Advances in
MICROBIAL PHYSIOLOGY
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Advances in
MICROBIAL PHYSIOLOGY Edited by
A. H. ROSE School of Biological Sciences Bath University, U K
Volume 34
ACADEMIC PRESS Harcourt Brace Jovanovich, Publishers London San Diego New York Boston Sydney Tokyo Toronto
ACADEMIC PRESS LIMITED 24-28 Oval Road London NWl 7DX US Edition published by ACADEMIC PRESS INC. San Diego CA 92101
Copyright 0 1993 by ACADEMIC PRESS LIMITED This book is printed on acid-free papei
All Rights Reserved No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers
British Library Cataloguing in Publication Data Advances in microbial physiology. Vol. 34 1. Micro-organisms-Physiology I . Rose, A. H. 576’.11 OR84 ISBN 0-12427734-4 ISSN 0065-291 1
Typeset by J&L Composition Ltd, Filey, North Yorkshire Printed in Great Britain by The University Press, Cambridge
Contributors D. J. Adams Department of Microbiology, University of Leeds, Leeds LS2 9JT, UK P. V. Dunlap Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA M. T. Elskens Laboratorium voor Analytische Scheikunde en Geochemie, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium G. W. Gooday Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen AB9 l A S , UK E. A. Meighen Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6 M. J. Penninckx Unit6 de Physiologie et Ecologie Microbiennes, FacultC des Sciences, UniversitC Libre de Bruxelles, CERIA, av. E . Gryson 1 , B-1070 Bruxelles, Belgium J. G. H. Wessels Department of Plant Biology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands P. K. Wolber D N A Plant Technology Corporation, 6701 San Pablo Avenue, Oakland, CA 94608, USA
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Contents Contributors
V
Physiological, Biochemical and Genetic Control of Bacterial Bioluminescence EDWARD A. MEIGHEN and PAUL V. DUNLAP I. Introduction 11. Biochemistry 111. Molecular biology IV. Physiological and genetic control of lux-gene expression V. Evolution and ecology VI. Acknowledgements References
2 6 24 35 48 58 58
Sex Hormones and Fungi GRAHAM W. GOODAY and DAVID J . ADAMS
I. Introduction 69 11. Endogenous hormones 70 103 111. Endogenous regulators of sexual development 105 IV. Interactions of mammalian hormones with fungi V. Signal transduction following interactions of ula mating factors 132 or mammalian hormones with yeasts VI. Conclusions 133 VII. Acknowledgements 134 References 134
Fruiting in the Higher Fungi JOSEPH G. H. WESSELS 1. Introduction 11. Development of emergent structures
147 149
111. IV. V. VI. VII. VITI. IX. X.
Control of fruiting by mating-type genes Accessory regulatory genes controlling fruiting Molecular and biochemical indices of fruiting Environmental control of fruiting Rapid expansion of fruit bodies Biotechnology Conclusions Acknowledgements References Note added in proof
155 170 175 180 185 190 192 194 194 20 1
Bacterial Ice Nucleation PAUL K. WOLBER 1. Introduction IT. Physical basis of ice nucleation 111. Bacterial ice-nucleation genes and proteins TV. Environmental significance of bacterial ice nucleation V. Applications of bacterial ice nucleation VI. Concluding remarks References
203 204 21 1 230 231 233 235
Metabolism and Functions of Glutathione in Micro-organisms MICHAEL J. PENNINCKX and MARC T. ELSKENS I . Introduction 11. Occurrence and distribution of glutathione and related compounds in micro-organisms 111. General outlines of glutathione metabolism in microorganisms IV. Interconversion of glutathione and glutathione disulphide V. Conjugation of glutathione: glutathione S-transferases VI. Other aspects of glutathione function VII. Concluding remarks VIII. Acknowledgements References
240
Author index Subject index
303 329
241 247 262 281 284 290 291 29 1
Physiological. Biochemical and Genetic Control of Bacterial Bioluminescence EDWARD A . MEIGHEN" and PAUL V . DUNLAPb aDepartment of Biochemistry. McGill University. Montreal. Quebec. Canada H3G I Y6. and bBiology Department. Woods Hole Oceanographic Institution. Woods Hole. M A 02543. USA
I . Introduction 11. Biochemistry
I11 .
IV .
V.
VI .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
A . Bioluminescence reaction . . . . . . B . Structure of luciferase . . . . . . . C . Aldehyde biosynthesis . . . . . . . D . Accessory lux proteins . . . . . . Molecular biology . . . . . . . . . A . Organization of lux genes . . . . . . B . Differential expression of lux genes . . . . C . Expression of lux genes in other organisms . . Physiological and genetic control of lux-gene expression A . Autoinduction . . . . . . . . B . CyclicAMP . . . . . . . . . C . Control by iron . . . . . . . . D . Control by oxygen . . . . . . . E . Control by osmolarity . . . . . . . F. Possible involvement of LexA, HtpR and FNR . Evolution and ecology . . . . . . . . A . Distribution of luminous bacteria . . . . B . Molecular approaches to identification and ecology of bacteria . . . . . . . . . . . . C . Comparisonsoflux-geneamino-acidsequences. . . D . Duplication of lux genes . . . . . . . . Acknowledgements . . . . . . . . . . . References . . . . . . . . . . . . .
ADVANCES INMICROBIAL PHYSIOLOGY .VOL.. 34 ISBN &I24277344
2 6 6 14 18 22 24 24 31 34 35 35 43 45 46 41 48 48 48 luminous
. . . . . . . . . . . . . . .
50 52 54 58 58
Copyright0 1993. by Academic Press Limited All rights of reproduction in any form reserved
2
E. A MEIGHEN A N D P V. D I J N I A P
I. Introduction
The bioluminescent bacteria comprise one of several groups of luminous organisms (Hastings and Morin, 1991). Significant differences exist between the bioluminescence reactions of different organisms, including the structure and properties of the luciferases and substrates. Molecular oxygen is the only common feature of bioluminescence reactions, indicating that the luminescent systems in most organisms may have evolved independently. Presently, at least 11 species in four genera (Vibrio, Photobacterium, Shewanella (Alteromonas) and Xenorhabdus) clustered in the enteric families Vibrionaceae and Enterobacteriaceae (Baumann and Baumann, 1981; Baumann et al., 1983) are known to produce visible luminescence TABLE 1.
Species and habitats of currently known bioluminescent bacteria"
Organism
Habitat
Gram-negative, facultatively anaerobic rods
Family Vibrionaceae Vibrio cholera (aome strains) Vibrio fischeri
Vibrio harveyi
Vibrio logei Vibrio orientalis Vibrio splendidus biotype 1 Vibrio vulnificus (some strains) Photobacterium leiognathi
Photobacterium phosphoreum Family Enterobacteriaceae Xenorhabdus luminescens
Brackish or freshwater ( V . albensis) Coastal seawater, intestines of marine animals, light organs of certain fishes and squids Coastal arid open seawater, surfaces and intestines of marine animals, sediments, coastal seawater' (psychotrophic) Exoskeleton lesions of crabs, intestines of marine animals, sediments Coastal seawater Coastal seawater Coastal seawater, surfaces of fish Coastal seawater, surfaces and intestines of marine animals, light organs of certain marine fishes and squids Surfaces and intestines of marine animals, light organs of certain marine fishes (psychrotrophic) Soil, nematode symbiont
Gram-negative, aerobic rods
Shewanella (Alteromonas) hanedai
Coastal seawater (psychrotrophic)
For details and references, see Baumann and Baumann (1981). MacDonnell and Colwell (1985), Nealson and Hastings (1991), Farmer and Hickman-Brenner (1991) and Palmer and Colwell (1991). P V. Dunlap (unpublished data).
FIG. 1. Light emission by luminescent bacteria on solid media. (A) Streaks of Photobacterium phosphoreum (NCMB 844). (B) Colonies of Vibrio harveyi (B392).
4
E A MFIGHFN A N D P V DUNLAP
(Table 1). Although found predominantly in the marine environment, luminescent bacteria are also present in freshwater and terrestrial habitats. They can occur as free-living forms, saprophytes, commensal symbionts, parasites of animals and specific light-organ symbionts (Hastings and Nealson, 1981; Hastings et al., 1987; Dunlap and Greenberg, 1991). The luminescence produced by these bacteria, in part because of its inherent beauty and ease of detection, has attracted scientific attention for over 300 years (Boyle, 1668). The dramatic brilliance of cultures of luminescent bacteria, as illustrated by the streaks of Photobacterium phosphoreum and the colonies of Vibrio harveyi (Fig. l), explains why researchers have been intrigued with the study and applications of the
FIG. 2. Dependence of bacterial luminescence on oxygen. A liquid culture of Photobacterium phosphoreum was shaken and then allowed to stand for a few minutes to let the bubbles of air rise to the top of the tube before exposure to film. As the air (oxygen) is more rapidly depleted at the bottom of the tube, the culture is brighter at the top.
RACTI-RIA1 BIOLUMINESCENCE
5
bacterial bioluminescent system both in the past and in the present. Robert Boyle in the 17th century observed the luminescence of rotting fish, presumably caused by luminescent marine bacteria growing in a saprophytic mode. By showing that the light from dead fish was extinguished in a vacuum, the requirement for air (oxygen) in bioluminescence was demonstrated for the first time (Boyle, 1668). As shown in Fig. 2, the effects of limiting oxygen on the luminescence of bacteria can readily be observed near the bottom of a liquid culture of P . phosphoreum from which air has been depleted. Indeed, the effects of oxygen on luminescence illustrate in all probability the first application of the bacterial luminescence system as a sensor of specific molecules that affect metabolic function and/or gene expression. Knowledge of the basic biochemistry, molecular biology and physiology of luminescent bacteria is thus not only of interest but of importance for future scientific endeavours. With the use of molecular approaches to study the luminescence systems of these bacteria, the past decade has seen a development of interest in all aspects of luminous bacteria, from population biology and ecology to molecular mechanisms of luminescence (lux) gene regulation. In this review, our goal is to provide a description of the current status of the bioluminescent systems of luminous bacteria, emphasizing the biochemistry, lux gene organization and the physiological and genetic regulation of lux gene expression. General aspects of the ecology and systematics of luminous bacteria and bioluminescent symbiosis will not be dealt with in detail here, having been covered in other recent contributions (Hastings et al., 1985, 1987; Hastings, 1986; Dunlap and McFall-Ngai, 1987; Campbell, 1989; Lee, 1989; Dunlap and Greenberg, 1991; Farmer, 1991; Farmer and Hickman-Brenner, 1991; Nealson and Hastings, 1991; Ruby and McFall-Ngai, 1992). Other recent reviews on the biochemistry or molecular biology of bacterial bioluminescence have also appeared (Meighen, 1988, 1991; Stanley and Stewart, 1990; Baldwin and Ziegler, 1991; Lee, J. et al., 1991a). The rapidly expanding area involving applications of bacterial luminescence will only be discussed briefly. The reader is referred to some recent reviews in this area (Engebrecht et al., 1985; Shaw and Kado, 1986; Ulitzer and Kuhn, 1986; Shaw et al., 1987; Kricka, 1988; Schauer, 1988; Stewart et al., 1989; Meighen, 1991) and specific examples of applications of the bacterial bioluminescent enzymes (Meighen et al., 1982; Balaguer et al. , 1989; Gautier et al., 1990) and genes (Elhai and Wolk, 1990; Schultz and Yarus, 1990; Nussbaum and Cohen, 1988; Burlage et al. , 1990; King et al. , 1990; Kamoun and Kado, 1990a,b).
6
F A MEIGHEN A N D P V D U N I A P
11. Biochemistry A . BIOLUMINESCENCE REACTION
Light emission in bioluminescent bacteria arises by the reaction of molecular oxygen with reduced flavin mononucleotide (FMNH2) and a long-chain aldehyde to give FMN, water and the corresponding fatty acid. During the reaction, molecular oxygen is cleaved, with incorporation of one atom of oxygen into a fatty acid and the other atom into water (Suzuki et al., 1983). The enzyme, luciferase, which catalyses the bioluminescent reaction is thus a mixed-function oxidase since both aldehyde (to fatty acid) and FMNHz (to FMN and water) are oxidized (Hastings et a f . , 1985). The energy generated by combined oxidation of these two substrates is more than sufficient to provide the 60 kcal mol-' necessary for emission of the blue-green light (490 nm).
+ +
FMNH2 O2 CH3-(CH2),-CHO + FMN
+ H 2 0+ CH,-(CH2),-COOH + light
I . Light Emission and Energy Requirements Several aspects of light emission suggest that it is energetically costly. Light emission represents an energy expenditure of approximately six ATP molecules for each photon, assuming an efficiency for the reaction of 100% (Hastings and Nealson, 1977). Estimates of the quantum yield for luciferase, however, range from 0.1 to 1 photon for each cycle of the enzymic reaction (Lee, 1972, 1985; Dunn et a f . , 1973; Karl and Nealson, 1980; Makemson, 1986) so, depending on the quantum yield in vivo, luminescence can account for six (quantum yield of 1) to 60 (quantum yield of 0.1) molecules of ATP for each photon of light emitted. Because fully induced cells can produce lo4 photons s-l cell-' (e.g. Dunlap, 1984b), light emission appears to account for a large number of ATP molecules in induced cultures. Furthermore, for induced cultures, luciferase comprises a few to several per cent of the soluble cellular protein (Hastings et a f . , 1965; Henry and Michelson, 1970; Wall et a f . , 1984b). Since other proteins are co-induced with luciferase (Michaliszyn and Meighen, 1976; Ne'eman et a f . , 1977; Boylan et a f . , 1985), a substantial amount of cellular energy is consumed in the synthesis of proteins. Moreover, up to 20% of the oxygen taken up by fully induced cells may be consumed in the luminescence reaction (Eymers and van Schouwenberg, 1937; Watanabe et a f . , 1975; Dunlap, 1984b; Makemson, 1986). These considerations suggest that the energetic cost of light emission is high. This might account for the tight regulation of the luminescence system (described in a later section) exhibited by many
BACTERIAL HIOLUMINbSCFNCb
7
species of luminous bacteria, in that energy is conserved by expressing luminescence only when physiologically important. In contrast, however, consumption of energy in bioluminescence, when experimentally measured, apparently is low, 0.01% or less of the total energy expended during growth (McIlvaine and Langerman, 1977; Makemson and Gordon, 1989). Possibly then, regulation of luminescence relates to factors other than its energetic cost. Luciferases have been purified from a number of different luminescent bacteria, including both terrestrial and marine species (Meighen, 1988, 1991; Baldwin and Ziegler, 1991). These enzymes are devoid of any prosthetic groups, metals and non-amino-acid residues (Hastings and Nealson, 1977) and do not appear to be subjected to post-translational modification. However, the probability that other proteins take part in, but are not necessary for, light emission is an area of active investigation (Prasher et al., 1990; Baldwin et al., 1990; Lee, J. et al., 1991a; O’Kane et al., 1991). In some luminescent bacteria, the colour and efficiency of light emission can be affected by other proteins. A yellow fluorescence protein shifts the peak intensity to 540 nm in one strain of Vibrio fischeri (Ruby and Nealson, 1977; Eckstein et al., 1990) whereas, in Photobacterium spp., the lumazine protein causes a small shift in the maximum intensity to lower wavelengths (470-480 nm) (Lee, 1985; Lee, J. et al., 1991a). 2. Flavin Specificity The flavin specificity of the bioluminescent reaction is quite restricted. Reduced FMN is by far the preferred substrate, with a K , value of less than 1 PM (Meighen and Hastings, 1971). Most alterations in the structure of FMNH2, including changes in the structure of the flavin ring or the ribose phosphate side-chain, significantly decrease the activity and/or binding. However, substitutions at position 8 of the flavin ring do not greatly affect activity (Chen and Baldwin, 1984; Macheroux et al., 1987) unless they alter the oxidation potentials of the reduced forms of the flavin analogues (J. Eckstein, J . W. Hastings and S. Ghisla, unpublished observations). A negative charge on the ribose residue of the flavin side-chain is important for activity (Meighen and Mackenzie, 1973). Elimination of the negative charge or decreasing the length of the side-chain lowers the luminescent response at least partly due to a decrease in binding affinity between the reduced flavin derivative and luciferase. Interestingly, reduced riboflavin can be used as a substrate with luciferase from V . harveyi if very high concentrations of phosphate or sulphate are added. Presumably, these anions act as autosteric effectors and substitute for the missing phosphate
8
E A M E I G H F N AND P V D U N L A P
on the ribose residue of the side-chain. Removal of hydroxyl groups from the ribose residue of the side-chain does not appear to affect interaction of luciferase with reduced flavin but does destabilize the enzyme intermediates formed during the reaction. By using high concentrations of reduced lumichrome, which corresponds to FMNH2 lacking the ribose phosphate side-chain, 10% of t h e activity of FMNH2 in the bioluminescent reaction can be obtained (Matheson and Lee, 1981). Binding of oxidized flavins to luciferase occurs much less readily. The binding affinity is at least 100-fold lower for the oxidized form compared with the reduced flavin (Baldwin et al., 1975) and there is much less selectivity for FMN (Paquatte and Tu, 1989). 3. Aldehyde Specijicity
Long-chain aliphatic aldehydes are required to obtain high levels of light emission in the bioluminescent reaction. Identification of the aldehyde factor was originally accomplished by Cormier and Strehler (1953) by demonstrating that the factor stimulating luminescence extracted from a rich source of nutrients (kidney cortex) was palmitaldehyde. Large amounts of light can be obtained with fatty aldehydes containing seven or more carbon atoms (Hastings et al., 1963; Hastings and Nealson, 1977). The response is dependent on the concentration of the aldehyde and the particular bacterial luciferase. In general, higher concentrations of aldehyde are required as the chain length of the aldehyde is decreased. At low nonsaturating aldehyde concentrations, luciferases appear to give the highest response with tetradecanal (Meighen et al., 1982), consistent with the proposal that tetradecanal is the natural aldehyde for the luminescent reaction. This latter conclusion is based on the isolation of tetradecanal from luminescent Photobacterium spp. (Shimomura et al., 1974) and preferential synthesis of this aldehyde by lux-specific enzymes responsible for supplying aldehyde to bioluminescent bacteria (Ulitzur and Hastings, 1979a; Rodriguez et al., 1985; Ferri and Meighen, 1991). The specificity for aldehyde chain length at high saturating concentrations of aldehyde is quite dependent on the particular luciferase. Luciferases from V . harveyi and Xenorhabdus luminescens give high activity with nonanal and decanal and lower intensities of light emission with octanal and dodecanal (Frackman et al., 1990; Szittner and Meighen, 1990). This effect is not observed, however, at non-saturating concentrations of fatty aldehyde. For luciferases from V . fischeri and Photobacterium spp., activities generally increase with increasing chain length up to tetradecanal under both saturating and non-saturating concentrations. However, some caution should be advised in interpretation of aldehyde specificity as it may
BACTFKIAL BlOl UMINESCbNCb
9
partially reflect aldehyde solubility under the assay conditions, particularly for aldehydes containing 14 or more carbon atoms. Alterations in aldehyde structure do not significantly decrease luminescence, providing substitutions are not located in close proximity to the carbonyl functional group (Hastings and Nealson, 1977). A series of unsaturated aldehyde pheromones have been shown to give a high level of luminescence (Meighen et al., 1982), including 11-tetradecenal, 11,13hexadecenedial, 14-methyl-ll-tetradecenal and 11- or 13-hexadecenal, among many others. These aldehydes are the primary pheromone components of many common insect pests, including the tobacco budworm, the corn-ear worm, the spruce budworm, the navel orangeworm and the dermestid beetles. In this regard, luciferases have much higher activity with the unsaturated C I 6aldehydes than with hexadecanal (Meighen and Grant, 1985). 4. Luciferase Assays
Knowledge of different luciferase assays is important both in interpretation of the kinetic parameters that have been reported, as well as in understanding the enzyme intermediates and the mechanism of the luminescence reaction. Three different types of assay have been used, namely the standard assay, the dithionite assay and the NAD(P)H-coupled assay. a. Standard Assay In this assay, FMNH, is injected into a solution containing luciferase and aldehyde. Light emission from this solution rises rapidly, reaching a maximum in less than a second and then decays in an exponential fashion as shown in Fig. 3. As excess FMNH, is oxidized chemically in the first second of the reaction, subsequent emission of light is due to turnover of a stable intermediate formed at the start of the reaction. Consequently, luciferase undergoes only a single turnover in this assay. The rate of decay of luminescence (turnover rate of the enzyme; k L ) is primarily dependent on the specific luciferase and aldehyde, with maximum light intensity (lo) depending on the amount as well as the specific bacterial luciferase and substrates. Total light emission corresponds to the integrated area under the curve (Zdk,) in Fig. 3 and is given in quanta if Zo is quoted in quanta per unit time. Luciferase activity, as for other enzymes, is defined as the rate of product formation. In luminescence assays, this activity corresponds to Zo, preferably given in quanta per second. Light intensity is dependent not only on relative amounts of the intermediates formed on the light-emitting pathway, as well
E A MFIGHFN A N D P V DlJNLAP
._ b > ._ c
9
Time (s)
FIG. 3 . Time-course of a standard assay for bacterial luciferase. FMNH2 (1.0 ml) was injccted into 1.0 ml of phosphate buffer, p H 7.0, containing 0.001% decanal and luciferase from Vibrio harveyi. Thc light was dctected by a photomultiplier tube and recorded graphically. Maximum light intensity and the rate of decay of luminescence are indicated by I,, and kL, respcctivcly.
as the rate of the slowest step in this process ( k L ) ,but also on the relative efficiency of conversion of these intermediates into light (i.e. whether the intermediate(s) decays via a light-emitting or dark pathway). A major advantage of the standard luminescence assay is that k , . can be measured from the decay of luminescence independent of total activity (lo).
b. Dithionite Assay An alternative assay that can be used involves injection of aldehyde into a solution of luciferase and FMNHz that has been reduced with sodium dithionite (Meighen and Hastings, 1971). Providing that the reducing agent is not in excess, there is sufficient oxygen in the aldehyde solution for maximum activity to be reached. In this assay, the kinetics of light emission are very similar to those in the standard assay (Fig. 3 ) . Activities and decay rates may be somewhat different in the dithionite and standard assays, depending to some degree on the nature of the luciferase. One reason for this difference is that oxidation of sodium dithionite produces a number of relatively undefined sulphur-containing products that may affect the bioluminescent reaction. However, the major difference between these two assays arises primarily from inhibition by high concentrations of aldehyde in the standard assay but not in the dithionite assay with some luciferases. In the dithionite assay, the enzyme-flavin complex is formed prior to interaction within the fatty-aldehyde substrate. This result is consistent with
11
B A C l E R l A l 6101 UMINFSCENCF
a mechanism in which the enzyme must interact with the reduced flavin before reacting with the aldehyde to form a functional light-emitting intermediate. Although it was proposed that the random order of addition of substrates (FMNH2 and aldehyde) could occur in the bioluminescence reaction (Holzman and Baldwin, 1983), initial work by Hastings etaf. (1965) showed that light could be obtained by adding aldehyde after reaction of the reduced flavin, oxygen and luciferase. Recent studies on the interaction of oxygen with the reduced flavin support an ordered mechanism with FMNHz binding before aldehyde (Baldwin and Ziegler, 1991). c. Coupled Assay Since FMNH2 is oxidized in less than a second in the presence of oxygen (Gibson and Hastings, 1962), continuous light emission in luminescence assays can only be maintained by continuous reduction of FMN using NAD(P)H and a NAD(P)H:FMN oxidoreductase. Such flavin reductases have been purified from luminescent bacteria (Jablonski and DeLuca, 1977; Michaliszyn et af., 1977). Alternatively, a suitable diaphorase capable of reducing free FMN can be used. E + FH,-
I
O2
F + H,O,
I(EFH,)
0,
A ll(EFHO0H)-llA IkD
F + H,O,
kL
- L i g h t
1
Dark
FIG. 4. Pathway showing common intermediates in the bacterial bioluminescence reaction. E indicates luciferase; F and FH2; oxidized and reduced forms of flavin mononucleotide, respectively; A , aldehyde.
5. Enzyme Mechanism
Due to slow turnover of the enzyme in the bacterial bioluminescence reaction, detection and isolation of enzyme intermediates have been readily accomplished (Fig. 4). In the first step, a single FMNH2 molecule is bound to each molecule of luciferase, based on both kinetic and physical studies (Meighen and Hastings, 1971; Becvar and Hastings, 1975), resulting in formation of the intermediate I (EFH2). As nuclear magnetic resonance (NMR) spectroscopy indicates that the N-1 nitrogen atom in the FMNH2 bound to luciferase is deprotonated (Vervoort et a f . , 1986), the correct representation of intermediate 1 may be EFH- rather than EFHz. Intermediate I as well as free FMNH2 react readily with oxygen and consequently, in the standard and dithionite assays already described, FMNHz or molecular oxygen is depleted in the first second of the reaction. Reaction
12
F.. A. MElClIEN A N D P. V. D U N L A P CH2-O-PO',
I
H-C-OH
I
H-C-OH
I
H-C-OH
FIG. 5. Structure of thc stable flavin-peroxy intermediate formcd during the
bacterial bioluminescent reaction.
of molecular oxygen with intermediate I results in formation of a highly stable enzyme-flavin-xygen intermediate (EFHOOH) often referred to as intermediate TI (Hastings and Gibson, 1963; Fig. 4). The resulting EFHOOH complex is stable enough to be resolved from the substrates and products of the reaction by low-temperature chromatography in organic solvents (Hastings et a f . , 1973). At room temperature, the half time for decay of this intermediate in the absence of aldehyde is about three seconds for the luciferase from V . harveyi and about 0.8 seconds for the enzyme from P. phosphoreum (Meighen and Bartlett, 1980). Under these conditions, decay occurs through a dark pathway (kD), producing FMN and hydrogen peroxide (Hastings and Balny, 1975). Intermediate IT has been shown to be a dihydro-4a-peroxy-FMN (Fig. 5) bound to luciferase, based on spectral studies (Hastings et a f . , 1973; Vervoort et a f . ,1986). Independent chemical support comes from the ability to react FMN and hydrogen peroxide with luciferase to form this intermediate (Hastings et al., 1979). Based on I3C NMR spectroscopy and resonance at position 4a of the flavin ring, the peroxyflavin intermediate appears to be in an almost planar configuration (Vervoort et al., 1986). Addition of aldehyde to this complex results in formation of an enzymeflavin-oxygen-aldehyde intermediate (IIA). Turnover of this intermediate is believed to be the rate-limiting step (k13 in the luminescent reaction and can be measured directly from the decay of luminescence. Recent experiments by J. Eckstein, J. W. Hastings and S. Ghisla (unpublished observations) have shown that the rate of luminescence emission is proportional to the oxidation potential of different 8-substituted flavin analogues, indicating that transfer of electrons from the peroxyflavin hemiacetal derivative is the rate-limiting step. The turnover rate is primarily dependent upon solvent conditions, the amount and chain length of aldehyde and the particular luciferase. However, accessory proteins, such as the lumazine protein (Gast and Lee, 1978) and the yellow fluorescence protein (Eckstein
BACTERIAL BlOI UMINbSCbNCk
13
et al., 1990), can affect the rate of light emission. Some caution must be exercised in interpretation of changes in rate of decay of luminescence since turnover can also be affected by aldehyde concentration (Ismailov et al., 1990), and long-chain alkyl compounds such as fatty alcohols and amides which interact with and stabilize dihydro-4a-peroxy-FMN (intermediate 11) can significantly lower the rate of light emission (Tu, 1979; J . C. Makemson, J . W. Hastings and M. E. Quirke, unpublished observations). Indeed, addition of dodecanol to intermediate TI sufficiently stabilizes it so that the peroxyflavin bound to luciferase can be resolved from reactants in an active form (Tu, 1986). As binding of aldehyde is believed to be reversible in many instances (Baumstark et al., 1979), secondary addition of another aldehyde during the course of the luminescence reaction can change the intensity and rate of luminescence emission. The intensity of light emission is of course also dependent on the amount of intermediate IIA as well as the relative efficiency (light as compared with dark pathways) in which intermediate IIA is converted to the excited state and light is emitted (Fig. 4). A number of mechanisms have been proposed by which the fatty aldehyde reacts with the 4a-peroxy-FMN intermediate. One of the first mechanisms postulated was that by Eberhard and Hastings (1972) in which the peroxy group reacts with the carbonyl of the aldehyde. The resulting intermediate then breaks down, with a hydride transfer from carbon to oxygen atoms, leaving the flavin or a derivative thereof in the excited state. However, some questions have been raised as to whether or not the flavin is the emitter since emission of light from free FMN in the excited singlet state occurs at lower energy (higher wavelength, 530 nm) than does light emission in the bioluminescence reaction (490 nm). Addition of the lumazine protein to luciferase also causes a further shift to lower wavelengths of light emitted during the bioluminescence reaction (Lee, 1985). Studies on intzraction of the yellow fluorescence protein with luciferase from V. fischeri have also shown that initial kinetics of yellow light emission (540 nm) indicate a slower emission than for “normal” blue-green light (at 490 nm) although the subsequent rate of decay of yellow and blue-green light is identical (Eckstein et al., 1990). Based on these and other observations, new mechanisms have been proposed. One mechanism involves formation of a radical ion pair from dehydro-4a-peroxy-FMN and aldehyde which in turn, on rearrangement by electron transfer, generates an excited state. A second possibility is that the primary excited singlet state may not be a flavin derivative but a compound with higher energy from which energy can be transferred to a suitable emitter (Raushel and Baldwin, 1989; Eckstein et al., 1990; Baldwin and Ziegler, 1991; Lee, J. et al., 1991a) However, other data support a model in which a flavin derivative forms the primary excited
14
F A MEIGHEN AND P V DUNLAP
state and which can transfer its energy to other emitters (Lee, J. et al., 1991a,b). Consequently, the identity of the high-energy intermediate formed initially from reaction of dihydro-4a-peroxyflavin and aldehyde is still an open question. B . STRUCTURE OF LUCIFERASE
I. Quaternary Structure All bacterial luciferases are 80 kDa heterodimers containing two nonidentical subunits (u and P) of 41 kDa and 37 kDa, respectively (Fig. 6). Based on analysis of mutants and formation of hybrid luciferases, the usubunit appears to dictate the primary kinetic properties, including light emission, aldehyde specificity and turnover rate (Meighen et al., 1971; Cline and Hastings, 1972). However, alterations in the P-subunit do appear to have some affect on interaction with the flavin (Meighen and Bartlett, 1980). The two subunits are homologous in sequence (Johnston et al., 1986), and both subunits appear to be essential for catalytic function. 2. Primary Structure Figure 7 compares the amino-acid sequences of the a- and P-subunits of the luciferase from V . harveyi. A gap of 28 amino-acid residues located after amino-acid residue 256 in the P-subunit is necessary to give best alignment of the sequences of the two subunits. The u- and P-polypeptides of the luciferase from V . harveyi have approximately 30% identity in aminoacid sequence, suggesting that the corresponding genes, luxA and luxB, respectively, arose by gene duplication.
@
41 kDa
@I37kDa
@
54kDa
@
33 kDa
FIG. 6. Diagram showing quaternary structure and subunits of the lux-specific enzymes, luciferase (left) and fatty-acid reductase (right). a indicates a-subunit; p, p-subunit; t, transferase subunit; s, synthetase subunit; r, reductase subunit.
15
B A C l b K l A L BIOLIJMINFSCFNCF
**
*
****
*
**
a**
**
*********
**
64 **
* *
MKFGNFUTYQPPELSQTEVMKRLVNLGKASEGCGFDTWLLEHHFl'EFGLLGNPYVAAAHLLG MKFGLFFLNFMNSKRSSDQVIEEMLDTAHYVDQLKFDTLAWENHFSNNGWGAPLTVAGFLU;
********
*
*
***
* * *** **
*
#
*** *****
** **
****
* *
# **** *******
128
**
ATETLNVGTAAIVLPTAHPVRQAEDVNLLDQMSKGRFRFRFGICRGLYDKD~VFGTD~NS~ HTKNAKVASLNHVITTHHPVRVAEEACLLDQMSEGRFAFGFSDCEXSADMRFFNRPTDSQFQLF
***
*******
**
****
* ***
*
**
b
*
*
* * : *
*
***
*
*
***
192 *
DCWYDLMKEGFNEGYIAADNEHIKFPKIQLNPSAYTQGGAPVYWAESASTTEWAAERGLPMIL
SECHKIINDAFTTGYCHPNNDFYSFPKISVNPHAFTEGGPAQ~ATSK~~~~LPLVF
*
* *
** **
**
**
***
* *
A
* ***
**
d
****
**
***
*
#
e
*
~
**
*
* * .,*
256 ** ** *
SWIINTHEKKAQLDLYNNATEHGYDVTKIDHCLSYITSVDHDSNRAKDICRNFLGHWYDSYVN RWDDSNAQRKEYAGLYHNAQAHGVDVSQVRHXLTLLVNQNVDGEAARAEAFA'YLEEFVRESYS
* *
** **
*
* ** ***
*
* *
*
* **
*
*
** * * *
*
******
*
** * * *
320
******
ATKIFDDSDQTKGYDFNXGQWRDFVLKGHKDT~ID~SYEINPVGTPEECIAIIQQDIDAT~I NTDFEQKMGELLSENAIGTYEESTQAARVAIECCGA * * * * f
******
**
**
*
*
**
355
DNICCGFEANGSEEEIIASMKLFQSDVMPYLKFXQ* ADLLMSFESMEDKAQQRAVIDWNANIVKYHS*
* *** *
*
#
* 324
FIG. 7. Alignment of the amino-acid sequences of the a- and P-subunits of Vibrio harveyi showing conserved residues in the a- o r P-subunits of all luciferases. Aminoacid residues in the a (upper line) and P (lower line) polypeptides have been aligned by introduction of a gap of 28 residues in the 0-subunit. Identical amino-acid residues in the a- or P-subunits of luciferases from all strains are indicated by the asterisks above and below the respective sequences. Positions of insertions o r deletions in specific strains arc denoted by the lower-case letters whereas aminoacid residues that have been changed in the luciferase from V. harveyi by sitespecific mutagenesis arc givcn by #. In addition, four histidine residues in the asubunit at positions 44, 45, 224 and 285 have recently been mutated (Xin et a l . , 1991). a indicates two-codon insertion in Vf, PI, Pp P-subunit; b , one-codon deletion in PI7*, P-subunit only; c, one-codon deletion in Vf, P1, PP a-subunit; d , two-codon deletion in PI P-subunit; e , three-codon insertion in Pp a-subunit; f, one-codon insertion in PI 0-subunit. See Table 4 for strain designation.
16
E A b l ~ I G l I F NAND P V DUNLAP
Sequences of the subunits of bacterial luciferases have been determined from species in the genera Vibrio, Photobacterium and Xenorhabdus, while the primary structure is known for at least 10 different luciferases (Meighen, 1991), although a few of the strains are very closely related. Only 44% of the amino-acid residues in the a-subunits of all luciferases so far sequenced have been conserved. Similarly, only 30% of amino-acid residues in the psubunits of all luciferases are identical. The idential residues in the a- or 0-subunits of all luciferases are indicated by the asterisks above or below, respectively, the primary structures of the a- and p-subunits of the luciferase from V . harveyi (Fig. 7). The relatively high conservation of the a-subunit sequences is consistent with the a-subunit primarily dictating the kinetic properties of the enzyme. By comparison of the conserved residues in both the a- and p-subunits of bacterial luciferases (asterisks, Fig. 7), it can be demonstrated that only 35 residues are identical in all luciferase subunits (both a and p) that presently have been sequenced.
3. Mutations and Active-Site Residues Earlier studies aimed at identifying key residues involved in the catalytic mechanism of luciferase were primarily based on chemical modification studies. A cysteine residue in the luciferase from V . harveyi at position 106 of the a-subunit (labelled # in Fig. 7) was initially thought to be essential for catalytic function (Nicoli et a f . ,1974). However, analysis of the sequence data shows that this residue is not conserved in all luciferases, and that substitution by other amino-acid residues using site-specific mutagenesis does not destroy activity (Baldwin et a f . , 1989b; Xi et af., 1990). A histidine residue on the a-subunit of the luciferase from V . harveyi has been implicated in the functional activity by modification with ethoxyformic anhydride. Mutagenesis experiments have implicated positions 44 and/or 45 (Xin et al., 1991). Because residues with pK, values of about 6.5 have been implicated in FMNH2 binding (Nicoli et al., 1974), it is possible that the histidine residue may be specifically involved in interaction with the flavin. However, other groups may have pK<,values in this range, including the phosphate on the ribose side-chain of FMN, the N-1 nitrogen atom of FMNH2 or even the a-amino group of the N-terminal residues in the luciferase subunits. Modification of a-amino groups in luciferase subunits with 2,4-dinitrofluorobenzene has been shown to inactivate luciferase (Welches and Baldwin, 1981). Since fusion of the N-terminus of the psubunit to the C-terminus of the a-subunit does not inhibit activity (Boylan et a f . , 1989b,c), only the a-amino group of the a-subunit is implicated in activity. Further evidence is required to implicate any residue in the active site of the enzyme.
BACTERIAL BlOtUMlNESCFNCE
17
Some site-specific mutagenesis experiments have focused on residues that have been identified in mutants with altered kinetic properties. Aside from the residue at position 102 in the enzyme from V . harveyi, two other residues have been investigated by site-specific mutagenesis of the a subunit of luciferase. These are the aspartate residue at position 113 (Baldwin et al., 1987) and the serine residue at position 227 (Chen and Baldwin, 1989) in the a-subunit of the luciferase from V . harveyi (labelled as # in Fig. 7). Both residues were identified by cloning and sequencing luciferase mutants, generated originally by Cline and Hastings (1972), that were defective in aldehyde or flavin binding. The aspartate residue at position 113 in the a-subunit is conserved in all luciferases, whereas the serine residue at position 227 is replaced by a threonine residue in some luciferases. Substitution of the aspartate by glutamate or cysteine residues maintains function whereas all other substitutions have much lower activities, suggesting that a negative charge may be important at this position. Replacement of the serine residue at position 227 had little effect on activity except for substitutions by larger amino-acid residues such as phenylalanine found in the original mutant. The effect of truncation of the C-terminus of the P-subunit of the luciferase from V . harveyi on folding of the enzyme has also been investigated (Sugihara and Baldwin, 1988). Deletion of the nine C-terminal residues (# below P-polypeptide, Fig. 7) resulted in a protein that could fold at 20°C but not at 37°C. More extensive deletions affected folding and activity at all temperatures. A similar result has been observed on fusion of the C-terminus of the a-subunit of luciferase from V . harveyi to the N terminus of the P-subunit. Folding could readily be accomplished at temperatures below 30°C whereas, at higher temperatures, very low levels of luciferase were detected (Escher et al., 1989a; Olsson et al., 1990). It should be noted that high levels of luciferase activity can only beeregained if both the a- and (3-subunits are present in the same cell, or removed from denaturant together, indicating that interaction of the two subunits may need to occur during the folding process (Waddle et al., 1987). This could arise, for example, if folding of the individual a- and P-subunits alone generated a hydrophobic subunit-contact surface that could cause aggregation of one subunit, thus diverting it into a non-productive pathway. Earlier studies by Gunsalus-Miguel et al. (1972) showed that recovery of luciferase activity was lower if urea was removed from the denatured P-subunit before complementation with the a-subunit, whereas high activity was obtained if the denaturant-free a-subunit was complemented with the P-subunit. These results suggested that the P-subunit may aggregate in the absence of the denaturant or of the a-subunit. At present, relatively little is known about the critical residues required
18
E. A . MEIGHEN AND P. V. DUNLAP
for generating a functional luciferase molecule, including residues specifically taking part in conversion of chemical into light energy or in the folding process. Clearly, further work on identifying specific residues and the threedimensional structure will be extremely advantageous in view of the expanding use of luciferase for various applications. Although some progress was made towards elucidating the crystallographic structure of luciferase (Swanson et al., 1985) and studies to determine the threedimensional structure are currently underway in a number of laboratories, the three-dimensional structure is not yet known. C. ALDEHYDE BIOSYNTHESIS
1. Structure of the Fatty-Acid Reductase Complex Synthesis of fatty aldehydes as substrates for the bioluminescence reaction is catalysed by a lux-specific fatty-acid reductase complex (Fig. 6). This multi-enzyme complex has been purified from P. phosphoreum and shown to contain three different protein components; these are an ATP-dependent synthetase subunit(s), a NADPH-dependent acylprotein (and acyl-CoA) reductase subunit(r) and an acyltransferase subunit(t) (Riendeau et al., 1982; Rodriguez et al., 1983a,b). The three different polypeptides, r, s and t, with molecular masses of 54, 42 and 33 kDa, respectively, interact to form a large molecular aggregate of approximately 500 kDa with the central core being composed of four reductase subunits (Wall and Meighen, 1986). Each reductase subunit interacts with a synthetase subunit which in turn interacts weakly with a transferase subunit, giving a complex with the structure r4s4tZ4. Since the subunit interactions are relatively weak and the protein complex readily dissociates, purification of the intact complex is relatively difficult and must be conducted at high concentrations of protein. However, the synthetase, reductase and transferase components can be resolved from each other, purified further and then complemented to reconstitute the activities catalysed by the fatty-acid reductase complex. 2. Mechanism of Aldehyde Biosynthesis The specific steps catalysed by the fatty-acid reductase complex and their connection with fatty acid biosynthesis and the bioluminescence reaction are outlined in Fig. 8. a. Transferase Subunit (t) The transferase is responsible for diversion of fatty acids from fatty-acid biosynthesis into the fatty-acid reductase reaction. The acyltransferase can
19
BACTERIAL BIOLUMINESCENCE
e
R-C-AMP
Transferase
B
Synthetadreductase s(r) -R-C-s-R-C-r-
Q
B
r/s
NADP’
Luciferase
H*O
FI
R-C-CoA
INAD
‘IATP
t
r
R-C -H
FMNH,, 0,
LIGHT
FIG. 8. Relationship of the fatty-acid reduction pathway to the luminescent reaction. Activated acyl groups (R-CO-X) are cleaved by the transferase (t) and then activated and reduced by the synthetase (s) and reductase (r), respectively, to form fatty aldehyde for the luminescence reaction. The fatty acid produced in the
luminescence reaction may be recycled. cleave a variety of different esters, including both oxy-esters and thio-esters (Carey et al., 1984; Byers and Meighen, 1985a). Among these esters are monoglycerides, acyl-CoA esters and acyl carrier proteins (ACPs). The ability to cleave a fatty-acyl ACP apparently is unique since the transferase is the only protein in extracts of luminescent bacteria with this activity while a similar activity cannot be detected in extracts of Escherichia coli. Tetradecanoyl derivatives have been demonstrated to be the preferred acyl substrates for the acyltransferases for both P. phosphoreum as well as V . harveyi (Ferri and Meighen, 1991). A preferential but transient accumulation of tetradecanoic acid has been demonstrated in wild-type V . harveyi but not in a transferase mutant of this strain (Byers, 1988). The initial step in the mechanism involves formation of a covalent bond between the acyl group and a serine residue on the transferase. The site of acylation has been identified at Ser79 as part of a characteristic motif, G - X - S - X - G, found in serine esterases (Table 2). Replacement of the serine residue by an alanine residue by site-specific mutagenesis blocks acylation of the enzyme and its acyltransferase activity (Ferri and Meighen, 1991). The acyl group is then transferred to water and other oxygen acceptors as well as thiol groups. Interestingly, the transfer to different acceptors is dependent to some degree on the donor, with transfer to water to form fatty acid more readily accomplished with fatty-acyl ACP than with acyl-CoA as the donor. Moreover, the relative rate of transfer of the acyl group to water to form a fatty acid can be stimulated to some degree by addition of the synthetase subunit. This particular effect is currently not
20
E. A. MEIGHEN AND P . V DUNLAP
TABLE 2.
Active site serine of transferases"
Photobacterium phosphoreum (NCMB 844), P. leiognathi (ATCC 25521), Vibrio Jischeri (ATCC 7744), V. harveyi (B392), Xenorhabdus luminescens (ATCC 29999). The position of the active-site serine residue in the amino acid-residue sequence of the transferase from P. phosphoreum is indicated.
understood although it may indicate that interaction between these two proteins could serve to channel the fatty acid directly to the synthetase subunit. b. Synthetase Subunit (s) The fatty acid is activated by the synthetase subunit to form an acyl-AMP along with the release of PP, (Rodriguez et af., 1985). This activity, which is catalysed by the synthetase subunit alone, can readily be measured by the fatty acid-dependent exchange of labelled PP, into ATP. The acyl-AMP remains tightly bound to the synthetase subunit and can be resolved from the substrates and PP, by gel filtration. Reaction of bound acyl-AMP to form the acylated synthetase intermediate only occurs at a very low level for the isolated subunits. However, in the presence of the reductase subunit in the complex, acylation of the synthetase readily occurs. Transfer of the fatty-acyl group to the synthetase can also be stimulated by the reductase when inactivated by reaction with N-ethylmaleimide, showing that a functional reductase is not required (Fig. 9) (Rodriguez et al., 1988). Interaction with the reductase apparently causes a conformational change in the synthetase so that the site of acylation in the synthetase can come directly into contact with the acyl-AMP. The site of acylation of the synthetase has been recognized as a conserved cysteine residue located in close proximity to the C-terminal (Table 3). Replacement of the cysteine residue at position 364 in the synthetase from P. phosphoreum by a serine residue blocked acylation and fatty-acid reductase activity. However, PP,-ATP exchange activity was fully retained (Soly and Meighen, 1991). In contrast, replacement of any of the other conserved cysteine residues by a serine residue did not affect acylation or activity. A model in which the C-terminal region of the synthetase containing the cysteinyl residue is forced into juxtaposition with the acylAMP bound to the synthetase on interaction with the reductase subunit has been proposed (Soly and Meighen, 1991). Moreover, this model would
21
BACTERIAL BIOLUMINESCENCE
FIG. 9. Dependence of acylation of the synthetase on the reductase subunit of the reductase complex from Photobacterium phosphoreum. Synthetase (2 pg per SO pl) was rcacted with 10 p~ [3H]tetradecanoic acid (27 Ci mmol-’) and 1 mM ATP in 0.05 M phosphate, pII 7.0, cithcr alone (s), with 2.5 pg of reductase (r/s) or Nethyl maleimide inactivated reductase (r’/s). Samples were run on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and the positions of the labelled reductase and synthetase detected by fluorography. TABLE 3. Active site cysteine of synthetascs 364
Ppa
7
R
E
Q
K
G
C
S
L
S
M
A
p1
-
-
-
-
-
-
-
-
-
-
-
-
Vf Vh XI
-
- , - - - A _ - _ _ - G M - - - A - - - S - I - - - A - L T
S G T S N V Q A
L K * - - * I Q K N G T A K
E
F
A
D
S
I N
K D
D ’ G G N *
*
‘See Table 2 for strain description. The position of the active-site cysteine residue in the amino acid-residue sequence of the synthetase from P. phosphoreurn is indicated.
be consistent with an acylation site on the reductase coming inio direct contact with the acylated cysteine residue (C-364) and transfer of the acyl group to the reductase (Wall et al., 1986). c. Reductase Subunit (r) Although the specific site of acylation on the reductase has noi yet been elucidated, since the acyl group on the reductase as well as the synthetase can readily be removed by hydroxylamine, it appears clear that the acyl group is transferred to a cysteinyl residue on the reductase. Alternatively, acyl-CoA can react directly with the reductase to form an acylated derivative, presumably on the same cysteinyl residue. In contrast to the transferase, however, the reductase cannot be acylated with either acylACP o r with acylglycerides, while transfer of the acyl group from the reductase can occur to thiol acceptors but not to oxygen acceptors.
22
E A MEIGHEN AND P V DUNLAP
Acyl-group transfer between the synthetase and the reductase is readily reversible (Wall et al., 1986). Consequently, acylation of the synthetase by acyl-CoA can occur by transfer of an acyl group from the reductase. The final step in formation of the aldehyde substrate for the luminescence reaction involves reduction of the acylreductase intermediate by NADPH. A K , value of 1 ~ L Mfor NADPH has been reported for the fatty-acid reductase reaction at saturating substrate concentrations of tetradecanoic acid and ATP. The stoicheiometry of the reaction has been demonstrated to be 1:l for the amount of fatty acid consumed and production of AMP, aldehyde and NADP', showing that the reactions are tightly coupled. Maximum activity has been measured using C,, fatty acids, which is consistent with tetradecanal being the natural substrate for the luminescence reaction (Rodriguez et al., 1985). The fatty aldehyde is then released into solution or possibly channelled directly to luciferase as a substrate for the luminescence reaction. Evidence for direct interaction between luciferase and the fatty-acid reductase complex has not yet been obtained. However, this putative interaction would have to be quite weak since the relative number of luciferase molecules in bacteria exceeds the number of fatty-acid reductase molecules by about 10-fold, at least in P. phosphoreum. Since the aldehyde is converted back into a fatty acid during the luminescence reaction, recycling of the fatty acid back through the fatty-acid reductase reaction may be possible (Fig. 8).
3. IdentiJication and Distribution of the Fatty-Acid Reductase Polypeptides Although the fatty-acid reductase complex containing the transferase, synthetase and reductase subunits has been isolated and purified only from P. phosphoreum, these polypeptides have been recognized easily in other luminescent bacteria, including Photobacterium leiognathi, V . harveyi, V . fischeri and X . lurninescens, by specific acylation with labelled fatty acids (Wall et al., 1984a; Boylan et al., 1985, 1989a; Lee, C. Y. et al., 1991). From these species, only the transferase from V. harveyi has been purified (Byers and Meighen, 1985b). However, the genes luxC, luxD and luxE, coding for the reductase, transferase and synthetase, respectively, have been cloned and sequenced from at least one strain of each of these five species. D. ACCESSORY lUX PROTEINS
1. Lurnazine and Yellow Fluorescent Protein Extensive studies have been conducted on the lumazine protein found in Photobacterium spp. (Lee, 1985; Vervoort et al., 1990). This protein is a
BACTtRIAL Bl OLUM l Nt SCt N C E
23
22 kDa monomer which has 1 mol of non-covalently bound 6,7-dimethyl8-ribityllumazine, the natural precursor to riboflavin. Mixing of the lumazine protein with luciferase shifts the peak of luminescence by about 1020 nm to a lower wavelength and stimulates the level of luminescence. These effects have been observed on mixing not only the lumazine protein with luciferases from Photobacterium spp. but also with luciferases from Vibrio spp. Analyses of the interaction by physical and spectral techniques have demonstrated that there is a weak interaction between luciferase and lumazine proteins. Since light emission is shifted to a higher energy level, it has been proposed that the flavin cannot be the initial excited state. However, the possibility that interaction of the lumazine protein and luciferase changes the micro-environment of the flavin sufficiently to cause emission at a lower wavelength must also be considered. A detailed analysis of the mechanism of the bioluminescence reaction and the role and interactions of the lumazine protein in energy transfer and the excited state are given in a recent review by Lee et al. (1991a). In one V . jischeri strain, a yellow fluorescent protein has been isolated and purified which causes a shift in the maximum of luminescence to a higher wavelength, and results in emission of yellow rather than blue-green light. The flavin bound to the enzyme in this complex appears to be FMN (Daubner et al., 1987) and not lumazine. The lumazine protein from P. phosphoreum and the yellow fluorescent protein from V.jischeri have been cloned and their amino-acid sequences determined (O’Kane et al., 1991; Baldwin et al., 1990). These proteins were found to have approximately 35% identity in sequence even though one protein bound FMN while the other bound lumazine. Moreover, both proteins were related in sequence to the a-subunit of riboflavin synthetase. Recently, a new gene coding for a protein homologous to riboflavin synthetase and closely linked to the lux genes in P. leiognathi has been identified which codes for a protein with about 30% identity in amino-acid sequence with both the lumazine protein from P. phosphoreum and the yellow fluorescence protein from V . jischeri (Lee and Meighen, 1992).
2. Non-fluorescent Flavoprotein Another protein which contains a tightly bound flavin derivative that does not readily fluoresce has been found in most but not all Photobacterium strains (Kasai et al., 1987; O’Kane et al., 1987). The flavin bound to this protein has been suggested to contain a residue of tetradecanoic acid covalently linked at position 6 through the p- or &-carbonatom of the fatty acid (Kasai et al., 1991). The sequences of the N-terminal residues of this protein from P. phosphoreum and P. leiognathi (O’Kane et al., 1987; Soly
24
E A MEIGHFN AND P V DUNLAP
et al., 1988; Kasai et al., 1991) correspond to the N-terminal sequence of the protein coded by the IuxF gene located immediately adjacent to the luciferase genes. A high degree of amino-acid residue sequence homology has been demonstrated between the luciferase subunits and the nonfluorescent flavoprotein, particularly with the I)-subunit of luciferase. 3. Other Lux-Related Proteins Other lux genes which code for proteins involved in regulation, or have unidentified functions, are described in the next section. Aside from proteins coded by lux genes, two other enzyme functions appear to be related to metabolism of substrates for the luminescence reaction. One group of enzymes are the NAD(P)H:FMN oxidoreductases that appear to be present in all luminescent bacteria, and of which some have been purified to homogeneity from V . harveyi (Jablonski and Deluca, 1977; Michaliszyn et al., 1977). Such enzymes are likely to be responsible for generating FMNH2 for the luminescence reaction and to be important for coupling the formation or level of NAD(P)H with the luminescent reaction in order to generate continuous light emission in coupled assays. An aldehyde dehydrogenase with a high specificity for NADP' and longchain fatty aldehydes has been purified from V . harveyi (Byers and Meigben, 1984). Although this enzyme is under different regulation from luciferase and the fatty-acid reductase polypeptides (Byers et al., 1988), it could still be involved in controlling the level of fatty aldehyde produced in this luminescent bacterium. 111. Molecular Biology A . ORGANIZATION OF
lUX
GENES
1. The luxCDAB(F)E Genes The number of lux genes cloned from bioluminescent bacteria has expanded remarkably in the last few years. Table 4 gives a list of over a dozen strains of luminescent bacteria from which lux-related genes have been isolated, representing at least five different species of bacteria. In those species examined ( P . leiognathi, P. phosphoreum, V . jischeri, V. harveyi and V . vuln@cus), the lux genes are chromosomal rather than plasmid-borne (Simon et al., 1982; Oliver et al., 1986). All bacterial luminescent systems so far analysed contain genes coding for the a- and I)-subunits of luciferase ( l u x A B ) and genes coding for the r-, t- and s-subunits of the fatty-acid reductase complex (luxCDE) responsible
25
BACTERIAL BIOLLJMINESCENCE
TABLE 4.
Sources of cloned lux genes in luminescent bacteria ~
~
~~~~
~~
Species
Strain"
Designation (this review)
Initial report"
Photo bacterium phosphoreum
NCMB 844 A13
PP Pp-A 13
Mancini et al. (1988) Prasher et al. (1990)
Photobacterium leiognathi
p1721, p1741 PI,,,
PI721 p1s54
ATCC 25521
PI
Delong et al. (1987) Illarionov and Protopopova (1987) Lee, C.Y. et al. (1991)
Vibrio fischeri
MJ1 ATCC 7744 Y1 ESll4'
Vf-MJ 1 Vf Vf-Y 1
Engebrecht et al. (1983) Boylan et al. (1985) Baldwin et al. (1990) Gray and Greenberg (1992)
Vibrio harveyi
B392 BB7
Vh Vh-BB7
Cohn et al. (1983) Belas et al. (1982)
Xenorhabdus luminescens
Hm ATCC 29999, Hb Hw
XI-Hm XI XI-Hw
Frackman et al. (1990) Szittner and Meighen (19%) Xi et al. (1991)
Kryptophanaron alfredi symbiont
Ka
Ka
Haygood and Cohn (1986)
~
~
~~
The lux gene sequences of most strains for a given species appear to be closely related (>97% identity in amino acid-residue sequence) except for the PI strain (ATCC 25521), which differs in the amino-acid residue sequences (23-26%) for the lux genes from those for the other Photobacterium leiognathi strains as well as XI-Hw, whose luciferase differs between 5 and 15% in amino acid-residue sequence from those encoded by the lux genes of the other Xenorhabdus luminescens strains. Nucleotide sequences and any additional lux genes cloned from these strains are generally given in subsequent reports. See references in Tables 5 and 6 for sources of nucleotide sequences. Eupryma scolopes symbiont. a
'
for synthesis of the aldehyde substrate. Table 5 gives the number of amino acids (codons) coded on the luxA-E genes from the different strains. In addition to the common lux genes, a number of other genes, either part of or closely linked to the lux operon and/or related to expression of luminescence, have been sequenced. Table 6 summarizes their properties, distribution and location in different luminescent strains. The different lux-gene organizations found in luminescent bacteria are outlined in Fig. 10. All luminescent bacteria contain the luciferase genes luxAB flanked upstream by luxCD and downstream by luxE with transcription from left to right. However, for P. phosphoreum (Mancini et al., 1988, 1989; Soly et al., 1988) and some strains of P. leiognathi (Illarionov et al., 1988; Baldwin et al., 1989a) the gene luxF is located between luxB and
26
E. A. MEIGHEN AND P. V . DUNLAP
TABLE 5.
Common lux genes from bioluminescent bacteria
Genes"
Codonsb
Protein product
Function and properties
luxA
354-360
a-Subunit
luxB
324-328
0-Subunit
Subunits of luciferase (ap), which catalyse the luminescence reaction. Approximately 30% identical in amino acidresidue sequence
luxC
477480
r (reductase)
lux D
305-3 14
t (transferase)
IuxE
37G378
s (synthetase)
Responsible for transfer (t), activation (s) and reduction (r) of fatty acids to generate aldehyde for the luminescence reaction. A multi-enzyme fatty-acid reductase complex (r4sqtZ), has been characterized from Photobacterium phosphoreum.
Nucleotide sequences of the luciferase genes (luxAB) have been reported for Pp (Ferri et al., 1991), PI,Z, (Baldwin et al., 1989a), PI,,, (Illarionov et al., 1990), P1 (Lee, C. Y. et al., 1991) Vf-MJ1 (Foran and Brown, 1988), Vf (Baldwin et al., 1989a) Vh (Cohn et al., 1985; Johnston etal., 1986),XI-Hm (Johnston etal., 1990), XI (Szittner and Meighen, 1990) and XlHw (Xi et al., 1991; Meighen and Szittner, 1992) as well as the nucleotide sequence of Ka luxA (Haygood, 1990). Nucleotide sequences for the fatty-acid reductase genes (luxCDE) have been reported for Vh (Miyamoto et al., 1988a,b; Johnston et al., 1989), Vf (Baldwin et al., 1989a), PI (Lee, C . Y . etal., 1991), XI (Meighen and Szittner, 1992) and XI-Hw (Xi e t a l . , 1991; Meighen and Szittner, 1992). In addition luxD from XI-Hm (Cochrum et al., 1990) and luxDE from Pp (Soly and Meighen, 1991; Ferri and Meighen, 1991) have been published. The number does not include the stop codon. The range is indicated for different bacterial species.
'
luxE whereas luxF is absent from Vibrio and Xenorhabdus spp. as well as in the neotype P . leiognathi strain (Lee C . Y. et al., 1991). 2 . Downstream D N A Downstream of luxE in all luminescent marine but not terrestrial bacteria is the luxG gene (Swartzman et al., 1990a,b; Lee, C. Y. et al., 1991). This gene is very closely linked to the ZuxE gene, with the ribosome-binding site being part of the ZuxE coding region in some species. Transposon insertions in this region of the genomes in V . harveyi or V . jischeri apparently do not block light emission (Engebrecht et al., 1983; Martin et al., 1989). Moreover, transfer into E . coli of lux D N A with the luxG gene
27
BACTERIAL BIOLUMINESCENCE
TABLE 6. The lux genes identified in specific luminescent strains Gene"
Codons
Protein function and properties
Gene location
luxFh
228-231
Non-fluorescent flavoprotein (27 kDa) with homology in sequence with the luciferase subunits
In most but not all Photobacterium strains between luxB and luxE
luxG
233-236
A 27 kDa protein related in sequence (40% identity) to a protein encoded by a gene in Escherichia coli (C. DiRusso, personal communication) with flavin reductase activity
Found after luxE in marine but not terrestrial luminescent bacteria
luxH
230
A 27 kDa protein related in sequence (64% identity) to a new protein (HtrP) involved in survival and growth of Escherichia coli above 37°C (Raina et al., 1991) and riboflavin synthesis (Richter et al., 1992)
Found in Vibrio harveyi after luxG
luxl
193
Regulatory protein (22 kDa) required for synthesis of autoinducer of Vibrio fischeri
Found in Vibrio fischeri upstream of luxC
luxR
250-252
Regulatory protein (29 kDa) in Vibrio fischeri required for response to autoinducer
Found in Vibriofischeri in a divergent operon immediately upstream of an operon containing the lux structural genes
luxR*
205
Regulatory gene locus controlling expression of lux genes in Vibrio harveyi. No homology in sequence to luxR in Vibrio fischeri
Gene locus in Vibrio harveyi not linked to the lux structural genes
lump
189
Lumazine protein (21 kDa) involved in spectral shift of luminescence emission. Homologous in amino acidresidue sequence to the a-subunit of riboflavin synthase (27% identity)
Found 600 bp upstream of luxC in Photobacterium phosphoreum'transcribed in the opposite direction. Possibly present in all Photobacterium strains
luxY
194
A yellow fluorescence protein (YFP; 22 kDa) that results in emission of yellow light in one Vibrio fischeri strain. Homologous in sequence (35% identity) to the lumazine protein
Found in one strain of Vibrio fischeri (Yl). Not linked to the lux structural genes
The sequences of the lux genes from the following strains have been reported; luxF from Pp (Soly et al., 1988), PI,,, (Illarionov et al., 1990) and PI,,, (Baldwin et al., 1989a); luxG from Vh (Swartzman et al., 1990a), Vf (Swartzman et al., 1990b) and PI (Lee, C. Y. et al., 1991); l u H from Vh (Swartzman et al., 1990a); luxIR from Vf-MJl (Engebrecht and Silverman, 1987) and Vf (Devine et al., 1988); luxR* from Vh-BB7 (Showalter e t a ! . , 1990); lump from Pp-A13 (O'Kane et al., 1991); and luxY from Vf-Y1 (Baldwin et al., 1990). The Pp luxF gene has been denoted as luxG in PI554 and as luxN in
28
-
E. A . MEIGHEN AND P . V. DUNLAP
*
P p - C I
0
1
2
3
D I
A
4
5
I
6 I F 5
6
7
E
8
I G I
9
1
0
Gene length (kb) FIG. 10. Organization of lux genes of bacterial luminescent systems. The different organizations are based primarily on the published nucleotide-sequence data for different luminescent bacteria (see Tables 5 and 6) and organization of the lux genes for PI, reported in a recent review (Baldwin and Ziegler, 1991). PI, corresponds to PI in Table 4. Extended non-coding regions are indicated by the diagonal lines and the 5' and 3' ends of mRNA identified by S1 nuclease andlor primer extension given by the solid circles. Large arrows indicate the direction of transcription whereas small arrows indicate the direction of transcription of other genes not yet identified. Names of bacteria are given in Table 4.
deleted was not accompanied by loss of luminescence (Miyamoto et al., 1987, 1988a). Recently, a gene Cfre) encoding a 26 kDa protein in E . coli (Spyrou et al., 1991) has been detected whose amino acid-residue sequence has 40% identity (Andrews et al., 1992) to that coded by the luxG gene (Table 6 ) . The protein in E. coli coded by the fre gene has been purified to homogeneity and shown to be a flavin reductase catalysing NAD(P)Hdependent reduction of flavins, including FMN and riboflavin (Fontecave et al., 1987). As the sequences of luxG and fre are related, it may be that the luxG gene codes for a similar function. This possibility is particularly interesting since NAD(P)H:FMN oxidoreductases have been purified from luminescent bacteria (Jablonski and DeLuca, 1977; Michaliszyn et al., 1977) and are likely to be responsible for generating FMNH2 for the luminescence reaction. As the luxG gene is not present immediately downstream of luxE in Xenorhabdus spp., it may be characteristic only of marine luminescent bacteria. Alternatively, it may be located further downstream or at another location on the genome in X . lurninescens. A gene that appears to be unique to the lux system in luminescent V . harveyi is luxH. The protein coded by a new gene (htrP) involved in stability
BACTERIAL BIOLUMlNESCENCb
29
and growth of E. coli abouve 37°C has been found to have a high degree of homology with the amino acid-residue sequence for the IuxHgene (Table 6). Both genes code for proteins of 27 kDa and have 64% identity extending across their entire sequence. Recently, the htrP gene has been found to synthetase, and enzyme code for a 3,4-dihydroxy-2-butanone-4-phosphate required for riboflavin synthesis (Richter et al., 1992). The high degree of homology suggests that these proteins have similar functions in the two bacteria. It is possible that luxH is required for light emission only under specific environmental, nutritional or physiological conditions. However, the possibility that riboflavin synthesis and luminescence are under the same regulation should be considered. A classical rho-independent terminator is present downstream of IuxH in V . harveyi and IuxG in V . jischeri (Swartzman et al., 1990a,b). In V . jischeri, the terminator functions bidirectionally and thus serves as a termination site for not only the IuxICDABEG operon but also for a convergent gene. Insertions of lux DNA from V. harveyi or V . jischeri between the lux promotor in V . harveyi (located upstream of luxC, see later) and a reporter gene (cat) coding for chloramphenicol transacetylase have demonstrated that, on transfer of the recombinant plasmid back into V. harveyi by transconjugation, only DNA that contains the termination site after IuxG in V. fischeri or IuxH in V . harveyi blocked expression of the cat gene (Miyamoto et al., 1990; Swartzman et al., 1990b). In contrast, other lux DNA in V . harveyi IuxCDABEGH or V . Jischeri LuxICDABEG did not prevent expression of the cat gene.
3. Upstream D N A Upstream of luxC in V . jischeri are the luxl and luxR genes, which are involved in regulation of luminescence expression in this species.“The luxl gene is part of the safie operon as the IuxCDA BEG genes and is believed to be responsible for synthesis of a small molecule (called an autoinducer) that causes induction of luminescence (Engebrecht et al., 1983; Engebrecht and Silverman, 1984). The IuxR gene is on a divergent operon and has been proposed to code for a protein involved as a receptor for the autoinducer. Transcriptional-start sites and open-reading frames for luxR and luxl genes flanking a 219 bp regulatory region have been defined by DNAsequence analysis, Slnuclease mapping and deletion mapping of the cloned lux DNA from V . Jischeri strains MJ-1 and ATCC 7744. This regulatory region contains two identified promoters. The promoter for IuxR contains -10 and -35 regions very similar to consensus sequences of “housekeeping” promoters in E. coli, whereas the promoter for ZuxICDABE contains a
30
E A MEIGHEN AND P V DUNLAP
-10 region but no -35 region similar to other consensus sequences seen in this bacterium. This intergenomic region has been implicated in a number of different regulatory functions (see Section 1II.B). In contrast, in the other luminescent bacteria, non-coding DNA highly enriched in A T content extending over 700 bp in some species is present immediately upstream of IuxC. Consistent with these data, a promoter upstream of luxC in V . harveyi was identified and found to be regulated by cell density and by glucose concentration (Miyamoto et al., 1990). The promoter contained a typical -10 region but no recognizable -35 region, and lux operon mRNA started 26 bases before the translation initiation codon for luxC (Swartzman et al., 1990a). Synthesis of mRNA encoded by this region was found to be inducible in a cell density-dependent manner. A gene thought to be analogous to IuxR (denoted here as luxR*) has also been detected in the genome of V . harveyi unlinked to the lux structural genes. The luxR* gene, however, is not related in sequence to the IuxR gene of V . fischeri (Showalter et al., 1990). Transposon mutagenesis of the luxR* gene blocks formation of lux mRNA and prevents expression of the luminescent system in V . harveyi (Martin et al., 1989). An additional difference between the systems in V .fischeri and V . harveyi is that the region upstream in luxC in V . harveyi bears only a slight resemblance to the IuxR-regulatory region-luxl arrangement in V.fischeri. A leftward-oriented open-reading frame of just over 600 bp occurs 640 bp upstream of luxC in V . harveyi (compared to approximately 850 bp for IuxR in V. fischeri). However, it does not appear to be involved in the luminescence system (Miyamoto et al., 1989). It is preceded by a potential CAMPreceptor protein (CRP)-binding site approximately 200 bp from the start of the open-reading frame (compared to approximately 100 bp in V. fischeri; Miyamoto et al., 1988b; Engebrecht and Silverman, 1987; Baldwin et al., 1989a). However, the region adjacent to luxCDABE which corresponds to the luxl gene of V . fischeri contains numerous stop codons on both strands in each reading frame (Miyamoto et al., 1988b), so a comparably located luxl gene apparently does not exist in V. harveyi (Miyamoto et al., 1988a). The lack of a luxl gene upstream of luxC is consistent with the location of the IuxCDABEGH promoter just upstream of luxC (Swartzman et al., 1990a). In Photobacterium spp., as in V . harveyi, extended AT-rich regions are also present above IuxC. In P. phosphoreum, however, the divergent gene 650 bp upstream of luxC (lumP) has been shown to code for the lumazine protein (Prasher et al., 1990; O’Kane et al., 1991) which is involved in affecting the emission spectrum of the luminescence system. The lumazine protein has been shown to be related in sequence to the a-subunit or
BACTERIAL BIOLUMINESCENCE
31
riboflavin synthetase (O’Kane et al., 1991), which is involved in conversion of lumazine into riboflavin. Analysis of the DNA extending 750 bp upstream of luxC in the neotype P. leiognathi strain (ATCC 25521) (Lee, C. Y. et al., 1991) did not reveal an extended open-reading frame for the lumazine protein. However, recent experiments have shown that a gene coding for a protein closely related to the a-subunit of riboflavin synthetase and to a lesser extent to the lumazine protein is closely related to the lux genes in this species (Lee and Meighen, 1992). Another gene (luxY), coding for a yellow fluorescence protein, is also related in sequence to lump (Baldwin et al., 1990). The ZuxY gene produces a protein involved in shifting the peak of luminescence to a higher wavelength, resulting in emission of yellow rather than blue-green light in a specific strain (Y-1) of V . jischeri.
B. DIFFERENTIAL EXPRESSION OF LUX GENES
The luxA-E genes are expressed to quite different levels although located on the same operon. The amount of luciferase coded by the luxA and luxB genes is at least five times higher than the amount of fatty-acid reductase coded by the luxC, luxD and luxE genes (Wall et al., 1984b; Meighen, 1988). In V . harveyi, these differences appear to be related to the levels of mRNA coding for the respective genes. A set of mRNAs, including a large polycistronic mRNA extending from the start of luxC to the end of luxH, has been detected by Northern blotting (Miyamoto et al., 1985, 1988a). The mRNAs most readily detected started before luxA or LuxD, and terminated after luxB, thus indicating that there were much higher levels of mRNA coding for luxA and luxB than mRNA for fatty-acid reductase genes, in particular luxC and luxE. This set of mRNAS appears to arise by processing, since only one strong promoter (before luxC) and terminator (after luxH) have been detected in the lux operon of this bacterium (Miyamoto et al., 1990). A potential stem-loop structure possibly involved in stability of the upstream mRNA coding for luxA and luxB occurs in the lux operon of V . harveyi between luxB and luxE (Sugihara and Baldwin, 1988). Removal of this region decreases luciferase expression. A similar stem-loop structure is also found in V . jischeri (Baldwin et al., 1989a) and the neotype strain of P . leiognathi (Lee C . Y . et al., 1991) also between luxB and luxE. Stemloop structures with a high degree of homology with that from the lux system of P . leiognathi (ATCC 25521) are also found in P . phosphoreum (Soly and Meighen, 1991) and in the other P . leiognathi strain (PIss4; Illarionov et al., 1990) between the luxF and luxE genes. Although not
T C A C G T A-T C A G-C T-A G-C A-T C-G G-C * G-C T-A A-T A-T T T T-A A-T TGCCATTA AAATTAAATAAGGGTTAACATG luxE
c
T T
c
G T G T T-A G-C
---
--
PI
PP
~~
T-A TCATTA ATTTTTTATTAAGGTGTAATD luxE
T A A T T A-T T-A T G T-A A-T .A-T C-G *G-C 'G-C TAATAAAATTAAG TTTTTTGCATTTCTGTTGATATTAG luxB
-
T T T A A A A T-A C-G G T T T G-C A-T 'C-G G-C G-C T-A A-T A-T T-A A-T GCCTCTT-A TTTAATTATATTTTAAATAGGATTAAACD
Vf
luxE
C G G A * A-T * C-G * G-C ~. * G-C G-C G-C A-T A-T WCGTTTAACTGATGCTG ATATCACCATTCTTTTCG luXB
Vh
FIG. 11. Intergenic stem-loop structures found before luxE genes in Vibrio and Photobacterium spp. Paired residues conserved in all stem structures are indicated by a dot with the boxed residues in being conserved in all Photobacterium spp. Names of bacteria arc given in Table 4. Reproduced by courtesy of C. Y. Lee.
BACTERIAL BlOl UMINFSCPNCF
33
found in P1721,the identical sequence of P1554can be found in this strain near the end of luxE (Baldwin et al., 1989a). Figure 11 depicts the stem-loop structures from five different strains of luminescent bacteria. The seven boxed pairs of residues are conserved in the stem of this structure in all Photobacterium strains and are located between 20 and 30 nucleotides in front of the luxE gene. The paired residues indicated with a dot are conserved in the stems of all structures. Clearly, there is a remarkable conservation of the stem-loop structure, particularly in Photobacterium spp., even though, for the neotype strain of P. leiognathi (ATCC 25521), it is located between luxB and luxE while for other Photobacterium strains it is located between luxF and luxE. If this structure is responsible for the stability of upstream mRNA then, in Photobacterium spp. containing the luxF gene, high levels of mRNA transcribed from this gene would be present. Consistent with this prediction are data showing that the non-fluorescent flavoprotein coded by luxF is synthesized coordinately with luciferase (Kasai et al., 1987). UGAACG G A AUAG A A A A A GA G A U GA A GGC GC CCCCAG GCAUAG U UUUCGAGWGC GC UAUACCU CCG CG GGGGUC UGUAUC A AAAGWCGACG CG AUAUGGG A G A A A A A U AG A AGUA A A A G AAC AGUGA
FIG. 12. Potential stem-loop structure formed from the ERIC sequcncc between the luxR and luxE genes in Xenorhabdus luminescens ATCC 29999.
A potential stem-loop structure with high stability has also been identified between luxB and luxE in X . luminescens (ATCC 29999) (Sziltner and Meighen, 1990). This structure has recently been recognized as part of a 126 bp enterobacterial repetitive intergenic consensus (ERIC) sequence found in genomes of enterobacteria including E. coli and Salmonella typhimurium (Hulton et al., 1991). These E R I C sequences have the potential to form extended stem-loop structures of high stability. Figure 12 shows the potential stem-loop structure for the E R I C sequence between luxB and luxE in X . luminescens ATCC 29999. Only 18 of the 126 bp nucleotides differ from the consensus sequence, which is an even closer relationship than most of the E R I C sequences from other enterobacteria (Hulton et al., 1991). A recent determination of the nucleotide sequence of the lux operon for a different strain of X . luminescens (Xl-Hw) revealed that there are multiple copies of the ERIC sequence. One copy of the E R I C sequence is between
34
k A MFlGHbN AND P V DUNLAP
IuxD and luxA, while DNA sequences equivalent to two copies of the ERIC sequence are between luxB and luxE, in contrast to the single ERIC sequence in the lux operon of X . lurninescens (ATCC 29999) (Meighen, 1992). The presence of multiple copies of the ERIC sequence in one bacterial operon flanking the luciferase genes raises some interesting questions concerning the role of these structures. C . E X P R E S S I O N OF l U X G E N E S IN O T H E R ORGANISMS
The lux genes have been transferred into both prokaryotes and eukaryotes. Recombinant DNA containing lux genes can readily be isolated (Miyamoto et al., 1986; Engebrecht and Silverman, 1986a; Shaw et al., 1987) and transferred into bacteria by transformation, transduction or transconjugation, depending on the species (Engebrecht et al.. 1985; Legocki et al., 1986; Schmetterer et al., 1986; Carmi et al., 1987; Hirooka et al., 1987; Rogowsky et al., 1987; Boivin et al., 2988; O’Kane et al., 1988; Shaw et al., 1988; Blissett and Stewart, 1989; Karp, 1989; Langridge et al., 1989; Park et al., 1989, 1991; Schauer et al., 1989; Jassim et al., 1990; Miyamoto et al., 1990). The lux genes have been transferred into Gram-positive and Gram-negative bacteria, including plant and human pathogens, encompassing bacteria from over 25 genera (Meighen, 1991). In most systems, only IuxAB genes coding for luciferase have been transferred, and light can be generated in vivo by addition of an aldehyde (preferably decanal or nonanal). The five lux genes ( l u x C D A B E ) coding for luciferase and the fatty-acid reductase complex responsible for aldehyde biosynthesis can also be transferred into bacteria using a single promoter. In these transfers, new luminescent species requiring no exogenous additions are created since precursors for aldehyde biosynthesis as well as sufficient FMNHz are present in most if not all prokaryotes. Among many applications of these new luminescent bacteria are: (a) measurement of the strength and regulation of promoters under different physiological, nutritional and environmental conditions; (b) ability to measure the distribution of a particular bacterium and its response to different molecules (i.e. inhibitors) that affect either the viability and/or metabolic functions of the cell thus disrupting luminescence; and (c) recognition of specific bacteria in the environment (e.g. Salmonella spp.) using species-specific vectors containing lux genes (Ulitzur and Kuhn, 1986). Fusion of IuxA and IuxB genes has resulted in monocistronic IuxA-B genes coding for active fused luciferases with the C-terminal of the asubunit linked by a short polypeptide to the N-terminal of the p-subunit (Boylan et al., 1989b,c; Escher et al., 1989a,b, 1991; Kirchner et al., 1989; Koncz et al., 1989; Olsson et al., 1989, 1990; Almashanu et al. , 1990; Costa
BACTERIAL BlOLtJMlNFSCENCE
35
et al., 1991). Consequently, the monocistronic fused bacterial luciferase can be expressed in eukaryotes without the necessity for having luxA and luxB genes under separate promoters (Koncz et al., 1987). The fused luciferases have been expressed in a variety of eukaryotic organisms, including yeast (Saccharomyces cerevisiae) , plants (Daucus carota, Nicotiana tabacum, N . plumbaginifolia), insects (Drosophila melanogaster, Spodoptera frugiperda) and mammalian cells (mouse fibroblasts) (Meighen, 1991). At present high expression can only be obtained at temperatures of 30°C or lower since the fused luciferase cannot readily fold at higher temperatures (Escher et al., 1989a).
IV. Physiologicial and Genetic Control of lux-Gene Expression
Both cellular and environmental factors control luminescence in bacteria. 'The ways in which the operative factors function at the genetic level are known to a greater or lesser extent in certain systems (e.g. autoinducer and CAMP in V . fischeri) but, for other factors (e.g. iron, osmolarity, oxygen), the mechanisms remain obscure. A. AUTOINDUCTION
In many luminous bacteria, luciferase synthesis and luminescence exhibit a distinctive pattern of cell density-dependent induction called autoinduction (Eberhard, 1972; Nealson, 1977; Rosson and Nealson, 1981). Strains expressing luciferase constitutively are known, however (Watanabe et al., 1975; Katznelson and Ultizur, 1977; Rosson and Nealson, 1981). Autoinduction is mediated by accumulation during growth (hence cell-density dependence) of an inducer molecule, called an autoinducer (Kempner and Hanson, 1968; Nealson et al., 1970; Eberhard, 1972). When autoinducer reaches a threshold concentration, which generally occurs above a cell density of lo7 cells ml-', it triggers synthesis of luciferase and other enzymes involved in luminescence (Fig. 13). Upon transfer of luminescent cells to fresh medium, autoinducer initially is diluted below this threshold level (Ulitzur and Hastings, 1979b; Barak and Ulitzur, 1981; Rosson and Nealson, 1981). Luciferase levels in each millilitre of batch culture remain constant for 2 4 hours (i.e. luciferase synthesis stops) and, during this time, luminescence levels decrease markedly (10-100-fold). After sufficient cell growth has occurred (i.e. after accumulation of sufficient autoinducer), luciferase synthesis is induced, leading to a 10G1000-fold higher level of luciferase and 1000-10,000-fold increase in luminescence over pre-induction levels (Coffey, 1967; Kempner and Hanson, 1968; Eberhard, 1972). The
36
E. A. MEIGHEN AND P. V. DUNLAP
1
0
0.1
n
0
0
2
4 6 Time (h)
8
10
FIG. 13. Cell density-dependent autoinduction of luciferase synthesis and luminescence in Vibrio fischeri MJ-1 . The culture was grown in a seawater-based complete medium (Dunlap, 1989) at 24°C. 0 indicates cell density; A , luciferasc luminescence (in quanta sC1mIC'). activity; 0,
lag in luciferase synthesis has been attributed to the need for metabolism of an inhibitor of luciferase synthesis that is present in rich media as well as for accumulation of sufficient autoinducer to activate transcription of genes encoding luciferase (Kempner and Hanson, 1968; Nealson et al., 1970; Eberhard, 1972). The decrease in luminescence before induction of luciferase synthesis has been attributed to a lack of sufficient cellular substrates for luciferase (FMNH2 and aldehyde) during the lag period
BACTERIAL BIOLUMINFSCENCE
37
(Watanabe et al., 1975; Middleton and Smith, 1976). The insufficiency of aldehyde could relate to turnover of polypeptides of the fatty-acid reductase complex (Nealson and Hastings, 1979). 1. Autoinducers of Luminous Bacteria
Autoinducers for luminescence can be thought of as bacterial pheromones in that they are specific extracellular signal molecules evoking a characteristic response from other cells (Eberhard, 1972; Eberhard et al., 1986; Silverman et al., 1989). Early studies using impure material showed that autoinducer for V . harveyi, but not that for V . jischeri, is heat-sensitive (Eberhard, 1972) and appears to be produced by some but not all other marine bacteria, including non-luminous species (it is not produced by E. coli) (Greenberg et al., 1979; Hada et al., 1985). Autoinducers for V .jischeri and V . harveyi have been purified from growth media and identified as N[3-oxohexanoyl]homoserine lactone and N-( P-hydroxybutyry1)homoserine lactone, respectively (Eberhard et al., 1981; Cao and Meighen, 1989) (Fig. 14). Autoinducer for V .jischeri is species-specific; other species of luminous bacteria neither respond to the pure molecule nor produce a compound that stimulates luminescence by V.jischeri (Eberhard, 1972; Nealson, 1977; Greenberg et al., 1979; Eberhard et al., 1981; Hastings and Nealson, 1981; Rosson and Nealson, 1981; Kaplan and Greenberg, 1985; Boettcher and Ruby, 1990). Recently, however, the terrestrial non-luminous Erwinia carotorora has been shown to synthesize autoinducer for V .jischeri and to use this molecule to control expression of genes involved in biosynthesis of the antibiotic carbapenem (Bainton et al., 1992). Other luminous species are
I
I 0
NH-CH
0
0
I
I
NH-CH I
OH
II
0
6
FIG. 14. Chemical structures of autoinducers of luminescence in (a) Vibriofischeri (N-[3-oxohexanoyl] homoserine lactone) (Eberhard et al., 1981) and (b) Vibrio harveyi (N-[P-hydroxybutyryl] homoserine lactone) (Cao and Meighen, 1989).
38
E A MEIGHEN AND P V DUNLAP
also likely to produce autoinducers of luminescence that are species-specific (e.g. Dunlap, 1984b). Studies with radiolabelled V . jischeri (Kaplan et al., 198.5) demonstrated that one to two molecules of autoinducer for each cell gave some induction of luminescence in V . jischeri, and that a maximal rate of induction requires only 40 molecules of autoinducer for each cell (Kaplan and Greenberg, 198.5). Therefore, besides being specific, the molecule for V.jischeri is effective at very low concentrations. This molecule has been demonstrated to be freely diffusible into and out of cells (Kaplan and Greenberg, 198.5); apparently, the rapid diffusion out of cells prevents the basal rate of autoinducer synthesis from leading to induction of 1uxICDABEG transcription at a low cell density. How autoinducers are synthesized is not well understood at present. In V. jischeri, the species for which most information on autoinducer is available, physiological studies with strain MJ-1 (from the light organ of the monocentrid fish Monocentris japonicus; Ruby and Nealson, 1976) indicated that autoinducer synthesis is under the same transcriptional control as luciferase synthesis and therefore is inducible (Friedrich and Greenberg, 1983). This has been borne out by molecular genetic studies that located a gene necessary for autoinducer synthesis (termed luxlfor inducer synthase gene) as the first gene of the lux operon (1uxlCDABE) (Engebrecht et al., 1983). Consistent with a positive-feedback model for autoinducer synthesis based in part on this location for luxl (Engebrecht et al., 1983), biochemical studies with V . jischeri grown in the presence of radioactively labelled methionine, a probable autoinducer prescursor, indicate that autoinducer is synthesized autocatalytically (Eberhard et a f . , 1991). Only the luxl gene from V. jischeri gene is necessary for E . coli to synthesize autoinducer (Engebrecht et al., 1983); therefore, substrates used by autoinducer synthase must be present in E. coli. These substrates may be S-adenosylmethionine and either 3-oxohexanoyl-CoA or 3-oxohexanoyl-ACP (Eberhard et al., 1991). Analogues of the autoinducer for V .jischeri exhibit a wide range of activities; some are strong agonists of the autoinducer while others are antagonists (Eberhard et al., 1986). Apparently, not all strains of V . jischeri produce autoinducer substrates under all conditions. Strains from the light organs of the Hawaiian squid (Eupryrnna scolopes) produce 1000-fold less light in laboratory culture than in the squid, due to a lower level of luciferase synthesis (Boettcher and Ruby, 1990). The E. scolopes strains produce little or no autoinducer in culture but respond to exogenously added autoinducer of V . jischeri. This suggests that autoinducer (or a precursor) is supplied to the bacteria by the squid or that, in culture, the level of autoinducer synthase is low in these strains (Boettcher and Ruby, 1990). The lux genes from E. scolopes strain ES114 have been cloned, and E. coli containing these genes on
BACTERIAL BlOl UMINESCFNCE
39
recombinant plasmids produces a high level of luminescence (Gray and Greenberg, 1992). Thus, a functional autoinducer synthase gene is apparently present in the cloned DNA. Possibly, the squid provides a precursor produced by E. coli but not produced by the V .fischeri strain isolated from the squid. Alternatively, the squid might in some way influence synthesis or activity of the luxl gene product (Boettcher and Ruby, 1990).
2. Mechanism of Autoinduction in the lux System in Vibrio fischeri In V .fifischeri, according to models of lux gene regulation (e.g. Engebrecht et al., 1983; Engebrecht and Silverman, 1984; Dunlap and Greenberg, 1991), autoinducer binds to the product of the luxR gene (the LuxR protein) forming a complex that then associates with the lux regulatory region. The autoinducer-LuxR protein complex is thought then to activate transcription from the lux operon promoter. Because the gene for autoinducer synthase (lux0 is part of the lux operon, autoinducer controls its own synthesis through a positive-feedback circuit (Engebrecht et al., 1983; Friedrich and Greenberg, 1983); so, once induction begins, the level of autoinducer increases rapidly. This leads to an exponential increase in luciferase synthesis and luminescence characteristic of autoinduction (see Fig. 13). Tritiated autoinducer with a high specific activity has been synthesized (Kaplan et al., 1985), but formation of an autoinducer-LuxR complex has not yet been demonstrated (Kaplan and Greenberg, 1987). However, results of point-mutational analyses are consistent with the hypothesis that the LuxR protein binds both autoinducer and lux DNA (Shadel et al., 1990b; Slock et al., 1990). Two domains essential for LuxR activity have been identified. One domain is proposed as the autoinducerbinding domain, based on activity of some, but not all, mutant LuxR proteins upon addition of autoinducer in vivo. The other domais, because of its sequence similarity to DNA-binding domains of several other transcriptional regulators, was designated as a DNA-binding or DNArecognition domain (Shadel et al., 1990b; Slock et al., 1990). However, in contrast to the transcriptional regulatory proteins that show sequence similarity with LuxR in the DNA-binding regions (a subclass of the twocomponent, sensor kinase-transcriptional regulators) (Deretic et al., 1989; Miller et al., 1989; Henikoff et al., 1990), LuxR does not show the Nterminal region similarity typical of the transcriptional regulators in this family of proteins (Henikoff et al., 1990; Slock et al., 1990). This N-terminal region is subject to phosphorylation by the sensor kinase component of the two-component systems, but LuxR is activated by autoinducer rather than by a phosphate group (Slock et al., 1990). In the luminescence system of V .fischeri then, the receiver and the transmitter of the signal are a single
40
F A MFIGHEN AND P V DUNLAP
molecule, LuxR. The region between the two domains might link them, with the N-terminal arm blocking transcriptional activation in the absence of autoinducer, and with autoinducer-binding functioning to alter folding of the N-terminal arm such that it does not hinder formation of a transcription-initiation complex (Choi and Greenberg, 1992). This hypothesis predicts that the N-terminal arm of the protein is dispensable. In its absence, autoinducer should not be required for lux transcriptional activation. Results of a fuxR 5' deletion analysis are consistent with these predictions. Thus, the luxR gene product may be a member of the superfamily of transcriptional regulators, the prokaryotic sigma factors (Choi and Greenberg, 1992). In this regard, the deduced amino acid-residue sequence of fuxR shows 27% identity with fasR, the transcriptional activator of elastase in Pseudomonas aeruginosa (Gambe!lo and Iglewski , 1991). Evidence that groEL is required for proper folding of LuxR has been obtained (Dolan and Greenberg, 1992). Besides these considerations, control of the luminescence system by autoinducer is known to the complex. Expression of luxR is negatively autoregulated by autoinducer and the LuxR protein at post-transcriptional (Engebrecht and Silverman, 1986b) and transcriptional levels (Dunlap and Greenberg, 1985, 1988; Dunlap and Ray, 1989). Furthermore, some evidence suggests that presence of the luxl gene suppresses synthesis of LuxR (Engebrecht and Silverman, 1984; Kaplan and Greenberg, 1987). Recently, even more complexity was indicated by the findings that the a region within fuxZCDAB E is required for autoinducer-dependent transcriptional-negative autoregulation of fuxR and that, at low levels of autoinducer, LuxR with autoinducer exhibits positive control over fuxR (Shade1 and Baldwin, 1991). A 20 bp inverted repeat, the putative LuxR protein-binding site (termed the lux regulon operator; Baldwin et a f ., 1989a; Devine et al., 1989; Shadel et a f . , 1990a), has been identified just upstream (-35 region) of the transcriptional start site for the fuxZCDABEG operon. Evidence for a cisacting element involved in repressing lux-operon transcription in the absence of LuxR and autoinducer was obtained with deletion analysis of the region upstream of the lux operator (Devine et a f . , 1989). In addition, approximately midway between the luxZ and luxR genes is a sequence identical with the consensus CRP-binding site of E. coli (Engebrecht and Silverman, 1987; Devine et al., 1988, 1989; Baldwin et al., 1989a). Control by CAMP and CRP is described below.
