, 130, 136, 143

Goos, R.D., 4 5 5 9 Gordon, I., 240, 241, 244, 248 Gordon, J.T., 276,298 Goto, O., 255, 265,297 Gottlieb, D., 38, 44, 58 Govindjee, 99, 101, 108, 109, 143, 147 Gram, C.S., 7,57 Gray, W.D., 223,225,249,255,270,276, 278,287,288, 290,297

Greenfield, P.F., 291,296 Gregg, I.I., 261, 262, 299 Griffin, D.H., 47, 58 Griffiths-Smith, K., 289, 291,298 Groh, K.R., 68, 71, 72, 81, 127, 128, 142 Grohmann, K., 265,300 Groos, G.A., 65, 139 Grootwassink, J.W.D., 54, 58 Gross, S.D., 85, 129, 144 Gruen, H.E., 177, 191 Guilliermond, A., 197, 249 Guiraud, J.P., 226, 249 Gunsberg, S., 294,299 Gustafsson, L., 171, 172, 188 H Haak, R.A., 279,297 Haavik, H.I., 7, 58 Hafiz, A., 154, 180, 191 Hagen, P.-O., 242, 249 Hagler, A.N., 243, 249 Hajny, G.J., 160, 191 Halberg, F., 62, 66, 67, 72, 74, 75, 76, 79, 91, 138, 141,143,146

Halegoua, S., 261, 262, 297 Hall, B.D., 270, 300 Halsey, Y.D., 157, 190 Halvorson, H.O., 116, 140 Hamilton, D., 4,57 Hamilton, T.C., 241,250 Hammond, J.B.W., 176, 177, 178, 183, 186, 190

Hankinson, O., 163, 190 Hanna, M.H., 45,58 Hansch, C., 272,298 Hansan, S.M., 260, 269, 297 Hansen, U.-P., 180, 182, 192 Harborne, J.B., 5, 38,58 Hardeland, R., 68, 73, 143 Harold, F.M., 255, 256, 269,297 Harris, S., 118, 140

310

AUTHOR INDEX

Harrison, J.S., 160, 192 Hars, R., 1 1 1, 147 Hartanowicz, P., 154, 158, 160, 192 Hartnoll, R.G., 65, 145 Hartwell, L., 116, 143 Haseltine, W.A., 261, 297 Haslam, E., 22, 58 Hastings, J.W., 65, 66, 67, 87, 88, 100, 101, 102, 103, 104, 105, 106, 107, 108, 110, 113, 114, 115, 123, 126, 129, 131, 132, 133, 135, 136, 140, 141, 143, 144, 145, 146, 147 Hattori, K., 161, 190 Haukeli, A.D., 215, 249 Haus, E., 72, 138, 143 Havsteen, B.H., 130, 141 Hawker, L.E., 160, 190 Haxo, F.T., 101, 102, 1 1 1 , 112, 147 Hayaishi, O., 65, 146 Hayashi, J.A., 155, 192, 193 Hayashida, S., 265, 274, 284, 285, 297, 298 Hayes, R.D., 226,248 Hayflick, L., 138, 142, 143 Hegeman, G.D., 45,60, 262, 263,299 Hegnauer, H., 49, 50, 59 Heinze, J.E., 261, 297 Hellebust, J.A., 108, 11 1, 143 Hendershot, W.F., 160, 191 Henry, L., 154, 180, 191 Henschler, D., 261, 298 Herbert, D., 218, 249 Herbert, R.B., 22, 58 Herman, E., 101, 143 Hermier, J., 244, 248 Herrero, A.A., 257, 265, 297 Herz, J.M., 101, 106, 107, 147 Hess, B., 130, 143 Hesse, M., 67, 126, 143 Heusel, H., 196, 250 Hinshelwood, C.N., 207,249 Hiramitsu, K., 275, 298 Hirsch, C.F., 45, 58 Hochberg, M.L., 118, 143 Hochuli, E., 155, 190 Hofer, M., 165, 180, 190 Hofmann, E., 289,296 Hoffmans, M., 95, 143 Holick, M., 154, 158, 160, 192 Holligan, P.M., 161, 165, 166, 179, 182, 185, 186, 190

Hollomon, D.W., 46, 58 Holz, G., 158, 189 Holzberg, I., 223, 249 Holzer, G., 265, 300 Holzer, H., 234, 236, 250 Homma, K., 101, 102, 136, 146 Hongo, M., 284,285,297 Hopfer, R.L., 151, 191 Hopwood, D.A., 45,57 Horecker, B.L., 152, 154, 163, 164, 189, 190, 192, 193 Horikoshi, K., 154, 156, 190 Horitsu, H., 152, 154, 163, 164, 190, 192 Hornby, A.P., 280,297 Hossack, J.A., 225, 251, 276, 286, 292, 299 HoSt’aIek, Z . , 7, 38, 41, 58, 60 Howe, H.B., Jr., 155, 170, 171, 192, 193 Hoyle, M.N., 119, 142 Hugo, W.B., 255,256,269,297 Hulme, M.A., 4, 57 Hult, K., 152, 156, 161, 162, 163, 185, 189, 190 Hunt, D.F., 271,298 Hunt, S., 178, 191 Humphrey, A.E., 10,60 Hunter, I.L., 197, 249 Hunter, K., 202, 244, 249 Hiitter, R., 22, 58 I Ichikawa, H., 245, 250 Igali, S., 261, 297, 298 Iidia, S., 154, 156, 190 Ikeda, Y., 154, 156, 190 Ikediugwu, F.E.O., 38,45,58 Ingraham, J.L., 196, 208, 249, 262, 263, 298 Ingram, J.M., 173, 190 Ingram, L.O., 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 275, 280, 282, 283, 291, 292,293, 294,296,297,298,299 Inouye, M., 261,262,296,297 Isakova, D.M., 216,223, 243,249 Isenberg, D., 179, 190 Ishita, Y., 51, 59 Isogai, A., 43, 59 Ito, K., 159, 192 Itoh, N., 151, 190

AUTHOR INDEX

Ives, D.J.G., 281, 297

J Jacobs, G.P., 255, 297 Jain, M.K., 268, 280, 282, 297, 299 James, A.P., 164, 191 James, A.T., 265, 267,297 James, T.W., 75, 80, 141 Janakidevi, K., 71, 143 Janoff, A.S., 254, 255,257, 278, 280,297 Jansen, D.H., 5,58 Jarrett, R.M., 75, 76, 81, 83, 84, 90, 126, 127, 142, 143 Jay, M.E., 75, 76, 81, 83, 90, 126, 142 Jeffery, J., 186, 190 Jefferys, E.G., 182, 189 Jennings, D.H., 161, 165, 166, 167, 168, 169, 171, 176, 177, 179, 18I , 182, 183, 185, 186, 189, 190, 191 Jensen, A., 67, 126, 145 Johnson, F.H., 201, 202, 203, 204, 205, 207, 208,239,249 Johnsson, A., 67, 145 Jones, E.B.G., 171, 191 Jornvall, H., 186, 190 Jones, S.D., 286, 296 Jukes, T.H., 280,297

K Kaback, H.R., 269,299 Kachau, A.F., 243,249 Kacser, H., 205, 208, 249 Kadis, S., 51, 56 Kalakoutskii, L.V., 46, 58 Kaltenborn, S.H., 118, 146 Kamiya, Y., 43, 58 Kammerer, J., 68, 73, 143 Kaneko, T., 255,265,297 Kanemasa, Y., 245, 250 Kanne, R., 46,47,58 Kappeli, O., 172, 174,190 Karakashian, M.W., 67, 73, 101, 107, 111, 112, 113, 114, 124, 143, 144 Karesch, H., 156, 177, 192 Kasai, R., 271, 275, 276, 298 Kates, M., 265, 267, 297 Katz, Y., 288, 297 Kauder, E.M., 158,189 Kauffman, S.A., 124, 129, 137, 146

31 1

Kauzmann, W., 130,145 Kavalenko, I.V., 43, 58 Kaye, M.A.G., 154, 160, 161,189 Keenan, M.H.J., 274,297 Keller, J., 154, 158, 160, 192 Kelln, R.A., 163, 191 Kemeny, G., 241,250 Kessell, R.H.J., 182, 189 Khalik, S., 45, 60 Khokhlov, AS., 43, 58 Khovrychev, M.P., 243,248 Khuller, G.K., 265, 267, 299 Kidder, G.W., 71, 143 Kiefner, G., 77, 144 Kieslich, K., 26, 58 Kim, H., 45,60 King, G.A., 8 5 , 88, 129, 144 Kirschstein, M., 75, 76, 94, 144 Kis, Z., 155, 190 Kiser, R.C., 152, 191 Kitada, C., 43, 59 Kitahara, K., 255, 265, 297 Kitajama, Y.,271, 272, 275, 276, 298 Kitamoto, Y., 177, 191 Kleinans, F.W., 279, 297 Kleiner, E.M., 43,58 Kleinkauf, H., 45, 46, 59 Klevecz, R.R., 85, 88, 124, 126, 128, 129, 134, 137, 144, 146 Klitzing, L. von, 11 1, 112, 144 Knight, S.G., 152, 154, 163, 189 Knotkova, A., 181, 192 Koch, A.L., 268,299 Kodama, K., 152, 158, 162,193 Kohlmann, A., 75,76,81,83,90, 126,142 Kolarov, J., 245, 251 Kolychexa, V.V., 262,300 Konetzka, W.A., 260,296,300 KOOP,H.-H., 11 I , 140 Kopperschlager, G., 289,296 Kornberg, T., 261, 297 Kornutskaya, E.Ya., 43,58 Kosaric, N., 226, 248 Kossel, A., 3, 58 Koster, J., 7, 57 Kosuge, T., 156, 192 Kotyk, A., 165, 180, 181, 190, 191, 192 Kovic, L., 245,251 Krasilnikova, O.I., 43, 58 Krasnow, R., 67, 87, 101, 102, 103, 107, 143,147

312

AUTHOR INDEX

Krause, M., 46, 59 Krebs, H.A., 186, 191 Kreuels, T., 76, 93, 144 Krieger, N.R., 101, 104, 146 Krinsky, N.I., 50, 58 Kros, J., 85, 129, 144 Krouwel, P.G., 210,215,249 Krumphanzyl, V., 22, 38,41,58,60 Kiihl, J.F.W., 137, 143 Kumagai, K., 154, 190 Kunkee, R.E., 285,300 Kushner, D.J., 265, 267, 297 Kvasnikov, E.I., 2 16, 223, 243, 249 L

Labuza, T.P., 241,249 Lafon-Lafourcade, S., 285, 297 Lakatua, D.J., 137, 143 Lang, M., 256,297 Lapp, H., 156, 173,191 Larcher, W., 196, 250 Lark, C., 260,297 Lark, K.G., 260,297 Larkin, J.M., 196, 249 Larue, F., 285,297 Laudrin, I., 291, 298 Laval-Martin, D.L., 75,76,8 1,83,84,85, 86, 87, 88, 90, 97, 98, 125, 133, 135, 137,139,142,144 Lavers, B.H., 291,298 Lawford, B.R., 291,298 Lazdunski, C., 261, 262, 298 LeBo, C., 225, 227, 228, 229, 230, 231, 232,243, 246,249,272,273, 277, 278, 298 Ledoigt, G., 80, 127, 144 Lee, K.J., 255, 291, 299 Lee, N.D., 279,297 Lee, P.P., 258, 260,298 Lefort-Tran, M., 90, 140 Leighton, T.J., 163, 191 Leisinger, T., 22, 58 Lemos-Carolino, M., 215, 216,249 Leo, A., 272,298 Leong, T.-Y., 115, 132,144 Leppik, R.A., 46, 58 Lester, G., 271, 272, 298 LeTourneau, D., 152,153,158, 161,163, 174, 175,189, 191, 193 Levandowsky, M., 130,144

Lewandoski, C., 154, 158, 160, 192 Lewis, D.H., 150, 154, 159, 160, 161, 163, 177, 185, 188,189,190, 191 Lewis, M.J., 243, 249 L’Haridon, R., 244,248 Lie, S., 215, 249 Liesch, J.M., 51, 58 Lilley, B.D., 260, 298 Lilly, M.D., 10, 60 Linden, J.C., 256, 269, 298,300 Lingappa, B.T., 270, 271,298, 300 Lingappa, Y ., 27 1,298 Liras, P., 54, 58 Liu, S.C., 75, 76, 81, 83, 90, 126, 142 Livingstone, L., 67, 145 Llorente, P., 225, 249, 289, 290, 298 Lloyd, D., 62, 63, 144 Lloyd, E.C., 182, 189 Lloyd, P.B., 182, 189 Lodder, J., 197, 249 Lonergan, T.A., 76,77,78,97,98,99,144 Lorenzen, H.G., 67, 126, 145 Lorowitz, W., 261, 298 Loureiro, V., 210,215,225,232,233,247, 249 Loureiro-Dias, M.C., 273, 298 Lovlie, A,, 96, 144 Lowe, D.A., 166, 167,191 Lozano, R., 286,299 Luard, E.J., 159, 171, 187, 188, 191 Lubochinsky, B., 257, 265,267, 295,296, 299 Lucas-Lenard, J.M., 261,298 Luckner, M., 3,45,58,60 Lumry, R., 240,249 Lutz, D., 261, 298 Lux, M., 266,296 Lyall, J., 256, 300 Lynen, F., 32, 35,57,58 Lysek, G., 168, 191

M McBride, R.J., 256, 300 McCann, P.A., 45,58 McCracken, L.D., 165, 190 McDaniel, M., 101, 105, 144 McDermott, J.C.B., 168, 169, 191 McGregor, A.N., 291,296 McGuinness, E.T., 154, 158, 160, 192 Mackay, V.L., 43,57