3. The Luminescence System of Vibrio harveyi Substantial similarities and differences exist between the luminescent systems in V . jischeri and V . harveyi with regard to lux gene organization
BACTFRIAI BlOLUMINtSCENCE
41
(described previously) and regulation. Vibrio harveyi is common in coastal waters and sediments, generally in areas somewhat warmer than those in which V . Jischeri is found (Nealson and Hastings, 1979; Baumann and Baumann, 1981). In V . harveyi, as in V .Jischeri, luminescence is controlled by a cell density-dependent autoinduction at the level of transcription (Nealson et al., 1970; Eberhard, 1972; Ulitzur and Hastings, 1979b; Barak and Ulitzur, 1981; Rosson and Nealson, 1981). During induction, other polypeptides, both cytoplasmic and membrane-associated, are co-induced with luciferase (Michaliszyn and Meighen, 1976; Ne’eman et al., 1977). In contrast to V . Jischeri, expression of luminsescence by E. coli containing the lux operon (luxCDABEGH) from V . harveyi is constitutive, and light levels are low except when the cloned insert is under the control of a strong promoter (Belas et al., 1982; Cohn et al., 1983; Baldwin et al., 1984; Gupta et al., 1986; Miyamoto et al., 1988a) or when the DNA is present in certain mutants of E. coli (Miyamoto et al., 1987). The low expression of luminescence by E. coli containing DNA from V . harveyi may relate to difficulties in recognition of promotors from V . harveyi by RNA polymerase from E. coli (Miyamoto et al., 1988a), which could reflect differences in the levels of regulatory proteins rather than intrinsic properties of the RNA polymerase. Attempts to identify regions of the chromosome in V . harveyi necessary for a high level of luminescence expression in E. coli, using complementation between a 13 kb fragment of DNA from V . harveyi encompassing the luxCDABE genes and shotgun-cloned genomic DNA, were unsuccessful (Miyamoto et al., 1988a). However, Martin et al. (1989), using transposon mutagenesis, identified two regions of the chromosome in V . harveyi involved in luminescence, namely region I, which encodes luxCDABE, and region 11, which lies at least 10 kb from 1uxCDABE. Expression of p-galactosidase from ZacZ fusions in region I shows an induction pattern and response to conditioned medium consistent with control by cell densitydependent accumulation of autoinducer during growth (Martin et al., 1989). Region IT is required for this induction and encodes a transacting regulatory function analogous to that of the luxR gene in V . Jischeri (Showalter et al., 1990). The luxR gene in V . harveyi contains a region with an amino acidresidue sequence similar to that of the DNA-binding domain of Cro-like proteins, but there is no DNA or amino acid-residue sequence similarity between the luxR gene of V . harveyi and that of V . Jischeri (Showalter et al., 1990). Cells containing transposon insertions in region I1 produce autoinducer (Martin et al., 1989), so the region I1 regulatory function is thought to be other than one involved in autoinducer synthesis (i.e. other than a luxl gene). Recently, luxR has been shown to function as a transcription-activation factor affecting initiation of lux mRNA (Swartzman et al., 1992). None of the 54 lux mutants of V. harveyi generated by
42
E A M E I G I I E N A N D P V DUN1 AP
transposon insertion mutagenesis (of approximately 50,000 mutants recovered) was defective in autoinducer synthesis or in synthesis of cAMP or CRP (Martin et al., 1989), although mutants defective in autoinducer synthesis and cAMP synthesis have been isolated using chemical mutagenesis (Ulitzur and Yasphe, 1975; Cao and Meighen, 1989; Cao et al., 1989). It is possible that the gene for autoinducer synthesis is located in a third region of the chromosome in V . harveyi. Recently, additional autoinducer mutants have been generated that emit light in the presence of relatively low concentrations of A'-@hydroxybutyry1)homoserine lactone. Addition of autoinducer to these V . harveyi mutants not only restores luminescence but appears to control other functions (J. Cao, unpublished data). Complementation of the autoinducer mutants using transconjugation of a shotgun library of genomic DNA resulted in identification of DNA that can restore the luminescence phenotype (C. Miyamoto, unpublished data). In addition, protein extracts of V . harveyi but not other luminescent bacteria cause a mobility shift on electrophoresis of a DNA restriction fragment just upstream of the lux promotor in V . harveyi (Swartzman et al., 1991), indicating the presence of a specific activator or repressor in V . harveyi. Regulation of luminescence in V. harveyi thus appears to be different and possibly more complex than in the lux system in V .fischeri. The possibility of pleiotropic effects by the lux autoinducer in V. harveyi as well as the ability of extracts of other bacterial species to excrete compounds that stimulate luminescence (Greenberg et al., 1979; Hada et al., 3985) would be consistent with autoinducer from V . harveyi acting as a more general environmental or nutritional signal (Eberhard, 1972; Eberhard et al., 1981; Silverman et al., 1989). 4. Luminescence Systems of Other Bacterial Species
Much less is presently known about lux gene regulation in other luminous bacteria. For P. phosphoreum, an involvement of the outer membrane in regulation of luminescence has been described (Lumen and Winkler, 1986). Luciferase and the fatty-acid reductase enzymes are induced at high cell density in some strains of P. leiognathi and P. phosphoreum (Wall et al., 1984a; Delong et al., 1987; Illarionov and Protopopova, 1987), but luciferase expression is constitutive in others (Katznelson and Ulitzur, 1977; Watanabe and Hastings, 1986). Using a T7 promoter system to express cloned DNA from P. phosphoreum, Mancini et al. (1988, 1989) demonstrated the presence of polypeptides of the fatty-acid reductase and luciferase components, together with a novel 26 kDa Lux polypeptide. The corresponding genes occurred in the sequence luxCDABFE, with luxF encoding the novel polypeptide. A similar arrangement of lux genes,
BACTERIA1 BIOLUMINESCENCE
43
including a gene analogous to luxF (IuxN), between luxB and luxE, was found for strains PIss4 and P1721of P. leiognathi (Illarionov et al., 1988; Baldwin et al., 1989a) whereas, in the neotype P. leiognathi strain, luxF was not present and the order of lux genes is luxCDABE (Lee, C. Y. et al., 1991). The location of the luxF or luxN genes and their sequence similarity to genes encoding the a- and b-subunits of luciferase suggest that they could play a role in the luminescence reaction in those strains in which they are found. It is known in this regard, however, that D NA without apparent function can be maintained and even amplified in bacteria. For example, P. phosphoreum contains repetitive apparently functionless pseudogenes for tRNA (Giroux and Cedergren, 1989). A detailed screening for distribution of these hitherto unknown genes in different strains of P. phosphoreum and P. leiognathi, and a survey for its presence in different species of luminous bacteria, would be valuable as a basis for evaluating lux gene evolution. For X . luminescens, a terrestrial luminous bacterium found free in soil and as a symbiont of nematodes that infect insect larvae, luminescence and luciferase exhibit a lag in expression followed by a rapid increase at high cell density, a pattern that is consistent with autoinduction (Colepicolo et al., 1989; Schmidt et al., 1989). Results of studies on lux-gene organization and expression in X . luminescens are consistent with the presence of the luxA BCDE genes, with the genes involved in aldehyde synthesis flanking those for luciferase (Frackman et al., 1990; Johnston et al., 1990; Szittner and Meighen, 1990; Xi et al., 1991). A number of other species of luminous bacteria have been described, including Vibrio logei, V. orientalis, V . splendidus, Shewanella (Alteromonas) hanedai and luminous strains of Vibrio cholerae and V . vulniJicus(Baumann and Baumann, 1981; Hastings and Nealson, 1981). At present, very little is known about the ecology of these bacteria or controf of their luminescence systems. They present fertile ground for comparative analyses of lux gene organization and regulation, and their study may provide additional insight into the physiological function and ecological role of bacterial luminescence. B. CYCLIC
AMP
Expression of luminescence in V . harveyi and V . jischeri, and possibly other species, requires cAMP and CRP. Early studies demonstrated that luminescence in V . harveyi and V .jischeri is subject to catabolite repression by glucose. In V . harveyi, glucose repression is permanent in batch culture and is reversible by cAMP (Nealson et al., 1972). A mutant of V . harveyi which requires cAMP for luminescence has been isolated (Ulitzur and
44
E A MFIGHFN AND P V DUN1 AP
Yashphe, 1975), while CRP from V . harveyi has been purified and characterized (Chen et a f . , 1985). Early studies on cAMP control in V . jischeri demonstrated that autoinduction of luminescence exhibits catabolite repression by glucose, but in an apparently atypical fashion. Glucose represses luminescence temporarily in batch culture. However, this repression is not reversed by addition of CAMP,and prior growth of V .jischeri in the presence of glucose eliminates the repression (Ruby and Nealson, 1976). By contrast, in phosphate-limited chemostat culture, glucose repression is permanent and addition of cAMP (or autoinducer) reverses that repression (Friedrich and Greenberg, 1983). Studies with cloned lux genes from V. jischeri in adenylate cyclase (cya) and CRP (crp) mutants of E. cofi demonstrated a requirement for cAMP and CRP in autoinduction of the luminescence system (Dunlap and Greenberg, 1985, 1988). However, as with V. jischeri, the temporary glucose repression of luminescence observed in the parent strain of E . cofi was not reversed by exogenous cAMP (Dunlap and Greenberg, 1985,1988). Studies with cya-like and crp-like mutants of V . jischeri confirmed a physiological requirement for cAMP and CRP in autoinduction of luminescence (Dunlap, 1989; Fig. 15), and use of Mu dI(fac2) fusions in the cloned
FIG. 15. Effect of cAMP on luminescence in cya-like and crp-like mutants of Vibrio fischeri MJ-1. Cells of the cya-like mutant (left half of plate) and the crp-like mutant (right half of plate) were inoculated onto a seawater-based complete medium (Dunlap, 1989) and allowed to grow up for approximately eight hours. Cyclic A M P ( 5 p1 of a 0.5 M stock solution) was then spotted onto both zones of growth. The platc was photographed four hours later in the light (left panel) and in the dark (right panel).
HACTFRlAl HIULUMINESCENCE
45
lux genes in E. coli revealed that cAMP and CRP activate transciption from the luxR promoter while decreasing transcription from the lux operon promoter (Dunlap and Greenberg, 1985, 1988; Dunlap and Ray, 1989). These results have been confirmed in V . jischeri with recently constructed chromosomal 1uxR::lacZ fusion mutants (Dunlap and Kuo, 1992). During activation of the lux operon transcription, the LuxR protein, along with autoinducer, counters the effect of CAMP-CRP by repressing transcription from the luxR promoter, thereby possibly modulating the level of LuxR protein in the cell (Dunlap and Greenberg, 1985, 1988; Dunlap and Ray, 1989). The level of LuxR protein in the cell is also modulated by a posttranscriptional luxR-negative autoregulation (Engebrecht and Silverman, 1986b). At the sequence level, a consensus CRP-binding site is present in the lux regulatory region (Devine et al., 1988; Engebrecht and Silverman, 1987), and a CAMP-dependent binding of the CRP from E. coli to this site has been demonstrated by in vitro footprinting (Shade1 et aZ., 1990a). The recent discovery of the novel ability of V . Jifischeri to grow on cAMP as a sole carbon and energy source by means of the cpdP gene product, a periplasmic 3':5'-CAMP phosphodiesterase, adds complexity to the relationship among CAMP,luminescence and symbiosis in this species (Dunlap et al., 1992; P. V . Dunlap, S. M. Callahan and A. Kuo, unpublished data). C. CONTROL BY IRON
Control of luminescence by iron presently is controversial in that physiological effects on luminescence have been documented for several species (e.g. Makemson and Hastings, 1982; Dunlap, 1984a; Haygood, 1984) but the mechanism for these effects remains obscure and may not involve a direct control over lux gene expression. The presence of exogenous iron can repress luminescence and luciferase synthesis in V. harveyi and can influence catabolite repression of luminescence by glucose (Makemson and Hasting, 1982). In V . Jischeri, addition of iron to a minimal medium containing a low concentration of iron delays induction of luciferase synthesis. Moreover, in complete medium, restriction of iron availability by addition of iron chelators results in slower growth and induction of luminescence and luciferase synthesis at a lower cell density (Haygood and Nealson, 1985a). Iron repression of luminescence in V . jischeri has been proposed to involve either interference by iron of autoinducer transport or thc activity of an iron-binding repressor protein that blocks ZuxICDABEG transcription (Haygood and Nealson, 1985b). However, iron is not likely to interfere with entry of autoinducer into cells since this compound can cross the bacterial membrane by simple diffusion (Kaplan and Greenberg, 1985). Furthermore, in studies with iron transport (tonB) and iron regulatory
46
E A MFIGHFN AND P V DUNLAP
(fur)mutants of E. cofi containing the lux genes of V.jischeri on recombinant plasmids, an effect of iron restriction on induction of the luminescence system analogous to that in V .jischeri was established but no evidence was obtained that indicated a direct control by iron over transcription from the fuxR of fuxZCDABEG promotors (Dunlap, 1992a). Evidence from studies on E. cofi and V . jischeri indicate that a CAMP-dependent but indirect control is operating, possibly at the level of DNA (i.e. lux gene) copy levels (Dunlap, 1992b; Dunlap and Kuo, 1992). At this point, then, the way in which iron affects luminescence is not yet well defined. Nonetheless, in the fish and squid symbioses, restriction of iron availability by the host animal could be an important way of limiting bacterial growth while maintaining a high level of bacterial light production (Haygood and Nealson, 1985a; Hastings et a f . , 1987). In X . luminescens, iron does not appear to control luminescence (Levisohn et al., 1988; Colepicolo et a f . ,1989) whereas, in P. phosphoreum, iron does regulate luminescence (Haygood, 1984) and this might involve a Fur-like protein (Knochelmann et a f . , 1991). D . CONTROL BY OXYGEN
Oxygen, like osmolarity (see below), is an environmental factor that can differentially affect luminescence and growth in some species of luminous bacteria. At low , growth rate-limiting oxygen tensions, luminescence in V . fischeri and P. phosphoreum is enhanced such that cultures of these species, incubated under conditions of low oxygen tension, contain more luciferase (Nealson and Hastings, 1977). However, X . luminescens shows no differential effect of low oxygen on luminescence (Colepicolo et a f . , 1989), while V. harveyi and P. feiognathiexhibit decreased luminescence under conditions of low oxygen tension (Nealson and Hastings, 1977). Under conditions of high oxygen tension, which can strongly inhibit growth of most species of luminous bacteria, growth is only slightly inhibited in X . luminescens while its luminescence and luciferase synthesis are stimulated (Colepicolo et a f . , 1988,1992). Based on the response of V .jischeri to oxygen, Nealson (1979) proposed that oxygen limitation may restrict growth of bacteria in the light organ of monocentrid fish. In contrast, for P. feiognathi in light organs of leiognathid fish, oxygen is delivered directly from the gas bladder and, therefore, is not likely to be limiting for bacteria in the leiognathid symbiosis (McFall-Ngai, 1983; Dunlap and McFall-Ngai, 1987). The mechanism by which growth under conditions of low oxygen tension affects luminescence is not yet known. With regard to oxygen, luciferase has been proposed to function as an alternative electron carrier for the cytochrome system (Ulitzur et al. , 1981; Hastings, 1983; Grogan, 1984). The high affinity of bacterial luciferases
BACTERIAL BIOLUMINESCENCE
47
for oxygen (Hastings et al., 1985; Lloyd et al., 1985) supports this proposal, and suggests that luciferase could promote survival and growth of luminous bacteria under conditions of low ambient oxygen tensions (Ulitzur et al., 1981). Evidence supporting this possibility has been obtained (Makemson, 1986; Makemson and Hastings, 1986a,b), as has evidence linking bioluminescence with proton-motive force (Guerrero and Makemson (1989). A role for the luminescence system of V .Jischeri in resistance to oxidative stress is suggested by the slow growth of lux deletion mutants (Kuo and Dunlap, 1991) and their poor survival in the presence of hydrogen peroxide (A. Kuo and P. V. Dunlap unpublished data). These and other hypotheses for the function of the bacterial luminescence system have recently been reviewed (Dunlap, 1 9 9 2 ~ ) . E. CONTROL BY OSMOLARITY
Several studies have demonstrated that osmolarity can influence bacterial luminescence (for early literature, see Harvey, 1952; MacLeod, 1968). In certain organisms the effects on luminescence and growth differ sharply. Farghaly (1950) found that luminescence was optimal in Achromobacter (V.)Jischeri grown in 3% sodium chloride, whereas growth was optimal in the presence of 1S%Osodium chloride. Differential effects of osmolarity or salt on luminescence and growth have been reported for other species and strains (Henry and Michelson, 1970; Kossler, 1970; Soli, 1970; Makiguchi et al., 1980), with different species exhibiting distinctly different responses. For example, strains of P. leiognathi isolated from light organs of leiognathid fish show the highest levels of luminescence but grow slowly in media osmotically equivalent to 40% seawater, and grow optimally but produce low levels of luminescence in media osmotically equivalent to 80% seawater (Dunlap, 1984a, 1985). The situation for P. phosphbreum is essentially reversed, with growth optimal at lower salt concentrations and luminescence optimal at salt concentrations similar to or higher than that in seawater (Dunlap, 1984a). In some species, such as V .fischeri, V . harveyi and X . luminescens, however, luminescence and growth are not differentially influenced by salt concentration (Dunlap, 1984a; Colepicolo et al., 1989). The effects for P. leiognathi and P. phosphoreum have been shown to be osmotic rather than caused by the concentration of a specific ion (Dunlap, 1984a, 1985). Nonetheless, P. phosphoreum exhibits a strong response to sodium chloride, addition of which triggers light emission in a matter of tens of minutes (Watanabe and Hastings, 1986). This response apparently is caused by an insufficiency of aldehyde, synthesis of which may be controlled by a potassium ion-mediated activation of a gyrase or a change in DNA supercoiling (Watanabe et al., 1991). Observed
48
b A MEIGHFN AND P V DUNLAP
osmotic effects on luminescence and growth of P . leiognathi, P. phosphoreum and V. jischeri might correlate with specific light-organ symbiosis of these species with marine animals (Dunlap, 1984a,b, 1985). F . POSSIBLE INVOLVEMENT OF
LexA, HtpR
AND
FNR
In addition to the genetic and physiological regulatory factors already described, other genetic regulatory factors might be involved in controlling expression of the lux genes. For example, in V . jischeri MJ-1, the putative LuxR protein-binding site, a 20 bp inverted repeat in the lux regulatory region, bears strong sequence similarity to the LexA recognition sequence in E. coli (Ulitzur and Kuhn, 1988; Baldwin et a f . , 1989a). Binding of the LexA protein of E. cofi to this region has been demonstrated using DNase I protection experiments (Shade1 et a f . , 1990a). It is possible that an interaction between the LuxR protein and a LexA protein from V. jischeri plays a role in controlling fux-operon transcription (Shade1 et al., 1990a). In addition to LexA, a possible promoter site typical of 032has been identified within the fuxl gene; it has been suggested that HtpR plays a role in fux-gene regulation (Ulitzur and Kuhn, 1988; Ulitzur, 1989). Using fuxl-deleted mutants of V . jischeri, it was found that the response to autoinducer was 1000-fold less in cells also containing a mutated HtpR protein (Adar et a f . , 1991) than in an HtpR' isogenic strain. In the latter strain only, the luminescence response to autoinducer was still dependent on growth phase, possibly due to an increase in HtpR activity in the later stages of growth. More recently, the Q~~ effects has been defined as a specific requirement for the products of groE in folding of the LuxR protein (Dolan and Greenberg, 1992). An involvement of the fumarate-nitrate reductase (FNR) protein has recently been implicated in luminescence (Muller-Breitkreutz et a f . , 1991).
V. Evolution and Ecology A. DISTRIBUTION OF LUMINOUS RACTERlA
Phylogenetically, luminous bacteria are clustered within four closely related genera, namely Vibrio, Photobacterium, Shewaneffn (Alteromonas) and Xenorhabdus, of the S-purple subdivision of the eubacteria. With the possible exception of Shewaneffa spp., these bacteria are classified in the facultatively aerobic, enteric families Vibrionaceae and Enterobacteriaceae. Presently, at least 11 species are known to produce luminescence (see Table 1). Each of the four genera also contains non-luminous species (Baumann
BACTERIA1 HIOLUMINESCENCF
49
and Baumann, 1981; MacDonnell and Colwell, 1985; Nearhos and Fuerst, 1987; Woese, 1987; Farmer, 1991; Farmer and Hickman-Brenner, 1991; Nealson and Hastings, 1991). The luminescence phenotype, because of its distinctive nature, has provoked and facilitated ecological studies of these bacteria. Furthermore, identification of many species of luminous bacteria requires only a simple set of phenotypic tests (Baumann and Baumann, 1981; Hastings and Nealson, 1981; Farmer, 1991; Farmer and Hickman-Brenner, 1991; Nealson and Hastings, 1991). Ecological and taxonomic studies have shown that luminous bacteria are distributed world-wide, with most species occurring in the marine environment but with some also being found in brackish waters, soil and possibly freshwater (see Table 1) (Baumann and Baumann, 1981; Nealson and Hastings, 1991). Like many non-luminous bacteria, luminous bacteria in marine environments occur free in seawater or associated with particulate matter (i.e. marine snow) and, in association with marine animals, as saprophytes, commensal (enteric) symbionts and parasites (Harvey, 1952; Singleton and Skerman, 1973; Baross et al., 1978; Nair et al., 1979; Orndorff and Colwell, 1980; Baumann and Baumann, 1981; Davis and Sizemore, 1982; West et al., 1983; Yang et al., 1983; Dilmore and Hood, 1986; Ramaiah and Chandramohan, 1987; Ramesh et al., 1987; Oritgosa et al., 1989). Unlike other bacteria, however, certain luminous species also form specific bioluminescent (light-organ) symbioses with marine fishes and squids (Ruby and Nealson, 1976; Fitzgerald, 1977; Hastings and Nealson, 1981; Dunlap, 1984b; Fukasawa and Dunlap, 1986; Dunlap and McFall-Ngai, 1987; Hastings et al., 1987; Boettcher and Ruby 1990; Dunlap and Greenberg, 1991; Nealson and Hastings, 1991). In the terrestrial environment, X . luminescens has established a symbiotic association with heterorhabditid nematodes that attack insect larvae (Poinar et al., 1980; Nealson and Hastings, 1991). Some strains of species of the human pathogens V . cholerae and V. vu1niJiCu.s can luminesce (Desmarchelier and Reichelt, 1981; West and Lee, 1982; Oliver et al., 1986). Recently, an agar-digesting luminous bacterium was described (Fukasawa and Kobayashi, 1987; Fukasawa et al., 1987), so association with marine algae is an additional niche for luminous bacteria shared with non-luminous forms. In the marine environment, distribution of luminous bacterial species relates to various environmental factors, including seasonally based temperature changes (Ruby and Nealson, 1978; Yetinson and Shilo, 1979), salinity (Shilo and Yetinson, 1979; Feldman and Buck, 1984), ability to grow under nutrient-poor conditions and sensitivity to photo-oxidation (Shilo and Yetinson, 1980), and depth (Ruby et al., 1980). The enteric habitat and light-organ symbiosis contribute to and might be dominating influences over the density and distribution of luminous bacteria (e.g. Reichelt et al.,
50
E. A . MEIGHEN AND P. V. DUNLAP
1977; O'Brien and Sizemore, 1979; Ruby and Morin, 1979; Haygood et al., 1984; Nealson et al., 1985; Ramesh et al., 1986; Ramesh and Venugopalan, 1988; Lee and Ruby, 1991). B. MOLECULAK APPROACHES TO IDENTIFICATION AND ECOLOGY OF LUMINOUS BACTERIA
The distribution of some species of luminous bacteria (e.g. V . harveyi) is reasonably well known due to the large number of isolates that have been characterized (Baumann and Baumann, 1981). Relatively little is known, however, about the distribution and ecology of two psychrotrophic species, namely V . logei and S. ( A . ) hanedai, because few isolates have been reported. Recently, waters off Woods Hole, Massachusetts, have yielded during the winter months psychrotrophic isolates that appear very similar to V . logei. These isolates ferment glucose, produce a yellow, cell-associated pigment, grow and produce luminescence at 4 and 15"C, and grow but do not produce visible luminescence at 24°C (P. V. Dunlap, unpublished data). From bioluminescent marine snow taken in oceanic water off Gibraltar, non-fermentative, psychrotrophic, luminous bacteria have been isolated that exhibit several differences from S. (A,) hanedai (J. C. Makemson, personal communication). With further study, the distribution and ecological significance of these psychrotrophic species will become better established. It is also possible that new species will be described as neglected habitats are examined for luminous bacteria. Traditionally, distributional and taxonomic studies of luminous bacteria have relied on the luminescence phenotype. However, it has been recognized for several years that genes for luminescence occur in a broader group of bacteria than those that produce visible light when grown in laboratory media. For example, luciferase has been demonstrated by enzyme assay and antibody methods in several non-luminous Vibrio spp. (Nealson and Walton, 1978; Makemson and Hastings, 1986a; Kou and Makemson, 1988), while several non-luminous and luminous bacterial species produce an inducer of luminescence in V . harveyi (Greenberg et al., 1979), and a luxbased D N A probe was shown to hybridize to the DNA of non-luminous bacteria from marine surface-water samples (Potrikus et al., 1984). Recently, also, Palmer and Colwell(l991) detected low-level light emission by visually dark strains of V. cholerae, a species that contains some luminous strains (Desmarchelier and Reichelt, 1981; West and Lee, 1982), and they demonstrated the presence of luxA-hybridizing DNA in strains that failed to produce detectable light (Palmer and Colwell, 1991). For V . jischeri, which can form light-organ symbioses with monocentrid fish (Ruby and Nealson, 1976) and certain sepiolid squids (Wei and Young, 1989;Boettcher
BACTERIAL BIOLUMINESCENCE
51
and Ruby, 1990; McFall-Ngai and Ruby, 1991; Ruby and McFall-Ngai, 1992), a form producing very low levels of light when grown in laboratory culture occurs in the light organs of Euprymna scofopes, where it produces a high level of luminescence (Boettcher and Ruby, 1990), and IuxA- and fuxR-based hybridization probes have been used to enumerate this form in Hawaiian coastal water (Lee and Ruby, 1991). Species-specific luxAbased probes have recently been developed for certain luminous bacterial species (Wimpee et a f . , 1991). However, in contrast to the studies already referred to, no non-luminous bacteria containing genes for luciferase-like proteins were identified in colony hybridizations of more than 40,000 field isolates (Wimpee et al., 1991). It is not yet clear, then, to what extent luxcontaining non-luminous bacteria are present in the environment. Use of lux-based probes should provide a rapid resolution of this issue, which relates not only to the ecology of this phenotype but also to a biochemical function for the luminescence system and the evolution of visible luminescence. A broad distribution of lux genes in non-luminous bacteria might imply a common biochemical function for the system not related to light emission but from which visible luminescence could have arisen (Seliger, 1987). Use of lux-based probes will rapidly expand knowledge of the ecology of visibly luminous and non-luminous lux-containing bacteria. Speciesspecific ZuxA-based probes for P. feiognathi, P. phosphoreum and V . fischeri, as well as a probe that identifies a small group of closely related species (V. harveyi, V . orientalis and V . vufnzjicus), have been developed (Wimpee et a f . , 1991). Besides these probes, species-specific probes for S. ( A . ) hanedai and V. spfendidus have also been developed and tested (K. H. Nealson, personal communication). For identification of field isolates, the probes have been effective already in identifying isolates of P. phosphoreum fram the Black Sea (Wimpee et al., 1991) and isolates of V. spfendidus from coastal waters of Kuwait (K. H. Nealson, personal communication). Species-specific probes should also be valuable in broadening knowledge of the distribution of as yet not cultured bacteria which inhabit the light organs of anomalopid and ceratioid fish. In this regard, the relatedness of anomalopid bacteria to other species of luminous bacteria has been examined with luxA-based probes (Haygood, 1990). This approach and that of restriction fragment-length polymorphism suggest that the anomalopid bacteria differ at the species level, depending on the genera of the host fish (Haygood, 1990; Wolfe and Haygood, 1991) as seen also for symbionts of two ceratoid fish (Haygood et a f . , 1992). The on-going cloning and sequencing of lux genes from different species of luminous bacteria has facilitated evolutionary and ecological considerations of the luminescence system as well as the bacteria that carry the lux
52
F A MElGHEN AND P V DUNLAP
genes. Sequence comparisons at the amino-acid and DNA levels can help reveal the process by which lux genes evolved and, by inference, these comparisons can provide an insight into how lineages of luminous bacteria may have arisen. These considerations assume, of course, that lux genes have not been spread by lateral gene transfer. While no evidence is available that suggests lateral transfer of lux genes (Nealson and Hastings, 1991), it remains an open question. Certain attributes of the lux system make it particularly advantageous for these studies. These include: (a) the presence in all species examined so far of a conserved set of five lux genes with an arrangement, in most cases, of ZuxCDABE; (b) the presence in some strains of additional lux-associated genes, which permits a clear recognition of these strains; and (c) a high degree of amino-acid sequence similarity between the luciferase a- and p-subunits and a good degree of similarity between the sequences of these subunits and the amino-acid sequence of the protein encoded by luxF, a gene found in most strains of Photobacterium spp.
c.
COMPARISONS OF
LUX-GENE
AMINO-ACID SEQUENCES
I . Luciferase Subunits For the six bacterial luciferases for which detailed sequence information is available (Table 7), the a-subunits exhibit between 54 and 88% identity in amino-acid sequences, and the P-subunits exhibit 45-77% identity. These high degrees of sequence similarity suggest that luxB arose by gene duplication of luxA, as suggested initially by Baldwin et al. (1979). Inspection of the amino-acid sequence identity percentages (Table 7) suggests that there are two groups, namely the V . harveyi and X . luminescens group, and the Photobacterium spp. and V. fischeri group. This grouping of luminous bacteria based on amino-acid sequence comparisons has been proposed by various other workers (Haygood, 1990; Baldwin TABLE 7. Vh Vh
XI Vf P1S54
PP PI
59 51 49 48 45
Amino-acid sequence identities of bacterial luciferase subunitsa
XI 85 52 51 47 47
Vf 64 66
65 61 57
PI554
61 63 77 77 74
PP 62 62 78 88
PI 54 56 65 77 72
68
Percentage identity between the sequences of the a-subunits (upper right) and between the sequences of the p-subunits (lower left). Bacterial strains are as designated in Table 4.
53
BACTERIAL BIOLUMINESCENCE
and Ziegler, 1991; Ferri et al., 1991). Moreover, specific deletions and/or insertions of codons in the luciferase genes are consistent with this grouping (Ferri et al., 1991). For example, there is a two-codon insertion in the Nterminal region (a in Fig. 7 ) in the p-subunit and a one-codon deletion in the central region (c in Fig. 7 ) in the a-subunit for Photobacterium spp. and V.fischeri which do not occur in the a- and P-subunits from V . harveyi and X . luminescens. Furthermore, the sequence of the luxA gene from V . fischeri is more similar to sequences of the luxA genes from Photobacterium spp. (Pp and P1 ss4) than is the neotype P. leiognathi strain although sequences coded by luxB are more similar among the P. leiognathi strains. In this regard, physiological differences among strains identified as P. leiognathi have been noted that might be sufficient to warrant separation into a new species of the non-light-organ symbiotic strains, which are deeper-welling, more psychotrophic and do not exhibit the characteristic differential growth and luminescence response to osmolarity (Dunlap, 1984a; P. V. Dunlap, unpublished data). 2. Fatty-Acid Reductase Subunits The amino-acid sequence identity for proteins of the fatty-acid reductase complex are shown in Table 8 (upper right, luxC; lower left, IuxD) and Table 9 (upper right, luxE). These comparisons reveal a clear similarity between sequences of the subunits from Photobacterium spp. Although the sequences coded by the luxD and luxE genes in V . jischeri are just as similar to the sequences coded by the corresponding genes from V. harveyi and X . luminescens as to sequences coded by genes from Photobacterium spp., the luxC gene in V . Jischeri does appear to be more similar to the luxC genes in Photobacterium spp. Further support for the notion that V . Jischeri is more closely related to Photobacterium spp. than to V . ’harveyi is obtained by comparison of the identity of the amino-acid sequences coded by the IuxG gene (Table 9, lower left) which has been found after TABLE 8. Amino-acid sequence identities between proteins coded by the luxC and luxD genes” Vh
Vh XI Vf PP PI
66 69 61 59
XI 61 61 61 61
Vf 57 58
67 65
PP 58 62 65
PI 57 63 66 80
74
Percentage identity between the amino-acid sequences coded by luxC genes (upper right) and between those coded by luxD genes (lower left). See Table 4 for strain designation.
a
54
E. A. MEIGHEN AND P. V. DUNLAP
TABLE 9.
Amino-acid sequence identities between proteins coded by the luxE and luxG genes" Vh
Vh
XI
Vf
67
67 64
XI
-
Vf
40
-
PP P1
(42Ib
-
39
PI
PP 59
60 65 61 81
62 67
(Wb 55
(7Wb
Percentage identity between the amino-acid sequences coded by the l u E gene (upper right) and the luxG gene (lower left). As Xenorhabdus luminescens lacks the luxG gene, a horizontal bar is given. See Table 4 for strain designation. Estimated identity based only on the 150 amino-acid residues at the N-terminal of Photobacterium phosphoreum. a
luxE only in marine bacteria and not in terrestrial bacteria. In this comparison, there is a much larger difference in the degree of sequence identity between the residues coded by the luxG gene in V . jischeri and the same gene in V . harveyi than between luxG genes in V. jischeri and Photobacterium strains. lUX
D. DUPLICATION OF
GENES
As already indicated, the IuxA and IuxB genes as well as the IuxF gene found in certain Photobacterium strains suggests that these genes arose by gene duplication as there is a high degree of sequence similarity in the proteins encoded by IuxA, luxB and luxF. Figure 16 shows the alignment
lux/=
-
1
(
69
I I
lux6
Amino
I
Carboxyl
I
1q9
256
lUXA
A
/
\
u FIG. 16. Representative alignment of the polypeptides coded by the luxA, luxB and luxF genes in Photobacterium strains. The N-terminal regions of the LuxA and LuxB proteins (a- and p-subunits of luciferase) are considered to extend to approximately residue 169 where alignment occurs with the LuxF protein starting from residue 69 and extending through the carboxyl region to the end of all three proteins. The positions of an additional 28 residues in the LuxA protein as well as a segment near the N-terminal of the LuxF protein that cannot be readily aligned are also depicted.
55
BACTERIA1 BIOLUMINESCENCE
*
*
192
MLHVNVNEAETVAKEELKLYIENYVACTQPSNFNGSIDSIIQS~GSYKDCLSYV~~GKFD -LF----DNP-Q--A--SI-L-D-LSY--AET ---E--N--AA-NFDT--HH--EM-QGLN
**
231 NTVDFLLCFESMQDQNKKKSVMIDL"QVIKFRQDNNLI*
-K----F----- K--EN---L--NFDKR--NY-KEH--N*
FIG. 17. Alignment of amino acid-residue sequences coded by the LuxF proteins from Photobacterium phosphoreum NCMB 844 ( P p ) and Photobacterium leiognathi (P1554).The upper sequence corresponds to LuxF from Pp. Identical residues in LuxF from are indicated by a horizontal line. Asterisks indicate the positions at which residues are identical in all LuxA, LuxB and LuxF proteins so far sequenced. The sequence of LuxF in PI,,, is not given as it has 97.4% identity with that in &4.
of the luxA, IuxB and IuxF genes in terms of their amino-acid sequence similarities. The sequences coded by luxA and luxB can be aligned through their entire length except for an insertion (deletion) of 28 residues at approximately position 256 in the u(P) subunit, thus accounting for the larger size of the a-subunit. The luxF gene codes for a protein much smaller than the luciferase a- and P-subunits but which is very similar in sequence to the C-terminal regions of the a- and P-subunits of luciferase (Fig. 16). Except for a short section at the immediate N-terminal, the first 70 aminoacid residues do not appear to be homologous in sequence. This finding is of interest since the N-terminal region of the LuxF protein from Photobacterium strains (Pp and PlSs4) has a high degree of sequence identity in this region. Figure 17 compares the amino acid-residue sequences of the LuxF proteins from Pp and P1s54.Although, the LuxF proteins have only 55% sequence identity, the degree of identity approaches about 85% near the N-terminal. Labelled with asterisks in Fig. 17 are amino-acid residues that are identical to residues at the same positions that have also been conserved in the a- and P-subunits of all luciferases (see Fig. 7). These residues correspond to 12 of the last 13 residues conserved in the C-terminal regions of luciferase subunits. In contrast, the 22 residues conserved in both luciferase subunits closer to the N-terminal could not be recognized in spite of the N-terminal region being highly conserved in the LuxF proteins.
56
E A MEIGHEN AND P V D U N L A P
TABLE 10. Amino acid-sequence identities between the C-terminal regions of the LuxF protein and the luciferabe subunits" LuxF LuxB
Average a
LuxF
PP
PIS54
LuxA
PP
PI554
32 31 31 31 32 31
34 35 31 31 27 29
Vh XI Vf Pk4 PP PI
22 24 22 20 24 22
21 23 21 21 20 22
34
31
22
21
Percentage identity extending over 157 amino-acid residues starting at residue 69 of LuxF
in Pp or PIss4 with LuxB (left) or LuxA (right) from different species aligned according to
Fig. 16. See Table 4 for strain designations. TABLE 11. Amino acid-sequence identities of the C-terminal regions of luciferase subunits from luminescent bacteria" Vh Vh XI Vf PI554 PP PI
49 42 42 40 37
XI 81 43 44 31 40
Vf 69 72
53 50 47
PI554 65 65 80 68 69
PP 65 64 19 83
PI 60 59 61 11 69
61
Percentage identity between sequences of the C-terminal regions of the a-subunits (upper right) and between sequences of the C-terminal regions of the p-subunits (lower left). See Table 4 for strain designations.
a
A comparison of the degree of sequence identity in the C-terminal region for proteins coded by the luxF genes from Pp and PlSs4 (Table 10) with the corresponding regions from the luciferase subunits coded by the luxB and luxA genes from different bacterial species demonstrates that the degree of identity of the sequences coded by the luxF and LuxB genes (3134%) is much greater than between the sequences coded by luxA and LuxF genes (21-22%) in this region. Moreover, the relative identity of the amino acid-residue sequences coded by the IuxF gene with those encoded by the luxB gene from species that do not contain the luxF gene (Vf, Vh, P1 and X1) is just as high or higher than between sequences encoded by the luxF and the LuxB genes in Pp and PlSs4.One scenario suggested by these results is that gene duplication resulting in the LuxB and LuxF genes occurred prior to divergence of the luminescent bacteria, and that the luxF gene was later lost from lineages giving rise to certain species.
57
BACTERIAL BIOLUMINtSCENCE
Comparison of the sequence identity between the C-terminal regions (24%) coded by the fuxA and fuxB genes of different species, as well as the N-terminal regions (30%), shows that the C-terminal regions of luciferase subunits have diverged to a much greater degree. Moreover, the fuxA gene from one species is just as closely related to the fuxB gene from a completely different species as it is to the fuxB gene from the same species, suggesting that gene duplication of the fuxA and fuxB genes occurred prior to the divergence of the different bacterial species. Comparison of the degree of sequence identities in the C-terminal regions, among sequences of the fuxA genes (Table 11, upper right), and between the sequences of the fuxB genes (Table 11, lower left), with that observed for the entire molecule (Table 7) reveals that the fuxA gene is just as or more highly conserved in the C-terminal region than in the Nterminal region, whereas the fuxB gene is much less highly conserved in the C-terminal region. Consequently the difference in sequence identity between the N - and C-terminal regions of proteins coded by luciferase genes (fuxA and fuxB) appears to be due to a more rapid divergence in the fuxB gene, particularly in the C-terminal region. Proteins coded by the fuxF and fuxB genes are much more similar in amino acid-residue sequences than the C-terminal regions of proteins coded
luxAB or luxAF
IuxABF
XI
Vh
Vf
PI
PI554
PP
FIG. 18. Gene duplication and evolution of lux systems in luminescent bacteria. The diagram represents the generation of luxABF genes and divergence of the different luminescent bacteria, taking into account differences in gene organization and the relationship between the amino acid-residue sequences coded by the lux genes from different strains. The luxF gene has only been retained in PI554 and Pp. Strain designations are given in Table 4.
58
E. A MEIGHEN AND P. V. DUNLAP
by luxB and luxA or LuxF and luxA genes. Consequently, divergence of the luxB and luxF genes apparently occurred after emergence of luxA. A general model incorporating these conclusions along with divergence of different luminescent species is given in Fig. 18.
VI. Acknowledgements The authors acknowledge the support of the Medical Research Council of Canada, the Woods Hole Oceanographic Institution and NSF grant DCB 9144653 (P. V. D.). We thank T. 0. Baldwin, C. DiRusso, J . J. Farmer, C. Georgopoulos, E. P. Greenberg, J . W. Hastings, M. G . Haygood, C. Kado, J. Lee, J . C. Makemson, K. H. Nealson, 0. Olsson, S. Raina, E. G . Ruby, A. Schauer, U. Winkler and M. Ziegler for generously providing information and data prior to publication, and P. Stanley for an extensive literature search of this area. We especially thank Joyce Herron for preparation of this manuscript as well as Marlene Gihooly and Gisele de Souza for their assistance. This is contribution No. 7794 from the Woods Hole Oceanographic Institution. REFERENCES
Adar, Y . Y . ,Kuhn, J. and Ulitzur, S . (1991). In “Bioluminescence and Chemiluminescence: Current Status” (P. E. Stanley and L. J. Kricka, eds), pp. 63-66. Wiley, Chichester. Alamashanu, S . , Musafia, B., Hadar, R., Suissa, M. and Kuhn, J . (1990). Journal of Bioluminescence and Chemiluminescence 5, 89. Andrews, S . C., Shipley, D., Keen, J . W., Findlay, J. B., Harrison, P. M. and Guest, J . R. (1992). FEBS Letters 302, 247. Bainton, N. J., Bycroft, B. W., Chlabra, S. R., Stead, P., Gledhill, L., Hill, P. J., Rees, C. E. D., Wilson, M. K., Salmond, G . P. C., Stewart, G. S. A . B. and Williams, P. (1992). Gene 116, 87. Balaguer, P., TCrouanne, B., Eliaou, J.-F., Humbert, M., Boussioux, A-M. and Nicolas, JC. (1989). Analytical Biochemistry 180, SO. Baldwin, T. 0. and Ziegler, M. M. (1991). In “Chemistry and Biochemistry of Flavoenzyrnes” (F. Muller, ed.). CRC Press, Boca Raton. Baldwin, T. O., Nicoli, M. Z., Becvar, J. E. and Hastings, J. W. (1975). Journalof Riologica/ Chemistry 250, 2763. Baldwin, T. O., Ziegler, M. M. and Powers, D. A. (1979). Proceedings of the National Academy of Sciences of the United States of America 76, 4887. Baldwin, T. 0.. Berends, T., Bunch, T. A . , Holzman, T. F., Rausch, S. K . , Shamansky, L., Treat, M. I,. and Ziegler, M. M. (1984). Biochemistry 23, 3663. Baldwin, T. O., Chen, L. H., Chlumsky, L. J., Devine, J. H . , Johnston, T. C., Lin, J. W., Sugihara J., Waddle, J. J. and Ziegler, M. M. (1987). In “Flavins and Flavoproteins” (E. E. Edmondson and D. B. McCormick, eds), pp. 621-631. Walter de Gruyter, Berlin. Baldwin, T. O., Devine, J. H., Heckel, R. C., Lin, J.-W. and Shadel, G. S. (1989a). Journal of Bioluminescence and Cherniluminescence 4, 326. Baldwin, T. O., Chen, L. H., Chlumsky, L. J., Devine, J . H . and Ziegler, M. M. (1989b). Journal of Bioluminescence and Cherniluminescence 4, 40.
BACTERIAL BIOLUMINESCENCE
59
Baldwin, T. O., Treat, M. L. and Daubner, S. C. (1990). Biochemistry 29, 5509. Barak, M., and Ulitzur, S. (1981). Current Microbiology 5, 299. Baross, J., Tester, P. and Morita, R. Y. (1978). Journal of the Fisheries Research Board of Canada 35, 1141. Baumann, P. and Baumann, L. (1981). In “The Prokaryotes. A Handbook on Habitats, Isolation and Identification of Bacteria (M. P. Starr, H. Stolp, H. G. Truper, A , , Balows and H. G. Schlegel, eds), pp. 1302-1331. Springer-Verlag, New York. Baumann, P., Baumann, L., Woolkalis, M. and Bang, S. (1983). Annual Review of Microbiology 37, 369. Baumstark, A. L., Cline, T. W. and Hastings, J. W. (1979). Archives of Biochemistry and Biophysics 193, 449. Becvar, J. E. and Hastings, J. W. (1975). Proceedings of the National Academy of Sciences of the United States of America 72, 3374. Belas, R., Mileham, A., Cohn, D., Hilmen, M., Simon, M. and Silverman, M. (1982). Science 218, 791. Blisset, S. J. and Stewart, G. S. A. B. (1989). Letters in Applied Microbiology 9 149. Boettcher, K. J. and Ruby, E. G . (1990). Journal of Bacteriology 172, 3701. Boivin, R., Chalifour, F-P. and Dion, P. (1988). Molecular and General Genetics 213, 50. Boylan, M., Graham, A. F. and Meighen, E. A. (1985). Journal of Bacteriology 163, 1186. Boylan, M., Miyamoto, C., Wall, L., Graham, A. F. and Meighen, E. A. (1989a). Photochemistry and Photobiology 49, 681. Boylan, M., Pelletier, J., Dhephlagnon, S., Trudel, S., Sonenberg, N. and Meighen, E. (1989b). Journal of Bioluminescence and Chemiluminescence 4, 310. Boylan, M., Pelletier, J. and Meighen. E. (1989~).Journal of Biological Chemistry 264, 1915. Boyle, R . (1968). Philosophical Transactions of the Royal Society of London 2, 581. Burlage, R. S . , Sayler, G . S. and Larimer, F. (1990). Journal of Bacteriology 172, 4749. Byers, D. M. (1988). Biochemistry and Cell Biology 66, 741. Byers, D. and Meighen, E. A. (1984). Journal of Biological Chemistry 259, 7109. Byers, D. M. and Meighen, E. A . (1985a). Proceedings of the National Academy of Sciences of the United States of America 82, 6085. Byers, D . and Meighen, E. A. (198%). Journal of Biological Chemistry 260, 6938. Byers, D. M., Bognar, A. and Meighen, E. A . (1988). Journalof Bacteriology 170, 967. Campbell, A. K. (1989). Essays in Biochemistry 24, 41. Cao, J.-G. and Meighen, E. A. (1989). Journal of Biological Chemistry 264, 21670. Cao, J., Swartzman, E., Miyamoto, C. and Meighen, E. (1989). Journal of Bioluminescence and Chemiluminescence 3, 207. Carmi, 0. A., Stewart, G . S. A. B., Ulitzur, S. and Kuhn, J . (1987). Journalof Bacteriology 169, 2165. Carey, L. M., Rodriguez, A . and Meighen, E. A. (1984). Journal of Biological Chemistry 259, 10216. Chen, L. and Baldwin, T. 0. (1984). In “Flavins and Flavoproteins” (R. C. Bray, D. C. Engel and S. G. Mayhew, eds), pp. 785-788. Walter de Gruyter, Berlin. Chen, L. H. and Baldwin, T. 0. (1989). Biochemistry 28, 2684. Chen, P.-F., Tu, S.-C., Hagag, N., Wu, F. Y.-H. and Wu, C.-W. (1985). Archives of Biochemistry and Biophysics 241, 425. Choi, S. H. and Greenberg, E. P. (1991). Proceedings of the National Academy of Sciences of the United States of America 88, 1115. Cline, T. W. and Hastings, J. W. (1972). Biochemistry 11, 3359. Cochrum, L., Hruska, K. S . , Rucker, E. B. and Johnston, T. C. (1990). Nucleic Acids Research 18, 5570. Coffey, J. J. (1967). Journal of Bacteriology 94, 1638. Cohn, D. H., Ogden, R. C., Abelson, J. N., Baldwin, T. O., Nealson, K. H., Simon, M. I. and Mileham, A. J. (1983). Proceedings of the National Academy of Sciences of the United States of America 80, 120. Cohn, D. H., Mileham, A. J., Simon, M. I., Ncalson, K. H., Rausch, S. K., Bonam, D. and Baldwin, T. 0. (1985). Journal of Biological Chemistry 260, 6139.
60
E. A MEIGHEN AND P. V. DUN1.AP
Colepicolo, P., Cho, K.-W. and Hastings, J. W. (1988). Photochemistry and Photobiology 47, 138. Colepicolo, P., Cho, K.-W., Poinar, G . 0. and Hastings, J. W. (1989). Applied and Environmental Microbiology 55, 2601. Colepicolo, P., Camarero, V. C. C. P., Eckstein, J. and Hastings, J. W. (1992). Journal of General Microbiology 138, 831. Cormier, M. J. and Strehler, B. L. (1953). Journal of the American Chemical Society 75,4864. Costa, S . , Douville, E., Bell, J. and Meighen, E. (1991). In “Bioluminescence and Chemiluminescence: Current Status” (P. Stanley and L. Kricka, eds), pp. 31-34. Wiley, Chichester. Daubner, S. C., Astorga, A., Leisman, G . and Baldwin, T. 0. (1987). Proceedings o f t h e National Academy of Sciences of the United States of America 84, 8912. Davis, J. and Sizemore, R. (1982). Applied and Environmental Microbiology 43, 1092. Delong, E. F., Steinhauer, D., Israel, A . and Nealson, K. H. (1987). Gene 54, 203. Deretic, V., Dikshit, R., Konyecsni, W. M., Chakrabarty, A . M. and Misra, T. K. (1989). Journal of Bacteriology 171, 1278. Desmarchelier, P. M. and Reichelt, J. L. (1981). Current Microbiology 5, 123. Devine, J. H., Countryman, C. and Baldwin, T. 0. (1988). Biochemistry 27 837. Devine, J . H., Shadel, G. S . and Baldwin, T. 0. (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 5688. Dilmore, L. and Hood, M. (1986). FEMS Microbiology Letters 35, 221. Dolan, K. M. and Greenberg, E. P. (1992). Journal of Bacteriology 174, 5132. Dunlap, P. V. (1984a). Ph.D. Dissertation: University of California, Los Angeles. Dunlap, P. V. (1984b). Biological Bulletin 167, 10. Dunlap, P. V. (1985). Archives of Microbiology 141, 44. Dunlap, P. V. (1989). Journal of Bacteriology 171, 1199. Dunlap, P. V. (1992a). Archives of Microbiology 157, 235. Dunlap, P. V. (1992b). Journal of Bioluminescence and Chemiluminescence 7, 203. Dunlap, P. V. (1992~).Photochemistry and Photobiology 54, 1157. Dunlap, P. V. and Greenberg, E. P. (1985). Journal of Bacteriology 154, 45. Dunlap, P. V. and Greenberg, E. P. (1988). Journal of Bacteriology 170, 4040. Dunlap, P. V. and Greenberg, E. P. (1991). In “Microbial Cell-Cell Interactions” (M. Dworkin, ed.). American Society for Microbiology, Washington, DC. Dunlap, P. V. and Kuo, A. (1992). Journal of Bacteriology 174, 2440. Dunlap, P. V. and McFall-Ngai, M. J . (1987). Annals of the New York Academy of Sciences 503, 269. Dunlap, P. V. and Ray, J. M. (1989). Journal of Bacteriology 171, 3549. Dunlap, P. V., Mueller, N., Lisa, T. A. and Lundberg, K. S. (1992). Journal of General Microbioloav 138. 115. Dunn, D. K.:Michaliszyn, G . A , , Bogacki, I. B. and Meighen, E. A. (1973). Biochemistry 12. 491 1. Eberhardi A. (1972). Journal of Bacteriology 109, 1101. Eberhard, A. and Hastings, J. W. (1972). Biochemical and Biophysical Research Communications 47, 348. Eberhard, A , , Burlingame, A. L., Eberhard, C., Kenyon, G . L., Nealson, K. H. and Oppenheimer, N. J. (1981) Biochemistry 20, 2444. Eberhard, A., Widrig, C. A , , McBath, P. and Schineller, J. B. (1986). Archives of Microbiology 146, 35. Eberhard, A, , Longin, T. L., Widrig, C. A . and Stranick, S. J. (1991). Archives of Microbiology 155, 294. Eckstein, J. W., Cho, K. W., Colepicolo, P., Ghisla, S . , Hastings, J. W. and Wilson, T. (1990). Proceedings of the National Academy of Sciences of the United States of America 87, 1466. Elhai, J. and Wolk, C. P. (1990). EMBO Journal 9, 3379. Engebrecht, J . and Silverman, M. (1984). Proceedings of the National Academy of Sciences of the United States of America 81 4154.
BACTERIAL BIOLUMINESCENCE
61
Engebrecht, J. and Silverman, M. (1986a). Methods in Enzymology 133, 83. Engebrecht, J. and Silverman, M. (1986b). Genetic Engineering 8, 31. Engebrecht, J. and Silverman, M. (1987). Nucleic Acids Research 15, 10455. Engebrecht, J., Nealson, K. H. and Silverman, M. (1983). Cell 32, 773. Engebrecht, J., Simon, M. and Silverman, M. (1985). Science 227, 1345. Escher, A, , O’Kane, D. J . , Lee, J. and Szalay, A. (1989a). Proceedings of the National Academy of Sciences of the IJnited States of America 86, 6528. Escher, A. P., O’Kane, D., Lee, J., Langridge, W. H. R. and Szalay, A. A. (1989b). New Methods in Microscopy and Low Light Imaging 1161, 230. Escher, A,, O’Kane, D. J. and Szalay, A. A. (1991). In “Bioluminescence and Chemiluminescence: Current Status” (P. E. Stanley and L. J. Kricka, eds), pp. 15-18. Wiley, Chichester. Eymers, J. G. and van Schouwenberg, K. L. (1937). Enzymologia 1 , 328. Farghaly, A. (1950). Journal of Cellular and Comparative Physiology 36, 165. Farmer, J. J., 111 (1991). In “The Prokaryotes, A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications” (A. Balows, H. G . Truper, M. Dworkin, W. Harder and K.-H. Schleifer, eds), 2nd edn. Springer-Verlag, New York. Farmer, J. J. and Hickman-Brenner, F. W. (1991). In “The Prokaryotes, A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications” ( A . Balows, H. G. Truper, M. Dworkin, W. Harder and K.-H. Schleifer, eds), 2ndedn. Springer-Verlag, New York. Feldman, K. A. and Buck, J. D . (1984). Estuaries 7, 93. Ferri, S. and Meighen, E. A. (1991). Journal of Biological Chemistry 266, 12852. Ferri, S. R., Soly, R. R., Szittner, R. B. and Meighen, E. A. (1991). Biochemical and Biophysical Research Communications 176, 541. Fitzgerald, J. M. (1977). Archives for Microbiology 112, 1.53. Fontecave, M., Eliasson, R. and Reichard, P. (1987). Journal of Biological Chemistry 262, 12325.
Foran, D. R. and Brown, W. M. (1988). Nucleic Acids Research 16, 777. Frackman, S . , Anhalt, L. M. and Nealson, K. H. (1990). Journal of Bacteriology 172, 5767. Friedrich, W. F. and Greenberg, E. P. (1983). Archives of Microbiology 134, 87-91. Fukasawa, S. and Dunlap, P. (1986). Agricultural and Biological Chemistry 50, 164.5. Fukasawa, S. and Kobayashi, H. (1987). Agricultural and Biological Chemistry 51, 269. Fukasawa, S . , Dunlap, P. V., Baba, M. and Osumi, M. (1987). Agricultural and Biological Chemistry 51, 265. Gambello, M. J. and Iglewski, B. H . (1991). Journal of Bacteriology 173, 3000. Gast, R. and Lee, J. (1978). Proceedings of the National Academy of Sciences of the United States of America 75, 833. Gautier, S. M., Blum, L. J. and Coulet, P. R. (1990). Journal of Bioluminescence and Chemiluminescence 5, 57. Gibson, Q. H . and Hastings, J. W. (1962). Biochemical Journal 83, 368. Giroux, S. and Cedergren, R. (1989). Journal of Bacteriology 171, 6446. Gray, K. M. and Greenberg, E. P. (1992). Journal of Bacteriology 194, 4384. Greenberg, E. P., Hastings, J. W. and Ultizur, S. (1979). Archives of Microbiology 120, 87. Grogan, D. W. (1984). Archives of Microbiology 137, 159. Guerrero, M. A. and Makemson, J. C. (1989). Current Microbiology 18, 67. Gunsalus-Miguel, A, , Meighen, E. A , , Nicoli, M. Z . , Nealson, K . H. and Hastings, J . W. (1972). Journal of Biological Chemistry 247, 398. Gupta, S. C . , Reese, C. P. and Hastings, J. W. (1986). Archives of Microbiology 143, 325. Hada, H. S., Stemmler, J., Grossbard, M. L., West, P. A,, Potrikus, C. J., Hastings, J. W. and Colwell, R. R. (1985). Systematic and Applied Microbiology 6, 203. Harvey, E. N. (1952). “Bioluminescence”. Academic Press, New York. Hastings, J. W. (1983). Journal of Molecular Evolution 19, 309. Hastings, J. W. (1986). In “Light Emission by Plants and Bacteria” (A. J. Govindgee and D. C. Fork, eds), pp. 363-398. Academic Press, New York. Hastings, J . W. and Balny, C. (1975). Journal ofBiologica1 Chemistry 250, 7288. Hastings, J. W. and Gibson, 0 . H. (1963). Journal of Biological Chemistry 238, 2537.
62
E. A. MEIGHEN AND P. V. DUNLAP
Hastings, J . W. and Morin, J. G . (1991). In “Neural and Integrative Anatomy and Physiology” (C. L. Prosser, ed.), pp. 131-170. Wiley-Liss, New York. Hastings, J . W. and Nealson, K. H. (1977). Annual Review of Microbiology 31, 549. Hastings, J . W. and Nealson, K. H . (1981. In “The Prokaryotes. A Handbook on Habitats, Isolation and Identification of Bacteria” (M. P. Starr, H . Stolp, H . G. Truper, A. Balows and H. G. Schlegel, eds), pp. 1332-1345. Springer-Verlag, New York. Hastings, J. W., Spudich, J. and Malnic, G . (1963). Journal of Biological Chemistry 238, 3100. Hastings, J. W . , Riley, W. and Massa, J . (1965). Journal ofBiologica1 Chemistry 240, 1473. Hastings, J. W., Balny, C., LePcuch, C. and Douzou, P. (1973). Proceedings of the National Academy of Sciences of the United States of America 70, 3468. Hastings, J. W., Tu, S-G., Becvar, J. and Presswood, R. P. (1979). Photochemistry and Photobiology 29, 383. Hastings, J. W., Potrikus, C. J., Gupta, S. C., Kurfurst, M. and Makemson, J. C. (1985). Advances in Microbial Physiology 26, 235. Hastings, J. W., Makernson, J. and Dunlap, P. V. (1987). Symbiosis 4, 3. Haygood, M. G. (1984). Ph.D. Thesis: University of California, San Diego. Haygood, M. G . (1990). Archives of Microbiology 154, 496. Haygood, M. G. and Cohn, D. H. (1986). Gene 45, 203. Haygood, M. G. and Nealson, K. G. (1985a). Symbiosis 1 , 39. Haygood, M. G. and Nealson, K. G. (1985b). Journal of Bacteriology 162, 209. Haygood, M. G., Tebo, B. M. and Nealson, K. H. (1984). Marine Biology 78, 249. Haygood, M. G., Distel, D. L. and Herring, P. J. (1992). Journal o f t h e Marine Biological Association of the United Kingdom 72, 149. Henikoff, S., Wallace, J . C. and Brown, J. P. (1990). Methods in Enzymology 183, 111. Henry, J . P. and Michelson, A. M. (1970). Compte Rendu de Z’Academie des Sciences, Paris 270, 1947. Hirooka, T. , Rogowsky, P. M. and Kado, C. I. (1987). Journal of Bacteriology 169, 1529. Holzman, T. F. and Baldwin, T. 0. (1983). Biochemistry 22, 2838. Hulton, C. S. J . , Higgins, C. F. and Sharp, P. M. (1991). Molecular Microbiology 5, 825. Illarionov, B. A. and Protopopova, M. V. (1987). Molekulyarnaya Genetika, Mikrobiologeya i Virusologiya 8, 41. Illarionov, B. A. , Protopopova, M. V., Karginov, V. A , , Mertvetsov, M. P. and Gitelson, I. I. (1988). Bioorganika Khimika 14, 412. Illarionov, B. A , , Blinov, V. M . , Donchenko, A. P., Protopopova, M. V., Karginova, V. A., Mertvetsov, N. P. and Gitelson, J. I. (1990). Gene 86, 89. Ismailov, A. D., Sobolev, A. Y. U. and Danilov, V. S. (1990). Journal ofBioluminescence and Chemiluminescence 5 , 213. Jablonski, E. and DeLuca, M. (1977). Biochemistry 16, 2932. Jassim, S. A. A,, Ellison, A , , Denyer, S. P. and Stewart, G. S. A. B. (1990). Journal of Bioluminescence and Chemiluminescence 5, 115. Johnston, T. C., Thompson, R . B. and Baldwin, T. 0. (1986). Journal of Biological Chemistry 261, 4805. Johnston, T. C., Hruska, K. S. and Adams, L. F. (1989). Biochemical and Biophysical Research Communications 163, 93. Johnston, T. C . , Rucker, E. B., Cochrum, L., Hruska, K. S. and Vandegrift, V. (1990). Biochemical and Biophysical Research Communications 170, 407. Kamoun, S. and Kado, C. I. (1990a). Journal of Bacteriology 172, 5165. Karnoun, S. and Kado, C. I. (1990b). Applied and Environmental Microbiology 56, 3855. Kaplan, H. B. and Greenberg, E. P. (1985). Journal of Bacteriology 163, 1210. Kaplan, H. B. and Greenberg. E. P. (1987). Proceedings ofthe National Academy of Sciences of the United States of America 84, 6639. Kaplan, H. B., Eberhard, A , , Widrig, C. and Greenberg, E. P. (1985). Journal of Labelled Compounds and Radiopharmaceuticals 22, 387. Karl, D. M. and Nealson, K. H . (1980). Journal of General Microbiology 117, 357. Karp, M. (1989). Biochimica et Biophysica Acta 1007, 84.
BACTERIAL BIOLUMINESCENCE
63
Kasai, S . , Matsui, K. and Nakamura, T. (1987). In “Flavin and Flavoproteins” (D. E. Edrnondson and D . B. McCorrnick, eds), pp. 647-650. Walter de Gruyter, Berlin. Kasai, S., Fujii, S., Miura, R., Odani, S . , Nakaya, T. and Matsui, K. (1991). In “Chemistry and Biochemistry of Flavoenzymes” (F. Muller, ed.). CRC Press, Boca Raton. Katznelson, R . and Ulitzur, S. (1977). Archives of Microbiology 115, 347. Kempner, E. S . and Hanson, F. E. (1968). Journal of Bacteriology 95, 975. King, J. M. H. , Digrazia, P. M., Applegate, B., Burlage, R., Sanseverino, J., Dunbar, P., Larimcr, F. and Sayler, G. S. (1990). Science 249, 778. Kirchner, G., Roberts, J. L., Gustafson, G. D. and Ingolia, T. D. (1989). Gene 81, 349. Knochelrnann, K., Lummen, P. and Winkler, U . K. (1991). In “Bioluminescence and Chemiluminescence: Current Status” (P. E. Stanley and L. J. Kricka, eds), pp. 55-58. Wiley, Chichester. Koncz, C., Olsson, O., Langridge, W. H. R., Schell, J. and Szalay, A. A. (1987). Proceedings of the National Academy of Sciences of the United States of America 84, 131. Koncz, C., Martini, N., Mayerhofer, R., Koncz-Kalman, Z . , Korber, H., Redei, G . P. and Schell, J. (1989). Proceedings of the National Academy of Sciences o f t h e United States of America 86, 8467. Kossler, R. (1970). Zeitschriftfur Allegemeine Mikrobiologie 10, 615. Kou, Y. S . and Makemson, J. C. (1988). Abstracts o f t h e Annual Meeting o f t h e American Society for Microbiology, Washington, DC, K-37. Kricka, L. J. (1988). Analytical Biochemistry 175, 14. Kuo, A. and Dunlap, P. V. (1991). Abstracts of the Annual Meeting of the American Society for Microbiology, Washington, D C , 1-24. Langridge, W. H . R., Fitzgerald, K. J., Koncz, C., Schell, J. and Szalay, A. A. (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 3219. Lee, C. Y. and Meighen, E. A. (1992). Biochemicaland Biophysical Research Communications 186, 690. Lee, C. Y., Szittner, R . and Meighen, E. A. (1991). European Journal ofBiochemistry 201, 161. Lee, J . (1972). Biochemistry 1 1 , 3350. Lee, J . (1985). In “Chemi- and Bioluminescence” (J. G. Burr, ed.), pp. 401-437. Marcel Dekker, New York. Lee, J. (1989). In “The Science of Photobiology” (K. C. Smith, ed.), pp. 391417. Plcnum Press, New York. Lee, J . , Matheson, I., Muller, F., O’Kane, D. J., Vervoort, J . and Visser, J. (1991a). In “Chemistry and Biochemistry of Flavoenzymes” (F. Muller, ed.). CRC Press, Boca Raton. Lee, J . , Wang, Y. and Gibson, B. G . (1991b). Biochemistry 30, 6825. Lee, K.-H. and Ruby, E. G . (1992). Applied and Environmental Microbiology 58, 942. Legocki, R. P., Legocki, M., Baldwin, T. 0. and Szalay, A. A. (1986). Proceedings of the National Academy of Sciences o f the United States of America 83, 9080. Levisohn, R., Beman, J . and Nealson, K. A. (1988). Abstracts ofthe Annual Meeting of the American Society for Microbiology, Washington, D C , 1-1. Lloyd, D., James, C. J. and Hastings, J. W. (1985). Journal of General Microbiology 131, 2137. Lummen, P. and Winkler, U. K. (1986). FEMS Microbiology Letters 37, 293. MacDonnell, M. T. and Colwell, R. R. (1985). Systematic and Applied Microbiology 6, 171. McFall-Ngai, M. J. (1983). American Zoologist 23, 907. McFall-Ngai, M. J. and Ruby, E. G . (1991). Science 254, 1491. Macheroux, P., Eckstein, J. and Ghisla, S . (1987). In “Flavins and Flavoproteins” (D. E. Edmondson and D. B. McCorrnick, eds), pp, 613417. Walter de Gruyter, Berlin. McIlvaine, P. and Langerman, N. (1977). Biophysical Journal 17, 17. MacLeod, R. A. (1968). Advances in Microbiology of the Sea 1 , 95. Makemson, J. C. (1986). Journal of Bacteriology 165, 461. Makemson, J. C. and Hastings, J. W. (1982). Current Microbiology 7, 181. Makemson, J. C. and Gordon, A. S. (1989). FEMS Microbiology Letters 57, 161.
64
F,. A . MEIGHEN A N D P. V. DUNLAP
Makemson, J. C. and Hastings, J. W. (1986a). Abstracts of the Annual Meeting of theAmerican Society for Microbiology, Washington, DC, 1-1. Makemson, J. C . and Hastings, J . W. (1986b). FEMS Microbiology Letters 38, 79. Makiguchi, N., Arita, M. and Asai, Y . (1980). Journal of General and Applied Microbiology 26, 75. Mancini, J. A , , Boylan, M., Soly, R . R . , Graham, A. F. and Meighen, E. A . (1988). Journal of Biological Chemistry 263, 14308. Mancini, J., Boylan, M . , Soly, R . , Ferri, S . , Szittner, R . and Meighen, E . (1989). Journal of Bioluminescence and Chemiluminescence 3, 201. Martin, M., Showalter, R . and Silverman, M. (1989). Journal of Bacteriology 171, 2406. Matheson, I. B. C. and Lee, J. (1981). Biochemical and Biophysical Research Communications 100, 532. Meighen, E. A . (1988). Annual Review of Microbiology 42, 151. Meighen, E. A. (1991). Microbiological Reviews, 55, 123. Meighen, E. A. and Bartlet, I. (1980). Journal of Biological Chemistry 255, 11181. Meighen, E. A . and Grant, G . G . (1985). In “Bioluminescence and Chemiluminescence: Instruments and Applications” (K. Van Dyke, ed.), vol. 11, pp. 253-268. CRC Press, Boca Raton. Meighen, E. A. and Hastings, J. W. (1971). Journal of Biological Chemistry 246, 7666. Meighen, E. A . and MacKenzie, R . E. (1973). Biochemistry 12, 1482. Meighen, E. A . and Szittner, R. B. (1992). Journal of Bacteriology 174, 5271. Meighen, E. A , , Nicoli, M. Z . and Hastings, J. W. (1971). Biochemistry 10, 4069. Meighen, E. A., Slessor, K. N. and Grant, G . G . (1982). Journal of Chemical Ecology 8,911. Michaliszyn, G. A. and Meighen, E. A . (1976). Journal of Biological Chemistry 251, 2541. Michaliszyn, G., Wing, S . and Meighen, E. (1977). Journalof Biological Chemistry 252,7495. Middleton, A . J . and Smith, E. B. (1976). Proceedings ofthe Royal Society of London, Series B 193, 159. Miller, J. F., Mekalanos. J . J. and Falkow, S . (1989). Science 243, 916. Miyamoto, C. M., Graham, A. F., Boylan, M., Evans, J . F., Hasel, K. W . , Meighen, E. A. and Graham, A. F. (1985). Journal of Bacteriology 161, 995. Miyamoto, C., Boylan, M., Graham, A. and Meighen, E. (1986). Methods in Enzymology 133, 70. Miyamoto, C. M., Byers, D., Graham, A. F. and Meighen, E. A. (1987). Journal of Bacteriology 169, 247. Miyamoto, C. M., Boylan, M . , Graham, A. F. and Meighen, E. A. (1988a). Journal of Biological Chemistry 263, 13393. Miyamoto. C. M., Graham, A. F. and Meighen, E. A . (1988b). Nucleic Acids Research 16,1551. Miyamoto, C., Boylan, M., Cragg, L. and Meighen, E. (1989). Journal of Bioluminescence and Chemiluminescence 3, 193. Miyamoto, C., Meighen, E. A. and Graham, A. F. (1990). Journalof Bacteriology 172.2046. Miiller-Breitkreutz, K., Abrahams, M. and Winkler, U. (1991), Abstracts of the 43rd Annual Meeting for the Deutsche Gesellschaj?fur Hygiene and Microbiologie, MunsterlWesifalen, FRG. Nair, G . B., Abraham, M. and Natarajan, R . (1979). Zndian Journal of Marine Science 8,46. Nealson, K. H. (1977). Archives of Microbiology 112, 73. Nealson, K. H. (1979). Trends in Biochemical Sciences 4 , 105. Nealson, K. H. and Hastings, J . W. (1977). Archives of Microbiology 112, 9. Nealson, K. H. and Hastings, J . W. (1979). Microbiological Reviews 43, 496. Nealson, K. H. and Hastings, J. W. (1991). In “The Prokaryotes, A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification. Applications” (A. Balows, H . G. Truper, M. Dworkin, W. Harder and K.-H. Schleifer, eds), 2nd edn. Springer-Verlag, New York. Nealson, K. H. and Walton, D. S. (1978). Abstracts of the Annual Meeting of the American Society for Microbiology, Washington, D C , 1-1. Nealson, K. H., Platt, T. and Hastings, J . W. (1970). Journal of Bacteriology 104, 313. Nealson, K. H . , Eberhard, A . and Hastings, J. W. (1972). Proceedings of the National Academy of Sciences of the United States of America 69, 1073.
BACTERIAL. BIOI.UMINESCENCF
65
Nealson, K. H., Haygood, M . G., Tcbo, B. M . , Roman, M., Miller, E. and McCosker, J. E. (1985). Microbial Ecology 11, 69. Nearhos, S. P. and Fuerst, J. A . (1987). Current Microbiology 15, 329. Ne’eman, Z . , Ulitzur, S . , Branton, D. and Hastings, J . W. (1977). Journal of Biological Chemistry 252, 5150. Nicoli, M. Z . , Meighcn, E. A . and Hastings, J. W. (1974). Journal of Biological Chemistry 249, 2385. Nussbaum, A. and Cohen, A . (1988). Journal of Molecular Biology 203, 391. O’Brien. C . H. and Sizemore, R. K. (1979). Applied and Environmental Microbiology 38, 928. O’Kane, D. J., Vervoort, J., Muller, F. and Lee, J. (1987). In “Flavins and Flavoproteins” (D. E. Edmondson and B. B. McCormick, eds), pp. 641445. Walter de Gruyter, Berlin. O’Kane, D. J . , Linglc, W. L., Wampler, J . E., Legocki, R. P. and Szalay, A . A . (1988). Plant Molecular Biology 10, 387. O’Kane, D. J., Woodward, B., Lee, J. and Prasher, D.C. (1991). Proceedings ofthe National Academy of Sciences of the United States of America 88, 1100. Oliver, J. D . , Roberts, D.M., White, V. K., Dry, M. A . and Simpson, L. M. (1986). Applied and Environmental Microbiology 52, 1209. Olsson, O., Escher, A , , Sandberg, G . , Schell, J., Koncz, C. and Szalay, A . (1989). Gene 81, 335. Olsson, O., Nilsson, 0.and Koncz, C. (1990). Journal of Rioluminescenceand Chemiluminescence 5, 79. Orndorff, S. A. and Colwell, R. R. (1980). Appliedand Environmental Microbiology 39,983. Ortigosa, M., Esteve, C. and Pujalte, J.-J. (1989). Systematic and Applied Microbiology 12, 316. Palmer; L. M. and Colwell, R . R . (1991). Applied and Environmental Microbiology 57, 1286. Paquatte, 0. and T u , S.-C. (1989). Photochemistry and Photobiology 50, 817. Park, S. F., Stirling, D. A . , Hulton, C. S., Booth, I. R . , Higgins, C. F. and Stewart, G. S. A. B. (1989). Molecular Microbiology 3, 1011 . Park, S . F., Nissen, U . andstewart, G. (1991). In: “Bioluminescence and Chemiluminescence: Current Status” (P. E . Stanley and L. J . Kricka, eds), pp, 35-38. Wiley, Chichester. Poinar, G. O., Thomas, G . , Haygood, M. G . and Nealson, K. H. (1980). Soil Biology and Biochemistry 12, 5. Potrikus, C. J . , Greenberg, E. P.. Hamlett, N. V . , Gupta, S. and Hastings, J . W. (1984). Abstracts of the Annual Meeting of the American Society for Microbiology, Washington, D C , N-42. Prasher, D. C., O’Kane, D., Lee, J. and Woodward, B . (1990). Nucleic Acids Research 18, 6450. Raina, S . , Mabey, L. and Georgopoulis, C. (1991). Journal of Bacteriology 173, 5999. Ramaiah, N . and Chandramohan, D. (1987). Indian Journal of Marine Science 16, 139. Ramcsh, A. and Venugopalan, V. K. (1988). FEMS Microbiology Letters 53, 27. Ramesh, A , , Nandakumar, R. and Venugopalan, V. K. (1986). Microbial Ecology 12, 231. Ramesh, A , , Nair, G . B., Abraham, M., Natarajan, R . and Venugopalan, V . K. (1987). Microbios 52, 151. Rauschcl, F. M . and Baldwin, T. 0. (1989). Biochemical and Biophysical Research Communications 164, 1137. Reichelt. J. L., Nealson, K. and Hastings, J. W. (1977). Archives of Microbiology 112, 157. Richter, G., Volk, R., Krieger, C., Lahm, M.-V., Rothlisburger. U . and Bachcr, A . (1992). Journal of Bacteriology 174, 4050. Riendeau, D., Rodriguez, A . and Meighen, E. A . (1982). Journal of Biological Chemistry 257, 6908. Rodriguez, A., Riendeau, D. and Meighen, E. (1983a). Journal of Biological Chemistry 258, 5233. Rodriguez, A., Wall, L., Ricndeau, D . and Meighen, E. A . (1983b). Biochemistry 22, 5604. Rodriguez, A . , Nabi, I. R. and Meighen, E. (1985). Canadian Journul of Biochemistry and Cell Biology 63, 1106.
66
E. A . MFIGHEN .AND P. V DUNLAP
Rodgriquez, A , , Wall, L., Raptis, S., Zarkadas, C. and Meighen, E. A. (1988). Biochimica et Biophysica Acta 964, 266. Rogowsky, P. M., Close, T. J . , Chimera, J . A . , Shaw, J. J. and Kado, C. I. (1987). Journal of Bacteriology 169, 5101. Rosson, R . A. and Nealson, K. H. (1981). Archives of Microbiology 129, 299. Ruby, E. G. and McFall-Ngai, M. J. (1992). Journal of Bacteriology 194, 4865. Ruby, E. G . and Morin, J. G. (1979). Applied and Environmental Microbiology 38, 406. Ruby, E. G . and Nealson, K. H . (1976). Biological Bulletin 151, 574. Ruby, E. G . and Nealson, K. H . (1977). Science 196, 432. Ruby, E. G . and Nealson, K. H . (1978). Limnology and Oceonography 23, 530. Ruby, E. G., Greenberg. E. P. and Hastings, J. W. (1980). Applied and Environmental Microbiology 39, 302. Schauer, A. T. (1988). Trends in Biotechnology 6, 23. Schauer, A , , Ranes, M . , Santamaria, R., Guijarro, J., Lawlor, E., Mendez, C., Chater, K. and Losick, R . (1989). Science 240, 768. Schmetterer, G . , Wolk, C . P. and Elhai, J. (1986). Journal of Bacteriology 167, 411. Schmidt, T. M., Kopecky, K. and Nealson, K. H . (1989). Applied and Environmental Microbio1og.v 55, 2607. Schultz, D . W. and Yarus, M. (1990). Journal of Bacteriology 172, 595. Seliger, H. H. (1987). Photochemistry and Photobiology 45, 291. Shadel, G . S. and Baldwin, T. 0. (1991). Journal of Bacteriology 173, 568. Shadel, G. S. and Baldwin, T. 0. (1992a). Journal of Biological Chemistry 267, 7690. Shadel. G. S. and Baldwin. T. 0. (1992b). Journal of Biological Chemistry 267, 7696. Shadel, G . S., Devine, J. H . and Baldwin, T. 0. (1990a). Journal of Bioluminescence and Chemiluminescence 5, 99. Shadel. G. S . , Young, R . and Baldwin, T. 0. (1990b). Journal of Bacteriology 172, 3980. Shaw, J . J . and Kado. C. I. (1986). Biotechnology 4, 60. Shaw, J. J . , Rogowsky, P., Close, T. J. and Kado, C. I. (1987). Plant Molecular Biology Reporter 5, 225. Shaw, J. J., Settles, L. G . and Kado, C . I. (1988). Molecular Plant-Microbe Interactions 1,39. Shilo, M. and Yetinson, T. (1980). Applied and Environmental Microbiology 38, 577. Shimomura, O., Johnson, F. H. and Morise, H. (1974). Proceedings of the National Academy of Sciences of the United States of America 71, 4666. Showalter, R. E., Martin, M. 0. and Silverman, M. R. (1990). Journal of Bacteriology 172, 2946. Silverman, M . , hfartin, M. and Engebrecht, J. (1989). in “Genetics of Bacterial Diversity” (D. Hopwood and K. F. Chater, eds), pp. 71-85. Academic Press, New York. Simon, R. D . , Shilo, M. and Hastings, J . W. (1982). Current Microbiology 7, 175. Singleton, R. J. and Skerman, T. M. (1973). Journal of the Royal Society of New Zealand 3, 129. Slock, J . , VanRiet, D . , Kolibachuk, D . and Greenberg, E. P. (1990). Journalof Bacteriology 172, 3974. Soh, G. (1970). Atti Societa Peloritana 16, 159. Soly, R. R. and Meighen, E. A. (1991). Journal of Molecular Biology 219, 69. Soly, R . R., Mancini, J. A . , Ferri, S. R . , Boylan, M. and Meighcn, E. A. (1988). Biochemical and Biophysical Research Communications 155, 35 1 . Spyrou, G., Haggard-Ljungquist, E., Krook. M., Jornvall, H., Nilsson. E . and Reichard, P. (1991). Journal of Bacteriology 173, 3673. Stanley, P. E. and Stewart, G. S. A. (1990). Journal of Bioluminescence and Chemiluminescence 5, 141. Stewart, G., Smith, T. and Denyer, S. (1989). Food Science and Technology Today 3, 19. Sugihara, J. and Baldwin, T. 0. (1988). Biochemistry 27, 2872. Suzuki, K., Kaidoh, T., Katagiri, M. and Tsuchiya, T. (1983). Biochimica et Biophysica Acta 722, 297. Swanson, R . F., Weaver, L. H., Remington, S. J., Mathews, B. W. and Baldwin, T. 0. (1985). Journal of Biological Chemistry 260, 1287.
BACTERIAL BIOLUMINESCENCE
67
Swartzman, E., Kapoor, S., Graham, A. and Meighen, E. (1990a). Journal of Bacteriology 112, 6797. Swartzman, E., Miyamoto, C., Graham, A . and Meighen, E. (1990b). Journal of Biological Chemistry 265, 3513. Swartzman, E., Cao, J . G., Miyamoto, C., Graham, A. and Meighen, E. (1991). In “Bioluminescence and Chemiluminescence: Current Status” (P. Stanley and L. J. Kricka, eds), pp. 27-30. Wiley, Chichester. Swartzman, E., Silverman, M. and Meighen, E. A. (1992). Journal of Bacteriology, in press. Szittner, R . and Meighen, E. (1990). Journal of Biological Chemistry 265, 16581. Tu, S. C. (1979). Biochemistry 18, 5940. Tu, S. C. (1986). Methods in Enzymology 133, 128. Ulitzur, S. (1989). Journal of Bioluminescence and Chemiluminescence 4, 317. Ulitzur, S. and Hastings, J. W. (1979a). Proceedings of the National Academy of Sciences of the United States of America 76, 265. Ulitzur, S. and Hastings, J. W. (1979b). Current Microbiology 2, 345. Ulitzur, S. and Kuhn, J. (1986). In: “Bioluminescence: New Perspectives” (Schomerich et a [ . , eds), pp. 46-72. Wiley, New York. Ulitzur, S. and Kuhn, J. (1988). Journal of Bioluminescence and Chemiluminescence 2, 81. Ulitzur, S. and Yashphe, J. (1975). Biochimica et Biophysica Acta 404, 321. Ulitzur, S., Reinhertz, A . and Hastings, J. W. (1981). Archives of Microbiology 129, 67. Vervoort, J., Muller, F., O’Kane, D. J., Lee, J. and Bacher, A . (1986). Biochemistry 25,8067. Vervoort, J . , O’Kane, D. J., Muller, F., Bacher, A . , Strobl, G. and Lee, J. (1990). Biochemistry 29, 1823. Waddle, J. J . , Johnston, T. C. and Baldwin, T. 0. (1987). Biochemistry 26, 4917. Wall, L. A . and Meighen, E. A. (1986). Biochemistry 25, 4315. Wall, L. A . , Byers, D. M. and Meighen, E. A. (1984a). Journal of Bacteriology 159, 720. Wall, L. A , , Rodriguez, A. and Meighen, E. (1984b). Journal of Biological Chemistry 259, 1409. Wall, L., Rodriguez, A . and Meighen, E. (1986). Journal of Biological Chemistry 261, 16018. Watanabe, H . and Hastings, J. W. (1986). Archives of Microbiology 145, 342. Watanabe, H., Mimurd, N., Takimoto, A. and Nakamura, T. (1975). Journal ofBiochemistry 11, 1147. Watanabe, H., Inaba, H. and Hastings, J. W. (1991). Archives of Microbiology 156, 1. Wei, S. L. and Young, R . E. (1989). Marine Biology 103, 541. Welches, W. R. and Baldwin, T. 0. (1981). Biochemistry 20, 512. West, P. A. and Lee, J. V. (1982). Journal of Applied Bacteriology 52, 435. West, P. A . , Lee, J . and Bryant, T. (1983). Journal of Applied Bacteriology 55, 263. Wimpee, C. F., Nadeau, T. L. and Nealson, K. H. (1991). Applied and Envtronmental Microbiology 51, 1319. Woese, C. R . (1987). Microbiological Reviews 51, 221. Wolfe, C. J. and Haygood, M. G . (1991). Biological Bulletin 181, 135. Xi, L., Cho, K.-W., Herndon, M. E. and Tu, S.-C. (1990). Journal ofBiologica1 Chemistry 265, 4200. Xi, L., Cho, K.-W. and Tu, S.-C. (1991). Journal of Bacteriology 173, 1399. Xin, X., Xi, L. and Tu, S.-C. (1991). Biochemistry 30, 11255. Yang, Y., Lee, P.-Y., Cao, Y., Baumann, L., Baumann, P., Tang, J. S. and Beaman, B. (1983). Current Microbiology 8, 95. Yetinson, T. and Shilo, M. (1979). Applied and Environmental Microbiology 37, 1230.
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Sex Hormones and Fungi GRAHAM W. GOODAY" and DAVID J. ADAMS' a Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen AB9 IAS, U K , and Department of Microbiology, University of Leeds, Leeds L S 2 9JT. U K
'
I . Introduction . . , . . . . , . . . . . . . TI. Endogenous hormones . . . . . . . . . . . . . A. Tsoprenoids . . . . . . . . . . . . . . B. Peptides . . . . . . . . . . . . . . . C . Uncharacterizcd hormone systems . . . . . . . . . D. Conclusions , . . . . . . . . . . . . . 111. Endogenousregulatorsofsexualdevelopment . . . . . . . IV. Ifiteractionsofmammalianhnrmoneswithfungi . . . . . . . A. Effects of hormones on fungal growth and morphogenesis in vitro . . B. Mammalian hormone-binding proteins and endogenous ligands in . , . . . . . . . . . . . . . fungi, C . Biochemical response . . . . . . . . . . . . D. Implications for pathogenesis . . , . . . . . . . E. Evolutionary aspects . . . . . . . . . . . . V. Signal transduction following interaction of ala mating factors or mammalian hormones with yeasts A. a- and a-mating factors . . . . . . . . . . . B. Mammalian hormones. . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . VIT. Acknowledgements. . . . . , . . . . . . . '. References . . . . . . . . . . . . . . . .
69 70 71 86 100 101 103 105 105 112 123 128 131 132 132 133 133 134 134
1. Introduction
It is self-evident that chemical communication is essential for co-ordination of activities in a multicellular organism. Hormones play an essential role in this communication as chemical signalling molecules. As defined by Huxley (1935), hormones are substances produced in one portion of an organism and transported by any means, including diffusion, to other ADVANCES IN MICRORIAI PHYSIOLOGY. VOL 34 ISBN G12427734-4
Copyright0 IY93, by AcademicPresslimited All rights ofreproduction in any form reserved
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portions of the same individual or other individuals of the same species where they induce specific responses. It is now abundantly clear that unicellular organisms have analogous systems of chemical communication. The hormones discussed here share common properties: they are of relatively low molecular weight; their production is specifically regulated, spatially, temporally and by environmental factors; they are produced in low concentrations and active at very low concentrations; and they elicit a specific set of responses. Here we describe the fungal sex hormone systems and compare and contrast these with the interactions of mammalian hormone systems with fungi. The growing number of examples of specific responses of fungi to mammalian hormones invites speculations on the evolution of hormone systems, and of their signal-transducing systems. Also raised are questions on the significance of hormones in the development of pathogenesis of fungi to mammals. The use of the term “pheromones” has become widespread for the fungal hormones, as chemicals (e.g. insect sex attractants) acting at a distance. In this review we retain the word “hormone” throughout, as this enables us to compare the fungal hormones with the mammalian hormones, and as it is a readily understandable term, as discussed by Raper (1967), Machlis (1972), Gooday (1974) and Bu’Lock (1976). Some examples discussed here, such as antheridiol-oogoniol and trisporic acid, would otherwise lead to confusion, as they are involved in intercolony communication in heterothallic strains (i.e. pheromones) but intracolony communication in homothallic strains (i.e. hormones). In contrast to the fungal sex hormones, which elicit complete switches in developmental pathways, we also consider substances which regulate the magnitude and kinetics of fungal sexual development, some of which may prove to be morphogens.
11. Endogenous Hormones
The fungus is a cornucopia of strange and wonderful chemicals. These secondary metabolites can be of value to us, notably as antibiotics. Only a few are of any apparent direct value to the organism producing them. One such group is the sex hormones-pheromones, which have precise functions in regulating sexual reproduction between two individuals of the same species. Chemical communication is clearly of paramount importance to fungi in their natural environments, and the compounds discussed here have the necessary properties of specificity and potency to enable potential mating partners to find each other and prepare themselves for sexual fusion. To date, only a few of the undoubted thousands of examples have been
SFX HOKMONES AND FUNGI
71
elucidated. Here we describe their roles in diverting the cells from other activities and bringing partners together in a physiological state for mating. They are chemically diverse but currently can be grouped in two classes, namely the isoprenoids comprising sesquiterpenes, sterols and apocarotenoids, and the hydrophobic peptides. Earlier reviews charting their discovery and characterization are by Gooday (1974, 1983), Bu’Lock (1976) and Ende (1976, 1983, 1984). A . ISOPRENOIDS
I. Chytridiomycetes Allomyces is a genus of chytrid water moulds, living saprophytically on submerged vegetable or animal matter. Haploid vegetative cells differentiate to give male and female gametangia, often on the same hypha. Thus, Allomyces macrogynus in culture forms a pair of multinucleate gametangia, the male being terminal and the female subterminal. In nutrient-poor conditions, gametogenesis occurs. The resulting gametes are similar, being uniflagellate and approximately spherical, but the males are smaller (about 8 pm in diameter) and bright orange with y-carotene, while the females are about 11 pm in diameter and colourless. They swim through exit pores that result from localized lysis of the gametangial walls. Their behaviours are different, the female cells swimming sluggishly with frequent changes in direction so that they tend to stay in one place, and the males swimming faster with fewer changes in direction so that they tend to cover much larger distances (Pommerville, 1987, 1981; Table 1). When they come into contact, plasmogamy can occur, resulting in formation of a biflagellate zygote (Pommerville, 1982; Pommerville and Fuller, 1976). This swimming cell eventually settles to germinate as the diploid vegetative thallm, which either produces diploid zoospores as propagules for further diploid thalli, or, via meiosis, haploid zoospores as propagules for the haploid vegetative thalli. Machlis (1958) showed that the meeting of male and female gametes is not random; rather, the female gametangium and the swimming female gametes produce a potent attractant for the male gametes. Machlis termed this attractant sirenin, after the mythical sirens whose singing lured sailors to their deaths. Sirenin is produced in small quantities and, in order to characterize it, Machlis el al. (1966, 1968) grew large-scale cultures of a predominately female hybrid that produced about 1 p~ sirenin in the culture filtrate. A n essential requirement for characterizing sirenin, as for the other hormones discussed in this review, is the development of a rapid specific and sensitive bioassay. Machlis (1973a,b) devised increasingly sensitive
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G . W . GOODAY AND D J ADAMS
TABLE 1. A comparison of male and female gametes of Allomyces macrogynus Property Pigmentation Size (pm in diameter) Swimming characteristics Velocity (pm s- ') Smooth-swimming distance (pm) Turn angle Number of turns per minute Production of sirenin Response to sirenin Uptake of sirenin Inactivation of sirenin Change in swimming Production of parisin Response t o parisin Specificities for plasmogamy and karyogamy
Male
Female
y-Carotene
None 11
8 100
60
50 60" 70 No
65 80" 100 Ycs
Yes Yes Yes Yes No To femalc
No No No No Yes To male
bioassays that involved counting the number of male gametes that settled on a membrane, the other side of which was the test solution, and he found that they were sensitive to sirenin over a very wide concentration range (100 PM to 10 p~).Pommerville (1977, 1987) has developed a microassay using wells cut in agar in immunodiffusion plates. Two wells of 1 mm in diameter are cut in the agar, 1.5 mm apart, and a 5 pl gamete suspension is added to one and 5 p1 of test solution to the other. Chemotaxis is observed as an accumulation of gametes on the side of the well adjacent to the test solution after 20 minutes, and the potency of the solution is assessed as a threshold value, i.e. the lowest concentration that causes chemoattraction. For sirenin this is 10 PM. Sirenin proved to be a remarkable bicyclic sesquiterpene (Nutting et al., 1968; Fig. I). Sirenin and a range of analogues and derivatives have been synthesized (Plattner et al., 1969; Bhalerao et al., 1970; Plattner and Rapoport, 1971; Harding et al., 1988; Pommerville et al., 1988). Synthetic (-)-sirenin, identical to the natural product, is fully active; (+)-sirenin has no activity (tested up to 100 nM), along with several other analogues (tested up to 1 p ~ (Bhalerao ) et al., 1970; Machlis, 1973b). Only one synthetic analogue has shown appreciable activity, equivalent to sirenin with a threshold value of 10 PM, and that is the monohydroxy analogue, lacking the hydroxymethyl group on the cyclohexene ring (Harding et al., 1988; Pommerville et al., 1988). From these results, Pommerville et al. (1988) suggest that the chemical features necessary for high activity are a terminal hydroxyl group on a chain with the hydrophobic portion of the bicyclic ring at the other end of the chain.
S F X H O R M O N t S ANI) ELINGI
73
CH ,OH
FIG. 1. Structurc of sirenin, the fcmale sex attractant of Aflomyces macrogynus.
The behavioural response of male gametes to sirenin has been analysed by Pommerville (1977, 1978, 1981). The male gametes show a smooth swimming pattern, interrupted by very brief jerks of the cell body, resulting in a change of swimming direction. The female gametes swim more slowly, and have a higher frequency of jerks. Female gametes introduced into the vicinity of males induce an immediate change in the male swimming pattern. Males move in helical paths towards females, with only a few jerks to reorient themselves. When very near to a female, a male swims in many very short runs and jerks until contact with the female is achieved, resulting in plasmogamy. The swimming pattern of the zygote is distinctive, being faster than that of either gamete, in smooth helices involving a continuous turning of the cell body. Addition of a pulse of sirenin solution to a suspension of male gametes results immediately in rapid jerking movements, like those seen in the immediate vicinity of a female cell, with very little net movement in any direction. Over the next 15-30 seconds, the cells change behaviour to swim in large circles or helices, like those seen at some distance from a female cell. About 6G90 seconds after addition of sirenin, the cells are reverting to their normal unstimulated pattern, but some are seen to swim in clusters, a behaviour never seen in the absence of sirenin. Sirenin produced by female gametes thus brings about a very precise orientation and attraction of male gametes, favouring efficient mating. The mechanism of sensing the concentration gradient of sirenin could be temporal, with an increase in concentration suppressing the frequency of jerks as a male swims towards a female, and a decrease in concentration increasing the frequency as a male swims away. The change in direction of swimming appears to involve temporary cessation of flagellar beating, resulting in a jerk (Pommerville, 1978). Of the five types of swimming cells produced by A . rnacrogynus, haploid and diploid zoospores, male and female gametes and zygotes, only male gametes respond to sirenin, and only female gametes produce it (Carlile and Machlis, 1965a). Zoospores and zygotes, but not gametes, are attracted
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G W GOODAY AND D J ADAMS
by some amino acids (Carlile and Machlis, 1965b). Both zoospores and gametes are repelled by some cations, but sirenin together with a cation attracts male gametes (Pommerville and Olson, 1987a). Calcium ions are required for the chemotactic response (Machlis, 1973a), and a calcium-ion influx may be part of the signal-transduction mechanism in response to sirenin (Pommerville and Olson, 1987a). Sirenin is rapidly inactivated by male gametes (Carlile and Machlis, 1965a,b; Machlis, 1973a), which presumably aids their sensing of the gradient in the medium. In addition, Klapper and Klapper (1977) describe a low molecular-weight inhibitor of the male chemotactic response. The female cells are attracted by a complementary hormone produced by the male cells, called parisin (again from Greek mythology) by Pommerville and Olson (1987b). Preliminary characterization suggests that parisin has similar chemical properties to sirenin, and may also be an isoprenoid. 2. Oomycetes Species in the genus Achlya (Oomycetes: Saprolegniales) are water moulds, living saprophytically on submerged vegetable and animal matter. Their vegetative hyphae are diploid, and sexual reproduction entails formation of antheridial and oogonial initials on the same mycelium or two closely growing mycelia. The antheridial initials grow towards the oogonial initials, to come into contact with them, when antheridia and oogonia mature by delimitation of the hyphal apices by septa and meiosis, to give egg cells and antheridial fertilization tubes. Most strains are homothallic, but some are heterothallic, self-sterile with three types of behaviour, solely male, solely female or male-female, according to the nature of the strain with which they are paired (Raper, 1951; Barksdale, 1967; Thomas and Mullins, 1967). The genetic mechanism of this mating system is unclear. In a series of elegant experiments, Raper (1951) showed that sexual differentiation in these fungi is regulated by complementary hormones, diffusing from hypha to hypha. The female cells release a hormone that switches the male cells from vegetative growth to production of many short narrow antheridial branches. The male cells then release a second hormone which diffuses back to the female cells, switching them in turn from vegetative growth to production of oogonial initials. Raper devised a reliable bioassay for the female hormone by showing that the number of antheridial branches produced is proportional to its concentration. In 1942, Raper and Haagen-Smit obtained 2 mg of active compound (called hormone A at that time) from 1440 litres of culture filtrate of female mycelium. This was far too little for characterization by contemporary chemical techniques, but the original sample was kept and later identified as the substantially
SEX HORMONES AND FUNGI
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OH
FIG. 2. Structures of (a) antheridiol and (b) oogoniol, the complementary sex hormones of Achlya spp. For oogoniol-1, R is an isobutyrate residue.
pure female hormone, antheridiol. Antheridiol was finally characterized as an oxygenated derivative of the common plant sterol fucosterol (Fig. 2) by McMorris and Barksdale (1967) and Arsenault et al. (1968). The yield was very low, at about 6 nM in the culture filtrate. A related metabolite, 23-deoxyantheridiol, was produced in about the same concentration (Green et al., 1971). Antheridiol and a range of isomers and derivatives have been synthesized and their biological activity assessed (Edwards et al., 1969, 1972; Barksdale et al. , 1974; McMorris et al. , 1974; Weihe and McMorris, 1978; Meyer et al. , 1987; McMorris, 1989). Besides antheridiol, only a few related sterols give a positive test in the bioassay with a male strain of Achlya ambisexualis. Antheridiol is active at 10 pg ml-'. The compound lacking the C-7 ketone has about 10% of its activity while 23-deoxyantheridiol and a trienoic acid intermediate have about 0.17'0, and isomers with different configurations at C-22 and C-23 have 0.1% or less. The complementary hormone produced by the male cells, oogoniol, proved more difficult to characterize. In most situations it was only produced in the presence of antheridiol, which complicated attempts at purification. It was finally purified from culture filtrates of a homothallic strain of Achlya heterosexualis, which produces it constitutively (Barksdale and Lasure, 1974). About 10 mg of a mixture of related metabolites was obtained from 200 litres of culture filtrate. The active compounds were
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G W G O O D A Y AND D J A D A M S
unesterified oogoniol and 24(28)-dehydro-oogoniol, and oogoniols-1, -2 and -3, the isobutyrate, propionate and acetate esters, respectively, and their (24)28-dehydro analogues (Fig. 2; McMorris et a f . , 1975; McMorris, 1978,1989; Preus and McMorris, 1979). Oogoniols and dehydro-oogoniols have been synthesized (McMorris et al., 1983, 1989; Moon et a f . , 1990). The bioassay involves counting the number of oogonial initials produced in a female strain of A . ambisexualis. The most active compound is the minor metabolite 24(28)-dehydro-oogoniol-1, active at 50 ng ml-'. Dehydrooogoniol has similar activity, whereas oogoniol-1 (the major metabolite) and oogoniol are about 100 times less active (McMorris et a f . , 1989). All of these hormones are derived from fucosterol, the major sterol of Achfya spp. (Poppelstone and Unrau, 1974; White and McMorris, 1978; Barrow and McMorris, 1982). It is clear, however, that antheridiol and the oogoniols are on two separate biosynthetic pathways from fucosterol, i.e. oogoniols are not derived from antheridiol (McMorris, 1978). Addition of antheridiol to receptive male mycelium of A. ambisexualis elicits a series of responses. These responses are only seen in nutrient-poor conditions (Mullins and Warren, 1975; Gow and Gooday, 1987). Riehl and Toft (1985a,b) report that the levels of cytosolic antheridiol-binding receptor vary inversely with the nutrient status of the culture. Thus, antheridiol-binding capacity fell rapidly on transferring a culture from a nutrient-poor to a nutrient-rich medium, and rose rapidly on transfer from a nutrient-rich to a nutrient-poor medium. Responses observed successively, with increasing concentrations of antheridiol, are: (a) Cessation of apical growth. Gow and Gooday (1987) grew hyphae out from a block of nutrient agar into a mineral salts medium. About an hour after addition of antheridiol, the hyphae had ceased to extend, while control hyphae extended for many hours (Fig. 3). This growth arrest is analogous to that observed when Phycomyes blakesleeanus and certain yeasts respond to their sex hormmes (see later). Gow and Gooday (1987) also observed that cessation of apical extension was accompanied by a diminution of the characteristic inward current associated with apices of vegetatively growing hyphae. This current probably represents the new inflow of cations, chiefly protons, involved in nutrient uptake, but the significance in its diminution is unclcar (Gow, 1989). (b) Antheridial branching. Antheridial initials are characteristic multiple narrow branches of the original hyphae, and their formation is the basis of the bioassay (Fig. 3 ; Barksdale,1967; Mullins and Ellis, 1974; Gow and Gooday, 1987). Gow and Gooday (1987) report that, under
SEX f i O R M O N E S AND FUNGI
77
FIG. 3 . Male hyphae of Achfyn arnbisexuulis growing from a block of nutrient agar into “artificial pond water”: (a) control culture showing continucd apical growth; (b) culture treated with anthcridiol, showing cessation of growth and profuse antheridial branching. Scale bar represents 200 pm.
their conditions, sites of antheridial branches were preceded and predicted by new sites of inward ion currents. Cooper-Palomar and Powell (1988) report accumulations of calcium ions, as detected by chlortetracycline fluorescence, in the apices of antheridial branches. (c) Chemotropism of antheridial branches. Oogonial initials secrete relatively large amounts of antheridiol, which acts as a chemotropic attractant for antheridial branches. Barksdale (1963, 1967) showed that antheridial branches grew towards plastic particles with adsorbed antheridiol, and wrapped around them. (d) Differentiation of antheridia. Finally, Barksdale (1963,1967) demonstrated that antheridiol treatment results in formation of a septum to delimit the developing antheridium, and meiosis to give the male gametic nuclei. Accompanying these morphological changes are a series of biochemical responses, most of which can be detected by 30 minutes after treatment: (a) Increase in synthesis and release of oogoniol. This increase is inhibited by cycloheximide (McMorris and White, 1977). (b) Induced metabolism of antheridiol to inactive metabolites (Musgrave and Nieuwenhuis, 1975; Musgrave et al., 1978). This process was inhibited by cycloheximide and actinomycin D. This inactivation may play a vital role in chemotropism, enabling the antheridial initials to detect the gradient of antheridioi. (c) Increase in the activity and release ofcellulase (Thomas and Mullins, 1969; Mullins, 1973). As this increase in cellulase accompanies
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G W GOODAY AND D I ADAMS
antheridial branching, it may play a direct role in softening of the lateral cellulosic hyphal wall, to allow the pushing out of the new branches. Freeze-fracture electron microscopic studies of male hyphae responding to antheridiol show aggregations of vesicles at the sites of branching, with clear indications of exocytosis into the wall (Mullins and Ellis, 1974; Mullins, 1979). Thomas (1970) showed that water stress, that is, loss of turgor pressure, inhibited both antheridial branching and cellulase secretion in response to antheridiol, providing more evidence that there is a direct relationship between these two phenomena. (d) Suppression of lipoxygenase activity, 24 hours after addition of antheridiol, assayed as oxidation of ['4C]arachidonic acid (Herman et al., 1989). ( e ) Marked enhancement of synthesis of rRNA, mRNA and protein, of histone acetylation and of transcription of the chromatin synthesized by Achlya spp. in the presence of cytosol from Achlya spp. (Silver and Horgen, 1974; Horgen et al., 1975; Timberlake, 1976; Horgen, 1981; Horgen et al., 1983, 1984; Timberlake and Orr, 1984). Induction of specific protein synthesis by antheridiol has proved difficult to characterize, and Rozek and Timberlake (1980) and Gwynne and Brandhorst (1980) concluded that the major changes in mRNA and protein synthesis are quantitative rather than qualitative. This view has been endorsed by Horten and Horgen (1989), who used differential screening of cDNA libraries constructed from poly(A)+ RNA isolated from antheridiol-induced and vegetative cultures of male A . ambisexualis to search for hormone-regulated sequences. Differential screening, dot-blot analysis and RNA hybridization experiments, however, revealed that only three out of the 36 clones isolated were steroid regulated, and all of these were downregulated by antheridiol . Earlier reports of a hormone-induced protein in the molecular-mass range 6 M 9 kDa (Groner et al., 1976; Michalski, 1978; Gwynne and Brandhorst, 1980; Horten and Horgen, 1985) have been clarified by Brunt and Silver (1986a,b, 1987). Using labelling with [35S]methionine, Brunt and Silver concluded that antheridiol regulates synthesis and/or accumulation of a number of proteins, including at least four distinct proteins in the molecularmass range 63-64 kDa. These four have different cellular localizations, being associated with the nucleus, cytoplasm, cell wall-cell membrane and cytoplasm-cell wallkell membrane, respectively. Brunt and Silver (1986a) also report qualitative and quantitative changes in secretion of a number of glycoproteins, as yet unidentified, in response to treatment with antheridiol. Brunt et al. (1990) further
SEX HORMONES A N D FUNGI
79
characterized a prominent antheridiol-induced protein, of 85 kDa, that they had earlier found both in the nuclear and cytoplasmic fractions (Brunt and Silver, 1986b). This has proved to be similar or identical to the 85 kDa heat-shock protein from A . ambisexualis (Pekkala and Silver, 1987) and to the non-hormone-binding component of the antheridiol receptor (Riehl et al., 1985; see below). Using [3H]7-deoxy-7-dihydroantheridiol as a ligand, Riehl et al. (1984) and Riehl and Toft (1984) discovered a specific binding protein in the cytosol of male cells. No specific binding was detected in cytosols from female cells. The equilibrium dissociation constant was 0.7 nM, and the maximum binding capacity was 1-2 pmol (mg protein)-'. These values are consistent with the bioassay results that male cells respond to a threshold concentration of 0.24 nM 7-deoxy-7-dihydroantheridio1, and the response increases within a 10-20-fold range above this concentration. Competition experiments with a range of sterols, including progesterone, oestradiol and testosterone, showed that the binding was specific to antheridiol and 7deoxy-7-dihydroantheridiol. The receptor had a molecular mass of 19.2 kDa. As already mentioned, the levels of receptor changed with the nutrient status of the culture. They were highest in nutrient-deficient media and scarcely detectable in rich media (Riehl and Toft, 1985a,b). Riehl et al. (1985) and Riehl and Toft (1986) showed that this antheridiol receptor is very similar to other steroid receptors: it is highly specific; it has a high affinity; its cellular concentration is very low, suggesting that a major portion of its binding sites would be occupied at physiological steroid concentrations; it exists in monomeric and multimeric states, with sedimentation coefficients 4 s and 8s; it has a relatively high frictional ratio, indicating a marked asymmetry of the receptor protein; and its structure and function are stabilized by sodium molybdate. Like other redeptors, such as chick oviduct progesterone receptor, the multimeric 8s form of the antheridiol receptor can be dissociated into an antheridiol-binding 4 s moiety and an 85 kDa receptor-binding protein (Riehl et al., 1985; Riehl and Toft, 1986). This protein shows immunological cross-reactivity with a range of 90 kDa vertebrate receptor-binding proteins (Riehl et al., 1985; Riehl and Toft, 1986), and with the 85 kDa heat-shock proteins detected in Achlya spp. (Brunt et al., 1990; Brunt and Silver, 1991). J. C. Silver (personal communication) also reports that two members of the multigene family encoding the 70 kDa heat-shock proteins in Achyla spp. are regulated by antheridiol. Thomas and McMorris (1987), working with a homothallic strain of A . heterosexualis, observed that antheridiol, while stimulating formation of antheridial initials, also inhibited formation of oogonial initials and asexual
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W ( i 0 0 D A Y AND D J
ADAMS
sporangiogenesis. They report that inhibition of asexual reproduction in a range of Achlya strains is directly proportional to the abundance of antheridiol receptor. They suggest that in nature antheridiol could play an allomonal role, that is, affecting individuals of species other than the producing species. There are certainly other equivalent hormone systems in other oomycete species, as yet uncharacterized. For example, Sherwood (1966) described a system very similar to that operating in Achlya spp. in species in the closely related genus Dictyuchus. Nes and Phu (1988) reported that the cholesterol analogue, 25-azacholesterol, when added to cultures of Saprolegnia ferax, had no effect on vegetative growth but interfered with sterol biosynthesis and abolished oogenesis. There has been much interest in possible involvement of sterol hormones in induction of sexual reproduction in the pythiaceous fungi, particularly those in the important plant pathogenic genera Phytophthora and Pythium. These fungi are unable to synthesize sterols, and require sterols for sexual reproduction, either as additions to the culturc medium, or from the host during pathogenesis (Elliott, 1983). A similar situation has been described for the mosquito parasitic oomycete Lagenidium gzganteum (Kerwin and Washino, 1983). Elliott (1972, 1979, 1983) and Nes et al. (1980) investigated the structural requirements for good activity of sterols in inducing oospore formation in the homothallic species Phytophthora cactorum. A finding, with as yet no explanation, is that oestradiol, oestrone and, to a lesser extent, progesterone will inhibit induction of oospores by cholesterol, albeit at high concentrations (Hendrix and Guttman, 1972; Elliott, 1983). The knowledge that antheridiol and oogoniol regulate sexual reproduction in species of the related genus Achlya has led to the hypothesis that sterols are exogenous precursors of sex hormones in pythiaceous fungi (Elliott, 1983). Consistent with this idea is that oogonia and antheridia form very close to each other, so that the putative hormones could be present in tiny amounts and/or be unstable. Against the idea is the repeated failure to characterize such metabolites, and the observation that the same responses can be elicited in some cases by non-sterol lipids such as phospholipids (KO, 1985, 1988) (but therz may have been trace sterol impurities or carry-over in these experiments). Certainly, there is good evidence for diffusible hormones controlling mating in heterothallic species where, for example, formation of antheridia and oogonia occurred in male and female strains of Pythium sylvaticum, respectively, when they were grown separated by a permeable membrane (Gall and Elliott, 1985). KO and other workers have documented a distinct hormone system, termed hormonal heterothallism, in heterothallic species. They have observed that both A1 and A2 mating types form oospores when paired with the opposite mating type of the same or different
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species on the opposite side of a polycarbonate membrane. This shows that a heterothallic strain is induced to become self-fertile by a mating typespecific hormone; A1 strains produce u l hormone which stimulates A2 but not A1 to produce oospores, while A2 strains produce u?, hormone with the complementary specificity (KO, 1983, 1988; KO et al., 1986; Shen et al., 1983; Chang et al., 1984; Guo and KO, 1991). This system is clearly separate from the putative sterol-hormone system. The chemical natures of u l and a2 remain unclear, but they seem to be small lipophilic molecules (KO, 1983, 1988). Elliott and Glen (1982), from experiments on the effects of sterols on mating of the heterothallic species Phytophthora parasitica, suggest that production of a2 hormone does not require sterol, but the response to it does, so that sterols are involved in oospore formation in the presence of a hormone. The effect of a1 hormone can be mimicked by volatile metabolites from Trichoderma spp. (Brasier, 197.5). Two metabolites, termed homothallins I and 11, have been characterized as novel isocyanides with cyclopentene rings (Sakata and Rickards, 1980).
3. Zygomycetes The involvement of diffusible compounds in mating of species of the Mucorales was shown by Burgeff (1924), who observed formation of characteristic gametic hyphae, the zygophores, in both mating types when (+) and (-) Mucor mucedo were grown on the same agar medium separated by a collodion membrane. Sutter and Jelinek (1983) studied formation of these sex hormones in (+) and (-) cultures of Blakeslea trispora separated by membranes, and concluded that its extent is proportional to the rate of diffusion of small molecules. Mesland et al. (1974) demonstrated that zygophore formation can also be induced when (+) and (-) mycelia are separated by an air gap, so that the diffusible agents involved may be volatile. That this hormone system is not species-specific, but universal, in the Mucorales is shown by mutual zygophore formation (but of course not zygospore formation) when (+) and (-) strains of different species are grown together (Blakeslee and Cartledge, 1927; Schipper, 1987). The zygophore-inducing hormones are trisporic acids (Figs 4 and 5 ) and they act on both (+) and (-) mycelia. This is especially clear with M . mucedo, which is used to bioassay trisporic acids, both mating types being sensitive in bioassay conditions to concentrations of the order of 10 PM (Jones et al., 1981; Sutter and Whitaker, 1981a; Ende, 1984). With P. blakesleeanus, Drinkard et al. (1982) have shown that cultures will not respond while actively growing, but need to be in a state of arrested growth before zygophores are formed. Trisporic acids B and C (Fig. 5 , VI and VII) have been identified from
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I
FIG. 4. Pathway showing collaborative biosynthesis of trisporic acid by crossfeeding of intermediates between (+) and (-) mating types of Blakeslea trispora. b-Carotene (I) is metabolized via retinal (11) to 4-dehydrotrisporol (111) by (+) and (-) strains. This last compound is metabolized by (+) strains to 4-dihydrotrisporic acid (IV) and by (-) strains to trisporol (V). Compounds IV and V are metabolized to trisporic acid (VI) only after diffusing to the (-) and (+) strains, respectively. This scheme is an over-simplification since the process generates many cometabolites (see the text for details).
mature cultures of M . mucedo, B. trispora and P. blakesleeanus (reviewed by Bu’lock et al., 1976; Sutter, 1977, 1987; Jones et al., 1981; Ende, 1983, 1984). Miller and Sutter (1984) and Sutter et al. (1989) found that trisporic acid E(3R), which accounts for about 30% of the trisporic acids produced
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VII
FIG. 5. Structures of the two major trisporic acids, B (VI) and C (VII), and of the inactive CI5 degradation product, trisporone (VII).
by P. blakesleeanus, and trisporic acids E(3R), E(3S) and D(2S) which are minor metabolites of B. trispora, are hydroxylated at positions 3R, 3s and 2S, respectively. Only negligible amounts of trisporic acids B and C are detectable in unmated cultures, since zygophores are not formed in unmated cultures. Sutter et al. (1973) did find small concentrations in cultures of (+) B. trispora. However, Sutter and Whitaker (1981b) and Sutter and Zawodny (1984) detected two CIScompounds, namely apotrisporal (trisporone; Fig. 5 , VIII) and the related dimethyl compound apotrisporin, in cultures of (-) B. trispora, which they regard as degradation products of trisporate precursors, preventing accumulation of trisporate in the unmated strain. The mechanism whereby trisporate synthesis is enormously enhanced in mated cultures is a remarkable example of metabolic collaboration between the two mating partners , whereby they transfer complementary predursors between each other and each produces trisporic acids. This process has been reviewed by Bu’lock (1975), Bu’lock et al. (1976), Sutter (1977), Ende (1978, 1983, 1984), Gooday et al. (1979), Jones et al. (1981) and Gooday (1983) and is summarized in Fig. 4. The trisporates are apocarotenoids, CIS oxidative metabolites of @-carotene (I). The initial steps common to both mating types are probably as follows: (a) @-carotene(I) is oxidatively cleaved to retinal (11) by carotene 14,15’-oxygenase; (b) retinal is oxidized to retinoic acid by a dehydrogenase; (c) retinoate is cleaved to give a @CIS ketone by a lyase via a CoA derivative; (d) the C-4 group in the cyclohexane ring is oxidized to an alcohol by a mono-oxygenase; (e) this product is reduced at C-11, C-12 to give 4-dihydrotrisporin; (f) both mating types can oxidize the 1-methyl group to an alcohol, to give 4-dihydrotrisporol (TI), catalysed by mono-oxygenase.
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G W GOODAY AND D J ADAMS
The (+) mating type has specific enzyme activities which oxidize the 1alcohol to a 1-carboxylate, catalysed by dehydrogenases, to give 4dihydrotrisporate (IV) and form the methyl ester of the 1-carboxylate. The (-) mating type also has specific enzyme activities. These include an NADPf-dependent dehydrogenase which oxidizes the C-4 hydroxyl group to a ketone group, to give trisporol (V), and an esterase which hydrolyses the 1-carboxylate methyl esters produced by (+) cells. The (+) mating type also has very low activities of these (-)-type enzymes (Werkman, 1976). The enzymes involved in these transformations have substrate specificities that are broad enough to give rise to a range of related metabolites with different states of oxidation, specifically at C-4 and of the C-1 methyl group, and of methylation. Interconversion between the B and C series of metabolites, i.e. the 13-keto and 13-hydroxyl compounds (Fig. 5 ) , is considered to be trivial. The key feature of this situation is that, when cells of complementary mating type grow near to one another, their mating type-specific metabolites (which are biologically inactive in themselves) can diffuse in advance of them, and enter the opposite mating-type cytoplasm. There they will be substrates for complementary enzymes, yielding the trisporic acids. The precursors are the same and the products are the same in each mating type, but the sequences of chemical transformations differ in (+) and (-) cells, giving a “metabolic grid” for the collaborative biosynthesis (Jones et al., 1981). Both mating types respond to trisporic acid by greatly amplifying the fluxes through the pathways so, as they approach each other, they produce trisporic acids at an ever increasing rate. The major recorded biochemical effect of trisporic acid is the many-fold stimulation of isoprenoid synthesis, primarily of p-carotene its precursor (Gooday, 1968,1978; Govind and Cerda-Olmedo, 1986), but also of sterols, polyprenols and ubiquinones (Bu’lock and Osagie, 1973; Gooday, 1978). The mechanism of this stimulation is unclear, except that it involves protein synthesis but no significant changes in specific activities of mevalonate kinase or hydroxymethylglutaryl-CoA reductase (Jones et al. ,1981). Bu’lock et al. (1976) also reported transient stimulations of RNA synthesis and CAMP levels, both peaking four hours after addition of trisporic acid. The major physiological effect of trisporic acid is formation of zygophores, and this forms the basis of its bioassay (Gooday, 1968, 1973, 1978). Zygophores of M . mucedo have distinctive cell-surface properties (Jones and Gooday, 1977, 1978), but mating-type specific agglutinins have not been detected (Gooday, 1992). Wurtz and Jockusch (1975) described mutants of M . mucedo that did not respond to exogenous trisporic acid. Some of these (+) mutants nevertheless could still mate, perhaps still being able to take up trisporate precursors. Zygophores exhibit zygotropism.
FIG. 6. Micrographs showing sexual interactions in the zone of meeting of a (+) colony (left-hand side of each micrograph) and a (-) colony (right-hand side) of Mucor mucedo. (a) Formation, zygotropism and fusion of two zygophores, photographed at four-minute intervals; (b) subsequent development of gametangia, eventually to produce zygospores over 30 minutes. The scale bar represents 200 pm.
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Two zygophores of M . mucedo of opposite mating type will unerringly grow towards each other to meet and fuse from a distance of at least 2 mm (Fig. 6). Just before meeting, a localized lateral bulging usually occurs at the site that will fuse. There are clearly two effects involved here, both mediated by mating type-specific volatile effectors; these are directed growth of each zygophore from a distance and then, when very close, a localized lateral branching. Zygotropism is mating type-specific not speciesspecific, as it occurs between zygophores of different species. Thus, Schipper (1987) recorded tall zygophores of Mucor flavus bending down to meet short zygophores of M . mucedo. While they are growing, zygophores of M . mucedo have a constant growth rate, irrespective of their zygotropic behaviour (Gooday, 1975), but a zygophore under the influence of zygotropic attractant grows much longer than an unexposed one (Plempel, 1960, 1962) and, when it meets a zygophore of opposite mating type, it stops growing immediately on contact and starts to form a progametangium (Gooday, 1975). Zygophores of M . mucedo are not geotropic or phototropic, but are negatively autotropic, that is, they grow away from their subtending vegetative mycelium, and away from neighbouring zygophores of the same mating type (Banbury, 1955). All evidence suggests that there are two complementary volatile compounds regulating zygotropism, one produced by each mating type and active on the opposite mating type (Banbury, 1955; Plempel, 1960, 1962; Gooday, 1973,1975; Mesland et al., 1974). Mesland et al. (1974) suggested that these two are, or are closely related to, particular mating type-specific precursors of trisporic acid. This is an appealing idea, as we do not have to invoke new biosynthetic pathways for biosynthesis and reception of zygotropic pheromones. It also explains observations such as that of Mesland et al. (1974), whose Fig. 2 shows (-) zygophores being attracted to a (+) sporangiophore since, in this experiment, the (+) mycelium would have been increasingly producing its trisporate precusors in response to the nearness of the (-) mycelium. Also relevant is Fig. 7 in Gooday (1975), which shows that zygophores of both mating types cease to be attractive on fusing with a compatible mate as, after fusion, the precursors that are the attractants are efficiently converted to trisporic acid by the partner, and so no longer can diffuse through the air. B . PEPTIDES
1. Ascosporogenous Yeasts
The best studied system of sex hormones and pheromones among fungi is that of Saccharomyces cerevisiae, on which there is now detailed genetic
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and biochemical information (Thorner, 1981; Cross et al. , 1988; Herskowitz, 1988; Yoshida et al. , 1989; Fields, 1990). The signal-transduction mechanisms in this system have been reviewed by Marsh (1991) and Marsh et al. (1991) and comparative aspects of these are considered in Section IV. Here, we consider chiefly some chemical and cell-biological aspects of the system. Haploid cells are of two mating types, a and a, determined by the regulatory alleles M A T a and M A T a . They will grow vegetatively by budding until brought together, when they fuse in mated pairs to form diploid zygotes. The zygotes in turn grow vegetatively by budding until induced to sporulate. Meiosis then occurs, with formation of four haploid ascospores. The involvement of diffusible factors regulating mating was first reported by Levi (1956), who observed the behaviour of yeast cells plated onto malt agar. Cells of (-) ( = a ) mating type put out elongated copulatory structures that grew towards (+) (=a) mating-type cells. If a/ a mating cells were removed from the agar surface and replaced by fresh a-cells, these a-cells responded to a factor in the medium by producing elongated copulatory structures. The factor could diffuse through a collodion membrane. Duntze et al. (1970) extracted this a-factor from culture filtrates of a-cells, and it was characterized as a mixture of hydrophobic peptides made up of two tridecapeptides, a1 and 1x3, and two dodecapeptides, a2 and a4 (Stotzler et al. , 1976). It is now clear that a1 (Fig. 7) is the major native a-factor, and that the other three are artefacts of purification resulting in formation of methionine sulphoxide from methionine and/or loss of an N-terminal tryptophan residue (Naider and Becker, 1986). A corresponding a-factor is produced by a-cells, and has reciprocal activity on a-cells (Michaelis and Herskowitz, 1988). a-Factor proved much more intractable to characterize, although it is now known that it is a very hydrophobic peptide and shows a strong propensity to associate with very highmolecular-weight mannans during purification (Betz et al., 1977; Betz and Duntze, 1979). Its hydrophobicity explains why its activity is not readily apparent in experiments with cells grown on agar (Levi, 1956; Sprague, 1. a-Factor
NH2-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln~Pro-Met-Tyr-COOH
NH~-Tyr-lle-lle-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys( S-farnesyl)-COOCH3
2. a-Factor 3. Trernerogen A-10
4. Tremerogen
a-13
5. Rhodotorucine A
NH~-Glu-His-Asp-Pro-Ser-Ala-Pro-Gly-Asn-Gly-Tyr-Cys(S-hydroxyfarnesyl)-COOCH~ NH~-Glu-Gly-Gly-Gly-Asn-Arg-Gly-Asp-Pro-Ser-Gly-Val-Cys(S-farnesyl)-COOH
NH~-Tyr-Pro-Glu-lle-Ser-Trp-Thr-Arg-Asn-Gly-Cys(S-iarnesyl)-COOH
FIG. 7. Amino acid-residue sequences of some hydrophobic peptide hormones. 1 and 2 are for Saccharomyces cerevisiae, 3 and 4 for Tremella mesenterica, and 5 for Rhodosporidium toruloides.
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1991). This very hydrophobic nature is due to its C-terminal cysteine residue being modified by carboxyl methylation and by farnesylation of its sulphur atom (Fig. 7; Betz et al., 1987; Anderegg et al., 1988). These peptides and a range of analogues have been synthesized and tested for biological activity (Baffi et al., 1984,1985; Shenbagamurthi et al. , 1985a,b; Naider and Becker, 1986; Raths et al., 1986, 1988; Xue et al., 1989a) and for a-factor (Becker et al., 1987; Xue et al., 1989b, 1990). Synthetic hormones elicit the entire array of biological effects attributed to natural compounds, showing that a-factor and a-factor are the sole primary signals for inducing mating programmes in a-cells and a-cells, respectively. a-Factor is produced constitutively by a-cells, and is the product of proteolytic processing of primary translation products of two unlinked genes, namely MFaI and MFa2 (Kurjan, 1985; Kurjan and Herskowitz, 1982; Singh et al. , 1983). The genes MFaI and MFa2 code for four and two copies of a-factor, respectively. Five of these copies are identical (Fig. 7), one (a product of MFa2) has two conservative changes (Gln4 to Asn and Lys to Arg), but both sequences have equivalent activities (Lipke, 1986; Raths et al., 1986). The translation products are processed by successive cleavage by membrane-bound proteases (Fuller et al., 1986, 1989; Bussey, 1988). The product of M F a l , prepro-a-factor, has 165 aminoacid residues, and consists of a 19 amino acid-residue signal peptide, a 64residue pro-region containing three sites for N-linked glycosylation, and four tandem repeats of the mature a-factor sequence preceded by a spacer peptide. The signal sequence ensures translocation into the endoplasmic reticulum, where it is cleaved to produce pro-a-factor (Waters et al., 1988). Core N-linked carbohydrate is added to the three sites within the proregion and outer-chain carbohydrate modification occurs in the Golgi apparatus (Emter et al., 1983; Julius et al., 1984a,b). By analysing a range of mfal mutants deficient in N-glycosylation, Caplan et al. (1991) concluded that this process expedites transport of pro-a-factor from endoplasmic reticulum to the Golgi apparatus. It is not essential, however, as one afactor is eventually secreted by these mutants. The major proteolytic cleavage then takes place in the Golgi apparatus, by a cathepsin B-like protease coded by KEX2 (Julius et al., 1983, 1984a,b; Achstetter and Wolf, 1985). The six products from the two MFa genes have two or three dipeptides (X-Ala-) on their N-termini, which are cleaved by a dipeptidyl aminopeptidase coded by STE13, while four products have -Lys-Arg dipeptides on their C-termini, which are cleaved by a serine protease related to carboxypeptidase Y and coded by KEXl (Dmochowska et al., 1987; Wagner and Wolf, 1987). Knowledge gained from these studies has led to exploitation of this unique pathway in secretion of heterologous polypeptides from Sacch. cerevisiae (Goodey et al., 1988).
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a-Factor (Fig. 7) is also coded for by two unlinked genes, with codons differing for one amino-acid residue (Val for Leu). Each gene codes for one copy of a-factor as a precursor with a highly conserved N-terminal leader, requiring proteolytic processing at Nand C-termini, and maturation as a result of methylation and farnesylation (Kuchler et al., 1989). This maturation is analogous to that of the ras proto-oncogene product, and the precursors for the two pathways have a consensus C-terminal sequence, namely -Cys-A-A-X (where A is an aliphatic amino-acid residue and X is any other amino-acid residue), which is the target for the final maturation (Marcus et al., 1990). Marcus et al. (1990) described a cell-free system from Sacch. cerevisiae that was able to mature the 21 amino-acid residue precursor, entailing peptide cleavage from both N- and C-termini, and modification of the resultant C-terminus by farnesylation with farnesyl pyrophosphate and methylation with S-adenosylmethionine. Farnesylation of both a-factor and RAS involves the gene RAMIDPRI (Powers et al., 1986; Goodman et al., 1988; Schafer et al., 1990). The resultant lipopeptide is actively secreted from a-cells through a protein with homology to the multidrug-resistance P-glycoprotein (McGrath and Varshavsky, 1989). a-Factor and a-factor are each recognized by a specific receptor. In each cell-type, the receptor, an integral membrane protein, is coupled with the same heterotrimeric G-protein, and binding of a-factor or a-factor to its receptor sets in train the signal-transduction process (Marsh and Herskowitz, 1988a; Marsh, 1991; Marsh et al., 1991). The a-factor receptor is encoded by the STE2 gene, which is expressed only in a-cells (Hartig et al., 1986; Marsh and Herskowitz, 1987, 1988b). The receptor has seven hydrophobic domains spanning the membrane, with the N-terminus outside the cell and the C-terminus inside (Burkholder and Hartwell, 1985; Nakayama et al., 1985), a typical structure for an integral membrane-protein receptor coupled to G-proteins. There are about 10,000 binding sites in each cell, as determined by binding of radioactive a-factor or competition studies, with an equilibrium dissociation constant 6 X lop9 to 2 X lo-' M (Jenness et al., 1983, 1986; Raths et al., 1988). The receptor is located on the surface of the cell, as shown by immunofluoresence of a STE2-Lac2 fusion protein that retains receptor activity (Marsh and Herskowitz, 1988a). The receptor-a-factor complex is internalized, to be replaced on the surface by new receptor, while the a-factor is degraded (Chvatchko et al. , 1986; Jenness and Spatrick, 1986; Singer and Riezmann, 1990). This process can be used to assay for endocytosis in Sacch. cerevisiae (Dulic et al., 1991). Dulic and Riezman (1990) reported that a functional vacuole is required for full response of a-cells to a-factor, as both vps mutants (defective in vacuolar protein sorting) and end mutants (defective in endocytosis) are unable to form shmoos, and class C vps mutants cannot
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degrade internalized a-factor. The a-factor receptor is encoded by STE3 and is expressed only in a-cells (Nakayama et a f . , 1985). The a-factor receptor also has seven membrane-spanning domains and a long hydrophilic C-terminus, but has little amino acid-residue sequence identity with afactor receptor (Clark et d . , 1988). Yeast gametes are unbudded single cells with one haploid genome. Mating pairs of cells grow towards one another with formation of elongating conjugation tubes. Their shape then resembles that of a cartoon character of A1 Capp, the “Shmoo”, hence they are termed shmoos. Development and behaviour of shmoos is regulated by a- and a-factor, in that they mediate courtship of two compatible cells so that two vegetative cells become two gametes. Heterozygous ala- diploid cells do not produce a- or a-factor and do not respond to them. Bioassays for both hormones are discussed by Sprague (1991). Most work on the biological activity of mating factors from Sacch. cerevisiae has been done with a-factor, primarily because it is readily available. In most aspects, however, it is apparent that a-factor elicits reciprocal effects. Genetic aspects are discussed by Cross et a f . (1988) and Marsh et a f . (1991). The range of physiological responses is as follows: (a) Conjugation tubes of both mating types quickly become covered with mating type-specific agglutinins, the nature and roles of which are reviewed by Gooday (1992), Yoshida et a f . (1989) and Cross et af. (1988). They are probably the fibrous material seen on the outer surfaces of developing shmoos in electron micrographs (Lipke et al., 1976; Baba et a f . , 1989; Fig. 8). This region stains brightly with fluorescein-labelled concanavalin A , consistent with localization of mannose units (Tkacz and MacKay, 1979). Watzele et a f . (1988) and Hauser et a f . (1990) used fluorescent antibodies to MA Ta and M A Ta agglutinins to demonstrate that they are localized on the developing conjugation tubes. a-Cells treated with a-factor showed immunofluorescent labelling for a-agglutinin, which developed exclusively on the elongating tips of the developing shmoos within 15 minutes. This corresponds to the time-scale for development of a-agglutinin as assayed by tight binding of purified a-agglutinin (Terrance and Lipke, 1987). Induction of a-agglutinin by a-factor is a particularly sensitive response, the K,, value being 60 PM,below values for celldivision arrest and conjugation-tube formation (Moore, 1983). The a- and a-agglutinins are complementary, as agglutination is only seen between haploid cells of opposite mating type (Yanagishima and Yoshida, 1981). They are unrelated mannoproteins. The a-agglutinin is large (20G300 kDa) with approximately half of its mass being
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N-linked carbohydrate which is not essential for activity. The protein moiety corresponds to 68.2 kDa and has been cloned and sequenced (Hauser and Tanner, 1989; Lipke et al., 1989). a-Agglutinin is much smaller (22 kDa) and its solely 0-linked carbohydrate is essential for activity (Orlean et af., 1986; Watzele et af., 1988). (b) There are other qualitative and quantitative changes in the cell wall as a shmoo develops. Notably, there is a large increase in the amount and location of chitin. In budding cells, nearly all of the chitin is confined to the division septum that becomes the bud scar, and there is only very little in the lateral wall. In contrast, newly synthesized cell wall of the elongating shmoo is rich in chitin, as shown by staining with primulin. Overall, small unbudded daughter a-cells, which start with no detectable chitin, make three times more chitin when grown in the presence of a-factor than do untreated exponentially growing cells (Schekman and Brawley, 1979). In Sacch. cerevisiae, chitin synthases are encoded by at least three genes, namely C H S l , CHS2 and CHS3 (encoding chitin synthases 1, 2 and 3, respectively). The genes CHSl and CHS2 have been characterized, as has CALI (resistance to Calcofluor) which is required for Chs3 activity, and they appear to have distinct roles. The product Chsl is present chiefly as a trypsin-activatable zymogen, with a role as a repair enzyme; Chs2 is specific for primary septum formation and also requires proteolysis for activation; Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall and is not proteolytically activatable (Bulawa et al., 1986; Bulawa and Osmond, 1990; Shaw et a f . , 1991; Valdivieso et al., 1991). Schekman and Brawley (1979) reported remarkable rises in trypsin-activatable, but especially in endogenous, chitin synthase activities in a-cell, peaking at four hours after treatment with a-factor. This was confirmed by Orlean (1987), who showed that activity of Chsl in a-cells doubled following treatment with a-factor. He further demonstrated, however, that chitin synthase 1is not required for a-factor response, as chsl mutants mated normally and showed the typical increase in chitin content on a-factor treatment. Appeltauer and Achstetter (1989), using Northern analysis, showed a more than six-fold elevation in levels of CHSl mRNA in a-cells treated with a-factor. They also established that the CHSl promoter region has a consensus sequence with the afactor-inducible promoter of the BAR1 gene, identified by Kronstad et af. (1987). Thus, chitin synthase 1 zymogen levels are elevated in shmoos, but are not responsible for the increased chitin content, perhaps instead having a repair function or a role in cell fusion. Instead, it appears that Chs3 is responsible for the increase, as it is
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(c)
(d)
(e) (f)
almost anulled in mutants of the CALI gene (Roncero et al., 1988; Valdivieso et al., 1991). There appears, however, to be no elevation in specific activity of chitin synthase 3 on treatment with a-factor (Orlean, 1987; whose “chitin synthase 2” activity can now be interpreted as chiefly due to chitin synthase 3; see Valdivieso et al. 1991). Watzele and Tanner (1989) reported a dramatic increase in expression of the gene encoding glutamine: fructose-6-phosphate aminotransferase in response to a-factor, with a 2-3-fold increase within 15 minutes. The product of this enzyme is glucosamine 6phosphate, the precursor of UDP-N-acetylglucosamine, which in turn is a precursor of chitin and glycoproteins. The resulting increase in the rate of supply of the UDP-N-acetylglucosamine may then account for the increase in chitin synthesis, by increasing the flux through the pathway. This increased rate of supply was reported by Orlean et al. (1985), who showed that the apparent inhibition of glycoprotein synthesis in response to a-factor reported by Orlean et al. (1984) was an artefact in their experimental technique, resulting from a dilution of their radioactive precursors by stimulation in availability of endogenous precursors. The role of the increased chitin content may be to strengthen the tubular conjugation wall of the shmoo, just as the hyphal wall of the mycelial form of the dimorphic yeast Candida albicans also has an increased chitin content. Lipke et al. (1976) reported that cell walls produced in response to a-factor also have an increase in g1ucan:mannan ratio from 1 : l to l.S:l, and the wall becomes more susceptible to digestion by glucanases. They also described changes in the nature of wall mannoproteins, which will include the mannoprotein agglutinins (see above). A further notable change to the cell surface is the 50-fold or more increase in transcription of the FUSl gene in cells of both mating types in response to their respective hormones. The FUSZ gene product appears to be a cell-surface protein that is involved in a late stage of cell fusion (McCaffrey et al., 1987; Trueheart et al., 1987). Responding vegetative cells are arrested at the START position in the GI phase of their cell cycle, with no initiation of DNA synthesis (Bucking-Throm et al. 1973; Wilkinson and Pringle, 1974). Possible mechanisms are discussed by Cross et al. (1988). This response results in formation of gametes which are unbudded single cells with one haploid genome. The nucleus of the gamete is prepared for karyogamy, or fusion with the nucleus of the mating partner (Rose et al. 1986). The conjugation tube of the shmoo is formed. Ford and Pringle
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FIG. 8. Section of an a-mating-type cell of Saccharornyces cerevisiae at an early stage of shmoo formation, 80 minutes after treatment with 3 p~ a-factor. Note the elongating conjugation tube with associated apical vesicles (Ves) and developing fibrous coat corresponding to agglutinin (A). The nucleus (N) is oriented with its spindle-pole body (SPB) and associated microtubule (MT) at its anterior side, and the nucleolus (Nu) at its posterior side. Also visible is the cell wall (CW) with bud scar (BS), vacuoles (V) and mitochondria (M). The scale bar represents 1.0 pm. The preparation was by freeze substitution as described by Baba et al. (1989). The micrograph was kindly provided by Misuzu Baba.
(1986) investigated the polarity of shmoo formation with respect to a cell’s previous budding history as assessed by Calcofluor staining of bud scars. The results were unequivocal; neither fusion sites nor shmoo tips were restricted to the budding pole, and they seemed to be located randomly on the cell surface. Perhaps polarity is established from the site of first perception of a- or a-factor by the responding cell. The conjugation tube is then chemotropically attracted to the source of the hormone (G. W. Gooday and B. Walmisley , unpublished results). Electron micrographs clearly show that the shmoo nucleus migrates with the growing tip with a spindlepole body attached at the front and the nucleolus to the rear (Lipke et al., 1976; Baba et al., 1989; Fig. 8). (8) Perlman et al. (1989) described a rapid transient intracellular alkalinization of a-cells responding to a-factor. Dissipation of this
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G. W. GOODAY AND D . J ADAMS
p H gradient, however, using 2,4-dinitrophenol or orthovanadate, does not inhibit other responses, and so this alkalinization appears to be an epiphenomenon as a consequence of the a-factor binding to its receptor. (h) Ohsumi and Anraku (1985) reported that a sustained calcium-ion influx is coupled with the initiation of shmoo formation. The influx and shmoo formation were inhibited by treatment with ionophore A23187, which had no effect on GI arrest or agglutination. Treatment with 5 mM EGTA had no effect on vegetative growth rate but inhibited zygote formation by 70%. In contrast, Tachikawa et af. (1987) described a rapid, transient uptake of calcium ions by a-cells responding to a-factor. Hiraga et af. (1991) suggested that this could be the result of a direct inhibition of the plasma-membrane Ca2+ATPase by a-factor. It may be interrelated with the apparently concomitant alkalinization. Preparations of Ca*+-ATPase from acells and a-cells lacking receptor (ste2 mutants), but not from a-cells, were inhibited by a-factor in a dose-dependent manner at concentrations ranging from 20 to 100 ng ml-', concentrations that would result in shmoo formation of the equivalent cells. Hiraga et af. (1991) suggest that the a-factor receptor is not necessary for this response, and it may represent an independent mechanism for a-factor signalling. A localized influx of calcium ions may polarize the cell, by interacting with the cytoskeleton so that the shmoo grows out at the site of initial exposure to a-factor. Hasek et al. (1987) and Baba et al. (1989) report actin accumulations in the contact areas of conjugating cells and in the projections of growing shmoos. Miyakawa et af. (1989) described a calcium ion- and calmodulin-dependent protein kinase, which they suggest may be involved in signal transduction of mating hormones, initially being activated by the transient calcium-ion influx, and then remaining active after the intracellular concentration of calcium ions returns to basal levels. (i) Cell-division arrest is accompanied by a subsequent decline in intracellular levels of CAMP (Watson and Berry, 1977; Perlman et al. , 1989). There is, however, no direct effect on adenylate cyclase activity (Casperson et af.,1983), contrary to an earlier report by Liao and Thorner (1980). (j) Production of each hormone is stimulated by the complementary hormone (Achstetter, 1989; Strazdis and MacKay, 1983; Michaelis and Herskowitz, 1988; Brake et af., 1985). This can be viewed as part of the courtship, so that as an a-cell responds to an a-cell, it increases output of a-factor to increase the response of the potential mate. By competition experiments, Jackson and Hartwell (1990a,b)
SEX HORMONES AND FUNGI
95
showed that both a- and a-cells preferentially mate with the potential partner that is producing the strongest hormone signal. (k) Production of each receptor is stimulated by its ligand (Burkholder and Hartwell, 1985; Nakayama et a f ., 1985; Hagen et a f . , 1986). Again this can be seen as part of the courtship. (1) As part of their response to a-factor, a-cells are induced to secrete a specific protease, the “barrier” activity, encoded by the BAR1 gene. This protease inactivates a-factor by cleaving between residues Leu6 and Lys, (Cejek and Thorner, 1979; MacKay et al., 1988). Kronstad et al. (1987) have shown that the BAR1 gene has an upstream activation site, which responds to stimulation by a 2-10fold induction of transcription. This specific destruction of a-factor serves to maintain its concentration gradient and receptivity of acells. It also allows regrowth in the absence of mating. a-Cells appear to have at least two systems that destroy a-factor. One is encoded by SSLl, and mutants are supersensitive to a-factor (Steden et a f . , 1989; Steden and Duntze, 1990). Marcus et a f . (1991), however, described “a-factorase”, a protease from a-cells that specifically degrades a-factor but is distinct from the SSLZ gene product. Further, they reported that the SSLl gene product, contrary to expectation, does not degrade a-factor. They suggest that the SSLZ gene is not the a-cell equivalent of the BAR2 gene, and instead that its product may be an a-factor-blocking or -binding agent. The a-factorase is membrane bound, as befits the lipophilic nature of a-factor, and its activity is increased by treatment of a-cells with a-factor. As well as the BAR1 gene product, a-cells also have another internal desensitization mechanism which allows them to resume growth (Moore, 1984). The length of time before budding is proportional to the number of occupied receptors (Moore, 1987). Both a- and a-cells have a further desensitization mechanism, involving the SST2 gene. Organisms with mutations in this gene are supersensitive to a- or a-factor. Expression of SST2 is greatly stimulated by exposure to the appropriate hormone (Dietzel and Kurjan, 1987). The biological activity of a-factor from Sacch. cerevisiae is not speciesspecific. a-Cells of the closely related species Sacch. kfuyveri, Sacch. exiguus, Hansenula wingei and H. anomafaare also induced to form shmoos (McCullough and Herskowitz, 1979; Yanagishima and Fujimura, 1981; Fujimura et a f . , 1982; Fujimura and Yanagishima, 1983; Sakurai et a f . , 1984; Yoshida et a f . , 1989). These species have their own a- and a-factors, and there is mutual cross-reactivity between their a-factors and a-cells, but not between their a-factors, and a-cells. The a-factors of Sacch. kluyveri
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G W GOODAY AND D J ADAMS
and Sacch. exiguus are closely related to that of Sacch. cerevisiae, with five and four amino-acid replacements, respectively (Sakurai et al., 1984, 1986; Yoshida et al., 1989). Sexual agglutinins formed in response to the hormones are, however, totally species-specific (Yoshida et al., 1989). Schizosaccharomyces pombe also has a sex-hormone system regulating mating of two compatible cells. Haploid cells are of two mating types, namely h+ and h- (or P and M) which, under conditions of nitrogen depletion, conjugate to form an h'lh- zygote. Evidence for hormonal control of conjugation came from experiments by Friedmann and Egel (1978), who showed that exchange of culture-medium filtrates between h f and h- cultures resulted in the same changes in DNA-binding proteins that they had observed during sporulation. During an investigation of mutants defective in the rasl gene, the homologue of mammalian ras oncogenes in Schiz. pombe, Fukai et al. (1986) observed mating responses of single cells plated onto nitrogen-free sporulation agar. The rasl- mutants were sterile, but h- rasl- cells showed an elongation response of nearby h+ rasl' cells, mimicking the first stage of conjugation. They could distinguish the cells by using an adenine auxotroph as one of the strains, micromanipulating the cells after observing which ones were responding and forming colonies on a complete medium, when the adenine auxotrophs formed red colonies and the prototrophs formed white colonies. They observed that only h' cells formed conjugation tubes. Leupold (1987) observed interactions between cells of opposite mating type inoculated on to thin blocks of nitrogen-free sporulation agar medium on microscope slides. He showed that hormone production and responses were reciprocal, each mating type producing a diffusible mating hormone that caused formation and chemotropic growth of conjugation tubes in cells of opposite mating type. The two hormones are termed M-factor, produced by h- (M-cells) and affecting h+ (P-cells), and P-factor, produced by P-cells and affecting M-cells (Fig. 9). Davey (1991) presented a preliminary characterization of M-factor, isolated from a homozygous h-lh' strain of Schiz. pombe. M-factor is a lipononapeptide (J. Davey , personal communication). Its characterization was possible after devising a bioassay based on the increase in cell volume of P-cells in a dose-dependent response to M-factor, as measured in a Coulter counter. As well as its role in conjugation, Leupold et al. (1989a) showed that P-factor could induce meiosis in diploid mutant strains lacking P-factor production. They suggested that, in these cells, addition of Pfactor activates the M-cell receptor, starting an intracellular cascade of signal transduction leading to activation of meiosis-specific genes. Egel et al. (1990) reviewed this coregulation of conjugation and meiosis in Schiz. pombe, and compared it with the situation in Sacch. cerevisiae where there
SEX HORMONES A N D FUNGI
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FIG. 9. Photomicrograph showing sexual interactions between two strains of Schizosaccharomyces pombe mediated by P-factor and M-factor. Both strains are diploid. The cells in the patch at the bottom are heterozygous at mat1 (PIMc-), where the Mc- mutation results in a requirement for external M-factor in order to undergo meiosis and sporulation. Response to M-factor from the other strain is seen as production of four-spored asci. The microcolonies at the top are homozygous at mat1 ( M I M ) , producing M-factor and responding to P-factor from the other strain by producing conjugation tubes elongating towards it. The videomicrograph was provided by R. Egel. The scale bar represents 20 pm.
is no cross-talk between hormones, receptor-signal processing and conjugation on the one hand, and nutrient depletion, meiosis and sporulation on the other, as there is in Schiz. pombe. In Schiz. pombe, sexual reproduction is inhibited by the patl protein kinase, activity of which is required for vegetative growth. Leupold et al. (1989b) showed that patl mutants of both mating types respond more strongly to the appropriate hormone than do wild-type cells. In their experiments, pat I P-cells were better producers of P-factor than wild-type cells. Using a range of sterile mutants, Leupold (1991) and Leupold et al. (1991) investigated further aspects of genetic control of production and response of M - and P-factors. They suggest that M-factor production is more readily expressed than P-factor, and that conjugation is initiated by M-cells. They also suggest that the meiotic response is triggered by a lower internal content of M - or P-factor than in the conjugation-tube response.
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G W GOODAY AND D J ADAMS
2. Basidiosporogenous Yeasts Some species of basidiomycetous yeasts have sex-hormone systems very similar to that of Sacch. cerevisiae. Tremella spp. arc jelly fungi and are members of the Tremellales. They can grow vegetatively as haploid budding yeast cells. Bandoni (1965) demonstrated hormonal regulation of mating in Tremella mesenterica by showing that yeast cells of each mating type, A and a , produce diffusible factors that cause cells of opposite mating type to cease budding and form conjugation tubes. Reid (1974) reported partial purification of the hormones and suggested that they were peptides. This was confirmed by the characterization of two lipopeptides, namely tremerogen A-I0 (from A-type cells) and tremerogen a-13 (from a-type cells) (Fig. 7; Sakagami et al., l979,1981a,b). Tremerogen A-10 is a dodecapeptide while tremerogen a-13 is a tridecapeptide. In both peptides, the sulphydryl group of the cysteine residue at the C terminus is S-farnesylated. In tremerogen A-10, one of the terminal methyl groups of the farnesyl moiety is hydroxylated, and the carboxyl group of the cysteine residue is methylated. The two peptides have no homology. A-Type cells of the closely related Tremella brasiliensis produce a closely related farnesylated hormone, namely tremerogen A-9291-1 (Ishibashi et al., 1983, 1984). Tremerogens A-10 and a-13 and a range of analogues have been synthesized and their biological activity determined (Fujino et al., 1980; Kitada et al., 1981). The minimum concentrations inducing mating-tube formation (equivalent to shmoo formation in Sacch. cerevisiae) for the natural products are of the order of 1 nM. For tremerogen A activity, a blocked carboxyl group and a lipophilic side-chain are essential for activity. Synthetic analogues with an amide group instead of a methyl ester and with longer isoprene sidechains (tetra- or pentaprenyl) were more active than the natural product. Similarly, for tremerogen a, a lipophilic side-chain was required for activity, and an analogue with an amide group at the C-terminus invoked more activity than the natural product. On treatment with tremerogen, receptive cells undergo GI arrest and produce a narrow (1.3-1.8 pm diameter) slowly elongating (1.5-2.0 pm h-') conjugation tube, which continues elongating in the presence of the tremerogen (Hirata et al., 1980a). On its removal, cells revert to budding. The single nucleus migrates into the elongating tube with its spindle-pole body attached at its anterior side. As the tube wall is formed as an outgrowth of the yeast cell, it ruptures the outer layer of the yeast wall (Hirata et al., 1980a,b). The composition of the wall of the conjugation tube is quantitatively different from that of the yeast cell, with more P-glucan and chitin but less a-glucan (Reid and Bartnicki-Garcia, 1976). The cell surface is also qualitatively different, with the presence of mating type-specific cellsurface proteins (Miyakawa et al., 1982a, 1984,1987a). These are the sexual
SEX HORMONES AND FUNGI
99
agglutinins, localized on the surface of the conjugation tubes and responsible for primary fusion of the conjugation tubes of a mating pair of cells. Other Tremella spp., and the related Sirobasidium magnum, have similar hormone systems, but there is no cross-reactivity between them so that they are species-specific (Flegel, 1981). Rhodosporidium is a genus of yeasts in the Ustilaginales or smuts. Abe et al. (1975) demonstrated that conjugation-tube formation in Rhodosporidium toruloides is regulated by diffusible hormones. Unlike the situation in Saccharomyces and Tremella spp., where both mating types constitutively produce their sex-specific hormones, only A-cells of Rhodosp. toruloides constitutively produce their hormone, namely rhodotorucine A ;a-cells then produce rhodotorucine a in response. Rhodotorucine A is a lipodecapeptide and, like the tremerogens, it has an S-linked farnesyl side-chain on the C-terminal cysteine residue (Fig. 7; Kamiya et al., 1978). It and some analogues have been synthesized and their biological activity assessed (Kitada et al., 1979). The natural and synthetic products were active down to about 5 nM, while the unfarnesylated undecapeptide had no activity. Three genes have been identified, namely R H A l , RHA2 and RHA3, encoding four, five and three copies of rhodotorucine A , respectively (Akada et al., 1987, 1989a,b). The predicted precursor sequences do not have obvious signal sequences at their N-termini, and the sites for subsequent cleavage are unusual, with a single lysine residue at the N-terminal side of each hormone peptide repeat. Thus, processing of the precursors to give active hormones must involve novel proteolytic processing steps. Genomic Southern-blot analysis of a-cells with R H A l DNA as a probe indicated the absence of RHAl genes from the a-type genome. Thus, genetic regulation of mating type is unlike that in Sacch. cerevisiae, where all mating type-specific genes are present in both mating types. The response of a-cells to rhodotorucine A includes inhibition of budding, inhibition of DNA synthesis (that is, GI arrest), formation of conjugation tubes and secretion of rhodotorucine a (Abe et al., 1975; Tsuchiya and Fukui, 1978). As for a- and a-factors from Sacch. cerevisiae, rhodotorucine A is specifically degraded by responding a-cells (Miyakawa et al., 1982b). The activity responsible is a surface-bound trypsin-type endopeptidase, highly specific for rhodotorucine A ; the peptide backbone, lacking the farnesyl side-chain, is not a substrate. This activity is not found in A-cells, or in some sterile mutant a-cells. Response to rhodotorucine A is inhibited by specific inhibition of the endopeptidase by a trypsin substrate and by a competing substrate, the S-oxide of rhodotorucine A . This proteolytic metabolism of rhodotorucine A , termed the trigger reaction, is necessary for response to the hormone. Miyakawa et al. (1982~)also reported a concomitant enhanced phosphorylation of membrane proteins, and suggested that this
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G W GOODAY AND D J ADAMS
was coupled with hydrolysis of rhodotorucine A in a novel transmembrane signalling system. This was further investigated by Miyakawa et al. (1985a), who demonstrated the involvement of surface-protein sulphydryl groups in the initial trigger reaction. The sulphydryl-blocking reagent, Ellman's reagent, strongly inhibited hydrolysis of rhodotorucine A and the response to it, while dithiothreitol enhanced these processes. The trigger peptidase has been characterized by Miyakawa et al. (1987b) as a membrane-bound thiol peptidase dependent on 1 mM calcium or manganese(r1) ions for maximum activity and requiring a phospholipid environment. Miyakawa et al. (1985b, 1986) reported a rapid transient influx of calcium ions, peaking at about four minutes, after addition of rhodotorucine A to receptive a-cells. This influx was followed by an efflux, then by a gradual increase of uptake. A sterile mutant, deficient in the early stages of mating, did not show these responses. The ionophore A23187 enhanced the calcium-ion responses and increased conjugation-tube formation, while trifluoperazine, an inhibitor of calmodulin, did not inhibit the initial signalling but suppressed conjugation-tube formation. Thus, two processes seem to be involved, initial signalling with rapid influx of calcium ions, and subsequent response, with involvement of calmodulin. Miyakawa et al. (1987~)reported that a membrane-bound Ca2+-ATPase from a-cells, but not that from A-cells, was specifically inhibited by rhodotorucine A and they suggested that this was responsible for the rapid transient calcium-ion influx. Liu et al. (1990) identified a range of calmodulin-binding proteins in Rhodosp. toruloides, and showed both quantitative and qualitative changes in them following treatment of a-cells with rhodotorucine A . They also described calcium ion-dependent and calmodulin-dependent protein kinase activity, and suggest that these may respond to the transient influx of calcium ions, as has been suggested by Miyakawa et al. (1989) for the hormone response of Sacch. cerevisiae. C. UNCHARACTEKIZED HORMONE SYSTEMS
In all taxonomic groupings of fungi there are reports suggesting hormonal control of sexual reproduction, and a few examples are now presented. In each example, advances are dependent on amenable experimental systems, as have been shown in the examples already presented. A good source of the chemical compounds, probably hyper-producing strains as normally the level of production is low, and a rapid and sensitive bioassay system are prerequisites for characterization of novel systems. In Neurospora crassa, Bistis (1981,1983) described the clear chemotropic attraction of the female filament (the trichogyne) of each mating type ( A and a) to the male fertilizing conidium. In these experiments, attraction of
SEX HORMONES AND FUNGI
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the a-trichogyne to the A male attractant was much stronger than its reciprocal. Also in N. crassa, chemical extracts have been reported to stimulate either fertility in poorly fertile crosses or development of protoperithecia in unmated strains (Islam, 1981; Vigfusson and Cano, 1974). The factor described by Islam (1981) is lipid in nature, perhaps a hydrocarbon, while that of Vigfusson and Can0 (1974) behaves like a protein. These factors, however, are probably best classified as regulators of sexual development rather than mating type-specific hormones (see later). In Arthroderma incurvatum, A . S . Donald and G . W. Gooday (unpublished results) obtained evidence for two reciprocal hormones, namely (+) factor and (-) factor, produced by (+) cells and (-) cells, respectively, which induce development of protocleistothecia in unmated cultures of (-) cells and (+) cells. They are small, diffusible, hydrophobic molecules, not yet characterized. Other hormonal systems in species of Ascomycetes ripe for reinvestigation are those in the genera Glomerella (Driver and Wheeler, 1955), Bombardiu (Zickler, 1952), Ascobolus (Bistis and Olive, 1968) and Nectria (Bistis and Georgopoulos, 1979). Sexual development in the cellular slime moulds involves small amoeboid cells which eventually form macrocysts. This process has been shown to be controlled by hormones, by O’Day and Lewis (1981) and O’Day et ul. (1987), but the chemical nature of the active compounds, which can act as volatile agents as well as in solution, remains unclear. D. CONCLUSIONS
The major senses of fungi are chemical. Thus, fungi use chemical compounds so that they can communicate to co-ordinate their sexual reproduction. Sexual reproduction is widespread and presumably important to fungi as it allows meiotic recombination, and also is often accompanied by formation of resistant resting stages, such as zygospores or ascospores. It requires, however, a diversion from vegetative growth and asexual sporulation. Thus, it must be an efficient and orderly process. The specificity of hormones ensures that sexual differentiation only occurs when there is a good chance of successful mating. The hormones are species-specific or specific to closely related species, in which case species specificity resides in cellkell fusion. The general properties of these hormones are summarized in Table 2. All are produced in tiny amounts (except for trisporic acid in mated cultures of B. trispora), as befits molecules with such powerful activities. Some are produced constitutively; synthesis of others such as oogoniol and rhodotorucine a requires induction by the complementary hormone. Their production tends to be favoured in nutrient-depleted conditions. They tend
P
0
t3
TABLE 2. Properties of fungal sex hormones Optimal yield (M)
Sensitivity of bioassay (M)
Hormone
Molecular structure
Probable precursor
Site and specificity of synthesis
Sirenin
Sesquiterpene (C&db)
Farnesyl pyrophosphate
Female gametes of Allomyces species, constitutive
10-10
Chemotaxis of male gametes
Antheridiol
Sterol (CZ9HdZ05)
Fucosterol
Female cells of Achlya species, constitutive
10-1'
Antheridia and oogoniol production by males
-
Oogoniol
Sterol ester (&HS4O6)
Fucosterol
Male cells of Achyla species, induced
Trisporic acid
Apocarotenoid (Ci&xOJ
Retinal
(+)/(-) Cells of species of Mucorales, collaborative
a-Factor
Tridecapeptide
Protein
a cells of Saccharomyces cerevisiae, constitutive
a-Factor
Farnesyl dodecapeptide
Protein, farnesyl pyrophosphate
a-Cells of Saccharomyces cerevisiae, constitutive
Tremerogen A
Farnesyl dodecapeptide
Protein, farnesyl pyrophosphate
A-Cells of Tremella mesenterica, constitutive
10-7
Tremerogen-a
Farnesyl tridecapeptide
Protein, farnesyl pyrophosphate
a-Cells of Tremella mesenterica, constitutive
10-8
Rhodotorucine A Farnesyl undecapeptide
Protein, farnesyl pyrophosphate
A-Cells of Rhodosporidium toruloides, constitutive
10-6
(Mucor mucedo); lo-' (Blakeslea trispora) 10-8
10-8
Response
Oogonia by females Zygophores by
(+) and (-) strains Shmoo formation by a-cells Shmoo formation by a cells
10-9
Conjugation tubes by a-cells Conjugation tubes by A-cells Conjugation tubes and rhodotorucine production by a-cells
n 3 0 $ P 0
2z 0
P P
U
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to be unstable molecules and to be destroyed in some specific manner by responding cells, both phenomena presumably helping to accentuate concentration gradients and responsiveness. As the two potential mating cells approach each other, there is increased production of hormone. This is a courtship “chemical display” clearly analogous to animal mating displays more familiar to us. All of the hormones are active at very low concentrations. Of those identified to date, all are isoprenoids or hydrophobic peptides, and they have clear chemical affinities with other hormones and biologically active compounds throughout biology (see later).
111. Endogenous Regulators of Sexual Development
In addition to specific fungal sex hormones, there are examples of compounds that can be considered as sexual morphogens, regulating development of the ensuing sexual structures, notably ascocarps of species of Ascomycetes. These compounds are detected by their ability to enhance sexual differentiation or to restore fertility to sterile strains or mutants. Champe et al. (1987) and Champe and El-Zayat (1989) described the psi factors (precocious sexual inducer) from the homothallic ascomycete Aspergillus nidulans which induce premature development of sexual sporulation. The psi activity was assayed by observing the response of mycelium growing from a uniform inoculum of conidia over an agar plate to filter paper discs impregnated with test samples. The activity resulted in formation of cleistothecia instead of conidiophores, and in the release of a yellow pigment into the medium. Three psi factors have been identified: psi C, the most active compound, is 5,8-dihydroxylinoleic acid; psi A , less active, is the corresponding cyclic S-lactone; while psi B, a minor metabolite, is 8-hydroxylinoleic acid, probably a biosynthetic intermediate (Mazur et al., 1990; S. P. Champe, personal communication; Fig. 10). The psi factors are overproduced by some mutants deficient in asexual and sexual sporulation (Champe et aE., 1987). They appear to be species-specific. A class of sexual morphogens (SF, sexual factors) have been described by Ilott et al. (1986) and Siddiq et al. (1989). These were isolated from mated cultures of the heterothallic ascomycete Pyrenopeziza brassicae, as diffusible factors that can promote development of apothecia and inhibit asexual sporulation in unmated cultures of both mating types. They also enhance the rate and extent of sexual reproduction in mated cultures. They are not species-specific, and have corresponding actions on several other ascomycetes and even some basidiomycetes. Their chemical nature is unclear, but the active water-soluble fractions are obtained from methanol
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Ci W. GOODAY AND D. J ADAMS
B o
OH
HO OH
c
OH FIG. 10. Sexual regulators: (A) zearalenone from Gibberella zeae; (B,C) psi factors C and A from Aspergillus nidulans.
extracts of freeze-dried mated mycelium. Other active factors that have been extracted from basidiomycetes include cerebrosides from Schizophyllum commune (Kawai and Ikeda, 1982) and similar factors from Agaricus bisporus (Rusmin and Leonard, 1978). A well-characterized metabolite whose role as a fungal sexual regulator is still unclear is zearalenone, an oestrogenic mycotoxin (see Section IV.B.l). This is a P-resorcyclic acid lactone (Wolf and Mirocha, 1973; Fig. lo), produced by Gibberella zeae and Fusarium spp. (Nelson, 1971; Plasencia and Mirocha, 1990). At low concentrations it will stimulate perithecial formation in G. zeae, but inhibits at high concentrations (Wolf and Mirocha, 1973). A putative specific receptor has been detected in extracts of G. zeae (Inaba and Mirocha, 1979). Windels et al. (1989), however, reported no correlation between zearalenone production and perithecial production in a range of isolates of Fusarium graminearum (= G. zeae). Nelson (1971) reported that low concentrations of zearalenone stimulated sexual reproduction in a wide range of fungi, suggesting that it can act as a broad-spectrum regulator.
SEX HORMONES AND FUNGI
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IV. Interactions of Mammalian Hormones with Fungi
Fossil evidence suggests that fungi evolved at least 300 million years ago while mammals emerged some 100 million years later (Margulis and Sagan, 1987). These facts make results published by a number of research groups during the last decade especially intriguing (Table 3). Their work suggests \ia\urixShfha~anhCill-men\oub\ungi p-rotmcecompounhsw'ri~chaxe sinihar (or identical) to mammalian hormones. Furthermore, fungi appear to interact with mammalian hormones in a manner closely analogous to the interaction of mammalian cells with these compounds. Such similarities may indicate a remarkable degree of conservation of messenger ligands and receptors during evolution. For the remainder of this review, we shall consider the interaction of mammalian steroid and polypeptide-protein hormones with fungi. Our original aim was to review the interaction of mammalian sex hormones (androgens, oestrogens, progesterone) with fungi. However, a considerable amount has now been published on the effects on fungal cells of hormones other than sex hormones, and aspects of these studies will also be discussed.
A . EFFECTS OF HORMONES ON FUNGAL GROWTH AND MORPHOGENESIS
In Vitro
I. Steroid Hormones The inhibition and, to a lesser extent, stimulation of fungal growth by M is well documented (Chattaway steroids at concentrations in excess of et al., 1963; Buetow and Levedahl, 1964; Hanifa Moursi and Refai, 1968; Capek and Simek, 1971; Mohr et al., 1973; Stevens, 1989; and references cited therein). More recently, steroid hormones have been shown to affect growth and morphogenesis in a number of dimorphic fungi at much lower steroid concentrations (10-'4-10-6 M), including those of physiological significance. These results are reviewed here, along with reports of inhibition of dermatophyte growth by progesterone and related hormones. It is important to emphasize that these effects at low steroid concentrations appear to occur following the specific interaction of a hormone with a fungus. The recent report of regulation of the cell cycle in the early GI phase of Saccharomyces cerevisiae by 17p-oestradiol (Tanaka et al., 1989) and the proposed underlying regulatory mechanism for this phenomenon will be discussed at a later stage. Mammalian steroid hormones influence growth of several fungi.
TABLE 3. Interactions of mammalian hormones with fungi Hormones Steroid hormones Androgens Corticosteroids Oestrogens
Aspergillus Candida Candida spp. Coccidioides Dermatophytes immitis fumigatusa albicans other than C. albicans
B B
Neurospora crassa
Paracoccidioides Saccharomyces brasiliensis cerevisiae
B,R B B.R
Progestins Protein-poly-peptide hormones Glucagon Human luteinizing hormonehuman chorionic gonadotrophin Insulin
-
R
B indicates that specific binding has been detected; R, a biochemical-growth response to the hormone; E, evidence exists for an endogenous ligand. M. Interactions with steroid hormones were noted at concentrations less than or equal to a LeRoith et al. (1980); for other references see text.
SEX HORMONES AND FUNGI
107
a. Paracoccidioides brasiliensis Paracoccidioidomycosis, a chronic systemic disease endemic to Central and South America, is caused by P. brasiliensis. The organism is dimorphic and is thought to exist outside the host in a saprophytic mycelial form while, in experimental animals and patients, it grows as a yeast which may exhibit a characteristic multipolar budding pattern (Fig. 11). In an early report of the effects of steroid hormones on P. brasiliensis, Muchmore et al. (1972) described inhibition of growth in vitro of mycelia and yeast cells by oestradiol, progesterone and the synthetic oestrogen stilboestrol. However, these results were recorded using hormone concentrations greatly in excess of those of physiological significance. More recently, Loose et al. (1983b) and Restrepo et al. (1984) demonstrated that oestrogens inhibited transformation from the mycelial to yeast form of this organism in a dosedependent manner, with significant inhibition noted at concentrations of 17P-oestradiol as low as 1 . lo-'" M . Diethylstilboestrol was also inhibitory but less potent than oestradiol. The effect was highly specific since 17a-oestradiol, a physiologically inactive stereoisomer of 17P-oestradio1, the oestrogen antagonist tamoxifen, corticosterone and testosterone were all inactive. Furthermore, the yeast-to-mycelium transformation, yeast growth and yeast reproduction by budding were all unaffected by 17poestradiol (Restrepo et al., 1984). In a subsequent study, Salazar et al. (1988) showed that 17P-oestradiol (lo-* M) and, to a lesser extent, an equimolar concentration of diethylstilboestrol inhibited conversion of conidia, purified from P. brasiliensis mycelia, into yeast cells. Other sex hormones and analogues, such as 17a-oestradiol, testosterone, tamoxifen and hydroxytamoxifen, had no effect on the conidium-to-yeast transformation.
b. Coccidioides immitis Coccidioidomycosis, a primarily pulmonary disease of major importance within endemic areas in the southwest USA and northern Mexico, is caused by the dimorphic pathogen C. immitis. The saprophytic mycelial form produces large numbers of arthroconidia which, when inhaled, develop in the lungs to form spherules containing numerous endospores (Fig. 12). On rupture of the spherule wall, endospores develop to form new spherules in adjacent tissue or, following dissemination, in other organs of the body. Drutz et al. (1981) examined the effects of human sex hormones and related compounds on growth and maturation of C. immitis at various stages of its life cycle. These authors reported stimulation of the rate of spherule growth and maturation, endosporulation and endospore release by 17poestradiol for three strains of C. immitis. Stimulation was demonstrable
108
G . W. GOODAY AND D.
J. ADAMS
Liberated aleuriospores
FIG. 11. Growth phases of Paracoccidioides brasiliensis.
with a starting inoculum of either arthroconidia or endospores. For example, enhancement of the rate of growth of the parasitic phase from arthroconidia was detectable at 17~-oestradiolconcentrations as low as M. A dose-related response extended through the physiological range for the normal non-pregnant female (approximately 7.4 10-13-2.4 . 10-l' M) into the range of unconjugated oestradiol encountered in late pregnancy (Le. approximately 3.7 . 10-7-8.8 . lo-' M). Progesterone and testosterone (both at concentrations of lop6M) were also highly stimulatory for growth of the parasitic phase of C. immitis, whereas cholesterol, ergosterol and 17a-oestradiol lacked such effects. Non-steroidal antioestrogens, namely tamoxifen and nafoxidine, had either stimulatory or inhibitory effects on the rate of spherule growth and maturation, depending on the fungal strain and experimental conditions. 17p-Oestradiol stimulated growth of the saprophytic mycelial phase of C . immitis, whereas tamoxifen and nafoxidine inhibited mycelial growth.
109
SEX HORMONES AND FUNGI
Art:iroconidia
Saprophytic phase
@@
-a&
1
Liberated arthroconidia
l
Parasitic phase Spherule rupture and release of endospores
I Spherule maturation
Endosporulation
FIG. 12. Growth phases of Coccidioides immitis.
c. Candida albicans The opportunistic human pathogen C. albicans, in common with many pathogenic fungal species, is dimorphic (Fig. 13), and the capacity for flexible interconversion between yeast cells and hyphae (and vice versa)
110
G. W. GOODAY AND D. J. ADAMS
FIG. 13. (a) Budding yeast and (b) mycelial growth phases of Curzdidu ufbicuns.
may be of importance during pathogenesis of candidosis (Odds, 1988). Kinsman et al. (1988) demonstrated that oestriol, pregnanediol and pregnanetriol increased the percentage of C. albicans yeast cells producing germ tubes in the presence of hormone concentrations of 1 * 10-9-1 * M . Other steroids (oestrone, 17P-oestradiol, progesterone) stimulated germination to a lesser extent or failed to stimulate germination. Therefore, Kinsman et al. (1988) argued that the capacity of steroids to promote germination of yeast cells of C . albicans was directly proportional to the degree of hydroxylation of these compounds. In a preliminary communication, Frey et al. (1988) also reported that oestradiol (in nanomolar to millimolar concentrations) stimulated germ-tube formation. In contrast, Loose et al. (1983a) found that progesterone, corticosterone and dexamethasone had no effect on growth of C. albicans (each incubation contained a single hormone at lo-* or lo-'" M), phase conversion M) or glucose oxidation M). d. Dermatophytes Inhibition of growth of Trichophyton rnentagrophytes by progesterone was described by Casas-Campillo et al. (1961), although the concentration of sterol used was not clearly stated. Capek and Simek (1971) also noted an inhibition of growth with 3.2 . M progesterone. More recently, Schar et al. (1986) and Clemons et al. (1988) reported that progesterone inhibited
111
SEX HORMONES AND FUNGI
growth of T. mentagrophytes, Arthroderma benhamiae ((+) and (-) mating types; a teleomorph of T. mentagrophytes), T. rubrum and Microsporum canis in a dose-dependent manner with 50% inhibition at concentrations of 5.5 . 9.8 . M , respectively. 1.2 . lo-', 2.7 . lo-' and 1.5 Clemons et al. (1989a) demonstrated that T. mentagrophytes can metabolize progesterone, providing at least a partial explanation for the relatively high concentration of this hormone required for a functional response. Earlier work indicated that deoxycorticosterone inhibited growth of T. mentagrophytes (Chattaway et al., 1959; Casas-Campillo et al., 1961; Capek and Simek, 1971) and, in two of these studies, deoxycorticosterone was more inhibitory than progesterone (Casas-Campillo et al., 1961; Capek and Simek, 1971). In contrast, Schar et al. (1986) and Clemons et al. (1988) noted that deoxycorticosterone and dihydrotestosterone inhibited growth of dermatophytes less effectively than progesterone. Dehydroepiandosterone and the synthetic progesterone promegestone (R5020) were almost as potent as progesterone in inhibiting growth of T. mentagrophytes (Clemons et al., 1988). Other steroids tested (hydrocortisone, corticosterone, tnamcinolone acetonide and dexamethasone) had insignificant or inconsistent effects on growth (Schar et al., 1986). 1
2. Protein and Polypeptide Hormones Human luteinizing hormone (hLH; 0.005-5 IU ml- ') increased the percentage of yeast cells of C. albicans producing germ tubes in a medium containing rat serum (10%) v/v, in saline; Kinsman et al., 1988). Similar results were obtained by Williams et al. (1990), who demonstrated that hLH (0.005-5 IU ml-l) promoted germination of yeast cells of C. albicans isolated from a late exponential-phase culture and grown subsequently in a defined medium. In contrast, Paveto et al. (1991) reported preliminary results which suggested that hLH and the closely related human chorionic gonadotrophin (hCG) inhibited germination of yeast cells of C. albicans isolated from a stationary phase culture. These results are intriguing since a similar biochemical response was noted in late exponential-phase (Williams et al. , 1990; Bramley et al., 1991a) and stationary phase (Paveto et al., 1991) yeast cells of C. albicans following incubation with hLH or hCG (see Section IV.C.2). Additional experiments should shed light on the apparent growth phase-dependent response of C. albicans cells to hLH. Paveto et al. (1990) reported that the pancreatic hormone glucagon ( 1 0 - 8 M) inhibited the yeast-to-hypha transition in C. albicans. The hormone also promoted a multibudded pattern of yeast-cell growth. Insulin promoted germination of yeast cells of C. albicans, apparently by stimulating uptake of proline (Nolting et al., 1982). Insulin also
112
C;.
W . GOODAY AND D. I. ADAMS
stimulated growth of a slime (wall-less mutant) of Neurospora crassa in a defined culture medium (McKenzie et al., 1988). Incubation with insulin also enhanced the long-term viability of stationary phase cells. Interestingly, cells grown in the presence of insulin exhibited marked morphological changes when compared to control cells. Indeed, these effects were reminiscent of insulin-induced morphological changes in mammalian cells. However, the authors pointed out that it was unclear whether the effects of insulin on the morphology of N . crassa occurred simply as a concomitant of accelerated growth or arose as a result of a more specific and undefined metabolic effect. The effects of the hormone on the growth and morphology of N . crassa occurred optimally at insulin concentrations (lW8-10-’ M) similar to those required for growth of mammalian cells in a defined culture medium. B. MAMMALIAN HORMONE-BINDING PROTEINS AND ENDOGENOUS LIGANDS IN FUNGI
In mammals, mediation of hormonal effects is dependent upon the interaction of these compounds with specific, high-affinity protein receptors on, or in, target cells. Steroid hormones can penetrate the plasma membrane to interact with cytoplasmic receptor molecules. However, protein and polypeptide hormones are generally unable to penetrate cell membranes. These molecules interact with receptors on the plasma membrane. Proteins which bind mammalian hormones with a high degree of affinity and specificity have been identified in a wide range of fungal species. Furthermore, the subcellular distribution of mammalian hormonebinding proteins in fungi appears to resemble closely the localization of their apparent mammalian counterparts.