AUTHOR INDEX

Macko, V., 38,44,46,47, 58,59 Maclean, D.J., 186, 191 McLeod, G.C., 67, 108, 111,143,147 McMeekin, T.A., 207,250 McMorris, T.C., 41, 58 McMurrough, I., 245,250 McMurry,L., 101,103,104,135,144,145 Macris, B., 234, 249 Madeira-Lopes, A., 210, 212, 2 13, 214, 215, 216, 217, 218, 219, 220, 221, 224, 225, 237, 238,239, 243. 245, 246, 247, 248,249,250,251 Magaiia-Schwencke, N., 242,250 Maleszka, R., 164, 191 Malik, V.S., 7,59 Malinowski, J.R., 87, 125, 144 Mallory, F.B., 276, 298 Mamouneas, T., 117, 134,147 Mann, J., 22, 59 Manners, J.M., 186, 191 Manney, T.R., 38,43,59 Mantle, P.G., 176, 189 Marahiel, M.A., 46, 59 Margaritis, A., 226, 250 Marmiroli, N., 245, 250 Maroudas, N.G., 270,300 Marr, A.G., 200,250, 262, 263,298 Martens, C.L., 118, 144 Martin, C.E., 275, 276, 298 Martin, J.F., 36, 54, 58, 59 Martins, S.A., 122, 123, 140 Matsumoto, T., 51,59 Matsushita, K., 185, 192 Mattern, D., 122, 123, 144 Marshall, J.H., 170, 171, 191 Martin, P.A., 198, 225, 248,251 Martinez-Peinado, J., 200, 250 Masker, W.E., 260,298 Mattox, S.M., 275, 276, 298 Maun, C.M., 165,190 May, J.W., 170, 171, 191 Meade, J.H., 38, 43,59 Meeson, B.W., 101, 108, 145 Meganathan, R., 20,57 Meinert, J.C., 68,70, 7 1,72,8 1, 127, 142, 144 Menaker, M., 65, 144 Menna, M. di, 197,248 Mergenhagen, D.M., 100, 106, 109, 111, 112, 113, 114, 131, 144, 145 Merkel, J.R., 151, 191

313

Merrett, M.J., 96, 98, 141 Merriam, V.H., 75, 76, 81, 83, 90, 126, 142 Metcalf, E., 168, 171, 191 Metcalfe, J.C., 282, 298 Meyer, F., 285,298 Meyer, S.A., 198,250 Michaelis, E.K., 255, 274, 277, 280, 282, 298 Michaelis, M.L., 255, 274, 277, 280, 282, 298 Miersch, J., 156, 173, 191, 192 Mihara, A., 159, 192 Millar, D.G., 289, 291, 298 Miller, K.W., 254,255,257,269,274,278, 280,297,299 Miller, M.W., 197, 198, 250 Minder, C., 111, 147 Mirocha, C.J., 7, 44,60 Misra, P.C., 180, 190 Mitchell, D.T., 176, 189 Mitchell, G., 198, 250 Mitchell, J.J., 261, 298 Mitchell, J.L.A., 75, 76, 81, 126, 144 Mitchison, J.M., 85, 124, 138, 144, 146 Miyamoto, H., 85, 144 Moat, A.G., 14, 16, 59 Moats, W.A., 201, 250 Modi, V.V., 158, 190 Mogi, K., 255, 265, 266, 300 Mnrller, U., 137, 145 Mollering, H., 158, 189 Monod, J., 199, 205, 217, 250 Montenecourt, B.S., 255, 299 Montie, T.C., 51, 56 Moore, D., 184, 191 Moore-Ede, M.C., 65, 145 Moorehead, P.S.,138, 143 Morehouse, L.G., 51, 60 Moreira, A.R., 256, 298 Moreira, J.R., 256, 269, 300 Morel, F.M.M., 127, 140 Moreno, M., 225,251 Mori, K., 43,59 Morrison, G.A., 255,296 Moses, V., 261, 299 Mothes, K., 3, 59 Moulin, G., 289, 298 Moustacchi, E., 202,250 Mrak, E.M., 197,250 Murray, J.B., 256,300

314

AUTHOR INDEX

Murrell, W.G., 46, 59

N Nagatani, M., 223, 248 Nagodawithana, T.W., 225, 250, 278, 279,289,298 Nakagawa, H., 65,146 Nakamura, K., 262,296 Nakashima, H., 118, 120, 122, 123, 145 Nandi, S., 46, 59 Nandini-Kishore, S.G., 271, 272, 275, 276,298 Nandini-Kishore, T.W., 275, 276, 298 Nash, C.H., 242,250 Nasmyth, K., 270,300 Navarro, J.M., 225, 233, 250, 278, 279, 289,290,298 Naylor, E., 65, 145 Neijssel, O.N., 11, 59 Neirinck, L.G., 164, 191 Neish, A.C., 173, 192 Nes, W.R., 286, 299 Nesmeyanova, M.A., 262,300 Neudecker, T., 261,298 Neuhaus, F.C., 258,260,298 Neway, J., 53, 59 Newell, P.C., 38, 44,59 Newman, E.I., 38,59 Nichols, R., 176, 177, 178, 183, 186, 190 Nicolis, G., 130, 143 Niederpruem, D.J., 154, 178, 179, 180, 187, 189, 190, 191, 192 Niehaus, W.G., Jr., 152, 156, 191 Nielsen, H.S., 243, 248 Ninnemann, H., 121, 145 Nishi, K., 245, 250 Njus, D., 101, 103, 106, 115, 131, 132, 136,145 Noguchi, F., 152, 158, 162, 193 Norkrans, B., 171, 188 North, M.J., 171, 191 Novak, B., 111, 113, 145 Nover, L., 3, 58 Novick, A., 257, 268, 297 Nozawa, Y., 271,275,276,298 Nuesch, J., 22, 58 Nunn, W.D., 263,266,298 Nyhlen, L.E., 49, 50, 59

0 Obaton, F., 160, 191 Ohta, K., 265, 274, 284, 285, 297, 298 Okabe, A., 245,250 Okamoto, K., 152, 158, 162, 193 Okuno, T., 51,59 Oliveira-Baptista, A., 215, 250 Oliver, R.M., 294, 299 Oliver, S.G., 225, 248, 270, 299 Olley, J., 207, 250 O’Malley, E., 181, 189 Onishi, H., 154, 171, 172, 191, 192 Onoprienko, V.V., 43,58 Opekarova, M., 181, 192 Osman, Y.A., 291,298 Osmond, C.B., 164, 191 Ostgaard, K., 67, 126, 145 Ostrowska-Krysiak, B., 7,59 Osztovics, M., 261, 298 Ough, C.S., 227, 250 Oura, E., 174, 191 Overath, P., 244,250 Oxender, D.L., 262, 269,296

P Pace, B., 239,250 Pages, J.-M., 261, 262, 298 Paietta, J., 121, 122, 134, 145 Painter, P.R., 200, 250 Palm, L., 45,59 Palmer, J.D., 62, 65, 67, 145, 146 Paltauf, F., 245, 248 Panchal, C.J., 279, 285, 290, 299 Parry, J.M., 202, 243, 249, 250 Pang, K.P., 269, 274,299 Parks, L.W., 285,299 Parsons, L.M., 254,296 Patel, L., 269, 299 Pathre, S.V., 7, 60 Pavlidis, T., 66,87,124,130, 131,136,145 Peace, J.N., 54, 59 Pedersen, A., 154, 171, 189 Peduzzi, R., 270,271,300 Peinado, J.M., 273, 298 Perlman, J., 118, 122, 123, 145 Peterson, W.H., 160, 191 Pfyffer, G., 150, 153, 159, 191 Phaff, H.J., 197, 249, 250 Pilgaokar, A.K., 261, 300

AUTHOR INDEX

Pinto, W.J., 286, 299 Piret, J.M., 46, 59 Pirone, J., 46, 59 Pirson, A., 67, 126, 145 Pirt, S.J., 220, 250 Pisano, M.A., 152, 156, 189 Pitt, J.I., 171, 191 Pittendrigh, C.S., 62, 66, 76, 85, 91, 92, 105, 134, 145 Pliner, S.A., 43, 58 Podo, F., 282,299 Podojil, M., 38, 41, 60 Pogell, B.M., 45, 58 Pohl, R., 76, 91, 145 Poivant, M., 261, 298 Polanshek, M.M., 85, 145 Polissar, M.J., 201, 202, 203, 204, 205, 207,208, 239,249 Polonovski, J., 266, 296 Poole, R.K., 62, 63, 144 Porter, J.W., 22, 59 Potter, V.R., 72, 142 Powell, A.J., 4, 57 Pozmogova, I.N., 216,223,243,248,250 Pradhan, D.S., 261, 300 Prasad, M., 271,298 Prazak, B.L., 69, 147 Precht, H., 196,250 Prelog, V., 155, 190 Preusser, H.J., 291, 296 Prbzelin, B.B., 99, 101, 108, 109,143,145, 147 Pringle, J.R., 116,143, 145,202,244,249 Provost, C., 261,299 Puglisi, P.P., 245, 250 Pugsley, A.P., 261, 262, 299 Pyle, A.L., 285, 296 Pyle, J.E., 155, 193

Q Quay, S.C., 262,269,296 Queiroz, O., 130, 131, 145 Queiroz-Claret, C., 130, 131, 145

R Raczynska-Bojanowska, K., 7,59 Raetz, C.R., 264, 299 Rahn, O., 287,299 Rajender, S., 240, 249

315

Ramgopal, M., 285,286,299 Rampini, C., 266, 296 Rapoport, S., 196, 248 Rasmussen, L., 85, 144 Rast, D.M., 49,50,59, 150, 153, 156, 159, 177, 190, 191, 192 Ratkowsky, D.A., 207,250 Raven, J.A., 180, 181,192 Rehacek, Z., 38,41, 60 Reich, J.G., 130, 146 Reid, B.T., 116, I43 Reinberg, A., 137, 146 Reinbothe, H., 156, 173, 191, 192 Remaley, A.T., 52, 54, 57 Renwick, J.A.A., 46, 58,59 Restivo, F., 245, 250 Reusch, R.N., 49,59 Reynolds, R., 285,296 Ribereau-Gayon, P., 285,297 Richards, O.W., 287,299 Richter, A. von, 171, 193 Rickes, E.I., 255, 294, 296 Rigomier, D., 265, 267, 299 Rines, H.W., 47, 59 Ristow, H., 45, 59 Roberts, M.F., 265, 297 Roberts, N.J., Jr., 196, 250 Rodriguez, R.J., 285, 299 Roeder, P.E., 122, 123, 146 Rogers, P.L., 255, 291,299 Rose, A.H., 22, 59, 196, 202, 225, 233, 242,244,245,248,249,250,251,270, 273, 274, 275,276, 278, 286, 289, 290, 292, 293, 294,296,297,299 Rose, H.M., 261, 279,299 Rosen, B.P., 260, 269,297 Rosenberg, B., 241, 250 Rosenberg, H., 38,47,48,59 Rosenkrauz, H.A., 261,299 Rosenthal, H.A., 196,248 Rosenthal, P.J., 68, 116, 117, 126, 134, 142, 147 Ross, I.K., 38, 59 Rostek, H., 291, 296 Roth, S., 281, 299 Rothwell, A., 182, 189 Rothwell, B., 182, 189 Rottenberg, H., 281,299 Round, F.E., 67,145, 146 Roxburgh, J.M., 172, 192 Rozynov, B.V., 43,58

316

AUTHOR INDEX

Rubin, E., 281, 299 Rubin, R.H., 11 1, 114, 147 Ruczaj, Z., 7, 59 Ruffner, H.P., 156, 177, 192 Russell, D.W., 7, 57 Rutten, H., 164, 191 Rye, R.M., 256,297 S

Sachsenmaier, W., 124, 146 Sa-Correia, I., 225, 226, 250 Sadoff, H.L., 49,59 Sager, R., 81, 146 Saito, H., 46, 56 Sajer, S.A., 151, 191 Sakagami, Y.,43,59 Sakurai, A., 43, 58 Salewski, L., 173, 192 Sallans, H.R., 172, 173, 192 Sami’s, H.V., Jr, 138, 146 Sand, F.E.M.J., 171, 192 Sanders, D., 180, 182, 183, 192 Sargent, M.L., 76, 77, 78, 93, 97, 98, 99, 118, 120, 121, 122, 123, 143, 144, 145, 146 Sasaki, I., 163, 190 Satoh, N., 138, 146 Saunders, D.S., 65, 146 Sawai, K., 51, 59 Sawnor-Korszynska, D., 7,59 Sawyer, W.H., 282,299 Schairer, H.U., 244, 250 Schazschneider, B., 45,59 Scheffer, R.P., 51,58,59 Schenberg-Frascino, A., 202,250 Scher, B.M., 154, 192 Scheving, L.E., 137, 146 Schimz, K.L., 234, 236,250 Schindler, J., 45, 60 Schindlbeck, W., 35, 58 Schliessmann, F., 77, 144 Schmidt, C.L.A., 280,297 Schmid, R., 111,140 Schmitz, K., 177, 192 Schnabel, G., 76, 83, 94, 95, 146 Schnaitman, C.A., 261,262,299 Schneider, H., 164, 191, 281,299 Schrempf, M., 132,142 Schrott, E., 121, 122, 140 Schuldiner, B. 269, 299

Schuytema, E., 155, 193 Schweiger, E., 11 1, 112, 146 Schweiger, G., 11 1, 140 Schweiger, H.G., 65,66,67,111, 112,113, 114, 115, 124, 129, 131, 132, 133, 136, 140, 143,144, 146 Schweiger, M.,66,67, 111, 113, 131, 132, 146 Schwencke, J., 242,250 Scopes, R.K., 289,291,298 Scott, A.I., 20, 21, 57 Scott, K.J., 186, 191 Scott, R.E., 137, 148 Scotti, T., 45, 57 Seddon, B., 46, 59 Seeman, P., 255, 281,299 Sekiya, T., 271, 275, 276, 298 Sekula, B.C., 286, 299 Sel’kov, E.E., 130, 146 Senger, H., 117, 121, 146 Seyffert, P., 35, 58 Shaffer, J.M., 260, 300 Shah, V.K., 158,190 Shalgovskaya, E.M., 243,248 Shaw, G., 50,57 Shaw, M.K., 210,250 Shen, W.K., 68, 116, 117, 126, 134, 142, 147 Shepherd, D., 4 , 5 7 Shepherd, M.G., 153, 158, 189 Sherman, F., 242, 245,250 Sherwood, W.A., 4 5 6 0 Sheys, G.H., 155, 192 Shimoide, M., 275, 300 Shinagawa, E., 185, 192 Shoda, M., 223,248 Shu, P., 172, 192 Shuch, D.J., 76, 81, 83, 85, 97, 135, 144 Shymko, R.M., 85, 124, 129, 137, 144, 146 Siegel, M.R., 237, 250 Sigler, K., 181, 192 Silver, S.A., 243, 250, 262, 269,299 Simeone, J.B., 5, 60 Simbes-Mendes, B., 215, 221, 243, 246, 247,250 Sinclair, C.G., 220, 251 Sinclair, N.A., 197, 242, 243, 250 Sinensky, M., 265, 299 Singer, S.J., 281, 296 Singh, B.N., 40, 46, 60

317

AUTHOR INDEX

Singh, K., 22, 60 Sironval, C., 11 1, 113, 145 Sisler, H.D., 237, 250 Sityta, B., 22, 58 Skofenko, A.A., 243,249 Skotnicki, M.L., 255, 291 299 Skriver, L., 275, 298 Slayman, C.L., 180, 182, 83, 192 Slayman, G.W., 181, 190 Sloan, J., 170, 171, 191 Slocum, W.S., 127, 140 Sloof, W. Ch., 198, 251 Smalley, H.M., 4, 57 Smith, B., 139, 148 Smith,D.C., 150, 163, 177, 184, 185, 188, 191,192 Smith, F.A., 180, 181, 192 Smith, G., 256,300 Smith, G.N., 4, 57 Smith, H.T.B., 85, 146 Smith, J.E., 22, 60 Smith, M. Th., 198, 250 Smith, S.E., 177, 192 Soifer, V.S., 43, 58 Sols, A., 171, 174, 175,190,225,249,289, 290,298 Sondheimer, E., 5,60 Sonnenborn, D., 74, 146 Sonnenborn, T.M., 74,146 Souza, K.A., 244,249 Sova, C., 278, 287,288, 290,297 Spencer, D.M., 172, 192 Spencer, J.F.T., 172, 173, 189, 192 Spencer-Martins, I., 215, 216, 221, 222, 25 1 Speth, J.L., 179, 192 Sprague, G.F., 175, 192 Spudich, J., 81, 146 Spurgeon, S.L., 22,59 Sreenivasan, A., 26 1, 300 Staffeld, G.D., 51,58 Stahly, D.P., 45, 60 Stanley, P.E., 200, 251 Staples, R.C., 46, 58, 59 Statzel, A., 197, 249 Stearn, A.E., 203, 204,251 Steele, J.A., 7, 60 Steffen, H., 289,296 Steinkraus, K.H., 223,225,249,250,278, 279,298 Stephens, K., 45,60

Sternberg, H., 75,76, 81,83,90, 126, 142 Stewart, G.G., 279, 285, 290, 299 Steyn, P.S., 20, 21, 60 Stickings, C.E., 176, 189 Stier, T.J.B., 285, 296 Stock, J.J., 163, 191 Stetler, D.A., 243, 251 Stoffel, W., 244,250 Stokes, H.W., 255, 299 Stokes, J.L., 196, 197, 249, 251 Stoop, J.K., 35,60 Stoops, J.K., 294,299 Storck, R., 271, 299 Straley, S.C., 109, 146 Strandberg, G.W., 152, 156, 157, 158, 163, 192 Straune, M., 151, 191 Streffer, C., 196,251 Strehaiano, P., 225, 251 Strobel, G.A., 156, 192 Strominger, J.L., 259, 260,296 Sturgeon, J.A., 257,299 Stussi, H., 49, 50, 59 Subik, J., 245, 251 Suda, M., 65, 146 Sugden, D.A., 270,299 Sullivan, K.H., 262, 263, 268, 299 Sullivan, P.A., 153, 158, 189 Sulzman, F.M., 65,76,77,78,83,99, 100, 101, 102, 104, 105, 106, 115, 131, 132, 136, 142, 144,145,146 Supanwong, K., 265,298 Sussman, M., 45, 60 Suzuki, A., 43, 59 Suzuki, T., 154, 161, 190, 192 Swain, T., 5, 60 Swait, J.C., 182, 189 Sweeley, C.C., 51, 58 Sweeney, B.M., 65, 66, 67, 88, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 111, 112, 113, 114, 115, 123, 125, 126, 128, 129, 131, 136, 140, 141, 143, 145, 146, 147, 148 Swings, J., 290, 294, 299 Switzer, R.C., 241, 250 Szabo, G., 45,46,57,60 Szyszko, A.H., 69, 147

T Taber, W.A., 7,57, 176, 193

318

AUTHOR INDEX

Takahashi, N., 43,58 Takashi, Y., 261,300 Tamura, S., 43, 58, 59 Tanaka, Y., 46,56 Tanaka, S., 152, 158, 162, 193 Taneja, R., 265,267,299 Tang, C.T., 266,298 Tani, Y., 271, 272, 275, 300 Tatum, E.L., 168, 189 Tay, D.E., 83, 85, 86, 97, 133, 142 Taylor, F.R., 285, 299 Taylor, K.E., 155, 190 Taylor, W., 87, 101, 102, 104, 105, 107, 123, 141, 147 Tedder, T.F., 268,269, 270,296 Tempest, D.W., 11, 59 Terborgh, J., 67, 108, 111, 143, 147 Tereda, O., 159, 170, 192 Terenzi, H.F., 271, 299 Terry, O.W., 70,75,76, 77,80,83,88,96, 109, 142, 147 Tessier, P., 176, 189 Thomas, D.S., 225, 251, 273, 274, 276, 279,286,292,299 Thompson, G.A., Jr., 271,272,275,276, 298 Thompson, J., 269,299 Thomson, R.H., 50,60 Thompson, R.O., 51,60 Thulborn, K.R., 282, 299 Tilley, F.W., 260, 300 Tobler, H., 156, 177, 192 Todosiychuk, S.R., 243, 249 Tom, G.D., 170, 171,192 Tomochika, K., 245,250 Tomoeda, M., 152, 154, 164, 190 Tomoyeda, M., 163, 190, 192 Topiwala, H., 220, 251 Tornabene, T.G., 265,300 Tovarova, I.I., 43, 58 Traverso-Rueda, S., 285, 300 Treick, R.W., 260, 300 Trevelyan, W.E., 160, 192 Tribe, D.E., 255, 291, 299 Tribhuwan, R.G., 261,300 Troke, P.F., 171, 192 Tropp, B.E., 266, 298 Troyer, J.R., 287, 300 Trucco, E.,71,73, 110, 130, 132, 133,142 Tsao, G.T., 165, 190 Tsuchiya, E., 43, 58

Tuffli,C.F., Jr., 111, 114, 147 Tunblad-Johansson, I., 154, 171, 189 Turian, G., 38, 41, 60, 270, 271, 300 Turner, W.B., 20, 21, 60 Tyagi, R.D., 225,249

U Uchida, K., 255, 265, 266, 292, 293, 300 Uchiyama, M., 46, 56 Uden, N. van, 197, 198, 199, 200, 201, 205, 208, 209, 210, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 224, 225, 226, 227, 228, 229, 230, 23 1, 232, 233, 234, 235, 236, 237, 238,239, 241, 243, 244, 245, 246, 247,248, 249,250, 251,272, 273,277,278,298,300 Ueng, P.P., 165, 190 Ueng, S.T.-H., 154, 158, 160, 192 Uhl, A., 234, 251 Ulaszewski, S., 117, 134, 147 Ulmer, D.C., 256,300 Umeda, K., 151,190 Uwajima, T., 159, 170, 192

V Vanden Driessche, T., 66, 67, 11 1, 112, 113, 114, 130, 147 Van der Baan, J.L., 155, 190 Vantk, Z., 7, 22, 38, 41, 58, 60 Varenne, S., 261, 298 Veghelyi, P., 261, 298 Veidi, A., 152, 161, 162, 163, 185, 190 Veiga, L.A., 152, 154, 163, 164, 189, 192, 193 Venzina, C., 22, 60 Vernon, C.A., 241,248 Vidal-Leiria, M., 198, 199, 208, 221,222, 224,241, 244,251 Villanueva, J.R., 54, 58 Vinina, L.C., 7, 59. 176. 193 Viswaiath-Reddy, M., 155, 170, 171, 192. 193 Vitalis, S., 45, 60 Vogel, H., 240, 241, 244, 248 Volm, M., 74, 126,147 Vreeland, N.S., 259, 280, 282, 283, 292, 29 7

319

AUTHOR INDEX

W Wada, C., 261, 269,300 Wagner, E., 130, 147, 148 Wakil, S.J., 35, 60, 294, 299 Wallraff, H.G., 11 I , 112, 146 Walsh, R.M., 198, 251 Walt, J.P., van der, 171, 192, 199, 251 Walter, H., 32, 57 Walther, W.G., 76, 77, 78,83, 96, 98,99, 142, 148 Walz, B., 101, 107, 123, 148 Wang, D.I.C., 10, 60 Wang, S.-Y.C., 152, 153, 158, 161, 163, 175, 193 Warden, K.A., 39,57 Waring, A., 281, 299 Watson, J.A., 155, 192, 193 Watson, K., 168, 193, 197, 245,248,251 Weber, D.J., 51, 58 Weber, K., 261,297 Webster, J., 38, 45, 58 Wehrli, W., 22, 58 Weiler, C.S., 67, 83, 84, 126, 139, 148 Weinbaum, G., 51,56 Weinberg, E.D., 36, 54, 60 Weinstein, L., 7, 60 Weintraub, W., 47, 58 Weiss, C.V., 121, 122, 140 Welker, N.E., 244,251 Wendt, L., 262, 269,299 Weppner, W.A., 258,260,298 Wever, R., 65, 148 White, D., 45, 60 Whitt, J.T., 225, 250 Whittaker, R.H., 5, 38, 60 Wickerham, L.J., 197,251 Widell, S., 121, 122, 140 Widelitz, R., 121, 122, 142 Wieland, F., 35, 58 Wieland, O., 158, 189 Wilke, C.R., 225, 248 Wilkie, D., 270, 300 Wille, J., 68, 71, 130, 139 Wille, J.J., 66, 67, 68, 69, 70, 71, 74, 126,

127, 128, 130, 137,139. 142.. 147., 148 Wille, J.J., Jr., 66, 67,68,69, 74, 100, 124, 126, 137, 138, 139,148 Williams, R.L., 255, 299 Williamson, V.M., 270, 300 Wilson, J.R.J., 256, 300 Winfree, A.T., 66, 87, 88, 124, 130, 136, 148 Wolf, J.C., 44, 60 Wong, D., 99, 101, 108, 143, 147 Wood, T.H., 201,242,251 Wood, W.A., 173,190 Woods, R.A., 279,297 Woodward, J.R., 68, 116, 117, 118, 120, 121, 122, 126, 134,142,146, 147, 148 Wright, J.R., 152, 158, 174, 189, 193 Wriston, J.C., Jr., 156, 190 Wu, N.M., 280,282,297 Wyllie, T.D., 51, 60

Y Yaacabi, M., 281,300 Yall, I., 243, 250 Yamada, H., 152, 158, 162,193 Yamashira, K., 271, 272, 275, 300 Yarrow, D., 198,250 Ycas, M., 242, 245,251 Yoder, O.C., 51,60 Young, E.T., 210,300 Young, I.G., 38, 47, 48,59 Yura, T., 269,300 Z

Zahner, H., 5, 38, 40, 46, 47, 48, 58, 60 Zakharov, I.A., 247,248,251 Zandern, Z., 45,60 Zemlyanukhina, O.A., 262,300 Zennaro, E., 245,250 Zeuthen, E.G., 85,144,148 Zichal, K.E., 68, 70, 71, 142 Zorn, G.A., 139, 148 Zusy, F.D., 67, 145

Subject Index A Absidia glauca, 160 mannitol dehydrogenase, 154, 155, 157 (table) mannitol kinase, 158 Acetabularia spp., circadian oscillator, 114 cycloheximide action on circadian rhythm, 114-15 polypeptide P39 (polypeptide X), 1 15 puromycin action on circadian rhythm, 114, 114-15 Acetabularia cremulata, 1 10, 1 12 Acetabularia major, 112 Acetabularia mediterranea, 11G15 chloroplasts, 11 1 circadian rhythms, 111 (table) biochemical aspects, 1 13-1 5 photosynthesis, 11 1-13 physiological, 11 1-13 electric potential rhythm in cells, 113 Acetaldehyde, 106, 261 Acetaldehyde, effect of on rhythms of Euglena gracilis, 106 Acetate, metabolites derived from, 17 Acetone, 107 Acetylcholine, 9-10 Acetyl-CoA, 11-12, 30 Acetylenes, 24 Acetylphloroglucinol, 30 Achyla genus, sex hormones, 41-2 Acrolein, 270 Actinomucor elegans, 159 Actinomycin D action on A . crenulata, A . mediterranea, 113 glow rhythm delay/suppression, 107 Adaptation, 61-2 Adenosine triphosphate, 36-8 circadian rhythm in Tetrahymena spp., 72 Aerobactin, 48 (table)

A-€actor, 43 Aflatoxins, 5 1 Agaricus bisporus, 177-8 glucose uptake, 184 hexose source, 183 mannitol dehydrogenase, 155 (table), 156 sporophore, 184 trehalose, 176, 177 Agaricus campestris, 155 (table), 156 Ageing, cell cycles clocks in, 138-9 Aggregation of cells, 36 cell-cell recognition, 36 Agmenellum quadruplicatum, alcohol effect, 257, 258 Alanine dehydrogenase, 99-1 00 Alcohols, 254 aliphatic, 2 6 6 7 allylic, 261 chain length, 262 phase shifts and, 106 effects on eukaryotic micro-organisms, 270-80 ethanol-induced leakage and excretion in yeasts, 278-80 growth/morphogenesis, 27G2 lipid composition/biosynthesis,274-7 membrane leakage, 274 thermal tolerance in yeasts, 277-8 transport systems, 2 7 2 4 effects on maltose transport, 273 effects on membrane organization, 28G2 effects on micro-organisms, 253-300 effects on prokaryotic microorganisms, 255-70 biosynthesis of outer membrane and secreted proteins, 261-2 cell morphology, 257 Alcohols effects on prokaryotic microorganisms, 255