I. Steroid Hormones Mammalian steroid hormones interact specifically with several fungi. a. Candida albicans The existence of fungal proteins which interact specifically with mammalian steroid hormones was demonstrated first by Loose et al. (1981). These authors described a cytosolic protein from yeast cells of C. albicans which bound [3H]corticosterone and [3H]progesterone with high affinity and selectivity. Radio-inert corticosterone competed for [3H]progesterone sites and radio-inert progesterone competed for [3H]corticosterone sites, suggesting that both probes were labelling the same site. However, higher
SEX HORMONES AND I-UNGI
113
levels of binding were found with [3H]corticosterone, and the authors used this glucocorticoid as their "-labelled probe to determine the characteristics of the binder in C. albicans. The results suggested that the binder was probably a protein of approximately 43 kDa, and that free sulphydryl groups were required for binding (Loose et al. , 1981; Loose and Feldman, 1982; Table 4). Scatchard plots yielded values for the equilibrium dissociation constant (Kd) and binding capacity (N,,,), which suggested the presence of a single class of low-capacity high-affinity binding sites in both yeast and mycelial forms of C. albicans (Loose et al., 1981; Loose and Feldman, 1982). Similar results were obtained by Powell and Drutz (1983) for yeast cells of C. albicans (Table 4). A number of steroids were tested for their ability to displace [3H]corticosterone from the yeast corticosteroid-binding protein (CBP; Loose and Feldman, 1982; Table 4). Interestingly, R5020, a synthetic progestin with a high affinity for mammalian progesterone receptors, had only 2% of the activity of progesterone while the potent synthetic glucocorticoid dexamethasone failed to bind. Prednisolone and llp-cortisol were moderate competitors. However, llu-cortisol, an inactive (in mammals) stereoisomer of 11p-cortisol, was devoid of activity. Other compounds tested had little or no competitive activity although Stover et al. (1983) demonstrated that the antifungal agent ketoconazole, which inhibits sterol biosynthesis in susceptible fungi, displaced [3H]corticosterone from the CBP in C . albicans in a competitive manner. Loose and Feldman (1982) concluded that the binder is remarkably steroid-selective and stereospecific in nature. In a subsequent study, Loose et al. (1983a) detected CBP in all of eight isolates of C. albicans of both A and B serotypes. They found that [3H]corticosterone entered intact yeast cells and specifically occupied CBPs. Corticosteronebinding protein was also demonstrated in all of six Candida spp., other than C. albicans, examined. Loose and his coworkers (Loose et al., 1981; Loose and Feldman, 1982) noted that the fungal CBP was comparable, in a number of respects, with the mammalian corticosteroid-binding globulin (CBG) and the classical glucocorticoid receptor. They suggested that the localization of the binder in the cytosol, and the fact that it is not secreted into the medium, indicates that it may function as a receptor rather than as a transport protein analogous to CBG. Loose et al. (1981) demonstrated that a crude ethanolic extract of yeast cells of C. albicans contained a factor which competed reversibly with [3H]corticosterone for the fungal binding site. The material, which appeared to be released into the growth medium, also displaced steroids from mammalian glucocorticoid receptors in a competitive manner. However, the yeast extract was considerably more potent in inhibiting binding to the fungal protein. Loose et al. (1981) concluded that the ethanolic extract of
TABLE 4. Specific binding sites for mammalian steroid hormones in fungi" Hormone
Species (number of strains tested is indicated in parentheses)"
K d (nM)
N,,, ( p o l Strong competitors (mg protein)-')
Weak competitors
Endogenous competitor
Corticosterone
Candida albicans mycelium'( 1) Candida albicans yeast'-5( 10)
6.3
0.65
ND
ND
ND
4.9C32.9
0.004-2.41
Corticosterone, progesterone
lla-Cortisol, prednisolone, R5020, ketoconazole, miconazole, econazole, ergosterol
Yes
Candida spp. (6) 6.CL38.0 other than Candida albicans (one strain of each specie^)^
0.08-6.53
ND
Candida albicans 6.CL110.0 yeast"'( 18) Candida glabrata6 (3) 23.0-25.9 Coccidioides immitis' 21 .O-37.0 (1)
0.04-13.24 0.6C5.4 0.5-1.56
17P-Oestradio1, oestrone, oestriol ND 17a-Oestradio1, oestrone, oestriol, dieth ylstilboestrol
0.078
17P-Oestradiol
0.12-0.59, 1.7-2.6
17p-Oestradiolb, oestrone, oestriol, progesterone
17p-Oestradiol
Paracoccidioides 13 brasiliensis mycelium"( 1) Paracoccidioides 8.5-17.0, brasiliensis yeast'@'' 150.0 (four strains for highaffinity binding site, one strain for low-affinity binding site)
ND
17a-Oestradiol
ND
ND 17a-Oestradiol, ND testosterone, progesterone, R5020, dexamethasone, corticosterone, ergosterol Diethylstilboestrol ND
17a-Oestradiol, dihydrotestosterone, testosterone, dieth ylstilboestrol
ND
Progestins Progesterone
R5020 Testosterone
Saccharomy ces cerevisiae13 (three strains: a- and a-haploids and d a diploid)
1.6-1.8
2.C-2.8
17p-Oestradio1, oestrone, oestriol
17a-Oestradiol, progesterone, tamoxifen, diethylstilboestrol
Not known
Arthroderma benhamiae ( + ) I 4 Arthroderma benhamiae (-)I4 Microsporum canis14 (1)
162.0
8.89
ND
ND
ND
173.0
14.38
ND
ND
ND
190.0
15.56
Progesterone, deoxycorticosterone
(1)
(1)
Trichophyton 95.0 mentagrophyte~'~.'~ (1)
4.98
Trichophy ton rubrum14 (1) Coccidioides immitis16 (3)
16.0
0.2
Dihydrotestosterone, ND deoxycortisol, corticosterone Progesterone, R5020, DehydroepiandoND androstenedione, sterone, deoxycorticosterone dihydrotestosterone, testosterone, 17ahydroxyprogesterone, 18-hydroxydeoxycorticosterone, aldosterone ND ND ND
1.2436.0
0.014-0.2
R5020
Progesterone, dihydrotestosterone
Yes
Coccidioides immitis' (I)
28.CL150.0
15.8-38.9
Testosterone dihydrotestosterone
Methyltrienolone, l7p-oestradiol
ND
Competitors are listed in approximate order of potency. The distinction between strong and weak competitors is arbitrary; the reader is referred to the original publications for a more accurate indication of competitive capacity. ND indicates not detected. a References: Loose and Feldman (1982), * Loose et al. (1981), Loose et al. (1983a), Stover et al. (1983), Powell and Drutz (1983), Powell et al. (1984), Othman et al. (1988), Skowronski and Feldman (1989), Powell and Drutz (1984), '"Stover et al. (1986), I' Loose et al. (1983b), Restrepo el al. (1984), l3 Burshell et al. (1984), l 4 Clemons et al. (1988), l5 Schar et al. (1986), I' Powell et al. (1983). Competition data are for high-affinity binder only.
'
'
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G W GOODAY AND D I ADAMS
C. albicans contained an endogenous ligand for the cytosol binder and proposed that C. albicans possesses both components of a hormonereceptor system, namely a ligand and a binding protein. Cytosolic proteins which bind oestradiol with high affinity and specificity have been identified in C. albicans by a number of research groups (Powell et al., 1984; Othman et al., 1988; Skowronski and Feldman, 1989; Table 4). However, the content of oestradiol-binding proteins in C. albicans was influenced markedly by the composition of the growth medium (Skowronski and Feldman, 1989), and this may explain why earlier attempts to identify oestrogen-binding proteins in this species were unsuccessful (Loose et al. , 1981; Loose and Feldman, 1982). Binding of oestradiol to cytosolic proteins in Candida spp. appears to be a species-dependent phenomenon in that, of the Candida spp. investigated (C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. pseudotropicalis, C. stellatoidea and C. tropicalis), only C. albicans, C. glabrata and C. tropicalis bound 17p-oestradiol specifically (Powell et al., 1984). Powell et al. (1984) also demonstrated specific uptake of oestradiol by viable yeast cells of C. albicans. There is some disagreement in the literature concerning a number of characteristics of the binder in C. albicans. For example, with regard to a requirement for the presence of free sulphydryl groups on the binding protein in C. albicans, Powell et al. (1984) found that incubation with the sulphydryl group-blocking reagent N-ethylmaleimide completely abolished oestradiol binding, whereas Othman et al. (1988) and Skowronski and Feldman (1989) found that the blocking reagent had little or no effect on binding. Estimates for the molecular mass of the binding protein also differ markedly. Powell et al. (1984) gave an M, value of approximately 85 kDa whereas Skowronski and Feldman (1989) described a protein with a value of approximately 46 kDa. Considerable variation was apparent in Kd and N,,, values detected for oestradiol-binding proteins from a total of 18 C. albicans strains (Powell et al., 1984; Othman et al., 1988; Skowronski and Feldman, 1989; Table 4). As was apparent for the CBP (Table 4), binding parameters were similar for oestradiol-binding proteins from both mycelial and yeast-growth forms, suggesting no major change in binding characteristics during morphogenesis (Skowronski and Feldman, 1989). However, Skowronski and Feldman (1989) did observe a pronounced increase in binding of ['H]oestradiol during the early logarithmic phase of growth of yeast cells. In the stationary phase of growth, binding fell to levels similar to those detected prior to the exponential phase. Similar results were obtained by Williams et al. (1990) using three independent assays for the oestradiol-binding protein (R. J. Williams, 0. S. Kinsman and D. J. Adams, unpublished observations). Saturation analysis suggested that increased binding was due to the
SEX HORMONES AND FUNGI
117
presence of an increased number of ligand-binding sites rather than to an increase in the affinity of the binder for oestradiol (Skowronski and Feldman, 1989). The binder in C. albicans was highly selective in that significant competition for oestradiol-binding sites was exhibited only by oestrogenic steroids (17~-oestradiol>oestrone>oestriol>l7a-oestradiol;Powell et al. , 1984; Othman et al., 1988; Skowronski and Feldman, 1989; Table 4). Clearly, the binding system is different from mammalian-binding proteins since diethylstilboestrol, a non-steroidal compound with strong oestrogenic potency in the mammalian receptor system, did not compete for binding to the protein in C. albicans. Similarly, the non-steroidal oestrogen antagonist tamoxifen, which has been shown to compete effectively with oestradiol for binding to the mammalian receptor, failed to displace [3H]oestradiol from the binding protein from C. albicans (Powell et al., 1984; Othman et al., 1988; Skowronski and Feldman, 1989). A further, interesting observation was that zearalenones, which are fungal products that act as oestrogens in mammalian systems (Katzenellenbogen et al. , 1979; see Section I11 and Fig. lo), were also devoid of competitive activity (Skowronski and Feldman, 1989).
b. Paracoccidioides brasiliensis Stover et al. (1986) demonstrated that cytosol from yeast and mycelial cultures of P. brasiliensis contained a high-affinity low-capacity binder for 17p-oestradiol (Table 4). In addition, a low-affinity binding moiety was detected following pre-incubation of yeast and mycelial cytosols at 37°C (Table 4). The high-affinity binder appeared to be protein in nature, with free sulphydryl groups necessary for hormone binding and an M , value of approximately 60 kDa (Loose et al. , 1983b). Binding to high-affinity sites in yeast and mycelial cytosols was highly specific in nature. Oestriol, oestrone and progesterone displayed about 25% of the apparent affinity of oestradiol for the yeast-binding protein, while 17a-oestradiol and the androgens dihydrotestosterone and testosterone were weak competitors (Loose et al., 1983b; Restrepo et al., 1984; Table 4). The non-steroidal oestrogen diethylstilboestrol competed weakly for yeast cytosol-binding sites. However, this oestrogen was moderately potent as a competitor for the mycelial oestrogen-binding protein. The nonsteroidal oestrogen antagonist tamoxifen failed to compete for binding to yeast or mycelial-binding proteins (Loose et al., 1983b; Restrepo et al., 1984; Stover et al. , 1986; Table 4).
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G W GOODAY AND D J ADAMS
c. Coccidioides immitis Specificbinding proteins for progestin (promegestone (R5020), progesterone), oestrogen (17p-oestradiol), androgen (dihydrotestosterone, methyltrienolone (R1881)), corticosterone and triamcinolone acetonide were identified in cytosols of the five strains of C. immitis investigated (Powell et al., 1983). Detailed analyses of progestin, oestrogen and androgen binding were undertaken (Powell et al., 1983; Powell and Drutz, 1984). The synthetic progestin R5020 bound specifically to a cytosolic protein from C. irnmitis, while progesterone and dihydrotestosterone were weak competitors for binding (Powell et al., 1983; Table 4). Scatchard plots of equilibrium binding data indicated apparently complex binding kinetics and suggested the presence of a low-affinity high-capacity progestin-binding system. However, Scatchard analysis of ammonium sulphate-precipitable cytosol fractions revealed, additionally, a high-affinity low-capacity binding system for R5020 (Table 4). Powell et al. (1983) proposed that the low-affinity binder may serve as a repository for a hormone within the fungus, prior to its attachment to the high-affinity binder and stimulation of growth. Preliminary studies with ethanol-extracted lipid from the cytosol of C. immitis revealed the presence of an inhibitor(s) of R5020 binding to crude cytosol (Powell et al., 1983). Thus, C . immitis may contain an endogenous ligand which interacts with the progestin binder. Scatchard analysis of binding data for l7p-oestradiol in crude cytosol preparations revealed a complex pattern of binding (to a proteinaceous binder with free sulphydryl groups required for binding), similar to that noted for progestin binding (Powell and Drutz, 1984). However, again, Scatchard analysis of ammonium sulphate-precipitable fractions indicated the presence of a high-affinity low-capacity binder (Table 4). A proteinaceous testosterone binder, with free sulphydryl groups apparently active in the binding mechanism, was detected in crude cytosol preparations (Powell and Drutz, 1984). In this case, Scatchard analysis of binding data was straightforward and identified a single-component binding system of low-affinity and high-capacity (Table 4). A low-affinity highcapacity binder was the only detectable species of androgen binder in ammonium sulphate-precipitable fractions. [3H]Testosterone binding was highly specific; only androgens competed strongly for binding (Table 4).
d. Dermatophytes Incubation of cytosol from T. rnentagrophytes with tritiated steroids identified specific binding sites for [3H]corticosterone and [3H]progesterone (Schar et al., 1986). Scatchard analysis of [3H]progesterone binding showed
SEX HORMONES AND FUNGI
119
a single class of relatively low-affinity high-capacity cytosolic binding sites in T. mentagrophytes, the (+) and (-) mating types of A. benhamiae and M . canis (Schar et al., 1986; Clemons et al., 1988; Table 4). The binder from T. rubrum had a higher affinity (Clemons et al., 1988; Table 4). Detailed characterization of the progesterone-binding protein in T. mentagrophytes demonstrated that progestins and androgenic compounds were the most active competitors for binding, although the mineralocorticoid deoxycorticosterone (which is structurally related to progesterone) was also potent (Schar et al., 1986; Clemons et al., 1988; Table 4). Similar results were obtained for the binder from M . canis (Clemons et al., 1988; Table 4). Competition studies also indicated that the progesterone binder from T. mentagrophytes differed markedly from the progesterone-corticosterone binder identified in C. albicans (Loose and Feldman, 1982; Loose et al., 1981, 1983a; Table 4). Mating types of A. benhamiae (a teleomorph of T. mentagrophytes) bound [3H]progesterone with equal affinity (Clemons et al., 1988). As Clemons et al. (1988) indicated, if one but not the other mating type had bound progesterone, then such a result might have suggested a role for the progesterone-binding protein of A. benhamiae, and endogenous ligands resembling progesterone, during sexual reproduction. However, these authors did not exclude the possibility that one or both mating types may produce natural ligands involved with sexual differentiation, which have much greater affinity than progesterone for the binding protein of A . benhamiae. e. Saccharomyces cerevisiae
A protein (approximate M, 60-70 kDa) which bound 17P-oestradiol with high affinity and specificity (Table 4) was detected in cytosol from Sacch. cerevisiae; free sulphydryl groups were required for hormone binding (Feldman et al., 1982; Burshell et al., 1984). Burshell et al. (1984) constructed a competition profile for the yeast 17P-oestradiol binder based on the capacity of various steroids to displace [3H]oestradiol from the binding protein (Table 4). These studies indicated that the oestradiol binder in Sacch. cerevisiae is unique and, like the binding proteins from C. albicans and P. brusiliensis, can be distinguished readily from mammalian oestradiolbinding proteins. Feldman et al. (1982) detected a compound in lipid extracts of Sacch. cerevisiae (grown in complex media) and culture filtrates (following incubation with yeast) which bound competitively to the [3H]oestradiolbinding sites in Sacch. cerevisiae and to mammalian oestrogen receptors. They suggested that the binding protein from Sacch. cerevisiae may be a
120
G W GOODAY A N D D J ADAMS
primitive hormone receptor, and that the lipid-extractable substance represented an endogenous ligand for this receptor. Partially purified extracts of the apparent endogenous ligand exhibited oestrogenic activity in mammalian systems (Feldman et al., 1984a) and the purified compound was identified as 17P-oestradiol (Feldman et al., 1984b). They concluded that the oestrogen was a yeast product. However, in subsequent experiments, Sacch. cerevisiae grown in a chemically defined medium was shown to contain extremely small amounts of oestradiol (Miller et al., 1986). These low levels could not be distinguished consistently from the low levels of oestradiol detectable in control media. The apparent explanation for the marked discrepancy between the amount of oestradiol detectable when Sacch. cerevisiae was grown in complex media (greater than 5 ng I-') compared with the amount produced in a simple, well-defined medium (less than 0.5 ng I-') was as follows. Components of complex media contain significant concentrations of oestradiol (e.g. molasses) and oestrone (e.g. Bactopeptone and molasses), and Sacch. cerevisiae metabolizes oestrone to oestradiol (Miller et al., 1986). Therefore, growth-medium oestradiol and metabolized oestrone may account for all of the oestradiol thought previously to have been synthesized by yeast (Feldman et al., l982,1984a,b). However, Miller et al. (1986) have detected agents in extracts of Sacch. cerevisiae cells and growth media, following incubation with yeast cells, which are neither oestrone nor oestradiol and which inhibit binding of [3H]oestradiol to the yeast oestradiol-binding protein and to the mammalian receptor. They propose, therefore, that other endogenous ligands besides oestradiol may be active in the yeast binding system. Pancreatic tissue contains an oestradiol-binding protein which may be distinguished from the oestradiol-binding protein from the uterus by its requirement for a coligand in the steroid-binding reaction (Boctor et al., 1981). The endogenous coligand appears to be the tetradecapeptide somatostatin. Grossman et al. (1986) demonstrated that yeast a-factor, the tridecapeptide pheromone that induces conjugation between haploid cells of opposite mating type (see Section II.B.l), was as effective as somatostatin in promoting specific binding of [3H]oestradiol to partially purified pancreatic protein. Furthermore, a-factor enhanced specific binding of [3H]oestradiol to the binding protein in Sacch. cerevisiae. However, although somatostatin, somatostatin analogues and an analogue of a-factor also enhanced binding of [3H]oestradiol to the yeast binder, these oligopeptides differed from afactor in that they did not inhibit cell growth or induce morphological changes in Sacch. cerevisiae. Grossman et al. (1986) concluded that coligand-requiring [3H]oestradiol-binding activity and mating in yeast are not directly related.
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2. Protein and Polypeptide Hormones High-affinity insulin-binding sites, similar to those provided by mammalian insulin receptors, were detected on the surface of intact N. crussa slime (wall-less) cells (McKenzie et ul., 1988). The protein (approximate M , 66 kDa) which was, apparently, responsible for cell-surface binding of insulin was purified to electrophoretic homogeneity from a solubilized membrane preparation (Kole et al., 1991). Unlike the much larger heterodimeric mammalian insulin receptor (Rosen, 1987), the binding protein from N. crassa lacked protein kinase activity against itself or against exogenous substrates, and did not contain phosphorylated amino-acid residues (Kole et al., 1991). However, phosphoproteins (approximate M , 50 kDa, Fawell and Lenard, 1988; approximate M , 38 kDa, Kole and Lenard, 1990) were identified in detergent-solubilized membrane fractions from N . crassa, using an antipeptide antibody (anti-P2) raised against a sequence from the autophosphorylation site in the kinase domain of the human insulinreceptor B-subunit. The 50 kDa protein had protein tyrosine kinase activity (Fawell and Lenard, 1988). A relationship between the insulin-binding protein and these phosphoproteins has not as yet been demonstrated. However, one possibility, suggested by Fawell and Lenard (1988), is that the binding protein and phosphoprotein(s) with protein tyrosine kinase activity may be components of a receptor in N. crassa, analogous to the mammalian insulin receptor (Rosen, 1987). Interestingly in this regard, cells of wild-type N. crussa contain a molecule that resembles insulin both immunologically and functionally (Le Roith et al., 1980), and Kole et al. (1991) proposed that the insulin receptor from N. crussa may interact specifically with this ligand. The structurally related glycoproteins hCG and hLH bound specifically to subcellular fractions of C. albicans, and specific hLH-binding sites were also demonstrated in C. tropicalis (Bramley et al., 1990b, 1991b). Similar hLH-binding sites have been identified in Sacch. cerevisiae, and in wildtype N . crassa and a wall-less mutant of this species (T. A. Bramley, G. S. Menzies, R . J. Williams, 0. S. Kinsman and D . J. Adams, unpublished observations). The fungal binding sites differed markedly from mammalian hCG-hLH receptors. For example, binding of hCG or hLH to cytosolic fractions was not generally noted for mammalian receptor systems (for references, see Bramley et al., 1991b). In contrast, although higher levels of binding were noted in fungal microsomal fractions, significant binding to cytosolic fractions was also detected. Similarly, sheep luteal membranes contained only a single high-affinity binder for '251-labelled hLH while cytosolic and microsomal fractions from C. albicans contained both highaffinity low-capacity, and low-affinity high-capacity, binding sites for this ligand (Bramley et al., 1990b, 1991b).
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The binding sites in C. albicans appeared to be hLH-hCG-specific in that gonadotrophin preparations containing hLH or hCG competed mutually for binding, whereas other hormones (epidermal growth factor, follicle-stimulating hormone, growth hormone, prolactin) neither bound to subcellular fractions nor competed with hCG-hLH for specific binding sites in this species (Bramley et a f . ,1990b). However, an unexpected observation was that, while '2s1-labelled hCG-hLH binding to membranes of C. albicans was displaceable by low levels (nanograms) of partially purified hCG preparations, much higher levels (micrograms) of highly purified hLH or hCG were required for significant displacement of radiolabelled hormone. The explanation for this result appeared to be the presence of a heat-labile glycoprotein ( M , 1 6 2 1 kDa) in crude (but not highly purified) gonadotrophin preparations which strongly inhibited '2sI-labelled hLH binding to membranes of C. albicans, but not to sheep or pig luteinizing-hormone receptors (Bramley et a f . , 1991a). The properties of this glycoprotein were similar to those of the p-core protein, a cleavage product of the p-subunit of hCG which is a contaminant of commercial gonadotrophin preparations (for references, see Bramley et a f . , 1991a). Of particular interest in this regard was the observation that highly purified p-core protein inhibited ['2sI]-labelled hLH binding to membranes of C. albicans but not to sheep luteal binding sites (Bramley et a f . ,1991a). Bramley et al. (1991a) proposed that endogenous hLH-hCG-p-core-like molecules may play a role in regulation of morphogenesis in C. albicans (see Section IV.C.2).
3. Conclusions Clearly, many fungi bind mammalian hormones with high affinity, selectivity and stereospecificity (Table 4). However, there are important caveats for research with these fungal binders. In particular, it is important to be aware of the marked effects that alterations in growth medium, or growth phase of the micro-organism, may have on the expression of binding proteins. Although all of the fungal binding proteins identified to date resemble their apparent mammalian equivalents in a number of respects, they are, nonetheless, distinct binding entities in their own right. For example, competition profiles obtained by competing a variety of ligands for radiolabelled hormone-binding sites in fungi were frequently unique, differing markedly from profiles for mammalian receptors or plasma binding proteins. Purification and sequence analysis of fungal binding proteins will enable a more direct comparison of these molecules with mammalian hormone receptors. A number of fungal species (C. albicans, C. immitis, N . crassa and Sacch. cerevisiae) contain factors which compete with radiolabelled hormone for
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specific binding sites. It is possible that such ligands may function as endogenous fungal hormones, although to date there is as yet no clear evidence of a physiological role for those molecules identified and characterized. C. BIOCHEMICAL RESPONSE
A number of fungi can metabolize steroid hormones. Indeed, mammalian hormones have been shown to induce steroid-hydroxylating enzymes in several fungal species (e.g. see Hudnik-Plevnik and Cresnar, 1990). This work will not be considered here. Instead, the work reviewed relates to stimulation of biochemical responses in fungi by mammalian hormones, which may follow specific interactions analogous to the hormone-receptor interactions of mammalian cells. 1. Steroid Hormones Biochemical responses have been characterized in several fungi. a. Saccharomyces cerevisiae The results of Tanaka et al. (1989) suggested that oestradiol regulates the cell cycle of Sacch. cerevisiae in the early G I growth phase by controlling the level of intracellular CAMP. This regulation is apparently achieved through oestradiol activating expression of the adenylate cyclase gene. Oestrogens interact with mammalian cells in many ways, and have been shown to act both as growth factors and transcriptional activators (for references, see Tanaka et a/., 1989). Interestingly, recent work suggests striking parallels between regulation of transcription activation by oestradiol in yeast and in mammals. Mammalian oestrogen-receptor proteins comprise both a DNA-binding domain and an independent domain responsible for hormone binding and transcription activation. Binding of oestradiol results in interaction of the hormone-receptor complex with an oestrogen-responsive element in the genome and stimulation of transcription (Yamornoto, 1985; Beato, 1989). The work of Pierre Chambon and his coworkers suggests a remarkable conservation of these regulatory mechanisms throughout evolution. For example, chimeric proteins, consisting of the hormone-binding domain of the human oestrogen receptor coupled to a DNA-binding domain from Sacch. cerevisiae, bound to DNA in a hormone-dependent manner and stimulated transcription in mammalian cells (Webster et al., 1988a). Furthermore, the DNA-binding domain of the human oestrogen receptor mediated stimulation of transcription in mammalian cells by activating
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regions of yeast transcriptional regulatory proteins (Webster et a f . ,1988b). In further studies, Metzger et a f . (1988) expressed the gene for the human oestrogen receptor in Sacch. cerevisiae, giving rise to a level of specific oestradiol-binding activity which was much higher than the endogenous oestradiol-binding activity found in this yeast (Feldman etaf., 1982; Burshell et a f . , 1984). Furthermore, oestrogen receptor synthesized by yeast bound to an oestrogen-responsive element in a hormone-dependent manner in vitro. Finally, Metzger and his coworkers demonstrated that the human oestrogen receptor stimulated transcription in yeast in vivo in a strictly hormone-dependent manner “indicating an amazing conservation of the molecular mechanisms underlying this activation across all eukaryotes” (Metzger et a f . , 1988). More recently, Wright et a f . (1990) expressed the C-terminal half of the human glucocorticoid receptor in Sacch. cerevisiae. They demonstrated that gene expression was activated by the presence of steroids with glucocorticoid activity, following interaction of the receptor with a glucocorticoidresponsive element fused to a yeast promoter upstream of a reporter gene. Furthermore, these workers proposed that their data were consistent with association of most of the expressed receptor with a yeast heat-shock protein ( M I 90 kDa). Thus, they suggested that the mechanism by which mammalian steroid-hormone receptors are sequestered in an inactive, non-DNA binding state in the absence of ligand may be functionally conserved in yeast.
b. Paracoccidioides brasifiensis In conjunction with its inhibition of the transition from the mycelium to the yeast form of P. brasifiensis, oestradiol (2.6 . lo-’ M) caused a concomitant block or delay in expression of several cytosolic proteins which appeared normally during the morphological transformation, or with development of the yeast growth phase (Clemons et al., 1989b). Oestradiol also altered methionine uptake during the phase transition. On the basis of these and earlier results (Loose et a f . , 1983b; Restrepo et a f . , 1984; Stover et al., 1986), Clemons et al. (1989b) proposed that the functional responses of P. brasiliensis to oestradiol were closely analogous to those detected following interaction of mammalian cells with steroid hormones (Yamomoto, 1985). Thus, they envisaged that the hormonal effects were mediated via the oestradiol-binding protein from P. brasifiensisfunctioning as a receptor which interacted with genomic DNA and regulated transcription of specific genes, leading ultimately to the expression of specific proteins.
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c. Candida albicans Frey et al. (1988) reported that oestradiol (in nanomolar to millimolar concentrations) stimulated protein synthesis and phospholipase activity in C. albicans.
2. Protein and Polypeptide Hormones Biochemical responses have been characterized in several fungi. a. Candida albicans
In vertebrates, the immediate response of target cells to interaction of the structurally related hormones hLH and hCG with specific cell-surface receptors is an increase in adenylate cyclase activity leading to an elevation of intracellular CAMP concentration (Hunzicker-Dunn and Birnbaumer, 1985; Levitzki, 1987). Recent results suggest the presence of a similar receptor-mediated mechanism for elevation of adenylate cyclase activity in C. albicans. Williams et al. (1990) demonstrated stimulation of adenylate cyclase activity following the specific binding of hLH to microsomes from C. albicans. These authors also provided the first demonstration of guanine nucleotide-binding proteins (G-proteins) in C. albicans. The regulation of mammalian adenylate cyclase following specific interaction of a hormone with a cell-surface receptor is mediated through the enhancement of GDPGTP exchange on a G-protein (Levitzki, 1988), Spontaneous hydrolysis of GTP by G-proteins ultimately causes cessation of their stimulation of the effector molecule, Williams et al. (1990) demonstrated that the nonhydrolysable GTP-analogue GTPyS stimulated adenylate cyclase activity in C. albicans, although to a lesser extent than its stimulation of mammalian adenylate cyclase activity. In addition, they showed that GTPyS promoted germination of yeast cells of C. albicans and proposed that stimulation of C. albicans morphogenesis by hLH is mediated by a receptor-coupled adenylate cyclase system involving G-proteins. More recently, Bramley et al. (1991a) demonstrated that hCG preparations could stimulate adenylate cyclase activity in membranes of C. albicans almost five-fold. They also showed that partially purified preparations of a glycoprotein which resembled the p-core protein of hCG, and which inhibited specific binding of hLH to membranes of C. albicans (see Section 1V.B.2), strongly inhibited basal adenylate cyclase activity (to 18% of control levels). Bramley et al. (1991a) proposed that endogenous hLHhCG-P-core-like factors may play a role in regulation of morphogenesis in C . albicans.
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Paveto et al. (1991) found that incubation of yeast cells of C . albicans with hCG or hLH, or the pancreatic hormone glucagon, resulted in a significant elevation of total (intracellular and extracellular) cAMP levels. Furthermore, these hormones stimulated Mg2+-GTP-dependentadenylate cyclase and CAMP-dependent protein kinase activity in permeabilized cells of C. albicans (Paveto et al., 1990, 1991). These effects appeared to be dependent upon exogenous administration of guanine nucleotides during cell permeabilization and enzyme-assay procedures. The authors' conclusions were similar to those of Williams et al. (1990). They emphasized that hormonally-induced elevation of intracellular cAMP most probably activates CAMP-dependent protein kinase activity, which may lead to phosphorylation of key enzymes involved in regulation of morphogenesis in C. albicans. b. Neurospora crassa Work on the effects of insulin on growth and morphogenesis of N. crassa and characterization of insulin-binding proteins in the fungus (described earlier) involved wall-less slime strains of this organism. Studies of the biochemical response of N . CYUSSU to insulin and glucagon also utilized slime strains. The results suggested that these hormones elicit strikingly similar responses in fungal and mammalian cells. The first reports were by Flawia and Torres (1972, 1973a,b), who demonstrated that glucagon and insulin modulated adenylate cyclase antagonistically in membrane preparations; inhibition of the enzyme by insulin was counteracted by glucagon. Flawia and Torres (1972) also demonstrated that glucagon increased the rate of glycogenolysis in whole cells; more specifically, the hormone stimulated glycogen phosphorylase but inhibited glycogen synthetase activity. The biochemical events following binding of insulin to a mammalian receptor and activation of receptor tyrosine kinase activity are incompletely understood (Rosen, 1987). Nonetheless, insulin is known to stimulate a large number of metabolic effects in mammalian cells, while the hormone has been shown to elicit closely comparable responses in N . crassa. Thus, in addition to inhibition of adenylate cyclase in membranes already noted, insulin induced the following effects in slime cells: enhanced consumption of glucose and enhanced production of glucose metabolites including carbon dioxide, ethanol, alanine and glycogen; and increased retention of intracellular sodium ions during glucose consumption (Fawell et al., 1988; Greenfield et al., 1988, 1990; McKenzie et al., 1988). A particularly interesting observation made by Fawell et al. (1988) was that insulin treatment of N . crassa caused activation of the enzyme glycogen synthase from a glucose 6-phosphate-dependent (D) form to a glucose
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6-phosphate-independent (I) form. These authors pointed out that this response is identical to that seen in mammalian cells in which the D-to-I conversion of glycogen synthase is achieved by dephosphorylation of the enzyme induced by an insulin-specific phosphatase. Fawell et al. (1988) also emphasized that the glycogen synthase in N. crassa is known to be a phosphoenzyme that can be activated by dephosphorylation. The similarities between fungal and mammalian systems, coupled with the effects of insulin on growth and morphogenesis of N . crassa (see Section IV.A.2) following specific interaction of the hormone with fungal binding proteins (see Section IV.B.2), have prompted workers in this field to suggest the probable existence of a signal-transduction pathway initiated by insulin (or an endogenous insulin-like molecule) binding to a plasma-membrane receptor protein in N . crama (Fawell et al., 1988; Kole et al., 1991). c. Saccharomyces cerevisiae An example of a fungal hormone interacting with a mammalian hormone receptor and eliciting a biochemical-biological response is provided by afactor, the tridecapeptide mating factor of Sacch. cerevisiae (see Section II.B.l). There is a significant degree of amino acid-residue sequence homology between a-factor and the hypothalamic decapeptide gonadotrophinreleasing hormone (GnRH; also known as luteinizing hormone-releasing hormone, LHRH; Hunt and Dayoff, 1979). Loumaye et al. (1982) demonstrated that both synthetic and natural preparations of a-factor bound specifically to GnRH receptors in homogenates of rat pituitary cells. Furthermore, these a-factor preparations caused a dose-dependent release of luteinizing hormone from cultured rat pituitary cells into the incubation medium. It should be noted that a-factor was less active than GnRH by four orders of magnitude both with respect to inhibition of '2sI-labelled GnRH binding to homogenates of cells and its capacity to stimulate release of luteinizing hormone from pituitary cells (Loumaye et al., 1982). On the basis of their interesting results, these workers proposed that the structural and functional properties of GnRH-related peptides may have been highly conserved during evolution. 3. Catecholamines
Human p,-adrenergic receptor (hp-AR) expressed in Sacch. cerevisiae displayed binding characteristics typical of the receptor found in mammalian cells (King et al., 1990). Partial activation of the yeast pheromone-response pathway by p-adrenergic receptor agonists was achieved in cells coexpressing hp-AR and a mammalian G-protein (Gsa) subunit. This
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demonstrated that components of a mammalian signal-transduction pathway could couple to each other and to downstream effectors when expressed in yeast. D. IMPLICATIONS FOR PATHOGENESIS
I . Coccidioidomycosis Normally, men are more likely than women to develop disseminated coccidioidal infection, and this may reflect their increased exposure to C. immitis in the environment (Drutz and Catanzaro, 1978). This situation is reversed during pregnancy (Harvey, 1980); the later in pregnancy that coccidioidomycosis is acquired, the more likely is dissemination to occur (Harris, 1966). The increased susceptibility of pregnant women to coccidioidal dissemination has been attributed to suppression of cell-mediated immunity that characterizes late stages of pregnancy. However, Drutz et al. (1981) noted that other infectious diseases show only a minor tendency to be aggravated by pregnancy. These authors therefore sought additional explanations for the high incidence of coccidioidal dissemination which accompanies the gravid state. As already noted (see Section IV.A.l), Drutz et al. (1981) demonstrated a dose-dependent stimulation of spherule growth and maturation, endosporulation and endospore release in vitro (Fig. 12) by physiologically significant concentrations of 170-oestradiol and progesterone. In subsequent studies, Powell et al. (1983) and Powell and Drutz (1984) detected specific binders in C . immitis for progestin and oestrogen, respectively (Table 4). These binders had sufficiently high affinities for the respective hormones to enable them to compete for levels of unbound 170-oestradiol and progesterone detected in sera of pregnant women. Thus, the effects of steroid hormones on growth and morphogenesis of C. immitis may be mediated by these apparently proteinaceous binding systems. These results are compatible with the proposal by Drutz et al. (1981) that the propensity of pregnant women to develop coccidioidal dissemination may be due to a direct stimulation of the growth and life cycle of C. immitis by the markedly elevated levels of 170-oestradiol and progesterone that occur during midto-late pregnancy, combined with a concomitant suppression of cellmediated immunity. 2. Paracoccidioidomycosis
Paracoccidioidomycosis, like coccidioidomycosis, is much more common in men than in women. For example, the male-female ratio of the disease
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in Colombia is approximately 48:l (Restrepo and Greer, 1983). Skin-test studies in endemic areas suggest that exposure to P. brasiliensis occurs to the same extent for males and females. Furthermore, males and females acquire the disease in equal numbers prior to onset of puberty. These data suggest that hormonal factors play an important. role in the pathogenesis of paracoccidioidomycosis (Loose et al., 1983b; Restrepo et al. , 1984). The transition from inhaled mycelial fragments or conidia of P. brasiliensis to the yeast form is a critical step in the establishment of infection (Fig. 11; for references, see Restrepo et al., 1984; Salazar et al., 1988). The inhibition of the mycelium conidium-to-yeast conversion by physiologically significant concentrations of oestradiol in vitro (Loose et al., 1983b; Restrepo et al., 1984; Salazar et al., 1988; Section IV.A.l), coupled with detection of a protein in the cytosol of P. brasiliensis which bound oestradiol with high affinity and specificity (Loose et al., 1983b; Restrepo et al., 1984; Stover et al., 1986; Table 4), prompted workers in this field to propose that similar interactions in vivo are, at least in part, responsible for the marked resistance of females to paracoccidioidomycosis (Restrepo et al., 1984; Salazar et al., 1988).
3. Candidosis Pregnancy and the use of oestrogen-containing oral contraceptives appear to be predisposing factors for vaginal candidosis (Sobel, 1985). The implication is that hormonal factors influence pathogenesis. Hormones may exert direct or indirect effects on C. albicans, and a number of potential interactions were discussed by Ryley (1986). Of most interest in the context of the present discussion is the possibility that hormones may stimulate growth and morphogenesis of C. albicans following a direct and specific interaction with the pathogen. Although both yeast and mycelial forms of C. albicans (Fig. 13) may be isolated readily from infected host tissue, it is possible that the adhesive and penetrative properties of hyphae play a particularly important role during the pathogenic process (for a review, see Odds, 1988). Kinsman et al. (1988) proposed that stimulation of germination of C. albicans by oestriol and other steroids at physiologically significant concentrations may explain the predisposition of pregnant women to infections by C. albicans. They also suggested that stimulation of germ-tube formation by steroid hormones, coupled with their demonstration of a stimulation of germination by luteinizing hormone, may help to explain recurrent bouts of vaginal candidosis in women who have particularly high levels of circulating steroid and protein hormones during specific phases of their menstrual cycle. Further work with C. albicans demonstrating promotion of morphogenesis
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by steroid and protein hormones, specific uptake of oestradiol by viable yeast cells, specific fungal binders for mammalian hormones and hormonal stimulation of protein synthesis and other biochemical events (Bramley et al.. 1990b, 1991a,b; Frey et al., 1988; Loose et al., 1981; Othman et al., 1988; Powell et al., 1984; Skowronski and Feldman, 1989; Williams et al., 1990) appear to lend weight to the above claims. However, it should be borne in mind that Paveto et al. (1990) found that hLH and hCG inhibited the yeast-to-mycelium transition of C. albicans. A further, interesting observation made by Powell and Drutz (1983) was that the incidence of disease caused by C. tropicalis and C. pseudotropicalis was not raised during pregnancy; unlike C. albicans, neither species contains specific binding proteins for progesterone. The susceptibility of patients receiving glucocorticoid therapy to C. albicans infections prompted Loose et al. (1981) to investigate the interaction of this species with corticosteroids. They proposed that their demonstration of a specific fungal binder for corticosterone (the binder also interacted with progesterone; Table 4) and interaction of an apparent fungal endogenous ligand with mammalian glucocorticoid receptors had potentially important clinical consequences. However, in a subsequent study, Loose et nl. (1983a) found that although [3H]corticosterone entered intact yeast cells and specifically occupied CBPs, the hormone had no effect on cell growth, morphogenesis or glucose metabolism and there was no correlation between the amount or affinity of CBP from eight C. albicans strains and the virulence of these strains in murine hosts. Stover et al. (1983) found that the antifungal agent ketoconazole competitively displaced [3H]corticosterone from the CBP of C. albicans at concentrations readily achieved in therapeutic settings. However, binding of ketoconazole and related drugs to this protein was not critical for the in vitro antifungal activity of these agents.
4. Dermatophytosis Dermatophyte infections have been recorded more frequently in males than in females. However, the incidence of infection in females appears to rise after the menopause (Jones, 1983). The results of Schar et al. (1986) and Clemons et al. (1988) may help to explain these observations. As already noted, these authors demonstrated inhibition of growth of T. mentagrophytes, (+) and (-) mating types of A . benhamiae, T. rubrum and M . canis by progesterone. They also detected specific binders for progesterone in each of these species (Table 4) and proposed that the hormone exerts an inhibitory effect on dermatophyte growth in the female host following an interaction with a fungal receptor protein.
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It should be noted that, in studies with dermatophytes and other fungal pathogens, a precise concordance between the Kd value concentration of hormone required to elicit a growth response in vitro and the concentration of the hormone in host tissues is not always apparent. A number of explanations have been suggested for these anomalies (Loose et al., 1983b; Restrepo et al., 1984; Schar et al., 1986; Clemons et al., 1988) which lend further weight to the proposal that hormone-fungus interactions may have important consequences for pathogenesis. E. EVOLUTIONARY ASPECTS
Mammalian hormone-like molecules and/or receptor-effector systems have been detected in both prokaryotic and eukaryotic micro-organisms, higher plants, insects and molluscs (for reviews, see LeRoith et al., 1986; Rosen, 1987; Bramley et al., 1990a; Grover et al., 1991). These and other related observations led Roth et al. (1982) to propose that biochemical elements of the endocrine system may have originated in unicellular organisms. These authors envisaged that, while increasingly complex anatomical units such as glands, differentiated target cells and a circulatory system evolved, the basic biochemistry of intercellular communication was conserved. Similarly, Pertseva (1990) and Janssens (1987, 1988) proposed that the signal-transduction systems of vertebrates had their origins in prokaryotic or eukaryotic micro-organisms, respectively. Recent work summarized in this review, detailing responses of C. albicans, N . crassa, P. brasiliensis and Sacch. cerevisiae to mammalian steroid or peptide hormones at the molecular level, lends weight to these hypotheses. These studies augment the growing body of evidence for specific hormone-fungus interactions which may lead to growth responses in fungal species (Table 3). Taken together, the data suggest that fungal and mammalian cells interact with mammalian hormones in a strikingly similar manner. Although a number of mammalian hormone-fungus interactions have been characterized in great detail, it should be emphasized that there has been no clear demonstration of a functional role for related endogenous ligand-receptor-effector systems in fungi. Such endogenous ligand-receptor interactions may have important regulatory roles during vegetative growth. Furthermore, the examples of A . ambisexualis (see Section II.A.2) and Sacch. cerevisiae (see Section II.B.l) illustrate that both steroidal and nonsteroidal fungal sex hormones have important physiological roles during mating. Thus, an intriguing possibility proposed by Drutz and Huppert (1983) is that mammalian hormones may mimic the effects of, as yet, undiscovered mating hormones in members of the Fungi Imperfecti such as C. albicans and C. immitis.
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One bizarre example of a fungal interaction with a mammalian sex hormone is the identification by Claus et a f . (1981) of the steroid 5-a-androst16-en-3-a-ol as a metabolite of the black truffle, Tuber melanosporum. This steroid has a musk-like odour and is a major component of the pheromone of the boar. Claus et al. (1981) suggest that this explains the avidity with which pigs root up truffles from as deep as 1 m in the soil. Other parallels that can be drawn, but with no obvious evolutionary significance, are the similarity between sirenin and insect juvenile hormones such as juvabione, and between trisporic acid and the plant growth regulator abscisic acid, and retinoic acid, the vertebrate morphogen.
V. Signal Transduction Following Interaction of ala Mating Factors or Mammalian Hormones with Yeasts A.
a-
AND
a-MATING FACTORS
An identical intracellular response appears to be triggered in Sacch. cerevisiae of a- and a-mating type following interaction of each pheromone with its namesake receptor (for a review, see Marsh et al., 1991). Both aand a-factor receptors seem to be coupled to the same multisubunit Gprotein composed of three types of subunit designated a , p and y (Kurjan, 1990). This heterotrimeric protein resembles well-characterized mammalian heterotrimeric G-proteins (Kaziro et al., 1991) in that activation of the receptor is thought to be associated with replacement of GDP with GTP on the a-subunit of the G-protein and separation of the G, subunit from the Ge,, subunit. However, a significant difference between yeast and most mammalian G-proteins is that, in yeast, it is the free G,, subunit rather than the G, subunit that is responsible for activating downstream components of the signalling pathway (for a review, see Marsh and Herskowitz, 1987). Although a number of gene products have been identified that are required for further signalling, the immediate target for the liberated GO, subunit has not yet been determined. Furthermore, there is as yet no evidence for involvement of second messenger molecules (e.g. CAMP) which play an important role in mammalian signal-transduction pathways (Iyengar and Birnbaumer, 1990). However, studies of genes that lie downstream of the G-protein suggest that protein kinases (STE7, S T E l l and FUS3) and a transcription factor that is subject to phosphorylation (STE12) are essential components of the mating-factor pathway in Sacch. cerevisiae (for references, see Marsh et al., 1991). Ultimately, stimulation of transcription of several genes and inactivation of GI cyclins lead to a range of physiological responses, as detailed in Section II.B.l.
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B. MAMMALIAN HORMONES
In mammals, heterotrimeric G-proteins couple plasmalemma-bound hormone receptors to internal effector systems such as adenylate cyclase (Iyengar and Birnbaumer, 1990; Kaziro et a f . , 1991). The possibility that mammalian hormones like hLH may signal to adenylate cyclase in yeasts such as C. afbicans (see Section IV.C.2) is intriguing in that, to date, those G-proteins of Sacch. cerevisiae involved in regulation of adenylate cyclase have been shown to be members of the monomeric RAS family (for references, see Kurjan, 1990). These RAS proteins appear to be involved in the transduction of environmental signals to the catalytic subunit of yeast adenylate cyclase. However, in the yeast system, no receptor or detector molecule that may respond to the environment has been identified. Furthermore, no ligand which might bind to the receptor and activate the cyclase system has been discovered (Engelberg et al., 1989). Therefore, it is important that any relationship between those G-proteins which may transduce hormonal signals in species such as C. albicans and the RAS G-proteins of Sacch. cerevisiae should be investigated in detail. Such work may provide valuable information concerning the role of signal-transduction mechanisms in regulation of fungal growth and morphogenesis by environmental conditions. Furthermore, it may be possible to exploit the dimorphic nature of C. albicans in studies of ras oncogenes. Thus, it should prove of interest to substitute normal and mutant mammalian ras genes for RAS genes from C. albicans, and so establish the effects of mutations in ras on adenylate cyclase activity and morphogenesis in this yeast. Interestingly, in this regard, we have recently identified homologues of RAS genes of Sacch. cerevisiae in C. albicans (D. J. Adams, S. Dutton, T. Ahmed and R. M. Walmsley, unpublished observation).
VI. Conclusions
It is clear that many parallels may be drawn between mating systems of fungi and well-characterized peptide/protein-receptor and steroid-receptor systems of mammalian endocrinology. Thus, a number of fungal messenger molecules and their receptors, and signal transduction mechanisms and other biochemical processes which mediate responses of fungal cells to endogenous hormones, bear close resemblance to their counterparts in mammalian cells. In addition, data summarized in Tables 3 and 4 demonstrate that a considerable number of fungal species interact with a broad range of mammalian hormones in a highly specific manner. Although some of these interactions may have important implications for the pathogenicity
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of certain fungi, their significance with regard to routine biochemistry and physiology of the fungal cell remains obscure. Further research in this area should prove valuable for a number of reasons. For example, investigations of fungus-mammalian hormone interactions may provide clues concerning the role of endogenous receptors and ligands in regulation of vegetative growth and/or mating in fungi. Furthermore, the strikingly similar responses noted following interaction of fungal and mammalian cells with mammalian hormones may be exploited in studies designed to characterize more fully biochemical mechanisms mediating responses of cells to hormones such as insulin. Thus, work with a genetically tractable organism such as N . crussu may permit novel investigations of the events induced following binding of insulin to its receptor.
VII. Acknowledgements We thank the authors who have supplied reprints and preprints, Richard Egel and Misuzu Baba for photographs, and Barry Causier for Figs 11-13. REFERENCES Abe, K., Kusaka, I . and Fukui, S. (1975). Journal of Bacteriology 122, 710. Achstetter, T. (1989). Molecular and Cellular Biology 9, 4507. Achstetter, T. and Wolf, D . H. (1985). EMBO Journal 4, 173. Akada, R., Minomi, K., Yamashita, I., Miyakawa, T. and Fukui, S. (1987). Agricultural and Biological Chemistry 51, 121 1 . Akada, R . , Minomi, K., Kai, J., Yarnashita, I., Miyakawa, T. and Fukui, S . (1989a). Molecular and Cell Biology 9, 3491. Akada, R., Kai, J., Yamashita, I., Miyakawa, T. and Fukui, S. (1989b). Archives of Microbiology 152, 484. Anderegg, R. J., Betz, R., Carr, S. A , , Crabb, J . W. and Duntze, IN. (1988). Journal of Biological Chemistry 34, 18236. Appeltauer, U. and Achstetter, T . (1989). European Journal of Biochemistry 181, 243. Arsenault, G. P., Biemann, K., Barksdale, A. W. and MeMorris, T. C. (1968). Journal of the American Chemical Society. 90, 5635. Baba, M., Baba, N., Ohsurni, Y., Kanaya, K. and Osumi, M. (1989). Journal of CeNScience 94, 207. Baffi, R. A., Shenbagamurthi, P . , Terrance, K., Becker, J . M., Naider, F. and Lipke, P. N. (1984). Journal of Bacteriology 158, 1152. Baffi, R. A , , Becker, J. M., Lipke, P. N. and Naider, F. (1985). Biochemistry 24, 3332. Banbury, G. H. (1955). Journal of Experimental Botany 6, 235. Bandoni, R. J . (1965). Canadian Journal of Botany 43, 627. Barksdale, A. W. (1963). Mycologia 55, 627. Barksdale, A. W. (1967). Annals of the New York Academy of Sciences 144, 313. Barksdale, A. W. and Lasure, L. L. (1974). Applied Microbiology 28, 544. Barksdale, A. W.,McMorris,T. C., Seshadri, R., Aranachalam,T.,Edwards, J. A., Sundeen, J. and Green, J . M. (1974). Journal of General Microbiology 82, 295. Barrow, S. E. and MeMorris, T. C. (1982). Lipids 17, 383. Beato, M. (1989). Cell 56, 335.
SEX HORMONPS AND FUNGI
135
Becker, J. M., Marcus, S . , Kundu, B . , Shenbagamurthi, P. and Naider, F. (1987). Molecular and Cellular Biology 7, 4122. Betz, R , and Duntze, W. (1979). European Journal of Bacteriology 95, 469. Betz, R . , MacKay, V. L. and Duntze, W. (1977). Journal of Bacteriology 132, 462. Betz, R . , Crabb, J. W., Meyer, H. E., Wittig, R. andDuntze, W. (1987). Journalo~Biological Chemistry 262, 546. Bhalerao, U. T., Plattner, J . J . and Rapoport, H . (1970). Journal of the American Chemical Society 92, 3429. Bistis, G . N. (1981). Mycologia 73. 959. Bistis, G . N. (1983). Experimental Mycology 7 , 292. Bistis, G . N. and Georgopoulos, S. G. (1979). Mycologia 71, 127. Bistis, G . N. and Olive, L. S . (1968). American Journal of Botany 55, 629. Blakeslee, A . F. and Cartledge, J . L. (1927). Botanical Gazette 84, 51. Boctor, A . M., Band, P. and Grossman, A . (1981). Proceedings of the National Academy of Sciences of the United States of America 78, 5648. Brake, A. J., Brenner, C., Najarian, R., Laybourn, P. and Merryweather, J. (1985). In “Current Communications in Molecular Biology” (M. J. Gething, ed.), pp. 103-108. Cold Spring Harbor Laboratory, New York. Bramley, T. A , , Menzies, G . S . and Jowett, T. (1990a). Molecular and Cellular Endocrinology 73, 93. Bramley, T. A., Menzies, G . S . , Williams, R. J . , Adams, D. J. and Kinsman. 0. S. (1990b). Biochemical and Biophysical Research Communications 167, 1050. Bramley, T. A , , Menzics, G. S . , Williams, R . J., Kinsman. 0. S . and Adams, D. J . (1991a). Journal of Endocrinology 128, 139. Bramley, T. A , , Menzies, G. S . , Williams, R . J., Kinsman, 0. S . and Adams, D. J. ( 199Ib). Journal of Endocrinology 130, 177. Brasier, C . M. (1975). New Phytologist 74, 183. Brunt, S . A . and Silver, J. C. (1986a). Experimental Cell Research 163, 22. Brunt, S. A . and Silver, J . C. (1986b). Experimental Cell Research 165, 306. Brunt, S . A . and Silver, J. C. (1987). Experimental Mycology 11, 65. Brunt, S . A. and Silver, J. C . (1991). Current Genetics 19, 383. Brunt, S. A , , Riehl, R. and Silver, J. C. (1990). Molecular and Cellular Biology 10, 273. Bucking-Throm, E., Duntze, W., Hartwell, L. H , and Manney, T. R. (1973). Experimental Cell Research 76, 99. Buetow, D . E. and Levedahl, B. H. (1964). Annual Review of Microbiology 18, 167. Bulawa, C. E. and Osmond, B. C. (1990). Proceedings of the National Academy of Sciences of the United States of America 87, 7324. Bulawa, C. E., Slater, E., Cabib, J., Au-Young, A , , Sburlati, A . , Adair, W. L. and Robbins, P. w. (1986). Cell 46, 213. Bu’lock, J. D . (1975). In “Proceedings of the Second International Symposium on Genetics of Industrial Microorganisms” (K. D. MacDonald, ed.), pp. 497-509. Academic Press, London. Bu’lock, J . D. (1976). In “The Filamentous Fungi”, (J. E. Smith and D. R. Berry, eds), vol. 2, pp. 345-368. Edward Arnold, London. Bu’lock, J . D. and Osagie, A. U. (1973). Journal of General Microbiology 76, 77. Bu’lock, J. D., Jones, B. E. and Winskill, N. (1976). Pure and Applied Chemistry 47, 191. Burgeff, H. (1924). Botanische Abhandlungen 4, I . Burkholdcr, A. C. and Hartwell, L. H. (1985). Nucleic Acids Research 13, 8463. Burshell, A , , Stathis, P. A . , Do, Y., Miller, S . C. and Feldman, D. (1984). Journal of Biological Chemistry 259, 3450. Bussey, H . (1988). Yeast 4 , 17. Capek, A . and Simek, A. (1971). Folia Microbiologica 16, 299. Caplan, S., Green, R . , Rocco, J . and Kurjan, J . (1991). Journal of Bacteriology 173, 627. Carlile, M. J. and Machlis, L. (1965a). American Journal of Botany 52, 478. Carlile, M. J. and Machlis, L. (1965b). American Journal of Botany 52, 484. Casas-Campillo, C., Balandrano, D. and Galarza, A. (1961). Journal of Bacteriology 81. 366.
136
G , W . GOODAY AND D. I. ADAMS
Casperson, G . F., Walker, N., Brasier, A. and Bourne, H . R . (1983). Journal of Biological Chemistry 258, 791 1 . Cejek, E . and Thorncr, J. (1979). Cell 18, 623. Champe, S. P . , Rao, P. and Chang, A. (1987). Journal of General Microbiology 133, 1383. Champe, S. P. and El-Zayat, A. A. E . (1989). Journal of Bacteriology 171, 3982. Chang, H. S., Shu, I. M. and KO, W. H . (1984). Annuls of the Phytopathological Society of Japan 50, 27. Chattaway, F. W . , Townsley, J . D . and Barlow, A . J. E. (1959). Nature 184, 1731. Chattaway, F. W., Townsley, J . D. and Barlow, A . J. E. (1963). Journal of General Microbiology 30, 261. Chvatchko, Y . , Howald, I. and Riezman, H. (1086). Cell 46, 355. Clark, K. L., Davis, N. G . , Niest, D. K., Hwan-Shum, J. J . and Sprague, G. F. (1988). Cold Spring Harbor Symposium of Quantitative Biology 53, 61 I . Claus, R., Hoppen. H. 0. and Karg, H. (1982). Experientia 37, 1178. Clemons, K. V . , Schar, G., Stover, E. P., Feldman, D . and Stevens, D. A. (1988). Journal of Clinical Microbiology 26, 21 10. Clemons, K. V., Stover, E. P . , Schar, G., Stathis, P. A., Chan, K., Tokes, L., Stevens, D . A. and Feldman, D . (1989a). Journal of Biological Chemistry 264, 11186. Clemons, K. V., Feldman, D . and Stevens, 6. A. (1589b). Journai of General Microbiology 135, 1607. Cooper-Palomar, J . L. and Powell, M. J. (1988). Mycologia 80, 783. Cross, F., Hartwell, L. H., Jackson, C. and Konopka, J. B. (1988). Annual Review of Cell Biology 4 , 429. Davey, J . (1991). Yeust 7 , 357. Dietzel, C. and Kurjan, J. (1987). Molecular and Cellular Biology 7, 4169. Dmochowska, A , , Dignard, D., Henning, D . , Thomas, D . Y. and Bussey, H. (1987). Cell 50, 573. Drinkard, L. C., Nelson, G . E. and Sutter, R. P. (1982). Experimental Mycology 6, 52. Driver, C. H. and Wheeler, H. E. (1955). Mvcologia 47, 311. Drutz, D . J . and Catanzaro, A . (1978). American Review of Respiratory Disease 117,559, 727. Drutz, D. J . and Huppert, M. (1983). Journal of Infectious Diseases 147, 372. Drutz, D . J., Huppert. M., Sun, S. H . and McGuire, W. L. (1981). Infection and Immunity 32, 897. Dulic, V. and Riezman, H. (1990). Journal of Cell Biology 97, 517. Dulic, V., Egerton, M., Elguindi, I., Raths, S., Singer, B. and Riezman, H. (1991). Methods in Enzymology 194, 695. Duntze, W., MacKay, V. and Manney, T. R. (1970). Science 168, 1472. Edwards, J. A , , Mills, J . S., Sundeen, J. and Fried, J . N. (1969). Journal of the American Chemical Society 91, 1248. Edwards, J. A , , Sundeen, J., Salmond, W., Iwadare, T. and Fried, J. H. (1972). Tetrahedron Letters 791. Egel, R., Neilson, 0. and Weilguny, D . (1990). Trend.s in Genetics 6, 369. Elliott, C . G . (1972). Journal of General Microbiology 72, 321. Elliott, C. G . (1979). Journal of General Microbiology 115, 117. Elliott, C. G. (1983). In “Phytophthora: Its Biology, Taxonomy, Ecology and Pathology” (D. C. Erwin, S. Bartnicki-Garcia and P. H. Tsao, eds), pp. 71-80. American Phytopathological Society, St. Paul. Elliott, C. G. and Glen, B. (1Y82). Journal of General Microbiology 128, 859. Emter, O., Mechler, B., Achstetter, T., Muller, H. and Wolf, D. U. (1983). Biochemical and Biophysical Research Communications 116, 822. Engelberg, D., Perlman, R. and Levitzki, A . (1989). Cellular Signalling 1, I . Fawell, S . E . and Lenard, J . (1988). Biochemical and Biophysical Research Communications 155, 59. Fawell, S. E., McKenzie, M. A , , Greenfield, N. J., Adebodun, F., Jordan, F. and Lenard, J. (1988). Endocrinology 122, 518. Feldman, D., D o , Y . , Burshell, A . , Stathis, P. and Loose, D. S. (1982). Science 218, 297.
SEX HORMONES AND FUNGI
137
Feldman, D., Stathis, P. A., Hirst, M. A., Stover, E. P., Do, Y. S. and Kurz, W. (1984a). Science 224, 1109. Feldman, D., Tokes, L. G . , Stathis, P, A , , Miller, S. C . , Kurz, W. and Harvey, D. (1984b). Proceedings of the National Academy of Sciences of the United States of America 81, 4722. Fields, S . (1990). Trends in Biochemical Sciences 15, 270. Flawia, M. M. and Torres, H. N. (1972). Proceedings of the National Academy of Sciences of the United States of America 69, 2870. Flawia, M. M. and Torres, H. N. (1973a). Journal of Biological Chemistry 248, 4517. Flawia, M. M. and Torres, H. N. (1973b). FEBS Letters 30, 74. Flegel, T. W. (1981). Canadian Journal of Microbiology 27, 373. Ford, S . and Pringle, J. (1986). Yeast 2, S114. Frey, C. L., McDonnell, D. P., Butt, T. R. and Drutz, D. J . (1988). 2Kth Interscience Conference on Antimicrobial Agents and Chemotherapy, Los Angeles, Abstract No. 60. Friedmann, K. L. and Egel, R. (1978). Zeitschrift fiir Nuturforschung 33c, 84. Fujimura, H. and Yanagishima, N . (1983). Archives of Microbiology 136, 79. Fujimura, H., Yanagishima, N., Sakurai, A , , Kitada, C., Fujino, M. and Banno, I. (1982). Archives of Microbiology 132, 225. Fujino, M., Kitadi, C., Sakagami, Y., Isogai, A., Taniura, S. and Suzuki, A. (1980). Naturwissenschaften 67, S. 406. Fukui, Y., Kaziro, Y. and Yamamoto, M. (1986). EMBO Journal 5 , 1991. Fuller, R., Brake, A , , Sterne, R., Kunisawa, R. and Barnes, D. (1986). In “Yeast Cell Biology” (J. Hicks, ed.), pp. 461476. Alan R . Liss, New York. Fuller, R. S., Brake, A. J. and Thorner, J. (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 1434. Gall, A. M. and Elliott, C. G. (1985). Transactions ofthe British Mycological Society 84,629. Gooday, G . W. (1968). New Phytologist 67, 815. Gooday, G . W. (1973). Symposium of the Society for General Microbiology 23, 269. Gooday, G . W. (1974). Anniiul Review of Biochemistry 43, 35. Gooday, G. W. (1975). In “Primitive Sensory and Communication Systems” (M. J . Carlile, ed.), pp. 155-204. Academic Press, London. Gooday, G. W. (1978). Philosophical Transactions of the Royal Society of London B 284,509. Gooday, G . W. (1983). In “Secondary Metabolism and Differentiation in Fungi” (J. W. Bennet and A. Ciegler, eds), pp. 239-266. Marccl Dekker, New York. Gooday, G. W. (1992). In “Perspectives in Plant Cell Recognition” (J. Callow and J. Green, eds), pp. 33-58. Cambridge University Press. Cambridge. Gooday, G . W., Jones, B. E. and Leith, W. H . (1979). In “Regulation of Secondary Product and Plant Hormone Metabolism (M. Luckner and K. Schreibcr, eds), pp. 221-230. Pergamon Press, Oxford. Goodey, A. R., Doel, S., Piggott, J . R., Watson, H. E. E. and Carter, B. L. A. (1988). In “Yeast Biotechnology” (D. R. Berry, I. Russell and G. G . Stewart, eds), pp. 401429. Allen and Unwin, London. Goodman, L. E., Peron, C. M., Fujiyama, A. and Tamanoi, F. (1988). Yeast 4, 271. Govind, N. S. and Ccrda-Olmedo, E. (1986). Journal of General Microbiology 132, 2775. Gow, N. A. R. (1989). Advances in Microbial Physiology 30, 89. Gow, N. A. R. and Gooday, G. W. (1987). Journal of General Microbiology 133, 3531. Green, D. M., Edwards, J. A , , Barksdale, A. W. and McMorris, T. C. (1971). Tetrahedron 27, 1199. Greenfield, N. J., MeKenzie, M. A , , Adebodun, F.. Jordan, F. and Lenard, J . (1988). Biochemistry 27, 8526. Greenfield, N . J., Cherapak, C. N., Adebodun, F., Jordan, F. and Lenard, J. (1990). Biochimica et Biophysica Acta 1025, 15. Groner, B., Hynes, N., Sippel, A. E. and Schutz, G. (1976). Nature 261, 599. Grossman, A , , Klein, H., Becker, J. M. andNaider, F. (1986). JournalofSteroid Biochemistry 25, 299. Grover, S . , McGee, Z . A. and Odell, W. D. (1991). Endocrinology 128, 3096. Guo, L. Y. and KO, W. H. (1991). Mycological Research 95, 452.
138
G W GOODAY AND D J. ADAMS
Gwynne, D. I. and Brandhorst, B. P. (1980). Experimental Mycology 4 251. Hagen, D. C., McCaffrey, G . and Spragne, G. F.(1986). Proceedingsofthe National Academy of Sciences of the United States of America 83, 1418. Hanifa Moursi. S. A. and Refai. M. (1968’1. Mvkosen 11. 747. . Harding, K. E.: Strickland, J. B. andPommerville, J . (1988). TheJournalof Organic Chemistry 53, 4877. Harris, R. E. (1966). Obstetrics and Gynecology 28, 401. Hartig, A, , Holly, J., Saari, G . and MacKay, V. L. (1986). Molecular and Cellular Biology 6, 2106. Harvey, R. P. (1980). “Coccidioidomycosis. A Text”, pp. 241-244. Plenum Medical, New York. Hasek, J., Ropes, I., Svobodova, J. andStreiblova, E. (1987). Journalof GeneralMicrobiology 133, 3355. Hauser, K. and Tanner, W. (1989). FEBS Ixtters 255, 290. Hauser, K., Cappelaro, C., Mrsa, V. and Tanner. W. (1990). Proceedings of the 4th International Mycological Congress, Regensburg, ID- 188/1. Hendrix, J. W. and Guttman, S . M. (1972). Phytopathology 62, 763. Herman, R. P., Luchini, M. M. and Herman, C. A. (1989). Experimental Mycology 13, 372. Herskowitz, I. (1988). Microbiological Reviews 52, 536. Hiraga, K., Tahara, H., Taguchi, N., Tsuchiya, E., Fukui, S. and Miyakawa, T. (1991). Journal of General Microbiology 137, 1. Hirata, A., Tsuchiya, E., Fukui, S. and Tanaka, K. (1980a). Archives of Microbiology 128, 215. Hirata, A., Tsuchiya, E., Fukui, S. and Tanaka, K. (1980b). Journal of General and Applied Microbiology 26, 429. Horgen, P. A , , (1981). In “Sexual Interactions in Eukaryotic Microbes” (D. H. O’Day and P. A. Horgen, eds), pp. 155-178. Academic Press, New York. Horgen,P. A , , Smith, R., Silver, J. C. andCraig, G. (1975). CanadianJournalofBiochemisfry 53, 1341. Horgen, P. A., Iwanochko, M. and Bcttiol, M. F. (1983). Archives of Microbiology 134,314. Horgen, P. A,, Nakagawa, C. H. and Irvin, R. T. (1984). Canadian Journal of Biochemistry and Cell Biology 62, 715. Horten, J . S. and Horgen, P. A . (1985). Canadian Journal of Biochemistry and Cell Biology 63, 355. Horten, J. S . and Horgen, P. A. (1989). Experimental Mycology 13, 263. Hudnik-Plevnik, T. and Cresnar, B. (1990). Journal of Steroid Biochemistry 35, 749. Hunt, L. T. and Dayoff, M. D. (1979). In “Peptides: Structure and Biological Function” (E. Gross and J. Meienhofer, eds), pp. 757-760. Pierce Chemical Co., Rockford, TI. Hunzicker-Dunn, M. and Birnbaumer. L. (1985). In “Luteinizing Hormone Action and Receptors” (M. Ascoli, ed.), pp. 57-134. CRC Press, Boca Raton. Huxley, J. S . (1935). Biological Reviews 10, 427. Ilott, T. W., Ingram, D. S. and Ralinson, C. J. (1986). Transactionsofthe British Mycological Society 87, 303. Inaba, T. and Mirocha, C. J. (1979). Applied and Environmental Microbiology 37, 80. Ishibashi, Y., Sakagami, Y., Isogai, A . , Suzuki, A. and Baudoni, R. J. (1983). Canadian Journal of Biochemistry and Cell Biology 61, 796. Ishibashi, Y., Sakagami, Y . , Isogai, A. and Suzuki, A. (1984). Biochemistry 23, 1399. Islam, M. S . (1981). In “Sexual Interactions in Eukaryotic Microbes” (D. H . O’Day and P. A. Horgen, eds), pp. 131-154. Academic Press, London. Iyengar, R. and Birnbaumer, L. (1990). “G proteins”. Academic Press, New York. Jackson, C. L. and Hartwell, L. H. (1990a). Molecular and Cellular Biology 10, 2202. Jackson, C . L. and Hartwell, L. H. (1990b). Cell 63, 1039. Janssens, P. M. W. (1987). Trends in Biochemical Sciences 12, 456. Janssens, P. M. W. (1988). Comparative Biochemistry and Physiology 90A, 209. Jenness, D. D. and Spatrik, P. (1986). Cell 46, 345. Jenness, D. D., Burkholder, A. C. and Hartwell, L. H. (1983). Cell 35, 521. I
,
SEX HORMONES AND FUNGI
139
Jenness, D. D., Burkholder, A. C. and Hartwell, L. H. (1986). Molecular and Cellular Biology 6, 318. Jones, B. E. and Gooday, G. W. (1977). Biochemical Society Transactions 5, 717. Jones, B. E. and Gooday, G. W. (1978). FEMS Microbiology Letters 4, 181. Jones, B. E., Williamson, I. P. and Gooday, G . W. (1981). In “Sexual Interactions in Eukaryotic Microbes” (D. H. O’Day and P. A. Horgen, eds), pp. 179-198, Academic Press, New York. Jones, H. E. (1983). In “Infectious Diseases” (P. D. Hoeprich, ed.), 3rd edn, pp. 966-978. Harper and Row, Philadelphia. Julius, D., Blair, L., Brake, A , , Sprague, G . and Thorner, J. (1983). Cell 32, 839. Julius, D., Brake, A , , Blair, L., Kunisawa, R. and Thorner, J . (1984a). Cell 37, 1075. Julius, D., Schekman, R. and Thorner, J . (1984b). Cell 36, 309. Kamiya, Y., Sakurai, S . , Tamura, S . , Takashi, N., Abe, K., Tsuchiya, E., Kitada, C. and Fujino, M. (1978). Biochemical and Biophysical Research Communications 83, 1077. Katzenellenbogen, B. S., Katzenellenbogen, J. A. and Mordecai, D. (1979). Endocrinology 105, 33. Kawai, G . and Ikeda, Y. (1982). Biochimica et Biophysica Acta 719, 612. Kaziro, Y., Itoh, H., Kozasa, T., Nakafuku, M. and Satoh, T. (1991). Annual Review of Biochemistry 60, 349. Kerwin, J . L. and Washino, R. K. (1983). Experimental Mycology 7, 109. King, K., Dohlman, H. G., Thorner, J., Caron, M. G . and Lefkowitz, R . J. (1990). Science 250, 121. Kinsman, 0. S . , Pitblado, K. and Coulson, C. J. (1988). Mycoses 31, 617. Kitada, C., Fujino, M., Tsuchiya, E . , Abe, K., Fukui, S . , Kamiya, Y . , Sakurai, A . , Tamwa, S . , Takashi, N., Sakagami, Y., Isogai, A . and Suzuki, A. (1979). In “Peptide Chemistry 1978” (N. Izamiya, ed.), pp. 155-200. Protein Research Association, Osaka. Kitada, C., Fujino, M., Sakagami, Y., Yoshida, M., Isogai, A. and Suzuki, A. (1981). Agricultural and Biological Chemistry 45, 1049. Klapper, B. F. and Klapper, M. H. (1977). Experimental Mycology 1, 352. KO, W. H. (1983). Journal of General Microbiology 129, 1397. KO, W. H. (1985). Journal of General Microbiology 131, 2591. KO, W. H. (1988). Annual Review of Phytopathology 26, 57. KO, W. H. , Lee, C. J. and Su, H. J. (1986). Mycologia 78, 134. Kole, H. K. and Lenard, J. (1990). Journal of Cell Biology 111, 472a. Kole, H. K., Muthukumar, G. and Lenard, J . (1991). Biochemistry 30, 682. Kronstad, J. W., Holly, J. A. and MacKay, V. L. (1987). Cell 50, 369. Kuchler, K., Sterne, R. E. and Thorner, J . (1989). EMBO Journal 8, 3973. Kurjan, J. (1985). Molecular and Cellular Biology 5, 787. Kurjan, J . (1990). In “G proteins” (R. Iyengar and L. Birnbaumer, eds), pp. 571-599. Academic Press, New York. Kurjan, J . and Herskowitz, I. (1982). Cell 30, 933. LeRoith, D., Shiloach, J., Roth, J. and Lesniak, M. A. (1980). Proceedings of the National Academy of Sciences of the United States of America 77, 6184. LeRoith, D., Roberts, C., Lesniak, M. A. and Roth, J . (1986). Experientia 42, 782. Leupold, U. (1987). Current Genetics 12, 543. Leupold, U. (1991). Current Genetics 20, 75. Leupold, U., Nielsen, 0. and Egel, R. (1989a). Current Genetics 15, 403. Leupold, U . , Nielsen, 0. and Egel, R . (3989b). Current Genetics 16, 465. Leupold, U., Sipiczki, M. and Egel, R. (1991). Current Genetics 20, 79. Levi, J. D. (1956). Nature 177, 753. Levitzki, A. (1987). Trends in Pharmacological Sciences 8, 299. Levitzki, A. (1988). Trends in Biochemical Sciences 13, 300. Liao, H. and Thorner, J . (1980). Proceedings of the National Academy of Sciences of the United States of America 77, 1898. Lipke, P. N. (1986). Journal of Bacteriology 168, 1472. Lipke, P. N., Taylor, A. and Ballou, C. E. (1976). Journal of Bacteriology 127, 610.
140
G. W. GOODAY AND D. J. A D A M S
Lipke, P. N., Wojciechowecz, D. and Kurjan, J. (1989). Molecular and Cellular Biology 9,3155. Liu, Y., Ohki, O., Azuma, Y., Tachikawa, T., Tsuchiya, E . , Fukui, S. and Miyakawa, T. (1990). Journal of General Microbiology 136, 131. Loose, D. S . and Feldman, D. (1982). Journal of Biological Chemistry 257, 4925. Loose, D. S., Schurman, D. J . and Feldman, D. (1981). Nature 293, 477. Loose, D. S . , Stevens, D. A , , Schurman, D. J. and Feldman, D. (1983a). Journal of General Microbiology 129, 2379. Loose, D. S., Stover, E. P . , Restrepo, A , , Stevens, D. A. and Feldman, D. (1983b). Proceedings of the National Academy of Sciences of the United States of America 80, 7659. Loumaye, E., Thorner, J. and Catt, K. J. (1982). Science 218, 1323. Machlis, L. (1958). Physiologia Planfarum 11, 845. Machlis, L. (1972). Mycologia 64, 235. Machlis, L. (1973a). Plant Physiology 52, 524. Machlis, L. (1973b). Plant Physiology 52, 527. Machlis, L., Nutting, W. H . , William, M. W. and Rapoport, H. (1966). Biochemistry 5,2147. Machlis, L., Nutting, W. H . and Rapoport, H. (1968). Journal of the American Chemical Society 90, 1674. McCaffrey, G., Clay, F. J., Kelsey, K. and Sprague, G. F. (1987). Molecular and Cellular Biology 7, 2680. McCullough, J. and Herskowitz, I. (1979). Journal of Bacteriology 138, 146. McGrath, J. P. and Varshavsky, A. (1989). Nature 340, 400. MacKay, V. L., Welch, S . K., Tnsley, M. Y . , Manney, T. R., Holly, J., Saari, G. C. and Parker, M. L. (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 55. McKenzie, M. A., Fawell, S . E., Cha, M. and Lenard, J . (1988). Endocrinology 122, 511. McMorris, T. C. (1978). Philosophical Transactions ofthe Royal Society of London B 284,459. McMorris, T. C. (1989). In “Analysis of Sterols and other Biologically Significant Steroids” (D. Nes and E . Parish, eds). pp. 61-79. Academic Press. New York. McMorris, T. C. and Barksdale, A. W. (1967). Nature 215, 320. McMorris, T. C . and White, R . M. (1977). Phytochemistry 16, 359. McMoms, T. C., Seshadri, R. and Aranachalam, T. (1974). Journal oforganic Chemistry 36,669. McMorris, T. C., Seshadri, R., Weihe, G. R., Arsenault, G. P. and Barksdale, A. W. (1975). Journal of the American Chemical Society 97, 2544. McMorris, T. C . , Lc, P. H., Preus, M. W., Schow, S . R. and Weihe, G. R . (1983). Journal of Organic Chemistry 48, 3370. McMorris, T. C., Le, P. H., Preus, M. W . , Schow, S . R . and Weiche, G. R . (1989). Steroids 53, 345. Marcus, S . , Caldwell, G. A , , Xue, C . B., Naider, F. and Becker, J . M. (1990). Biochemical and Biophysical Research Communications 172, 1310. Marcus, S . , Xue, C. B., Naider, F. and Bcckcr, J. M. (1991). Molecular and Cellular Biology 11, 1030. Margulis, L. and Sagan, D. (1987). “Microcosmos. Four Billion Years of Microbial Evolution”. Allen and Unwin, London. Marsh, L. (1991). In “Sensory Receptors and Signal Transduction” (J. Spudich and B. Satir, eds), pp. 203-225. Wilcy-Liss, New York. Marsh, L. and Herskowitz, I. (1987). Cell 50, 995. Marsh, L. and Herskowitz, I. (1988a). Cold Spring Harbor Symposium of Quantitative Biology 53, 557. Marsh, L. and Herskowitz, I. (1988b). Proceedings of the National Academy of Sciences of the United States of America 85, 3855. Marsh, L., Neiman, A. M. and Herskowitz, I. (1991). Annual Review of Cell Biology 7, 299. Mazur, P., Meyers, H . V., Nakanishi, K., El-Zayat, A. A. E. and Champe, S. P. (1990). Tetrahedron Letters 31, 3837. Mesland, D. A. M., Huisman, J. G. and van den Ende, H . (1974). Journal of General Microbiology 80, 111. Metzger, D., White, J. H . and Chambon, P. (1988). Nature 334, 31.
SEX HORMONES AND FUNGI
141
Meyer, M. D., Carlson, G. L., Toft, D. O., Greaves, A. M., Ng, K. M. E. and MeMorris, T. C. (1987). Journal of Labelled Compounds and Radiopharmacology 24, 171. Michaelis, S. and Herskowitz, I. (1988). Molecular and Cellular Biology 8, 1309. Michalski, C. J. (1978). Biochemical and Biophysical Research Communications 84, 417. Miller, M. L. and Sutter, R. P. (1984). Journal of Biological Chemistry 259, 6420. Miller, S. C., Bottema, C. D. K., Stathis, P. A , , Tokes, L. G . and Feldman, D. (1986). Endocrinology 119, 1362. Miyakawa, T., Okubo, Y . , Hatomo, T., Tsuchiya, E . , Yamashita, I . and Fukui, S. (1982a). Agricultural and Biological Chemistry 46, 2403. Miyakawa, T., Nishihara, M., Tsuchiya, E. and Fukui, S. (1982b). Journal of Bacteriology 151, 1184. Miyakawa, T., Maruyama, H . , Tsuchiya, E. and Fukui, S . (1982~).Agriculturaland Biological Chemistry 46, 2179. Miyakawa, T., Kadota, T., Okubo, Y . , Hataro, T., Tsuchiya, E. and Fukui, S. (1984). Journal of Bacteriology 158, 814. Miyakawa, T., Kaji, M., Yasataka, T., Jeong, Y . K., Tsuchiya, E. and Fukui, S. (1985a). Journal of Bacteriology 162, 294. Miyakawa, T., Tachikawa, T., Jeong, Y. K . , Tsuchiya, E. and Fukui, S. (1985b). Journal of Bacteriology 162, 1304. Miyakawa, T . , Tachikawa, T . , Akada, R., Tsuchiya, E. and Fukui, S. (1986). Journal of General Microbiology 132, 1453. Miyakawa, T., Azurna, Y., Tsuchiya, E. and Fukui, S. (1987a). Journal of General Microbiology 133, 439. Miyakawa, T., Kaji, M . , Jeong, Y . K., Tsuchiya, E. and Fukui, S. (1987b). Journal of Bacteriology 169, 1626. Miyakawa, T., Tachikawa, T . , Jeong, Y. K . , Tsuchiya, E. and Fukui, S. (1987~).Biochemical and Biophysical Research Communications 143, 893. Miyakawa, T . , Oka, Y., Tsuchiya, E. and Fukui, S . (1989). Journal ofBacteriology 171,1417. Mohr, J. A.,Tatem,B. A,, Long, H.,Muchmore,H. G. andFelton,F. G . (1973). Sabouraudia 11, 140. Moon, S. S., Stubmiller, L. M., Chadha, R. K. and MeMorris, T. C. (1990). Tetrahedron Letters 7, 2287. Moore, S. A. (1983). Journal of Biological Chemistry 258, 13849. Moore, S. A. (1984). Journal of Biological Chemistry 259, 1004. Moore, S. A. (1987). Experimental Cell Research 171, 411. Muchmore, H . G., McKown, B. A. and Mohr, J. A. (1972). “Proceedings of the First Pan Amcrican Symposium on Paracoccidioidomycosis” (Pan Amcrican Health Organization Scientific Publication No. 254), pp. 30(M04. Pan American Hcalth Organization, Washington DC. Mullins, J. T. (1973). Mycologia 65, 1007. Mullins, J. T. (1979). Tissue and Cell 11, 585. Mullins, J . T. and Ellis, E. A. (1974). Proceedings of the National Academy of Sciences of the United States of America 71, 1347. Mullins, J. T. and Warren, C . 0. (1975). American Journal of Botany 62, 770. Musgrave, A. and Nicuwenhuis, D. (1975). Archives of’ Microbiology 105, 313. Musgrave, A., Ero, L. and Schcffer, R. (1978). Acta Botanicu Neerlandica 27, 397. Naider, F. and Becker, J. M. (1986). CRC Critical Reviews in Biochemistry 21, 225. Nakayarna, N., Miyajima, A. and Arni, K. (1985). EMBO Journal 4, 2642. Nelson, R. R. (1971). In “Morphological and Biochemical Events in the Plant-Parasite Interaction” (S. Akai and S. Ouchi, eds), pp. 181-205. The Phytopathological Society of Japan, Tokyo. Nes, W. D . and Phu, H. L. (1988). Pesticide Biochemistry and Physiology 30, 87. Nes, W. D., Patterson, G. W. and Bean, G. A. (1980). Plant Physiology 66, 1008. Nolting, S . , Hagemeier, H. H . and Fcgeler, K . (1982). Mykosen 25, 36. Nutting, W. H., Rapoport, H . and Machlis, L. (1968). Journal of the American Chemical Society 90, 6434.
142
C . W. GOODAY AND D. J . ADAMS
O’Day, D. H. and Lewis, K. E. (1981). In “Sexual Interactions in Eukaryotic Microbes” (D. H . O’Day and P. A. Horgen, eds), pp. 122-199. Academic Press, New York. O’Day, D. H., Rama, R . A. and Lydan, M. A. (1987). Experientia 43, 619. Odds, F. C. (1988). “Candida and Candidosis”, 2nd edn. Baillikre Tindall, London. Ohsumi, Y. and Anraku, Y. (1985). Journal of Biological Chemistry 269, 10482. Orlean, P. (1987). Journal of Biological Chemistry 262, 5732. Orlean, P., Schwiager, H., Appeltauer, U., Haselbeck, A. and Tanner, W. (1984). European Journal of Biochemistry 140, 183. Orlean, P., Arnold, E. and Tanner, W. (1985). FEBS Letters 184, 313. Orlean, P . , Ammer, H., Watzele, M. and Tanner, W. (1986). Proceedings of the National Academy of Sciences of the United States of America 83, 6263. Othman, Y. H. S., Adams, D . J., Hitchcock, C. A. and Oakey, R. E. (1988). Biochemical Society Transactions 16, 789. Paveto, C., Egidy, G., Galvagno, M. A. and Passeron, S . (1990). Biochemical and Biophysical Research Communications 167, 1177. Paveto, C., Mallo, G . , Egidy, G . , Galvagno, M. A. and Passeron, S. (1991). Cell Biology International Reports 15, 169. Pekkala, D. and Silver, J . C. (1987). Experimental Cell Research 168, 325. Perlman, R., Eilam, Y., Padan, E., Simchen, G. and Levitzki, A . (1989). Cellular Signalling 1, 577. Pertseva, M. N. (1990). Journal of Evolutionary Biochemistry and Physiology 26, 384. Plasenicia, J. and Mirocha, C. J . (1990). Phytopathology 80, 1019. Plattner, J. J. and Rapoport, H. (1971). Journal of the American Chemical Society 93, 1758. Plattner, J. J., Bhalerao, U. T . and Rapoport, H. (1969). Journal of the American Chemical Society 91, 4933. Plempel, M. (1960). Planta 55, 254. Plempel, M. (1962). Planta 58, 509. Pommerville, J. (1977). Experimental Cell Research 109, 43. Pomrnerville, J. (1978). Experimental Cell Research 113, 161. Pommerville, J. (1981). In “Sexual Interactions in Eukaryotic Microbes” (D. H. O’Day and P. A. Horgen, eds), pp. 53-92. Academic Press, New York. Pommerville, J. (1982). Journal of Cell Science 53, 193. Pommerville, J . (1987). In “Zoosporic Fungi in Teaching and Research” (M. S. Fuller and A. Jaworski, eds), pp. 274-275. Southeastern, Athens, GA. Pommerville, J. and Fuller, M. S. (1976). Archives of Microbiology 109, 21. Pommerville, J . C. and Olson, L. W. (1987a). Journal of General Microbiology 133, 2573. Pommerville, J. and Olson, L. W. (1987b). Experimental Mycology 11, 245. Pommerville, J. C., Strickland, J. B., Romo, D . and Harding, K. E. (1988). Plant Physiology 88, 139. Popplestone, C. R. and Unrau, A. M. (1974). Canadian Journal of Chemistry 52, 462. Powell, B. L. and Drutz, D. J. (1983). Journal of Infectious Diseases 147, 359. Powell, B. L. and Drutz, D. J. (1984). Infection and Immunity 45, 784. Powell, B. L., Drutz, D. J., Huppert, M. and Sun, S. H. (1983). Infection and Immunity 40,478. Powell, B. L., Frey, C. L. and Drutz, D. J. (1984). Experimental Mycology 8, 304. Powers, S., Michaelis, S . , Broek, D., Santa Anna-A. S . , Field, J . , Herskowitz, I . and Wigler, M. (1986). Cell 47, 413. Preus, M. W. and McMorris, T. C. (1979).Journal ofthe American Chemical Society 101,3066. Raper, J. R. (1951). American Scientist 39, 110. Raper, J. R. (1967). In “Handbuch der Pflanzenphysiologie” (W. Ruhland, ed.), vol. 18, pp. 214-234. Springer, Berlin. Raper, J. T. and Haagen-Smit, A . G. (1942). Journal of Biological Chemistry 143, 311. Raths, S., Shenbagamurthi, P., Naider, F. and Becker, J . M. (1986). Journal of Bacteriology 168, 1468. Raths, S. K., Naider, F. and Becker, J. M. (1988). Journal of Biological Chemistry 263,17333. Reid, I. D. (1974). Canadian Journal of Botany 52, 521. Reid, I. D. and Bartnicki-Garcia, S. (1976). Journal of General Microbiology 96, 35.
SEX HORMONES AND FIJNGI
143
Restrepo, A. and Greer, D . L. (1983). In “The Occupational Mycoses” (A. F. DiSalvo, ed.), pp. 43-64. Lea and Febiger, Philadelphia. Restrepo, A., Salazar, M. E., Cano, L. E . , Stover, E. P., Feldman, D. and Stevens, D . A. (1984). Infection and Immunity 46, 346. Riehl, R. M. and Toft, D. 0. (1984). Journal of Biological Chemistry 259, 15324. Riehl, R. M. and Toft, D. 0. ( 1 9 t h ) . Journal ofsteroid Biochemistry 23, 483. Riehl, R. M. and Toft, D. 0. (1985b). In “Molecular Mechanism of Steroid Hormone Action” (U. K. Moudgil, ed.), pp. 733-753. Walter de Gruyter, Berlin. Riehl, R. M. and Toft, D . 0. (1986). In “The Receptors” (P. Conn, ed.), vol. 4, pp. 149-169. Academic Press, Orlando. Riehl, R . M., Toft, D . O., Meyer, M. D., Carlson, G. L. and McMorris, T. C. (1984). Experimental Cell Research 153, 544. Riehl, R. M., Sullivan, W. P., Vroman, B. T., Bauer, V. J . , Pearson, G. R. and Toft, D. 0. (1985). Biochemistry 24, 6586. Roncero, C . , Valdivieso, M. H . , Ribas, J. C. and Duran, A. (1988). Journal of Bacteriology 170, 1950. Rose, M. D., Price, B. R . and Fink, G. R . (1986). Molecular and Cellular Biology 6, 3490. Rosen, 0. M. (1987). Science 237, 1452. Roth, J . , LeRoith, D., Shiloach, J., Rosenzweig, J. L., Lesniak, M. A. and Havrankova, J. (1982). New England Journal of Medicine 306, 523. Rozek, C. E. and Timberlake, W. E. (1980). Experimental Mycology 4, 34. Rusmin, S. and Leonard, T. J. (1978). Plant Physiology 61, 538. Ryley, J. F. (1986). Journal of Medical and Veterinary Mycology 24, 5. Sakagami, Y., Isogai, A , , Suzuki, A , , Tamura, S., Kitada, C. and Fujino, M. (1979). Agricultural and Biological Chemistry 43, 2643. Sakagami, Y., Yoshida, M., Isogai, A. and Suzuki, A. (1981a). Science 212, 1525. Sakagami, Y., Yoshida, M., Isogai, A. and Suzuki, A. (1981b). Agricultural and Biological Chemistry 45, 1045. Sakata, K. and Rickards, R. W. (1980). In “23rd Symposium on Chemistry of Natural Productions”, pp. 165-172. Nagoya, Japan. Sakurai, A., Sakamoto, M., Tanaka, H., Esumi, Y., Takahashi, N., Fujimura, H., Yanagishima, N. and Banno, I. (1984). FERS 1,etters 166, 339. Sakurai, A., Tanaka, H . , Esumi, Y., Takahashi, N., Hisatomi, T., Yanagishima, N. and Banno, I. (1986). FEBS Letters 203, 285. Salazar, M. E., Restrepo, A . and Stevens, D. A . (1988). Infection and Immunity 56, 711. Schafer, W. R., Trueblood, C. E., Yang, C. C., Mayer, M. P., Rosenburg, S., Poulter, C. D., Kim, S. H . and Rine, J. (1990). Science 249, 1133. Schar, G., Stover, E. P., Clemons, K. V., Feldman, D . and Stevens, D. A. (1986). Infection and Immunity 52, 763. Schekman, R. and Brawley, V. (1979). Proceedings of the National Academy of Sciences of the United States of America 76, 645. Schipper, M. A. A. (1987). In “Evolutionary Biology of the Fungi” (C. Brasier, A. D . M. Rayner, and D. Moore, eds), pp. 261-269. Cambridge University Press, Cambridge. Shaw, J. A., Mol, P. C., Bowers, B., Silverman, S. J., Valdivieso, M. H., Duran, A. and Cabib, E. (1991). Journal of Cell Biology 114, 111. Shen, C. Y., Bower, L. A., Erwin, D. C. and Tsao, P. H . (1983). Canadian Journal of Botany 61, 1462. Shenbagamurthi, P., Kundu, B., Becker, J. M. and Naider, F. (1985a). International Journal of Peptide and Protein Research 25, 187. Shenbagamurthi, P., Kundu, B., Raths, S., Becker, J. M. and Naider, F. (1985b). Biochemistry 24, 7070. Sherwood, W. A. (1966). Mycologia 58, 215. Siddiq, A. A., Ingram, D. S., Johnstone, K., Friend, J. and Ashby, A. M. (1989). Aspects of Applied Biology 23, 417. Silver, J . C. and Horgen, P. A. (1974). Nature 249, 252. Singer, B. and Riezman, H . (1990). Journal of Cell Biology 110, 1911.
144
G W. GOODAY A N D 1) J ADAMS
Singh, A , , Chen, E. Y., Lugovoy, J. U. M., Chang, C. N., Hitzman, R . A. and Seeburg, P. H . (1983). Nucleic Acids Research 11, 4049. Skowronski, R. and Feldman, D. (1989). Endocrinology 124, 1965. Sobel, J. D . (1985). American Journal of Obstetrics and Gynecology 152, 924. Sprague, G. F. (1991). Methods in Enzymology 194, 77. Steden, M. and Duntze, W. (1990). FEMS Microbiology Letters 68, 229. Steden, M., Betz, R. and Duntze, W. (1989). Molecular and General Genetics 219, 439. Stevens, D. A. (1989). Journal of Medical and Veterinary Mycology 27, 133. Stotzler, D., Kiltz, H. H. and Duntze, W. (1976). European Journal of Biochemistry 69,397. Stover, E. P., Loose, D. S . , Stevens, D . A. and Feldman, D. (1983). Biochemical and Biophysical Research Communications 117, 43. Stover, E. P., Schar, G., Clemons, K. V., Stevens, D . A. and Feldman, D. (1986). Infection and Immunity 51, 199. Strazdis, J. R . and MacKay, V. L. (1983). Nature 305, 543. Sutter, R. P. (1977). In “Eukaryotic Microbes as Model Developmental Systems” (D. H. O’Day and P. A. Horgen, eds), pp. 251-272. Marcel Dekker, New York. Sutter, R. P. (1987). In “Phycomyces” (E. Cerda-Olmedo and E. D. Lipson, eds), pp. 317336. Cold Spring Harbor Laboratory, New York. Sutter, R. P. and Jelinek, B. G. (1983). Experimental Mycology 7, 188. Sutter, R. P. and Whitaker, J. P. (1981a). Journal of Biological Chemistry 256, 2334. Sutter, R. P. and Whitaker, J. P. (1981b). Naturwissenschaften 68, 147. Sutter, R . P. and Zawodny, P. D. (1984). Experimental Mycology 8 , 89. Sutter, R. P., Capage, D. A., Harrison, T. L. and Keen, W. A . (1973). Journalof Bacteriology 114, 1074. Sutter, R . P., Dadok, J., Bothner-By, A. A , , Smith, R. R. and Mishra, P. K. (1989). Biochemistry 28, 4060. Tachikawa. T., Miyakawa, T., Tsuchiya, E . and Fukui, S. (1987). Agricultural and Biological Chemistry 51, 1209. Tanaka, S . , Hasegawa, S . , Hishinuma, F. and Kurata, S . (1989). Cell 57, 675. Terrance, K. and Lipke, P. N. (1087). Journal of Bacteriology 169, 4811. Timberlake, W. E. (1976). Developmental Biology 51, 202. Timberlake, W. E. and Orr, W. C. (1984). In “Biological Regulation and Development. Hormone Action” (R. F. Goldberger and K. R. Yamamoto, eds), vol3B, 255-283. Plenum Press, New York. Thomas, D. D. S . and Mullins, J. T. (1967). Science 156, 84. Thomas, D. S . (1970). Canadian Journal of Botany 48, 977. Thomas, D. S . and McMorris, T. C. (1987). Journal of Chemical Ecology 13, 1131. Thomas, D. S . and Mullins, J. T. (1969). Physiologia Plantarum 22, 347. Thorner, J . (1981). In “The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance” (J. N. Strathern, E. W. Jones and J. R. Broach, eds), pp. 143-180. Cold Spring Harbor Laboratory, New York. Tkacz, J . S. and MacKay, V. L. (1979). Journal of Cell Biology 80, 326. Trueheart, J . , Boeke, J. D . and Fink, G. R. (1987). Molecular and Cell Biology 7, 2316. Tsuchiya, E. and Fukui, S. (1978). Biochemical and Biophysical Research Communications 85, 473. Valdivieso, M. H., Mol, P. C., Shaw, J. A . , Cabib, E. and Duran, A. (1901). Journal of Cell Biology 114, 101. van den Ende, H. (1976). “Sexual Interactions in Plants. The Role of Specific Substances in Sexual Reproduction”. Academic Press, London. van den Ende, H. (1978). In “The Filamentous Fungi” (J. E. Smith and D . R. Berry, eds), vol. 3, pp. 257-273. Edward Arnold, London. van den Ende, H . (1083). In “Fungal Differentiation: A Contemporary Synthesis” (J. E. Smith, ed.), pp. 449479. Marcel Dekker, New York. van den Ende, H. (1984). In “Encyclopedia of Plant Physiology” (H. F. Linskens and J. Heslop-Harrison, eds), vol. 17, pp. 333-349. Springer-Verlag, Berlin. Vigfusson, N. V. and Cano, R . J. (1974). Nature 249, 381.
SEX HORMONES A N D FUNGI
145
Wagner, J . C. and Wolf, D. H . (1987). FEBS Letters 221, 423. Waters, M. G., Evans, E. A. and Blobel, G . (1988). Journal of Biological Chemistry 263,6209. Watson, D. and Berry, D. R. (1977). FEMS Microbiology Letters 1 , 83. Watzele, G . and Tanner, W. (1989). Journal of Biological Chemistry 264, 8753. Watzele, M., Klis, F. and Tanner, W. (1988). EMBO Journal 7, 1483. Webster, N. J. G., Green, S., Jin, J. R. and Chambon, P. (1988a). Cell 54, 199. Webster, N., Jin, J. R., Green, S., Hollis, M. and Chambon, P. (1988b). Cell 52, 169. Weihe, G. R. and McMorris, T. C. (1978). Journal of Organic Chemistry 43, 3942. Werkman, T. B. A. (1976). Archives of Microbiology 109, 209. White, R. H. and McMorris, T. C. (1978). Phytochemistry 17, 1800. Wilkinson, L. E. and Pringle, J . R. (1974). Experimental Cell Research 89, 175. Williams, R. J., Dickinson, K., Kinsman, 0. S., Bramley, T. A., Menzies, G. S. and Adams, D. J. (1990). Journal of General Microbiology 136, 2143. Windels, C. E., Mirocha, C. J., Abbas, H. K. and Weiping, X . (1989). Mycologia 81, 272. Wolf, J . C. and Mirocha, C. J. (1973). Canadian Journal of Microbiology 19, 725. Wright, A . P. H., Carlstedt-Duke, J. and Gustafsson, J.-A. (1990). Journal of Biological Chemistry 265, 14697. Wurtz, T. and Jockusch, H . (1975). Developmental Biology 43, 213. Xue, C. B., Eriotou-Bargiota, E . , Miller, D., Becker, J . M. and Naider, F. (1989a). Journal of Biological Chemistry 264, 19161. Xue, C. B., Caldwell, G . A., Becker, J . M. and Naider, F. (1989b). Biochemical and Biophysical Research Communications 162, 253. Xue, C. B., Ewenson, A , , Becker, J. M. and Naider, F. (1990). International Journal of Peptide and Protein Research 36, 362. Yamomoto, K. R. (1985). Annual Review of Genetics 19, 209. Yanagishima, N. and Fujimura, H . (1981). Archives of Microbiology 129, 281. Yanagishima, N. and Yoshida, K. (1981). In “Sexual Interactions in Eukaryotic Microbes” (D. H. O’Day and P. A. Horgen, eds), pp. 261-295. Academic Press, New York. Yoshida, K., Hisatomi, T . and Yanagishima, N. (1989). Journal of Basic Microbiology 2,99. Zickler, H. (1952). Archiv fiir Protistenkunde 98, 1.
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Fruiting in the Higher Fungi JOSEPH G . H . WESSELS Department of Plant Biology. University of Groningen. Kerklaan 30. 9751 N N Haren. The Netherlands
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Introduction . . . . . . . . . . . . Development of emergent structures . . . . . . . Controloffruitingbymating-typcgcncs . . . . . . A . Mating-typcgcncsasmasterregulators . . . . . B . Molccular structure of mating-type genes . . . . C . R N A and protein regulation duringvegetative growth . D . Biochemical changes during formation of the dikaryon . E . R N A and protein regulation in the dikaryon duringfruiting Accessory regulatory genes controlling fruiting . . . . A . Haploid fruiting genes . . . . . . . . . B . Othcrpotentialregulatorygenes . . . . . . Molecular and biochemical indices of fruiting . . . . A . Hydrophobins . . . . . . . . . . B . Cyclic AMP . . . . . . . . . . . C . Phenol oxidases . . . . . . . . . . Environmentalcontroloffruiting . . . . . . . A . Fruiting-inducingsubstances . . . . . . . B . Light, temperature and carbon dioxide . . . . . Rapid expansion of fruit bodies . . . . . . . . A . Metabolicchanges . . . . . . . . . B . Hyphal-wall expansion . . . . . . . . Biotechnology . . . . . . . . . . . . Conclusions . . . . . . . . . . . . Acknowledgements . . . . . . . . . . References . . . . . . . . . . . . . Note added in proof . . . . . . . . . . . I
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I Introduction
The large elaborate fruit bodies of the ‘higher’ fungi. variously called mushrooms. brackets or toadstools. have attracted attention from naturalists and epicures alike and they have stimulated the imagination of writers and artists since the start of history (Ainsworth. 1976). These fruit bodies are ADVANCESINMICROBIAL.PIIYSIOI.OGY.VOI.. 74 ISRN l&IM)277344
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also objects of many taxonomic and morphological studies, many by gifted amateurs such as Reijnders (Reijnders, 1963; Reijnders and Moore, 1985). However, surprisingly few experimental biologists have been drawn to a study of the development of these structures, possibly because those species best known for culinary reasons do not readily produce fruit bodies in the laboratory under controlled conditions. In evolutionary terms, fruit bodies are adaptations for dissemination of sexual spores. In the fruit bodies, specialized cells are generated in which genetically different haploid nuclei, derived from a mating and co-existing in a common cytoplasm (a heterokaryon), fuse to form diploid nuclei. These diploid cells do not propagate but undergo meiosis to form haploid spores. These spores arc generated within (Ascomycotina) or outside (Basidiomycotina) the once diploid cells. After discharge, the haploid spores can germinate and generate recombinant homokaryotic mycelia which, depending on an often complex system of mating-type genes, can fuse to produce a fertile heterokaryotic mycelium. In the basidiomycetes, species behaving according to this scheme are in the majority and are called heterothallic. In some species also the homokaryon can fruit and produce normal basidiospores (primary homothallism). In others, for example the commonly cultivated white button mushroom Agaricus bisporus, the basidiospores are mostly heterokaryotic and the germinated mycelium can proceed to form fruit bodies without mating (secondary heterothallism). Within the Ascomycotina, only members of the Discomycetes produce large fruit bodies, called ascocarps (e.g. morels and truffles). On the other hand, most members of the Basidiomycotina, except for those belonging to the Teliomycetes (e.g. the smuts and rusts), produce large fruit bodies, also called basidiocarps or basidiomes. In species belonging to the Hymenomycetes, the basidia develop on an exposed hymenium. The species considered in this review all belong to this class. In species belonging to the Gasteromycetes (e.g. puff balls and stink horns) the basidia and basidiospores develop inside the basidiocarps. For a taxonomic overview, the reader is referred to Webster (1980). Among the Hymenomycetes the species best known for culinary reasons, and thus commercially most valuable, can only be fruited with difficulty or not at all under laboratory conditions, as in the case of mycorrhizal fungi (see Section VIII). Therefore, basic knowledge of fruiting has mainly come from studying a few economically worthless species, notably Schizophyllum commune and Coprinus cinereus (referred to as Coprinus lagopus in older literature and as Coprinus macrorhizus in much Japanese literature; Moore et al., 1979) and to a lesser extent from studies with Coprinus congregatus and some other species. Within two weeks, these species complete their life cycles on simple synthetic media. Consequently, S. commune and C. cinereus are also the only two Hymenomycetes well studied genetically
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(Burnett, 1975; Fincham et al., 1979; Raper, 1988). Fortunately, these two species occupy different habitats in nature and belong to different taxonomic groups. Coprinus cinereus is a coprophilous fungus thriving on dung and belonging to the Agaricales. It forms fruit-body primordia containing completely differentiated tissues. These primordia then rapidly expand into mature fruit bodies by cell enlargement (Moore etal. , 1979). After shedding the black spores (ink cap) the fruit bodies rapidly lyse. Alternatively, sclerotia can be formed which probably serve vegetative survival under unfavourable conditions. Attractive features of this species include formation of asexual spores (oidia) by homokaryons and the occurrence of synchronous meiotic divisions in basidia (Lu, 1982). In contrast to the ephemeral fruit bodies of ink caps, the fruit bodies of S. commune are very long-lasting and can shed basidiospores upon wetting after being kept dry for years (Ainsworth, 1962). Schizophyllum commune is a wood-inhabiting fungus belonging to the Aphyllophorales, despite the possession of gills which, however, bear no relationship ontogenetically to those of the Agaricales (Webster, 1980). The fruit-body primordia grow into large fanshaped structures by indeterminate proliferation of hyphae which appear to grow by apical extension (Wessels, 1965; van der Valk and Marchant, 1978; Raudaskosi and Vauras, 1982). Fruit-body development is thus more akin to that of the polypores or bracket fungi, many of which, like S . commune, are active in lignocellulose degradation (Odier, 1987; Broda et al., 1989; Gold et al., 1989). A disadvantage of S. commune is the absence of asexual spores, except for chlamydospores (Koltin et al., 1973), which are, however, of no use in isolating uninucleate cells.
11. Development of Emergent Structures
Emergence of fruit bodies in basidiomycetes can only be understood with reference to the mycelium as a whole. Clearly, growth of all parts of a fungus, except for expansion growth in the fruit bodies of agarics, depends on growth of individual hyphae extending only at their apices (Wessels, 1986, 1990). Although the fruit bodies consist of a dense mass of hyphae forming a pseudoparenchymous tissue (plectenchyma) they show no evidence for the occurrence of meristems as found in plant tissues. The vegetative mycelium colonizes the substrate, and its growth depends on regularly branching hyphae such that the total length of hyphae divided by the number of tips, known as the hyphal growth unit, remains constant (Trinci, 1974). Another useful concept developed by Trinci and his coworkers (see Bull and Trinci, 1977) is that extension of the tip of an individual hypha is supported by a certain volume of protoplasm which remains constant as the hypha moves forward; this hyphal region involved
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in tip growth is called the peripheral growth zone of the colony. In addition to the apical hyphal compartment, the peripheral growth zone may comprise many subapical compartments which are thought to form a continuum because the septa between hyphal compartments contain pores. As the peripheral growth zone of constant length moves forward, non-proliferating mycelium is left behind and does no longer appear to participate in growth. Although the aforementioned concepts well describe the initial growth phase of a fungal colony on nutritive medium, they fall short in describing the full potential of the colony, particularly under growth-limiting conditions. Not only do tips contribute to growth, they also participate in formation of anastomoses between hyphae. Thus, the colony does not consist of radially advancing hyphae but rather represents a network of hyphae allowing for translocations in different directions (hyphal anastomoses are not formed in the vegetative mycelium of fungi belonging to the Zygomycotina, which are of no concern here). In addition, hyphae at the advancing front of basidiomycete mycelia may grow over non-nutritive surfaces for considerable distances, their growth being supported by transport of water and nutrients from a food base (Jennings, 1984) or by an autolysing part of the colony (see below). So, cell death and cell turnover may become important parameters in growth. Such secondary processes often accompany generation of new foci of hyphal growth, away from the advancing front. Such focal points of hyphal growth may be related to production and secretion of idiophase enzymes (S. Moukha, H. A. B. Wosten and J. G. H. Wessels, unpublished data) but also to generation of reproductive structures such as fruit bodies. Mathematical models have been constructed that take most of these activities of the whole mycelium into account (Edelstein and Segel, 1983). Such models can, for example, explain the occurrence of rhythmic increases in hyphal densities (rings) in fungal colonies. Of particular relevance to the present discussion is the notion that initiation of fruit bodies can be viewed as the development of focal points of high-density growth of branching hyphae with their growth axis away from the substrate (Edelstein, 1982). It is clear that growth of these emergent structures, particularly when developing into large fruit bodies, requires massive transport of materials from the substrate mycelium. A mathematical model describing initiation and growth of Agaricus bisporus (white button mushroom) crops (Chanter and Thornley, 1978) assumes that initiation of fruit bodies only occurs when the substrate mycelium has attained a certain threshold density. Although mathematical models are valuable in describing integration of various activities in developing mycelia, and can suggest important parameters to be considered in experimental research, they do not, of course, reveal the physiological factors involved. Unfortunately, experimental data have not yet provided much insight with respect to mechanisms that
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integrate these activities. For instance, only in the zygomycetes (Gooday, 1983) and the ascomycetes Neurospora crassa (Bistis, 1983) and Aspergillus nidulans (Champe and El-Zayat, 1989) have hormonal systems been indicated. In the basidiomycetes no hormonal systems have been found to operate in the mycelium; only for stipe elongation of agaric fruit bodies has some evidence been presented suggesting involvement of a hormonelike substance produced in the caps (Gruen, 1982). With respect to transport of water and solutes over long distances in the colony, Jennings (1984) has suggested that mass flow occurs through hyphae because of the existence of a gradient in hydrostatic potential created by sources and sinks of assimilates, akin to the mechanism held responsible for phloem transport in plants. In the assimilating part of the colony, water enters the mycelium osmotically; in the growing but not assimilating part, water and assimilates are drawn out of the conducting system. This concept is mainly based on observations on the basidiomycete Serpula lacrymans (dry rot) in which the water flow manifests itself by extrusion of drops when the mycelium grows over a non-nutritive surface extending from a food base. Droplet exudation generally occurs from fruit-body primordia in a variety of basidiomycetes, and has received special attention in Lentinus edodes (Leatham, 1985). However, at the moment too little is known to generalize and to attribute all transport phenomena within mycelia, including possible movement of whole cytoplasmic masses (Gregory, 1984; but see Ingold, 1986), to turgor-driven mass flows. Though uncertainty exists as to the exact mechanisms of translocation, the concept of sinks and sources of assimilates regulating transport appears intuitively correct, particularly when development of emergent structures is considered. These non-assimilative massive structures must draw heavily on resources in the substrate mycelium. Molecular studies (Wessels, 1991; see Section V) have implicated wall-bound hydrophobic proteins (hydrophobins) in generating emergent structures such as aerial hyphae and fruit bodies. Non-proteinaceous compounds, such as (+)-torreyol, have also been suggested to confer hydrophobicity on the cell surface of emergent hyphae in some basidiomycetes (Ainsworth et al., 1990). Such hydrophobic wall coatings could effectively shield hyphae from the environment and effect their emergence into the air where the water potential is generally much lower than in the substrate. Given a high growth potential for hyphae in emerging fruit bodies, these structures would act as powerful sinks. Redistribution of active cytoplasms has been considered as most characteristic for fungal mycelia (Gregory, 1984). This was recently clearly demonstrated in fruit-body formation in Schizophyllum commune by in situ hybridization with a 32P-labelled clone of the gene for 18s rRNA (Ruiters and Wessels, 1989a). As shown in Fig. 1, in a five-day-old darkgrown dikaryotic colony, fruit bodies are absent and 18s rRNA, equated
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with the occurrence of active cytoplasm, is found throughout the colony, although more concentrated in an outer zone of the expanding colony where protein synthesis is highest (Yli-Mattila et al., 1989a). This zone probably represents the peripheral growth zone (Bull and Trinci, 1977) already referred to. When such a colony is brought into light, fruit bodies are initiated a few millimetres behind the advancing front and can be clearly seen microscopically six hours after illumination (Raudaskoski and Vauras, 1982). These fruit-body initials are characterized by intensified branching of hyphae with short hyphal compartments which tend to adhere to each other and grow in parallel (Raudaskoski and Viitanen, 1982; van der Valk and Marchant, 1978; Fig. 2). In situ hybridizations (Fig. 1) show the activity of fruiting genes (see Section 1II.A) in the initials and 12 hours after illumination. After 24 hours, rRNA is seen concentrated at sites where primordia develop and at the still-advancing colony front. Between 24 and 96 hours, the advance of this front gradually decreases and nearly all rRNA, and thus cytoplasm, apparently moves into the developing primordia. At the same time, there is competition between the primordia themselves because, at the 96-hour stage, only about 60% of the primordia show an 18s rRNA signal. Apparently there was also translocation of cytoplasm from abortive to developing primordia. At the moment, it is not clear how much of this apparent translocation of cytoplasm is due to movement of cytoplasm. Because of the presence of complex dolipore septa between hyphal compartments, I and my colleagues assume a major role for cell turnover, involving breakdown of cytoplasmic components, translocation of the breakdown products and resynthesis in growing primordia. Cell turnover, involving translocation of breakdown products, becomes very evident in the later stages of fruit-body development which can occur in the absence of external nutrients. Due to substrate limitation in most laboratory cultures of S. commune, only a few primordia can grow into the typical fan-shaped fruit bodies, which can measure a few centimetres across. During enlargement, in this situation based on proliferation of hyphae and not on hyphal inflation as in agarics, nitrogenous compounds and carbohydrates are retrieved from FIG. 1. In situ hybridization of five-day-old colonies of Schizophyllum commune after transfer to light. Column (a) shows the morphological appearance of colonies at various times after transfer. The extreme margin of the colonies is indicated by stippling in columns (a) and (b). Column (b) shows autoradiographs of the same colonies after hybridization of 30" sectors with 32P-labelledclones for mRNAs Scl7, Sc9 and Sc14, and 18s rRNA (R), the vector without insert (-), and a blank without DNA during hybridization (0). Column (c) shows autoradiographs of the same sectors shown in column (b) but after rehybridization with an 18s rRNA
clone. From Ruiters and Wessels (1989a).
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FIG. 2. Scanning electron micrographs of fruit-body primordia of Schizophyllum commune. (a) Early stage of fruit-body formation, showing aggregation and upward growth of parallcl hyphae. Note that the hyphae are directed to the apical centre of the primordium. (b) Fruit-body primordium at a more advanced stage of development. Note the inward growth of peripheral hyphae and the presence of swollen cell parts at thc outside of the primordium. Bars represent 100 pm. From van der Valk and Marchant (1978).
preformed substrate mycelium and from abortive primordia (Wessels, 1965; Niederpruem and Wessels, 1969; Wessels and Sietsma 1979). After depletion of a typical storage polymer, namely glycogen, degradation of p-(1+3)/(3-(1-+6)-glucan occurs. This glucan occurs as a jelly-like material around substrate hyphae and in the medium (Wessels et al., 1972). In addition, a glucan (R-glucan) of similar structure, but occurring as a major alkaliinsoluble component of hyphal walls (Sietsma and Wessels, 1977, 1979), is degraded in substrate hyphae and abortive in primordia to provide for the needs of growing fruit bodies. What remains of the supporting structures are empty hyphae with walls mainly containing u-(1+3)-glucan, a polymer not degraded in this fungus. A mutant of S. commune, in which R-glucan degradation is blocked, is deficient in outgrowth of primordia (Wessels, 1965, 1966). In the wild-type strain, high concentrations of carbon dioxide lead to synthesis of an altered R-glucan which is less susceptible to enzymic degradation and thus cannot sustain outgrowth of primordia (Sietsma et al., 1977). Also, spore production in S . commune depends on degradation of previously synthesized polymers (Bromberg and Schwalb, 1976). Even in the presence of a carbon source in the medium, 30% of the total material in spores discharged from mature fruit bodies derives from previously synthesized material. In agarics such as Coprinus cinereus (Madelin, 1960), Flammulina velutipes (Kitamoto and Gruen, 1976; Gruen and Wong, 1981) and Coprinus congregatus (Robert, 1977), fruit-body formation has also been
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shown to be associated with breakdown of polymers in the substrate mycelium. The dramatic degradation processes associated with fruit-body formation, already referred to, may only occur when these fungi are grown on a limiting concentration of substrate. Under nutritive conditions the needs of the emerging fruit bodies are probably largely provided for by the on-going assimilative capacity of the substrate mycelium. However, the occurrence of fruit-body development in the absence of external nutrients shows that the concept of a sink-source relationship must be broadened to include controlled degradation of previously grown structures as a source of biosynthetic materials for emergent structures. This poses special problems in translocation and in regulation of various activities within the mycelium. How can autolysing parts of the mycelium provide for the build-up of a turgor-driven mass flow postulated for translocation of materials towards growing fruit bodies? Possibly at this stage there is also a major contribution from transport through transpiration of water from fruit bodies (Jennings, 1984). With respect to regulation of hydrolytic activities in the mycelium as a whole, it was found that, in S . commune, synthesis of the wall-degrading enzyme R-glucanase was induced, as a result of glucose depletion in the medium, both in the substrate mycelium and in fruit bodies. But, in emerging fruit bodies, newly synthesized R-glucan was protected against degradation by an unknown mechanism (Wessels, 1966). However, such a mechanism can hardly be envisaged for all polymers subjected to local degradation. Local synthesis or activation of hydrolytic enzymes probably also occurs. Initiation of fruit bodies is primarily controlled by genetic factors and secondly by the developmental status of the mycelium and by environmental factors. It appears that fruit-body initials, consisting of a dense mass of actively growing hyphae, create massive sinks for water and assimilates. In the absence of evidence for the occurrence of long-range signalling systems such as provided by the growth regulators in plants (phytohormones), this may be the major determinant for ensuing processes. Developing fruitbody primordia would draw on materials provided by the assimilating substrate mycelium. Depletion of external nutrients may induce autolysis in those parts of the colony less effective in sink activity, including less vigorous fruit-body primordia, which then become sources of materials to be used by the most competitive fruit bodies, that is, those most advanced in development. Failure to produce mature fruit bodies may thus result, inter alia, from defects in initiation of primordia, in build-up of sufficient reserves, in breakdown of these reserves, or in the translocating system. 111. Control of Fruiting by Mating-Type Genes A . MATING-TYPE GENES AS MASTER REGULATOKS
In the heterothallic basidiomycetes, fruiting is most regularly or exclusively observed in the heterokaryon, also called the secondary mycelium, which
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arises from a mating between two compatible homokaryons. From a teleological point of view this makes senbe because it ensures that diploid cells (basidia) which are formed in fruit bodies produce recombinant meiotic progeny (basidiospores). It is less clear why a minority of basidiomycetes (about 10%) are homothallic, that is, mycelia which grow from basidiospores are capable of directly forming fruit bodies. However, homothallic forms can arise from heterothallic forms by mechanisms such as: (a) the presence of constitutive mutations in the mating-type genes (see below); (b) the presence of alleles which by-pass control by mating-type genes (haploid fruiting; see Section IV. A); and (c) formation of heterokaryotic basidiospores as in the white button mushroom Agaricus bisporus (Raper et al., 1972). Formation of a stable heterokaryon in basidiomycetes is controlled by what has been called “homogenic incompatibility” (Esser, 1971); homokaryotic mycelia which carry identical incompatibility factors are intersterile. There is a group (about 45% of the species) in which a single genetic factor regulates mating and, therefore, species in this group, for example Coprinus comatus, Agaricus bitorquis and probably A. bisporus, are called unifactorial or bipolar. The majority belong to a group in which two unlinked genetic factors regulate mating, and species belonging to this group, e.g. Schizophyllum commune, Coprinus cinereus and Pleurotus ostreatus, are called bifactorial or tetrapolar. It should be stressed that in none of these fungi is there any form of sexual differentiation; mating occurs between morphologically identical homokaryons by hyphal fusions and (reciprocal) exchange of nuclei. With respect to morphological differences between homokaryons and derived heterokaryon, there is a great deal of variation. The most regular pattern is that exemplified by the two most intensively studied species, namely S . commune and C. cinereus. Here, the homokaryon contains one nucleus in each hyphal compartment, and is therefore called a monokaryon. The established heterokaryon contains two (genetically different) nuclei in each hyphal compartment and is therefore called a dikaryon. In these species, the dikaryon is typified by the presence of a clamp connection at each septum which is formed during synchronous mitotic division of the two nuclei (Fig. 3 ) . To cite a few deviating examples: in the occasionally cultivated A. bitorquis, the homokaryon is multikaryotic while the fertile heterokaryon is dikaryotic but without clamp connections (Raper, 1976); secondly, in the commonly cultivated A. bisporus, a secondary heterothallic form, the fertile heterokaryon which grows directly from a basidiospore is multikaryotic without clamp connections (Raper et al., 1972). Returning to the genetic system that controls establishment of the heterokaryon possessing a propensity for fruiting, the incompatibility
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homokaryons (nionokaryons )
!!
meiosis
T fruit- body formation
dikaryosis
genotype
nuclear migration
mYcelial type
nuclear migration
Ax
Bcon
homokalyon
+
Ax Ay
BX
heterokaryon
-
Acon
BX
homokaryon
-
hyphal morphology
fruit- body formation
+ ]-
+ FIG. 3 . Diagram showing the life cycle of Schizophyllum commune and the effects of constitutive mutations in the mating-type genes on hyphal morphology and fruitbody formation.
factors represent one of the most remarkable systems of genetic interactions. A detailed discussion is outside the scope of this review, and those readers interested are referred to a number of excellent reviews (Raper, 1966, 1978, 1983, 1988; Koltin et al., 1972; Koltin, 1978; Casselton and Economou, 198s). However, a brief discussion is necessary as an introduction to molecular studies related to control of heterokaryon formation and fruiting exercised by the incompatibility factors. In the tetrapolar S . commune, and probably also in C. cinereus, each of
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the two incompatibility factors, A and B , comprise two genes, namely a and B. For the occurrence of a compatible mating between monokaryons, leading to formation of a dikaryon, an allelic difference must exist in either the a-gene or the b-gene of each of the two factors. In S . commune, an extensive series of alleles for these genes has been shown to exist; the estimate is nine A a , 32 AD, nine Ba and nine BP alleles. Because the agenes and P-genes of each factor are equivalent, each combination is unique and, thus, there are theoretically 288 different A-factors and 81 different B-factors which give compatible matings in all combinations. In C. cinereus, the situation is less clear because the assumed a-genes and P-genes within a factor are so tightly linked that recombination is a rare event. As pointed out by Casselton and Economou (1985), it is the genetic difference that sets things into motion. Diploids or aneuploids with phenotype AxAy are compatible with both Ax and Ay haploids; similarly, BxBy is compatible with both Bx and B y . A similar conclusion can be drawn from matings with strains in which a different A has been introduced by transformation (see later). Rather than assigning to this system the term “homogenic incompatibility” (Esser, 1971), it would thus seem more appropriate to use the term “heterogenic compatibility” and to consider genes within the Afactor and the B-factor as mating-type genes. Henceforth, this terminology will be used and, when referring to the A and B mating-type genes the reference is to one of the genes within the A and B gene complexes. Control of fruiting by mating-type genes can only be appreciated when it is realized that the primary control concerns formation of the dikaryon (Fig. 3). As shown by the morphology of the heterokaryon that arises from the hemicompatible mating A = B# , interaction of different B-genes makes possible dissolution of septa and migration of nuclei. Septa1 dissolution and nuclear migration have in fact become constitutive in this heterokaryon. In the hemicompatible mating A+ B = , septa1 dissolution and nuclear migration do not occur but nuclei in fusion cells undergo synchronous nuclear divisions with formation of hook cells that do not fuse (pseudoclamps). In the fully compatible A # B+ mating, nuclear migration does occur but ceases once a foreign nucleus reaches an apical cell, where it associates with the resident nucleus; henceforth, the two divide synchronously with formation of clamp connections. Because fusion of hook cells does not occur in A+ B= heterokaryons, such fusion is another process controlled by the B-gene. The regulatory activities of mating-type genes are clearly indicated by the effects of constitutive mutations in these genes (Fig. 3). These mutations, Acon and Bcon (mutations in one of the constituent genes), have been induced in both S. commune (Parag, 1962; Raper et al., 1965; Raper, 1966) and C. cinereus (Day, 1963; Haylock et al., 1980; Swamy et
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al., 1984). In S. commune, phenotypes of the mutants completely mimic heterokaryons in which the functions of mating-type genes are switched on by heterogenic conditions, that is, Acon Bx mimics A+ Bx, A x Rcon mimics A x B f , and Acon Bcon mimics A f B f . The Acon Bcon homokaryon is a dikaryon which shares with the heterokaryotic dikaryon the propensity to form fruit bodies in which meiosis occurs (with formation of identical Acon Bcon basidiospores). The presence of these two constitutive mutations thus effectively converts the fungus from being heterothallic to homothallic. In C. cinereus, these mutations have similar effects with the exception that an Ax Rcon homokaryon is morphologically indistinguishable from a wild-type homokaryon. Although the mutation does not elicit septa1 disruption and nuclear migration in this fungus, it does effect clamp-cell fusion in an Acon Bcon dikaryon (Haylock et al., 1980; Swamy et al., 1984). The homokaryotic dikaryon of C. cinereus also fruits normally but, in contrast to the A f B f heterokaryotic dikaryon, it forms as many oidia as the wild-type homokaryon (Swamy et al., 1984). B . MOLECULAR STRUCTURE OF MATING-TYPE GENES
What is the molecular nature of the interactions between mating-type alleles and of constitutive mutations that mimic interactions between these alleles? Within the basidiomycotina, the hymenomycetes S. commune and C. cinereus and the non-fruiting teliomycete Ustilago maydis are being investigated to solve this question. Ullrich’s group has cloned and sequenced three alleles of the Aa matingtype gene of S. commune (Giasson et al., 1989; Ullrich et al.. 1991). Cloning of Aa4 was achieved by a chromosome walk with a cosmid library constructed with D N A inserts from a homokaryon carrying Aa4. At each step of the walk, cosmids were tested by transformation into an Aal recipient and transformants were examined for the presence of clamp connections, the diagnostic feature of A-activated development. The walk started from the previously cloned PABl gene which lies in between A a and AP at 0.3 cM from A a . At about 50 kb from P A B l , the Aa4 gene was encountered. By probing genomic libraries from AaZ and Au3 strains with the Aa4 probe, these two alleles were recovered. Unexpectedly, the fragments in these cosmids, active in switching on morphogenesis in a strain with an alternative allele, did not hybridize to each other and Southern blots of genomic D N A from different strains showed hybridization only with strains from which the particular A u allele was obtained, even at low stringency of hybridization. The different Aa alleles thus show considerable sequence heterogeneity and also strongly differ in sequence from the AD, Ba, and BP alleles. In addition, these hybridizations showed that there are no silent copies of
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these alleles elsewhere in the genome, which is a prominent feature of the mating-type systems of Saccharomyces cerevisiae (Hicks et al., 1979; Nasmyth and Thatchel, 1980) and Schizosaccharomyces pombe (Beach and Nurse, 1982). Sequence analyses of the A a l , Aa3 and Aa4 alleles of S. commune, however, did reveal sequence homologies between a number of different putative open-reading frames (ORFs) contained within these alleles, some of them apparently coding for amino-acid sequences reminiscent of homeodomains (Ullrich et al., 1991). In the absence of comparisons between transcripts and genes, uncertainty exists as to the nature of the proteins encoded by these genes. However, a significant finding was that a 1.2 kb fragment from Aa4, encompassing a particular O R F (ORF l), could activate an A d recipient that does not have a sequence allelic to O R F 1. Also, another part of Aa4, containing ORFs 2, 3 and 4, could activate all other A m except A d , which does contain alleles of ORFs 2, 3 and 4. This challenges the whole concept of interactions occurring between different alleles of mating-type genes. Such interactions between (portions of) non-homologous mating-type genes in S. commune do resemble presumed interactions between the A and a mating-type genes of the ascomycete Neurospora crassa, which show no homologies of either their DNA or predicted amino-acid sequences and therefore have been called “idiomorphs” instead of alleles (Metzenberg and Glass, 1990). In C. cinereus, A mating-type genes have been isolated (Casselton et al., 1989; Mutasa et al., 1991) using the same strategy as outlined for S. commune. Here, the PA BI gene is flanking Aa and AD, which are closely linked. By walking the chromosome, both Aa3 and AD3 were recovered on separate cosmids. Again, by hybridization analysis, different alleles appeared to have unique sequences and occurred only once in the genome. Sequence data were not reported but transcripts from the cloned genes were detected (Kues et al., 1991), which should allow isolation of cDNA clones and a better prediction of the encoded proteins than those based on genomic sequences. While data for mating-type genes in S . commune and C. cinereus do not yet permit construction of detailed models for their action, this is less so for mating-type genes of U. maydis. In this basidiomycete, there are two mating-type genes, a and b , that control progression through the life cycle. Fusion of haploid yeast cells (products of meiosis) requires the presence of two different forms of the a-gene, namely a l and a2. These have been cloned and shown to be very different in nucleotide sequence (S. A. Leong, personal communication). These alternative mating-type genes may thus act similarly to the way proposed for the “idiomorphic” mating-type genes A and a of N. crassa (Metzenberg and Glass, 1990). Formation of a pathogenic dikaryotic mycelium in U. maydis, however, also requires the
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presence of different b-genes for which an extensive series of alleles (at least 23) exist. A few of these b-alleles have been cloned by functional analysis, that is, by selecting a clone from a genomic library of a strain on the basis of its ability to transform yeast cells of a strain with another ballele to the mycelial mode of growth (Kronstad and Leong, 1989; Schulz et al., 1990). A comparison of the sequence of four different b-alleles showed that they all contained a single O R F of 410 amino-acid residues with a variable N-terminus encompassing 110 amino-acid residues (60% identity). The remainder was constant in all four alleles (93% identity) and contained a motif related to a homeodomain (Schultz et al., 1990). On the basis of these findings, simple models were constructed to explain how, by formation of heterodimers from proteins encoded by different b-alleles, active regulatory molecules could arise. However, a realistic view of the regulatory interactions has to await isolation of target genes involved in hyphal morphogenesis and pathogenesis in this fungus. C.
RNA
AND PKOTEIN REGULATION DURING VEGETATIVE GROWTH
Mutational analysis of S. commune indicated that some 30 genes, divided into 12 classes according to phenotype, are controlled during the monokaryondikaryon transition (Raper, 1966, 1983). These mutations, called modifier mutations, are not expressed in monokaryons but modify several aspects of the morphological sequences switched on by the interactions of the A and B mating-type genes. Although this would suggest at least a similar number of target genes under control of mating-type genes, few differences were found in a molecular analysis. D e Vries et al. (1980) compared young mycelia of S. commune (20-30% of the cells being apical and thus executing morphogenesis) of co-isogenic monokaryons with derived dikaryons with respect to protein patterns, using two-dimensional electrophoresis. Among 700 proteins analysed, 20 were seen only in the monokaryon while 23 proteins appeared unique for the dikaryon. Many of these dikaryon-specific proteins were also synthesized in co-isogenic Acon Bcon, Acon Bx and Ax Bcon strains. If the observed difference of 6% extends to the whole range of proteins synthesized in S. commune (10,00&13,000), and were due to differential mRNA synthesis, this should be revealed by nucleic-acid hybridizations. However, quantitative hybridizations of poly(A)-containing RNAs and polysomal RNAs with genomic DNA failed to detect any difference between the monokaryon and the dikaryon (Zantinge et al., 1979; Hoge et al., 1982). Also poly(A)RNA::cDNA hybridizations and in vitro translations of poly(A)RNA and polysomal RNA failed to detect differences in RNA composition (Zantinge et al., 1981; Hoge et al., 1982a). Raper and Timberlake (1985), using a technique called cascade hybridization,
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similarly failed to detect differences between the monokaryon and the dikaryon except for an apparently unique RNA of 580 nt in the dikaryon. This was not investigated further but this mRNA probably represents the transcript of the Sc4 gene which becomes very abundant in the dikaryon after prolonged growth (see later). It is thus doubtful whether regulation of protein patterns in young cultures of the established monokaryon and dikaryon by mating-type genes involves extensive transcriptional regulation. Rather, post-translational control is inferred. In a recent study (Salvado and Labarkre, 1991a) a comparison was made of protein patterns in a large number of non-isogenic vegetative monokaryons and dikaryons of Agrocybe aegerita. Although large variations existed among strains, not a single difference was found in protein-stained gels that could be attributed to the dikaryotic state. On gels reacted with concanavalin A , a single glycoprotein was found to be specifically present in all dikaryons. Although differences in transcript composition were not detected in young cultures of monokaryons compared with established dikaryons, these two mycelial types start to express different genes after prolonged growth. These genes were originally detected and cloned on the basis of their high expression in the dikaryon during fruiting in surface cultures (see Section 1II.E). However, it was found that the dikaryon, but not the monokaryon, also expresses these genes when fruiting is suppressed as in shaken cultures or in surface cultures growing in the dark or in the presence of high concentrations of carbon dioxide (Wessels et al., 1987). An example is the differential regulation of the homologous hydrophobin genes S c l , Sc3 and Sc4 (Schuren and Wessels, 1990). After 3-4 days in shaken cultures Sc3 mRNA rises to very high levels (up to 2% of the total mRNA) in both the monokaryon and the dikaryon but, at this time, the Scl and Sc4 mRNAs become abundant only in the dikaryon (Wessels et al., 1987). Similarly, de Vries et al. (1986) showed that 2-3-day-old cultures of the dikaryon, but not of the monokaryon, grown at 30°C in the dark (completely suppressing fruiting) produced an extracellular laccase. Using an antiserum raised against this laccase, the enzyme could be detected on Western blots of proteins synthesized in vitro on total R N A and separated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis (0.M. H. de Vries, unpublished data). This shows that an abundant mRNA for this laccase was present in the dikaryon but not in the monokaryon. There are thus clear differences in the RNA populations synthesized by the monokaryon and the dikaryon, related to activities of the mating-type genes. However, because these differences in gene expression are not manifested in young cultures of the monokaryon and the dikaryon, which already fully express their differences in cellular morphologies, they bear no relationship to the monokaryon-dikaryon transition proper. Nevertheless, transcriptional
FRUITING IN I'HF HIGHER P U N G I
163
regulation may be important during formation of the dikaryon, that is during septal dissolution and nuclear migration before dikaryotic cells are established. Because many biochemical differences have been noted between the monokaryon and both the common-A heterokaryon (A= B f ; ) and the Ax Bcon mutant which constitutively express septal dissolution and nuclear migration, it would be of interest to compare these mycelia at the RNA level. D. BIOCHEMICAL CHANGES DURING FORMATION OF THE DIKARYON
A brief discussion of biochemical events that take place during establishment of the dikaryon is appropriate because of the similarities of degradative processes during fruit-body expansion in the dikaryon (see Section 11) and those occurring during septal dissolution and nuclear migration in the common-A heterokaryon and the Ax Bcon homokaryon (Wessels and Niederpruem, 1967; Niederpruem and Wessels, 1969; Wessels, 1969a, 1978; Wessels and Sietsma, 1979). Of the lytic enzymes that show high activities in the fruiting A-on B-on mycelium (dikaryon) during carbon depletion and in the A-off B-on mycelium on fully nutritive medium, most attention has been given to the enzyme R-glucanase. This enzyme, probably a p(1+6)-glucan glucanohydrolase (Wessels, 1969b), solubilizes alkali-insoluble (1+3)-/P-(1+6)-glucan, a major component of the hyphal wall and septum of S . commune (van der Valk et al., 1977). Degradation of this wall glucan in the dikaryon mobilizes this glucan as a major reserve compound during outgrowth of fruit bodies (Wessels, 1965). When only the B-sequence is expressed, a high R-glucanase activity (together with chitinase) effects dissolution of septa which are susceptible to degradation in the monokaryon but not in the dikaryon (Wessels and Marchant, 1974). High activity of Rglucanase and other lytic enzymes in A-off B-on mycelia is accompanied by decreased levels of R-glucan, glycogen and triacylglycerols which probably are being synthesized at a normal rate (Wessels, 1978). This could explain the low growth efficiency of these mycelia without a decrease in the capacity to synthesize ATP, as noted by Hoffman and Raper (1971, 1974). A hypothetical scheme of events during formation of the dikaryon is shown in Fig. 4. It is based on the assumption that, after entry of a compatible nucleus, the kinetics of formation of the products of A and B mating-type genes in the foreign cytoplasm are different. First, the product of the B-gene would accumulate and its interaction with the product of the resident B-gene activates lytic processes leading to septal dissolution and permitting nuclear migration. Only after the foreign nucleus is trapped in an apical cell would the product of the A-gene accumulate, while its
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.I.G. H WESSELS
Ax Bx
1
RELEASE OF CATABOLITE REPRESSION
FIG. 4. Hypothetical scheme depicting presumed changes in R-glucanase-R-glucan interactions during and aftcr dikaryon formation in Schizophyllum commune. The R-glucan-chitin complex in lateral walls and cross walls is indicated in black; the hatched area indicates dissolution of R-glucan; the dotted area represents Rglucanase activity. Ax Bx and Ay By represent mating types of two fusing monokaryons. Cytoplasmic products of the mating-type genes, (ax) and (bx), present in the cytoplasm of the acceptor strain can only interact with those specified by the donor nuclei, (ay) and (by), after the latter products have accumulated in the cytoplasm of the acceptor strain. The product (by) accumulates first and its interaction with (bx) leads to an increase in R-glucanase and other proteins related to operation of the B-sequence which includes septal degradation and nuclear migration. Interaction between (ax) and (ay) in apical cells leads to switching on processes related to the A-sequencc and to blockage of part of the B-sequence, namely septal dissolution, by suppressing R-glucanase and by effecting synthesis of septa re5istant to R-glucanase. When glucose in the medium is depleted, R-glucanase is again formed. Products released by degradation of R-glucan in walls of the dikaryon are then translocated to, and used by, growing fruit bodies. From Wessels (1978).
interaction with the product of the resident A-gene would activate the Amorphogenetic sequence, including synchronous nuclear divisions, clamp formation and synthesis of degradation-insensitive septa. Interaction between the A-genes would also suppress lytic activities activated by interaction between the B-genes; the only lytic activity remaining would be concerned
FRUITING IN THE HIGHFR FUNGI
165
with hook-cell fusion to complete clamp formation during divisions of dikaryotic cells. However, suppression of general lytic activities by A-genes only seems to work as long as a carbon source is available in the medium. In the absence of a carbon source, lytic activities again increase and now effect net degradation of polymers leading to outgrowth of fruit bodies. None of the changes in enzyme activities referred to has bcen related to differences in protein patterns as observed by de Vries et al. (1980) between a wild-type homokaryon and a mutant with constitutive nuclear migration. Moreover, no study has yet been made regarding possible changes in mRNA populations accompanying constitutive operation of the B-sequence. An interesting phenomenon which could be explored in such studies is that the constitutive mutants Acon Bcon and Ax Bcon both show a delay in expression of their phenotypes, growing as normal wild-type monokaryons for the first few days (Koltin, 1970; Marchant and Wessels, 1974). Recently, Ross et al. (1991) reported that an arginine-requiring dikaryon of Coprinus congregatus grew as a monokaryon when starved of arginine. E.
RNA
AND PROTEIN REGULATION IN THE DIKARYON DURING FRUITING
Monokaryons and dikaryons which differ genetically only in their matingtype alleles can be grown separately, under identical conditions, so that their proteins and RNAs can be compared. Any detected difference in gene expression is then likely to be related to differences in morphogenesis (e.g. only aerial hyphae in the monokaryon and fruit bodies in the dikaryon) and, at the same time, any differences found can be attributed to direct or indirect control by mating-type genes. This procedure would seem to cancel out any changes in gene expression which are not relevant to morphogenesis as such, but occur as a consequence of increasing mycelial age or changes in the environment brought about by the developing system, such as depletion of nutrients and accumulation of staling factors. Although comparison of protein patterns in young co-isogenic vegetative mycelia of monokaryons and dikaryons of S. commune revealed few differences (de Vries et al., 1980), clear differences were observed when proteins synthesized by four-day-old surface cultures of the monokaryon (aerial hyphae only) and the dikaryon (few aerial hyphae, surface covered with fruit bodies) were compared (de Vries and Wessels, 1984). Among 400 proteins, pulse labelled with ["S]sulphate and analysed on twodimensional gels, only eight of them appeared to be synthesized exclusively in the monokaryon while the fruiting dikaryon synthesized 37 abundant proteins not detected in the monokaryon (Fig. 5 ) . Of these, 15 proteins
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were found only in fruit bodies while the others were synthesized both in fruit bodies and supporting vegetative mycelium. Of the latter, nine were secreted in abundance into the culture medium. These differences in the ability to synthesize different proteins in vivo correlated with the occurrence of different abundant mRNAs as detected by in vitro translations of total RNA preparations followed by separation of protein products by twodimensional electrophoresis (Hoge et al., 1982b). Few differences, if any, could be found after two days of culture when emergent structures had not yet appeared in either the monokaryon or the dikaryon. However, after four days of culture, 22 unique abundant mRNAs were found in the fruiting dikaryon while only a few unique mRNAs were detected in the monokaryon, which produced only aerial mycelium. These results were verified and extended by homologous and heterologous RNA: :cDNA hybridizations (Hoge et al., 1982b). The fruiting dikaryon essentially contained all RNA sequences present in the monokaryon, but RNA from the monokaryon failed to hybridize about 5% of the cDNA synthesized on RNA from the dikaryon. It was deduced from the results of these hybridizations that the difference concerned only a few dozen abundant rnRNAs. The apparent absence of regulation in the class of rare rnRNAs (about 10,000) was confirmed by competition hybridizations using genomic DNA (Hoge et al.,
MONOKARYON
DIKARYON
FIG. 5. Diagram showing the number of abundant polypeptides specifically synthesized in a monokaryon and a co-isogcnic fruiting dikaryon of Schizophyllum commune after four days in surface culture. Cultures were labelled with [3sS]sulphate during the last day of growth and proteins examined by two-dimensional electrofocusingkodium dodecyl sulphate-polyacrylamide-gel electrophoresis. Of 400 polypeptides examined, most were the same. Each symbol refers to a single polypeptide differentially synthesiLed. 0 indicates polypeptides exclusively synthesized in the monokaryon; 0 ,polypeptides synthesized in both the monokaryon and in vegetative mycelium of the dikaryon but not in fruit bodies; A , polypeptides not synthesized in the monokaryon but synthesized in both vegetative mycelium and fruit bodies of the dikaryon; A , polypeptides exclusively synthesized in fruit bodies. From Wessels et al. (1985); based on de Vries and Wessels (1984).
I..RUITING IN THF HIGHER FUNGI
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1982b). However, such hybridizations cannot detect differences in fewer than about a hundred mRNAs (Zantinge et al., 1981). Complementary DNA synthesized on poly(A)RNA of the fruiting dikaryon of S. commune was cloned and clones containing sequences expressed in this dikaryon, and not in a similarly grown co-isogenic monokaryon, were selected (Dons et al., 1984a; Mulder and Wessels, 1986). In approximate agreement with results obtained by RNA::cDNA hybridizations (Hoge et al., 1982b), 3.75% of the clones were dikaryon-specific; 40 clones encompassed nine different sequences (Mulder and Wessels, 1986). These nine different fruiting cDNA clones, together with a few vegetative cDNA clones, were used to measure the contents of specific mRNAs in the developing system. The results can be summarized as follows. Fruiting mRNAs are scarce in young vegetative mycelia of both the monokaryon ( A x B x ) and the dikaryon ( A + B f . ) but, while remaining so in the monokaryon, their contents increase in the dikaryon, concomitant with fruiting (Mulder and Wessels, 1986). This is most noticeable for Scl and Sc4 mRNAs which may rise to 0.5 and 3.5%, respectively, of the total mRNA. When development of most of the fruit bodies ceases in these cultures, the content of fruiting mRNAs drops but remains high in the few fruit bodies that continue development. Of the vegetative mRNA, the Sc3 mRNA is noteworthy because it increases to high abundance (about 1% of the total mRNA mass) in both the monokaryon and the dikaryon during emergence of aerial hyphae, and then declines again. In contrast to fruiting mRNAs it is very low in developing fruit bodies. Fruiting mRNAs are clearly regulated by mating-type genes. They accumulate in both A B+ heterokaryons and A c o n Bcon homokaryons but not in A = B f a n d A + B = heterokaryons nor in A x Bcon and Acon Bx homokaryons (Ruiters et al., 1988). Apparently, both mating-type genes are involved in regulation. At least part of this regulation occurs at the transcriptional level as shown by run-on experiments with isolated nuclei (F. H. J. Schuren and J. G. H. Wessels, unpublished data). In radially growing colonies of the dikaryon, fruiting mRNAs accumulate at the place and time of fruit-body formation. This was shown by measuring mRNA contents in concentric rings cut from colonies (Ruiters et al., 1988) as well as by in situ hybridizations of whole colonies (Ruiters and Wessels, 1989a; see Fig. 1). By performing in situ hybridizations on sections of developing fruit bodies (Ruiters and Wessels, 1989b), it became clear that fruiting mRNAs were concentrated in the growing apical part of fruit bodies but that they were also present in supporting vegetative mycelium (Fig. 6). Fruiting mRNAs also accumulate in the dikaryon when fruiting is inhibited, either by growing the mycelium in submerged culture or by growing surface cultures in the presence of high concentrations of carbon
+
p ,*ma
::.::: .'.. . - .... ..-. .I . . . . .I I 4 8 . . .
flel (t?
,l;*m!"L
:: *
. . . . . I. . . . .I I I ..................
. II . .. . :...:.:.-.-. :.;x:...>::!.
(I. %
:
..
.:p. 18 S
sc 1
sc 2
sc
sc 4
3 RNA
sc 5
Sc 6
sc 7
sc 9
Sc 14
species
FIG. 6. Diagram showing intensitics of signals obtaincd by in situ hybridizations of 18s r R N A and various mRNAs derived from genes Scl, Sc2, etc., at different stages of development of fruit bodies of Schizophyllum commune. Sections were made at various times after transfer of four-day-old dark-brown colonies to light to inducc fruit-body formation. The probes werc biotinylated plasmids containing 18s r D N A or cDNA inscrts and hybridization was revealed by alkaline-phosphatase reaction. From Ruiters and Wesscls (198Yb).
IXUI'I'ING IN I H E HIGHER FlJNGl
169
dioxide or in darkness (Wessels et ul., 1987). Thus, the probed mRNAs thought to be involved in fruiting may be necessary but are not sufficient for the occurrence of fruiting. Several fruiting genes have been sequenced and, in conjunction with sequence data from the cDN As, encoded proteins have been predicted. The fruiting genes Sc7 and Sc14 are homologous and closely linked. They encode proteins of about 20 kDa containing signal sequences for secretion (F. H. J . Schuren, E. Kothe and J. G. H. Wessels, unpublished data). Functions for these proteins are not yet known. The genes Scl and Sc4, which are most abundantly expressed during fruiting, are also homologous to each other and, in addition, they share homology with the Sc3 gene, one of the most abundantly expressed genes in both monokaryons and dikaryons (Dons et al., 1984b; Schuren and Wessels, 1990). The proteins encoded by these three genes (Fig. 7) clearly belong to a family of hydrophobic proteins with eight cysteine residues at conserved positions, each containing a signal sequence for excretion. We call these proteins hydrophobins. They are excreted into the medium as small proteins but, in the walls of emergent structures, they form highly insoluble complexes: pSc3 in walls of aerial hyphae, pScl and pSc4 in walls of fruit bodies (Wessels et ul., 1991a,b; see Section V . A ) . Referring to Agrocybe uegeritu, Salvado and Labarkre (1991b) have also suggested limited regulation of mRNAs during fruit-body formation. 'They
H2N
H2N
CGQH
COOH
FIG. 7. Comparison of the coding sequcnccs of hydrophobin gencs from Schizophyllum commune. The shaded blocks indicate stretches with identical amino-acid residues or conservative substitutions (small interruptions in the sequcnccs arc introduced to obtain optimal alignment). Vertical bars undcr the blocks indicate positions of cysteine residucs. Asterisks indicate positions of possible N-glycosylation sites. Locations of introns in the genes are shown by triangles within which the lengths of the introns are indicated by the number of base pairs. Dashed lines in the N-terminal parts of the sequences indicate approximate cleavage sites of signal peptides. Data from Dons et al. (1984b) and Schuren and Wessels (1990).
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H WESSELS
isolated eight cDNA clones corresponding to mRNAs of unknown functions expressed late in primordium development. However, their comparison of isolated fruit-body primordia with whole stationary phase vegetative mycelia does not seem to exclude the possibility of expression of (some of) these mRNAs at other stages of development. A study by Yashur and Pukkila (1985) suggested extensive regulation of mRNAs during fruit-body formation in C. cinereus. However, their strategy was entirely different. First, they compared RNA isolated from a dikaryon grown vegetatively in shaken culture with RNA from fruit bodies plucked from surface cultures. Second, they used hybridizations to genomic clones to compare RNAs. Of these clones, at least 9% contained sequences expressed in both fruit bodies and vegetative mycelium, 11% hybridized to RNA sequences preferentially present in vegetative mycelium, and 9% to RNA sequences preferentially present in fruit bodies. These results, which suggest regulation of thousands of mRNAs during fruiting, are similar to those reported for conidiation in Aspergillus nidulans using the same strategy (Timberlake, 1980). Apart from the fact that differential probing of genomic clones is more likely to reveal differences in low-abundance mRNAs, we surmise that the discrepancy compared with the results obtained with S. commune stems from a comparison of fungal structures raised under totally different conditions and that most of the genes differentially expressed code for enzymes of metabolic pathways, and other proteins, not directly related to morphogenesis. As far as we know, none of the regulated mRNAs in C. cinereus has been studied in detail.
IV. Accessory Regulatory Genes Controlling Fruiting A . HAPLOID FRUITING GENES
Haploid fruiting, also called monokaryotic or homokaryotic fruiting, has been seen in laboratory cultures of many basidiomycetes (for a review, see Stahl and Esser, 1976). The phenomenon also occurs in a few, sometimes many, haploid progeny of fruit bodies collected in the wild, depending on the species. Haploid fruit bodies are abnormal or nearly normal in morphology and may be sterile or produce basidiospores by mitosis in the basidium, the spores often having a low germination potential. Haploid fruiting may occur spontaneously, only under stress conditions such as transfer to nutritionally deficient media or injury, or only after applying fruit-body formation-inducing substances. Unexplained is a case in which nutritional stress apparently induced dikaryotic fruiting in originally monokaryotic cultures of Coprinus cinereus with the occurrence of normal meiosis
FRUITING IN THF HIGHER FUNGI
171
and formation of basidiospores of one mating type (Verrinder-Gibbins and Lu, 1984). Haploid fruiting, in which control of fruiting by mating-type genes is apparently by-passed, must be distinguished from homokaryotic fruiting as it occurs in A c o n Bcon homokaryons and in diploid monokaryons. In both of these homokaryons, mating-type genes do control fruiting. Diploids of Schizophyllum commune arise in A f: B+ matings in which mates each carry the recessive mutation dik (Koltin and Raper, 1968). The precocious fusion of nuclei in the mycelium is accompanied by formation of a monokaryotic mycelium but, in monokaryotic fruit bodies, normal meiosis occurs. By studying segregation of naturally occurring alleles for haploid fruiting in the tetrapolar Polyporus ciliatus (Stahl and Esser, 1976) and the bipolar Agrocybe aegerita (Esser and Meinhardt, 1977), Esser and his coworkers have implicated the alleles ji' and Fb', operating in sequence. In the presence ofji', only stipes were formed; the additional presence of fb+ led to formation of normal but small fruit bodies. In P. ciliatus the presence offif also led to earlier fruiting in the dikaryon whereas, in A. aegerita, at least one dose of both ji' and fa' was necessary for dikaryotic fruiting to occur. In S. commune (Esser et al., 1979) the allelesjifil' andfi2-' were each found to lead to formation of fruit-body initials, The presence of both alleles led to formation of stipes without spores. In addition to fil-' and Ji2-+, the presence of a third allele, fh', led to formation of abnormally shaped but gilled fruit bodies with two-spored basidia. The presence of exclusively j i , , ji2 and fa alleles permitted fruiting in the A f Bf dikaryon, but the presence of the fruiting alleles tended to shorten the time required for fruiting. Fruiting alleles, by-passing control by mating-type genes, have also been studied by Leslie and Leonard (1979a,b). These authors also implicated two alleles, h a p 5 and hap-6, working in sequence and promoting spontaneous initiation of haploid fruit bodies in S. commune. In addition, in the absence of these alleles, they implicated four other hap alleles in haploid fruiting as it occurred after mechanical injury and, again, two other hap alleles, in the absence of any of the others, in the occurrence of haploid fruiting after adding (unidentified) fruiting substances. Haploid fruiting was also studied by Yli-Mattila et al. (1989b). These authors found that inbreeding of haploid fruiters led to enhancement of haploid fruiting, indicating the polygenic character of this trait. A cross between inbred strains resulted in a dikaryon that could even fruit in the dark. In accordance with an earlier observation (Ruiters et al., 1988), they found that mRNAs characteristic for dikaryotic fruiting also accumulated during haploid fruiting. Nothing is known about the molecular mechanism of haploid fruiting.
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( 1
H Wt55tL5
It has been suggested that the haploid fruiting pathway operates independently from that operating during dikaryotic fruiting (Raper and Krongelb, 1958), but the aforementioned clear effects of the presence of haploid fruiting alleles on dikaryotic fruiting argue against this. One possibility is that these naturally occurring alleles represent relaxed versions of regulatory DNA sequences within regulatory circuits by which mating-type genes control normal dikaryotic fruiting. For instance, haploid fruiters could contain secondary regulatory genes which are no longer rigorously controlled by mating-type genes. This can be likened to mating-type control of sporulation in Succhuromyces cerevisiue. In this yeast, mutations in a secondary regulatory gene ( R M E ) (Mitchell and Herskowitz, 1986) and a tertiary regulatory gene (ZME) (Kassir et ul., 1988) resulted in sporulation in the absence of control by mating-type genes. €3. O T H E R POTENTIAL REGULATORY GENES
An ideal genetic background to identify genes involved in dikaryotic fruiting, i.e. genes controlled by mating-type genes, is provided by the (haploid) Acon Bcon homokaryon which mimics the Af. B f . heterokaryon in most or all respects. This approach has been taken by Kanda and Ishikawa (1986) who, after ultraviolet irradiation of oidia of an Acon Bcon strain of C. cinereus, identified several recessive mutations of morphogenesis. Among the mutants were those that were called knotless, since they were unable to form any aggregates. Others did form knots but failed to make genuine primordia (primordialess) or were blocked at a later stage of development (maturationless). Many did form apparently normal fruit bodies but failed to produce spores (sporeless). The Acon Bcon homokaryon would also appear an ideal genetic background for attempting gene disruptions to study the function of isolated genes thought to be involved in fruiting. In attempting such gene disruptions in S. commune, however, an extremely frequent spontaneous mutation (jbf)was encountered which converted up to 10% of regenerated mycelia from an Acon Bcon strain into sterile mycelia forming abundant aerial hyphae (Springer and Wessels, 1989). Contrary to data already reported, this mutation has never been seen to revert. It also often occurs in Acon Bcon colonies, causing sectors of sterile mycelia which have a slightly higher growth rate than the fruiting progenitor. All independently isolated fbf mutations were allelic and recessive; only when present in a double dose did they prevent fruiting in an A+ B+ dikaryon. Althoughfbfproduced no noticeable effect on hyphal morphology in Acon Bcon strains (clamps and pseudoclamps), it behaved as a (semi)dominant character with respect to hyphal morphology in matings. In A f . B+ matings involving a single dose of fbf, pseudoclamps
I.KlJlTING IN THE HlCiHFR FUNGI
173
were seen in addition to clamps but when homozygous for fbf only pseudoclamps were seen. It was concluded that f h f may belong to a class of previously described spontaneous mutations called modifier mutations of class I1 (Raper and Raper, 1966) which likewise ceased fruiting when the mutation was present in a double dose. There may also be a re!ationship to the spontaneous recessive mutation cohl (Perkins and Raper, 1970) because no complementation occurred in an [bf X cohl cross (Springer and Wessels, 1989). Importantly, the ,fbfmutation prevented accumulation of mRNAs of all fruiting genes for which clones were available, including the Scl and Sc4 hydrophobin genes, but it did not affect accumulation of mRNA for the Sc3 hydrophobin gene, which appears to be associated with formation of aerial hyphae (Springer and Wessels, 1989). It was concluded that FBFis an unstable locus involved in regulation of a battery of structural genes associated with fruiting. Another frequently occurring spontaneous mutation in S. commune is thin (thn), which suppresses formation of all emergent structures (Raper and Miles, 1958; Schwalb and Miles, 1967; Wessels et al., 1991b). This recessive mutation is expressed in monokaryons where it gives a phenotype characterized by absence of aerial hyphae while submerged hyphae appear wavy or corkscrew-like. A double dose of thn in an A + B+ dikaryon leads to suppression of formation of both aerial hyphae and fruit bodies. In conjunction with these effects, thn prevents accumulation of the Sc3 hydrophobin mRNA in the monokaryon and, in the homozygous condition, accumulation of both Sc3 mRNA and the whole set of mRNAs found associated with fruiting in the dikaryon (Wessels et al., 1991b). It is concluded that THN is an unstable locus involved in regulation of a battery of structural genes necessary for construction of all emergent structures. The F B F and T H N genes could belong to a class of secondary regulatory genes controlling structural fruiting genes, with mating-type genes as primary regulatory genes. Alternatively, they could code for transcription factors which, in combination with products of mating-type genes, are necessary to switch on these genes, the product of the THN gene also being required for activation of genes involved in emergence of aerial hyphae. The reason for the frequent occurrence of mutations in these regulatory genes is unknown. The mutation flf appears to occur in single cells, as shown by the appearance of sectors in Acon Bcon colonies. The thn mutation, however, mostly seems to occur throughout the colony and may be due to some epigenetic mechanism such as DNA methylation. An interesting control element, called F R T I , was recently selected from a genomic cosmid library of an Acon Bcon strain of S. commune by its ability to cause fruiting when transformed into non-fruiting monokaryons (Horton and Raper, 1991; Raper and Horton, 1991). It also accelerated
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fruiting in dikaryons derived from these transformants. Curiously, when FRTI was transferred to monokaryons by conventional crosses, it did not elicit fruiting. It was therefore postulated that the cloned version of FRTI was devoid of a repressor element which normally switches off its activity in monokaryons. In A f : B+ heterokaryons and Acon Bcon homokaryons the activities of mating-type genes would turn this repressor off. In monokaryons receiving a cloned copy of FRTI without its repressing element by transformation, the FRTl gene product would be formed and activate fruiting genes. A complication is that different alleles and multiple copies of FRTI exist and that FRTI did not activate fruiting in a monokaryon containing the same allele of FRTI. This needs further investigation but, for the moment, it would appear that FRTI could be a control element positioned in between the mating-type genes and the structural fruiting genes. Mutations in FRTI ,for example those obliterating the repression function, could constitutively activate the gene and induce fruiting in monokaryons (haploid fruiting). Mutations that inactivate FRTI would lead to inability to fruit. The interesting observation was made that, in Southern blots of DNA from sterile sectors of the Acon Bcon strain from which FRTl was isolated, the fragment hybridizing to FRTI was missing (Raper and Horton, 1991). Therefore, the possibility arises that FRTI is related to FBF identified by Springer and Wessels (1989). Other genes of fruit-body development have been indicated but not yet studied at the molecular level. In an early study, Raper and Krongelb (1958) detected dominant alleles in a natural population of S . commune affecting the morphology of fruit bodies, described as corraloid (highly involuted hymenium), medusoid (long stipes) and bug’s ear (numerous small fruit bodies without gills). Of these, bug’s ear (bse) was shown to contain about twice the normal amount of CAMP, while a phenocopy of M this genotype could be obtained by treating wild-type strains with CAMP (Schwalb, 1978a,b). The presence of bse also promoted haploid fruiting, resembling the presence of the allele jisc in C. cinereus which causes haploid fruiting and a continuously high concentration of CAMP, probably due to decreased activity of a phosphodiesterase (Uno and Ishikawa, 1971, 1973, 1982). By mutagenesis of an A t B+ dikaryon of C. cinereus and regrowth from hyphal fragments, Takemaru and Kamada (1972) isolated a remarkably high number of variants, many of them similar to those later isolated by mutagenesis of an Acon Bcon mycelium by Kanda and Ishikawa (1986). Many of the mutations were recessive but genetic analysis of an elongationless variant (Eln)and expansionless variant ( E x p ) , mutants affected in fruit-body expansion, showed that these traits were controlled by single dominant genes (Takemaru and Kamada, 1971). Many of the mutants isolated by these workers were defective in sporulation.
FRUITING IN THE H I C H t K I UNGI
175
Temperature-sensitive sporulation mutants of S. commune have also been isolated (Bromberg and Schwalb, 1977).
V. Molecular and Biochemical Indices of Fruiting
To detect molecular and biochemical processes which are uniquely used in fruit-body formation, and thus are targets for developmental regulation, essentially two approaches are possible. One is to search for anonymous genes which are specifically expressed during fruiting and then try to identify the proteins that they code for and to elucidate their functions. This approach has led to the discovery of hydrophobins in the hyphal walls of emergent structures, and is likely to yield more information on hitherto unknown proteins involved in development. A powerful adjunct to this approach is the possibility of disrupting the gene for which a clone has been obtained and to examine the effect of the targeted mutation on development. The second approach is more classical and attempts to establish changes in (known) biochemical parameters associated with fruiting. Once found, the occurrence of the changes can be tested for genetic regulation in parallel with fruiting. This approach has been followed in studies on the roles of CAMP and laccase in fruiting. Cyclic AMP was suspected to play an important role because it acted as a fruiting-inducing compound. Laccase is an easily assayed enzyme which is often observed to rise during fruiting. A. HYDKOPHOBINS
Among the most abundant mRNAs expressed during emergent growth of Schizophyllum commune are those transcribed from the genes S c l , Sc3 and Sc4, a gene family encoding small hydrophobic proteins each with eight cysteine residues at conserved positions (hydrophobins, see Fig. 7). Table 1 summarizes the accumulation of mRNAs for these genes in co-isogenic strains that differ only in specific genetic elements that regulate emergence of aerial hyphae and fruit bodies. Formation of aerial hyphae is clearly associated with expression of the Sc3 gene, which appears to be regulated only by the T H N gene. Formation of fruit bodies is associated with expression of the ScZ and Sc4 genes, which are regulated by a combination of mating-type genes, FBF and T H N . The proteins encoded by the Sc3 and Sc4 genes (Schuren and Wessels, 1990) have been detected in vivo (Wessels et al., 1991a,b). The mRNAs for these hydrophobins are formed both in submerged supporting mycelium and in emergent structures but submerged hyphae excrete hydrophobins into the medium whereas, in
176
J . G. t l . WESSF1.S
TABLE 1. Abundance of hydrophobin mRNAs in various strains of Schizophyllum commune, correlated with the occurrence of fruit bodies and aerial hyphae Genotype"
A41 B41 A43 R43 A41 B4/!A43 B43 Aeon Bcon Aeon Bcon fbf A41 R41 thn A43 R43 thn A41 B41 thnlA43 8 4 3 thn
Fruit bodies
~
+ + ~
-
Aerial mycelium
+
+ i ?
+ ~
mRNA abundance' Scl
sc4
sc3
ND" ND 30.6 34.2 ND ND ND ND
3.6 5.1 98.4 96.2 2.7 ND ND ND
49.5 63.5 41.4 36.6 84.0 2.8 ND ND
All strains were co-isogenic cxcept for the genes indicated and grown under the same conditions for four days in surface culture. Abundance of mRNA is given as percentage of the total R N A (. lo3). The maximum variation in mRNA values for replicate cultures was 5%. Indicates that the mRNA was not detectcd. i.e. an abundance of less than 0.5 . lo-'%.
''
emergent structures, hydrophobins accumulate in the hyphal walls as sodium dodecyl sulphate-insoluble complexes. The Sc3 hydrophobin accumulates in walls of aerial hyphae, while the Sc4 hydrophobin (and probably the Scl hydrophobin) accumulates in walls of hyphae that constitute the fruit body. Solubilization and dissociation of hydrophobin complexes from walls could only be achieved by extraction with agents such as formic acid-performic acid and trifluoroacetic acid. This emphasizes the importance of hydrophobic interactions between hydrophobins in keeping them insoluble in the wall; however, other interactions cannot be excluded Hydrophobins constitute some 6 8 % of all proteins synthesized at the time of emergent growth and are likely to play important morphogenetic roles. The most water-repellent hydrophobin, namely pSc3, could be necessary for formation of a water-impermeable hydrophobic coating on aerial hyphae. Possibly it is a constituent of a layer composed of small rods, spaced about 10 mm, seen at the surface of hyphal walls in S . commune (Wessels et al., 1972). Such rodlets are generally seen on air-exposed surfaces and are thought to consist of protein (Cole, 1973; Cole et al., 1979). They are believed to confer hydrophobicity to the spore surface, which is necessary for spore dispersal, because conidiospores of a mutant of Neurospora CYUSSU without rodlets were easily wettable (Beever and Dempsey, 1978). Such a mutant lacking spore rodlets was recently obtained from Aspergillus nidulans by disruption of a gene for which a developmentally regulated transcript had been cloned (Stringer et al., 1991). Interestingly,
I RUITIN(i IN T H F I I I C H F R I l J N G I
177
the gene inactivated was shown to encode a protein with a clear homology to hydrophobins from S. commune. Hydrophobin genes thus seem to be evolutionarily conserved in basidiomycetes and ascomycetes, which points to their biological importance. Unlike the Sc3 gene, the Scl and Sc4 genes are only activated in the dikaryon, and at least the Sc4 hydrophobin accumulates as an insoluble complex in walls of fruit-body hyphae which, however, contain very little Sc3 hydrophobin (Wessels et al., 1991a). The functional significance of this differential distribution of hydrophobins is not yet clear. With regard to protein structure, pSc3 is most hydrophobic and lacks N-glycosylation sites whereas pScl and pSc4 are somewhat less hydrophobic and contain putative Nglycosylation sites. If the Sc3 hydrophobin forms an impermeable coating on individuallygrowing aerial hyphae, then perhaps the Scl and Sc4 hydrophobins fulfil an additional task in aiding interactions between hyphae in fruit bodies. Aerial hyphae and fruit bodies are completely dependent on translocation of water and building materials from submerged assimilating mycelium or from reserves stored therein. Inactivity of hydrophobin genes during the early growth phase of mycelia (Mulder and Wessels, 1986) may ensure that at least a minimum amount of assimilating mycelium is formed before emergent structures can appear. The first developmental switch leading to emergent growth would be activation of these genes, Sc3 in the monokaryon and the dikaryon, Scl, Sc4 and other known fruit-body associated genes in only the dikaryon. Small hydrophobins are excreted into the culture medium and I and my colleagues hypothesize that this potentiates mycelium to form aerial structures. Hydrophobins are possibly excreted at hyphal tips (Wessels, 1990) and, when these tips break through the substrate-air interface, hydrophobins can no longer diffuse into the medium and therefore accumulate in the wall, where they form insoluble complexes. The wall would thereby acquire a hydrophobic surface and affected hyphae would be destined for growth into the air. Alternatively, or in addition, some other gene product and/or some environmental signal, such as a high redox potential, is required to make the switch from free diffusion of hydrophobins into the medium to incorporation of hydrophobins into walls. After emergence of aerial structures, the level of Sc3 mRNA drops but Scl and Sc4 mRNAs remain at high levels in developing fruit bodies (Mulder and Wessels, 1986; Ruiters and Wessels, 1989b). Therefore, in emerging fruit bodies, a third molecular switch must operate to silence the Sc3 gene. B. CYCLIC
AMP
Roles for cAMP in fungal metabolism have been reviewed by Pall (1981). A role for cAMP in fruiting was discovered by Uno and Ishikawa (1971,
178
1 G
H WFSSFIS
1973) because it acted as a substance inducing fruiting in Coprinus cinereus. This early work (reviewed by Uno and Ishikawa, 1982) concerned monokaryons, some of which could be induced to fruit by addition of low concentrations (greater than 2 . 10 M) of CAMP, the active principle in fruiting-inducing extracts from fruit bodies of various species. Monokaryons of C. cinereus which could be induced to fruit were calledfis+ to distinguish them fromfis' strains which fruited without addition of cAMP and@- strains which never fruited. The fis+ strains contained low levels of endogenous cAMP compared with fisc strains, apparently due to their having a high phosphodiesterase activity, whereas fis- strains seemed to lack both adenylate cyclase and phosphodiesterase activities. In all strains a high level of a CAMP-dependent protein kinase activity was found to be associated with the fruiting response. Protein phosphorylation, induced by CAMP, was linked to activation of glycogen phosphorylase and inhibition of glycogen synthase, as in animal systems. This would mobilize glycogen as a carbohydrate reserve in the mycelium to activate growth of fruit bodies. Evidence for such a view has also been contributed by Kuhad et al. (1987). In a later series of papers, it was shown that cAMP metabolism is also important in dikaryotic fruiting and that CAMP-dependent protein phosphorylation is controlled by mating-type genes (Swamy et al., 198Sa). At the time of fruiting in the A+ B{ dikaryon of C. cinereus, levels of cAMP were 2-3 times higher than in the parent non-fruiting monokaryons. The fruiting Acon Bcon dikaryon mimicked the heterokaryotic dikaryon in this respect. Neither the A#=Bx and A x B f nor Acon Bx and A x Bcon heterokaryons fruited, and they all resembled non-fruiting monokaryons with respect to cAMP levels and activities of adenylate cyclase and phosphodiesterase. In addition, of all of these strains only A#=B{ and Acon Bcon strains contained a CAMP-receptor protein and a high CAMPdependent protein kinase. Only in cell-free extracts of these dikaryons and in the fis' monokaryons could a 46 kDa protein be detected which was phosphorylated in the presence of cAMP (Swamy et al., 198%). In these papers, a role for the kinase in glycogen metabolism was not stressed, but the CAMP-dependent kinase activity was linked to inactivation of NAD+dependent glutamate dehydrogenase. However, in contrast to NADP+dependent glutamate dehydrogenase, this NAD+-dependent enzyme had not been shown to play a role in fruiting of C. cinereus (Moore, 1984). Cyclic AMP has also been reported to stimulate fruit-body formation in Phanerochaete chrysosporium (Gold and Cheng ,1979) while high endogenous levels of cAMP were found to be associated with fruiting in Lentinus edodes (Takagi et al., 1988). In S . commune, a relation was found between high levels of endogenous cAMP and fruiting (Schwalb, 1978a) but exogenously added cAMP did not stimulate fruiting. Instead, high concentrations
FRIJITING IN T H E H I G H E R FIJNGI
179
M) resulted in abnormal fruit bodies, phenocopies of the dominant bse mutant (Schwalb, 1978b). In agreement with a role for cAMP in fruiting, Yli-Mattila (1987) saw an increase in the level of cAMP after inducing fruiting in dikaryons of S. c o m m u n e with UV irradiation ( 3 2 0 0 nm).
C. PHENOL OXIDASES
Fungal phenol oxidases (Bell and Wheeler, 1986) have been well studied in relation to developmental processes in As. nidulans (a subject reviewed by Clutterbuck, 1990). With respect to fruiting in basidiomycetes, attention has been given to a very active tyrosinase (monophenol oxidase) in fruit bodies of Agaricus bisporus, an enzyme which is responsible for browning after bruising and probably for pigmentation of spores. Its natural substrate in A . bisporus has been identified as y-glutaminyl-4-hydroxybenzene(Stiissi and Rast, 1981; Rast et al., 1981). However, with respect to fruit-body formation, attention has primarily focused on laccases (diphenol oxidases, EC 1.10.3.2). In S. c o m m u n e (Leonard and Phillips, 1973; Phillips and Leonard, 1976) and L. edodes (Leatham and Stahmann, 1981; Leatham, 1985), extracellular and intracellular laccase activities have been seen to rise during fruiting. Ross (1982b), working with C. congregatus, failed to detect extracellular laccase but noted a general increase in intracellular laccase just before fruiting. However, detailed measurements of laccase activities in diffcrent growth zones indicated that laccase is probably not involved in development of fruit-body primordia although an involvement in the light-induction process was suggested. Also, de Vries et al. (1986) failed to find laccase in the medium during fruiting of a dikaryon of S. commune grown on minimal medium. However, in contrast to the parent monokaryons, this dikaryon produced high laccase activities in the medium when grown at 30°C in the dark, conditions which prevent fruiting. An antibody raised against this laccase reacted with a laccasc present in hyphal walls from fruit-body primordia, which raised the possibility that laccase may be involved in oxidative cross-linking of hyphae in fruit bodies (Wessels et al., 1985). This explanation was proposed by Leatham and Stahmann (1981), who, likewise, detected laccase activity in hyphal walls of fruit bodies of L. edodes. It is possible that phenolic compounds become covalently linked to cell-wall constituents or to mucilaginous material seen interposed between fruit-body hyphae (van der Valk and Marchant, 1978) and that oxidative cross-linking of these compounds helps to strengthen interactions between hyphae. Cross-linking of polysaccharides by oxidation of phenolic compounds generally occurs in plant cell walls (Fry, 1986). In basidiomycete fruit bodies, a role for laccases in oxidative cross-linking was first proposed for polypores (Bu’Lock, 1967; Bu’Lock and Walker,
180
J G I[
WFSSI-LS
1967), which become pigmented and woody by oxidation of phenolic compounds, and which may be synthesized by the fungus or derived from degradation of plant lignin. Nothing is known about the natural substrate for wall-bound laccases in S . commune and L. edodes. Considering a role for laccases in the culture medium, it should be noted that so little is known about the mechanism of lignin degradation that, besides lignin peroxidase and manganese peroxidase (Odier, 1987; Rroda et al., 1989; Gold et al., 1989), there is still a possible role for laccase in this process (Kawai et al., 1988). High laccase activities in the medium following growth of many wood-rotting basidiomycetes could thus be related to the ability of these fungi to degrade lignin. For instance, it has been shown that A . bisporus, grown on compost or synthetic media, excretes up to 2% of total cellular protein as laccase (Turner, 1974; Wood and Goodenough, 1977). Laccase activity increased until fruit-body formation and then dropped, mainly due to enzyme inactivation (Wood, 1980). This was correlated with active lignin degradation in compost, during vegetative growth and before onset of fruiting (Wood et al., 1990; Durrant et al., 1991). During cropping, lignin degradation slowed down while cellulose degradation became prominent, possibly providing carbohydrates for growth of fruit bodies. VI. Environmental Control of Fruiting
Numerous studies have been devoted to the effects of environmental conditions on initiation and further development of fruit bodies in basidiomycetes, particularly with respect to the effects of light, temperature and ambient carbon dioxide concentrations. These studies are, of course, relevant for establishing optimal conditions for cultivation of edible mushrooms, but often they have also been conducted in the hope that they would lead to a fundamental understanding of fruit-body development. Unfortunately, as in plants, a study of the modulation of development by changing environmental conditions or by adding chemical compounds has rarely led to a better understanding of the internal processes involved. Exceptions are the discovery of some growth regulators in plants and the role of CAMP in basidiomycete fruiting. As can be expected, ecophysiological parameters are very much species-specific, sometimes strain-specific, because they probably arose as adaptations to meet specific requirements of the organism in its habitat. A . FRUITING-INDUCING SUBSTANCES
Much effort has gone into identifying the chemical nature of compounds in crude extracts from various sources that stimulate fruiting, in the hope
FRUITIN(; IN T H F HIGHFR FUNGI
181
that finding such a compound would lead to a better understanding of the fruiting process. The only instance in which this hope has been fulfilled appears to be the identification of CAMP and AMP (an inhibitor of phosphodiesterase) as active principles in extracts from various fruit bodies which induced fruiting in monokaryons of Coprinus cinereus (Uno and Ishikawa, 1971, 1982). Extracts from Cladosporium cladosporioides and fruit bodies of Agaricus bisporus induced fruit-body formation in monokaryons of Schizophyllum commune but the active principle was not identified (Leonard and Dick, 1968; Rusmin and Leonard, 1978). On the other hand, Kawai and Tkeda (1982, 1983) reported that cerebrosides from various sources, including S. commune, stimulated dikaryotic fruiting in S. commune. A detailed analysis of these natural and synthetic cerebrosides identified the functional moieties as sphingolipids (Kawai et al., 1986) but how these substances interact with the fungus to elicit the fruiting response remains unknown. Possibly, they interact with the plasma membrane, as has been suggested for the effects of phospholipids and sterols on sexual reproduction in oomycetes (Kenvin and Duddles, 1989). Also, the mechanism by which anthranilic acid stimulates fruiting in Flavolus arcularius and replaces the stimulating effect of light (Murao et al., 1984) is unknown. B . LIGHT, TEMPERATURE AND CARBON DIOXIDE
Light has been most intensively studied as a modulating factor. The immense literature on fungal photobiology has been reviewed by Tan (1978) and Durand (1985). Aspects specifically related to fruit-body development are treated in, among other publications, Eger-Hummel (1980), Durand (1985), Manachere (1980, 1988) and Manachere et al. (1983). Although some species, notably A . bisporus and some variants of S . commune (Yli-Mattila et al., 1989b), d o not require light for fruiting, light is mostly necessary for fruit-body development. The effect of light on S. commune is limited to induction of primordia (Perkins, 1969; Raudaskoski and Yli-Mattila, 1985); often illumination for a few minutes suffices. This inducing effect of light can sometimes be by-passed, such as by lowtemperature treatment (Tsusue, 1969) and nutrient starvation (VerrinderGibbins and Lu, 1984) in C. cinereus. In, for example, Coprinus congregatus (Manachere, 1970), C . cinereus (Tsusue, 1969; Kamada et al., 1978) and F. arcularius (Kitamoto et al., 1974), light is also required for normal stipe and pileus (cap) development; in darkness, long stipes without caps develop. However, continuous light inhibits elongation of stipes, normal pileus development and sporulation; an interrupting dark period is required for normal morphogenesis. This is why these fungi are mostly cultivated in an alternating light-dark regime (Fig. 8). The requirement for a dark
182
J . G . H. WESSELS
I I Phases of development
, II
111
1
I
IV
,
10 Days P. S. D.
d3 1e
I
II
no initiation
i '
1
'
4
'
5
'
6
'days
FIG. 8. Effects of light-dark periods on development of fruit bodies of Coprinus congregatus. After vegetative growth in the dark for 10 days, fruit-body primordia are induced by light. Normal development occurs if, at the previous 36 hour stage (the end of phase I), a dark period is provided followed by light (a, b, c). If the primordia are placed in continuous darkncss before the dark-sensitive phase (phase TI), no caps develop; only elongation of stipes occurs (d, e). If the dark pcriod in phase I1 is not followed by a light period in phase 111, cap development occurs but there is no normal sporogenesis o r autolysis (f, g). In continuous light, formation of fruit-body primordia is induced but stipe elongation, cap dcvclopmcnt, meiosis and sporogenesis are all inhibited (h, i). In continuous darkness, fruit-body primordia are not formed (j). A t thc cnd of phase I , basidia are still binucleate. Normal progression through diploidization and meiosis also rcquircs dark-light periods after phase I. Only phase IV, in which sporulation occurs, is indifferent to light. This diagram is reproduccd by courtesy of G. ManachCrc.