320

SUBJECT INDEX

Alcohols (cont.) fatty acid composition/biosynthesis, 262-6 growth/survival, 255-7 macromolecular synthesis, 26& 1 membrane-bound enzymes and transport systems, 268 membrane leakage, 269-70 peptidoglycan synthesis, 257-60 phospholipid composition/biosynthesis, 2 6 6 7 fermentation inhibition, 254-5 fermentation prolongation, 255 hydrocarbon tail, 280 hydroxyl function, 280 localization in membranes, 282 long chain, 262-3, 280 monohydric, inhibitory effects, 288 phospholipid synthesis, 267 polyhydric, inhibitory actions, 288 short chain, 262, 280 tolerance and increased membrane sterol content, 254 toxicity, 256 Aldehyde dehydrogenase, 170 Alditol dehydrogenase (NADP+), 153-4 Algae circadian rhythms, 67-8 (table), 74-1 15 hatching, 126 (table) n-Alkane, yeasts grown on, 161 Alkanols, 232-3, 277-8 Alkyl resorcinols, 48-9 Allelochemicals, 5 Allomones, 5 Allomyces, sex hormones in, 41 Allomyces arbusculi, 8,9 Ally1 alcohol (propenol), 261 Alternaria alternata, 161-3 mannitol cycle, 162 Alternariolide, 51 Amatoxins, 51 , Amethopterin, clock function (glow rhythm), 107 Amillaria mellea, 116 Amino acids, metabolism, 12 metabolites derived from, 19 u-Aminoisobutyric acid uptake in marine pseudomonads, phenylethyl effect, 269 y-Aminoisobutyric acid, 274

32 I

2-Amin0-4-methylpyramidine, 92 Anisomycin, 107 Antheridol, 41-2 Anthranilate, 19 Antibiotics aminocyclitol, 25 aminoglycoside, 46 peptide, 25 Antichaeotropic salts, 260 Antiluciferase, 104 L-Arabinose, 154 L-Arabinose ketol-isomerase, 163 Arabitol in Debaryomyces hansenii, 172 in Dendryphiella salina, 165, 166 increase with medium osmotic potential, 169 in Mastigomycotina, 159 in Serpula lacrimans, 177 synthesis, 166, 179 trehalose converted into, 177 D-Arabitol, 172 L-Arabitol, 154 Aranotins, 21 (table) Arsenate, 105 Ascomycetes, mannitol cycle in, 163 Ascomycotina, 16&1 Aspartate carbamoyltransferase, 10 Aspergillic acids, 21 (table), 25 Aspergillus candidus, mannitol cycle, 163 mannitol dehydrogenase in, 155 (table), 156 mannitol 1-phosphatase in, 158 mannitol I-phosphate in, 152, 153 (table) Aspergillus japonicus, 159 Aspergillus niger, 152, 153 (table), 154, 158 citric acid production, 11 mannitol cycle, 162 Aspergillus oryzae, high concentration alcohol-producing factor, 284 mannitol dehydrogenase (NAD+) in, 1567, 157 (table) mannitol dehydrogenase (NADP+), 154, 155 (table) Aspergillus parasiticus, 155, 156 molecular weight, 157 Asperphenamate, 21 (table), 55

322

SUBJECT INDEX

Astronomical time measurement, 62 Austamides, 20 (table), 21 (table) Auto-inhibition (self-inhibition), 44 Azotobacter vinelandii, alkyl resorcinols, 48-9

B Bacillaceae, dipicolinic acid and calcium salt in, 46 (table) B a c i l h brevis, 45 Bacillus cereus, 265, 267 Bacillus subtilis alcohol effect on, 257 ethanol effect on, 267 ethanol-resistant mutants, 295 fatty-acyl residues, 265 Bacillus thuringiensis, 45 (table) Bacitracin, 7 Bacteria amorphous structural components, 49-50 aromatic amino acids and, 2Cb1 extracellular defense and operations functions, 38 (table), 40 Bassiamin, 20 (table) Basidiomycotina, polyol metabolism, 176-80 Benzyl alcohol, bacterial growth toxicity, 256 Bethanol, 261 Bioconversion, 10 Biological clock, 65 Biomembranes, 244-8 Biopolymers, 10 Biotransformation, 10 Blue light, 117 photoreceptors, 121 Borneol, 27 Botrytis cinerea, 162 Bovinone, 20 (table) Brevianamides, 20 (table), 21 (table) A and B, 54 Butanol, 227, 230 (table), 263 Butan-1-01, 287 Butan-2-01, 287 Butenol, 261 Butyraldehyde, 106 C

Calcium chloride, 105

Caldariomycin, 25 Campesterol, 9 Cancer research, chronobiology, 137-8 Candicidin, 54 Candida sp., 241 (table) grown on n-alkane, 161 T,,, value, 198 Candida albicans, associative temperature profile, 215, 216 phenethyl alcohol effect, 271 polyunsaturated fatty acids, 245 thermal death, 209 (fig) Candida bovina, 197 Candida curiosa, 221, 222 (fig), 223, 224 (fig) Candidafrigida, 209 (fig) Candida kefyr, 2 15 Candida (Pichia) guilliermondii, 1 1 3 4 ribitol 5-phosphate dehydrogenase in, 156 Candida parapsilosis, 245 glycerol oxidase in, 159 Candida lusitaniae, associative temperature profile, 2 15 Candida macedoniensis, associative temperature profile, 21 5 temperature range, 197 (fig) Candida marina, thermal death, 209 (fig) Candida mycoderma, 158 Candida nivalis, 241 (table) thermal death, 209 (fig) Candida parasilosis, 245 Candida sakP, 198 Candida sloo&, 245 temperature range, 197 (fig) Candida tropicalis, 161, 242 (table) Candida utilis, carbohydrate metabolism pathways, 164 (fig) circadian rhythms, 126 (table) glycerol metabolism, 171 glycerophosphate dehydrogenase, 157 lipid composition changes, 245 mannitol cycle not present, 162 pentose sugar utilization, 163-5 polyol dehydrogenase in, I54 supraoptimal/supramaximal temperature effects, 242 (table) thermal death, 209 (fig) xylitol dehydrogenase in, 152 Candida zeylanoides, 161

SUBJECT INDEX

Carbonic anhydrase, 98 Carbonylcyanide m-chlorophenylhydrazone, 106, 262 Cardiolipin, 266, 267 Carlosic acid, 20 (table), 23 Carotenoids, 20, 27, 50 Catechol, 50 Celestial orientation, 62 Cell-cell recognition, 36 Cellkycle clocks: ultradian, circadian, infradian interfaces, 124-9 Cephalosporin(s), 7, 21 (table), 25 Cephalosporin C, 25 Cephalosporium chrysogenum, 152, 155 (table) atomic weight, 157 Ceratium furca, circadian rhythms, 126 (table) Ceratocystis multiarmulata, 163 Cetoleic acid, 286 Chaeotropic salts, 260 Chaetomium globosum, 161 glycerol production increase, 171 mannitol dehydrogenase (NAD+) in, 154, 157 (table) Chinese hamster lung fibroblasts, 88 Chlarnydomonas reinhardii, 95 circadian rhythms, 109-10, 126 (table) metabolic deficiency strains, 110 Chloramphenicol, 238, 239 (fig) action on A . mediterranea, 113-14, 114 glow rhythm and, 107 Streptomyces strain produced, 7 Chlorella pyrenoidosa, circadian rhythms, 126 p-Chloromercuribenzoate, 105 Chlorophenyl- I, 1-dimethylurea, 105 Chlorophyll, 9 Cholesterol, 9 Chromobacterium violaceum, 46 (table) Chronogenes, 133 Chronopharmacology, 136-8 Chronotherapy, 136-8 Chrysosporium fastidium, 187 Circadian clocks see also Circadian rhythms cell-cycles clocks in development/ageing, 138-9 ultradian, circadian, infradian interfaces, 124-9 cell-division cycle control, 75-91

323

chronogenes, 133 circadian oscillators interaction with cell division cycle, 125-6 coupled oscillators, 136 cytochron, 91, 133 insertions/deletions of time segments into cell cycles, 133 intracellular clock shops, 135 life cycle clock, 138 molecular models, 129-33 feedback-loop (“network”), 130 (table), 131 membrane, 131 (table), 132 molecular, 129-3 1 transcriptional (“tape reading”), 110, 132 multiple cellular oscillators, 135-6 nature, 129-33 Sacch. cerevisiae, 1 15 see also circadian rhythms Circadian rhythms, 61-148 see also Circadian clocks A . mediterranea, 1 10- 15 acrophase chart, 75, 79 (fig) Aschoff s rules, 64 (fig) cell-division cycle control, 73 cell motility, 91-6 cell settling rhythm, 96 dark mobility rhythm, 93-5 phototaxis rhythm, 91-3 chronopharmacoIogy/chronotherapy, 136-8 chronotypic death, 70 circadian-infradian rule, 69, 72, 126-8 conditional arrhythmicity, 103 critical pulse, 87-8 cytochron, 91 damping out, 83-4 energy reserve escapement mechanism, 72 entrainability, 64 (fig), 80-1 Euglena gracilis see Euglena gracilis evolution, 134-5 free-running, of cell division, 8 1-3 fungi, 68 (table), 115-24 gating, 125 genetic basis, 64 (fig) G-E-T effect, 74-5 glucose 6-phosphate effect, 95 Gymnodinium splendens, 126 hatching, 125, 126 (table)

324

SUBJECT INDEX

Circadian rhythms (cont.) Hymenomonas carterae, 126 (table) initiation, 83-4 intercellular communication, 136 ion transport across membranes in limit-cycle membrane model of clock, 106 membrane involvement, 106 modulation of free-running period by light intensity, 64 (fig) Olisthodiscus sp., 126 (table) organization in micro-organisms, 66 oscillatory enzymic activities, 99-100 Paramecium aurelia, 73-4 Paramecium bursaria, 73, 74, 126 (table) Paramecium multimicronucleatum, 74, 126 (table) persistence, 64 (fig), 81-3 phase perturbation, 85 phase-shiftability, 64 (fig), 84-7 photosynthetic capacity, 96-9 phototactic response, 91 Pittendrigh’s theory, 85 properties, 63-6 Prorocentrum sp., 126 (table) protozoa, 66-74 Pyramimonas sp., 126 (table) quanta1 cell cycles, 128-9 Sacch. cereuisiae, 126 (table) secondary arrhythmicity, 84 singularity point, 87-8 Skeletonema costatum, 126 (table) skeleton photoperiods, 80 temperature-compensation, 64 (fig), 88-9 Tetrahymena spp., 72 Tetrahymena pyriformis, 68-9, 71, 126 (table) time cues (Zeitgeber), 64, 80, 8 1 time domains, 63 (fig) tyrosine aminotransferase activity, 73 urea effect, 95 Circinella sp., 159 Circinella naumorii, 159 Citric acid, 11, 23 Cladosporium cladosporoides, I 62 Claviceps purpurea, 176 Clostridium acetobutylicum, 257 alcohols effect on ATPase, 269 ethanol effects, 267

Clostridium botulinum, toxin, 5 1 Clostridium thermocellum, 257, 265 Cobalamin, 39 Compound A23187, 106 Coprinus spp., polyol deficient sporophores, 184 Coprinus cummatus, 159 Coprinus heptemerus, principle of, 45 (table) Coprogen, 48 (table) Corynebacterium diphtheriae, toxin, 5 1 Coupled oscillators, 136 Cryptococcus sp., 241 (table) Cryptococcus albidus, 242 (table) Cryptococcus difluens, 242 (table) Cryprococcus neoformans, 221, 22 1-3, 224 (fig) “Crytomycin”, 20 (table) Cyanide, 105 Cyclic AMP (adenosine 3’,5’-monophosphate), 44 circadian clock function, N. crassa, 121 circadian rhythm in Tetrahymena spp., 72 Cycloartenol, 13, 18 (fig) Cycloheximide, 93, 178, 179, 237, 238 (tables) action on Acetabularia sp., 114, 114-15 G. polyedra pulsed with, 107 Cyclopiazonic acid, 20 (table) Cyniclomyces guttulatus, 197 (fig), 197 Cytochalasins, 20 (table) Cytochron, 91, 133

D Dactylorchis purpurella, 177 Darwin, C., 35 Debaryomyces hansenii, 172, 197 (fig) Decanol, 263 Decyl citrate, 23 Decylcitric acid, 20 (table) Dendryphiellasalina, 161, 165-9, 179, 183 arabitol, 165, 167 (fig), 178 erythritol, 165 glucose uptake, 184 glycerol, 165, 169 growth and pH increase, 182 mannitol, 165, 166 (fig) 3-O-methylglucose, 168, 169

SUBJECT INDEX

Dendryphiella salina (cont.) pentose phosphate pathway, 166, 168, 178 sorbitol bypass, 186 L-sorbose, 168, 169 Deoxyribonucleic acid (DNA), circadian synthesis, 71 Depsipeptides, 25 heavy water), Deuterium oxide (ZH~O, 92-3, 105 Deuteromycotina, 160-1 Development, cell-cycle clocks in, 138-9 a, y-Diaminobutyrate, 19 2,6-Diaminopurine sulphate, 92 10 pM Dichloromethylurea, 109 Dichlorophenyl-1,l-dimethylurea,105 Dictyostelium discoidium, 45 (table) Dictyuchus monosporus, 45 (table) Dihydroxyacetone kinase, 171 5,6-Dihydroxyindole, 49 1,8-Dihydroxynaphthalene,50 3,5-Dihydroxyphenylaceticacid, 30 3,4Dihydroxyphenylalanine (dopa), 49, 50

Diketopiperazines, modified, 25 Dionethyl suphoxide, 107, 261 2,CDinitrophenol (DNP), 105 Diphenylbenzoquinones, 23 Dipicolinic acid, 46 (table) calcium salt, 46 (table) Diptodia viticola, 155 (table) Discadenine, 44 Disferal (ferrioxamine), 48 (table) Diterpenes, 27, 28 DNA polymerase 111, 261 Dopa, 49,50 Drechselera halodes, 45 (table) Drosophila spp., 105 Drug courses, sinusoidally varying, 137

E Echinulin, 20 (table), 21 (table), 25 Elaidic acid, 284 Embden-Meyerhof pathway, 287,289 Emeliania huxleyi, circadian rhythms, 126 Enniatin, 7 Enniatin B, 25 Enterochelin (enterobactin), 48 (table)

325

Entner-Doudoroff pathway, 290, 291, 294 Ergosterol, 9, 286 synthesis as evolutionary trait, 293 in yeast membranes, 275 Ergot alkaloids, 7 Erythritol, 172, 176, 179 activity value, 154 L-Erythritol (threitol), 176 Erythritol dehydrogenase, NAD+ dependent, 179 D-Erythrose, 154 Erythroskyrin, 20 (table) Escherichia coli alcohols (short-chain) effect on peptidoglycan synthesis, 263 ally1 alcohol-resistant mutants, 261 bethanol effects on, 261 ethanol as fermentation product, 294 ethanol effect on, 255, 256 (fig), 256 cell morphology changes, 257 fatty-acyl composition, 264 membrane functions, 268 ethanol-resistant mutants, 295 fatty-acyl composition, 262, 263, (fig), 264,294 manipulation by lipid mutants and fatty-acid supplements, 282-4 hexanol effect on fatty-acyl composition, 264 lactose permease system, 268 ethanol effects, 268 lipid supplements on alcohol tolerance, growth, survival, fermentation, 282-4 lipid synthesis, 294 long-/short-chain alcohols effect on fatty-acyl composition, 280 non-synthesizing vaccenic acid mutants, 283 phenethyl alcohol effect on biosynthesis of outer membrane and secreted proteins, 261-2 on membrane leakage, 269 phenethyl alcohol-resistant mutants, 269 relative rate of multiplication as function of temperature, 207 (fig) Ethanol alcohol tolerance value, 287 central nervous system depression, 254

326

SUBJECT INDEX

Ethanol (cont.) effect on temperature profile of yeasts, 223-33 Embden-Meyerhof pathway, 287, 289 experimental measurement of intracellular concentrations, 279-80 glucose utilization inhibition in Z. mobilis, 29 1 glycolytic enzyme inhibition, 289-90 G . polyedra exposed to, 106 growth-inhibiting properties, 287 hydrocarbon tail, 292 mechanism of inhibition of fermentation, 287-91 membrane interactions, 283 (fig), 293 (fig) microbial fermentation production, 254 octane enhancer, 254 Sacch. cerevisiae: effects on, 223-33 temperature profile shift, 2 4 6 7 thermal death, 230 (table) staling effect on fermentation, 255 Ethylenediaminetetra-acetate (EDTA), 105 Euglena spp. acrophase chart, 79 (fig) circadian rhythms, 74-100 Euglena gracilis, 74-5 circadian rhythms, 75-126 alcohols and mobility rhythm, 106 biochemical, 77-8 (table) cell-division cycle control, 75-91 cell motility, 91-6 cell settling rhythm, 96 chlorophyl content/photosynthetic capacity, 97, 98 dark mobility rhythm, 93-5 Dunkelbeweglichkeit, 93-5 entrainability, 80-1 enzymic variation/photosynthetic capacity, 98 gating, 126 G-E-T effect, 7 4 5 hatching, 126 (table) initiation, 8 3 4 nutritional change effects, 93, 94-5 oscillatory enzymic activities, 99-100 persistence, 81-3 phase-shiftability, 84-7 photosynthetic capacity, 96-9 photosystems I, II/photosynthetic

capacity, 98-9 phototaxis rhythm, 91-3 physiological, 7 6 7 (table) singularity point, 87-8 temperature compensation, 88-9 1 photosynthetic mutants, 80-1, 83 P4ZUL mutant, 81, 83, 88, 90 temporal differentiation, 75 vitamin BIZdeprivation effect, 90 wild-type (Z strain), 78 (table, n.), 80 WbZHL mutant, 83 Eukaryotes, unicellular, sterol composition modification, 254 Eukaryotic cell clock, 72

F Factor C, 45 (table) Fatty acids, structural requirements for increased ethanol tolerance, 284 Fatty-acyl chain length and ethanol resistance, 286 in yeast membranes, 275 Ferric chloride, 105 Ferrichromes, 48 (table) Ferrioxamine (disferal), 48 (table) Fibroblasts, 139 replication capacity, 138 Flammulina velutipes, 177 Flavin adenine dinucleotide, 157 Fluorodeoxyuridine (FUdR), 105 p-Fluoro-DL-phenylalanine,178 Formaldehyde, 106 Fructose, 160 Fructose 6-phosphate, 160, 161, 174 Fumagillin, 27 Functional biochronometry, 62 Fungi amorphous structural components, 49-50 aromatic amino acids and, 20-1, 21 (table) circadian rhythms, 68 (table), 115-24 coprophilous, principle of, 45 (table) external pH effect on internal pH, 182 extracellular defence and operations functions, 38 (table), 40 glycerol metabolism, 170 (fig) hatching, 126 osmotolerant, 171

321

SUBJECT INDEX

Fungi (cont.) osmotophilic, 171 osmototrophic, 171 polyol-deficient, 184 polyol metabolism, 149-93 regulation of cytoplasmic pH values, 18M spore germination stimulators, 47 Fusaric acid, 20 (table) Fusarium roseum, 44

G Galactilol, activity value, 154 D-Galactose, 154 Geldanomycin, 20 (table) Gene-action clock mechanism, 72 General metabolism, 5 Genes ageing, 138 cell clock longevity, 138 Geosmin, 20,27 Geotrichium candidum, 151, 187 G-E-T effect, 7 4 5 Gibberella fujikuroi, 182 Gibberella zeae, 44 Gibberella zene, 163 Gibberellic acid, 27, 29, 92 Gibberellin, 105 Gliotoxin, 21 (table) Glucitol, 177 activity value, 154 bypass, 186-7 Glucitol (sorbitol) dehydrogenase (NAD+), 158 Glucokinase, 291 Gluconobacter suboxydans, 185 Glucose 6-phosphate, 39 Glucose 6-phosphate dehydrogenase, 99, 161 1-(4-Glutaminyl)-3,4-dihydroxybenzene, 50 Glyceraldehyde, 12 D,L-Glyceraldehyde, 154 Glyceraldehyde kinase, 170 Glyceraldehyde phosphate dehydrogenase, 289 Glyceraldehyde 3-phosphate dehydrogenase, 98

Glycerol activity value, 154 metabolism in fungi, 170 (fig) synthesis, 171-2 Glycerol dehydrogenase (NAD+), 151, 170 Glycerol dehydrogenase (NADP+), 155-6 Glycerol kinase (glycerokinase), 157-8, 170-1 Glycerol oxidase, 159 Glycerol 3-phosphate dehydrogenase (NAD+), 151, 17&1 sn-Glycerol3-phosphate, 266 Glycerophosphate dehydrogenase, 157 Gonyaulax polyedra, 99, 100-9 acid-stimulated bioluminescence, 103 bioluminescence rhythms, 1 W 7 aliphatic aldehydes effect, 106 biochemical aspects, 1 0 4 7 conditional arrhythmicity, 103 induced flashing rhythm, 100 physiological characteristics, 1 O M spontaneous glow rhythm, 100, 101-2 stimulated bioluminescence, 101 (table), 102 transducing mechanisms, 104 circadian rhythms, 101 (table), hatching, 126 (table) cycloheximide effect, 107 ethanol pulse, 106 fluorescence transients, 108 inhibitors of macromolecular synthesis, clock mechanism effect, 107 photosynthesis rhythms, 107-9 photosynthetic capacity, 108 transducing mechanisms, 108 Gramicidin, 106 Gramicidin S , 25, 46 Gram-positive bacteria, fatty-acyl residues, 265 Griseofulvin, 4, 6 (table) Guanosine tetraphosphate, 261 Gymnodinium splendens, circadian rhythms, 126

H Hansenula sp., T,,, value, 198 Hansenula polymorpha, thermal death, 209 (fig)

328

SUBJECT INDEX

HC-toxin, 51 Heavy water (2H~O), 92-3, 105 Helminthosporal, 27 Helvolic acid, 23 Hexanol, 259 effect on fatty-acyl composition of E. coli, 264 hydrocarbon tail, 280, 292 membrane interactions, 283 (fig), 293 (fig) Hexokinase, 161, 289 Hispidin, 20 (table), 50 Homoeoviscous adaptation, 244 Host tolerance, 137 Hyalodendrins, 21 (table) Hydroxamate formation, 25 3-Hydroxyanthranilic acid, 50 Hymenomonas carterae, circadian rhythms, 126 (table)

Lactose permease, 268 Lanosterol, 13, 18 (fig), 22 Leucosporidium sp., T ,, value, 198 Leucosporidium frigdum, 245 Leucosporidium scottii, 197 (fig) Leucosporidium stokesii, 242 (table) Life cycle clock, 138 Linoleic acid, 286 Lipid metabolism, 11-12 Lipid supplements effects on alcohol tolerance growth, survival, fermentation, 282-6 Lipomyces sp., T,,, value, 198 Lipomyces kononenkoae, 221, 222 (fig), 223 Luciferase, 104 Luciferin, 104 Lysergic acid, 20 (table), 25 Lysophosphatidic acid, 266

I

M Magnesium ions, 39 Malonate, 33 Malonyl-CoA, 11 Maltose transport, alcohols effects on, 273 Mannitol, 165, 169 Absidia glauca growth on, 160 in Agaricus bisporus, 177-8 cycle, 162 (fig), 163 in Pyrenochaeta terrestris, 174 in Pythium debaryanum, 159 in Sclerotinia sclerotiorum, 175 sink for protons, 183 synthesis from glucose, 166 (fig) in Zygomycotina sp., 159 Mannitol dehydrogenase (NAD +), 154-5, 161, 162 Mannitol dehydrogenase (NADP+), 161, 175 D-Mannitol dehydrogenase, 179 Mannitol kinase, 158 Mannitol 1-phosphatase, 158, 161, 162, 175 Mannitol 1-phosphate, 162, 185 Mannitol 1-phosphate dehydrogenase (NAD'), 152-3, 161 Mastigomycotina, 159 Matricaria ester, 24 Melamspora lini, 152, 155, 156

L-Iditol dehydrogenase, 158 Illudin M, 27, 28 Infradian growth mode, physiological rhythms in, 69-70 Intercellular communications, 136 Intracellular clock shops, 135 Iron chelation, 47, 48 (table) Itaconate, 23 Itaconic acid, 19

K Kairomones, 5 Kinetin, 92, 105 Kloeckera africana, 241 (table) Kloeckera apiculata, 289 Kluyveromyces fragilis, 242 (table), 243 (table) ethanol effects on, 225 (fig), 226 temperature range, 197 (fig) L

Lactic dehydrogenase, 99 Lactobacillus heterohiochii, 255-6,265-6, 293 ethanol requirement, 294 Lactobacillus homohiochii, 255, 265-6

SUBJECT INDEX

329

Melanins, 49-50 Mucor javanicus, 155-6 Membrane fluidity, 280 Mucor moelleri, 159 alcohols effect on, 280 Mucor racemosus, 159 Membranes Mucor rouxii, 153 acidic phospholipid increase, 295 phenethyl alcohol effect, 271 alcohols effect on organization, 280-2 phenylpropanol effect, 271 alcohols localization, 282 Mycelianamide, 21 electrostatic interactions, 28 1 Mycobacterium smegmatis, 265, 267 ethanol effects on permeability proper- Mycobacterius, 48 (table) ties, 281 Mycophenolic acid, 7, 55 hydrophobic interactions, 281 Mycosporines, 38-9 involvement in alcohol tolerance, 292-5 non-bilayer conformation stability, 280 phospholipid: protein content, 295 widths of phase transitions, 280 N Meso-erythritol, 161 Metabolites, prefunctional, 40 Nadsonia elongata, temperature range, primary see Primary metabolites 197 (fig) Metal-chelating agents, 47, 48 (table) Nematospora sp., Tmaxvalue, 198 Metal ions, stabilization of polyoxometh- Nematospora coryli, dissociative temylene chains, 32 perature profile, 221 Methanol, 287 Neurospora spp., 117-24 hydrocarbon tail, 292 Neurospora crassa, 118, 170-1 phase shift ability, 106 biochemical mutants, altered clock proMethyl-4-dihydrotrisporate B, 42 perties, 120-4 Methyl cis-3,4-dimethoxycinnamate,46 cel mutant, 121-3 3-O-Methylglucose, 168, 169 clock mutants, 119 (table) 6-Methylhept-5-en-2-one,47 isolation/characterization, 1 18-20 2-Methylisoborneol, 20 cyclic AMP in circadian clock funcMethylmalonate, 33 tion, 121 Methyl cis-3-methoxy-4 hydroxy cinnadihydroxyacetone kinase, 171 mate, 46 drug-resistant mutants, altered clock 6-Methylsalicylate, 35 properties, 1 2 3 4 6-Methylsalicylic acid, 52, 53 (table) glycerol dehydrogenase, 155-6 biosynthesis, 32 glycerol metabolism, 170-1, 185 6-Methylsalicylic acid synthase, 32 glycerophosphate dehydrogenase, 157 Methymycin, 33 internal pH regulation, 180, 182 Metschnikowia sp., T,,, value, 198 mannitol cycle, 163 Mevalonate, 17 mycelial mass doubling, 127 Micro-organisms phenethyl alcohol effect on: mesophilic, 197 glucose and amino-acid uptake, 272 psychrophilic, 197 membrane leakage, 274 thermophilic, 197 mycelial growth/differentiation, Microsporum gypseum, 163 270-1 Mitochondria, 2 4 4 8 proton pump, 181 Mitomycin, 20 (table) Nicotinamide adenine dinucleotide phosMitomycin C, clock function (glow phate, 36-8 rhythm), 107 Nonanal, 47,263 Morphogenesis, hormones involved in, Nonapeptides, 19 43-4 Novobiocin, clock function (glow .Mortierella rammaniana, 159 rhythm), 107

330

SUBJECT INDEX

Nucleic acid bases, 92 Nucleotide metabolism, 12 Nybomycin, 20 (table)

0 Oleic acid, 186, 284 Oligomycin, 123 Oligoprenyl pyrophosphates, 27 Olisthodiscus sp., circadian rhythms, 126 (table) Oogoniol, 41 Oosporidium sp., T ,,, value, 198 Ophiobolin A, 27 Organism, growth/adaptation/reproduction, 36-9 Ornithine, 19 Orsellinic acid, 30 Osmoregulation, 188 Osmotolerant fungi, 171 Osmotophilic fungi, 171 Osmototrophic fungi, 171 Outer-membrane proteins, biosynthesis, 261-2 Overflow metabolism, 11 Oxaloacetate, 23 /3-Oxohexanoate, 23 Oxosuccinate, 39