PRLJITING IN THF H1GHk.K PUNGI
183
period can, however, be by-passed by lowering the temperature (Tsusue, 1969; Robert and Durand, 1979). Action spectra for the light effects have been established for S. commune (Perkins and Gordon, 1969; Yli Mattila, 1985), F. arculurius (Kitamoto et al., 1972, 1974), Psilocybe cubensis (Badham, 1980), C. congregatus (Durand and Furuya, 1985) and Pleurotus ostreatus (Richartz and MacLellan, 1987). The spectra show differences, but all exhibit peaks in the UV ( 3 2 W 0 0 nm) and blue (400-520 nm) regions, suggesting that a flavin is acting as a photoreceptor (Tan, 1978). With no fungus has progress been made in isolating the photoreceptor or in elucidating the transduction pathway. It is a general finding (Perkins, 1969; TsusuC, 1969; Ross, 1982a) that light induction of fruit-body primordia cannot occur before cultures are a few days old, whether grown from a mycelial homogenate as a confluent lawn or from a single inoculum as an expanding colony. In this regard, it may be significant that, in S. commune, it has been found that genes for hydrophobins, thought to play a decisive role in emergent growth (Wessels et al., 1991a,b), are delayed in their expression until the mycelium has grown for a few days (Mulder and Wessels, 1986). In colonies of S. commune (Perkins, 1969; Raudaskoski and Vauras, 1982) and C. congregatus (Ross, 1982a; Durand, 1983), it was shown that light induction of primordia occurs in the youngest growth zone, just behind the advancing front of the colony. In S. commune, induction leads to the immediate appearance of primordia. In C. congregatus, some additional stimulus emanating from the whole mycelium is required to realize formation of primordia. Ross (1982a) noted that primordia formed in the growth zone in which they were induced but only after the colony front had reached the edge of the Petri dish. In contrast, Durand (1983) saw immediate formation of primordia after light induction in the growth zone of a half-colony growing on non-nutritive medium while the other part had already fully colonized a nutrient medium. It is thus possible that, in S. commune, induced initials can immediately act as a sink for translocation of materials from the vegetative mycelium (Fig. 1) whereas, in C. congregatus, vegetative mycelium has to be checked in its growth before such a translocation system becomes operative. In other words, the two species could differ in the competitive value of vegetative mycelium as compared with fruit-body primordia for translocated materials. With respect to the immediate cytological effects of environmental factors, the studies of Raudaskoski and her colleagues on formation of fruit-body primordia in S. commune (Raudaskoski and Viitanen, 1982; Raudaskoski and Salonen, 1984) are noteworthy. Light caused formation of short heavily branched hyphal compartments, an effect completely absent from sealed cultures which do not develop primordia, possibly due to
184
J . G H. WFSSELS
accumulation of carbon dioxide. The effect of sealing cultures on fruitbody formation in S . commune was originally detected by Niederpruem (1963). However, some caution is necessary because this fungus also releases large amounts of methylmercaptan (Birkinshaw et al., 1942) to the extent that nearly all of the sulphate in the medium that is not assimilated is converted into this volatile compound (0.M. H. de Vries, unpublished data). In C. cinereus, hyphal aggregates, which arise in complete darkness, all develop into sclerotia, unless illumination induces a few of these hyphal aggregates to embark on a route to formation of fruit-body primordia (Moore et al., 1979). Important questions in both instances are which hyphae perceive the light stimulus and why only certain hyphae seem to react. Experiments using narrow-beam illumination, as conducted by Galun (1971) with Trichoderma sp., have, as far as I know, not been carried out with these basidiomycete systems. It may be that only hyphae that start the morphogenetic process contain the light receptors or that other parts of the mycelium perceive the light and relay the stimulus to the morphogenetically active part, as in flower induction by photoperiod in plants. Because numerous hyphal aggregates arise, many more than can eventually form mature fruit bodies, it is possible that stochastic processes and competition between emerging structures for translocated materials determine which initials will grow into primordia and subsequently into mature fruit bodies. The light stimulus, combined with sufficient aeration, probably leads to activation of specific genes. Only in S . commune have genes been identified that play a role in fruiting. Between 6 and 24 hours after illumination of dark-grown colonies, levels of mRNAs for these genes rise (Yli-Mattila et al., 1989a). However, because these genes are also activated in the dikaryon when fruiting is suppressed by darkness or by a high concentration of carbon dioxide (Wessels et al., 1987) while fruit-body primordia maintain high concentrations of these mRNAs, in contrast to the vegetative mycelium (Mulder and Wessels, 1986; Ruiters and Wessels, 1989b), increases seen after illumination were probably a consequence rather than a cause of formation of fruit-body primordia. In Neurospora crassa fast reactions of blue light on gene expression have been observed (within 2-45 minutes) for some genes with unknown functions (Sommer et al., 1989; Nawrath and Russo, 1990) and for genes encoding enzymes of carotenoid biosynthesis (Nelson et al., 1989; Schmidhauser et al., 1990). In basidiomycete fruitbody initiation the most rapid effects of blue light detected were increases in contents of CAMP in C. cinereus (Uno et al., 1974) and S. commune (Yli-Mattila, 1987).
~KLlI'~ING IN 'IHF HIGHFR FUNGI
185
VII. Rapid Expansion of Fruit Bodies A . METABOLIC CHANGES
In Schizophyllum commune, expansion of the cup-shaped fruit-body primordia (Wessels, 1965; van der Valk and Marchant, 1978; Raudaskoski and Vauras, 1982) is entirely due to continued apical growth and differentiation of hyphae in the primordium. As shown by micro-autoradiography, radioactive N-acetylglucosamine was incorporated only into chitin at hyphal apices and no evidence for diffuse extension growth was obtained (M. S . Manocha and J. G. H. Wessels, unpublished data). Little is known about biochemical processes that take place during expansion of fruit bodies. During transition from vegetative growth to fruit-body development, the respiratory quotient of cultures changes from above two to around unity, indicating the operation of purely oxidative metabolism in the fruit bodies but some fermentative activity in the substrate mycelium (Wessels, 1965). Of enzymes involved in respiratory activity, Schwalb (1974) noted a marked decrease in the activity of phosphoglucomutase in fruit bodies. This was linked to the appearance of specific proteases in fruit bodies which inactivated the enzyme (Schwalb, 1977). The significance of these processes for outgrowth of fruit bodies remains unknown. In agarics, fruit-body primordia differentiate all tissues present in mature fruit bodies and outgrowth of primordia is mainly due to rapid expansion of existing structures (Reijnders, 1963; Reijnders and Moore, 1985). For C. cinereus, cytological details of formation and expansion of fruit-body primordia have been described (Matthews and Niederpruem, 1973; Moore et al., 1979). An attempt has also been made to monitor electrophoretic protein patterns during fruit-body development in C. cinereus (Moore and Jirjis, 1981) but this was less than successful, probably due to problems with proteolytic activities in the protein extracts (de Vries et al., 1980). Rapid elongation of stipes of agaric fruit bodies has received much attention. The upper or upper-middle parts of the primordial stipe elongate to a much greater extent than the base, and this has been correlated with a parallel increase in cell length in Coprinus radiatus (Haffner and Thielke, 1970; Eilers, 1974). However, cell elongation may be followed by septation, maintaining a constant unit cell length as in Agaricus bisporus (Craig et al., 1977) and Flammulina velutipes (Wong and Gruen, 1977). Hyphal growth, of course, depends on growth of hyphal walls. Although, during stipe elongation, there may be some decrease in the dry weight of the expanding wall for each unit length of stipe (Kamada and Takemaru, 1977), there is continuous addition of wall material along the whole length of the elongating hyphae as shown by autoradiography of chitin synthesis in A.
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bisporus (Craig et al., 1977) and C. cinereus (Gooday, 1982). In both A . bisporus (Wood and Hammond, 1977) and C. cinereus (Gooday et al., 1976) stipe elongation was effectively prevented by applying polyoxin D, an inhibitor of chitin synthase. Although both the wet and dry weight of the expanding fruit bodies have been observed to increase, implying that not only water enters the fruit bodies, it has been shown for some species that detachment from the mycelium of the fruit bodies, beyond a critical stage of development, still permits stipe elongation and cap expansion, as shown for A . bisporus (Turner, 1977), C . radiatus (Eilers, 1974) and C. cinereus (Gooday, 1974; Cox and Niederpruem, 1975). This capacity may depend on the ability of young fruit bodies to accumulate reserve polymers that can be converted into osmotically active solutes needed for expansion. Accumulation of glycogen at the base of the primordial stipe of C. cinereus and its disappearance during expansion have been demonstrated (Moore et al., 1979). In most fungi, stipe elongation has been shown to depend on the presence of the pileus, at least during the early stages of elongation. Numerous surgical studies with different species showing this dependence, and the failing attempts to isolate growth regulators from the pilei, have been reviewed adequately by Gruen (1982). Coprinus cinereus is remarkable because decapitated stipes, measuring 5-10% of their final lengths, can elongate normally even when detached from the mycelium (Gooday, 1974; Cox and Niederpruem, 1975). As to the identity of the osmoticum that causes influx of water and maintenance of turgor pressure during fruit-body expansion, variation exists among species. In A . bisporus, mannitol is most important; it may contribute 4&50% to the dry weight of the mushroom (Hammond and Nichols, 1975; Ruffner et al., 1978). Mannitol is synthesized from fructose and NADPH by mannitol dehydrogenase, which has been recently purified (Pfyffer et al., 1989). The NADPH is produced in the first two steps of the pentose phosphate pathway (Dutsch and Rast, 1972), which appears to be more active in fruit bodies than in vegetative mycelium (Hammond, 1977, 1985). In C. cinereus, evidence has been presented for different osmotica in the stipe and the cap. In the elongating stipe, trehalose accumulates to the extent of 18% of the dry weight (Rao and Niederpruem, 1969). In the cap, however, urea has been implicated as the main osmoticum, its accumulation in the cap being accompanied by high activities of enzymes of the urea cycle and low activity of urease (Moore, 1984). Also, an NADPH-dependent glutamate dehydrogenase was shown to be specifically present in the cap, the NADH-dependent enzyme being present in the stipe and vegetative mycelium (Moore, 1984). The NADPH-dependent
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enzyme was thought to play a role as a scavenger for ammonium ions, which are a powerful inhibitor of pileus expansion and sporulation (Moore et al., 1987). B . HYPHAL-WALL EXPANSION
Fungal hyphae generally grow by apical extension of their walls (Wessels, 1986, 1990) so that diffuse extension growth of walls in expanding fruit bodies is exceptional and more like wall growth in plant tissues. Diffuse extension growth was also noted in the subapical part of elongating sporangiophores of Phycomyces sp. Roelofsen and Houwink (1953) showed that primary walls of such sporangiophores and of plant hairs have a similar fibrillar architecture on which they based their multi-net growth theory. According to this theory, new microfibrils of cellulose (plants) or chitin (fungi) are deposited on the inner surface of the wall in a transverse (circumferential) direction. During elongation of the wall, these microfibrils are re-oriented in a longitudinal (axial) direction while new microfibrils continue to be deposited on the inside in the transverse direction. The general occurrence of this mechanism has been questioned (Roland and Wan, 1979) but it is still believed to hold for at least some growing plant cells. In these plant cells, auxin is believed to effect acidification of the wall, activating hydrolytic enzymes which loosen the matrix thereby allowing expansion of the wall (Cleland, 1980). However, the general occurrence of enzymic breakage of bonds in the matrix polymers serving as the basis for auxin-induced elongation in plant cell walls has also been questioned (Kutschera et al., 1987). It is thus understandable that even more uncertainty exists as to the mechanism of wall extension in agaric fruit bodies, which has received much less attention than the mechanism of wall extension in plant cells. Kamada and Takemaru (1977) investigated mechanical properties of hyphal walls derived from elongating stipes of C. cinereus. They found that the osmotic potential of the cytoplasm remained constant, but that there was a positive correlation between elasticity of the walls (measured by the extent of shrinkage after plasmolysis), extensibility and minimum stressrelaxation time of the walls (measured by mechanically stretching the walls) and the rate of elongation which these walls could sustain in the growing stipe. Apart from being visco-elastic, these walls must expand more in the longitudinal than in the transverse direction, although stresses in the wall due to turgor pressure are expected to be twice as large in the transverse than in the longitudinal direction. One may therefore expect to find an anisotropic deposition of stress-bearing fibrils, as in plant cells. Indeed, in stipes of C. cinereus, Gooday (1979) observed a strong anisotropic
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component in the wall using polarization microscopy while, in the electron microscope, extracted walls showed predominantly transversly oriented microfibrils. However, no change was seen in microfibril orientation before and after stipe elongation, refuting a mechanism as proposed by the multinet growth theory. In A . bisporus, Mol et af. (1990) also observed an anisotropic component in the walls of stipe hyphae, albeit much weaker than that in C. cinereus. In contrast to substrate hyphae, chitin in the wall of stipe hyphae of A . bisporus was mainly non-fibrillar but, nevertheless, chitin chains appeared to run predominantly in the transverse direction. Enzymic dissolution of the p-glucan component of the wall caused longitudinal contraction, indicating the presence of this glucan in between circumferentially running chitin chains. Chitin and p-glucan were also much more susceptible to enzymic degradation in stipe walls than in walls from substrate hyphae, suggesting the presence of fewer hydrogen bonds between individual glucan chains and chitin chains. However, although the wall architecture of stipe hyphae and substrate hyphae with their randomly running chitin microfibrils was very different, the gross chemical composition and the presence of covalent linkages between p-glucan and chitin were much the same (Mol and Wessels, 1990). In contrast, Marchant (1978) showed a four-fold increase in the content of chitin in walls from stipes of C. cinereus when compared with walls from the substrate mycelium. On the basis of their observations, Mol et af. (1990) proposed a model of diffuse wall extension in fruit-body stipes as shown in Fig. 9. Walls of substrate hyphae grow by apical extension only. Chitin and P-glucan chains, after being extruded into the wall at the apex, become tightly linked to each other by covalent linkages and hydrogen bonds (Wessels, 1986,1990). It is assumed that, in fruit-body primordia, covalent linkages between chitin and p-glucan are likewise formed but that hydrogen bonding between pglucan chains is weaker, possibly due to the higher incidence of p-(1+6) linkages relative to p-(1+3) linkages in this glucan in walls from stipe hyphae as compared with walls from substrate hyphae (Mol and Wessels, 1990). Due to turgor pressure and the prevailing stress pattern in the wall, hydrogen bonds between glucan chains in stipe hyphal walls would easily break and reform, allowing the originally randomly deposited chitin chains to become transversely aligned. Under turgor pressure, the wall would now yield further, mainly in a longitudinal direction, while hydrogen bonds between glucan chains break and form and new chitin and glucan chains are intercalated in the wall. Such a mechanism would ensure maintenance of transversely oriented chitin chains during elongation. After elongation ceased, it was seen that, at the inner surface, a wall had been deposited with an architecture similar to that of substrate hyphae. This layer probably prevented further elongation.
II e
t
extensive hydro en bonding among glucan c L s
weak hydrogen bonding amona alucan chains
t
EXPANSION
addition of new wall polymers reorientation
AXIAL EXPANSION ONLY
FIG. 9. A hypothetical scheme showing possible interactions between P-glucan chains (thin lines) and chitin chains (thick lines) in walls of apically growing substrate hyphae and in walls of diffusely elongating hyphae in the mushroom stipe of Agaricus bisporus. Weak hydrogen bonding betwcen glucan chains in the stipe hyphal walls enables chitin chains to reorient under turgor pressure and permits expansion in the axial direction only. From Mol et al. (1990).
In hyphal walls derived from the stipe of C. cinereus, the existence of covalently linked chitin-glucan complex has also been demonstrated (Kamada and Takemaru, 1983). A relationship between wall metabolism as it occurs during apical extension growth and during diffuse extension growth in the stipes was suggested by the finding that many temperaturesensitive mutants restricted in apical extension also exhibited diminished stipe elongation (Kamada et al., 1984). However, Kamada and his
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coworkers (Kamada et al., 1980, 1982) also showed a relationship between stipe elongation and the activities of wall-lytic enzymes. Such enzymes, including chitinase, had previously been shown to be deposited in vacuoles (lysosomes) and to be released into the walls at the time of autolysis of the fruit body of C. cinereus (Iten and Matile, 1970). In contrast to A . bisporus, fruit bodies of coprini lyse rapidly after elongation has occurred. Invoking lytic activity in the cutting of glucan cross-links in growing stipes of C. cinereus, Kamada et al. (1991) postulated a model otherwise similar to that described in Fig. 9. It is possible that, in C. cinereus and in agarics in general, wall-lytic enzymes play a role in loosening the matrix between chitin chains, allowing realignment of these chains as envisaged in Fig. 9. However, any theory involving wall-lytic enzymes in wall elongation requires the establishment of a mechanism that controls the activity of such enzymes.
VIII. Biotechnology
Unlike zygomycetes and ascomycetes, basidiomycetes have played a minor role in industrial mycology, unless cultivation of mushrooms, an agricultural activity with a high technical input, is also considered an industrial activity. All cultivated mushrooms, among which Agaricus bisporus and A . bitorquis (white button mushrooms), Lentinus edodes (shii-take), Pleurotus ostreatus (oyster mushroom), Volvariella volvacea (straw mushroom) and Flammulina velutipes are commercially most important, are grown on lignocellulose substrates and currently provide the only means of converting this abundant material into food. Methods and problems concerned with cultivation of these and other commercially important mushrooms have been extensively reviewed (Chang and Hayes, 1978; Flegg et al., 1985; Wuest et al., 1987) and will not be further considered here. A major research effort is directed towards elucidation of the unique capacity of these fungi to degrade lignocellulose but essentially only one non-edible basidiomycete has been studied in this respect, namely Phanerochaete chrysosporium (Odier, 1987; Broda et al., 1989; Gold et al., 1989). The aim of this research is to develop biotechnological procedures for wooden-fibre processing, including industrial use of the enormous amounts of lignin, which at present is merely a waste product of the pulping industry. Although genes for lignin peroxidase and manganese peroxidase, believed to play a pivotal role, have been cloned, lignin degradation is still incompletely understood. The commercially most valuable mushrooms-those mostly appreciated for their taste-are borne on mycelia that live in mutual symbiosis with the roots of trees. These mycorrhizal mycelia can be grown, albeit slowly,
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without any association with trees but attempts to fruit them under such conditions have all failed. This is not due to any lack of effort by experimenters. Success in cultivating the chanterelle (Cantharellus cibarius), ctpe (Boletus edulis), matsutake (Tricholomn matsutake) or truffles (Tuber melanosporum, T . rnagnatum) would yield considerable financial revenues. However, the relationship between trees and these biotrophic fungi is so poorly understood at the moment that it appears impossible to imitate the favourable conditions of the symbiotic association; in addition, the trial and error methods employed have led to much frustration. However, it should be realized that even the fruit bodies of saprotrophic A . bisporus, which has been cultivated on compost since the days of Louis XIV of France, and now has an annual world-wide production exceeding lo6 tonnes (Hayes and Nair, 1975; Flegg et al., 1985), cannot be obtained in agar cultures although, under these conditions, some strains do form small aggregates, presumably fruit-body primordia (Wood, 1976; Elliot and Wood, 1978). Formation of mature mushrooms is possible in axenic cultures, but then the fungus must be grown in compost and a casing layer must be applied as in commercial practice (Durrant et al. , 1991). Conceivably, basic studies as reported in this review will ultimately show how to overcome problems with cultivating mycorrhizal mushrooms. The recognition that formation of mycorrhiza (ectomycorrhiza) is of crucial importance for growth of trees in temperate forests (Harley and Smith, 1983; Read, 1984) also opens as yet unexplored possibilities for optimizing this association (Kendrick and Berch, 1985), particularly in view of the decreasing vitality of forest trees in polluted areas. The relatively slow advent of molecular genetics as applied to fruiting basidiomycetes, and thus the possibility of performing genetic manipulations with these organisms, can be illustrated by the fact that only recently have DNA-mediated transformation systems become available for Schizophyllurn commune (Munoz-Rivas et al., 1986), C. cinereus (Binninger et al., 1987), the lignolytic P . chrysosporiurn (Alic et al., 1989) and the mycorrhizal Laccaria laccata (Barrett et al., 1990). In these fungi, with the exception of L . luccata, homologous nutritional genes were used to select for transformants. For biotechnological work it would be more useful to avoid nutritional mutants and to be able to select for transformants as, for example, on the basis of antibiotic resistance. This has apparently been possible with L. laccata (Barrett et al., 1990) by introducing hygromycin B resistance, using the bacterial hygromycin phosphotransferase ( H P T ) gene fused to the glyceraldehyde-3-phosphate dehydrogenase (GPD) promotor from Aspergillus nidulans constructed by Punt et al. (1987). In S . commune, however, use of this plasmid led to heavy methylation of the HPT gene and flanking vector sequences, and was accompanied by very poor expression of this gene (Mooibroek et al., 1990).
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The commercially most important mushroom, A . bisporus, is notoriously recalcitrant to the application of genetic methods. Because it is a secondary heterothallic species, a homokaryotic stage is missing; spores are heterokaryotic and grow directly into a fruiting mycelium. However, by isolating and regenerating protoplasts, it is now possible to recover homokaryons routinely, for the purpose of hybridizations between strains (Castle et al., 1987; Sonnenberg et al., 1988). It would be particularly useful if a transformation system for this species was available. However, all attempts so far to develop such a system have failed (Challen et al., 1991; Royer and Horgen, 1991). Because good nutritional markers are not available, attempts to obtain expression of a foreign gene, such as that encoding hygromycin B resistance, have formed the general approach. One reason for the lack of success may be that, as in S . commune, heterologous DNA becomes heavily methylated and is poorly expressed, unless sufficient homologous sequences are included in the transforming DNA (Mooibroek et al., 1990). Possibly, the use of homologous strong promotors, such as that from a recently cloned G P D gene of A . bisporus (Harmsen et al., 1991a), will lead to successful expression of genes introduced into this species. Molecular studies with A . bisporus have also dealt mainly with the structure of the mitochondria1 genome (Hintz et al., 1988), use of restriction fragment length polymorphism for identifying strains and hybrids (Summerbell et al., 1989), and molecular analysis and sequencing of a double-stranded RNA virus (Marino et al., 1976; Harmsen et al., 1989, 1991b) which can cause a large loss of crop. This work has led to the development of a diagnostic test for early detection of the disease by molecular hybridization.
IX. Conclusions Formation of large fruit bodies in basidiomycetes and some members of the ascomycetes can be considered as a special case of emergent growth in fungi. As in other fungi, development starts with growth of an extensive mycelium that colonizes its substrate, followed after some time by emergence of aerial hyphae. In fruit-body formation, such emergent hyphae cease growing as individual hyphae and become engaged in multicellular morphogenesis. They continue to grow as a branching mycelium, but now away from the substrate, forming a pseudotissue in which cell differentiation occurs and shape is generated. As in other multicellular systems, next to nothing is known about the cause of morphogenesis. Therefore, this review has been mainly concerned with initiation of fruiting, the dependence of
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developing fruit bodies on the substrate mycelium for mass increase, and the rapid expansion of some types of fruit bodies, based on cell enlargement rather than formation of new tissues. Evidence has been presented for the role of mating-type genes as master regulators within a regulatory circuit that provides the genetic basis for fruiting potential. Some of these regulatory genes are being cloned and sequenced, and this work will probably lead to a detailed description of the genetic mechanisms that operate in determining different developmental potentials of two cell types (monokaryon and dikaryon) that essentially contain the same genetic information. Among the genes that are ultimately regulated by the mating-type genes, the family of hydrophobin genes may be of particular importance because they have been related to emergent growth. Also, some of them possibly play a role in interactive growth of hyphae within fruit bodies. Members of this gene family are differentially regulated, and their protein products are deposited into the walls where they may be effective in isolating hyphae from the environment and to enable them to act as powerful sinks for assimilates. Within the mycelium as a whole there appears to be competition for assimilates. Emergence of fruit bodies may slow down growth of the colonizing substrate myceliuni. Sometimes, a factor inhibiting growth of this mycelium may be necessary to effect translocation towards the developing fruit-body initials and permit their growth. After depletion of nutrients, not only is there mobilization of reserve compounds accumulated in the substrate mycelium but also structural components of this mycelium and of stunted fruit-body primordia may be degraded to provide for the needs of the developing fruit bodies. The rise in levels of CAMP often observed to accompany fruit-body initiation may be related to switching on of these degradative processes. Although extensively studied, the effects of environmental factors on fruiting are poorly understood. Factors such as light and concentration of carbon dioxide do not seem to regulate directly the genes, such as those for hydrophobins, which up to now have been implicated in the fruiting process. Environmental stimuli may have a direct effect on hyphal branching of emerging hyphae in formation of hyphal aggregates. Apart from often triggering initiation of fruit-body development, light and dark periods have also been observed to be necessary for further development of agaric fruit bodies, but often these effects depend on temperature. All studies agree on the effectiveness of blue-UV radiation but nothing is known about the mechanisms of photomorphogenesis. Expansion of agaric fruit bodies is mainly based on diffuse extension growth of hyphae. In the fruit-body hyphae a low osmotic potential is maintained by formation of low molecular-weight compounds while the
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walls yield to turgor pressure over their whole length. The architecture of the walls of these elongating hyphae principally differs from that of substrate hyphae, which can only grow by apical extension. A model was proposed to explain how the special architecture of the walls of elongating stipes is related to their preferential axial expansion. Only a brief reference has been made to cultivation of edible mushrooms. This is an ancient art, still dominated by empirical methods, which, however, have yielded impressive results. The advent of molecular genetics applied to basidiomycetes promises new ways of breeding and introduction of desirable traits by genetic manipulation. This would be particularly useful for further improvement of the widely cultivated white button mushroom, which offers little genetic diversity and is difficult to breed using classical approaches.
X. Acknowledgements
The author acknowledges illuminating discussions with Alan D. M. Rayner on the problem of emergent growth in fungi. He is also grateful to his colleagues in the Plant Molecular Biology Laboratory for many stimulating discussions and for permission to quote unpublished results. REFERENCES
Ainsworth, G . C. (1962). Nature 195, 1120. Ainsworth, G. C. (1976). “Introduction to the History of Mycology”. Cambridge University Press, Cambridge. Ainsworth, A. M., Rayner, A . D. M., Broxholme, S . J., Beeching, J. R., Pryke, J. A., Scard, P. R., Berriman, J., Powell, K. A , , Floyd, A. 3. and Branch, S . K. (1990). Mycological Research 94, 799. Alic, M.. Kornegay, J. R . , Pribnow, D . and Gold, M. H. (1989). Applied and Environmental Microbiology 55, 406. Badham, E. R . (1980). Mycologia 72, 186. Barrett, V . , Dixon, R. K. and Lemke, P. (1990). Applied Microbiology and Biotechnology 33, 313. Beach, D . and Nurse, P. (1982). Nature 296, 682. Beever, R. E. and Dempsey, G. P. (1978). Nature 272, 608. Bell, A. A. and Wheeler, M. H. (1986). Annual Review of Pkytopatkology 24, 411. Binninger, D. M., Skrzynia, C., Pukkila, P. J. and Casselton, L. A. (1987). E M B O Journal 6, 835. Birkinshaw, J. H., Findlay, W. P. K. and Webb, R. A. (1942). Biochemical Journal 36,526. Bistis, G. N. (1983). Experimental Mycology 7 , 292. Broda, P.. Sims, P. F. G. and Mason, J. C. (1989). Essays in Biochemistry 24, 824. Bromberg, S. K. and Schwalb, M. N. (1976). Journal of General Microbiology 96, 409. Bromberg, S. K. and Schwalb, M. N. (1977). Canadian Journal of Genetical Cytology 19,477. Bull, A. T. and Trinci, A. P. J. (1977). Advances in Microbial Physiology 15, 1 . Bu’Lock, J . D. (1967). In “Essays in Biosynthesis and Microbial Development, E. R. Squibb Lectures on Chemistry and Microbial Products”, pp, 1-18. Wiley, New York.
FRUITING IN TlfE HIGHER FLING1
195
Bu’Lock, J. D. and Walker, D. C. (1967). Journal of The Chemical Society Section C, 336. Burnett, J. H. (1975). “Mycogenetics”. Wiley, London. Casselton, L. A. and Economou, A . (1985). In “Developmental Biology of Highcr Fungi” (D. Moore, L. A. Casselton, D. A. Wood and J . C. Frankland, eds), pp. 213-229. Cambridge University Press, Cambridge. Casselton, L. A,, Mutasa, E. S . , Tymon, A,, Mellon, F. M., Little, P. F. R . , Taylor, S . , Bernhagen, J . and Stratmann, R. (1989). In “Molecular Biology of Filamentous Fungi” (H. Nevalainen and M. Penttila, eds), vol. 6, pp. 139-148. Foundation for Biochemical and Industrial Research, Helsinki. Castle, A. J., Horgen, P. A. and Anderson, J. B. (1987). Applied and Environmental Microbiology 53, 816. Challen, H. P., Rao, B. G . and Elliot, T. J. (1991). In “Genetics and Breeding of Agaricus” (L. J. L. D . van Griensven, ed.), pp. 129-134. Centre for Agricultural Publishing and Documentation, Wageningen. Champe, S. P. and El-Zayat, A. A. E. (1989). Journal of Bacteriology 171, 3982. Chang, S. T. and Hayes, W. A. (eds) (1978). “The Biology and Cultivation of Edible Mushrooms”. Academic Press, New York. Chanter, D. 0. and Thornley, J. H . M. (1978). Journal of General Microbiology 106, 55. Cleland, R. E. (1980). In “Plant Growth Substances 1979” (F. Skoog, ed.), pp. 71-78. Springer-Verlag, Berlin. Clutterbuck, A. J. (1990). Journal of General Microbiology 136, 1731. Cole, G. T. (1973). Canadian Journal of Botany 51, 2413. Cole, G. T., Takashi, S . , Kasai, R., Yokoyama, T. and Nozawa, Y. (1979). Experimental Mycology 3, 132. Cox, J. and Niederpruem, D. J. (1975). Archives ofMicrobiology 105, 257. Craig, G. D . , Gull, K. and Wood, D . A. (1977). Journal of General Microbiology 102, 337. Day, P. R. (1963). Genetical Research 4, 55. de Vries, 0. M. H. and Wessels, J. G. H. (1984). Journal of General Microbiology 130, 145. de Vries, 0. M. H . , Hoge, J. H. C. and Wessels, J. G . H . (1980). Developmental Biology 74, 22. de Vries, 0. M. H., Kooistra, W. H. C. F. and Wessels, J. G . H. (1986). Journal of General Microbiology 132, 2817. Dons, J. J. M., Springer, J., de Vries, S . C. and Wessels, J. G . H. (1984a). Journal of Bacteriology 157, 802. Dons, J. J. M., Mulder, G. H . , Rouwendal, G. J. A , , Springer, J., Bremer, W. and Wessels, J . G. H. (1984b). EMBO Journal 3, 2101. Durand, R. (1983). Transactions of the British Mycological Society 81, 553. Durand, R. (1985). Physiologie Vigttale 23. 935. Durand, R. and Furuya, M. (1985). Plant and Cell Physiology 26, 1175. Durrant, A. J., Wood, D. A. and Cain, R. B. (1991). Journal of General Microbiology 137, 1751. Diitsch, G. A. and Rast, D. M. (1972). Phytochemistry 11, 2677. Edclstein, L. (1982). Journal of Theoretical Biology 98, 679. Edelstein, L. and Segel, L. A. (1983). Journal of Theoretical Biology 104, 187. Eger-Hummel, G . (1980). In “The Blue Light Syndrome” (H. Senger, ed.), pp. 555-562. Springer-Verlag, Berlin. Eilers, F. I. (1974). Archives of Microbiology 96, 353. Elliot, T. J. and Wood, D. A. (1978). Transactions of the British MycologicalSociety 70, 373. Esser, K. (1971). Molecular and General Genetics 110, 86. Esser, K. and Meinhardt, F. (1977). Molecular and General Genetics 155, 113. Esser, K., Saleh, F. and Meinhardt, F. (1979). Current Genetics 1 , 85. Fincham, J. R. S . , Day, P. R. and Radford, A. (1979). “Fungal Genetics”. Blackwell, Oxford. Flegg, P. B., Spencer, D. M. and Wood, D. (1985). “The Biology and Technology of the Cultivated Mushroom”. Wiley, Chichester. Fry, S. C. (1986). Annual Review of Plant Physiology 37, 165. Galun, E. (1971). Plant and Cell Physiology 12, 779.
196
J. G . H. WESSk1.S
Giasson, L., Specht, C. A., Milgrim, C., Novotny, C. P. and Ullrich, R. C. (1989). Molecular and General Genetics 218, 72. Gold, M. H. and Cheng, T. M. (1979). Archives of Microbiology 121, 37. Gold, M. H., Wariishi, H. and Valli, K. (1989). American Chemical Society Symposium 389, 127. Gooday, G. W. (1974). Transactions of the British Mycological Society 62, 391. Gooday, G . W. (1979). I n “Fungal Walls and Hyphal Growth” (J. H. Burnett and A. P. J. Trinci, eds), pp. 203-223. Cambridge University Press, Cambridge UK. Gooday, G. W. (1982). I n “Basidium and Basidiocarp” (K. Wells and E. K. Wells, eds), pp. 157-173. Springer-Verlag, Berlin. Gooday, G. W. (1983). In “Secondary Metabolism and Differentiation in Fungi” (J. W. Bennett and A. Ciegler, eds), pp. 239-266. Marcel Dekker, New York. Gooday, G. W . , De Rousset-Hall, A. and Hunsley, D . (1976). Transactions of rhe British Mycological Society 67, 193. Gregory, P. H. (1984). Transactions of the British Mycological Society 82, 1. Gruen, H. E . (1982). In “Basidium and Basidiocarp” (K. Wells and E. K. Wells, eds), pp. 125-155. Springer-Verlag, New York. Gruen, H. E. and Wong, W. M. (1981). Canadian Journal of Botany 60, 1342. Haffner, L. and Thielke, C. (1970). Berichte der Deutsche Botanische Gesellschaft 83, 27. Hammond, J. B. W. (1977). Journal of General Microbiology 102, 245. Hammond, J. B. W. (1985). Journal of General Microbiology 131, 329. Hammond, J. B. W. and Nichols, R. (1975). Journal o f t h e Science of Food and Agriculture 26, 835. Harley, J. L. and Smith, S. E . (1983). “Mycorrhizal Symbiosis”. Academic Press, London. Harmsen, M. C., van Griensven, L. J. L. D. and Wessels, J. G. H . (1989). Journal of General Virology 70, 1613. Harmsen, M. C., Scheer, J., Schuurs, T. A . and Wessels, J. G. H . (1991a). In “Genetics and Breeding of Agaricus” (L. J . L. D. van Griensven, ed.), pp. 14@144. Centre for Agricultural Publishing and Documentation, Wageningen. Harmsen, M. C., Tolner, B., Krdm, A , , Go, S. J., de Haan, A. and Wcssels, J. G. H. (1991b). Current Genetics 20, 137. Hayes, W. A. and Nair, N. G . (1975). In “The Filamentous Fungi” (J. E. Smith and D. R. Berry, eds), vol. 1, pp. 212-248. Edward Arnold, London. Haylock, R. W., Economou, A. and Casselton, L. A. (1980). Journalof General Microbiology 121, 17. Hicks, J., Strathern, J. N. and Klar, A. J. S. (1979). Nature 282, 478. Hintz, W. E. A , , Anderson, J. B. and Horgen, P. A. (1988). Current Genetics 14, 43. Hoffman, R. M. and Raper, J. R. (1971). Science 171, 418. Hoffman, R. M. and Raper, J . R. (1974). Journal of General Microbiology 82, 67. Hoge, J. H. C., Springer, J., Zantinge, B. and Wessels, J. G. H. (1982a). Experimental Mycology 6, 225. Hoge, J. H. C., Springer, J. and Wessels, J. G . H. (1982b). Experimental Mycology 6 , 233. Horton, J. S. and Raper, C. A. (1991). Genetics 129, 70. Ingold, C. T. (1986). Transactions of the British Mycological Society 86, 349. Iten, W. and Matile, P. (1970). Journal of General Microbiology 61, 301. Jennings, D. H. (1984). I n “The Ecology and Physiology of the Fungal Mycelium” (D. H. Jennings and A . D. M. Rayner, eds), pp. 143-164. Cambridge University Press, Cambridge. Kamada, T. and Takemaru, T. (1977). Plant and Cell Physiology 18, 831. Kamada, T. and Takemaru, T. (1983). Journal of General Microbiology 129, 703. Kamada, T., Kurita, R. and Takemaru, T. (1978). Plant and Cell Physiology 19, 263. Kamada, T., Fujii, T. and Takemaru, T. (1980). Transactions of the Mycological Society (Japan) 21, 359. Kamada, T., Hamada, Y. and Takemaru, T. (1982). Journal of General Microbiology 128, 1041. Kamada, T.. Katsuda, H . and Takemaru, T. (1984). Current Microbiology 11, 309. Kamada, T., Takemaru, T . , Prosser, J. I. and Gooday, G. W. (1991). Protoplasma 165, 64. Kanda, T. and Tshikawa, T. (1986). Journal of General and Applied Microbiology 32, 541.
FKUI’IING IN THE HIGHEK FlJNGl
197
Kassir, Y., Granot, D . and Simchen, G. (1988). Cell 52, 853. Kawai, G. and Ikeda, Y. (1982). Biochimica et Biophysica Acta 719, 612. Kawai, G. and Ikeda, Y. (1983). Riochimica et Biophysica Acta 745, 243. Kawai, G.. Onishi, M., Fujino, Y. and Ikcda, Y. (1986). Journal of Biological Chemistry 261, 779. Kawai, S . , Umezawa, T. and Higuchi, T. (1988). Archives of Biochemi.stry and Biophysic~ 262, 99. Kendrick, B. and Berch, S. (1985). In “Comprehensive Biotechnology” (M. Moo-Young, ed.), vol. 4, pp. 110k152. Pergamon Press, Oxford. Kenvin, J . K. and Duddles, N . D. (1989). Journal of Bacteriology 171, 3831. Kitamoto, Y . and Gruen, H. E. (1976). Plant Physiology 58, 485. 49, 338. Kitamoto, Y., Suzuki, A. and Furukawa, S . (1972). Plant Physi~il~igy Kitamoto, Y . , Horikoshi, T. and Suzuki, A. (1974). Planta 119, 81. Koltin, Y. (1970). Archiv fur Mikrobiologie 74, 123. Koltin, Y. (1978). In “Genetics and Morphogenesis in the Basidiomycetes” (M. N. Schwalb and P. G. Miles, cds), pp. 31-54. Academic Press, New York. Koltin, Y. and Raper, J. R. (1968). Science 60, 85. Koltin, Y., Stamberg, J. and Lemke, P. A. (1972). Bacteriological Reviews 36, 156. Koltin, Y., Wessels, J. G. H. and van der Valk, P. (1973). Archiv fur Mikrobioiogie 91, 179. Kronstad, J . W. and Leong, S. A. (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 978. Kues, U., Richardson, W., Mutasa, E., Tymon, A , , Gaubatz, S. and Casselton, L. (1991). Abstracts of the EMBO Workshop on the Molecular Biology of Filamentous Fungi, p. 32. Kuhad, R. C . , Rosin, I. V. and Moore, D. (1987). Transactions o f t h e British Mycological Society 88, 229. Kutschera, U., Bergfcld, R. and Schopfer, P. (1987). Plantu 170, 168. Leatham, G. F. (1985). In “Developmental Biology of Higher Fungi” (D. Moore, L. A. Casselton, D. A. Wood and J. C. Frankland, eds), pp. 403427. Cambridge University Press, Cambridge. Leathani, G. F. and Stahmann, M. A. (1981). Journal of General Microbiology 125, 147. Leslie, J. F. and Leonard, T. J. (197%). Molecular and General Genetics 171, 257. Leslie, J. F. and Leonard, T. J. (1979b). Molecular and General Genetics 175, 5 . Leonard, T. J. and Dick, S . (1968). Proceedings of the National Academy of Sciences of the United States of America 59, 745. Leonard, T. J. and Phillips, I,. E. (1973). Journal of Bacteriology 114, 7. Lu, B. C. (1982). In “Basidium and Basidiocarp” (K. Wells and E. K. Wells, eds), pp. 93-112. Springer-Verlag, New York. Madelin, M. F. (1960). Transactions of the British Mycological Society 43, 105. Manachere, G . (1970). Annales des Sciences Naturelles, Botaniyue et Biologie Vigktale, Paris 11, 1. Manachere, G. (1980). Transactions of the British Mycological Society 75, 255. Manachere, G. (1988). Cryptogamie, Mycologie 9, 291. Manachere, G., Robert, J. C., Durand, R., Bret, J. and Fevre, M. (1983). In “Fungal Differentiation, a Contemporary Synthesis” (J. E. Smith, ed.), pp. 481-514. Marcel Dekker, New York. Marchant, R. (1978). Journal of General Microbiology 106, 195. Marchant, R. and Wessels, J. G . H . (1974). Archives of Microbiology 96, 175. Marino, R., Saksena, K. N., Schuler, M., Mayfield, J. E. and Lemke, P. A. (1976). Applied and Environmental Microbiology 31, 433. Matthews, T. R. and Niederpruem, D. J. (1973). Archiv fur Mikrobiologie 88, 169. Metzenberg, R. L. and Glass, N. L. (1990). BioEssays 12, 53. Mitchell, A. P. and Herskowitz, I. (1986). Nature 319, 738. Mol, P. C. and Wessels, J. G. H. (1990). Mycological Research 94, 472. Mol, P. C., Vermeulen, C. A. and Wessels, J. G . H . (1990). Mycological Research 94, 480. Mooibroek, H., Kuipers, A. G . J., Sietsma, J. H., Punt, P. J. and Wessels, J. G. H. (1990). Molecular and General Genetics 222, 41.
198
J . G . H. WESSFLS
Moore, D. (1984). Experimental Mycology 8,283. Moore, D. and Jirjis, R . (1981). New Phytologist 87, 101. Moore, D., Elhiti, M. M. Y. and Butler, R. D. (1979). New Phytologist 83, 695. Moore, D., Homer, J. and Liu, M. (1987). FEMS Microbiology Letters 44,239. Mulder, G. H. and Wessels, J. G . H . (1986). Experimental Mycology 10, 214. Munoz-Rivas, A., Specht, C. A , , Drummond, B. J., Froeliger, E., Novotny, C. P. and Ullrich, R. C. (1986). Molecular and General Genetics 205, 103. Murao, S . , Hayashi, H. and Tarui, N. (1984). Agricultural and Biological Chemistry 48, 1669. Mutasa, E. S . , Tymon, A. M., Gottgens, B., Mellon, F. M., Little, P. F. R. and Casselton, L. A. (1991). Current Genetics. 18, 223. Nasmyth, K. A. and Tatchell, K. (1980). Cell 19, 753. Nawrath, C. and Russo, V. E. A. (1990). Journal of Photochemistry and Photobiology B: Biology 4, 261. Nelson, M. A. , Morelli, G., Carattoli, A , , Romano, N. and Machino, G. (1989). Molecular and Cellular Biology 9, 1271. Niederpruem, D. J. (1963). Journal of Bacteriology 85, 1300. Niederpruem, D. J. and Wessels, J. G. H. (1969). Bacteriological Reviews 33, 505. Odier, E. (ed.) (1987). “Lignin Enzymic and Microbial Degradation”. INRA, Paris. Pall, M. L. (1981). Microbiological Reviews 45, 462. Parag, Y. (1962). Proceedings of the National Academy of Sciences of the United States of America 48, 743. Perkins, J. H. (1969). Plant Physiology 44, 1706. Perkins, J. H. and Gordon, S . A. (1969). Plant Physiology 44, 1712. Perkins, J. H . and Raper, J. R . (1970). Molecular and General Genetics 106, 151. Phillips, L. E. and Leonard, T. J. (1976). Mycologia 68, 268. Pfyffer, G. E. , Hiibscher, U. and Rast, D. M. (1989). Experimental Mycology 13, 321. Punt, P. J., Oliver, R. P., Dingemanse, M. A , , Pouwels, P. H. and van den Hondel, C. A. M. J. J. (1987). Gene 56, 117. Rao, P. S. and Niederpruem, D . J. (1969). Journal of Bacteriology 100, 1222. Raper, C. A. (1976). Journal of General Microbiology 95, 54. Raper, C. A . (1978). In “Genetics and Morphogenesis in the Basidiomycetes” (M. N. Schwalb and P. G. Miles, eds), pp. 3-29. Academic Press, New York. Raper, C. A. (1983). In “Secondary Metabolism and Differentiation in Fungi” (J. W. Bennett and A. Ciegler, eds), pp. 195-236. Marcel Dekker, New York. Raper, C. A. (1988). In “Genetics of Plant Pathogenic Fungi” (G. S. Sidhu, ed.), pp. 51 1-522. Academic Press, London. Raper, C. A. and Horton, J. S. (1991). In “Genetics and Breeding of Agaricus” (L. J. L. D. van Griensven, ed.), pp. 120-125. Centre for Agricultural Publishing and Documentation, Wageningen. Raper, C. A. and Raper, J. R. (1966). Genetics 54, 1151. Raper, C. A. and Timberlake, W. E. (1985). Experimental Mycology 9, 269. Raper, C. A., Raper, J. R. and Miller, R. E. (1972). Mycologia 64, 1088. Raper, J . R. (1966). “Genetics of Sexuality in Higher Fungi”. Ronald Press, New York. Raper, J. R. and Krongclb, G. S . (1958). Mycologia 50, 707. Raper, J. R. and Miles, P. G . (1958). Genetics 43, 530. Raper, J. R., Boyed, D. H. and Raper, C. A. (1965). Proceedings of the National Academy of Sciences of the United States of America 53, 1324. Rast, D . M., Stiissi, H . , Hegnauer, H. and Nylhltn, L. E. (1981). In “The Fungal Spore: Morphogenetic Controls” (G. Turian and H. R. Hohl, eds), pp. 507-531. Academic Press, London. Raudaskoski, M. and Salonen, M. (1984). In “The Ecology and Physiology of the Fungal Mycelium” (D. H. Jennings and A. D . M. Rayner, eds), pp. 291-322. Cambridge University Press, Cambridge. Raudaskoski, M. and Vauras, R. (1982). Transactions of the British Mycological Society 78, 475. Raudaskoski, M. and Viitanen, H. (1982). Transactions ofthe British Mycological Society 78,89.
FRUITING IN THE HIGHER FUNGI
199
Raudaskoski, M. and Yli-Mattila, T. (1985). Transactions o f t h e British Mycological Society 85, 145. Read, D. J. (1984). In “The Ecology and Physiology of the Fungal Mycelium” (D. H. Jenning and A. D. M. Rayner, eds), pp. 215-240. Cambridge University Press, Cambridge. Reijnders, A. F. M. (1963). “Les problbmes du DCvelopmcnt des Carpophores des Agaricales et de quclques Groupes Voisin”. Junk, The Hague. Reijnders, A. F. M. and Moore, D. (1985). In “Developmental Biology of Higher Fungi” (D. Moore, L. A. Casselton, D. A. Wood and J. C. Frankland, cds). Cambridge University Press, Cambridge. Richartz, G . and MacLcllan, A . (1987). Photochemistry and Photobiology 45, 815. Robert, J. C. (1977). Transactions of the British Mycological Society 68, 389. Robert, J. C. and Durand, R. (1979). Physiologia Plantarum 46, 174. Roelofsen, P. A. and Houwink, A . L. (1953). Acta Botanica Neerlandica 2, 218. Roland, J. C. and Vian, €3. (lY79). International Review of Cytology 61, 129. Ross, I. K. (1982a). Journal of General Microbiology 128, 2755. Ross, 1. K . (l982b). Journal of General Microbiology 128. 2763. Ross, I. K., Loftus, M. G. and Foster, L. M. (1991). Mycological Research 95, 776. Royer, J. C. and Horgen, P. A. (1991). In “Genetics and Breeding of Aguricus” (L. J . L. D . van Griensven, ed.), pp. 135-139. Centre for Agricultural Publishing and Documentation, Wageningen. Ruffner, H . P., Rast, D., Tobler, H. and Karesch, H. (1978). Phytochemistry 17, 865. Ruiters, M. H. J. and Wessels, J. G . H. (1989a). Journalof General Microbiology 135, 1747. Ruiters, M. H. J. and Wessels, J. G . H. (1989b). Experimental Mycology 13, 212. Ruiters, M. H. J., Sietsma, J. H. and Wesscls, J. G . H. (1988). ExperimentalMycology 12, 60. Rusmin, S. and Leonard, T. J . (1978). Plant Physiology 61, 538. Salvado, J. C. and Labarbre, J. (1991a). Applied and Environmental Microbiology 57, 2714. Salvado, J. C. and Labarcre, J . (1991b). Current Genetics 20, 205. Schmidhauser, T. J., Lauter, F. R . , Russo, V. E. A. and Yanofsky, C. (1990). Molecularand Cellular Biology 10, 5064. Schulz, B., Bannuctt, F., Dahl, M., Schlessinger, R., Schafer, W., Martin, T., Herskowitz, I . and Kahmann, R. (1990). Cell 60, 295. Schurcn, F. H. J. and Wessels, J. G H. (1990). Gene 90, 199. Schwalb, M. N. (1974). Developntental Biology 40, 84. Schwalb, M. N. (1977). Journal of Biological Chemistry 225, 8435. Schwalb, M. N. (1978a). FEMS Microbiology Letters 3, 107. Schwalb, M. N. (197%). In “Genetics and Morphogenesis in the Basidiomycetes” (M. N. Schwalb and P. G . Miles, cds), pp. 135-165. Academic Press, New York. Schwalb, M. N. and Miles, P. G . (1967). American Journal of Botany 54, 440. Sietsrna, J. H. and Wessels, J. G. H. (1977). Biochimica et Biophysicu Acta 496, 225. Sictsrna, J. H. and Wessels, J. G . H. (1979). Journalof General Microbiology 114, 99. Sietsma, J. H., Rast, D. and Wessels, J. G. H. (1977). Journal of General Microbiology 102, 385. Sommer, T., Chambers, J. A . A , , Eberle, J., Lauter, E. R. and Russo, V. E. A. (1989). Nucleic Acids Research 17, 5713. Sonnenberg, A. S . M., Wessels, J. G . H. and van Gricnsven, L. J . (1988). Current Microbiology 17, 285. Springer, J. and Wessels, J. G . H. (1989). Molecular and General Genetics 219, 486. Stahl, U. and Esser, K. (1976). Molecular and General Genetics 148, 183. Stringer, M. A., Dean, R. A , , Sewall, T. L. and Timberlake, W. E. (1991). Genes and Development 5, 1161. Stiissi, H. and Rast, D. M. (1981). Phytochemistry 20, 2347. Summerbell, R. C., Castle, A. J., Horgen, P. A. and Anderson, J. B. (1989). Genetics 123.293. Swamy, S., Uno, I. and Ishikawa, T . (1984). Journal of General Microbiology 130, 3219. Swamy, S., Uno, I. and Ishikawa, T. (1985a). Journal of General Microbiology 131, 3211. Swamy, S., Uno, I. and Ishikawa, T. (1985b). Journal of General and Applied Microbiology 31, 339.
200
.I G. H . WESSE1.S
Takagi, Y.. Katayose, Y. and Shishido, K. (1988). FEMS Microbiology Letters 55, 275. Takemaru, T. and Kamada, T. (1971). Reports of the Tottori Mycological Institute (Japan) 9, 21. Takemaru, T. and Kamada. T. (1972). Botanical Magazine (Tokyo) 85, 51. Tan, K . K. (1978). In “The Filamentous Fungi. Developmental Mycology” (J. E. Smith and D. R. Berry, eds), vol. 3, pp. 334-357. Edward Arnold, London. Timberlake, W. E. (1980). Developmental Biology 78, 497. Trinci, A. P. J . (1974). Journal of General Microbiology 81, 222. Tsusue, Y. M. (1969). Development, Growth and Differentiation 11, 164. Turner, E. M. (1974). Transactions of the British Mycological Society 63. 541. Turner, E . M. (1977). 7ransactions of the British Mycological Society 69, 183. Ullrich, R. C.. Specht, C. A , , Starikis, M. M., Yang, H., Giasson, L. and Novotny, C. P. (1991). “Genetic Engineering, Principles and Methods”, vol. 13, pp. 279-306. Plenum Press, New York. Uno, I. and Ishikawa, T. (1971). Molecular and General Genetics 113, 228. Uno, I. and Ishikawa, T. (1973). Journal of Bacteriology 113, 1240. Uno, I. and Ishikawa, T . (1982). I n “Basidium and Basidiocarp” (K. Wells and E . K. Wells, eds), pp. 113-123. Springer-Verlag, New York. Uno, I., Yamaguchi, M. and Ishikawa, T. (1974). Proceedings ofthe National Academy of Sciences of the IJnired States of America 71, 479. van der Valk, P. and Marchant, R. (1978). Protoplusmu 95, 57. van der Valk, P., Marchant, R. and Wessels, J. G. H. (1977). Experimental Mycology 1 , 69. Verrinder-Gibbins, A . M. and Lu. B. C. (1984). 7ransaction.s of the British Mycological Society 83, 331. Webster, J. (198O). “Introduction t o Fungi”. Cambridge University Press, Cambridge. Wessels, J. G. H. (1965). Wentia 13, 1. Wessels, J . G. H. (1966). Antonie van Leeuwenhoek. Journal of Microbiology and Serology 32, 341. Wessels, J. G. H. (196%). Journal of Bacteriology 98, 697. Wessels, J. G. H. (1969b). Biochimica et Riophysica Acta 178, 191. Wessels, J. G. H. (1978). I n “Genetics and Morphogenesis in the Basidiomycetes” (M. N. Schwalb and P. G. Miles, eds), pp. 81-104. Academic Press, New York. Wessels, J. G. H. (1986). International Review of Cytology 104, 37. Wessels, J. G. H . (1990). In ”Tip Growth in Plant and Fungal Cells“ (I. B. Heath, ed.), pp. 1-29. Academic Press, San Diego. Wessels, J. G. H. (1991). In “Frontiers in Mycology” (D. L. Hawksworth, ed.), pp. 2748. CAB International, Kew. Wessels, J . G. H. and Marchant, R. (1974). Journal of General Microbiology 83, 359. Wessels, J. G. H. and Niederpruem, D. J. (1967). Journal of Bacteriology 94, 1594. Wessels, J. G . H . and Sietsma, J . H. (1979). I n “Fungal Walls and Hyphal Growth” (J. H. Burnett and A. P. J. Trinci, eds), pp. 2 7 4 8 . Cambridgc University Press, Cambridge. Wessels, J. G. H., Krcger, D. R., Marchant, R., Regensburg, B. A. and de Vries, 0. M . H. (1972). Biochimica et Biophysica Actu 273, 346. Wessels, J. G. H., Dons. J. J. M. and de Vries, 0. M . H . (1985). In “Developmental Biology of Higher Fungi” (D. Moore, A. Casselton, D. A. Wood and J. C. Frankland, eds), pp. 485497. Cambridge University Press, Cambridge. Wessels, J. G. H., Mulder, G. H . and Springer, J. (1987). Journal of General Microbiology 133, 2557. Wessels, J. G. H . , de Vries, 0. M. H., Asgeirsdbttir, S. A . and Schuren, F. H. J. (1991a). The Plant Cell 3, 793. Wessels, J . G. H . , de Vries, 0. M. H., Asgeirsdottir, S. A. and Springer, J. (1991b). Journal of General Microbiology 137, 2439. Wong, W. M. and Gruen, H. E. (1977). Mycologia 69, 899. Wood, D. A. (1976). Journal of General Microbiology 95, 313. Wood, D . A. (1980). Journal of General Microbiology 114; 161. Wood, D . A. and Goodenough, P. W. (1977). Archives of Microbiology 114, 161. Wood, D. A . and Hammond, J. B. W. (1977). Journal of General Microbiology 98, 625.
FRUIIIIUG I N IIIE HIGlIER b l 1 N G l
201
Wood, D. A , , Perry, C., Thurston, C. F., Matcharn, S. E . , Dudley, K . , Claydon. N. and Allan, M . (1990). I n “Biotechnology in Pulp and Paper Manufacture” (T. K. Kirk and H.M . Chang, eds), pp. 659-666. Bulterworth-Heineman, Boston. Wuest, P. J., Royce. D. J. and Beelrnan, R. B. (eds) (1987). “Cultivating Edible Fungi”. Elsevier, Amsterdam. Yashar, B. M. and Pukkila, P. J. (1985). Transactionsofthe British MycologicalSociety84,215. Yli-Mattila, T. (1985). Physiologiu Plunturum 65, 287. Yli-Mattila, T. (1987). Physiologiu Plunturum 69, 451, Yli-Mattila, T., Ruiters, M. H. J. and Wessels, J. G. H. (1989a). Current Microbiology 18,289. Yli-Mattila, T., Ruiters, M . H. J.. Wessels, J. G. H. and Raudaskoski, M . (1989b). Mycological Research 93, 535. Zantinge, B., Dons, H. and Wessels, J. G. H. (1979). European Journal of Riochernistry 101, 251. Zantinge, B., Hoge, J . H. C. and Wessels, J. G. H. (1981). EuropeunJournulofHiochemistry 113, 381.
Note Added in Proof
After this review paper had gone to press, considerable progress was reported concerning the molecular structure of mating-type genes in basidiomycetes (Section 1II.B). ‘The two forms, a1 and a2, of the a matingtype locus of U. maydis each contain two genes encoding the precursor for a specific lipopeptide pheromone and a receptor for the pheromone secreted by cells of the opposite mating type (Bolker et al., 1992). More relevant to the structure and operation of the mating-type genes of the fruiting basidiomycetes is the structure of the multi-allelic b locus of I/. maydis which controls dikaryon formation after cell fusion. Instead of containing one multi-allelic gene (Schultz et ul., 1990), this locus has now been shown to contain two multi-allelic genes designated b E and bW (Gillissen et al., 1992). Both genes contain homeodomains, and crosses between strains in which one of the genes is disrupted show that activation of development by b involves interaction of bE and bW gene products contributed by different alleles. Similar findings have been reported for the A a matingtype locus of S . commune. Most Au loci contain not one but at least two different genes, designated Z and Y , containing homeobox-like sequences named HD1 and HD2, respectively (Stankis et al., 1992). Transformation analyses (Specht et al., 1992) show that, after mating, Z from one mating partner interacts with Y from the other partner to activate the Au-regulated pathway of development, possibly through formation of a heteromeric protein activator of gene transcription. Similar results have been reported for the AulJ mating-type locus of C. cinereus which contains at least four specificity genes (Kiies et al., 1992; Kiies and Casselton, 1992). Contrary to earlier beliefs, the effective interactions after mating, which switch on dikaryon formation and fruiting, are thus not between proteins encoded
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J G H WFSSELS
by two di. '?t alleles of mating-type genes but between proteins encoded by two cktfibrknt mating-type genes or idiomorphs (Metzenberg and Glass, 1990), each represented by a number of alleles. The constitutive mutation Aco" which switches on development without mating was shown, in C. cinereus, to arise from a deletion in the A@ locus fusing a gene containing the HD1 homeodomain with a gene containing the HD2 homeodomain (Kiies and Casselton, 1992). REFERENCES
Bolker, M., Urban, M. and Kahmann, R. (1992). Cell 68, 441. Gillissen, B., Bergemann, J., Sandmann, C., Schroeer, B., Bolker, M. and Kahmann, R. (1992). Cell 68, 647. Kues, U. and Casselton, L. A. (1992). In "Genetic Engineering, Principles and Methods" (J. K. Setlow, ed.), vol. 14. Plenum Press, New York (in press). Kues, U., Richardson, W. V. N., Tymon, A . M., Mutasa, E. S . , Gottgens, B., Gaubatz, S., Gregoridas, A. and Casselton, L. A . (1992). Genes and Development 6, 568. Metzenberg, R. L. and Glass, N. L. (1990). BioEssays 12, 53. Specht, C. A , , Stankis, M. M., Giasson. L., Novotny, C. P. and Ullrich, R. C. (1992). Proceedings of the National Academy of Sciences of the United States of America 89,1174. Stankis, M. M., Specht, C. A . , Yang, H., Giasson, L., Ullrich, R. C. and Novotny, C . P. (1992). Proceedings of the National Academy of Sciences of the United States of America 89, 7169.
Bacterial Ice Nucleation PAUL K . WOLBER D N A Plant Technology Corporation. 6701 San Pablo Avenue. Oakland. CA 94608. USA
I . Introduction . . . . . . . . . . . IT . Physical basis of ice nucleation . . . . . . . . . . . . . . A . Types of ice nucleation . B . Heterogeneousnucleation bycoherenttemplates C . Measurementofice-nucleationactivity . . . . D . Temperatureclassesofbacterialicenuclei . . . 111. Bacterialice-nucleationgenesandproteins . . . . A . Surveyofgeneandinferredproteinsequences . . B . Protein sequence and domain structure . . . . C . Biochemistry and immunology of ice-nucleation proteins D . Modelsof bacterial-nucleation protein structure . . E. Evolution of ice-nucleation genes . . . . . IV . Environmental significance of bacterial ice nucleation . . A . Frost damage to plants . . . . . . . . B . Meteorological significance . . . . . . V . Applications of bacterial ice nucleation . . . . . . . . . . . . A . Artificial snow-making B . Freezing control . . . . . . . . . C . Ice-nucleation reporter genes . . . . . . VI . Concluding remarks . . . . . . . . . References . . . . . . . . . . . .
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203 204 204 206 208 209 211 211 212 221 225 228 230 230 231 231 231 232 233 233 235
.
I Introduction
Metastability is a phenomenon which surrounds us . Clouds (which consist of supercooled water). organic compounds (which oxidize exothermically). diamonds (which are less stable than graphite) and hydrogen (which undergoes exothermic nuclear fusion) are all commonplace examples of matter trapped in states far from equilibrium . Often. matter escapes such traps through the action of catalysts. which continuously intervene to lower ADVANCESINMICROBIALPHYSIOJ~OGY. VOL . 34 ISBN &I24277344
Copyright 01993. by Academic PressLimited All rights of reproduction in any form reserved
204
P K WOLRER
the energy barrier preventing a process, or initiators which briefly provide a lower energy route to a spontaneous process. Biological catalysts, such as enzymes, are well studied. However, the ability of certain biological systems to initiate physical processes in metastable systems has only recently been appreciated and investigated. Perhaps the best characterized biological initiators are bacterial ice nuclei, which trigger crystallization of ice from supercooled water. Ice-nucleation systems have been characterized in detail in certain strains of Erwinia ananas, E. herbicola, Pseudomonas puorescens, P. syringae and Xanthomonas campestris. Generally, ice-nucleating bacteria are members of plant epiphytic communities. In all of the strains investigated so far, the phenotype is encoded by a single gene (Green and Warren, 1985; Warren et al., 1986; Abe et al., 1989; Warren and Corotto, 1989; Zhao and Orser, 1990). During the last 20 years, the study of bacterial ice nucleation has grown from the esoteric pursuit of a few dedicated scientists (Vali, 1971; Schnell and Vali, 1972, 1973; Maki et al., 1974; Arny et al., 1976; Hirano et al., 1978, 1982; Lindow et al., 1978b; Maki and Willoughby, 1978; Yankofsky etal., 1981;Lindow, 1982) into a mature branch of science with conferences, periodic scholarly reviews (Lindow, 1983b,d; Warren, 1987; Warren and Wolber, 1987,1991 ; Wolber and Warren, 1989; Margartis and Bassi, 1991), and practical applications (Margartis and Bassi, 1991). The purposes of this review are to provide a foundation for the reader who is unfamiliar with the phenomenon of bacterial ice nucleation, to integrate the considerable data and theory which have been published, to show how basic research in the field has resulted in practical applications, and to catalogue sources of more detailed information, databases and biomaterials.
11. Physical Basis of Ice Nucleation A . T Y P E S OF I C E N U C L E A T I O N
Three mechanisms of ice nucleation are commonly recognized (Fletcher, 1970). These are (a) homogeneous ice nucleation (self-nucleation of ice crystallization in pure supercooled water), (b) heterogeneous ice nucleation (initiation of ice crystallization through binding of supercooled water to some non-water material), and (c) secondary ice nucleation (seeding of ice crystallization by a pre-existing ice crystal). Homogeneous ice nucleation can only take place at temperatures below -35°C (Franks, 1985); secondary ice nucleation can take place at any temperature below freezing, but can only occur after a “seed” ice crystal has been initiated through another
205
RACTFRlAl ICF NUCI FATION
mechanism. Heterogeneous nucleation does not require pre-existing ice, and can take place at temperatures as warm as -2°C. Initiation of most ice crystals can eventually be traced to a heterogeneous nucleation event (usually through a chain of secondary nucleation events). Why is supercooled water metastable? The inability of pure water to freeze spontaneously at temperatures between 0 and -35°C is explained by the observation that very small crystals of ice melt at temperatures lower than “bulk” ice. This change in melting point is due to the existence of an interfacial layer with increased free energy between the water and ice phases. The total change in free energy (AG) as i water molecules form a small, submerged ice crystal is the sum of the change in free energy of the surface layer (which increases in proportion to i2’3) and the change in the free energy of the crystalline phase (which decreases in proportion to i). The surface energy dominates when the number of molecules, i, is small.
.................................
AG*
NetAG
\ \ \ \
\
Volume Free Energy
\ \ \ \
Number of Molecules in Embryo, i FIG. 1. Free energy relationships governing initiation of ice crystallization. The surface, volume and net free-energy changes (AG) associated with formation of ice embryos containing various numbers ( i ) of water molecules are denoted graphically. For each temperature (7)below O’C, there exists a critical number of water molecules, i * , which must enter an ice embryo before crystallization becomes spontaneous (i.e. AG decreases as more water molecules are added).
206
P K WOLBER
However, at any temperature below 0°C there is a critical crystal size beyond which the volume term decreases faster than the surface term increases. This crystal size is characterized by the number of water molecules it encloses, namely i*( 7) (Fletcher, 1970; Franks, 1985). Crystals of this size and larger can grow spontaneously, while smaller crystals are less stable than the supercooled water surrounding them, and melt. All three types of nucleation operate by producing a “critical ice embryo” which is capable of expanding spontaneously. The free-energy relationships which govern initiation of ice-crystal growth are shown graphically in Fig. 1. Since both homogeneous and heterogeneous nucleation depend upon a probabilistic event (the fortuitous clustering of water molecules into a critical ice embryo), does the concept of a “temperature of nucleation” make sense? Thermodynamic models of the nucleation phenomenon (Fletcher, 1970; Franks, 1985) predict for pure water that the rate of critical - ~ is approximated by embryo formation, J ( ~ m s-l)
J
- loz7 exp(-AG*lkT),
where k is the Boltzmann constant, T the absolute temperature, and the “free energy of activation” for critical ice-embryo formation, AG*, is given (for a spherical embryo) by 16x0’ AG*
=
3(
A7)*
where o is the surface free energy between ice and water, AT the supercooling range (i.e. the difference between the temperature of the supercooled water and the freezing point of “bulk” water), and the average entropy of fusion of water over the supercooling range AT. The value of J predicted by this formula depends very strongly upon the degree of supercooling. This prediction is supported by experiment (Fletcher, 1970). Ice nucleation is observed to exhibit a sharp temperature threshold, above which supercooled water is metastable, and below which freezing is rapidly initiated. Theoretical models of heterogeneous nucleation yield similar predictions (vide infru), and these predictions are again confirmed by experiment (Fletcher, 1970). It therefore makes good physical sense to speak of a “nucleation-threshold temperature” at which a nucleation site becomes active.
B. HETEROGENEOUS NUCLEATION BY COHERENT TEMPLATES
Heterogeneous nucleation of ice crystallization is most frequently observed in crystalline materials with arrangements of atoms which mimic symmetries
BACTFRIAL ICb NUCI FATION
207
and atomic spacings found in ice (Vonnegut, 1947; Fletcher, 1970; Franks, 1985). Such materials are sometimes capable of binding water in arrangements similar to tiny ice crystals, and are said to nucleate ice formation through a coherent template mechanism. Examples of such compounds are silver and lead iodides, and copper sulphide. If the freeenergy change associated with water binding is sufficiently negative, a critical ice embryo csn form spontaneously at much warmer temperatures than would be possible in pure water. Theoretical models (Fletcher, 1970; Franks, 1985) of the rate of ice nucleation by heterogeneous nuclei produce an overall expression similar to that for homogeneous nuclei (vide supra), except that the free energy of activation AG* is replaced by
AGL
=
AG* f ( m , R ) .
The function f ( m , R ) depends upon the geometry of the nucleation site, and is always less than unity (i.e. f ( m , R ) defines the degree to which a heterogeneous nucleation site lowers the homogeneous free-energy barrier to nucleation). The parameter m is given by m =
(+L-%P)/%L,
where o p L , ~and s p c~~~ are the free energies of the nucleating particleliquid water interface, the particle-solid ice interface and the ice-water interface, respectively. The parameter m defines the degree to which the surface organizes water into an ice-like configuration, and ranges between -1 (for a surface incompatible with ice) to +1 (for a surface which interacts strongly with ice). Effective heterogeneous nucleators interact strongly with ice, but weakly with water, and may even be moderately hydrophobic. The parameter R represents some characteristic length of the nucleus geometry (e.g. the radius of a disc-shaped nucleation site). Generally, the larger the characteristic length, the warmer the temperature of nucleation. However, a coherent template introduces an additional complication; any mismatch between the lattice spacings of the template and ice will introduce strain in the ice embryo. As the embryo grows, so will the strain until it eventually overwhelms the propensity of water to bind in an ice-like configuration. Therefore, R should be thought of as the smaller of the actual size of the nucleation site and the “coherence length”, that is, the maximum distance over which the site can organize water before the accumulated strain of lattice mismatch prevents further binding. The effects of lattice mismatch produce a counter-intuitive effect. Sometimes, lattices with defects (e.g. a silver bromide-silver iodide coprecipitate) are more effective nucleators than the corresponding “perfect” lattice (Vonnegut and Chessin, 1971). This may be due to relief of strain in the
208
P K WOlBER
ice embryo as it crosses a dislocation or other imperfection in the template lattice. C. MEASUREMENT OF ICE-NUCLFATION ACTIVITY
Ice-nucleation activity is usually measured via a method (Vali, 1971) analogous to the most probable number method (Finney, 1964) used in bacteriology. Nucleation activity is calculated from the frequency of freezing observed in multiple, small volumes (often droplets) of a sample diluted in water or a weak buffer so that, at the measurement temperature, some but not all of the replicate volumes freeze. The system is then governed by Poisson statistics, and the concentration of nuclei active at or above a temperature T , Ina(T), is given by (Vali, 1971) Ina(T) = Dv,-'
I~[N,I(N,-N~)],
where D is the dilution factor (i.e. D = 100 for a 1in 100 dilution), Vd is the volume of an individual droplet, Nt is the total number of droplets tested, and Nf is the number of droplets frozen at the temperature T. Generally, freezing is scored visually, either by monitoring the change from transparency to translucence which accompanies freezing, or by inclusion of a fluorescent freezing indicator dye (Warren and Wolber, 1988). Just as the most probable number method assumes no background (i.e. sterile media), measurement of ice nucleation assumes that the diluent and test surface are free from ice nuclei active at or above the assay temperature. In practice, autoclave- or filter-sterilized buffers are free from nuclei at temperatures above - 12"C, and nucleus-free surfaces can be easily prepared by, for example, spray-coating aluminium foil with a 2% solution of paraffin in xylene, and then baking the foil at 100°C. Such buffers and surfaces are sufficient for measurement of the activity of most bacterial ice nuclei. There are two important differences between the measurement of icenucleation activity and the most probable number measurement for bacterial populations. First, ice-nucleation activity is a function of temperature, and the calculated activity, Ina(T), is cumulative; the assay measures all nuclei active at or above the test temperature. More complicated formulae (or simple subtraction) can be used to approximate the differential frequency of ice nuclei, that is, the concentration of nuclei active in a small range { T , T + A T } . However, such calculations are of limited accuracy, due to the second difference from the most probable number method. A particular droplet can only freeze once. Therefore, any additional nuclei present in the droplet which are active at temperatures lower than the nucleation site which first initiates ice formation are hidden from
209
BACTERIAL ICE NUCLFATION
experimental measurement (Vali, 1971). If the differential frequency of nucleation increases strongly with decreasing temperature and all nuclei sort into droplets in an independent manner, this effect is negligible. However, small numbers of independently sorting, colder threshold nuclei can be detected in the presence of a higher concentration of warmer threshold nuclei only by testing a very large number of droplets at a dilution factor high enough such that the probability of a droplet containing two nuclei active at different temperatures is very low. If the nuclei do not sort independently (e.g. multiple nuclei bound to a bacterial cell), then only the nucleus active at the warmest temperature is experimentally observable. All measurements of bacterial ice-nucleation activity should be interpreted with this fact in mind. D. TEMPERATURE CLASSES OF BACTERIAL ICE NUCLEI
Several experimentally measured bacterial ice-nucleation spectra are shown in Fig. 2. As is customary, the temperature axis is plotted with increasing 100, I
10 - 1 1
0 L
10-4
Q)
0
-
'"1
10 -7
10 -8
2
I
I
I
I
4
6
8
10
AT ("Cbelow zero) FIG. 2. Typical bacterial ice-nucleation spectra. Data are shown for Pseudomonas syringae PS31 grown on nutrient agar-glycerol plates at 24°C (W) and in Luria broth at 24°C (O), and for Erwinia herbicola MH3000 grown on nutrient agar-glycerol plates at 24°C (0)and in Luria broth at 24°C (0).From Deininger et al. (1988).
210
P K WOLRER
degrees of supercooling (i.e. lower temperatures) to the right. The cumulative frequency of nucleation is displayed on a logarithmic scale, and has been normalized by dividing the cumulative concentration of nuclei by the concentration of bacteria (measured by direct count, turbidity or plating assay). It is obvious from Fig. 2 that bacterial ice nuclei are active at widely varying frequencies and temperatures, and that the variation is modulated by bacterial strain and growth conditions. Traditionally, this variation has been used to define three classes of bacterial ice nuclei (Yankofsky et al., 1981): type 1 nuclei, which are active at temperatures between -2 and -5°C; type I1 nuclei, which are active between -5 and -7°C; and Type 111 nuclei, which are active below -7°C. One of the central quests of bacterial ice-nucleation research has been to discover the mechanisms responsible for the perplexing variation observed in nucleation temperature and frequency. The source of the variation does not appear to be genetic; it is observed in clonal populations and at all stages of bacterial growth (Warren and Wolber, 1991). The theory of heterogeneous nucleation (see Section 1T.B) predicts that such variation might be caused by variations in the value of m, which defines the intrinsic quality of the nucleating surface, variations in the size of the nucleation site, or variations in the coherence length of the nucleation site if nucleation occurs by coherent template matching. Some insight has been gained into the source of nucleation-site variations by means of two simple physical experiments. First, frozen or freeze-dried ice nucleation-active (Ina’) cells of P.syringae were irradiated with varying doses of 6oCoy-radiation, the effects on ice-nucleation activity at various temperatures measured, and the results interpreted according to target theory (Govindarajan and Lindow, 1988b). Such experiments have shown that the target size varies log-linearly with nucleation temperature. When coupled with biochemical data which indicate that the nucleation site is membrane bound and protease-sensitive (Phelps et al., 1986; Govindarajan and Lindow, 1988a), the data have been interpreted to indicate that the different classes of nucleation sites are not of the same masses, varying from greater than 2 . lo7 D a (at -3°C) to -10’ Da at -13°C. It should be noted that variz..tionsin coherence length (or effective sizes of the nucleation sites) are also compatible with the data; however, variations in the intrinsic quality of the nucleating surface are incompatible with these experiments. It is interesting to note that the measured target sizes agree with theoretical predictions for the size of a disc-shaped heterogeneous nucleation site with “perfect” surface properties (i.e. m= 1) (Burke and Lindow, 1990). The second set of physical experiments (Lindow, 1983b) is based on the observation that bacterial ice nuclei are temperature-labile. Nuclei with warmer threshold temperatures are preferentially destroyed by briefly
B A C l k R I A L ICb NUCI F A rlON
211
heat shocking the bacteria, but reappear upon subsequent incubation of cells at the normal growth temperature (around 20°C). This reappearance is independent of new protein synthesis. The destruction kinetics are first order, while reappearance kinetics are of mixed order. These data have been used to argue that the ice-nucleation sites are membrane-bound aggregates, which disaggregate at higher temperatures by sequentially losing individual molecules, and reaggregate at normal growth temperatures by a co-operative assembly process. Again, the data are also compatible with changes in the effective size of the nucleation site, if individual molecules in an aggregate undergo independent, reversible denaturation to yield an inactive conformation, and co-operative renaturation to yield an active conformation. In either case, the data are difficult to explain through variations in the intrinsic quality of the nucleation site, and point towards a structure which is an aggregate of parts which can, under certain circumstances, behave independently of one another.
111. Bacterial Ice-Nucleation Genes and Proteins A . SURVEY OF GENE AND INFERRED PROTEIN SEQUENCES
The study of bacterial ice nucleation was greatly aided by the discovery that the Ina' phenotype could be transferred to Escherichia coli by cloning a single stretch of chromosomal DNA from an Ina' bacterial species (Orser et al., 1985). To date, at least nine D N A fragments capable of imparting the Ina' phenotype to E. coli have been cloned (see Table l ) , and the DNA sequences of five of the fragments have been published. The five sequenced clones share several properties: (a) all contain one long open-reading frame of -3600 bp; (b) about 80% of every open-reading frame consists of a series of hierarchically organized, imperfectly repeated DNA sequences with lengths of 24, 48 and 144 bp; (c) the DNA sequences of the open-reading frames are highly homologous, while the DNA sequences of the regions outside the open-reading frames are usually not homologous; (d) protein sequences inferred from D N A sequences are more strongly conserved than the D N A sequences themselves (i.e. many of the base changes among genes are silent differences at redundant positions in the codons of the open-reading frames). Studies of transposon-insertion mutations of ice-nucleating clones (Orser et al., 1985; Corotto et al., 1986), sequencing of the N-terminus of the
212
P K WOLBER
TABLE 1.
ina gene (reference)
Cloned inn genes
Source species
Gene sequence Reference
Unnamed (Orser et al., 1985) iceC (Orser et al., 1985) inaY" (Amy et al., 1976) inaZ (Green and Warren, 1985) inaW (Corotto et al., 1986)
Erwinia herhicola 26SR62 Pseudomonus syringae Cit75 1 Pseudomonas syringue PS31 Pseudomonas Jyringae S203
Pseudomonas fluorescens MS16.50 IceE (Yankofsky et al., 1983) Erwinia herhicola M 1
inuA (Arai et al., 1989) Erwinia anunas IN-10 inaX (Zhao and Orser, 1990) Xanthomonas campestris pv. translucens X56S inaC? (Anderson and Pseudomonas viriflava Ashworth, 1986; Hasegawa KUTN-2 et al., 1990)
Not sequenced Not sequenced Not sequenced Green and Warren (1985) Warren et al. ( 1986) Warren and Corotto (1989) Abe et al. (1989) Zhao and Orser ( 1990) Not sequenced
Database
GenBank GenBank GenBank
EMBL
a This gene has been cloned into Escherichia coli and partially sequenced (G. Warren, unpublished observation).
protein product of another such clone (Wolber et al., 1986) and studies of the effects of heterologous promoters on expression of the h a f phenotype (Wolber et al., 1986; Southworth et af., 1988) have provided additional evidence that, in every micro-organism, the Ina+ phenotype is the result of expression of a single ice-nucleation ( i m ) gene, to yield a single icenucleation (Ina) protein. The names of the cloned ina genes, their source species, and references to their sequences (if available) are summarized in Table 1. B . PROTEIN SEQUENCE AND DOMAIN STRUCTURE
The five sequenced ina genes encode five proteins with highly homologous inferred amino acid-residue sequences. The sequences can be organized into a series of domains, based upon the absence or presence of particular types of repeated amino acid-residue sequences (Green and Warren, 1985; Warren etal., 1986,1987a; Warren and Corotto, 1989; Wolber and Warren, 1991). The consensus domain structure inferred from the five sequenced ice-nucleation genes is outlined in Fig. 3. A comparison of the five inferred protein sequences is shown in Fig. 4. An ice-nucleation protein contains three domains: an N-terminal unique
213
RACTERIAL ICE NUCI F A IION
Repeating Domain
I
Repeat-?
Repeat-2
Repeaf~3
8-mers
8-mers 16-mers 48-mers
8-mers 16-men 48-mers
-
-
-
-
unique
-COOH
\ \ \
/
48
8-mers
\
/ / / /
I
Repeat-4
\ \
H 48 H 48 H 48 H 48 )-1 48 H 48
t AGYGST-TAG--SSLI
xN
HAGYGSTQTAG--S-LTHAGY
t
x3
AGYGST-TA---S---
t
x2
A---S---
FIG. 3. Consensus organization of ice-nuclcation proteins. The proteins arc organized into three domains: the N-terminal uniquc domain, thc rcpeating domains and the C-tcrminal unique domain. Four repeating subdomains have been defined on the basis of the hicrarchy of repetition and consistent patterns of amino acidresidue occurrence in particular repeat positions. The repeating domains appear to have evolved through repeated duplication and elaboration of a primitive octapeptide unit.
region, a central region rich in alanine, glycine, serine and threonine residues that contains nested sets of repeated sequences, and a C-terminal unique region. The first 20 residues of the N-terminal unique region possess some of the properties of a membrane-insertion signal sequence (Heijne, 1984, 1985). The extreme N-terminus is positively charged (net +1 to +3 for residues 1-10, not counting the N-terminal amino g r o u p ) , and the predicted secondary structure is an a-helix followed by a turn (Warren et al., 1986). However, the helix is not as hydrophobic as a typical signal sequence. The remainder of the N-terminal unique domain provides few clues as to its function. The nucleotide and predicted protein sequences of the first 60% of this domain are much better conserved between genes than the last 40%. This last point is demonstrated by the comparison between inferred protein sequences shown in Fig. 4(a). The central, repeating domain can be divided into four subdomains, which are, in turn, composed of nested repeated sequences of 8, 16 and
214
P K WOLBER
48 amino-acid residues, in increasing order of fidelity, in subdomains 2 and 3. The repetition in subdomains 1 and 4 lacks the 48 amino acid-residue periodicity. These subdomains are outlined in Fig. 3, and are shown in greater detail in the sequence comparisons, Fig. 4(b-e). The fidelity of repetition increases from the C-terminal subdomain 4 to subdomain 2 (where the 48 amino acid-residue repeat is nearly perfect). Subdomain 1 is usually an incomplete 32-residue repeat, which may serve as a transition region between the N-terminal and repeating domains. The transition region between subdomains 2 and 3 is marked by a loss of conservation of primary sequence between different ice-nucleation proteins, and is sometimes marked by the presence of a break in the phase of the 48 amino acidresidue repeat. There is also a consistent substitution of basic residues in subdomain 2 for corresponding acidic residues in subdomain 3 , or vice versa. This may indicate that these two domains form salt bridges with each other. The transition between subdomains 3 and 4 is marked by loss of the 48-residue repeat. Subdomain 4 also usually substitutes cysteine for serine in position 5 of the third from last 16 amino acid-residue repeat. The C-terminal unique domain is strongly positively charged at its Nterminal end (consensus net + 3 ) , and strongly negatively charged at its Cterminal end (consensus net -5 to -7). The secondary structure of this domain is predicted to form a turn between these highly charged regions (Warren et al., 1986); the domain may therefore fold back on itself to form internal salt bridges or a charged binding site. The amino acid-residue sequences predicted from ice-nucleation genes are strongly homologous. The homologies between the five inferred h a protein sequences have been calculated for pairwise sequence alignments (Needleman and Wunsch, 1970; Devereux et al., 1984), and are shown in Table 2. Two numbers are reported for each alignment, namely the percentage of identical amino-acid residues and the percentage of similar residues. The rules for assigning similarity of residues are those of Schwartz and Dayhoff (1979), as modified by Gribskov and Burgess (1986), and are FIG. 4. Aligned protein amino-acid sequences inferred from the five sequenced ina genes. The InaW, IceE, InaA and InaX sequences have all been aligned (Devereux et al., 1984) to the InaZ sequence (shown underlined, in single letter code), using the algorithm of Needleman and Wunsch (1970). Dots denote amino acid-residue identities, upper-case letters denote conservative (Schwartz and Dayhoff, 1979; Gribskov and Burgess, 1986) amino acid-residue substitutes, lowercase letters denote non-conservative substitutions, tildes denote gaps inserted by the algorithm to improve alignments, and asterisks denote stop codons. The alignment is shown by domain: (a) N-terminal unique domain; (b) repeat domain I ; (c) repeat domain 2; (d) repeat domain 3; (e) repeat domain 4; (f) C-terminal unique domain. The repeating domains are shown with intramolecular homologies aligned.
BACTERIAL ICE NUCLEAIION
N-Terminal Uniaue Block
Z inaW iceE inaA inaX ~
Z inaW iceE inaA inaX ~
& Z ~
inaW iceE inaA inaX inaZ __ inaW iceE inaA inax
(a)
Repeatinv Block 1
21 5
216
P . K. WOI-BER
Repeatinv Block 2 Z-__
inaW i ceE inaA inaX k Z inaW iceE inaA inaX ~
inaZ inaW iceE inaA inaX
& Z inaw i ceE inaA inaX ~
Z~
inaW iceE inaA inaX & Z inaW iceE inaA inaX inaZ __ inaW iceE inaA inaX & Z ___ inaW iceE inaA inaX
AGYGSTQTAGGDSALT AGYGSTQTAREGSNLT
BACTERIA1 ICF NUCl FATION
217
jnaZ
inaW ic e E inaA
inaX
Z& inaW i ceE inaA inaX jnaZ __
AGYGSTGTAGADSSLI
i n aW
..........S.....
iceE
..........S...I.
inaA inax
..........S...I. .............T..