P P+ prohormone, 42 P- prohormone, 42 Pachysolen tannophilus, 164 Palmitic acid, 4, 5-8 biosynthesis steps, 6 (table) distribution, 5-8 occurrence, 6 (table) physiological role, 6 (table) synthesis through producer’s lifetime, 6 (table) turnover, 6 (table) Palmitoleic acid, 286 Pamamycin, 45 (table) Paraffins (high molecular weights), bioconversion, 216 Paramecium aurelia, 73-4 Paramecium bursaria, 73, 74, 126 (table)

Paramecium multimicronucleatum, 74, 126 (table) Patulin, 35, 52, 53 (table) Paxilline, 20 (table) Pebrolide, 27 Penicillin(s), 2 1 (table) anneleation reaction, 25 Penicillin N, 25 Penicillium brevicompactum, 54-5 Penicillium chrysogenum, 152, 154, 155 (table), 156-7 molecular weight, 157 polyol concentration increase, 187 Penicillium cyclopium, 45 (table) Penicillium frequentans, 162 Penicillium islandicum, 162 Penicillium notatum, 158 Penicillium patulum, 35, 52, 53 (table) 6-methylsalicylic acid production, 5 2 4 Pentanol, 263 Pentan- 1-01, alcohol tolerance value, 287 Pentilol, 164, 175 Pentilol dehydrogenase (NAD+), 176 Pentilol dehydrogenase (NADP+), 176 Pentose metabolism, 163-4, 165 Pentose sugars, utilization by Candida utilis, 163-5 Peptidoglycan synthesis, 257-60 long-/short-chain alcohols effect on, 280 Petroselenic acid, 284 Phenethan-2-01 (phenethyl alcohol), 256, 26C1 Phenethyl alcohol (phenethan-2-01), 256 effect on plasma-membrane phospholipid composition, 266 inhibition of biosynthesis of outermembrane proteins and secreted proteins, 261-2 inhibition of macromolecular synthesis, 2 6 C1 Phenethyl alcohol-resistant mutants, 261 Phenylpyruvic acid, 23 Phenylurethane, 92 Phosphatidylcholine, 284 Phosphatidylethanolamine, 266, 267 Phosphatidylglycerol, 266, 267 Phosphatidylglycerol synthetase, 266 Phosphatidylserine, 266 Phospho-N-acet ylmuram y lpentapeptide translocase, 258

33 1

SUBJECT INDEX

Phosphoglycerate kinase, 289 Phosphoglycerokinase, 29 1 Physiology materials of limited taxonomic distribution role, 39-40 meaning of term, 36-9 Phytophthora cinnamoni, 159 Pichia sp., T,,, value, 198 Pichia guilliermondii, 156, 173-4 Pichia haplophyla, 159 Pichia membranaefaciens, 197 (fig) Pichia miso, 172 Pichia pastori, 215 Pichia quercuum, 154 Plasmalemma proton pump, 95 Pleuromotilin, 27 Polyketide(s), 17, 19-20, 26 secondary metabolism requirements, 30-4 stabilization, 32-3 Polyketide synthases, 32, 3 3 4 Polymerization mechanisms, 13-1 5 Polymers bacterial/fungal, 49-50 status, 10 Polyol(s), 150 enzymes, 150-9 functions, 18S-6, 188 NAD(H)/NADP(H) concentration in fungi, 186 osmoregulatory role, 185, 187-8 physiological buffering agents, 188 synthesis as sink for protons, 184 Polyol dehydrogenase (NADP'), 153-4 Polyoxomethylene chains, stabilization, 32 Polypeptide P39 (polypeptide X), 1 15 Polyporenic acid A, 22 Polyporenic acid C, 22 Polyporic acid, 23 PolysphondyIium violaceurn, 45 (table) Potassium cyanide, 92 Potassium sorbate, 236 Potassium sulphate, 260 Prefunctional metabolites, 40 'renyl pyrophosphates, 17 'rimary metabolism, 3 biochemistry, 11-17 comparison with secondary metabolism, 34-5 canonical forms of reactions on pre-

formed carbon skeletons, 16 (table) non-conforming reactions, 17 (table) carbon-carbon bond-forming reactions, 12-13 condensation reaction, 14 (table) retrocondensation, 14 (table) substitution reaction, 15 (table) polymerization mechanisms, 13-15 Primary metabolites, 3, 9 characteristics, 3, 4 covalently bound, 15 distribution-based criteria, 5 Prodigiosin, 20 (table) Propanol, 227, 230 (table) Propan-2-01, 227, 230 (table) Propenol (ally1 alcohol), 261 Prorocentrum sp., 126 (table) Protein synthesis on 80s cytoplasmic ribosomes, 107 Proton pump, 180-1 Protozoa circadian rhythms, 66-74 hatching, 126 (table) Pseudan, 20 (table) Pseudomonad, marine, phenylethyl effect on a-aminoisobutyric acid uptake, 269 Pseudomonas aeruginosa, 270 Puccinia graminis, sorbitol bypass, 186 Puromycin action on Acetabularia sp., 114-15 action on A . mediterranea, 113-14 glow rhythm delay, 107 Pyramimonas sp., 126 (table) Pyrenochaeta terrestris, 152, 153 (table), 153, 158, 174 Pyricularia oryzae, 152, 153 (table), 158, 162 Pyrocystis fusijormis, 125, 126 (table), 128 4-Pyrone, 30 Pyruvate decarboxylase, 289,29 1 Pythium debaryanum, 159

Q Quiesone, 46

R Redox reactions, 183

332

SUBJECT INDEX

Rhizopus arrhizus, 271 Rhizopus japonicus, 159 Rhizopus oryzae, 159 Rhodosporidium toruloides, 43 Rhodotorucine A, 43 Rhodotorula sp., 155-6 Rhodotorula gracilis, 165 internal pH regulation, 180 Rhythms, persistent, 62 Ribitol, 154, 159 Ribitol 5-phosphate dehydrogenase (NADP+), 156 Ribonucleic acid (RNA), circadian synthesis, 71 transcriptotypes, 71 D-Ribose, 154 L-Ribdose, 163 Ribulose 1,5-biphosphate carboxylase, 98, 108 Rifampicin, action on Acetabularia sp., 114 Rifamycin, 7, 20 (table), 33 Roquefortine, 20 (table), 21 (table) Rosenonolactone, 27, 28 Rubratoxin, 20 (table) Rust uredospores, 47 S

Saccharomyces spp., 1 15-1 7 alcohol resistance, 270, 274 chromophores, 117 [14C]histidineuptake, 116 lipid composition, 274-5 [14C]lysineuptake, 116 transmembrane gradients as energy source for transport systems, 274 Saccharomyces bayanus, 241 (table) dissociative temperature profile, 22 1 Saccharomyces carlsbergensis, 199, 241 (table) associative temperature profile, 21 5 Saccharomyces cerevisiae, 43, 174-5 alcohols effect on: growth and sugar/amino acid uptake in anaerobic conditions, 273 lipid environment of plasma membrane, 272-3 solute accumulation, 272 D-xylose uptake, 272, 273 (fig)

alcohol-sensitive/-resistant mutants, 270 alkanols effects on, 227-33 model, 232-3 associative temperature profile, 210-16 respiration-deficient strain, 215, 216 (fig) chemostat cultures, 217-20 glucose-limited growth, 220 chloramphenicol effects, 238, 239 (fig) circadian clock, 1 15 circadian rhythms, 126 (table) cycloheximide effects, 237, 238 (table) cytoplasmic respiratory deficiency, 270 ergosterol biosynthesis-defective mutant, 286 ethanol effects on, 223-7, 246-7 maximal growth temperature, 277 (fig) ethanol, fermentation products, 294 fatty-acyl membrane composition alteration, 275 glycerol metabolism, 171, 174-5, 185 production, 174-5 glycerol 3-phosphate dehydrogenase (NAD+), 151, 174 glycerol-utilizingdefectivemutants, 175 glycogen production, 160 growth on galactose, anaerobic/aerobic conditions, 172 heat resistance of exponentially growing populations, 202 homoeoviscous adaptation, 244 inhibition of fermentation by alcohols, 288 (fig) inner mitochondria1 membrane, 245, 247 internal pH regulation, 180, 181-2 lipid supplements effects on alcohol tolerance, growth, survival, fermentation, 284-6 lipid synthesis, 294 mechanism of inhibition of fermentation by ethanol, 287-90 mitochondria, 245-6 petite mutation, 241 (table), 245-7 passim, 270 phenethyl alcohol effect, 270 polyol production, 160 proton pump, 181

SUBJECT INDEX

Saccharomyces cerevisiae (cont.) semitheoretical isotherms, specific growth rates/specific death rates, 217-18 sorbic acid effect, 23C7 staling effect, 287-90 sulphur dioxide-induced death, 234-6 supraoptimal/supramaximal temperature effects, 241 (table), 242 (table) survival plots at various temperatures, 201 (fig) thermal death, 209 (fig) thermal tolerance, 277 transport regulation, 115 Saccharomyces chevalieri, 241 (table) associative temperature profile, 2 15 Saccharomyces fermentati, 241 (table) Saccharomyces~orentinus,241 (table) Saccharomyces heterogenicus, 241 (table) Saccharomyces italicus, 215, 241 (table) Saccharomyces kloeckerianus, 22 1 Saccharomyces mellis, 197 (fig) Saccharomyces pastorianus, 241 (table) Saccharomyces pombe, 171 Saccharomyces rosei, 241 (table) Saccharomyces rouxii, 172 arabitol production, 173 glycerol synthesis, 172 growth in absence/presence NaCI, high concentrations, 172 polyol production, 172 ribose production, 173 Saccharomyces sake, 271 Saccharomyces steineri, 241 (table) Saccharomyces telluris, 245 Saccharomyces telluster, 197 Saccharomyces uvarum, 199 Saccharomyces veronae, 241 (table) Sake production, 284-5 Salmonella typhimurium, 26 1 Schizophyllum commune, 154,157 (table), 178-80 Schizosaccharomyces, Tmax value, 198 Schizosaccharomyces octosporus, 197 (fig) Schizosaccharomyces pombe, 171, 242 (table) Sclerotina sclerotiorum, 161, 175-6 mannitol cycle enzymes, 163 mannitol 1-phosphatase, 158 mannitol 1-phosphate dehydrogenase, 152, 153 (table), 153

333

xylitol dehydrogenase, 152 Sea urchin embryos, central timer mechanism, 138 Secondary metabolism, 1-60 biochemistry, comparison with primary metabolism, 34-5 canonical reactions not commonly found in primary metabolism, 24 (table) definition, 3-4 Secondary metabolites, 3 acetate derived, 17, 26 allelochemicals, 5 allomones, 5 amino acids derived, 19 augmented primary reaction battery derived, operating under basic primary metabolism principles, 23-6 augmented primary reaction battery derived, without total regard for basic operational principles of primary metabolism, 2 6 3 4 biosynthesis, 22 characteristics, 3-4, 4-5 distinguishing criteria, 6 (table) distributionally defined, key physiological roles, 41-51 distribution-based criteria, 5 division into groups, 21-2 fungal, formed from two amino acids, 20-1, 21 (table) kairomones, 5 physiological roles ascribed to, 51-5 reaction battery derived, and basic operational principles of primary metabolism, 22-3 sugar metabolism components derived, 19-22 terpenoid, 20 Secreted proteins, biosynthesis, 26 1-2 Self-inhibition (auto-inhibition), 44 L-Serine deaminase, 99 Serpula lacrimam, 176, 177 Serratia marcescens, 46 (table) Sesquiterpenes, 27, 28 Sesterterpenes, 27 Sex hormones, 41-3 Sideramines, 40, 47, 48 (table) Siderochromes, 47 Siderophores, 47

334

SUBJECT INDEX

Silver nitrate, 105 Sirenin, 8, 41 Sirodesmins, 21 (table) Sitosterol, 9 Sketetonema costaturn, 126 (table) Skeleton photoperiods, 80 Sodium chloride, 260 Sodium perchlorate, 260 Sodium thiocyanate, 260 Sorbic acid, 236-7 Sorbitol (glucitol), bypass in fungi, 186-7 Sorbitol (glucitol) dehydrogenase (NAD+), 158 L-Sorbose, 168, 169 Sphingosine, 15 Spore germination/outgrowth, agents influencing, 44-7 Spore-germination stimulators, 47 Sporodesmins, 20 (table), 21 (table) Sporopollenin, 50 Squalene oxide, 23 conversion: into cycloartenol, 13, 18 (fig) into lanosterol, 13, 18 (fig) Squalene oxjdocyclase, 23, 26, 28 Staling effect, 255, 287-90 Staphylococcus aureus, 258 Sterols alcohol tolerance in yeasts, 2 7 6 7 structural requirements for alcohol tolerance, 286 Stigmasterol, 9 Stigmatella aurantiaca, 45 Streptimodone, 107 Streptomyces spp. aminoglycoside antibodies, 46 (table) diffusible substance, 45 (table) monomers of sheaths of aerial hyphae, 46 Streptomyces alboniger, 45 (table) Streptomyces coelicolor, 45 (table) Streptomyces griseus, 43 candicidin production, 54 macrotetrolides, 46 (table) proteinaceous factor (Factor C), 45 (table) Streptomyces viridochromogenes, 45 (table) Streptomycin, 25, 46 (table) Streptose, 19 Streptovaricin, 20 (table)

Sugar metabolism, I2 components, metabolites derived from, 19-22 Sulphur dioxide, Sacch. cerevisiae, death induced, 234-6

T Temperature profiles of yeasts, 195-25 1 activation parameters, 196, 202-6 associative profiles, 210-20 batch culture, 210-16 chemostat cultures, 21 7-20 cardinal, 195, 196-9 cells affecting, 196 dissociative profiles, 22 1-3 drug effects, 196, 223-9 metabolites affecting, 196 specific rates of growth and thermal death, 195-6, 199-202 growth, 199-200 thermal death, 20&2 targets of temperature effects, 23948 biomembranes, 244-8 mitochondria1 membranes, 245-8 thermodynamic compensation, 2 4 W types, 206-23 Temporal organization, 6 1-3 Tenellin, 20 (table) Tenuazonic acid, 20 (table) Terpenes, 17 non-lanosterol-derived, 26, 27-30 Terreic acid, 35 Tertiary butanol, 288 Tetra-acetic acid, 30 Tetracycline(s), 4, 6 (table), 20 Tetrahymena spp., 66-73 circadian rhythms in metabolism and biochemistry, 70-3 glycogen metabolism, 7 1-2 physiological rhythms during infradian growth mode, 69-70 rhythms of cell division, 66-9 Tetrahymena pyriformis, 68 alcohols effect on, 271-2, 275-6 circadian rhythms, 68-9, 71, 126 (table) epinephrine in, 7 1 glycogen metabolism, 71

335

SUBJECT INDEX

Tetrahymena pyriformis (cont.) serotonin in, 71 tetrahymenol production, 276 Tetrahymenol, 276 Tetraketides, 17, 20, 30 possible planar conformations, 3 1 (fig) Thermomyces lanuginosus, 162 Thiamin, in tl-0x0 acid decarboxylation, 13 in transketolase reaction, 13 Threitol (L-erythritol), 176 L-Threonine deaminase, 99 Time sense (Zeitgeduchtnis),62 Torulopsis sp., T,,, value, 198 Torulopsis bovina, 245 Torulopsis candida, 209 (fig), 2 15 Torulopsis colliculosa, 22 1 Torulopsis dattila, 221 Torulopsis fugisanensis, 209 (fig) Torulopsis glabrata, 2 15 Torulopsis haemulonii, 209 (fig) Torulopsis holmii, 2 15 Torulopsis magnoliae, 172, 173 Torulopsis pintolopesii, 197 Toxins, host-specific, 5 1 Transcriptotypes, 71 Transmembrane gradients as energy source for transport systems, 274 Trehalose, 160, 1767, 183, 186 Tremella mesenterica, 43 Tremerogen A- 10, 43 Tremorgens, 21 (table) Tricarboxylic acid, 11 Trichodermin, 27, 28 Trichosporon sp., T,,, value, 198 Trichothecins, 5 1 Trichothecium roseum, 162 Trigonopsis sp., T,, value, 198 Triketides, 17 Trisporic acid, 42 Trisporin B, 42 Trisporol B, 42 Tuberculostearic acid, 14-1 5 Turgor regulation, 188 Tylosin, 33 Tyrocidin, 45 (table) Tyrosine aminotransferase, 72, 73

U

V

Vaccenic acid, E. coli mutants unable to synthesize, 283 cis-Vaccenic acid, 284 trans-Vaccenic acid, 284 Valinomycin, 106 Verruculogens, 2 1 (table) Verticillins, 21 (table) Viridin, 22

W Wickerham’s test, 197

X Xanthocillin, 21 (table) Xerophilic fungi, 171 Xerotolerant fungi, 171 Xylitol, 154 Xylitol dehydrogenase (NAD+), 152 deficiency, 164-5 Xylose, 163 D-Xylose, 154 uptake, alcohols effect, 272, 273 (fig) D-Xylose isomerase, 165 D-Xylose ketol-isomerase, 163 Xylulose reductase, 173 D-Xylulose, 163 D-Xylulose reductase (NAD+), 152, 179

Y Yeast(s) Arrhenius plot of growth rates, 195 Arrhenius plots of thermal death, 209 (fig) ethanol-induced leakage and excretion, 278-80 grown on N-alkane, 161 linoleyl residue-enriched cells, 279 mesophilic, 197 oleyl residue-enriched cells, 279 osmophilic, 171-3 plasma membrane as diffusion barrier, 279 premature inhibition of fermentation, 179

336

SUBJECT INDEX

Yeast(s) (cont.) psychrophilic, 197 psychrophobic, 197 “rapid fermentation”, 278 sterols and alcohol tolerance, 2 7 6 7 temperature profiles see Temperature profiles thermal tolerance, 277-8 thermophilic, 197 thermotolerant, 223 T,, values, 197-9 Wickerham’s test, 197 Yeast mating hormone, 43 2

Zahner notion, 41

Zearalenone, 7, 3 4 , 4 Zeitgeber (time cues), 64 Zeitgeduchtnis (time sense), 62 Zygomycotina, 159-60 Zygorrhincus moelleri, 159, 27 1 Zymomonas mobilis, 290 alcohol intracellular concentration, 29 1 alcohol production, 290-1 Entner-Doudoroff pathway, 294 ethanol effect, 255, 256 (fig), 256, 257 on fatty-acyl composition, 265 on glucose utilization, 291 on phospholipid composition, 267 ethanol production, 255, 256 (fig), 256, 257 fatty-acyl composition, 294 and ethanol production, 295 membrane, 293

Cumulative Index of Contributors Volumes 1-25 Volume numbers are indicated by bold type

Amy, N. K., 18, 1 Archibald, A. R., 11, 53 Atkinson D. E., 15,253 Attwood, M. M., 17,303 Ball, P., 23, 183 Ballou, C., 14, 93 Bater, A. J., 16, 279 Benemann, J. R., 5, 135; 8, 59 Beran, K., 2, 143 Borgia, P. T., 18, 67 Braun, R., 21, 1 Brennan, P. J., 17,47 Brown, A. D., 17, 181 Brown, C. M., 11, 1 Bull, A. T., 15, 1 Burge, R. E., 7, 1 Bussey, H., 22, 93 Buttke, T. M., 25,253 Cacciapuoti, A. F., 20, 251 Campbell, I. M., 25, 1 Campbell, L. L., 3, 83 Carter, B. L. A., 6, 47; 17, 243 Chapman, A. G., 15,253 Chopra, I., 23, 183 Clarke, P. H., 4, 179 Coakley, W. T., 16, 279 Cole, J. A., 14, 1 Crandall, M., 15, 307 Dagley, S., 6, 1 Dawes, E. A., 10, 135 Doolittle, W. F., 20, 1 Drews, G., 22, 1 Dring, G. J., 11, 137 Duffus, J. H., 23, 151

Dundas, I. E. D., 15,85 Dworkin, M., 9, 153 Eberhardt, U., 7,205 Eddy, A. A., 23, 1 Edmunds, L. N., 25,61 Egel, R., 15, 307 Elliott, C. G., 15, 121 Ellwood, D. C., 7,83 Farrell, J., 3, 83 Fiechter, A., 22, 123 Finnerty, W. R., 18, 177 Fisher, D. J., 9, 1 Forrest, W. W., 5, 213 Fuhrmann, G. F., 22, 123 Galbraith, J. C., 5, 45 Garrett, R. H., 18, 1 Gest, H., 7,243 Glover, S. W., 18,235 Goldfine, H., 8, 1 Gould, G. W., 11, 137 Griffiths, A. J., 4, 105 Guffanti, A. A., 24, 173 Halvorson, H. O., 6, 47 Hamilton, W. A., 12, 1 Hammes, W. P., 13,245 Harder, W., 17, 303; 24, 1 Harley, J. L., 3, 53 Harold, F.M., 4, 45 Harrison, D. E. F., 14,243 Holland, I. B., 12, 55 Holwill, M. E. J., 16, 1 Huang, J.C.-C., 8, 215 Hughes, D. E., 3, 197

337

338

COMULATIW INDEX OF CONTRIBUTORS

Ingram, L. O’N., 25, 253 Ippen, K. A., 3, 1

Oelze, J., 22, 1 Ornston, L. N., 9,89

Jannasch, H. W., 11, 165 Jennings, D. H., 25, 149 Jones, C. W., 11,97

Paigen, K., 4, 251 Payne, J. W., 13, 55 Paznokas, J. L., 18,67 Postgate, J. R., 1, I; 10, 81

Kandler, O., 13, 245 Kappeli, O., 22, 123 Kelley, W. S., 2, 89 Kjeldgaard, N. O., 1, 39 Klug, M. J., 5, 1 Koch, A. L., 6, 147; 16, 49; 24, 301 Koffler, H., 6, 219 Konings, W. N., 15, 175 Krulwich, T. A., 173 Kubitschek, H. E., 12, 247 Kulaev, I. S., 24, 83 Larsen, H., 1, 97 Lascelles, J., 2, 1 Le Gall, J., 10, 81 Levi, C., 23, 151 Lewis, D. H., 3, 53 Linnane, A. W., 16, 157 Lipmann, F., 21, 227 Ljungdahl, L. G., 19, 149 Lloyd, D., 16, 279 Losel, D. M., 17, 47 Lysko, P. G., 20, 251 Macdonald-Brown, D. S., 11, 1 MacKay, V. L., 15, 307 Mandelstam, J., 20, 103 Manners, D. J., 23, 151 Markovetz, A. J., 5, 1 Marquis, R. E., 14, 159 Mateles, R. I., 1, 25; 11, 165 Matsushita, T., 12, 247 Meers, J. L., 11, 1 Mobach, H., 3, 1 Morris, J. G., 12, 169 Morse, S. A., 20, 251 Mortlock, R. P., 13, 1 Moseley, B. E. B., 2, 173; 16, 99 Murrell, W. G., 1, 133 Nagley, P., 16, 157 Nakae, T., 20, 163 Nikaido, H., 20, 163

Quayle, J. R., 7, 119 Ratledge, C., 13, 115 Raven, J. A., 21, 47 Razin, S., 10, 1 Reaveley, D. A., 7, 1 Richmond, D. V., 9, 1 Richmond, M. H., 2, 43; 9, 31 Rittenberg, S. C., 3, 159 Rogers, H. J., 19, I Salton, M. R. J., 11, 213 Sargent, M. G., 18, 105 Schaechter, M., 2, 89 Schlegel, H. G., 7, 205 Schleifer, K. H., 13, 245 Seebeck, T., 21, 1 Senior, P. J., 10, 135 Shaw, N., 12, 141 Silverman, P. M., 3, 1 Sinclair, P. R., 5, 173 Smith, J. E., 5, 45 Smith, R. W., 6, 219 Sommerville, J., 4, 131 Sriprakash, K. S., 16, 157 Stanier, R. Y., 9, 89 Starr, M. P., 8, 215 Stevens, L., 19, 63 Stouthamer, A. H., 14, 315 Strange, R. E., 8, 105 Sudo, S. Z., 9, 153 Sutherland, I. W., 8, 143; 23, 79 Sykes, R. B., 9, 31 Sypherd, P. S., 18, 67 Tauro, P., 6,47 Tempest, D. W., 4, 223; 7, 83 Trinci, A. P. J., 15, 1 Trudinger, P. A,, 3, 1 1 1 Vagabov, V. M., 24,83 Valentine, R. C., 3, 1; 5, 135; 8, 59 Van Dijken, J. P., 24, 1

COMULATIVE INDEX OF CONTRIBUTORS

Van Uden, N., 25, 195 Veenhuis, M., 24, 1 Venema, G., 19,245 Walker, D. J., 5, 213 Warth, A. D., 17, 1 Weinberg, E. D., 4, 1 Weitzman, P. D. J., 22, 185 White, D. C., 5, 173 Williams, B., 4, 251 Williams, E., 16, 99

Wimpenny, J. W. T., 3, 197 Winther, M. D., 19, 63 Wogan, G. N., 1 , 2 5 Wolf, D. H., 21, 267 Wolfe, R. S., 6, 107 Yates, M. G., 11, 97 Young, M., 20, 103 Zeikus, J. G., 24, 215

339

Cumulative Subject Index Volumes 1-25 Volume numbers are indicated by bold type

Acellular slime moulds, transcription in, 21, 1

Acidophilic and alkalophilic bacteria, physiology, 24, 173 Active transport of solutes in bacterial membrane vesicles, 15, 175 Adaptive responses of Escherichia coli to a feast and famine existence, 6, 147 Adenine nucleotide concentrations and turnover rates: their correlation with biological activity in bacteria and yeast, 15, 253 Aflatoxins, 1, 25 Alcohols, effects on micro-organisms, 25, 253

Aliphatic amidases of Pseudornonas aeruginosa, 4, 179

Amoebae, encystment in, 4, 105 Antibiotic polypeptides, bacterial production by thiol-linked synthesis on protein templates, 21, 227 Antibiotics, transport into bacteria, 23, 183

Antimicrobial agents and membrane function, 4,45 Aromatic compounds, catabolism by micro-organisms, 6, 1 Aspects of genetic engineering in microorganisms, 18, 235 Assimilatory and dissimilatory metabolism of inorganic sulphur compounds by micro-organisms, 3, 111 Azotobacter, respiration and nitrogen fixation in, 11,97 Bacterial exopolysaccharides, 8, 143 Bacterial flagella, 6, 219

Bacterial glycolipids and glycophospholipids, 12, 141 Bacterial photosynthetic apparatus, 2, 1 Bacterial production of antibiotic polypeptides by thiol-linked synthesis on protein templates, 21, 227 Bacterial transformation, 19, 245 Bdellovibrios, physiology, 8, 215 Biochemical aspects of extreme halophilism, 1, 97 Biochemical and physiological aspects of differentiation in the fungi, 5,45 Biochemistry of the bacterial endospore, 1, 133

Biochemistry of dimorphism in the fungus Mucor, 18,67 Biochemistry and genetics of nitrate reductase in bacteria, 14, 3 15 Biogenesis of the wall in bacterial morphogenesis, 19, 1 Biology, physiology and biochemistry of hyphomicrobia, 17, 303 Biophysical aspects of ciliary and flagellar motility, 16, 1 Biosynthesis of microbial exopolysaccharides, 23, 79 Biosynthesis of secondary metabolites: roles of trace metals, 4, 1 Branched electron-transport systems in bacteria, 5, 173 Budding of yeast cells, their scars and ageing, 2, 143 Carbohydrate utilization, catabolite repression and other control mechanisms in, 4, 25 1