*Z
AGYGSTQTSGSESSLT
inaW
iceE
inaA inaX (c)
Weatinp Block 3 4
inaZ -
inaW iceE inaA inaX
AGYGSTGTAGSGSSLI AGYGSTQTASYRSMLT AGYGSTQTAREHSDLV
218
k
P K WOLBER
Z
~
i n aW iceE
inaA inaX inaz inaW iceE
inaA inax a
Z
~
inaW ic e E inaA inaX ~
inaZ inaW i ceE inaA inaX
aZ
inaW iceE
inaA inax k
Z
~
inaW i ceE inaA inaX
T G Y G S T S T A G Y A S L AGYGSTQTAGYECTLT AGYGSTQTAQENSSLT ______
BACTERIAL ICE NUC1,EATION
Repeatinv Block 4 inaZ
inaW iceE
inaA inaX bZ
inaW ic e E inaA inaX
inaZ __ inaW ic e E inaA inaX dZ
inaW ic e E inaA inaX
C-Terminal Unique Block
~
inaZ inaW ic e E inaA inaX
219
220
P K WOlBER
based on observed tolerated substitutions in families of homologous proteins. In all Ina protein domains, the degree of conservation is high, averaging about 77% identity and 85% similarity. Amino acid-residue changes are also clustered rather than being randomly distributed through the sequences. An exceptionally close relationship is implied by the gene pair formed by iceE from E. herbicola and inaA from E. ananas (see Table 1). For these bacteria, the inferred amino acid-residue sequences are nearly identical, as shown in Fig. 4. Analysis of the corresponding gene sequences (Wolber and Warren, 1991) has shown that nucleotide conservation is very good at all codon positions and in most regions, including the 5' upstream region of the genes; this latter region is poorly conserved between all other ice-nucleation genes. These data imply that selection for conservation of the primary protein sequence, and presumably the protein function, has taken place. The striking sequence conservation between iceE and inaA suggests that divergence of the source species is extremely recent, despite the distance between the points of isolation (Israel and Japan, respectively). Alternatively, both species may have acquired ice nucleation rather recently, from the same third species, as a result of conjugation. TABLE 2.
Homology matrix of inferred h a protein sequences in ice-nucleation bacteria Comparison sequence"
Reference sequence
InaW
IceE
InaA
InaZ
77.2 (84.0)
InaW IceE InaA
-
77.2 (85.1) 78.9 (87.0)
-
-
76.9 (85.2) 78.7 (86.7) 97.7 (98.4)
-
-
-
a
InaX
75.2 75.7 74.9 74.7
(83.3) (84.1) (83.1) (82.5)
Percentage residue identity, or similarity (in parentheses)
Ice-nucleation genes have been mutagenized by several methods in order to study the roles of various protein domains. Early experiments with transposon mutagenesis yielded an unexpected result: none of the mutants was a complete null. This result was eventually understood to derive from a combination of the extreme sensitivity of ice-nucleation assays and the ability of rare recombinational events to reconstruct functional ice-nucleation genes from transpositionally inactivated genes (Corotto et al., 1986). These experiments also showed that recombinational repair of the repeating portion of ice-nucleation genes is particularly effective; this may explain why there is improved conservation of third, silent bases in the repeating portion of ice-nucleation genes (Warren et al., 1986; Wolber and Warren, 1991), and why there is sometimes insertion or deletion of repeats
BACI ERIAL ICE NUCLEATION
22 1
between otherwise highly conservative pairs of nucleation proteins (see Fig. 4(b-e)). Experiments in which in-frame sequences have been deleted from or, in certain cases, inserted into ice-nucleation genes have more precisely defined the functions of various protein domains (Green et al., 1988; Abe et al., 1989). The effects of a given class of changes on the Inaf phenotype are generally consistent within a particular protein domain, but vary qualitatively and quantitatively between domains. Deletions from the N-terminal unique domain degrade or abolish type I and I1 nucleation, but have little or no effect on type I11 nucleation. Those from the C-terminal unique domain severely diminish or abolish ice-nucleation activity at all temperatures. Deletions from or insertions into the repeating domain produce a variety of effects. The effects are most deleterious to nucleation activity when the length of the deletion-insertion is not a multiple of one of the orders of periodicity. This finding indicates that the folding pattern of an icenucleation protein can accommodate, to some extent, addition or deletion of repeated modules. The data are consistent with a model in which the C-terminal unique region is a protein-folding nucleus. The repeated domain binds water into an ice template, and the N-terminal unique region promotes aggregation of ice-nucleation protein monomers into large arrays (Wolber and Warren, 1991). It has also been suggested that the C-terminal unique region may play some role in aggregate formation (Abe et al., 1989). C. BIOCHEMISTKY AND IMMUNOLOGY OF ICE-NUCLEATION PROTEINS
The biochemistry and immunology of bacterial ice nuclei have been extensively studied, in both naturally occurring Ina' bacteria and recombinant bacteria. The goals of such studies have included: (a) localization of ice-nucleating sites in or on the bacterial cell; (b) confirmation that homologous proteins of the proper size are present in h a ' bacteria and absent from Ina- bacteria; (c) demonstration of the presence or absence of secondary chemical modifications to ice-nucleation proteins; (d) analysis of the assembly and structure of the ice-nucleation site; (e) analysis of the dose-response relationship between levels of icenucleation protein and levels of ice-nucleation activity. Analysis of ice nuclei shed by certain species of E. herbicola (Phelps et al., 1986) and fractionation of natural Ina' bacteria (Lindow et al., 1989a) have demonstrated that natural bacterial ice nuclei are localized on the outer membrane of Gram-negative bacteria. Studies of overexpression in E. coli have also demonstrated that active ice nuclei can be assembled on
222
P . K WOLHFR
the inner membrane of bacteria (Wolber et al., 1986). Lipid extraction and reconstitution experiments have shown that membrane lipids are required for ice-nucleation activity (Govindarajan and Lindow, 1988a). However, lipopolysaccharide, a component found only in the outer membrane of Gram-negative bacteria, is not required, and many different lipids can reconstitute activity. Thus, the requirement for lipid may be generic rather than specific. Proteolysis studies (Phelps et al., 1986) have shown that bacterial ice nuclei contain an essential protein component, and that type TI1 nuclei are more protease-resistant than type I or TI nuclei. This last result supports the hypothesis that nucleation temperature is governed by real or effective nucleus size (see Section 1I.D). Several chemical agents known to disrupt membranes have also been shown to decrease or abolish bacterial ice-nucleation activity. These agents include dyes which stain bacterial cell walls (Maki et a f . , 1974), cationic detergents (Maki et al., 1974; Watanabe et al., 1988) and membranefluidizing agents such as 2-phenylethanol (Lindow, 1983b). Other agents, such as the protein denaturant urea (Lindow, 1983c), sulphydryl-modifying reagents and carbohydrate-reactive compounds such as lectins and borates (Kozloff et al., 1983) have also been shown to decrease bacterial icenucleation activity. However, in some experiments, chemical-inactivation studies have been conducted with whole bacteria as opposed to cell-free ice nuclei. These earlier studies did not adequately control for cell death after treatment with a reagent. Subsequent experiments with cell-free nuclei have shown that, with at least one reagent (the sulphydryl-modifying reagent N-ethylmaleimide), the observed effects were entirely due to cell death and were not observed when cell-free ice nuclei were substituted for whole cells (Phelps et a f . , 1986). Comparison of patterns of protein expression from recombinant Ina' strains of E. cofi and their Ina- ancestors have demonstrated that transformation of E. coli to the h a + phenotype is accompanied by appearance of a new protein on sodium dodecyl sulphate-polyacrylamide gels (Wolber et al., 1986; Lindow et a f . , 1989a). The new protein generally exhibits an apparent molecular size 25-50% larger than the size predicted from the expected mass of the corresponding ice-nucleation protein (predicted molecular masses for Ina proteins are generally around 120 kDa). The observed size anomaly may be evidence for post-translational chemical additions to ice-nucleation proteins. However, the observed anomaly is dependent upon electrophoresis conditions and may simply indicate that ice-nucleation proteins bind sodium dodecyl sulphate poorly (Deininger et a f . , 1988). Polyclonal antisera have been raised against the product of the inaW gene (Deininger et al., 1988), against the protein product of a fusion
RACTERIAI. ICE NUCLEAI'ION
223
between the iceC and lacZ genes (Lindow et al., 1989a) and against several synthetic peptides drawn from the repeating domain of ice-nucleation proteins (Mueller et al., 1990; Ruggles et al., 1991). Recently, three monoclonal antibodies raised against the InaW protein have also been characterized (C. A. Vance, N. M. Watanabe and P. K. Wolber, unpublished data). In all of the experiments conducted to date, each of the antisera cross-reacts with several characterized ice-nucleation proteins. In all analyses, the apparent molecular masses of ice-nucleation proteins on Western blots are identical for proteins expressed in either their natural bacterial source or recombinant E. coli. These results indicate that, if the anomalous apparent molecular masses of ice-nucleation proteins are caused by secondary chemical modification, then the same modification must take place after expression in very different hosts. The antiserum against the InaW protein also cross-reacts with large proteins which appear in concert with the Ina' phenotype in other ice-nucleating organisms (Deininger et al., 1988). The only other proteins which cross-react with such antisera are proteolytic degradation products of ice-nucieation proteins (Deininger et d.,1988). At this time, there are no known bacterial proteins associated with Ina- bacteria which cross-react with anti-InaW antisera. Several different antisera raised against ice-nucleation proteins or their component peptides have been shown to inhibit ice nucleation by cell-free ice nuclei shed by E. herbicola (Ruggles et al., 1991). The concentration of antibody needed to inhibit nucleation varied with the nucleationthreshold temperature. Type I nuclei were most sensitive to inhibition while type 111 nuclei were least sensitive. These results have been interpreted according to a model in which antibody binding breaks a large coherent template into smaller templates by physically blocking part of the waterbinding surface (Ruggles et al., 1991). Such blockage would be predicted to lower the nucleation-threshold temperature by decreasing the real or effective template size, and would be expected to affect type I nuclei most severely (since they afford a larger target for binding), in agreement with the results observed. In a related experiment, antifreeze glycopeptides, which are believed to prevent ice crystallization by binding to the surface of ice-crystal embryos, have been shown to inhibit ice nucleation by cellfree ice nuclei shed by E. herbicola (Parody-Morreale et al., 1988). The pattern of inhibition closely paralleled that seen with antibodies. Warmer threshold nucleation sites were more sensitive than colder threshold sites. This result provides strong evidence that bacterial ice nuclei function by a coherent template mechanism, since the only property relating bacterial ice nuclei and antifreeze glycopeptides is their shared postulated affinity for ice. Examination of the effects of substituting D 2 0 for H 2 0 on ice-nucleation
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P K WOIRER
spectra (Turner et al., 1990) has shown that type I, I1 and I11 nuclei can be differentiated on the basis of their isotope-induced shifts in nucleation threshold. These results, coupled with the differential effects of phospholipase CII (Kozloff et al., 1984; Turner et al., 1990) and other enzymic and chemical probes of glycosylation (Kozloff et al., 1991; Turner et al., 1991) on various classes of ice nuclei, have been used to argue that the ice-nucleation protein in type I nuclei has been modified by addition of a phosphatidylinositol membrane anchor. Unfortunately, no one has yet produced direct biochemical confirmation of this intriguing hypothesis. Such confirmation may be difficult to obtain, since ice nuclei appear to be homo-aggregates of ice-nucleation protein, and chemical modification of a minority population of protein monomers may be sufficient to change the nucleation threshold by such means as increasing the coherence length of a template array. As stated previously, studies of y-ray inactivation of ice nuclei have demonstrated that the target size for radiation damage increases log-linearly with temperature of nucleation, and is always greater than or equal to the average predicted size of ice-nucleation proteins (about 120 kDa) (Govindarajan and Lindow, 1988b). Studies of the dependence of icenucleation frequency on the concentration of ice-nucleation protein have shown that nucleation frequency at all temperatures increases non-linearly with this concentration, indicating that 2-3 monomers must assemble cooperatively to initiate formation of an active nucleus (Southworth et al., 1988; Lindgren et al., 1989). Measurements of rates of assembly of ice nuclei have confirmed cooperative initiation of nucleus assembly, and have shown that nuclei active at warmer temperatures are assembled more slowly than nuclei active at colder temperatures (Watanabe et al., 1990). This difference in assembly rates may indicate that type I nuclei are formed by addition of monomers of ice-nucleation protein to type I1 or I11 nuclei. Measurements of rates of destruction of ice nuclei assembled in heterologous hosts have demonstrated that, in these hosts, type I nuclei are turned over much more slowly than type 11or I11 nuclei (Watanabe et al., 1990). Since heterologously expressed ice nuclei appear on both the inner and outer membranes of Gram-negative bacteria, this may simply be evidence that various temperature classes of nucleation sites are assembled at different locations in unnatural hosts. However, this may also be additional indirect evidence that type I nuclei contain post-translationally modified ice-nucleation protein. Ice-nucleation proteins have been visualized in situ in E. coli strains transformed to express the TnaZ or InaW proteins (Mueller et al., 1990) by indirect immunofluorescence microscopy. The visualized proteins showed several interesting features:
BACTERIAL ICE NUCLEATION
225
(a) the immunofluorescence was tightly clumped into multiple, cellassociated patches; (b) the patches appeared to be associated with the bacterial membrane; (c) patches on a given cell were of different sizes; (d) both the largest patch size and the number of patches observed in a given clonal population of bacteria correlated directly with the frequency of higher-temperature threshold nucleation observed in that population. These data afford the most direct evidence to date that the variety of nucleation-threshold temperatures and nucleation frequencies observed in natural and constructed Ina' bacterial strains is a stochastic effect caused by dynamic assembly and disassembly of multiple, membrane-bound aggregates of ice-nucleation proteins. Taken together, the available data on nucleus structure indicate that icenucleation proteins must associate with a membrane, and then self-associate in order to become active. The degree of self-association appears to be directly related to nucleation-threshold temperature. The selective pressure which has preserved the homology between ice-nucleation proteins has also preserved the ability of these proteins to form co-operatively membranebound arrays. If this selection is for the ability to nucleate ice, then the need for aggregation is dictated by the physics of ice nucleation (Burke and Lindow, 1990). D . MODELS OF BACTERIAL-NUCLEATION PROTEIN STRUCTURE
If bacterial ice nuclei function as a result of a coherent template mechanism, then some part of the protein which forms such nuclei must fold to form a surface which is capable of binding water in an array that closely approximates a small ice crystal. The leading candidate for this template is the section of the molecule containing repeated sequences. The assignment of a template function to this domain is based on several observations: (a) the repeating domain is naturally suited to formation of a repeating template structure (Warren et al., 1986; Mizuno, 1989); (b) this domain is rich in amino-acid residues with neutral hydrophilic side-chains capable of forming hydrogen bonds with water molecules (Warren et al., 1986, 1987a; Mizuno, 1989); (c) removal of several 48-amino-acid repeating blocks lowers the nucleation-threshold temperature but does not abolish nucleation activity (Green et al., 1988); (d) the hierarchy of lengths of repeating motifs (8, 16 and 48 aminoacid residues) forms the multiplicative sequence { 1,2,6}. The
226
P K WOlBFR
symmetry of this sequence is similar to that of certain crystal planes of ice (Warren et al., 1986; Warren, 1987). The sequences of amino-acid residues in the repeating domain correlate strongly with sequences which form structures dominated by p-sheets and turns, and are anticorrelated with sequences which form a-helices (Warren et al., 1986). However, the proposed secondary and tertiary structure models based on p-sheets and turns (Warren et al., 1986) are unusual, and have no counterparts among known protein structures (Mizuno, 1989). By contrast, models of the repeating domain based on a-helices conform to recognized structural motifs (Mizuno, 1989), but completely ignore predictions of correlative methods (Chou and Fasman, 1978; Garnier et al., 1978; Cohen et al., 1986; Warren et al., 1986). The question as to which model of the repeating domain (if any) is closer to the truth will have to await results from some physical method (e.g. X-ray diffraction, electron diffraction, neutron diffraction or nuclear magnetic resonance) capable of providing data about the spatial arrangement of the amino-acid residues in active bacterial ice-nucleation proteins. Given that bacterial ice nuclei are membrane bound, and that even highly active bacteria probably contain only a few thousand copies of the protein in each cell (Wolber et al., 1986; Deininger et al., 1988; Watanabe et al., 1990), preparation of active samples of suitable purity for structural determination will not be an easy task. It should be noted that all published models (Warren et al., 1986; Mizuno, 1989) of the repeating domain predict surfaces which are capable of organizing water into an ice-like structure in three dimensions. Most models of heterogeneous nuclei consider only two-dimensional templates (Fletcher, 1970; Franks, 1985; Burke and Lindow, 1990); it will be interesting to see if nature has been more imaginative than human theorists. All of the models also rationalize the surprising observation that the structure of the repeating domain can accommodate subtraction or addition of 48-amino-acid repeating blocks. Any new models of the repeating domain will also have to contend with this observation. The secondary and tertiary structures of the N- and C-terminal domains have not been modelled in any detail. Such modelling is very difficult, since there are neither structural data nor constraints imposed by a periodic sequence to limit their possibilities. One aspect of ice-nucleation proteins which seems particularly mysterious when all of the domains are considered together is their strong association with the bacterial membrane and a lipid requirement for activity. The amino-acid sequence of an ice-nucleation protein is rather hydrophilic (Warren, 1987). The only portion of the molecule which contains hydrophobic stretches which are candidates for conventional transmembrane a-helices is the N-terminal unique domain
BACTERIAL ICE NUC’LEATION
227
(Warren, 1987). Thus, the N-terminal domain may contain the membrane anchor for ice-nucleation proteins, and the template formed by the repeating domain may be associated only with the membrane surface. Such a location would fit the postulated function of the repeating domain, namely organizing water. Alternatively, ice-nucleation proteins could be anchored to lipid molecules by some form of secondary chemical modification (Turner et al., 1990, 1991; Kozloff et al., 1991). There is a general consensus on the quaternary structure of ice-nucleation proteins. Most investigators now believe that the protein forms homoaggregate arrays which serve as water-binding templates for ice formation (Warren et al., 1986; Warren, 1987; Govindarajan and Lindow, 1988b; Kozloff et al., 1991; Ruggles et al., 1991). A schematic representation of this model is shown in Fig. 5; the probable points of attack of various agents which inhibit or destroy bacterial ice nuclei are also noted. The remaining point of disagreement in this model is the balance of forces which drive aggregate formation, and the molecular sources of those forces.
Changes in temperature cause disaggregation Proteases Protein denaturants
FIG. 5. A model of the quaternary structure of bacterial ice nuclei. Copies of the ice-nucleation protein are shown as hexagonal prisms in order to emphasize their role as an ice template and their propensity to aggregate into (presumably periodic) membrane-bound arrays. The postulated sites of action of various treatments which degrade bacterial ice nuclei are also noted. The stippling indicates bound water, an ice embryo.
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P K WOLBER
E . EVOLUTION OF ICE-NUCLEATION GENES
Perhaps the most striking aspect of the five ice-nucleation genes sequenced so far is the strong conservation of inferred amino-acid sequences (see Figs 3 and 4 and Tables 1 and 2). Not only particular sequences but also the organization of these sequences into domains are conserved. One possible explanation for such strong conservation is that ice-nucleation genes are of relatively recent origin and have been laterally dispersed by bacterial conjugation. However, the generally poor conservation of non-coding sequences upstream and downstream of the structural genes argues against this explanation (Wolber and Warren, 1991). There appears to have been selection for preservation of the primary sequence of ice-nucleation proteins, and presumably for preservation of the h a + phenotype. The serendipitous fact that the repeating domain of ice-nucleation proteins is rich in serine residues has provoked the suggestion that all of the ice-nucleation genes sequenced so far probably evolved from a common ancestor (Warren et al., 1986). The argument hinges on the fact that serine is encoded by two families of codons: AG(Py) and TCN. It is impossible to move from one family to another by a single nucleotide change. In all ice-nucleation proteins, the serine residues at positions 8N-3 of the 48 amino acid-residue repeat (for N = 1,2,. . . ,6) are encoded by the AG(Py) family, while serine residues at positions 11, 14 and 30 are of the TCN family. If ice-nucleation genes and proteins evolved by convergent evolution of dissimilar ancestors, it is highly unlikely that equivalent patterns of codon bias would have emerged in all genes. Analyses of the structure of the repeating domain has provided strong evidence that this domain evolved as a result of recursive duplication of sequences (Green and Warren, 1985; Warren et al., 1986, 1987a; Warren, 1987; Warren and Corotto, 1989; Wolber and Warren, 1991) which can eventually be traced to an ancestral octapeptide repeat (see Fig. 3). Data from mutagenesis (Green et al., 1988; Abe et al., 1989) provide support for two additional hypotheses about evolution of ice-nucleation genes. First, the three protein domains appear to have distinct functions, which link together to effect ice nucleation. The autonomous mutability of the N-terminal and repeating domains suggests that folding of these domains is independent, and that they may have evolved with relative independence. The dependence of ice nucleation on the presence of the acidic portion of the C-terminus suggests that folding of one or both of the other domains is dependent on the C-terminal domain, or that this terminus provides some other function essential for ice nucleation. Second, recombination is enhanced in the repeating portion of an ice-nucleation gene (Warren et al., 1986). This lends support to the notion that this region evolved by
BACTERIAL ICE NUCLEATION
229
repeated duplication of sequences, and that correction of mutations in this region is aided by intragene recombination. Finally, homology searches of databases of published gene sequences have so far yielded no clues concerning other bacterial genes which might be related to bacterial ice-nucleation genes (Wolber and Warren, 1991). Such searches are hampered by having a large percentage of an ice-nucleation gene devoted to repeating sequences. Such sequences completely stymie the computer algorithms usually used to locate homologies. In addition, genes encoding outer-membrane proteins from plant epiphytic Gramnegative bacteria are under-represented in current databases. The observed conservation of ice-nucleation genes implies that there exists a positive selection for preservation of such genes, and for preservation of the encoded phenotype. There is experimental evidence for two possible selective forces. First, the Ina' phenotype may assist dispersion and deposition of plant epiphytes onto plant surfaces in raindrops (Lindemann et al., 1982; Constantinidou et al., 1990) by nucleating ice formation in clouds around air-borne bacteria. This hypothesis can also be modified to explain t h e existence (Fall and Schnell, 1985) of Ina' oceanic organisms. Second, the h a ' phenotype may provide the basis of a unique form of opportunistic pathogenesis. When mild frosts occur in the spring or autumn, microcolonies of h a + epiphytic bacteria nucleate ice formation, causing lesions which release nutrients. This release enhances reproduction of h a + strains (Buttner and Amy, 1989). Circumstantial support for this hypothesis is also provided by the pattern of regulation of ice-nucleation genes (Lindow et al., 1978b; Lindow, 1982; Deininger et al., 1988; Lawless, 1988) which resembles the pattern of regulation of known pathogenesis genes in related organisms (Huynh et af., 1989; Wolber and Warren, 1991). Molecular and ecological data from ice-nucleating bacteria have been synthesized into a model of evolution of such genes (Wolber and Warren, 1991). A version of this model is shown in Fig. 6. In this model, the key event in the evolution of ice-nucleation genes is hypothesized to have been accidental nucleation of ice formation by some aggregated, periodic ancestral membrane protein, at temperatures above the threshold temperature of other nucleation sites available in that environment. At that point, any positive selection(s) for the h a + phenotype would have become active, and the gene would have been recruited by selective forces into a programme of evolutionary improvement of the Ina' phenotype. Such improvement would have been greatly facilitated by the protein's periodic structure, which would have opened a facile path to enlargement of the nucleation site by internal duplication of portions of the encoding gene. Finally, the gene is thought to have undergone horizontal dispersion
230
P. K. WOLBER
Aggregation in membrane
\
Periodic structure
/
Protein
I
Pseudomonas spp.
threshold temperature for selection of nucleation phenotype Rapid improvement of ice-nucleation phenotype by sequence duplication and
Erwinia spp. Xanfhomonas spp.
-
Horizontal dispersal by conjugation
selection for plant damage? dispersion? FIG. 6. A model for evolution of bacterial ice-nucleation proteins.
through the phyllosphere as a result of bacterial conjugation in mixed epiphytic microcolonies.
IV. Environmental Significance of Bacterial Ice Nucleation A. FROST DAMAGE TO PLANTS
Ice-nucleating bacteria are the chief initiators of frost damage to many economically important crop plants. This non-intuitive effect is the result of the ability of many air-exposed plant parts to supercool to temperatures of -6°C or lower before nucleation sites intrinsic to the plant material become active (Lindow et al., 1978a, 1982a,b; Lindow, 1983a,c,d, 1985a; Margartis and Bassi, 1991). Agricultural losses in the USA due to frost injury have been estimated at over $1000 million yearly (White and Haas, 1975; Lindow, 1983d). Canadian losses are also believed to be significantly affected by frost damage (Margartis and Bassi, 1991). In some situations, frost injury appears to open the way for attack by plant pathogenic bacteria; in other situations, ice-nucleating bacteria do
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not cause plant disease in the classical sense (Lindow, 1983d; Buttner and Amy, 1989). In recent years, the use of non-nucleating (Ina-) mutants of non-pathogenic Ina' bacteria as biocontrol agents for preventing plant frost damage has been explored (Lindow, 1983c, 1985b,c, 1987, 1989; Lindow et al., 1983, 1989b; Lindemann and Suslow, 1987; Warren et al., 1987a,b; Margartis and Bassi, 1991) and patented (Lindow and Amy, 1977, 1980; Lindow, 1984, 1989). In the USA, a family of biocontrol agents based on Ina- mutants of Ina' bacteria has been licensed under the registered tradename of FrostBan. B. METEOROLOGICAL SIGNIFICANCE
The initial discovery that plant surfaces shed airborne (bacterial) ice nuclei was prompted by the general interest by atmospheric scientists in ice nucleation (Schnell and Vali, 1972, 1973). Since then, there has been an on-going investigation of the incidence and importance of bacterial ice nucleation in the atmosphere (Schnell, 1976, 1977; Schnell and Vali, 1976; Vali et al., 1976; Sands et al., 1982; Snider et al., 1985). To date, the meteorological importance of bacterial ice nuclei remains unproven, although the data hint at an important role for them in some situations. Definitive proof of a link between airborne h a + bacteria and rainfall amounts will probably not appear until the relative roles of primary and secondary nucleation by shattered ice in storm clouds are fully defined.
V. Applications of Bacterial Ice Nucleation A. ARTIFICIAL SNOW-MAKING
The first commercial application of h a ' bacteria was their addition to water used in artificial snow-making for recreational skiing. The bacteria, which are sold as a freeze-dried, radiation-sterilized powder, act as a source of ice nuclei in the small water droplets that are sprayed into the air by a conventional snow-making machine. Addition of nuclei increases the percentage of droplets which begin to freeze before hitting the ground, and increases the amount of freezing of a given droplet by initiating freezing at an earlier point in the droplet's trajectory. The process was invented by M. D. Woerpel (Woerpel, 1980), developed into a commercial prototype by Advanced Genetic Sciences, Inc. (now a part of DNA Plant Technology Corp.), and then scaled-up and commercialized by the bioproducts division of Eastman Kodak (this division is now a part of Genencor, International) (Kocak and VanGemert, 1988). The product, which is sold under the
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registered trade-name of Snomax, is based on P. syringae PS31 (see Table 1). Each gram of freeze-dried powder contains about 10" ice nuclei with nucleation thresholds of -5°C or warmer. Snomax thus serves as a very efficient source of ice-nucleation sites active at the temperatures at which artificial snow-making is usually performed. The chief advantages imparted by Snomax are a reduction in energy consumed by snow-making machines for each unit of snow produced, and an improvement in the recreational quality of artificial snow (Margartis and Bassi, 1991). The product therefore improves the economics of snowmaking, since most of the operating costs of artificial snow-makers are energy-based (Margartis and Bassi, 1991). Recently, the use of Snomax as an additive to seawater used to assemble ice structures in the arctic has also been explored (Kocak and VanGemert, 1988; Margartis and Bassi, 1991).
B . FREEZING CONTROL
Freezing is frequently used as a processing step in manufacture of foods. Bacterial ice nuclei can be used as a controlled source of nucleation sites in freeze processing. So far, three uses have been investigated. In one set of experiments, Watanabe and Arai (1987) studied the effect of addition of Ina' bacteria on freeze drying of foods. They reported that addition of ice-nucleating sites led to savings in refrigeration costs, shorter freezing times and improved efficiency of production. In another set of experiments, Arai and Watanabe (1986) used Ina' bacteria as a source of nucleation sites for freeze texturing of foods. Raw egg-white, bovine blood, soya bean curd, milk curd and aqueous dispersions of soya-bean protein isolate were all successfully textured by anisotropic ice-crystal growth at relatively warm (equal to or above -5°C) temperatures. Finally, the effects of bacterial ice nuclei on freeze concentration are beginning to be explored (Watanabe et al., 1989; Margartis and Bassi, 1991). A difficulty which potentially limits use of bacterial ice nuclei in foods is the nature of the organisms which naturally express the h a + phenotype. All of the natural h a + organisms are Gram-negative bacteria. While none of the h a ' organisms examined has shown mammalian toxicity, the mere presence of compounds characteristic of Gram-negative bacteria (e.g. lipopolysaccharide) could confound assays commonly used to screen foods for bacterial contamination. Therefore, application of bacterial ice nuclei to foods will probably require either purification of active nuclei in a biochemically simpler form or isolation or construction of an h a + food-grade organism.
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C. ICE-NUCLEATION REPORTER GENES
The first use of bacterial ice-nucleation genes as reporters for linked events was accidental. The initial investigation of cloned ice-nucleation genes by transposon mutagenesis turned up the curious result of seemingly “leaky” mutants (Corotto et al., 1986). These results were eventually explained by the isolation of rare excision revertants from a population of transposon mutants (Corotto et al., 1986). However, the ease and sensitivity with which reconstruction of bacterial ice-nucleation genes could detect rare events did not go unnoticed. Since that time, ice-nucleation genes have been exploited as reporter genes in two separate applications. The inaZ gene of P. syringae has been used to construct a remarkably sensitive, quantitative promoter probe (Lindgren et al., 1989). This probe system places a promoterless inaZ gene in a cassette which isolates it from other promoters present in the plasmid. Random pieces of DNA from the organism to be investigated are inserted into a cloning site at the 5’ end of the inaZ gene. The constructs are then screened for ice-nucleation activity; active clones contain promoters. These constructs have subsequently been used to identify and characterize inducible promoters involved in bacterial pathogenesis of plants. Ice-nucleation genes have also been used as the basis of a rapid, sensitive assay for pathogenic bacteria, named the bacterial ice nucleation diagnostic (BIND) assay (Wolber and Green, 1990a,b). The principle of operation of the BIND assay is outlined in Fig. 7. Basically, the assay exploits the ability of a target organism-specific bacteriophage to transform transiently that organism to the Ina’ phenotype. The success of the prototype assays constructed to date is the result of a combination of the exquisite sensitivity of ice-nucleation assays and the empirical fact that most samples routinely examined for human-pathogenic bacteria are essentially ice nucleus-free at temperatures equal to or greater than -10°C. A prototype assay based on the Salmonella phage P22 has been used to detect as few as 25 CFU of Salmonella sp. per gram in a variety of food samples in less than 24 hours. Coupling of the freezing process to a fluorescent freezing indicator dye (Warren and Wolber, 1988) has allowed development of a simple, homogeneous (i.e. washless) assay format. Currently, the BIND assay is undergoing further development by the DNA Plant Technology Corporation. VI. Concluding Remarks
The last 15 years have witnessed an explosive growth of the body of knowledge concerning bacterial ice nuclei. Several former mysteries, such as the genetic basis of the Ina’ phenotype, sizes and locations of the
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+ \
Bacterium
';I
Transducing Phage
Transduction
n
\
Expression
FIG. 7. Diagram describing operation of the BIND assay for pathogenic bacteria. Genetically engineered bacteriophages infect their target bacterial species if it is present in a sample being tested. The infected bacteria are transiently transformed to the Ina' phenotype by cxpression of ina genes carried by the engineered phage. The resulting ice nuclei can be rapidly detected with great sensitivity.
nucleation sites, and the source of the heterogeneity of nucleation-threshold temperatures exhibited by clonal populations of bacteria, are essentially solved. However, these old questions have been replaced by new questions. These include the following: are some of the copies of ice-nucleation protein
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in an assembled nucleus secondarily modified? What are the secondary and tertiary structures of an ice-nucleation protein? What are the selective forces which result in conservation of ice-nucleation gene sequences and ice-nucleation protein structure? Finally, are ice-nucleation proteins related to any other bacterial proteins? It is the author’s sincerest hope that, within 10 years, someone will write another article which answers these questions, and then proposes a new set of mysteries to be solved. After all, we would not wish our children to think their parents lazy. But neither would we wish their intellectual lives to be boring!
REFERENCES
Abe, K., Watabe, S., Emori, Y., Watanabe, M. and Arai, S. (1989) FEBS Letters 258, 297. Anderson, S. A. and Ashworth, E. N. (1986). Plant Physiology 80, 956. Arai, S . , Abe, K., Watabe, S . , Emori, Y. and Watanabe, M. (1989). FEMS Microbiology Letters 61, 53. Arai, S. and Watanabe, M. (1986). Agricultural and Biological Chemistry 50, 169. Amy, D. C., Lindow, S. E. and Upper, C. D . (1976). Nature 262, 282. Burke, M. J . and Lindow, S. E. (1990). Cryobiology 27, 80. Buttner, M. P. and Amy, P. S. (1989). Applied and Environmental Microbiology 55, 1690. Chou, P. Y. and Fasman, G. D. (1978). Annual Review of Biochemistry 47, 251. Cohen, F. E., Abarbanel, R . M., Kuntz, I. D. and Fletterick, R. J . (1986). Biochemistry 25, 266. Constantinidou, H. A., Hirano, S. S . , Baker, L. S. and Upper, C . D. (1990). Phytopathology 80, 934. Corotto, L. V., Wolber, P. K. and Warren, G . J. (1986). EMBO Journal 5, 231. Deininger, C. A , , Mueller, G. M. and Wolber, P. K. (1988). Journal of Bacteriology 170,669. Devereux, J., Haeberli, P. and Smithies, 0. (1984). Nucleic Acids Research 12, 387. Fall, R. and Schnell, R . C. (1985). Journal of Marine Research 43, 257. Finney, D. J. (1964). “Statistical Methods in Biological Assay”, pp. 570-586. London. Fletcher, N. H. (1970). “The Chemical Physics of Ice”, pp. 73-103. Cambridge University Press, Cambridge. Franks, F. (1985). “Biophysics and Biochemistry at Low Temperature”. Cambridge University Press, Cambridge. Gamier, J., Osguthorpe, D. J. and Robson, B. (1978). Journal of Molecular Biology 120,97. Govindarajan, A. G. and Lindow, S. E. (1988a). Journal of Biological Chemistry 263, 9333. Govindarajan, A. G. and Lindow, S. E. (1988b). Proceedings of the National Academy of Sciences of the United States of America 85, 1334. Green, R. L. and Warren, G. J . (1985). Nature 317, 645. Green, R. L., Corotto, L. V. and Warren, G. J. (1988). Molecular and General Genetics 215, 165. Gribskov, M. and Burgess, R. R. (1986). Nucleic Acids Research 14, 6745. Hasegawa, Y., Sakai, N., Yoshitome, H., Kawate, S., Obata, H . and Tokuyama, T. (1990). Journal of Fermentation and Bioengineering 70, 143. Heijne, G. v. (1984). EMBO Journal 3 , 2315. Heijne, G. v. (1985). Journal of Molecular Biology 184, 99. Hirano, S. S . , Maher, E. A , , Kelman, A. and Upper, C. D. (1978). In “Proceedings of the 4th International Conference on Plant Pathogenic Bacteria” (S.d.P.V.e. Phytobactkriologie, ed.), pp. 717-724. Institut National de la Recherche Agronomique, Beaucouze.
236
P. K. WOI.BER
Hirano, S. S . , Nordheim, E. V., Amy, D. C. and Upper, C. D. (1982). Applied and Environmental Microbiology 44, 695. Huynh, T. V . , Dahlbeck, D . and Staskawicz, B. J. (1989). Science 245, 1374. Kocak, R . and VanGemert, H . (1988). Journal of Biotechnology 2, 37. Kozloff, L. M., Lute, M. and Wcstaway, D. (1984). Science 226, 845. Kozloff, L. M., Schofield, M. A. and Lute, M. (1983). Journal of Bacteriology 153, 222. Kozloff, L. M., Turner, M. A . and Arellano, F. (1991). Journal of Bacteriology 173, 6528. Lawless, R . J . , Jr (1988). European Patent Application G281-145. Lindemann. J., Constantinidou, H. A., Barchet, W. R . and Upper, C. D. (1982). Applied and Environmental Microbiology 44, 1059. Lindemann, J. and Suslow, T . V. (1987). Phytopathology 77, 882. Lindgren, P. B., Frederick, R., Govindarajan, A. G . , Ponopoulos, N. J . , Staskawicz, B. J. and Lindow, S. E. (1989). EMBO Journal 8, 1291. Lindow, S . E. (1982). In “Phytopathogenic Prokaryotes” (M. S . Mount and G . H. Lacy, eds), pp. 335-362. Academic Press, New York. Lindow, S . E. (1983a). Current Topics in Plant Biochemistry and Physiology 2, 119. Lindow, S. E. (1983b). Phytopathology 73, 809 (Abstract 338). Lindow, S . E. (1983~).Plant Disease 67, 327. Lindow, S . E. (1983d). Annual Review of Phytopathology 21, 363. Lindow, S . E. (1984). US Patent 4,432,160. Lindow, S . E. (1985a). Citrograph 70, 136. Lindow, S . E. (1985b). In “Biological Control in Agricultural IPM Systems” (M. Hoy and D . Herzog, eds), pp. 395413. Academic Press, New York. Lindow, S . E. (1985~). In “Microbiology of the Phyllosphere” (N. Fokkcma, ed.) pp. 293-31 1. Cambridge University Press, Cambridge. Lindow, S . E. (1987). Applied and Environmental Microbiology 53, 2520. Lindow, S. E. (1989). US Patent 4,855,230. Lindow, S. E. and Arny, D. C. (1977). US Patent 4,045,910. Lindow, S . E. and Arny, D . C. (1980). US Patent 4,161,084. Lindow, S. E., Arny, D. C., Barchet, W. R . and Upper, C. D. (1978a). In “Plant Cold Hardiness and Freezing Stress” (P. Li, cd.), pp. 249-263. Academic Press, New York. Lindow, S . E., Amy, D. C. and Upper, C. D. (1978b). Phytopathology 68, 523. Lindow, S . E., Arny, D. C. and Upper, C. D. (1982a). Plant Physiology 70, 1084. Lindow, S . E., Arny, D. C. and Upper, C. D. (1982b). Plant Physiology 70, 1090. Lindow, S. E., Amy, D. C. and Upper, C. D. (1983). Phytopathology 73, 1097. Lindow, S . E., Lahue, E., Govindarajan, A . G., Panopoulos, N. J. and Gies, D. (1989a). Molecular Plant-Microbe Interactions 2, 262. Lindow, S . E., Panopoulos, N. J. and McFarland, B. L. (1989b). Science 244, 1300. Maki, L. R. and Willoughby, K. J. (1978). Journal ofApplied Meteorology 17, 1049. Maki, L. R., Galyan, E. L., Chang-Chien. M. and Caldwell, D. R. (1974). Applied Microbiology 28, 456. Margartis, A. and Bassi, A. S. (1991). Critical Reviews in Biotechnology 11, 277. Mizuno, H. (1989). Proteins: Structure, Function, and Genetics 5, 47. Mueller, G. M., Wolber, P. K. and Warren, G. J. (1990). Cryobiology 27, 416. Needleman, S. B. and Wunsch, C. D. (1970). Journal of Molecular Biology 48, 443. Orser, C., Staskawicz, B. J., Panopoulos, N. J., Dahlbeck, D. and Lindow, S. E. (1985). Journal of Bacteriology 164, 359. Parody-Morreale, A . Murphy, K. P., DiCera, E., Fall, R., DeVries, A. L. and Gill, S . J. (1988). Nature 333, 782. Phelps, P., Giddings, T. H., Prochoda, M. and Fall, R . (1986). Journal of Bacteriology 167, 496. Ruggles, J . A., Nemecek-Marshall, M. and Fall, R. (1991). Cry0 Letters 12, 245. Sands, D. C., Langhans, V . E . , Scharen, A. L. and deSmet, G. (1982). Journal ofthe Hungarian Meteorological Service 86, 148. Schnell, R. C. (1976). Bulletin o f t h e American Meteorological Society 57, 1356. Schnell, R . C. (1977). Journal of Atmospheric Science 34, 1299. ~~
HACI’bKIAL ICE NIJCLEATION
237
Schnell, R. C. and Vali, G . (1972). Nature 236, 163. Schnell, R. C. and Vali, G. (1973). Nature 246, 212. Schnell, R . C. and Vali, G. (1976). Journal of Atmospheric Sciences 33, 1554. Schwartz, R. M. and Dayhoff, M. 0. (1979). In “Atlas of Protein Sequence and Structure” (M. 0. Dayhoff, ed.), pp. 35S358. National Biomedical Research Foundation, Washington, D. C. Snider, J . R . , Layton, R. G . , Caplc, G . and Chapman, D. (1985). Journal de Recherches Atmosphiriques 19, 139. Southworth, M.W . , Wolber, P. K. and Warren, G. J. (1988). Journal oJBiologica1 Chemistry 263, 15211. Turner, M. A . , Arellano, F. and Kozloff, L. M. (1990). Journal of Bacteriology 172, 2521. Turner, M. A, , Arellano, F. and Kozloff, L. M. (1991). Journal of Bacteriology 173, 6515. Vali, G . (1971). Journal of Atmospheric Sciences 28, 402. Vali, G . , Cristensen, M., Fresh, R. W., Galyan, E. L . , Maki, L. R. and Schnell, R. C. (1976). Journal of Atmospheric Sciences 33, 1565. Vonnegut, B. (1947). Journal of Applied Physics 18, 593. Vonncgut, B. and Chcssin, H. (1971). Science 174, 945. Warren, G. J. (1987). In “Biotechnology and Genetic Engineering Reviews” (G. E. Russell, ed.), pp. 107-135. Intercept, Wimborne. Warren, G . and Corotto, L. (1989). Gene 85. 239. Warren, G . J. and Wolber, P. K. (1987). Cryo-Letters 8, 204. Warren, G. J. and Wolber, P. K. (1988). US Patcnt 4,784,943. Warren, G. and Wolber, P. (1991). Molecular Microbiology 5, 239. Warren, G., Corotto, L. and Wolber, P. (1986). Nucleic Acids Research 14, 8047. Warren, G., Wolber, P. and Green, R. (1987a). In “Plant Pathogcnic Bacteria” (E. L. Civerolo, A. Collmer, R. E. Davis and A. G. Gillaspie, eds), pp. 1013-1017. Martinus Nijhoff, Boston. Warren, G. J., Lindemann. J., Suslow, T. V. and Green, R . L. (1987b). In “Biotechnology in Agricultural Chemistry” (H. M. LeBaron, R. 0. Mumma, R. C. Honcycutt and J. H . Duesing, eds), pp. 215-227. American Chemical Society, Washington, DC. Watanabc, M. and Arai, S . (1987). Agricultural and Biological Chemistry 51, 557. Watanabe, M., Watabe, S . and Arai, S . (1988). Agricultural and Biological Chemistry 52, 1869. Watanabe, M., Watanabe, J., Kumcno, K., Nakahama, N. and Arai, S. (1989). Agricultural and Biological Chemistry 53, 2731, Watanabe, N. M., Southworth, M. W., Warren, G. J. and Wolber, P. K. (1990). Molecular Microbiology 4 , 1871. White, G . F. and Haas, J. E . (1975). “Assessment of Rcscarch on Natural Hazards”. MIT Press, Cambridge, MA. Wocrpel, M. D . (1980). US Patcnt 4,200,228, Wolber, P. K. and Green. R. L. (1990a). Trends in Biotechnology 8, 276. Wolbcr, P. K . and Green, R. L. (1990b). Food Science and Technology I , 80. Wolber, P. and Warren, G. (1989). Trends in Biochemical Sciences 14, 179. Wolber, P. K. and Warren, G. J. (1991). I n “Microbial Ecology of Leaves: Proceedings of the 5th International Symposium on Microbiology of the Phyllosphere” (J. Andrews and S. Hirano, eds), pp. 315-330. Springer-Verlag, New York. Wolber, P. K . , Deininger, C . A,, Southworth. M. W . , Vandekerckhove, J., van Montagu, M. and Warren, G . J. (1986). Proceedings of the National Academy of Sciences of the United States of America 83, 7256. Yankofsky, S. A . , Levin, Z . , Berfold, T. and Sandlerman, N. (1981). Applied Meteorology 20, 1013. Yankofsky, S. A , , Nadler, T. and Levin, 2 . (1983). Current Microbiology 9, 263. Zhao, J. and Orser, C . S. (1990). Molecular and General Genetics 223, 163.
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Metabolism and Functions of Glutathione in Micro-organisms MICHEL J. PENNINCKX" and MARC T. ELSKENS' Unite' de Physiologie et Ecologie Microbiennes, Faculte' des Sciences, Universite' libre de Bruxelles, Instut Pasteur Rrabant, 642 rue Engeland, B-I 180-Brussels, Belgium, and (present address) Laborutorium voor Analytische Scheikunde en Geochemie, Vrije Universiteit Rrussel, Pleinlaan 2, B-I050 Brussels, Belgium
a
Introduction . . . . . . . . . . . . . . . Occurrence and distribution of glutathione and related compounds in micro, . . . . . . . . . . . . . . organisms , 111. General outlines of glutathione metabolism in micro-organisms . . . A. Biosynthesis: y-glutamylcystcine synthetase and glutathione synthet ase . . . . . . . . . . . . . . B. Degradation:y-glutamyltranspeptidase . . . . . . C. Regulation of the y-glutamyl cyclc . . . . . . . . D. Glutathione metabolism mutants . . . . . . . . . E. Physiological roles of y-glutamyltranspeptidase . . . . . . IV. Interconvcrsion of glutathione and glutathionc disulphide . . . . . A. Glutathione transhydrogcnases . . . . . . . . . B. The glutaredoxin system . . . . . . . . . . . C. Glutathione peroxidase and the antioxidant defence system in microorganisms , . . . . . , , , , . . . . D. The glutathione redoc cycle . . . . . . . . . . V . Conjugation of glutathione: glutathionc Stransferases . . . . . A. Occurrence and distribution in micro-organisms . . . . . . B. Substrates and physiological functions . . . . . . . . VI. Otheraspectsofglutathionefunction . . . . . . . . . A. The glyoxalase pathway . . . . . . . . . . . B. Methanol dissimilation . . . . , . . C. Heavy-metal detoxification . . , , , , VII. Concluding remarks . . . . . . . . . . . . . VIII. Acknowledgements. . . . . . . . . . References . . . . . . .
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I. Introduction
A century ago, the French scientist de Rey-Pahlade (1888) observed that an ethanolic extract of brewer’s yeast reacted with elemental sulphur to provide hydrogen sulphide. This amazing property was attributed to the presence of a “sulphur-loving” compound first called philothion. The compound was later isolated from yeast and renamed glutathione (GSH) by the English biochemist Frederick Gowland Hopkins (1921). The structure of GSH was established as a tripeptide, y -glutamylcysteinylglycine (y-Glu-Cys-Gly), by chemical analysis, acid-base titration, degradation and synthesis (Kendall et al., 1929; Price and Pinhey, 1929; Harington and Mead, 1935). Since then, it has been demonstrated that GSH is present in high concentration in most living cells from micro-organisms to man. The elucidation of GSH metabolism and its physiological significance in cells has slowly evolved by studies on a variety of biological systems and biochemical reactions. The accelerating rate of data collection on the physiological functions of GSH is reflected by the frequency of symposia regarding this peptide (Colowick et al., 1954; Crook, 1959; FlohC et al., 1974; Arias and Jacoby, 1976; Elliott and Whelan, 1981; Cohen and Friedman, 1982; Larsson et al., 1983; Monks et al., 1990). The biological importance of GSH is mainly related to the free sulphydryl moiety of the cysteine residue which confers unique redox (E’” = -0.24 V for thiol-disulphide exchange) and nucleophilic properties on the tripeptide. The biosynthesis of GSH is remarkable in two ways: it is mRNAindependent, and the glutamic residue is joined in an unusual peptide linkage of the y -carbon atom to the cysteine residue. Due to this structural peculiarity, GSH is protected against proteolytic cleavage. It follows that a variety of functions have been attributed to GSH. Obviously, GSH research has ever become more specialized. A computer research of the Index Medicus indicates that more than 10,000 papers on, or quoting, GSH have appeared in the period since the publication of the Fifth Karolinska Institute Nobel Conference on the functions of this compound (Larson et al., 1983) till mid 1991. Most of them deal with studies on animal cells and, consequently, cover multidisciplinary fields, such as biochemical, physiological, toxicological and clinical aspects. In comparison, fewer studies were devoted to GSH in micro-organisms or plants (see, however, Penninckx and Jaspers, 1982; Rennenberg, 1982). Nevertheless, as a result of exchange of ideas, many aspects of GSH metabolism and its functions demonstrated or claimed in animal tissues were also found to apply to micro-organisms. However, substantial differences exist and it is the aim of the following sections to give an up-to-date picture of the development of knowledge on GSH metabolism in prokaryotes and
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microbial eukaryotes. We hope that assessment of the facts given may foster new endeavours and inform microbiologists as to the multiple aspects of this fascinating molecule. We discuss briefly the biologically relevant chemistry of GSH and its occurrence in microbial cells. The GSH-related biochemical reactions and the (possible) physiological roles of GSH are summarized.
11. Occurrence and Distribution of Glutathione and Related Compounds
in Micro-organisms As pointed out by Kosower and Kosower (1978), the GSH status of cells is defined by the total concentration of GSH and the nature and distribution of the possible forms in which the tripeptide can occur in the cell. The most important forms of this compound include reduced GSH, oxidized GSSG and mixed disulphides, mostly GSS-protein or GSSR (R represents a suitable residue such as cysteine or CoASH). Other possibilities are thiol esters which function as intermediates in metabolism of certain compounds such as methylglyoxal and formaldehyde (see Section V1.A). In addition, cellular compounds which behave chemically like GSH or GSSG, such as cysteine, y-glutamylcysteine and reactive disulphides, or which are produced by transpeptidation reactions (see Section 1II.B) like y-glutamylpeptides, should also be considered in assessing the GSH status of the cell. Glutathione has long been thought to be the principal low-molecularweight thiol in many biological systems, but the experimental basis for this generalization has been rather weak, owing to limitations in the available analytical methods. A systematic screening of the occurrence of GSH and related compounds in micro-organisms started at the end of the 1970s with the introduction of a powerful technique based upon the use of bromobimanes, fluorescent labelling agents developed by Kosower and his coworkers (1978, 1979, 1983). Glutathione and soluble non-protein thiol contents were examined by Fahey and his coworkers in a broad spectrum of micro-organisms. In bacteria, hydrogen sulphide was found in all species and was a major compound of many species. The general occurrence of sulphide in bacteria is not surprising and most likely originates from ironsulphur proteins rather than free hydrogen sulphide (Fahey and Newton, 1983). Glutathione appeared to occur primarily in facultative and aerobic Gram-negative bacteria, but not in strict anaerobes (Fahey et al., 1978). Thiol analysis of bacteria lacking GSH has indicated that CoASH was a major thiol in a number of species, both in Gram-negative and Grampositive bacteria (Fahey and Newton, 1983) whereas y-glutamylcysteine
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appeared mainly in halobacteria (Newton and Javor, 198.5). Glutathione, on the contrary, was found to be the major low-molecular-weight thiol in many microbial eukaryotes, including, fungi, protozoa and algae (Fahey and Newton, 1983; Fahey et a f . , 1987; Fairlamb, 1990). The fact that GSH occurred primarily in organisms with an aerobic lifestyle suggests that GSH metabolism might have evolved during or after the development of oxygenic photosynthesis. The finding that many bacteria, especially anaerobes, do not produce GSH and that a wide range of phototrophic organisms, purple bacteria, cyanobacteria and eukaryote algae are able to synthesize GSH seems to be consistent with this view (Fahey et a f . , 1987). The only phototrophic bacteria that tolerate oxygen and lack GSH are halobacteria (Newton and Javor, 1985). However, these bacteria, members of the archaebacteria lineage (Fox et a f . ,1980), produce y-glutamylcysteine in large amounts and have a disulphide reductase that maintains it in reduced state (Newton and Javor, 1985). Green bacteria are another group where occurrence of GSH is questionable, but most green bacteria are obligate anaerobes carrying out anoxic photosynthesis. So, it has been suggested that endosymbiotic processes giving rise to mitochondria and chloroplasts might represent a plausible mechanism for acquisition of GSH synthesis in eukaryotes (Fahey et al., 1987). Entomoeba histolytica, which lacks both chloroplasts and mitochondria, is indeed the only eukaryote that has been clearly demonstrated not to produce GSH (Fahey et a f . , 1984). The radioprotective effect of GSH as well as the correlations found between radiosensitivity variations of cells and their GSH contents were put forward as an argument for the concept that the tripeptide can be an intrinsic cellular radioprotector of special importance (Fuchs and Warner, 1975; Kosower and Kosower, 1978; Revesz and Malaise, 1983). It is therefore tempting to postulate that the initial function of GSH, when oxygen became a significant component of the atmosphere, was protection of cells against oxygen toxicity by destruction of thiol-reactive oxygen byproducts (Fahey el a f . , 1987). However, there is little other evidence to support this view. The finding that many bacteria, including some strict aerobes, lack GSH but contain other thiols suggests that more than one thiol-based protection system could have evolved in prokaryotes. Moreover, the apparent absence or virtual absence of GSH transferase and GSH peroxidase, some of the key enzymes involved in oxygen detoxification (see Sections 1V.B and V) in Escherichia coli (Smith and Shrift, 1978; Lau et al., 1980) and in Saccharomyces cerevisiae (Smith and Shrift, 1978; Aisaka et al., 1983), raises serious questions about the role of GSH in these organisms. It is possible, therefore, that GSH played entirely different functions in early bacteria, and that the oxygen detoxification
243
GLU~IAI'IIIONE IN MICRO-ORGANISMS
C/"\ k C-N
c'
0
,CH3
I y,/ \
c\ CH, C-OH
d
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I
I
NHZ C-N
o*
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'L H'
(b)
FIG. 1. Comparison of the structurc of (a) isopcnicillin N and (b) the p-lactam form of GSH (glutacillin). From Spallholz (1987).
function evolved only later. In this connection, the hypothesis of Spallholz (1987) should be considered. Glutathione is structurally similar to the precursor of the antibiotics produced in fungi in the genera Penicillium and Cephalosporiurn. Its potential conversion to the penicillin-like derivative glutacillin, a p-lactam form of GSH, raises the intriguing question whether glutathione was once a universal penem-like precursor of antibiotics in cells of many life forms (Fig. 1). The loss of the ability to convert GSH (if it ever existed) is, of course, open to speculation. It should be noted that the emergence of cellular immune systems with an apparent role for GSH and specific phagocytosis in higher organisms may have evolutionarily displaced the need for formation of natural antibiotics in higher organisms. The oral activity of glutathione against post-tumour induction in rats was found to be common with the oral activity of many penicillin derivatives against bacteria (Spallholz, 1987). The intracellular content of GSH is variable according to its distribution and occurrence in micro-organisms. Under normal, unstressed
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physiological conditions, much of the tripeptide is present in the free reduced form. In Sacch. cerevisiae (Penninckx et al., 1980) and E. coli (Newton and Fahey, 1990), the GSH content is very high and accounts for more than 1% of cell dry weight. The concentration of the oxidized form, GSSG, is usually much smaller, with reported values for the GSH-GSSG ratio generally being greater than 50. The balance between the thiol and disulphide groups is essentially maintained by the widely distributed GSH reductase (see Section 1V.D) ensuring a cellular environment in which essential sulphydryl groups of key enzymes and co-enzymes are protected. Modest changes in the rather low concentration of GSSG may be critical for regulation of certain physiological processes (Kosower and Kosower, 1978). Refined analytical methods are now available for very sensitive determinations of GSH and GSSG pools (Meister, 1985; Eyer and Podhradsky, 1986; Fahey and Newton, 1986) and considerable efforts have been undertaken to optimize extraction procedures in several kinds of organisms (Fahey and Newton, 1983; Fahey et al., 1987). Mixed disulphides have not been studied as extensively in microorganisms (see, however, Fahey et al., 1975). The mixed disulphide GSSprotein represents, in most organisms, intermediate forms with enzymes. These associations could reflect either a regulation of enzyme activity as with inorganic pyrophosphatase in Steptoccoccus faecalis (Lahti and Suonpaa, 1982) or modulation of protein conformation by thiol-disulphide exchange reactions (Pryor, 1962; Freedman and Hillson, 1980). It has been shown that the mixed disulphide CoASSG is a major component of the CoA pool in yeast (Stadtman and Kornberg, 1953) and E. coli (Loewen, 1981). The disulphide inhibits RNA polymerase and its reduction is catalysed by a specific enzyme in E. coli. The pool of CoA does not change much in mutants affected in GSH biosynthesis, but strains deficient in yglutamylcysteine synthetase (gshA) produce only the CoA dimer whereas mutants impaired in GSH synthetase (gshB) produce the mixed disulphide of CoA and y-Glu-Cys (Loewen, 1981). Another interesting and important derivative is the covalent adduct GSHspermidine formed at the end of exponential growth by E . coli (Tabor and Tabor, 1979) and in trypanosomatids (Fairlamb et al., 1985, 1986). In E coli, the product probably undergoes a rapid turnover and, therefore, may exist at a very low steady-state level in exponentially growing cells. Two specific enzymes, catalysing synthesis of the product from spermidine, GSH, ATP and magnesium ions and its hydrolytic degradation were, respectively, present during the entire growth stage. Glutathionylspermidine may play a role in regulation of growth and nucleic acid metabolism (Tabor and Tabor, 1975). In trypanosomes and leishmania, about 80% of GSH is present as N'-glutathionylspermidine and N' , fl-bis(glutathiony1)
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spermidine; the latter compound is unique to trypanosomatids and was once called trypanothione (Fairlamb et al., 1985). The biosynthetic pathway to trypanothione has been established by radiolabelling and inhibitor studies (Fairlamb et al., 1986, 1987; Bellofato et al., 1987). A single enzyme catalysing both N’-mono-, N8-monoglutathionylspermidine and trypanothione biosynthesis from ATP-Mg2+, GSH and spermidine has been purified approximately 14,500-fold to homogeneity in an overall yield of 40% (Henderson et al., 1990). The enzyme was active in monomeric form ( M r 87,000) and has a turnover number of 1700 min-’ with GSH and spermidine. It has been suggested that trypanothione has important physiological functions in trypanosomatids. In the first place, due to the absence of glutathione reductase (Fairlamb and Cerami, 1985), GSSG is reduced non-cnzymically by thiol-disulphide exchange with hydrotrypanothione (T[SH],). The resulting cyclic trypanothione disulphide (TS,) is reduced in turn by an NADPH-dependent flavoenzyme, trypanothione reductase (Shames et al., 1986; Jockers-Sherubl et al., 1989). Secondly, a trypanothione peroxidase activity that could contribute to protection of cells against oxygen damage was identified in Trypanosoma brucei and Crithidia fasciculata (Henderson et al., 1987; see also Section 1V.B). The importance of the trypanothione system is also of considerable interest in the development of chemotherapy against tropical diseases caused by parasitic trypanosomes (African sleeping sickness and Chagas’ disease) and leishmania (cutaneous and visceral leishmaniasis). A number of existing drugs have already been shown to interact with this important area of~metabolism(see Fairlamb, 1989, 1990). Amohg the different forms of GSH-related compounds are the peptides (y-Glu-Cys),-Gly produced in the presence of cadmium salts by Schizosaccharomyces pombe (Grill et al, 1985) and Candida glabrata (Mehra et al., 1988). These compounds are presumably involved in heavymetal detoxification (see Section V1.C). In addition, several low-molecularweight y-glutamyl compounds, including dipeptides and more complex mglCcules, were shown to be produced by micro-organisms. Little is known about their physiological roles (see, however, Section 1II.E) and their link with GSH metabolism is not always demonstrated. While there is some evidence that synthesis of y-glutamylpeptides occurs in vivo by transpeptidation reactions in Sacch. cerevisiae after growth on glutamate as the nitrogen source (Jaspers et al., 1985), and in Corynebacterium glutamicum, during the L-glutamic acid fermentation (Hasegawa and Matsubana, 1978), yglutamyl compounds are also produced by other pathways lacking direct relationship with GSH metabolism. For example, in the koji mold Aspergillus oryzae (Tomita et al., 1989) and Bacillus natto (Noda et al., 1980), a glutaminase (or transamidase) catalysed formation of y-glutamyl compounds
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from glutamine and amino acids. Several analogues of GSH were also reported. Ophthalmic acid (y-L-glutamyl-L-a-aminobutyrylglycine) and norophthalmic acid (y-L-glutamyl-L-alanylglycine) were isolated from the algae Undaria pinnatijida (Ogawa et al., 1990). The precursors of ophthalmic acid, a-aminobutyrate and y-glutamyl-r,-a-aminobutyrate, were often substituted to cysteine and y-glutamylcysteine, respectively, in the in vitro assays of y-glutamylcysteine synthetase (y-GCS) and GSH synthetase activities (Mooz and Meister, 1967). N-(N-y-Glutamyl-3-sulphoL-alany1)glycine was found in the mushroom Flammulina vetupilis (Ogawa et al., 1987) and l-y-glutamyl-2-(2-carboxyphenyl)hydrazineis produced by Penicillium oxalicum (Minato, 1979). The latter compound, trivially named anthglutin, was also synthesized as the result of a rational chemical design for y-glutamyltranspeptidase (y-GT) inhibitors (Griffith and Meister, 1979a). y-Glutamyltranspeptidase
-
Amino-acid transport
integrity
FIG. 2. Diagrammatic representation of the intcrrelationship of GSH with other cellular biochemical systems (Mitchell, 1988).
GI UTATHIONF IN MICRO ORGANISMS
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The present data are a good illustration of the diversity regarding occurrence of GSH and related compounds in the microbial world, and provide some insight into the complexity of thiol biochemistry in microorganisms. A precise expression of GSH status, as proposed by Kosower and Kosower (1978), involving measurements of GSH contents, a quantitative description of the different chemical forms of the tripeptide and related compounds and an assessment of their spatiotemporal variations within the cell, is still difficult to establish for micro-organisms due to a lack of knowledge of certain reactions. Nevertheless, one should bear in mind that the term “status” does not imply a fixed or constant value for GSH contents, but refers rather to a description of a dynamic system with a shifting of the equilibrium among different forms in response to natural or artificial perturbations. Although GSH appears not to be essential in prokaryotes, its uniform distribution in eukaryotes suggests that it might serve essential functions. Amid the complex machinery of cellular biochemistry, the tripeptide assumes a pivotal role in numerous bioreductive reactions, transport, enzyme activity, protection against harmful oxidative species, and detoxification of xenobiotics. Having such functional diversity, GSH is interrelated with a number of biochemical systems (Fig. 2). As for animal cells, GSH-related enzymes in micro-organisms can be grouped into those concerned with biosynthesis, degradation, reduction, oxidation, conjugation and those in which GSH serves as a cofactor.
111. General Outlines of Glutathione Metabolism in Micro-organisms
The scheme given in Fig. 3 outlines the biochemistry of GSH and associated pathways that were identified or claimed to exist in micro-organisms. Many investigators in this field were largely influenced by studies on animal tissues and tried firstly to identify similar pathways in micro-organisms. Differences appeared in the course of these investigations, and some major features regarding enzymes and biochemical phenomena involved have emerged only quite recently. Several lines of evidence indicate that GSH metabolism proceeds in higher eukaryotes through the y-glutamyl cycle (Meister, 1981a; Rennenberg, 1982; Meister and Anderson, 1983). In its original version, the cycle accounts for six reactions whose three steps are ATP-dependent, namely the two consecutive reactions of GSH biosynthesis (equations (1) and (2)) and conversion of 5-oxo-~-prolineto i,-glutamate (equation (6)). Modifications of the y-glutamyl cycle have been discussed in detail elsewhere (Meister, 1981b). y-Glutamyltranspeptidase, following hydrolysis and transpeptidation reactions, provides an alternative pathway in
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\ /\
GLY
\
5-Oxoproline
\
ATP
FIG. 3. The y-glutamyl cycle (Meister, 1983). The enzymes involved are (1) yGlutamylcysteine synthetase, (2) glutathione synthetase, (3) y-glutamyltranspeptidase, (4) cysteinylglycine dipeptidase, (5) y-glutamylcyclotransferase and (6) 5-oxoprolinase.
which activities of y-glutamylcyclotransferase and 5-oxoprolinase are excluded. Until now, only little evidence has been presented for the existence of a complete y-glutamyl cycle in micro-organisms (Jaspers et al., 1985), even though a first report for baker's yeast was published in 1976 (Mooz and Wigglesworth, 1976). In strains of Saccharomyces cerevisiae, GSH catabolism appears to be mediated by y-GT and cysteinylglycine dipeptidase only, and it was observed that the latter activity is shared by several peptidases constitutively produced by this organism (Jaspers et al., 1985). Both y-glutamylcyclotransferase and 5-oxoprolinase activities were undetected in crushed or permeabilized yeast cells. Direct labelling experiments have shown that ['4C]5-oxoproline (pyroglutamic acid) was taken up intact, but not further metabolized into glutamate (tllz being 1000 minutes). y-Glutamyltranspeptidase was described long ago in many bacteria (Milbauer and Grossowicz, 1965) and was even suggested as a useful indicator for identification of members of the Enterobacteriaceae (Giammanco et al., 1980). A dipeptidase was also shown in Bacillus cereus (Cheng et al., 1973). More recently, a typical 5-oxoprolinase from a Pseudomonas putidu strain was purified and characterized (Li et ul., 1988). As far as is known, this remains an isolated but substantiated observation in the
249
GLUTATHIONE IN MICRO-ORGANISMS
microbial world. Hence, the current picture emerging from these investigations into micro-organisms suggests that, for most of them, a truncated version of the y-glutamyl cycle, involving the biosynthesis enzymes y-GT and cysteinylglycine dipeptidase, would exist. Quite apart from this debate, it is clear that the cycle concept introduced by Meister as a result of observations on animal tissues has been very useful as a working hypothesis for a number of investigations on the biochemical function of GSH. This concept has led to many findings about glutathione and has radically altered the understanding of metabolism of the thiol tripeptide in living cells. A.
BIOSYNTHESIS:
y-GLUTAMYLCYSTEINE SYNTHETASE
AND
GLUTATHIONE SYNTHETASE
Glutathione is synthesized intracellularly by the consecutive action of y-GCS (L-y-glutamate-L-cysteine y-ligase (ADP); EN 6.3.2.3) and GSH synthetase (L-y-glutamy1cysteine)-glycine y-ligase (ADP); EN 6.3.2.3) (Snoke and Bloch, 1954). Both enzymes require ATP and magnesium ions for activity. There is some evidence that enzyme-bound y-glutamylphosphate and yglutamylcysteinylphosphate are formed in these reactions, whose mechanisms are thus similar to those catalysed by glutamine synthetase (Meister 1983). y-Glutamylcysteine synthetase (y-GCS) has been isolated from several sources, but only recently purified and characterized from a microbial source (Watanabe et al., 1986). The enzyme from Escherichia coli consists of a single polypeptide chain ( M , 55,000) differing from the rat-kidney enzyme (Orlowski and Meister, 1971), which dissociated into two non-identical subunits ( M , 74,000 and 24,000) and from the enzyme from Proteus mirabilis, which separated into three subunits with respective molecular weights of 30,000, 11,000 and 13,000 on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis (SDS-PAGE) (Kumagai et al. , 1982). However, the possible presence of proteolytic contaminants was apparently not checked in the latter report. The complete nucleotide sequence of the gene coding for y-GCS in E. coli has been reported (Watanabe et al., 1986). The polypeptide deduced from the open-reading frame has a molecular weight that agrees with values+reviously determined by SDS-PAGE and gel filtration (Murata and Kimura, 1990). As already shown for the rat-kidney enzyme (Richman and Meister, 1975) the bacterial enzyme is also inhibited by physiological concentrations of GSH with a Ki value of 2.5 mM (Murata and Kimura, 1990). Presumably, this indicates a physiologically significant feedback mechanism. Molecular cloning of the y-GCS gene from Sacch. cerevisiae has shown that the GSHA gene comprises a segment of 2034 bp that encodes for a protein with about 678
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amino-acid residues (Ohtake and Yabuuchi, 1991). The deduced aminoacid sequence presented 45% homology with the rat-kidney enzyme (Yan and Meister, 1990), but only 26% with the enzyme from E. coli (Watanabe et al., 1986). L-Methionine (S)-sulphoximine is an inhibitory analogue of the enzymebound y-glutamylphosphate intermediates formed in reactions catalysed by both glutamine synthetase and y-GCS (Meister, 1983). The rational design and synthesis of new analogues which produce selective inhibition of these enzymes have been developed by Meister and his coworkers (Griffith et al., 1979; Griffith and Meister, 1979b). Buthionine (S,R)-sulphoximine (BSO) is an analogue inhibitor of the transition state of y-GCS-bound substrates (y-glutamylphosphate and cysteine) and is at least 200 times more active than methionine sulphoximine. Since the S-butyl moiety of BSO prevents its interaction with glutamine synthetase, the new inhibitor is very selective. Depletion of glutathione by BSO has proved to be a very useful method for decreasing GSH levels in many organisms (Meister, 1988) and has several advantages over the use of oxidizing agents and compounds that react with GSH itself. The second enzyme, GSH synthetase, was purified from yeast 25 years ago (Mooz and Meister, 1967). An apparent molecular weight of 123,000 was first deduced from ultracentrifugation experiments. Further investigations using gel filtration and SDS-PAGE indicated molecular weights of, respectively, 152,000, 147,000 and 152,000 for enzymes from yeast (Meister, 1974), P. mirabilis (Nakayama, 1984) and E. coli (Gushima et al., 1983). The purified enzyme from E. coli is apparently composed of four identical subunits ( M , 38,000). Crystallization and preliminary X-ray studies were recently reported. Rather amazingly, a strong homology between GSH synthetase from E. coli and mammalian and bacterial dihydrofolate reductases was shown over 40 amino-acid residues, in spite of the fact that these enzymes differ in their reaction mechanisms and ligand requirements (Kato et al., 1987). Some current studies are following the trend of elucidating evolutionary pathways of these enzymes (Murata and Kimura, 1990). B.
DEGRADATION: y-GLUTAMYLTRANSPEPTIDASE
y-Glutamyltranspeptidase (y-GT; E C 2.3.2.2.) is an enzyme of major importance in GSH metabolism although its physiological role is not yet fully understood. As previously stated, y-GT is widely distributed in bacteria and has also quite recently been isolated from Mycobacterium smegmatis, a representative species of the actinomycetes (Kumar et al., 1990). In microbial eukaryotes, the enzyme was detected in yeast (Mooz
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and Wigglesworth, 1976; Penninckx et al., 1980), in the moulds Tricholoma shimeji (Iwami et al., 1978) and Aspergillus oryzae (Tomita et al., 1988), and in the epimastigotes of the protozoan Trypanosoma cruzi (Repetto et al., 1987). In E. coli and P. mirabilis, y-GT is localized in the cell walls and periplasmic space or both, as shown by studies with lysozyme-EDTA and fluorescent antibodies (Nakayama et al., 1984; Suzuki et al., 1986). In Sacch. cerevisiae, the enzyme appears as a membrane-bound entity localized mainly in vacuoles (Jaspers and Penninckx, 1984) and/or in the plasmalemma (Payne and Payne, 1984). Fully cytosolic forms were reported for T. cruzi and M . smegmatis (Repetto et al., 1987; Kumar et al., 1990). An excreted form was also detected in A . oryzae (Tomita et al., 1988). The enzyme was extensively purified from P. mirabilis (Nakayama et al., 1984), E. coli (Suzuki et al., 1986) and from Sacch. cerevisiae (Jaspers and Penninckx, 1985). In all instances, the enzyme was dissociated into two different subunits as previously shown for the mammalian form. However, in mammals, y-GT appears to be a glycoprotein and this was only once reported for the enzyme from Sacch. cerevisiae. The DNA sequence of the gene in E. coli has been determined recently (Suzuki et al., 1989). The sequence contained a single open-reading frame, encoding the signal peptide and both subunits, which suggests a post-translational processing of y-GT. Purified microbial y-GTs can catalyse, as the mammalian enzyme, three types of reactions: (a) hydrolysis in which the y-glutamyl moiety is transferred to water; (b) transpeptidation in which the y-glutamyl moiety is transferred to an amino-acid or peptide acceptor; and (c) autotranspeptidation in which the y-glutamyl moiety is transferred to GSH. Glutathione (GSH and GSSG), S-substituted GSH and numerous yglutamyl compounds are potential substrates for y-GT. L-Cystine, methionine and glutamine are among the most active acceptors, but other amino acids, as well as many dipeptides, especially aminoacylglycines, also participate significantly in transpeptidation (Meister and Anderson, 1983; Penninckx and Jaspers, 1985). y-Glutamyltranspeptidase is inhibited specifically and competitively by L- and D-isomers of y-glutamyl(o-carb0xy)phenylhydrazine and related compounds (Griffith and Meister, 1979a; Minato, 1979). A combination of L-serine and borate apparently inhibits y-GT by forming an analogue of the transition state (Tate and Meister, 1978). Several glutamine analogues, such as L-azaserine, 5-oxo-~-norleucineand ~-[aS,5S]-u-amino-3-chloro4,5-dihydro-5-isoxazole acetic acid were also described as non-specific but potent irreversible inhibitors of y-GT (Tate and Meister, 1978; Allen et al., 1980; Griffith and Meister, 1980).
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C. REGUCATION OF T H E y-GLUTAMYL CYCLE
The intracellular concentration of GSH reflects the equilibrium between biosynthesis and degradation. In Sacch. cerevisiae, y-GT synthesis was found to be regulated by at least two apparently distinct pathways (Table 1). In the presence of ammonium ions as a nitrogen source, the transpeptidase level is low in the wild-type strain (about 40 nmol h-l (mg protein)-'). In the presence of glutamine, arginine, ornithine or citrulline, the enzyme level rises to an intermediate value (8&100 nmol h-' (mg protein)-') and the highest specific activities are observed with proline, urea or glutamate (200-225 nmol h-' (mg protein-'). When cells of Sacch. cerevisiae are starved for two hours by transfer to a medium devoid of a nitrogen source, the level of cellular enzyme rises to the highest value (200 nmol h-' (mg protein)-'). A study of the rate of y-GT synthesis showed that, after transfer from a proline-supplemented medium to a medium containing ammonium TABLE 1. Regulation of the y-glutamyltranspeptidasc in Sacchuromyces cerevisiae. The enzyme specific activity (as rate of release of p-nitroaniline; see Penninckx et al., 1980) was estimated in crude extracts from exponential-phase cells growing on the nitrogen sources listed in thc table ~
Strains and genotypes
Nitrogenous nutrients
Enzyme specific activity (nmol h-' (mg protein)-')
-~
Z1278b (wild type)
gshA
gdhA
gdhCR
gap
-
apf argp
-
argp
-. gap-.
argp-, lysp-
Ammonium ions Proline Urea Glutamate Ammonium ions Ammonium ions with GSH Glutamate Glutamate with CSH Ammonium ions Glutamate Urea Ammonium ions Glutamatc Urea Ammonium ions Proline Ammonium ions Urea Ammonium ions Urea Ammonium ions Urea Ammonium ions Urea
<40 200 225 225 98 60 430 270 45 225 240 50 210 235 <40 200 <40 t40 <40 240 <40 260 <40 240
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ions, the enzyme is expressed at the low rate characteristic of the minimal medium. This result is consistent with a repression mediated by ammonium ions (Pennickx et al., 1980). The repressive effect of ammonium ions on the transpeptidase was found to be unaffected in mutants (gdhA, gdhCR) which have lost the regulatory circuits governed by glutamine and the glutamate dehydrogenase-NH4" complex (Table 1 ) . Taking into account that most of the nitrogen metabolic pathways in Sacch. cerevisiae were found to respond to at least one of the two above-mentioned signals (Dubois et al., 1977), it seems that regulation of the transpeptidase level depends on a different mechanism. The second regulation mechanism was discovered in the course of an investigation into the biochemical characterization of a GSH-deficient yeast mutant (gshA) impaired in the first step of glutathione biosynthesis. As shown in Table 1, GSH deficiency has a noticeable influence on the specific activity of y-GT. In the Gsh- phenotype, the specific activity of y-GT was about twice (9C100 nmol h-' (mg protein)-') that in the wild-type strain growing on the same ammonia-containing medium. Addition of GSH to a culture of a deficient strain decreased the differential rate of y-G1' synthesis to a value close to that of the wild type. In contrast, derepression of y-GT synthesis was observed when the GSH level in the wild-type strain was lowered by the use of BSO. It is interesting to note that the rate of y-GT synthesis increases drastically when the intracellular concentration of GSH falls below 4 nmol (mg dry weight)-'. The enzyme derepression which appears constitutive in gshA mutants results, therefore, most likely from an alteration of the thiol status and correlates best with a decrease in the GSH pool among the sulphur-containing compounds examined (Elskens et al., 1991). The repression mediated by ammonium ions is still present in GSHdeficient strains and the highest specific activity is again observed with glutamate, proline or urea as the sole nitrogen source (Table 1). Promotion of the y-GT synthesis caused by GSH deficiency and the absence of a better nitrogen source results from a simple cumulative effect. The extent of derepression is relatively weak, with a 2-10-fold increase in activity when both effects are combined. Derepression of y-GT synthesis in Sacch. cerevisiae is correlated with a strong increase in GSH turnover rate. From a value of about 900 minutes on a minimal medium containing ammonium ions the half-life of intracellular GSH is decreased to approximately 200 minutes with glutamate as the only nitrogen source (Jaspers et al., 1985). Since the increase of turnover rate does not lower the intracellular GSH content under normal unstressed physiological conditions (Jaspers and Penninckx, 1981; Robins and Davies, 1981b; Jaspers et al., 1985), a net increase in the GSH biosynthesis rate is expected.
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In yeast, the intracellular level of GSH synthetase is apparently unaffected by the nature of the nitrogen source (Jaspers et al., 1985), but y-GCS is feedback inhibited by GSH and is apparently the rate-limiting step in biosynthesis of the tripeptide (Ohtake et al., 1989). Biotechnological investigations dealing with GSH biosynthesis by genetically engineered cells of E. coli support this view (Murata and Kimura, 1990). Molecular breeding experiments with hybrid plasmids having the genes encoding GSHA and GSHB have shown that the first step in glutathione biosynthesis is ratelimiting, and that a simultaneous increase in activities of both the GSHA and GSHB enzymes resulted in a higher production of glutathione. It could be suggested, therefore, that GSH might be able to regulate its own formation and maintain a steady-state equilibrium between synthesis and degradation by acting on y-GCS only (see Kumagai et al., 1982). However, the situation might be more complicated. In Penicilliurn chrysogenum, a stimulatory effect of ammonium ions on GSH formation has been observed (Schwartz et al., 1988). High concentrations of the ion stimulated not only formation of GSH, but also increased the intracellular pools of glutamate, alanine and glutamine. The induction effect of glutamate on GSH synthesis by stimulating y-GCS activity was shown in resting cells of the wild-type strain Pe. chrysogenurn. In yeast, it would not be very surprising to find that the availability of nitrogenous nutrients plays a leading role in determining rates of GSH formation because synthesis of enzymes involved in the amino-acid biosynthetic and catabolic pathways is regulated (Jones and Fink, 1982). Nevertheless, as specific regulatory controls in aminoacid biosyntheses are not common in yeast (Messenguy et al., 1980), the possible inductive effect of glutamate on y-GCS remains to be demonstrated for this organism. The carbon source has been found to affect GSH production in yeast. A recent study, emphasizing optimal production of GSH in which the specific growth rate of Sacch. cerevisiae was controlled in glucose-fed batch culture, has shown that, when the growth rate was kept at sufficiently low values, ethanol was not produced and the intracellular level of GSH was about 2540% higher than under conditions where ethanol is produced (Shimizu et al., 1991). It is predictable, therefore, that GSH biosynthesis may be under the influence of the so-called glucose effect (Pasteur and Crabtree effects) which is observed in a larger number of yeast species. One should stress, however, that glucose regulation, especially in yeasts, includes an extensive number of enzymes and is probably based on a rather complicated regulatory situation in terms of underlying mechanisms. It seems, indeed, that catabolite repression represents only one of the mechanisms involved, and it could be accompanied by other regulatory mechanisms, specific controls of protein synthesis and modification as well as catalytic turnover of enzymes (Fiechter
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et al., 1981). So far, it would appear that the effect of glucose limitation on GSH biosynthesis may be direct or indirect and needs to be investigated in greater detail before the question is definitively answered. Besides the effects of nitrogenous and carbon nutrients, perturbation of the intracellular GSH status has been reported in relation to the cell cycle, growth and development. During the exponential growth phase of Sacch. cerevisiae, the intracellular concentration of GSH remains almost constant, but increases drastically in a reproducible way within fairly narrow ranges when measured under the same conditions at the beginning of the stationary phase of growth. Changes in the intracellular GSH content during induction of the yeast-to-mycelial transition in Candida albicans was also reported. As emphasized by Kosower and Kosower (1978), variations, both in time and space, are natural for the GSH status of cells. Serious deviations from usual or normal values can occur as a consequence of (a) normal physiological situations, (b) genetic defects, and (c) chemical reactions resulting from addition of exogenous agents. In the last case, most of the perturbations decrease the GSH content by chemical reactions and biochemical processes, such as oxidation and conjugation. Given the current GSH status for a cell, a perturbation can be either reversible or irreversible. Reversible situations mostly involve oxidation of the reduced form into GSSG and mixed disulphides, but also include acylation of GSH. Irreversible perturbations take place when GSH synthesis fails, if GSH is converted into covalently bound derivatives or when GSH or GSSG is eliminated from the cell. Unlike animal cells, GSH and its derivatives are not normally excreted into the surrounding culture medium by whole microorganisms, but leakage was reported for some strains of Sacch. cerevisiae growing in the presence of selenite or ethanol (Izuka et al., 1988) and in filamentous forms of Candida tropicalis (Yamada et al., 1984). Similarly, inhibition of y-GT by serine-borate causes leakage of GSH from whole P. mirabilis (Nakayama et al., 1984) and in some mutants of E. coli (Murata et al., 1980). D . GLUTATHIONE METABOLISM MUTANTS
y-Glutamylcysteine synthetase (gshA)- and GSH synthetase (gshB)deficient mutant strains have been isolated and characterized in bacteria (Apontoweil and Behrends, 1975b; Fuchs and Warner, 1975; Murata et al., 1981; Fuchs et al., 1983; Kerklaan et al. , 1985; Bouter et al., 1988) and in yeast (Kistler et al., 1986; Glaeser et al., 1991). Apontoweil and Behrends (1975a,b) isolated a mutant of E. coli impaired in y-GCS by selecting cells with increased diamide sensitivity. The gshA mutant contained no detectable glutathione in a trichloroacetic acid
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M J. PENNINCKX AND M. T. EISKENS
extract of [35S]sulphate-labelled cells. Comparative assays for y-GCS indicated that the mutant had only 4% of the parental activity. Although the growth rate of the Gsh- phenotype appeared unaltered by glutathione deficiency, the cells were 3-10-fold more sensitive to sulphydryl reagents, antibiotics, chemotherapeutic agents, food additives, pesticides and other miscellaneous chemicals. The gshA mutation mapped at 57 minutes on the E. coli chromosome (Apontoweil and Berends, 1975b). Concurrently, with isolation of a y-GCS-defective E. coli mutant, Fuchs and Warner (1975) isolated a gshB mutant in an attempt to obtain thioredoxin reductase mutants (the thioredoxin pathway provides to some extent an alternative pathway for reactions that may otherwise require glutathione; see Section 1V.B). Glutathione was not detected in the gshB mutant, neither when the classical enzymic assay was used for GSH determination nor when the trichloroacetic acid fraction was labelled with ['4C]N-ethylmaleimide, a thiol alkylating reagent. However, the mutant had a significantly elevated pool of y-glutamylcysteine. Assays for GSH synthetase indicated that the mutant had less than 10% of the parental enzyme activity. The gshB mutant grew at the same rate as the parental strain, but its growth was inhibited at a three-fold lower concentration of diamide or methylglyoxal. It was found to have an enhanced sensitivity to X-rays in the absence of oxygen (Morse and Dahl, 1978), but not when oxygen was present (Fuchs and Warner, 1975). The gshB mutation mapped near xyl by Hfr matings (Fuchs et al., 1983). To diminish the possibility that a small but significant amount of glutathione might be synthesized in either the gshA and the gshB mutant, Fuchs and his coworkers (1983) constructed a (gshA gshB) double mutant. A TnlO transposon was introduced into a gene required for utilization of sorbitol (srl) by Csonka and Clark (1980). From the mapping data of Apontoweil and Berends (1975b), the gshA gene should cotransduce with this sr1:TnlO with a frequency of about 66%. A sr1:TnlO gshA strain was thus constructed and subsequently used to transduce the gshB mutant to tetracycline resistance. The double mutant did not differ in growth characteristics from the gshB parent, which suggests that GSH is not required by E. coli under laboratory growth conditions (Fuchs et a l . , 1983). As previously stated, a strain of E. coli enriched in its content of y-GCS (GSHA) and GSH synthetase (GSHB) activities by recombinant DNA techniques was also characterized (Moore et al., 1989; Murata and Kimura, 1990). The proficient enriched strains had a higher GSH content than their corresponding wild type and appeared much more resistant to the lethal effects of y-irradiation. The radioresistance, however, was shown to correlate better with increased ability of the gene-enriched strain to synthesize GSH rather than to the cellular level of the tripeptide per se. It
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follows that resistance can be abolished by BSO, a specific y-GCS inhibitor that decreases the rate of GSH biosynthesis but does not react directly with the tripeptide (Moore et al., 1989). Glutathione-deficient mutants (gshA) of Sacch. cerevisiae were isolated after ultraviolet irradiation using the N'-nitro-N-nitrosoguanidines as selective agents (Kistler et al., 1986). Since the nitrosoguanidines (MNNG or ENNG) are activated by GSH in bacteria, yeast and mammalian cells (Mohn et a f . , 1983), selection was based upon isolation of the Gshphenotype as MNNG- and/or ENNG-resistant clones. All isolates were impaired in at least the first step of GSH biosynthesis (gshA). Residual activity of y-GCS was found to be about 5-10% of the parental activity (Kistler et al., 1990). The mutants exhibit considerably lower residual GSH contents, varying from 2 to 8% of the wild-type strain. All isolates show a 2:2 segregation of the Gsh-: GSH' phenotypes, suggesting a monogenic recessive mutation. Complementation analysis furthermore indicated that all mutants belong to one complementation group. Deficiency of GSH leads to pleiotropic phenotypes of the mutants. Lower GSH levels in the haploid Gsh- strains are correlated with an extension of the lag phase of growth and a decrease in growth rate when cells were cultivated on rich or synthetic media (Kistler et al., 1986). Similar conclusions were reached by Elskens et a f . (1991) from experiments with BSO. Cells containing more than about 1 nmol GSH (mg dry weight)-' are apparently able to grow normally. At present, it is unknown if a complete lack of GSH is lethal in Sacch. cerevisiae as the growth rate decreases dramatically below this value. Kistler et al. (1986) reported that a residual GSH level of 1%"of the wild-type value, as found in some gshA mutants, leads to serious impairments in the viability and respiratory ability, accompanied by loss of mitochondria1 DNA. Prototrophic Gsh- derivatives (for filiation of the strains, see Elskens et al., 1991) show in various degrees, as already described for the original isolates, the pleiotropic effect of the mutation on growth and sexual reproduction. But it was also demonstrated that they presented striking differences in contents of the sulphur-containing amino acids and thiol status when compared with wild-type strains growing on the same minimal medium containing ammonium ions. Similar to gshA mutants of E. coli, GSH-deficient strains of Sacch. cerevisiae were found to be more sensitive to sulphydryl reagents such as diazene derivatives (e.g. diamide), dithiocarbamate pesticides and chloro- or fluorodinitrobenzene compounds. It is important to note that the selection procedure may influence isolation of GSH-deficient mutants. According to Kistler and his colleagues (1990), yGCS-deficient strains have been much more frequent among selected Gshmutants of E. coli. Furthermore, when nitrosoguanidine (MNNG, ENNG) resistance was the selected phenotype, only y-GCS-deficient
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mutants were isolated from Salmonella typhimurium and E. coli (Bouter et al., 1988). There is substantial evidence that GSH-mediated activation of nitrosoguanidines is performed in bacteria by dipeptide y-glutamylcysteine as well, especially when it is accumulated as a result of a GSH synthetase deficiency. Therefore, it has been suggested that MNNG selection would probably be inappropriate to detect GSH synthetasedefective mutants (Kistler et al., 1990). It appears, however, that such mutants of Schizosaccharomyces pombe were obtained by this procedure (Glaeser et al., 1991). Genetic defects related to GSH biosynthesis are considered to be a useful tool in determining some of the roles of GSH in micro-organisms. In spite of the fact that GSH is apparently not essential for growth of E. coli under usual laboratory conditions, it is likely that E. coli and probably many other prokaryotes require the tripeptide as protection against chemical challenges (e.g. reactive free radicals, oxidative stress, genotoxic agents and miscellaneous xenobiotics) . Similar conclusions can be drawn for microbial eukaryotes, although a minimum level of GSH might be required for growth, as previously shown for Sacch. cerevisiae. Detailed analyses of the effects of GSH deficiency on growth and respiration of yeast cells will probably lead to enlightenment of some of the regulatory functions of GSH in this organism. E. PHYSIOLOGICAL ROLES OF y-GLUTAMYLTRANSPEPTIDASE
1. Transport of A m i n o Acids and Potassium Ions In animal cells, y-GT is membrane bound and highly concentrated at sites of extensive amino-acid transport (Meister, 1983). The hypothesis that amino acids might be transported as y-glutamyl derivatives has been suggested by Meister and his coworkers. Recent findings indicate that formation and transport of y-glutamyl-amino acids across the cellular membrane is most likely associated with transport of amino acids by kidney cells, although the relative contribution of this pathway is still unknown (Meister, 1988). The possible involvement of the y-glutamyl cycle in uptake of amino acids by Sacch. cerevisiae and Candida utilis has been proposed (Mooz, 1979; Osuji, 1980) but is further debated. A number of significant observations have been made regarding permeation system of amino acids and dipeptides into Sacch. cerevisiae (Wiame et al., 1985). Although several systems that mediate amino-acid transport in yeast exist, the mechanism by which some operate is not yet known. In this connection, the idea that certain of these solutes might be transported into yeast cells as y-glutamyl
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derivatives was appealing. To support this assessment, investigations have emphasized a requirement for studies of y-GT regulation, using selective inhibitors and different yeast mutants impaired in the amino-acid permeation system. As shown in Table 1, the intracellular level of y-GT remained unaffected in mutants which lost the general amino-acid permease (Gap-) and the specific systems for L-arginine (Argp-) and L-lysine (Lysp-), but only very low activity was detected in the Apf mutant growing on urea, a nitrogen source promoting high levels of y-GT in the wild-type strain (Penninckx et al., 1980). The Apf mutation most likely affects a common element shared by all of the amino-acid permeation systems (Grenson and Hennaut, 1971). This latter result supported the idea that y-GT could be involved in a group-translocation mechanism similar to the HPr or protein I elements of the bacterial phosphotransferase system (Roseman, 1969; Kunding, 1976). However, the effect of the Apfmutation on the level of y-GT activity was further shown to be only indirect (Jaspers, 1985). No correlation can thus be established between the level of y-GT activity and mutations impairing bulk uptake of amino acids in yeast and no relation was found with turnover of GSH (Robins and Davies, 1981a,b). From the GSH content of yeast, its turnover and rate of L-glutamate uptake, it has been estimated that about 30-100 molecules of L-glutamate are transported for every degraded GSH molecule. Similar conclusions were reached by Jaspers and his colleagues (1985) using a different methodology for determination of GSH turnover. In addition, specific chemical inhibition of y-GT in whole Sacch. cerevisiae using 6-diazo-5-oxo-~-norleucinedid not affect uptake rates of several amino acids, dipeptides and y-glutamyl compounds (Payne and Payne, 1984). Finally, progress in the field of aminoacid transport in micro-organisms, especially in Sacch. cerevisiae, has highlighted the role of ionic pumps (Eddy, 1982). To conclude, all of these results militate against a role for the y-glutamyl cycle in transport of amino acids in yeast, but d o not rule out, for instance, possible involvement of y-GT in a vacuolar-facilated transport (Jaspers and Penninckx, 1984) or in generation of y-glutamyl derivatives to act as signals in regulation of transmembrane processes (Payne and Payne, 1984). Discrete pools of y-glutamylglutamine and y-glutamylglutamate were indeed detected in Sacch. cerevisiae growing on glutamate as a nitrogen source (Jaspers et al., 1985). The rate of y-GT biosynthesis is derepressed in such a medium and L-glutamate, as well as L-glutamine, was shown to be a good acceptor in the transpeptidation reaction catalysed by the yeast enzyme (Penninckx and Jaspers, 1985). Substantial portions of intracellular amino acids and glutathione are compartmentalized within the vacuole. Redistribution of amino acids between compartments has been demonstrated in response to metabolic signals (Messenguy et al., 1980; Wiemken,
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M J P F N N I N C K X AND M
r
FISK~NS
1980; Kitamoto et al., 1988). To what extent y-glutamyl derivatives might play a role in such processes is unknown, and further research work along these lines is needed. A role for GSH in transport of potassium ions in E. coli has been discovered (Meury and Robin, 1985, 1990). Mutants deficient in GSH, unlike the wild type, rapidly leak potassium ions and grow very slowly on a medium containing a low concentration of potassium ions. Addition of GSH, ophthalmic acid or y-(glutamylcysteiny1)bisglycine to the culture medium decreases the leak of potassium ions and restores a wild-type growth rate. A protein with a highly apparent affinity to GSH (Kd 50 n M ) was identified as a cytoplasmic element regulating the circulation of potassium ions. For each of three peptides, the Kd value €or binding is similar to the minimal concentration achieving maximal decrease of the leak. The possible physiologicai effectors might be y-glutamyl derivatives generated by periplasmic y-GT. 2. Mobilization of Glutathione as a Sulphur Source Glutathione was long ago cited as a growth factor for micro-organisms but, meanwhile, few detailed reports have further addressed the subject (Brown, 1974; Penninckx and Jaspers, 1982). Glutathione also appeared to be an alternative source of sulphur for morghogenesis of spores of Bacillus cereus (Cheng et al., 1973) and vegetative cells of a species of cyanobacterium (Giddings et al., 1981). In Sacch. cerevisiae, substantial evidence indicates that the y-glutamyl cycle can function to meet an intracellular requirement for cysteine, and is plausibly involved in overall regulation of sulphur metabolism in this organism (Elskens et al., 1991). Potentially, two pathways exist for synthesis of cysteine (Jones and Fink, 1982; Ono et al., 1988). The first (Fig. 4, reactions (1)-(2)) proceeds by acetylation of serine to yield O-acetylserine (OAS), followed by a sulphydrylation. The latter enzyme also acts as O-acetylhomoserine (OAH) sulphydrylase (reaction (4); Yamagata, 1980). This pathway is analogous to the cysteine biosynthetic pathway in enteric bacteria (Soda, 1987) and plants (Giovanelli, 1987) and has been referred to as direct sulphuration (Ono et al., 1991). In Sacch. cerevisiae, OAS-OAH sulphhydrylase was shown to be repressed by methionine (Cherest et al., 1969); the same has been shown for Neurospora crassa (Wiebers and Garner, 1967), Aspergillus nidulans (Paszewski and Grabski, 1974, 1975) and Saccharomycopsis lipolytica (Morzycka and Paszewski, 1982). The second pathway proceeds from homocysteine and involves the cystathionine biosynthesis cycle (reactions (5)-(8); Masselot and de Robichon-Szulmajster, 1975; Masselot and SurdinKerjan, 1977). This pathway is analogous to the mammalian cysteine
26 1
GLUTAT1 IIONE IN MICKO-ORGANISMS
SAM
methionine
(13)
c ystathionine
(3)
(5)
y O-acetylhomoserine +homoserine
t
so 2-
serine
(1)
+
+ 0-acetylserine glycine
P+
cysteine
cysteinylglycine
and"f-glutamylglutamate peptides
4,
glutamate
(14) "f -glutamylcysteine
(16)
\
glutathione
r/
FIG. 4. Pathways showing the main fluxes of sulphur-containing compounds in Saccharomyces cerevisiae (Elskens et ul., 1991). (1) indicates serine acetyltransferase, (2) cysteine synthase (OAS sulphydrylasc). ( 3 ) homoscrinc acctyltransferase, (4) homocystcine synthase (OAH sulphydrylase), ( 5 ) y-cystathionine synthase, (6) ycystathionase (C7T y-lyase), (7) p-cystathionasc, (8) p-cystathioninc synthase, (9) homocysteine methyltransferase, (10) S-adenosylmethionine (SAM) synthetase, (1 1) S-adenosylmethionine dcmcthylasc, (12) adcnosylhomocystcinase, (13) a sulphatc-reducing pathway, (14) y-glutamylcysteine synthetase, (15) glutathione synthetase, (16) y-glutamyltranspcptidasc and (17) cysteinylglycinc dipeptidase.
biosynthetic pathway (Reed et ai., 1983; Griffith, 1987) and has been referred to as reverse transulphuration (Soda, 1987). Ono et al. (1991) have shown that the second enzyme, y-cystathionase (reaction (6); CTT y-lyase) is derepressed nearly 15-fold when Sacch. cerevisiae was starved for both inorganic and organic sulphur-containing compounds. Growing evidence supports, furthermore, that wild-type strains of Sacch. cerevisiae utilized methionine preferentially over sulphate if both were available in the environment. The enzyme CTT y-lyase is repressed in methioninegrown cells as well as in sulphate-grown cells, while derepression takes place following an extensive depletion of cysteine. From these observations, the authors inferred that CTT y-lyase is the target for regulation and that cysteine is very likely the effector in this regulation. Almost simultaneously, a working model for the main fluxes of sulphur-containing compounds in
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M J PENNINCKX AND M T FI S K F N S
Sacch. cerevisiae involving GSH metabolism was proposed (Elskens et af., 1991). It suggests that, in the presence of sulphate as a nutrient, most excess sulphur is incorporated into GSH (reactions (13)-(15)). Under these conditions, y-GT activity is low and the turnover rate of GSH is exceedingly slow. When cells are deprived of sulphate, GSH is apparently able to serve as an internal sulphur source until it reaches a residual concentration of about 10% of its normal value. An increase in the specific activity of y-GT is attained by derepressing synthesis of the enzyme and, as a consequence, the turnover rate of GSH increases (reactions (16)-(17)). Regulatory controls of y-GT and enzymes of cysteine biosynthesis, i.e. CTT y-lyase (reverse transulphuration) and OAS-OAH sulphydrylase (direct sulphuration), appeared distinct and it was only under conditions of total sulphate deprivation that all these enzymes were derepressed. As already described, derepression of y-GT correlated best with a decrease in the GSH pool among examined sulphur compounds, which was not so for C 7 T y-lyase and OAS-OAH sulphydrylase. The model requires, moreover, that derepression of the bidirection transulphuration pathway leads to a more active transfer of sulphur atoms from cysteine to methionine and vice versa (Elskens et al., 1991). Hence, it could be argued that Sacch. cerevisiae is able (a) to avoid excess biosynthesis of cysteine by direct sulphuration by regulation of OAS-OAH sulphydrylase (Cherest et al., 1969), (b) to prevent back-flow from methionine through reverse transulphuration by regulation of CTT y-lyase (On0 et af., 1991), and (c) to utilize GSH as a physiological reservoir (source and sink) for cysteine. Yeast y-GT is a vacuolar enzyme and, since about 50% of the total cellular GSH is concentrated in the central vacuole (Jaspers and Penninckx, 1984), it has been suggested that the tripeptide might have a storage function, similar to that for glycogen, trehalose, polyphosphates and lipids (Gancedo and Serrano, 1989), and be mobilized during starvation andlor reproduction (Elskens et a f . , 1991).