340

CUMULATIVE SUBJECT INDEX

Carbohydrates, unnatural, catabolism by micro-organisms, 13, 1 Catabolism of aromatic compounds by micro-organisms, 6, 1 Catabolism of unnatural carbohydrates by micro-organisms, 13, 1 Catabolite repression and other control mechanisms in carbohydrate utilization, 4, 251 Cell cycle, synthesis of enzymes during, 6, 47 Cell cycle in prokaryotes, surface extension and, 18, 105 Cell division, is it regulated by initiation of chromosome replication?, 16,49 Chemolithotrophic bacteria, physiology, roles of exogenous organic matter, 3, 159 Chemotrophic anaerobes, metabolism of one-carbon compounds by, 24,215 Chromosome replication, does its initiation regulate cell division?, 16, 49 Ciliary and flagellar motility, biophysical aspects, 16, 1 Circadian rhythms in micro-organisms, physiology, 25, 61 Citric acid cycle, bacterial enzymes, unity and diversity in, 22, 185 Colicin action, physiology, 12, 55 Comparative aspects of bacterial lipids, 8, 1 Comparative biology of prokaryotic resting cells, 9, 153 Compatible solutes and extreme water stress in eukaryotic micro-organisms, 17, 181 Continuous culture, experimental bacterial ecology studied in, 11, 165 Continuous culture, place in microbiological research, 4, 223 Control of metabolism in yeast and other lower eukaryotes through action of proteinases, 21, 267 Control mechanisms, in carbohydrate utilization, 4, 251 Cyanobacterial genome, its expression and the control of that expression, 20,l Dimorphism in the fungus Mucor, biochemistry of, 18, 67 Disruption of micro-organisms, 16,279

34 1

DNA in bacteria, damage and repair, 16, 99 DNA in bacteria, repair of irradiation damage, 2, 173 DNA replication in bacteria, 12, 247 DNA, yeast mitochondrial, structure, synthesis and genetics, 16, 157 Does the initiation of chromosome replication regulate cell division?, 16, 49

Early events during bacterial endospore formation, 20, 103 Ecology, bacterial, studied in continuous culture, 11, 165 Ectotrophic mycorrhizas, physiology, 3, 53 Effect of endogenous and exogenous factors on the primary structures of bacterial peptidoglycan, 13, 245 Effects of alcohols on micro-organisms, 25,253 Effects of environment on bacterial wall content and composition, 7,83 Electrons, high-energy, in bacteria, 5,135 Electron-transport systems, branched, in bacteria, 5, 173 Electrophoretic mobility of microorganisms, 9, 1 Encystment in amoebae, 4, 105 Endospore, bacterial, biochemistry, 1, 133 Endospores, bacterial, early events during formation, 20, 103 Energy, generation and utilization during growth, 5,213 Energy conversion and generation of reducing power in bacterial photosynthesis, 7, 243 Energy coupling in microbial transport, 12, 1 Energy reserve polymers, role and regulation in micro-organisms, 10, 135 Environment, effects on bacterial wall content and composition, 7, 83 Enzymes, synthesis during the cell cycle, 6, 47 Enzymes, citric acid cycle, bacterial, unity and diversity in, 22, 185 Enzymes, membrane associated in bacteria, 11, 213

342


Escherichia coli, adaptive responses to a feast and famine existence, 6, 147 Escherichia coli, F-pilus of, 3, 1 Eukaryotes, lower, metabolism in, control through action of proteinases, 21, 267 Eukaryotic micro-organisms, compatible solutes and extreme water stress in, 17, 181 Eukaryotic micro-organisms, mechanisms of solute transport in, 23, 1 Exogenous organic matter, roles in physiology of chemolithotrophic bacteria, 3, 159 Exogenous and endogenous factors, effect on primary structures of bacterial peptidoglycan, 13, 245 Exopolysaccharides, bacterial, 8, 143 Exopolysaccharides, microbial, biosynthesis, 23, 79 Experimental bacterial ecology studied in continuous culture, 11, 165 Flagella, bacterial, 6, 219 Flagellar and ciliary motility, biophysical aspects, 16, 1 F-pilus of Escherichia coli, 3, 1 Fungal development, oligoamines in, 19, 63 Fungal growth, physiology and metabolic control, 15, l Fungal lipids, physiology, 17,47 Fungi, differentiation in, biochemical and physiological aspects, 5, 45 Fungi, function of sterols in growth and reproduction, 15, 121 Fungi, nitrate assimilation in, 18, 1 Fungi, polyol metabolism in, 25, 149 Fungus Mucor, biochemistry of dimorphism in, 18, 67 Gas metabolism, microbial, 14, 1 Generation and utilization of energy during growth, 5, 213 Genetic engineering in micro-organisms, 18,235 Genome, cyanobacterial, expression and its control, 20, 1 Glucans of yeast cell wall, 23, 151 Glucose metabolism in growing yeast cells, regulation, 22, 123

Glycolipids and glycophospholipids, bacterial, 12, 141 Gram-negative bacteria, 8-lactamases of, and their possible physiological role, 9, 31 Gram-negative bacteria, outer membrane, 20, 163 Halobacteriaceae, physiology, 15, 85 Halophilism, extreme, biochemical aspects, 1, 97 Heat resistance of spores, mechanisms, 11, 137 High-energy electrons in bacteria, 5, 135 High-pressure microbial physiology, 14, 159 Hydrocarbons, aliphatic, utilization by micro-organisms, 5, 1 Hyphomicrobia, biology, physiology and biochemistry, 17, 303 8-Ketoadipate pathway, 9, 89 Knallgasbacteria, metabolism, regulatory phenomena, 7,205 8-Lactamases of Gram-negative bacteria and their possible physiological role, 9, 31 “Life cycle” of bacterial ribosomes, 2, 89 Lipids, bacterial, comparative aspects, 8, 1

Lipids, fungal, physiology, 17, 47 Mannan component of yeast cell envelope, structure and biosynthesis, 14, 93 Mating in three yeasts, physiology of, 15, 307 Mechanisms of solute transport in selected eukaryotic micro-organisms, 23, 1 Mechanisms of spore heat resistance, 11, 137 Membrane function, antimicrobial agents and, 4,45 Membrane vesicles of bacteria, active transport of solutes in, 15, 175 Membrane, outer, of Gram-negative bacteria, 20, 163 Membranes of phototrophic bacteria, organization and differentiation, 22, 1 Membranes, walls and, in bacteria, 7, 1


Membrane-associated enzymes in bacteria, 11, 213 Metabolism of one-carbon compounds by chemotrophic anaerobes, 24,215 Metabolism of one-carbon compounds by micro-organisms, 7, 119 Metabolism, secondary, and microbial physiology, 25, 1 Metabolites, secondary, biosynthesis, 4 , l Methane, microbial formation, 6, 107 Microalgae, nutrient transport in, 21, 47 Microbial formation of methane, 6, 107 Microbial gas metabolism, 14, I Molecular structure of the bacterial spore, 17, I Morphogenesis, bacterial, biogenesis of the wall in, 19, 1 Morphogenesis, microbial, surface stress theory, 24, 301 Mycobacteria, physiology, 13, 1 I 5 Mycoplasmas, physiology, 10, 1 Mycorrhizas, ectotrophic, physiology, 3, 53 Neisseria gonorrhoeae, physiology, 20, 25 1 Nitrate assimilation in fungi, 18, 1 Nitrate reductase in bacteria, biochemistry and genetics, 14, 315 Nitrogen fixation, pathways, 8, 59 Nitrogen fixation, and respiration, in Azotobacter, 11, 97 Nitrogen, inorganic, physiological aspects of microbial metabolism, 11, 1 Nucleic acid and protein formation in bacteria, regulation, 1, 39 Nucleotide, adenine, concentrations and turnover rates. correlation with biological activity in bacteria and yeast, 15.253 Nutrient transport in microalgae, 21, 47

Obligate anaerobiosis, physiology, 12, 169 One-carbon compounds, metabolism by chemotrophic anaerobes, 24, 2 15 One-carbon compounds, metabolism by micro-organisms, 7, I19 One-carbon compounds, metabolism in yeasts, significance of peroxisomes, 24, 1

343

Organization and differentiation of membranes of phototrophic bacteria, 22, 1 Outer membrane of Gram-negative bacteria, 20, 163 Oxygen metabolism by micro-organisms, 3, 197 Paramecium, serotype expression in, 4, 131 Pathways of nitrogen fixation, 8, 59 Peptides and micro-organisms, 13, 55 Peptidoglycan, bacterial, effect of endogenous and exogenous factors on primary structures, 13, 245 Peroxisomes, significance in metabolism of one-carbon compounds in yeasts, 2471 Phospholipid metabolism, bacterial, physiology and biochemistry, 18, 177 Photosynthesis, bacterial, energy conversion and generation of reducing power, 7,243 Photosynthethic apparatus, bacterial, 2, 1 Phototrophic bacteria, membranes, organization and differentiation, 22, 1 Physiological aspects of microbial inorganic nitrogen metabolism, 11, 1 Physiology of acidophilic and alkalophilic bacteria, 24, 173 Physiology of the bdellovibrios, 8, 215 Physiology of circadian rhythms in micro-organisms, 25,61 Physiology of colicin action, 12, 55 Physiology of ectotrophic mycorrhizas, 3, 53 Physiology of fungal lipids, selected topics, 17, 47 Physiology of halobacteriaceae, 15, 85 Physiology of killer factor in yeast, 22,93 Physiology of mating in three yeasts, 15, 307 Physiology of the mycobacteria, 13, 115 Physiology of mycoplasmas, 10, 1 Physiology of Neisseria gonorrhoeae, 20, 25 1 Physiology of obligate anaerobiosis, 12, 169 Physiology of sulphate-reducing bacteria, 10, 81

344


Physiology of thermophilic bacteria, 19, 149 Physiology and biochemistry of bacterial phospholipid metabolism, 18, 177 Physiology and metabolic control of fungal growth, 15, 1 F-Pilus of Escherichia coli, 3, 1 Place of continuous culture in microbiological research, 4, 223 Plasmids of Staphylococcus aureus and their relation to other extrachromosoma1 elements in bacteria, 2, 43 Polymers, energy reserve, role and regulation in micro-organisms, 10, 135 Polyol metabolism in fungi, 25, 149 Polypeptides, antibiotic, bacterial production by thiol-linked synthesis on protein templates, 21, 227 Polyphosphate metabolism in microorganisms, 24, 83 Populations, sparse microbial, rapid detection and assessment, 8, 105 Pressure, effect on microbial physiology, 14, 159 Prokaryotes, cell cycle in, surface extension and, 18, 105 Prokaryotic resting cells, comparative biology, 9, 153 Protein and nucleic acid formation in bacteria, regulation, 1, 39 Pseudomonas aeruginosa, aliphatic amidases of, 4, 179 Rapid detection and assessment of sparse microbial populations, 8, 105 Regulation of glucose metabolism in growing yeast cells, 22, 123 Regulation of nucleic acid and protein formation in bacteria, 1, 39 Regulation of respiration rate in growing bacteria, 14, 243 Regulatory phenomena in metabolism of knallgasbacteria, 7, 205 Repair of damaged DNA in bacteria, 16, 99 Repair of damaged DNA in irradiated bacteria, 2, 173 Replication of DNA, in bacteria, 12, 247 Respiration and nitrogen fixation in Azotobacter, 11, 97

Respiration rate, regulation in growing bacteria, 14, 243 Resting cells, prokaryotic, comparative biology, 9, 153 Ribosomes, bacterial, “life cycle”, 2, 89 Role and regulation of energy reserve polymers in micro-organisms, 10, 135 Roles of exogenous organic matter in the physiology of chemolithotrophic bacteria, 3, 159 Secondary metabolism and microbial physiology, 25, 1 Secondary metabolites, biosynthesis, 4, 1 Serotype expression in Paramecium, 4, 131 Significance of peroxisomes in the metabolism of one-carbon compounds in yeasts, 24, 1 Slime moulds, acellular, transcription in, 21, 1

Some biophysical aspects of ciliary and flagellar motility, 16, 1 Spermine, spermidine and putrescine in fungal development, 19, 63 Spore, bacterial, molecular structure, 17, 1 Spore heat resistance, mechanisms, 11, 137 Staphylococcus aureus, plasmids of, 2,43 Sterols in fungi: their functions in growth and reproduction, 15, 121 Structure, biosynthesis of the mannan component of the yeast cell envelope, 14,93 Structure, biosynthesis and function of teichoic acid, 11, 53 Structure, synthesis and genetics of yeast mitochondria1 DNA, 16, 157 Sulphate-reducing bacteria, physiology, 10,81

Sulphur compounds, inorganic, assimilatory and dissimilatory metabolism, 3, 111 Surface extension and the cell cycle in prokaryotes, 18, 105 Surface stress theory of microbial morphogenesis, 24, 301 Survival of microbes under minimum stress, and viability, 1, 1


Synthesis of enzymes during the cell cycle, 6, 47 Teichoic acid, structure, biosynthesis and function, 11, 53 Temperature profiles of yeasts, 25, 195 Thermophilic bacteria, physiology, 19, 149 Thermophilic bacteria and bacteriophages, 3, 83 Trace metals, roles in biosynthesis of secondary metabolites, 4, 1 Transcription in acellular slime moulds, 21, 1 Transformation, bacterial, 19, 245 Transport of antibiotics into bacteria, 2& 183 Transport of nutrients, in microalgae, 21, 47 Transport of solutes, mechanisms in selected eukaryotic micro-organisms, 23, 1 Transport, active, of solutes in bacterial membrane vesicles, 15, 175 Transport, microbial, energy coupling in, 12, 1 Unity and diversity in some bacterial citric acid cycle enzymes, 22, 185 Utilization of aliphatic hydrocarbons by micro-organisms, 5, 1 Viability measurements and the survival of microbes under minimum stress, 1,1

345

Wall, bacterial, effects of environment on content and composition, 7, 83 Wall biogenesis, in bacterial morphogenesis, 19, 1 Walls and membranes in bacteria, 7, 1 Water stress, extreme, and compatible solutes in eukaryotic micro-organisms, 17, 181 Yeast growing cells, regulation of glucose metabolism in, 22, 123 Yeast, killer factor in, physiology, 22,93 Yeast cells, budding, scars and ageing, 2, I43 Yeast cell envelope, structure and biosynthesis of the mannan component, 14, 93 Yeast cell-wall glucans, 23, 151 Yeast mitochondria1 DNA, structure, synthesis and genetics, 16, 157 Yeast nucleus, 17, 243 Yeast and other lower eukaryotes, metabolism in, control through action of proteinases, 21, 267 Yeasts, metabolism of one-carbon compounds in, significance of peroxisbmes, 2491 Yeasts, physiology of mating in three species, 15, 307 Yeasts, temperature profiles of, 25, 195


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