IV. Interconversion of Glutathione and Glutathione Disulphide Intracellular GSH is converted to GSSG by selenium-containing GSH peroxidase, which catalyses destruction of hydrogen peroxide and organic peroxides (see Section 1V.B). A selenium-independent form, active with numerous organic hydroperoxides, was also found and is related to certain GSH S-transferases, a group of cytosolic isoenzymes with overlapping substrate specificity (see Section V.A). Glutathione transhydrogenases (protein disulphide isomerase, thiol-transferase, thiol-disulphide oxidoreductase), a group of proteins involved in thiol-disulphide exchanges,
GLUTATHIONE IN MICRO-ORGANISMS
263
catalyse also formation of GSSG. A number of reactions of this type will be further considered (see Section 1V.A). There is substantial evidence that chemical oxidation of GSH occurs within the cell by non-enzymic processes. Amongst the most useful agents to oxidize intracellular GSH are diazene derivatives introduced by the Kosowers (1978a). Formation of GSSG by reactions with free radicals, and oxidation of GSH mediated by trace metals, have been reported as well, although the exact mechanism still remains obscure (see Section 1V.C). Finally, reduction of GSSG is catalysed in vivo by the widely distributed GSH reductase, whose role in the GSH redox cycle is particularly important (see Section 1V.C). A . GLUTATHIONE TRANSHYDKOGENASES
By virtue of its thiol group and its relatively high concentration in cells, GSH has long been thought to play a leading role in the thiol status of many cell constituents, including proteins with cysteine residues (Baron, 1951; Jocelyn, 1972). Several proteins depend upon disulphide bonds for stability of their folded conformation, while many metabolic and physiological functions involve thiol-disulphide exchanges, e.g. protein synthesis and degradation, activation and inactivation of enzymes, synthesis of deoxyribose intermediates, mitosis, and alterations in the dormant state of bacteria (Flohe and Gunzler, 1976; Fahey, 1977; Kosower and Kosower, 1978; Freedman, 1979; Buchanan, 1980; Mannervik and Axelsson, 1980). Regulation of enzymes by thiol-disulphide exchanges was recognized a long time ago (Baron, 1951). The simplicity of the mechanism for covalent modification of enzymes is especially appealing since the reaction is readily reversible and quite well characterized (Szajewski and Whitesides, 1980; Creighton, 1983). Formation of the mixed disulphide of an enzyme can either increase or decrease catalytic activity, and regulation of several enzymes by this mechanism has been previously reported (see Ziegler et al. , 1983). However, since the initial model studies of these processes in vitro were much slower than expected from in vivo kinetics of synthesis, folding and secretion of disulphide-bonded extracellular proteins (Anfinsen, 1973), there was little doubt that thiol-disulphide exchanges were most likely catalysed in vivo by thiol transferase or transhydrogenase enzyme(s). In the course of research for a potential catalyst for disulphide formation in proteins, a disulphide isomerase activity (PDI: E C 5.3.4.1) was discovered in studies on reactivation of reduced pancreatic RNAase (Goldberger et al., 1963). Concurrently, with Anfinsen’s group, Tomizawa (1962) purified an enzyme from bovine liver that catalyses reductive cleavage of disulphide bridges in insulin in the presence of GSH. The essential similarity of these two thiol-protein disulphide exchange reactions was noted by several
264
M J PENNINCKX AND M T ELSKENS
investigators, and the question of identity was raised when Ansorge and his coworkers demonstrated that both activities copurified in their rat-liver preparations (Ansorge et al., 1973a,b). Nevertheless, because so many varied substrates participate in thiol-disulphide exchange reactions, there may be several thiol transferases or transhydrogenases. In an attempt to identify the enzyme(s) responsible for thiol-protein exchange, Freedman (1979) considered the idea that there may be separate enzymes with different but overlapping specificities. Although a great deal of work has been carried out since then on details of folding and oxidation of reduced proteins in vitro, while special attention has been directed towards systems catalysing such reduction and rearrangement of protein disulphide bonds in vivo (see Freedman et a f . , 1988), the physiological functions associated with these proteins are not fully understood. In prokaryotes, Sundquist and Fahey (1989) demonstrated that GSH thiol transferase was produced by Escherichia coli and Beneckea alginolytica in substantial amounts (higher than in rat liver) but was absent from other investigated GSH-producing bacteria such as Rhodospirillum rubrum, Chromatium vinosum and Anabaena sp. In halobacteria, which produce the dipeptide y-glutamylcysteine instead of GSH, the presence of significant thiol transferase activity was also reported. Based upon these results, the authors have suggested that GSH and the thiol transferase may play a significant, although not universal, role within cells in alleviating disulphide formation. Interestingly, numerous heterotrophic bacteria isolated from soil, water, sediment and vegetation, and marine algae were found to methylate sulphide. Production of methanethiol was shown to depend on a thiol methyltransferase activity that uses organic thiols as substrates. The enzyme is widespread in bacteria and may contribute to biogenic emission of methylated sulphur gases and production of methyl thioethers (Drotar et al., 1987). The specificity of the protein(s) towards glutathione is, however, unknown. Other possible functions of significant ecological importance for bacterial protein-disulphide transhydrogenase have been recently proposed (Pel and Gottschal, 1989). Fermentation of chitin by mixed cultures of a chitinolytic Clostridium sp. and various non-chitinolytic bacteria was shown to proceed up to eight times faster than in pure cultures of the Clostridium strain. The agent responsible for this stimulation was found either in spent media of the mixed culture or in cell-free extracts of the non-chitinolytic bacteria and was thought to be a thioredoxin or a thioldisulphide transhydrogenase. This stimulation thus emphasized a possible new type of interspecies interaction in anaerobic cultures. A thiol-disulphide transhydrogenase with a relatively high affinity and reaction rate to cystine was purified from yeast several years ago (Nagai and Black, 1968). The enzyme was inactive towards insulin and other
Glutamate+ cysteine
I
y GLUTAMYL C Y S ” E SYNlHETASE(Gslul )
y-Glutamylcysteine Glycine
GLUTATHIONE
7-
GLUTATHIONE S Y I V l W T m (GshB)
m u c r (~G ~~1K-
GLUTAREDOXIN (Grx 1
>
Ribonucleotide reduction Sulphate reduction Disulphide reduction Metliionine sulphoxide reduction
NADPH
THIoREDoxlN REDuCrksE(Tr*B)
L
THIoREDoxlN
( T d)
FIG. 5. Pathway showing transfer of electrons in Escherichia coli by the thioredoxin and the glutaredoxin systems. From Fuchs et ul. (1983).
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M . J . PENNINCKX A N D M T. ELSKENS
proteins containing disulphide bridges. Quite recently, the nucleotide sequence of the yeast YCL313 gene, localized on the left arm of chromosome 111, was determined (Scherens et al., 1991). This gene encodes for a protein with 522 amino-acid residues (MT 58,300) which presents large homologies with the human, mouse, chicken, bovine and rat PDI proteins. Furthermore, all of these proteins contain two regions, defined as a and a', with strong similarity to the thioredoxin active sites. It was thus suggested that the YCL313 gene encodes for a yeast PDI protein. Gene disruption of YCL313 leads to a lethal phenotype, indicating that this gene is essential for cell survival. Although GSH transhydrogenases probably have important functions related to synthesis, structure, degradation and folding of proteins, other systems that affect the thiol-disulphide status of cells are also of considerable importance. These include the widely distributed thioredoxin and glutaredoxin systems (Holmgren, 1983, 1985; Holmgren et al., 1986; Gleason and Holmgren, 1988). R . THE GLUTAREDOXIN SYSTEM
Figure 5 illustrates transfer of electrons by thioredoxin and glutaredoxin systems in micro-organisms. Ribonucleotide reductase catalyses the first step in DNA synthesis by reducing four different ribonucleotides to the corresponding deoxyribonucleotides (Reichard and Thelander, 1979; Reichard, 1987). Reduction of the ribose moiety of a ribonucleotide requires a hydrogen-donor system, and thioredoxin from E. coli was the first possible physiological hydrogen donor for the ribonucleotide reductase described by Reichard and his coworkers (Mathews et al., 1987). Thioredoxin is a small ( M , 12,000) ubiquitous redox protein with the conserved active-site structure of -Trp-Cys-Gly-Pro-Cys- (Gleason and Holmgren, 1988). The oxidized form (TRX-S2) contains a disulphide bridge which is reduced to the dithiol form by NADPH and the FAD enzyme thioredoxin reductase. The reduced form (TRX-(SH),) is a powerful protein-disulphide oxidoreductase. Thioredoxin has been characterized from a wide variety of prokaryotes, microbial eukaryotes, plant, and animal tissues and appears universal (Holmgren, 1981; Holmgren et al., 1986; Gleason and Holmgren, 1988; Meyer et al., 1991). Further discussion on the biochemical aspects of this protein is outside the scope of this review. During studies of thioredoxin mutants in E. coli that were killed by phage T7 infection (Mark and Richardson, 1976), glutaredoxin, another hydrogen donor system for ribonucleotide reduction requiring GSH, was discovered (Holmgren, 1976). A remarkable account of the relative contribution, structural relation and distribution of these two hydrogen-donor systems
267
GLUTATHIONE IN MICRO-ORGANISMS
in E. coli was made by Holmgren and his coworkers. Isolation and characterization of E. coli mutants which are impaired either in the thioredoxin (Fuchs, 1977; Mark et al., 1977; Fuchs et a f . , 1983) or glutaredoxin (Kren et al., 1988; Russel and Holmgren, 1988; Sandberg et al., 1991) system have, furthermore, extended the field of investigation from previous in vitro to in vivo studies. Glutaredoxin from E. coli is a heat-stable acidic protein with about 85 amino-acid residues with a cystine disulphide bridge and a molecular weight of 9674 for the reduced form, making it one of the smallest known enzymes (Hoeoeg et al., 1983). The single active-centre disulphide has the structure -Gly-Cys-Pro-Tyr-Cys-, with the half-cystine residues located at positions 11 and 14 in the polypeptide chain (Holmgren, 1983). Reduction of glutaredoxin is readily obtained with dithiothreitol and it is thereby similar to thioredoxin (Holmgren, 1979). However, neither protein shows immunological cross-reactivity , and both are structurally unrelated and are apparently different gene products (Holmgren, 1976, 1983; Hoeoeg et al., 1986). Unlike thioredoxin, which requires a dithiol (dihydrolipoic acid or dithiothreitol) as a reductant, reduction of the disulphide bridge in glutaredoxin is obtained by GSH in the presence of NADPH and glut at hione reductase :
+ 2GSH- dCDP + GSSG + H20 GSSG + NADPH + H+-+ 2GSH + NADP' CDP
(glutaredoxin) (glutathione reductase)
Chemically reduced glutaredoxin is enzymically active in conversion of each of the four ribonucleoside 5'-phosphates in the presence of ribonucleotide reductase (Holmgren, 1979). Oxidized glutaredoxin is not a substrate for NADPH and thioredoxin reductase, and thioredoxin does not catalyse GSH reduction, demonstrating that thioredoxin and glutaredoxin systems operate independently (Fig. 5). However, since the structure of glutaredoxin was shown to be quite similar to phage T4 thioredoxin, with respect to the size of the polypetide chain and its amino acid-residue sequence, it was suggested that the T4 protein-encoding gene might have evolved from an early glutaredoxin gene (Hoeoeg et al., 1983). The mixed properties and functional similarities of these two proteins are compatible with such a view. Given present knowledge of the structure of thioredoxin and glutaredoxin, an attempt was made to deduce some common features at both the primary and tertiary structural levels that might reflect common functional properties. There are great differences between the sequences of thioredoxins from E. coli and bacteriophage T4, but the tertiary structures of both proteins are quite similar in spite of their poor sequence homology (Branden et al., 1983). A comparison of the
268
M. I. PENNINCKX A N D M . '1. ELSKENS
tertiary structure gave a common fold of 68 a-carbon atoms with a mean square root difference of 2.6 A (Eklund et al., 1984). Assuming that glutaredoxin has the same common fold, the amino acid-residue sequence of glutaredoxin has been aligned to those of thioredoxins from E. coli and phage T4. A model of the glutaredoxin molecule was built on a vector display using this alignment and the phage T4 tertiary structure (Eklund et al., 1984). By comparison of this model with those of thioredoxins, the authors have identified a common molecular surface area on one side of the redox active disulphide bridge which might represent the binding region for redox interactions with other proteins (e.g. ribonucleotide reductase). In spite of the fact that there are some arguments for the existence of a glutathione-binding site on ribonucleotide reductase (Hoeoeg et al., 1982), the functional organization with glutaredoxin and (or) thioredoxin in cells remains largely unknown. While deoxyribonucleotides are apparently formed by ribonucleotide reductase, the nature of the in vivo hydrogen donor is still questionable (Holmgren, 1988). It should be stressed that, if the apparent concentration of thioredoxin in wild-type E. coli is about SO-fold higher than that of glutaredoxin, the molecular activity of the glutaredoxin system seems greater (Holmgren, 1979, 1983). Quite possibly, both thioredoxin and glutaredoxin might function during normal growth and serve as substitutes for each other. Studies with E. coli mutants impaired in the thioredoxin or glutaredoxin system support this view. A strain deficient in thioredoxin reductase ( T r x B ) , which was unable to use methionine sulphoxide as a methionine source, was isolated by Fuchs (1977). In permeabilized whole cells of the mutant, the ribonucleotide reductase assay revealed only 5% of the parental activity. However, when the cell preparation was supplemented with GSH, reduction of uridine diphosphate was observed at the same rate as that of the parental strain, indicating that glutaredoxin could replace thioredoxin reductase. The TrxB mutant grew like the wild type under laboratory conditions, and the mutation mapped between 14 and 16 minutes on the chromosome of E. coli (Fuchs et al., 1983). A thioredoxin (TrxA)-defective mutant showing no altered growth characteristics was also isolated (Mark et al., 1977). The mutation mapped at 84 minutes on the Chromosome and was 34"% contransducible with the MetE gene. However, transduction experiments using this TrxA strain have indicated the existence of several genetic defects linked with the mutation. Due to close linkage of deleterious mutations, and possibly a structural alteration in the region containing the TrxA gene, it has proved difficult so far to interpret the phenotypes of thioredoxin and GSH-deficient double mutants. A glutaredoxin-negative mutant of E. coli was recently constructed by
G I IJ I A l H I O N F IN MI( KO ORGANISMS
269
inactivation and insertion of a 2 kb kanamycin-resistance fragment into the coding sequence of the glutaredoxin gene in E. coli (Hoeoeg et al., 1986). The inactivated gene was inserted into the chromsome of E. coli and mapped at about 18 minutes. A gene-replacement technique was then used to obtain a mutant that lacked glutaredoxin, as tested by radio-immunossay and ribonucleotide reductase assay (Russel and Holmgren, 1988). While obtaining Grx- or Trx- mutants showed that glutaredoxin or thioredoxin, taken alone is not essential for viability of E. coli, attempts to construct a double mutant lacking both glutaredoxin and thioredoxin have been unsuccessful. This indicates, at least, that the presence of one of the two electron donors is essential for growth (Russel and Holmgren, 1988). Glutaredoxin-defective mutants of E. coli were also characterized by Fuchs and his coworkers (Kren et al., 1988). The mutants have a detectable but decreased glutaredoxin-ribonucleotide reductase activity in either crushed or permeabilized cells. The mutants appeared deficient in sulphste and ribonucleotide reduction, suggesting that in vivo glutaredoxin is the preferred cofactor for ribonucleotide and adenosine-3’-phosphate-5’phosphosulphate reductases. C . GLUTATHIONE PEROXIUASE AND THE ANTIOXIDANT DEFENCE SYSTEM I N MICRO-ORGANISMS
Hydrogen peroxide is a ubiquitous biological compound, formed as the enzymic product of numerous oxidases (e.g. superoxide dismutase) present in the cell cytosol, plasma membranes, peroxisomes and mitochondria1 matrix, as well as by auto-oxidative reactions of haemoproteins, ilavoproteins and other cell components (Chance et al., 1979; Fridovitch, 1982; Kappus, 1986). Disposal of hydrogen peroxide is of primary importance with regard to oxidative injuries, since it is a component of both the Fenton and Haber-Weiss cycle reactions (Kappus, 1985; Halliwell and Gutteridge, 1990), which are possible sources of the reactive hydroxyl radical. This radical is one of the most reactive oxygen metabolites and is thought to be responsible for serious damage that can occur during redox cycling processes, e.g. peroxidation of membrane lipids, protein and DNA. Lipid peroxidation disrupts membrane functions and yields toxic reactive byproducts, such as malonic dialdehyde or 4-hydroxynonenal (Kappus, 1987; Halliwell, 1991). Protein damage leads to amino-acid oxidation, resulting in structural changes and enzyme inactivation (Dean et al., 1986). Damage to DNA leads tostrand breakage, deoxykbosefragmentationand extensive chemical alteration of purine and pyrimidine bases (Von Sonntag, 1987; Halliwell and Aruoma, 1991). Thus, intracellular generation and metabolism of hydrogen peroxide has to be limited by the presence of very
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M J PENNINCKX AND M T ELSKFNS
efficient systems in cells for its decomposition. Cellular destruction of hydrogen peroxide was found to be catalysed by different enzymic systems, among which the most studied are catalase (hydrogen-peroxide oxidoreductase (EC 1.11.1.6) and GSH peroxidase (EC 1.11.1.9); Kappus, 1986; Halliwell and Gutteridge, 1989). A study of inborn errors of metabolism strongly suggests, furthermore, that GSH peroxidase is most important in removing hydrogen peroxide from human tissues, probably because it is located in the same subcellular compartments as superoxide dismutase (Halliwell, 1991). The chemical and physical properties of GSH peroxidase have been reviewed by Wendel (1980). The enzyme fails to display saturation kinetics with respect to GSH concentration, and the extrapolated V,,, value is consequently infinite. Lack of a defined K , value agrees with the fact that the apparent maximum velocity for infinite peroxide concentration is a linear function of GSH concentration, thereby supporting a ter uni pingpong mechanism (Wendel, 1980). In biological systems, glutathione peroxidase activity is expressed by at least two enzymes (Lawrence and Burk, 1976). These are the seleniumdependent form containing a selenocysteine residue at the active site and which is able to reduce hydrogen peroxide and organic hydroperoxides (Flohe et al., 1980), and the selenium-independent form which acts mainly on organic hydroperoxides and is related to certain GSH S-transferase izoenzymes (Lawrence and Burk, 1978; Prohaska, 1980). In addition, GSH peroxidation reactions can occur by non-enzymic processes. Several low molecular-weight compounds (e.g. organoselenium compounds and dithiocarbamates) were found to have a GSH peroxidase-like activity. Confusion may occur if unfounded generalizations are made, and a number of reactions of this type will be further discussed. According to Ziegler and his colleagues (1983) , peroxidation of glutathione is capable of altering the intracellular thiol-disulphide balance, and is undoubtedly a major source of cellular disulphide, although most of the GSSG formed is reduced in turn by GSH reductase and NADPH. Consequently, under oxidative challenge, these bioreduction reactions are able to consume a significant fraction of NADPH reducing equivalents in the cell. As described later, GSH can be assigned a regulatory role in these processes due to the high demand that can be placed on cellular capacity to generate NADPH (Reed, 1986). While GSH peroxidase is widely distributed in animal tissues, its occurrence in micro-organisms is still questionable. Representative species of the most important GSH-producing bacteria, e .g. the purple bacteria and cyanobacteria, ( B . alginolytica, R. rubrum, C. vinosum and Anaebaena sp. strain 7119) and E. coli were found to lack any significant GSH peroxidase and transferase activities. Similarly, GSH peroxidase could not
GI U IAI'lIIONE IN MICRO-ORGANISMS
271
be detected in halobacteria which produce the dipeptide y-glutamylcysteine (Sundquist and Fahey, 1989). The virtual absence of GSH peroxidase from photosynthetic bacteria raises, of course, serious questions concerning the antioxidative potential of cells to scavenge hydroperoxides formed as by-products of photosynthetic activity. It has been suggested that in cyanobacteria (Nostoc muschorum and Synechococcus spp.) the effective mechanism for removal of hydroperoxides involves ascorbate peroxidase and recycling of GSH and ascorbate (Tel-Or et al., 1985). That GSH might sustain production of the reducing equivalents in these processes through action of a GSH reductase-NADPH-catalysed reaction was also emphasized by Dupouy et al. (1985). The presence of a GSH peroxidase activity was demonstrated in strains of Pseudomonas putida, especially in cells oxidizing trivalent arsenite. Arsenite is thought to initiate free-radical lipid peroxidation yielding malonic dialdehyde. Accumulation of malonic dialdehyde was shown in experiments with cell homogenates, while the activity of antioxidant enzymes (superoxide dismutase, catalase, GSH peroxidase and reductase) rose in bacteria grown on an arsenite-containing medium. It was, therefore, suggested that these enzymes may interfere with the free-radical processes allowing metabolization of arsenite (Abdrashitova et al., 1990). Unlike bacteria, GSH peroxidase appears to be much more widely distributed in micro-algae (Overbaugh and Fall, 1982). Euglena gracilis was shown to possess both types of GSH peroxidase activity. The enzymes were apparently not induced in response to stimulation of cellular processes that generate oxidant species, such as p-oxidation or photosynthesis, but the peroxidation activity increased in autotrophic cultures containing the herbicide N'-(3,4-dichlorophenyl)-N,N-dimethylurea(Overbaugh, 1985). A selenium-independent peroxide was purified to electrophoretic homogeneity from a permanently bleached strain of E. gracilis. The native enzyme has a molecular weight of 130,000,as measured by gel-permeation chromatography, and consists of four identical subunits ( M , 31,500), as indicated by SDS-PAGE (Overbaugh and Fall, 1985). A seleniumdependent form, whose enzymic properties were closely similar to the GSH selenium peroxidase found in animal tissues, was demonstrated in the green alga Chlamydomonas reinhardtii. (Yokota et al., 1988). Growth of the alga in a sodium selenite-containing medium increases the level of GSH peroxidase at the expense of ascorbate peroxidase. In contrast, two unicellular marine algae (Dunaliella primolecta and Porphyridium cruentum) have been found to contain a selenium-inducible, but nonenzymic, GSH peroxidase activity when cultured in the presence of selenite (Gennity et al., 1985a,b). Since part of the hydrogen peroxide and t-butylhydroperoxide (t-BuO0H)-dependent oxidation of glutathione in cell
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homogenates was shown to be heat- and cold-stable, it was suggested that this contribution could be non-enzymic in nature and probably mediated by a variety of small molecules such as ascorbate, selenocystine, selenomethionine, dimethylselenide and copper nitrate (Gennity et al. , 1985a). Crushed cells of the red alga Po. cruentum also contained, however, a heat- and cold-labile t-BuOOH-dependent reductase activity which accounted for about 90% of the total peroxidation activity. This possible enzymic activity remained unaltered by growth in the presence of selenite (Gennity et al., 1985a). To what extent both non-enzymic and enzymic processes might contribute to in vivo GSH peroxidation is still unknown, and there is no current evidence for an in vivo functioning of selenium as an antioxidant in these algae (Gennity et al., 1985b). The antioxidant defence system of protozoa has also been investigated (Murray et al., 1981; Penketh and Klein, 1986; Fairfield et al., 1988; Fairlamb, 1990). In the human malarial parasite Plasrrzodium falciparum, three oxidant defence enzymes whose activity changed with the growth stages have been characterized (Fairfield et al., 1988). Isolated early intraerythrocytic stages of P1.falciparum were shown to contain mainly catalase, superoxide dismutase and little, if any, GSH peroxidase activities, while late intra-erythrocytic stages were shown to possess much more of the dismutase and GSH peroxidase, and slightly less catalase. It was suggested, furthermore, that P1. faciparum and PI. berghei probably acquired most of their dismutase from the host, since the parasite-associated enzyme was predominantly cyanide-sensitive like the host enzyme, while parasites grown in red cells that had been partially depleted of superoxide dismutase were most sensitive to exogenous superoxide. Most trypanosomatids apparently lack GSH peroxidase (Penketh and Klein, 1986; Penketh et al., 1987). Instead, an analogous trypanothione peroxidase activity has been identified in Trypanosoma brucei and in Crithidia fasciculata (Henderson et al., 1987). It is thus possible that, in these organisms, the trypanothione reductase-peroxidase system has assumed the role of the GSH reductaseperoxidase system of mammalian cells. The situation is, however, less clear in Trypanosoma cruzi, where unstable ascorbate and low alkyl peroxidase activities have been detected, the latter probably being a GSH S-transferase (Yawetz and Agosin, 1981). Glutathione peroxidase was found in cell-free extracts of Mucor spp., where enzyme production was shown to be almost completely associated with mycelial growth (Aisaka et al., 1983), and in Pyricularia oryzae, where the antioxidative systems (superoxide dismutase, GSH peroxidase and catalase) were thought to be involved in parasite tolerance (Nikolaev et al., 1989). Controversial results were, however, reported regarding the presence of GSH peroxidase in yeasts. Aisaka and his colleagues (1983)
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performed a systematic screening for the occurrence of GSH peroxidase in numerous micro-organisms and did not find any significant activities of this enzyme in any one of 31 yeast strains. In Saccharomyces cerevisiae, the enzyme was not detected by Smith and Shrift (1978) or Penninckx and Jaspers (1982), but was reported by Galiazzo and his colleagues (1987). Other investigations have emphasized the presence of GSH peroxidase activities involving both selenium-dependent and -independent forms in different yeast species, such as members of the genera Candida, Saccharomyces, Schizosaccharomyces, Hansenula and Sporobolomyces (Casalone et al., 1988). Nevertheless, the physiological contribution of GSH peroxidase to the defence systems of yeasts against peroxidative attacks needs to be clarified. Owing to its ability to grow either anaerobically or aerobically, Sacch. cerevisiae has been considered long ago as a particularly suitable biological model for investigating the cytotoxic effects of oxygen. A role for GSH peroxidase in these processes was apparently not previously envisaged (Sels and Brygier, 1980; Van Huffel and Sels, 1987). To identify some of the physiological parameters that can modulate cellular defences in Sacch. cerevisiae, Sels and his coworkers used different redox compounds known to potentiate in situ oxygen toxicity. From these results, it was obvious that both catalase and superoxide dismutase appeared essential for survival of the yeast under aerobic conditions. Quite recent studies have, however, demonstrated that, besides catalase, other peroxidase(s) might play a significant role in the removal of hydrogen peroxide (Verduyn et al., 1988). A catalase-negative mutant of Hansenula polymorpha was found to dismutate hydrogen peroxide generated intracellularly during oxidation of methanol and to destroy exogenous peroxides added to the culture medium. Destruction of hydrogen peroxide was apparently not attributable to any GSH peroxidase activity, but rather to a cytochromec peroxidase (EC 1.11.1.5) that increased to very high levels in cells growing on a glucose-hydrogen peroxide medium. A similar trend was observed with the wild-type strain of H . polymorpha, where an increase in the level of both cytochrome-c peroxidase and catalase activities was shown. By comparison, when Sacch. cerevisiae was grown in the glucose-hydrogen peroxide medium, the activity of catalase remained low because of the repressive effect mediated by glucose in this strain, while the cytochromec peroxidase activity rose with increasing rates of hydrogen peroxide utilization. Therefore, according to Verdyun et al. (1988), the peroxidase might be a key enzyme in detoxification of hydrogen peroxide in yeast, and catalase and the peroxidase might effectively compete for peroxidation reactions. However, cross-reactivity would probably be limited because of subcellular compartmentalization of the enzymes, as catalase is mainly located in peroxisomes whereas cytochrome-c peroxidase is mainly
27 4
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mitochondrial. Although it appears that GSH peroxidase is apparently not implicated in the above-mentioned scavenging mechanisms, its possible involvement in a cellular defence system against lipid peroxidation cannot be ruled out. So far, except with some eukaryotic algae (Overbaugh, 1985), only very few studies have been devoted to the role of GSH peroxidase in microbial antioxidant defence systems. D . TH E GLUTATHIONE REDOX CYCLE
I . Glutathione Reductase
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Glutathione reductase (EC 1.6.4.2; GSSG NADPH Hf+ 2GSH NADP') is one of an important group of flavoenzymes about which a considerable amount of mechanistic and structural information is available (Ghisla and Massey, 1989). This group of enzymes includes dihydrolipoamide dehydrogenase, GSH reductase, thioredoxin reductase, trypanothione reductase and mercuric reductase. Early work on the first three of these enzymes has been described in detail in a review by Williams (1976). With the exception of thioredoxin reductase, which appears to have a quite different protein structure, there are remarkable similarities between the known enzymes of the group (Ghisla and Massey, 1989). In the first place, a two-electron reduction of the enzymes yields a spectrally characteristic red intermediate which is a charge-transfer complex between a thiolate anion of one of the nascent cysteine residues and the oxidized flavin. Secondly, these enzymes have similar amino acid-residue sequences and chain folding, resulting in overall comparable three-dimensional structures. In spite of the fact that this considerable homology suggests that these proteins have evolved from a common gene ancestor, they currently fulfil distinct physiological functions and present different substrate specificities with little, if any, cross-reactivity (Williams, 1976). Glutathione reductase has been characterized in a wide variety of microorganisms and appears to be of universal occurrence (Williams, 1976; Ondarza et al., 1983; Serrano et al., 1984; Scrutton et al., 1987; Montero et al., 1988; Sundquist and Fahey, 1989). However, two different GSH reductases have been isolated from bacteria: a NADH-specific enzyme from C. vinosum (Chung and Hurlbert, 1975) and a NADPH-specific enzyme from E. coli (Williams and Arscott, 1971). A reductase that is specific for NADH, the disulphides of pantetheine 4' ,4"-diphosphate and CoA has also been detected in many Gram-positive eubacteria and has been purified from Bacillus megaterium (Swerdlow and Setlow, 1983). Although these enzymes demonstrate different substrate specificities, they catalyse analogous reactions and share several physical characteristics. It
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has been suggested, therefore, that GSH reductase might have evolved in bacteria from lipoamide dehydrogenase, perhaps subsequent to the appearance of an oxidizing atmosphere (Fahey, 1977; Fahey et al., 1987). Lipoamide dehydrogenase has, indeed, a much wider distribution among bacteria than any other disulphide reductase (Danson et al., 1984) and displays a considerable sequence homology with GSH reductase (Williams, 1976). It follows that substrate specificities of GSH reductase might type different stages in evolution of GSH metabolism, as C. vinosum appeared to be the least oxygen-adapted organism able to produce GSH in substantial quantities (Fahey et al., 1987). On the contrary, GSH reductase purified from photosynthetic cyanobacteria (Serrano et al., 1984) and, in a more general way, from eukaryotes (Williams, 1976), exhibits a considerable preference for NADPH over NADH and is quite specific towards GSSG. For example, the preference of the enzyme from Sacch. cerevisiae for the NADPH-GSSG reductase reaction was shown to be kinetically related to the high catalytic efficiency and low dissociation constants of the substrates (Tsai and Godin, 1987). Cloning of the gor genes encoding GSH reductase in E. coli (Greer and Perham, 1986) and Pseudomonas aeruginosa (Perry et al., 1991), and site-directed mutagenesis, have further shown the similarities (and differences) between GSH reductases from several sources and also allowed determination of the amino acid-residue sequence involved in substrate specificity. The three-dimensional structure of the enzyme from E. coli, which was solved at the 3 A level, displayed a considerable homology with the well-investigated human enzyme (Pai and Schulz, 1983; Karplus and Schulz, 1987) and also showed some spectacular effects of site-directed mutations, such as a change in the cofactor specificity from NADPH to NADH (Ermler and Schulz, 1991). Among other related proteins most likely having a similar physiological function to GSH reductase are a bis-y-glutamylcysteine reductase, purified from Halobacterium salinarum (Sundquist and Fahey, 1988) and trypanothione reductase (Shames et al., 1986; Henderson et al., 1987; Krauth-Siege1 et al., 1987). The gene coding for the latter enzyme has also been isolated and cloned from the cattle pathogen Trypanosoma congolense and Leishmania donovani (Shames et al., 1988; Taylor et al., 1989). In both studies the two disuphide-specific reductases were thought to play an important role in maintaining cellular thiol groups in a reduced state in organisms that are either lacking GSH, but producing the dipeptide yglutamylcysteine such as H . salinarum, or exhibiting a peculiar and unique metabolic trypanothione pathway (see Section 11). Steps involved in the reaction catalysed by GSH reductase have been dissected in detail for the enzyme from baker’s yeast by a combination of classical spectroscopic examination, fast kinetics, isotope effects and by
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specific chemical modifications (Ghisla and Massey, 1989). A hybrid kinetic mechanism (bi-bi ordered sequential and ping-pong) has been proposed for the enzymes from yeast (Mannervik, 1973), E. coli (Perham et al., 1988), Anabaena sp. (Serrano et al., 1984) and Phycomyces blakesleeanus (Montero et al., 1990). It is particularly impressive that the balance flux between the ping-pong and ordered sequential pathways can be infl7ienced by a mutation of just one amino-acid residue, probably a tyrosine residue, as shown with the protein from E. coli (Perham et al., 1987). As with other reductases, such as nitrate, nitrite and NADP' reductases, GSH reductase is inactivated after reduction by its own electron donor, NADPH, and other reductants. Inactivation of the purified enzymes from Sacch. cerevisiae (Pinto et al., 1984, 1985) and E. coli (Mata et al., 1985a) was shown to be a time-temperature and pH-dependent process. Since both active and inactive forms of the enzymes had similar molecular weights, inactivation was attributed to intramolecular modification(s). The purified enzyme from Sacch. cerevisiae displayed protection against redox inactivation in the presence of GSSG, ferricyanide, GSH and dithiothreitol. High concentrations of NADP+ and GSSG effectively protected the enzyme at even lower concentrations than that required by GSH. It has been suggested that this auto-inactivation of GSH reductase by NADPH and its subsequent reactivation by GSSG has an important in vivo regulatory role (LopezBarea and Lee, 1979; Pinto et al., 1983). Redox interconversion of the enzyme was further demonstrated using crushed and permeabilized E. coli, treated with different reductants, and with intact cells incubated with compounds known to alter the intracellular redox state. In both sets of experiments, the results indicated that the interconversion mechanism was most likely controlled by intracellular NADPH and GSSG concentrations (Mata et al., 1985b). The level of GSH reductase activity may thus reflect the physiological need of the cells and could, presumably, regulate its own requirement for NADPH. It is known that reducing equivalents needed in the NADPHdependent reactions of anabolism are derived from substrates of several dehydrogenases. In yeasts, Bruinenberg et al. (1983a) have shown that the NADPH requirement for biomass formation is strongly dependent on available sources of carbon and nitrogen and it was inferred that the carbon flow towards NADPH-producing pathways should vary accordingly. Most likely, the hexose monophosphate pathway, and possibly NADP+-linked isocitrate dehydrogenase, would be the major sources of NADPH in Candida utilis (Bruinenberg et al., 1983b). Glucose-6-phosphate dehydrogenase, which catalyses the first reaction on the hexose monophosphate pathway, is inhibited by NADPH (Bonsignore and De Flora, 1972; Llobell et al., 1988). Regulation of the enzyme was expected since
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the ratio of NADPH to NADP' was found to vary with respect to the physiological status of the cell. Efforts to understand regulation of the hexose monophosphate pathway led Eggleston and Krebs (1974) to consider the ability of GSSG to counteract inhibition of NADPH on glucose-6phosphate dehydrogenase, and this possibility was further debated by Levy and Christoff (1983). More recently, Lopez-Barea and his coworkers have demonstrated that the glucose-6-phosphate dehydrogenase from Sacch. cerevisiae is inhibited by low concentrations of NADPH in cell-free extracts and that this inhibition is relieved by addition of GSSG in the presence of GSH reductase (Llobell et al., 1988). It follows that low intracellular levels of NADPH might inactivate GSH reductase in the absence of GSSG and subsequently decrease the glucose metabolism via the hexose monophosphate pathway (Reed, 1986). The physiological GSH-GSSG ratio should, however, provide sufficient GSSG at this level to permit retention of a significant GSH reductase activity by preventing total inactivation (Lopez-Barea and Lee, 1979). When the intracellular content of GSSG increases (e.g. under an oxidative stress), GSH reductase is reactivated and catalytic reduction of GSH is able to lower the cellular NADPH to levels that relieve inhibition of glucose-6-phosphate dehydrogenase by NADPH. As described below, reducing equivalents contained in NADPH and GSH can provide a very dynamic response during drug bioreduction processes. Arguments supporting this view were obtained in experiments with cells submitted to an oxidative challenge (Reed, 1986; Elskens and Penninckx, 1986). 2. Modulation of Radiation and Chemical Sensitivity Manipulation of the intracellular GSH content (or GSH-dependent enzyme systems) drastically modulates the toxicity of numerous chemicals. This provides strong biological evidence that GSH is responsible for protection against or activation of these compounds. Conclusions concerning the mechanism of toxicity cannot, however, be drawn without additional investigations correlating biological data with chemical studies in a highly integrated research effort (Smith et al., 1983). Bioreduction in activation of drugs appears to incur potential hazards for prodrugs. Prodrugs undergo conversion to active drugs essentially by two mechanisms. These are either by direct conversion or formation of an instable intermediate that undergoes a usually spontaneous reaction to yield the drug (Gorrod, 1980). These processes are not exclusive, and the intermediates may have potential for greater toxicity than the drug itself. It is apparent that a major protective role against reactive drug intermediates, generating reactive oxygen species, is provided by the
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ubiquitous GSH redox cycle (Kappus, 1986; Reed, 1986). This cycle utilizes NADPH in the mitochondria1 matrix, as well as in the cytoplasm, to provide a recycled supply of GSH by GSH reductase-catalysed reduction of GSSG. However, as previously described in Section TV.C, these processes involving GSH peroxidase activity are far from being universal in micro-organisms, and in many organisms remain to be demonstrated. Examples of drugs that induce superoxide formation are the pesticides paraquat and diquat, and aromatic and heterocyclic nitro compounds (Hewick, 1982; Mason and Josephy, 1985; Reed, 1986). The GSH redox cycle can also directly sustain bioreduction of thioloxidizing agents. Diazenecarbonyl derivatives were found to oxidize intracellular GSH rapidly by a well-defined set of chemical reactions (Kosower and Kanety-Londner, 1976; Kosower and Kosower, 1978). There is generally a simple stoichiometric relationship between GSH lost and the concentration of added reagent. These reactions can result in either activation or inactivation of the compound, according to the reactivity of the drug intermediate. For example, phenyldiazenecarboxylate (azo-ester) was found to oxidize GSH to GSSG on a minute scale. Excess reagent leads, however, to formation of free radicals by hydrolysis, decarboxylation and reaction of the phenyldiazene intermediate with oxygen. On the contrary, diazenedicarboxylic acid bis(N,N-dimethylamide) (diamide) was shown to penetrate cells rapidly and oxidize intracellular GSH within seconds or less at room temperature. Diamide is less susceptible to hydrolysis and quite stable in aqueous solution. Its reduction product is a stable, relatively non-toxic hydrazide. The reaction course is thus similar to that of azo-ester, but does not give rise to free radicals. Possibly, this pathway could be a protection against oxidation of cellular proteins. Nevertheless, it should be pointed out that the situation is often more complex. For example, the fungicide tetramethylthiuram disulphide (thiram), was shown to oxidize GSH in vivo and in vitro by two-step chemical reactions whose kinetics are discussed by Elskens et al. (1988a,b). For thiram concentrations below the minimal inhibitory concentration, sustained production of high levels of GSH-reducing equivalents was shown in a wild-type strain of Sacch. cerevisiae. The rates of thiram elimination displayed elements of saturation kinetics and were, in turn, regulated by the intracellular content of GSH, the ability of the yeast to provide NADPH and the specific activity of GSH reductase. Because the sensitivity to thiram was greatly enhanced in GSH-deficient mutants and in cells artificially depleted in GSH by pharmacological manipulation, bioreduction of thiram was initially thought to be involved in protection against the oxidant properties of the compound (Elskens and Penninckx, 1986). However, several lines of evidence indicated that dimethyldithiocarbamate (DMDT),
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the reduction product of thiram, and the analogue diethyldithiocarbamate (DEDT), can undergo a variety of chemical reactions (Kumar et al., 1986). In the first place, the compounds can be re-oxidized by interaction with components of the respiratory chain, thus initiating under aerobic conditions a futile redox cycle mediated by GSH. Evidence of such processes in vitro was obtained by Kumar and his colleagues (1986). In vivo, the oxidative challenge induced by thiram in the yeast was found to decrease uptake of oxygen by galactose-induced cells, and interaction with the respiratory chain was demonstrated by in situ analysis of the redox state of electron carriers (M. T. Elskens and M. J. Penninckx, unpublished results). Secondly, oxidation of DMDT or DE DT by peroxides, and its reversal by GSH, indicated that the drugs might engage a GSH peroxidase mimic in a cyclic reaction (Kumar et al., 1986). Indeed, if the compounds might substitute GSH peroxidase in detoxifying peroxides in vivo, the GSH redox cycle would, therefore, play a significant role in chemical modification of radiation sensitivity. The mechanism of radioprotection and chemoprotection by DMDT or D E D T may have common elements with other sulphydryl radioprotection systems (Evans, 1985). On the other hand, dithiocarbamates were also found to inhibit superoxide dismutase activity because of copper-ion chelation (Heikkila et al., 1976). This effect may explain their radiosensitizing properties (Evans, 1985) as well as their role in potentiation of toxicity of drugs generating superoxide radical anions. In this connection, it would not be very surprising to find both radioprotection and radiosensitization by dithiocarbamates within the same organism. To what extent these compounds might contribute to the reaction scheme proposed is probably determined by the physiological status of the cell. For example, it appears that, in yeast, the intrinsic toxicity of thiram is modulated by the GSH redox cycle, as shown by experiments with GSHdeficient strains, but this toxicity may also vary according to the ability of yeast to grow either anaerobically or aerobically. As already stated, GSH peroxidase mimics have been detected in cellfree extracts of two unicellular marine algae (Gennity et al., 198%). This non-enzymic activity, mediated by GSH, has been attributed to the presence of endogenous compounds such as selenocystine, selenomethionine or dimethylselenide. It was hypothesized that these compounds could enhance the antioxidant defence of algae when cells were cultivated in a selenite-containing medium. Therefore, the GSH redox cycle might be therapeutically useful in protecting against oxidative damage and have a general cytoprotective role (Halliwell, 1991). Ebselen (2-phenyl-l,2benzisoselenazolin-3(2h)-one) is an organoselenium compound which has been developed as an antioxidant in disease therapy (Halliwell, 1991). It catalyses removal of peroxides by a cyclic reaction with intracellular GSH
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and was shown to have anti-inflammatory effects in a number of small mammals. Inactivation of GSH reductase has been used in several studies to assess the key importance of the GSH redox cycle during oxidant stress generated by intracellular bioreductive processes. Amongst the best known inhibitors of GSH are the nitrosourea compounds, such as 1,3-bis(2-chIoroethyl)-lnitrosourea (BCNU) and l-(2-chloroethyl)-3-(2-hydroxyethyl)-l-nitrosourea (HeCNU). Studies by Babson and Reed (1978) indicated that inactivation of the reductase occurs only when the enzyme is in the reduced state (EH2). Inactivation appears to involve a thiocarbamate adduct, presumably with the distal thiol group of the active site. The fungicide thiram was also found to inactivate the enzyme from Sacch. cerevisiae in vivo after incubation of cells with the pesticide, in vitro in cell homogenates or with a purified enzyme preparation. The mode of action is probably similar to BCNU and also involved the reduced form of the enzyme (M. T . Elskens and M. J. Penninckx, unpublished results). Thiram was, however, not a selective inhibitor and exhibits, as already described, a variety of pharmacological activities. Nitrofurans were other compounds shown to inhibit the GSH reductase from baker’s yeast by acting as non-competitive inhibitors for NADPH and GSH. The quinone-substituted nitrofurans were found to be the most effective inhibitors (Cenas et a f . , 1991). A search for selective inhibitors of GSH reductase is proving useful in attempts to improve current treatments against some human parasites. For example, since malarial parasites are believed to be more susceptible to oxidative stress than their host, BCNU and HeCNU were found to be efficient in preventing growth of Pf.falciparum in the early and late intraerythrocytic stages (Zhang et a f . , 1987). Similarly, the flavin analogue 10(4’-chlorophenyl)-3-methylflavin, which inhibits the antioxidant reductase from human erythrocytes by acting as a competitive inhibitor for GSSG, was shown to have antimalarial activity (Becker et a f . , 1990). In trypanosomatids, more effective and selective inhibitors of trypanothione reductase were recently obtained by substituting the nitrofuran moiety of nifurtimox, an existing trypanocidal drug with a relatively broad-activity spectrum (Henderson et al., 1988). These compounds were shown to undergo futile redox cycling by trypanothione reductase, and were thought to kill trypanosomes by subverting the normal antioxidant role of the enzyme (Fairlamb, 1990). The evidence is overwhelming that the GSH redox cycle has a vital role in the cellular response to bioreduction and activation of various classes of compounds. In many instances, it is only after inactivation of GSH reductase that it is possible to observe the degree of cell injury induced by the absence of this protective system.
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V. Conjugation of Glutathione: Glutathione S-Transferases A. OCCURRENCE AND DISTRIBUTION IN MICRO-ORGANISMS
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Glutathione S-transferases (EC 2.5.1.18; GSH RX + GSR X) were identified in 1961 and have been extensively studied since then. Numerous reviews of these enzymes have been published (Boyland and Chasseaud, 1969; Jacoby, 1978; Mannervik, 1985; Mannervik and Danielson, 1988; Boyer, 1989; Pickett et al., 1989; Waxman, 1990; van Bladeren and van Ommen, 1991) and one can estimate that the number of research articles on the enzyme, or quoting it, most likely exceeds 4000. Because of its assumed central role in biotransformation of xenobiotics, this group of enzymes has attracted much attention and new isoenzyme species are still being described. Multiple forms of GSH S-transferases may occur in living cells and the establishment of such multiplicity was, in most instances, based on chromatographic and electrophoretic separations, combined with activity measurements. For example, six major GSH S-transferases were characterized in rat liver (Jacoby et al., 1984; Mannervik, 1985). In animal tissues, the isoenzymes appear as dimeric proteins, composed of four different subunits, so that homodimers and heterodimers can exist. The enzyme was named on the basis of its constituent subunits (Jacoby et al., 1984; Mannervik, 1985; Boyer, 1989). In reactions catalysed by the transferase isoenzymes, the sulphur atom of GSH provides electrons for nucleophilic attack on or reduction of the second electrophilic substrate. The GSH conjugate thus formed is further metabolized, and residues excreted by a well-defined sequence of reactions, the best known of which is the mercapturic pathway in animal tissues (Mannervik, 1982, 1985; Mannervik et al., 1983). The first survey of microbial GSH S-transferases appeared at the beginning of the 1980s (Lau et al., 1980) and the enzyme was detected in numerous micro-organisms, including bacteria, protozoa, algae and fungi. Since then, several reports have been published on the occurrence and distribution of the isoenzymes in prokaryotes and microbial eukaryotes, but most of them deal principally with their evolutionary and structural relationships, rather than with their physiological functions. Glutathione S-transferases were purified from and characterized in several members of the Enterobacteriaceae (Di Ilio et al., 1988,1991; Izuka et al., 1989; Piccolomini et al., 1989; Arca et al., 1990), in Pseudomonas aeruginosa (Piccolomini et al., 1989; Dierickx, 1991) and in Methylobacterium organophilum (Sysoev et al., 1990). The bacterial enzymes appeared to be composed of two identical subunits ( M , 22,500) and, in some instances, in the form of isoenzymes with different isoelectric points. An N-terminal
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residue sequence analysis of GSH S-transferases from Proteus mirubilis showed no obvious homology with the sequence of a-, 7c- and p-classes of the mammalian enzymes or of those of plants (Di Ilio et af., 1989). Low levels of GSH transferase activity were detected in Succharomyces cerevisiae (Shishido, 1981; Jaspers and Penninckx, 1982), but further studies have revealed the presence of substantial amounts of the enzyme in other yeast species (Casalone et al., 1988; Kuniagai et al., 1988). The enzyme was also purified from Mucor japonicus (Ando et al., 1988), Issatchenkia orientalis (Tamaki et al., 1989) and several protozoa species (Yawetz and Agosin, 1981; Overbaugh et al., 1988; Dierickx et al., 1990). Unfortunately, only very scarce information is currently available on the molecular characteristics of the transferases from microbial eukaryotes. The first reported purified microbial enzyme was for the epimastigotes of Trypanosoma cruzi, the agent of Chagas’ disease (Yawetz and Agosin, 1981). The enzyme appeared as a heterodimer with subunits of molecular weights 20,000 and 17,000, and was apparently related to the animal B forms. The enzyme from M . japonicus was shown to be made up of two identical subunits ( M , 22,000), as measured by SDS-PAGE (Ando et al., 1988), and only one major form, with a molecular weight of 35,000 estimated by gel filtration and of 33,000 by SDS-PAGE, appears to occur in Tetrahyrnena therrnophila (Overbaugh et al., 1988). B. SUBSTRATES AND PHYSIOLOGICAL FUNCrIONS
Comprehensive descriptions of the various compounds that can serve as substrates for GSH S-transferases have been published (Chasseaud, 1979; Jacoby and Habig, 1980). An important question is whether there are endogenous substrates for GSH S-transferases in the organism or if the function of the enzymes is to detoxify xenobiotics. Later, some examples will be given of substrates that are known to arise in the metabolism of endogenous, rather than exogenous, cornpounds. Epoxides constitute a group of possible substrates that have received considerable attention. It is well established that endogenous compounds, as well as xenobiotics, may form epoxides and that GSH conjugation is a significant route in their biotransformation (Mannervik, 1985). Another group of substrates that may arise in metabolism are sulphate esters. It has been demonstrated that arylalkyl sulphates, such as benzyl sulphate, are substrates for GSI-I Stransferases. Such substrates may arise by oxidation of an alkyl group followed by sulphation (Mannervik, 1985). As stated earlier, a possible significant biological function for GSH S-transferases is protection of cell membranes against lipid peroxidation. Aldehydes, such as 4-hydroxyalkenals and malonic dialdehyde produced by lipid peroxidation,
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were shown to give rise to GSH conjugates. In addition, it should be noted that, in animal tissues, certain transferase isoenzymes were also characterized as species-binding steroids, bilibirubins and azo-dyes, and they appear to be involved in biosynthesis of a number of important arachidonic acid metabolites, such as prostaglandins and leukotrienes (Mannervik, 1985; van Bladeren and van Ommen, 1991). If these findings logically suggest that GSH S-transferases may be specific enzymes designed for endogenous substrates with defined biological functions, the question about their primary cellular role still remains unanswered. In view of the fact that the individual isoenzymes demonstrate differential though overlapping substrate selectivities, the extent to which biotransformation occurs is dependent on the profile of the isoenzyme present. Consequently, both genetic and external factors causing changes in the level or activities of individual isoenzymes are of relevance with respect to the individual susceptibility towards electrophilic compounds. In many instances, cells were able to modulate their intracellular level of transferases in response to natural and artificial perturbations (Mannervik, 1985; Pickett, 1989; Vos and van Bladeren, 1990). This adaptation mechanism may thus be responsible for the acquired (de n o w ? ) role of GSH S-transferases in drug resistance. In this context, one could expect to have multiple forms of such an enzyme with a broad specificity to accommodate different types of potentially toxic agents as substrates. As described in Section 11, GSH in trypanosomatids is mainly in the form of spermidine conjugates. This contribution does not, for instance, rule out a possible involvement of free GSH in cellular detoxification of protozoa. Recent investigations, focusing on the effect of intracellular free GSH on the susceptibility of T. cruzi to trypanocidal drugs such as nifurtimox and benzomidazole, have shown a positive correlation between GSH content and resistance to drugs (Moncada et al., 1989). Furthermore, BSO which was used to lower the intracellular level of GSH in T . cruzi, was found to potentiate the toxicity of both drugs. The implication of GSH S-transferases in the mechanism of resistance of Te. thermophila and Beweria sp. to isosorbide dinitrate was recently discussed (Ropenga and Lenfant, 1987; Ropenga et al., 1989). It was shown that the compound induced transferase activity and that this activity correlated well with the rate of drug bioconversion into the 5- and 2-mononitrate forms. The organochlorine pesticide captan was shown to inhibit growth of the non-symbiotic nitrogen-fixing bacterium Azospirillum brasilense. A mutant that contained high levels of GSH and GSH S-transferases was isolated and found to be resistant to the pesticide (Gallori et al., 1988). Similarly, captan-resistant strains of Botrytis cinerea were apparently able to regulate their biosynthesis of GSH and GSH S-transferase activity when grown in the presence of the pesticide (Barak and Edgington, 1984).
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The plasmid conferring resistance to fosfomycin in Escherichia coli was shown to encode for a GSH S-transferase protein (Arca et al., 1988). Mutants deficient in GSH were more susceptible to the antibiotic, indicating that the tripeptide is crucial for the detoxification pathway. The enzyme which mediates resistance was further purified and characterized as a typical GSH S-transferase, catalysing opening of the epoxide ring of the antibiotic to form an inactive adduct (Arca et al., 1990). In Salmonella typhimurium which is a tester strain for the Ames test, GSH and the transferase isoenzymes were identified as possible factors affecting mutagenicity of xenobiotics (Summer et al., 1980). Possible roles for GSH and the transferase system were also reported in degradation of methidathion by Bacillus coagulans (Gauthier et al., 1988), in the resistance of Aspergillusfiavus to endogenous aflotoxin (Saxena et al., 1988), and in detoxication of aromatic xenobiotics by Cunninghamella elegans (Wackett and Gibson, 1982) and by a Fusarium sp. (Cohen et al., 1986). Although these data illustrate the role played by GSH and the transferase isoenzymes in detoxification of or resistance to several xenobiotics in microorganisms, it should be stressed that, in a number of micro-organisms, biosynthesis and biotransformation of GSH S-conjugates lead to formation of toxic metabolites (Kerklaan et al., 1985; Anders, 1988; Anders et al., 1988). For example, bacterial GSH might activate numerous mutagens, including l-chloro-2,4-dinitrobenzene, substituted nitrosoguanidines, styrene-7,8-oxide, 1,2-dibromoethane, methyl methane sulphonate and, quite possibly, halogenated alkenes.
VI. Other Aspects of Glutathione Function A. THE GLYOXALASE PATHWAY
Hydration and rearrangement of methylglyoxal(2-oxopropanal) to D-lactic acid are catalysed by a GSH-dependent system, termed glyoxalase, consisting of two distinct enzymes (Racker, 1951), namely glyoxalase I (lactoylglutathione lyase; E C 4.4.1.5) and glyoxalase I1 (hydroxyacylglutathione hydrolase; E C 3.1.2.6). The reactions involved are: methylglyoxal
+ GSH
hemithioacetal
(non-enzymic)
+ S-D-lactoylglutathione (glyoxalase I) + H 2 0 + D-lactic acid + GSH (glyoxalase 11)
hemithioacetal S-D-lactoylglutathione
Despite numerous efforts, the biological function of the system remains puzzling, especially in micro-organisms. Recent advances in this field with
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animals and plants have, however, suggested that the glyoxalase system may be associated with regulation of cell proliferation and maturation, vesicle mobilization and disease processes, such as tumour growth and diabetes mellitus (Thornalley, 1990). Glyoxalases were initially thought to be involved on a major metabolic pathway for breakdown of glucose to Dlactate via methylglyoxal (Harden, 1932). However, the discovery of phosphorylated glycolytic intermediates, and the finding that L-lactate was the major product of glycolysis in animal tissues, have disclaimed the glyoxalase system as belonging to mainstream hexose catabolism. Glyoxalase I was studied mainly in animal tissues and Saccharomyces cerevisiae, but was also identified in prokaryotes (Thornalley, 1990). Unlike the mammalian enzyme, which is a homodimer, microbial glyoxalases I exist in the form of monomeric entities with a fairly broad substrate specificity towards a-ketoaldehydes; they have high in vitro K , values for the hemithioacetal adduct (Rhee et al., 1986; Douglas et al., 1986). The encoding genes for the enzymes from Sacch. cerevisiae and Pseudonzonas putida have been cloned and characterized (Rhee et al., 1988). Several lines of evidence indicate that the origin of bacterial glyoxalase I might be essentially different from that of its eukaryotic counterpart. Much less is known about glyoxalase 11, although the enzyme has been purified from Sacch. cerevisiae (Murata et al., 1986b). Determination of its molecular weight by gel filtration and SDS-PAGE gave a value of about 19,000, which is within the range observed for mammalian enzymes (Thornalley 1990). Amongst several thiol esters tested, the yeast enzyme appeared quite specific and hydrolysed only S-lactoylglutathione with a K,,, value of 7 PM. This value is extraordinarily low in comparison to those measured for mammalian enzymes, which ranged between 180 and 440 PM. It should be pointed out that hydrolysis of S-lactoylglutathione is also shared in Sacch. cerevisiae by a GSH thiol esterase (Murata et al., 1987). Nevertheless, after purification, this protein appears totally distinct from glyoxalase 11. Early research work on the physiological role of the glyoxalase pathway logically first focused on mechanisms of methylglyoxal production and its susequent fate. A reaction sequence providing a by-pass to the glycolytic pathway was discovered in Escherichia coli by Cooper and his coworkers (Cooper and Anderson, 1970). This sequence of reactions, which is a combination of phosphorylated and non-phosphorylated pathways for breakdown of glucose, involves a methylglyoxal synthase activity and the GSH-dependent glyoxalase system. However, the physiological function of the by-pass is still a matter for conjecture. Methylglyoxal synthase and the glyoxalase system in E. coli belong to a constitutive pathway. At the beginning, it was suggested that this pathway could be operative under
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phosphate limitation, a situation that impairs action of glyceraldehyde-3phosphate dehydrogenase and prevails often under ecological conditions (Cooper, 1984). Since methylglyoxal synthase is strongly inhibited by inorganic phosphate in vitro, limitation of inorganic phosphate could allow a significant metabolic carbon flow through the non-phosphorylated pathway. Unfortunately, no experimental evidence has been presented to support this hypothesis. Another interesting, but related, supposition is that an operative glyoxalase by-pass would permit dissociation of glucose breakdown from synthesis of ATP (Cooper, 1984). In effect catabolism of dihydroxyacetone phosphate, which is mediated by methylglyoxal synthase, might provide the inorganic phosphate required to trigger the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase that leads to phosphoenolpyruvate biosynthesis. It is noteworthy that resting cells of E. coli, energetically disconnected, still utilize about 50% of the glucose used in the medium (Roberts et al., 1963). Methylglyoxal has also been proposed to serve as a precursor of D-lactate, which can be subsequently used to energize transport systems for sugars and amino acids in E. coli (Kaback, 1974). Despite all of these hypotheses, it is obvious that the glyoxalase pathway in E. coli is still enigmatic and apparently inadequate for channelling of large amounts of 2-oxoaldehydes (Hopper and Cooper, 1971). Indeed, challenging cells fully derepressed for glycerol catabolism and having lost feedback control on glycerol kinase with fructose 1,6-bisphosphate resulted in production of lethal amounts of methylglyoxal. Resistance to methylglyoxal in E. coli can, however, be achieved by a mutational or a genetically engineered increase in the intracellular content of GSH and the level of glyoxalase activities (Murata et al., 1980; Murata and Kimura, 1990). In a Pseudomonas sp, it was suggested that gluconate metabolism may proceed by a glyoxalase by-pass which involves glyceraldehyde 3-phosphatase and glyceraldehyde dehydratase catalysing formation of methylglyoxal (Rizza and Hu, 1973). Arguments supporting this view were supplied using differently labelled gluconate substrates. The proposed pathway, at variance with the classical Entner-Doudoroff scheme, implies that substrate-level phosphorylation may not occur in conversion of gluconate to pyruvate and could account for the strictly aerobic life-style of the pseudomonad. The status of the glyoxalase pathway in Sacch. cerevisiae appears completely different. More precisely, enzyme production seems to be regulated. For example, growth on glycerol as the carbon source or on glucose in the presence of methylglyoxal induced glyoxalase activities. The system was also shown to respond to the glucose effect (Penninckx et al.,
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1983). Furthermore, it was demonstrated that the activity of the glyoxalase system is related to the overall growth status of Sacch. cerevisiae (Dudani et al., 1984). High levels of activity were detected in actively growing cells, whereas low levels were expressed in resting cells. It should be noted that a similar phenomenon was encountered in plants (Ramaswamy etal., 1983). Interestingly, this observation raises the question of the existence of a constitutive methylglyoxal production in Sacch. cerevisiae and recalls an old hypothesis put forward by Szent-Gyorgyi and his coworkers (Egyud and Szent-Gyorgy, 1968). These authors suggested that methylglyoxal acted as a physiological growth inhibitor whose effect might be relieved by the glyoxalase system, growth resulting from the balance between both effects. In this connection, certain yeast species which dissimilate methanol were shown to have a complete and constitutive glycolytic by-pass similar to that of E. coli (Babel and Hofrnan, 1981). A mutant fully defective glyoxalase I , bearing only one nuclear mutation, has been isolated from Sacch. cerevisiae (Penninckx et al., 1983). This strain, which is killed by exposure to glycerol, was found to accumulate about 10 times more intracellular methylglyoxal than the wild type when cells were transferred from a glucose-containing medium to a glycerolcontaining medium. Since methylglyoxal synthase activity was not detected in the strain, it was suggested that the oxoaldehyde formed was derived from spontaneous decay of intracellular glyceraldehyde 3-phosphate which accumulates during growth on glycerol. Therefore, glyoxalase I could play a leading role in detoxification of methylglyoxal that accumulates as a consequence of non-regulated glycerol catabolism. In essence, the system shows some resemblance to formation of catalase and superoxide dismutase which function in a similar relationship to oxidative metabolism. Glyoxalase I from yeast is not absolutely specific for methylglyoxal and may utilize numerous 2-oxoaldehydes. In this connection, other possible sources of oxoaldehyde in yeast might be aminoacetone and 1-hydroxyacetone formed during catabolism of threonine, isoleucine and valine (Murata et al., 1986a,b). To conclude, it could be said that, in spite of progress in understanding the nature of the reactions catalysed by the glyoxalase system, the role and fate of methylglyoxal in living cells remain largely unexplained. Current views suggest that the glyoxalase pathway can function as a possible detoxification pathway for endogenous oxoaldehydes or in intermediary metabolism. Recent observations in this field with mammalian cells have focused attention on the multiple effects of S-D-lactoylglutathione, including potential therapeutic uses (Gillespie, 1979; Thornalley, 1990). Genetically engineered cells of E. coli, which carry the gene (glol) encoding for glyoxalase I biosynthesis in P. putida, were suggested as a useful
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tool for commercial production of S-D-lactoylglutathione (Murata and Kimura, 1990). B . METHANOL DISSIMILATION
Methylotrophs perform their total cellular biosynthesis from C1 compounds. Diversity in metabolic strategies for C, compounds, and particularly methanol catabolism, have been extensively described for bacteria (Anthony, 1982; Sysoev et al., 1990). By contrast, quite homogeneous pathways were observed among microbial eukaryotes, especially in methylotrophic yeasts, with respect to the mechanism of methanol dissimilation and assimilation (Sahm, 1977; van Dijken et al., 1981). When grown on methanol, Candida boidinii and Hansenula polymorpha synthesized crystalline cytoplasmic peroxisome inclusions containing methanol oxidase and catalases (Fukui and Tanaka, 1979). Formaldehyde, produced during catabolism of methanol, is exported to the cytosol, where it apparently reacts spontaneously with GSH to form S-hydroxylmethylglutathione, a hemimercaptal adduct. The NADf-linked formaldehyde dehydrogenase (EC 1.2.1.1) catalyses subsequent biotransformation of the compound into S-formylglutathione, which is rapidly hydrolysed to formate and GSH by a separate enzyme, S-formylglutathione hydrolase (EC 3.1.2.12). Formate is then oxidized to give carbon dioxide by a second dehydrogenase system (Fig. 6).
I
FORMALDEHYDE DEHYDROGENASE
S-FORMYIGLLITATHIONE HYDROIASE
*GsH
G9I4
02
OXIDASE
\
H
C H @ H ~ s H C M
GHZo2
H20 1R 0 2 i
I'EROXISOME
CYTOSOL
FIG. 6. Pathway describing oxidation of methanol to carbon dioxide in methylotrophic yeasts.
Formaldehyde dehydrogenase is as ubiquitous as glyoxalase in living cells (Uotila and Koivusalo, 1983). The enzyme was detected and partially purified 30 years ago from baker's yeast (Rose and Racker, 1962) and was snown to be present in considerable amounts in methanol-utilizing yeasts
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(Sahm, 1977; Egli et al., 1980). Several but not all methylotrophic bacteria contain the enzyme, which is also found in E. coli (Cox and Quayle, 1975; Ben-Bassat and Goldberg, 1977). Formaldehyde dehydrogenase from C . boidinii was shown to be a dimer consisting of two identical subunits with a molecular weight of about 40,000 (Schiitte et al., 1976). This value is similar to those reported f m mammalian enzymes (Uotila and Koivusalo, 1974). S-Formylglutathione hydrolase, which catalyses irreversible hydrolysis of S-formylglutathione, was originally discovered and purified to homogeneity from human liver (Uotila and Koivusolo, 1983). Homogeneous preparations have also been described for the methylotrophic yeasts Kloeckera sp. and C. boidinii (Kato et al., 1980; Neben et al., 1980). The enzyme from these yeasts appears as a heterodimer with a molecular weight of about 60,000 and is highly specific for S-formylglutathione (van Dij ken et al., 1976). Formaldehyde dehydrogenase and S-formylglutathione hydrolase participate in metabolism of methylotrophic bacteria and yeasts, on the pathway for complete oxidation of methanol to carbon dioxide (Sahm, 1977). Formaldehyde, S-formylglutathione and formate are intermediates on this pathway, which provides energy for growth. In many micro-organisms, synthesis of formaldehyde dehydrogenase is apparently controlled by derepression-repression processes (Sahm, 1977; Neben et al., 1980). In non-methylotrophic organisms, such as Sacch. cerevisiae and E. coli, it is quite possible, however, that formaldehyde dehydrogenase has a role in detoxifying formaldehyde. Substantial evidence supporting this view was reported for animal tissues (Uotila and Koivusalo, 1983). Formaldehyde can be formed on several metabolic pathways, such as catabolism of methionine and choline, oxidation of methanol by an alcohol dehydrogenase and catalase or by other minor metabolic reactions. Methylglyoxal is a good substrate for formaldehyde dehydrogenase. Utilization of methylglyoxal by the action of glyoxalase or formaldehyde dehydrogenase is thus dependent on GSH and results in GSH thiol-ester iormation. Therefore, besides functioning in detoxification of formaldehyde and methylglyoxal, formaldehyde dehydrogenase may have some functions associated with thiol-ester products. As already indicated, specific GSH esterases that catalyse hydrolysis of the products have a!so been demonstrated in yeasts (Murata et al., 1987). C. HEAVY-METAL DETOXIFICATION
A higher sensitivity of GSH-deficient mutants of E. coli towards heavy metals has been reported (Apontoweil and Berends, 1975b). On a chemical
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J PI-NNINCKX AND M T ELSKENS
basis, it was previously expected that GSH would be able to form chelation complexes with heavy metals and may, in this way, be involved in a detoxification process. Recent progress in this field was obtained by studies with Schizosaccharornyces pombe and Candida (Torulopsis) glabrata (Grill et al., 1985; Dameron et al., 1989). When exposed to cadmium salts, these yeasts were shown to synthesize peptide derivatives that involved cadmium ions and multiple moieties of GSH (Grill et al., 1985; Hayashi et al., 1988; Mehra et al., 1988). The so-called cadystins or phytochelatins (Grill et al., 1987) were apparently also found in some plant species (Jackson et al., 1987), but remained undetected in bacteria, Sacch. cerevisiae and animal tissues. It was further shown that the pathway for heavy-metal inactivation was apparently dependent on growth conditions. When cultivated in nutrient broth, Schiz. pombe and T. glabrata, exposed to cadmium salts, form cadmium sulphide particles coated with GSH and y-glutamylcysteine whereas when grown in minimal media the cadmium adduct was coated with peptides having the general structure (y-Glu-Cys),-Gly (Dameron et al., 1989). Two pathways for biosynthesis of this compound were found in cell-free extracts of Schiz. pombe (Hayashi et al., 1991). The first involves a transfer of y-Glu-Cys from both GSH and cadystins to GSH and cadystins, whereas the second is a polymerization of y-Glu-Cys from (y-Glu-Cys), and GSH to give (y-Glu-Cys),+,, followed by addition of a glycine residue catalysed by GSH synthetase. Mutants of Schiz. pombe deficient in GSH were recently described (Glaeser et al., 1991). The Gsh- mutants have lost the ability to excrete cadmium and were also shown to be more susceptible to other heavy metals like bismuth, copper, lead, zinc and silver.
VII. Concluding Remarks
It is now clearly established that GSH and related compounds are widespread in the microbial world, especially amongst organisms with an aerobic life-style. This observation emphasizes the role of GSH in cellular protection against by-products generated by oxidative metabolism, but it does not in any way limit its functions to this role. Glutathione has, indeed, been shown to act as an enzyme cofactor, transport component, nucleophilic substrate and sulphur reservoir, and it also participates in key cellular processes such as protein synthesis and degradation, regulation of enzyme activity, synthesis of DNA, and maintenance of the integrity of cell membranes and organelles. Having such functional diversity, GSH is interrelated with a number of metabolic pathways and its intracellular modulation could obviously have an impact on the entire cell, making it
GLUTATHIONE IN MICRO-ORGANISMS
29 1
extremely difficult to associate directly a given cellular end-point with one molecule or system. Nevertheless, genetic or pharmacological manipulations that alter GSH status were found to be useful for investigating the role of the tripeptide in detoxification of xenobiotics and its function as a scavenger of externally and possibly internally formed radicals. One might be astonished to note that, both in Escherichia coli and Saccharomyces cerevisiae, GSH appears to play an important role in cellular protection during chemical stresses in spite of the fact that key enzymes of detoxification, such as GSH peroxidase and GSH S-transferase, remain at a low level. This protective effect could be attributed to the GSH redox cycle and also highlights the chemical reactivity of the tripeptide. It is appropriate to mention the profound influence exerted by investigations of Meister and the Kosowers on the early development of microbial GSH research. Since then, further knowledge has been gained on the specific metabolism and functions of GSH in micro-organisms, and mammalian physiologists take an ever greater interest in the use of microbial models for understanding peculiar aspects of metabolism in animal tissues. It is expected that, in the future, the GSH network will be extended in all possible directions. VIII. Acknowledgements
This work was supported in part by research grants from the Fonds National de la Recherche Scientifique (FNRS) to M. J. P., and an Actionde Recherche Concertee (ARC) financed by the Belgian State. The skilful assistance of Anne Wies and Fernand-Pierre Wies was very much appreciated. REFERENCES
Abdrashitova, S. A., Mynbaeva, B. N., Aidarkhanov, B. B. and Ilyaletdinov, A. N. (1990). Mikrobiologiya 59, 234. Aisaka, K., Uwajima, T. and Tereda, 0. (1983). Agricultural and Biological Chemistry 47, 1269. Allen, L., Meck, R. and Yunis, A. (1980). Research Communications in Chemical Pathology and Pharmacology 21, 175. Anders, M. W., Lash, L., Dekant, W., Elfarra, A. A. and Dohn, D. R. (1988). Critical Reviews in Toxicology 18, 31 1. Ando, K., Honma, M., Chiba, S., Tahara, S. and Mizutani, J . (1988). Agricultural and Biological Chemistry 52, 135. Anfinsen, C . B. (1973). Science 181, 223. Ansorge, S., Bohley, P., Kirschke, H., Langgner, J., Marquardt, L., Wiederanders, B. and Hanson, H. (1973a). FEBS Letters 37, 238. Ansorge, S., Bohley, P., Kirschke, H . , Langger, J., Wiederanders, B. and Hanson, H. (197313). European Journal of Biochemistry 32, 27. Anthony, C. (1982). In “The Biochemistry of Methylotrophs”. Academic Press, London.
292
M. J . PENNINCKX AND M. T. ELSKENS
Apontoweil, P. and Berends, W. (1975a). Biochimica et Biophysica Acta 399. 1. Apontoweil, P. and Berends, W. (1957b). Biochimica et Biophysica Acta 399, 10. Arca, P . , Rico, M., Brana, A . F., Villar, C. J., Hardisson, C. and Suarez, J . E . (1988). Antimicrobial Agents and Chemotherapy 32, 1552. Arca, P., Hardisson, P. and Suarez, J. E . (1990). Antimicrobial Agents and Chemotherapy 34, 844. Arias, I. M. and Jacoby, W. B. (eds) (1976). “Glutathione: Metabolism and Function” (Kroc Foundation Series, vol. 6 ) . Raven Press, New York. Babel, W. and Hofmann, K. H. (1981). FEMS Microbiology Letters 10, 133-136. Babson, J. R. and Reed, D. J . (1978). Biochemical and Biophysical Research Communications 83, 754. Barak, E. and Edgington, L. V. (1984). Pesticide Biochemistry and Physiology 21, 412. Baron, E. S. (1951). Advances in Enzymology 11, 201. Becker, K., Christopherson, R. I., Cowden, W. B., Hund, N. H. and Schirmer, R. H. (1990). Biochemical Pharmacology 39, 59. Bellofatto, V., Fairlamb, A. H., Henderson, G . B. and Cross, G . A. (1987). Molecular and Biochemical Parasitology 25, 227. Ben Bassat, A. and Goldberg, I. (1977). Biochimica et Biophysica Acta 497, 586. Bonsignore, A. and De Flora, A. (1972). Current Topics in Cellular Regulation 6, 21. Bouter, S . , Kerklaan, P. R. M., Zoetemelk, C. E. M. and Mohn, G. R. (1988). Biochemical Pharmacology 37, 577. Boyer, T. D. (1989). Hepatology 9, 486. Boyland, E. and Chasseaud, L. F. (1969). Advances in Enzymology 32, 173. Branden, C. I., Eklund, H. and Soderberg, B. 0. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 251. Raven Press, New York. Brown, R . W. (1974). Journal of Dairy Science 57, 797. Bruinenberg, P. M., van Dijken, J. P. and Scheffers, W. A. (1983a). Journal of General Microbiology 129, 953. Bruinenberg, P. M., van Dijken, J. P. and Scheffers, W. A. (1983b). Journal of General Microbiology 129, 965. Buchanan, B. B. (1980). Annual Review of Plant Physiology 31, 314. Casalone, E. , Di Illio, C., Federici, G . and Polsinelli, M. (1988). Antonie van Leeuwenhoek 54, 367. Ctnas, N. K., BironaitC, D. A , , Kulys, J. J . and Sukhova, N. M. (1991). Biochimica et Biophysica Acta 1073, 195. Chance, B., Sies, H. and Boveris, A. (1979). Physiological Reviews 59, 527. Chasseaud, L. F. (1979). Advances in Cancer Research 29, 175. Cheng, H., Aronson, A . and Holt, S. (1973). Journal of Bacteriology 113, 1135. Cherest, H., Eichler, F. and de Robichon-Szulmajster, H. (1969). Journal of Bacteriology 97, 328. Chung, Y. C. and Hurlbert, R. E. (1975). Journal of Bacteriology 123, 855. Cohen, E., Gambiel, A. and Katan, J . (1986). Pesticide Biochemistry and Physiology 26, 1. Cohen, G. M. and Friedman, R. B. (1982). Biochemical Society Transactions 10, 78. Colowick, S . , Lazarow, A., Racker, E., Schwarz, D. R., Stadtman, E. and Waelsh, H. (eds) (1954). “Glutathione”. Academic Press, London. Cooper, R. A. (1984). Annual Review of Microbiology 38, 49. Cooper, R. A. and Anderson, A. (1970). FEBS Letters 11, 273. Cox, R. B. and Quayle, J. R. (1975). Biochemical Journal 150, 569. Creighton, T. E. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and €3. Mannervik eds), p. 205. Raven Press, New York. Crook, E. M. (1959). Biochemical Society Symposium 17, 116,. Csonka, L. N. and Clark, A. J. (1980). Journal of Bacteriology 143, 529. Dameron, C. T., Reese, R. N., Mehra, R. K., Kortan, A. R., Caroll, P. J., Steigenvald, M. L., Brus, L. E. and Winge, D . R . (1989). Nature 338, 596.
GLUTATHIONE IN MICRO-ORGANISMS
293
Danson, M. J., Eisenthal, R., Hall, S . , Kessel, S. R. and Williams, D. L. (194X). Biochemical Journal 218, 811. de Rey-Pahlade, J. (1888). AcadCmie des Sciences (Paris) 107, 43. Dean, R. T., Thomas, S. M. and Garner, A. (1986). Biochemical Journal 240, 489. Di Ilio, C., Aceto, A , , Piccolomini, R., Allocati, N., Faraone, A ,, Cellini, L., Ravagnan, G. and Federici, G. (1988). Biochemical Journal 255, 971. Di rho, C., Aceto, A , , Piccolomini, R., Allocati, N., Faraone, A , , Bucciarelli, T., Barra, D . and Federici, G . (1991). Biochimica et Biophysica Acta 1077, 1431. Dierickx, P. J. (1991). Archives Internationales de Physiologie et de Biochimie 100, 10. Dierickx, P. J., Almar, M. M. and De Jonckheere, J. F. (1990). Biochemistry International 22, 593. Douglas, K. T., Seddon, A. P. andNakagawa, Y. (1986). International Journal of Biochemistry 18, 549. Drotar, A , , Burton, G., Tavernier, J. E. and Fall, R. (1987). Applied and Environmental Microbiology 53, 1626. Dubois, E., Vissers, S . , Grenson, M. and Wiame, J.-M. (1977). Biochemical and Biophysical Research Communications 75, 233. Dudani, A. K., Srivastava, L. K. and Prasad, R. (1984). Biochemicaland Biophysical Research Communications 119, 962. Dupouy, D., Conter, A , , Croute, F., Murat, M. and Planel, H. (1985). Environmental and Experimental Botany 25, 339. Eddy, A. A. (1982). Advances in Microbial Physiology 23, 1. Eggleston, L. V. and Krebs, H. A. (1974). Biochemical Journal 138, 425. Egli, T., van Dijken, J. P., Vcenhuis, M., Harder, W. and Fiechter, A. (1980). Archives of Microbiology 124, 115. Egyud, L. G. and Szent-Gyorgy, A . (1968). Science 160, 1140. Eklund, H., Cambillau, C., Sjoeberg, B. M., Holmgren, A ,, Joernvall, H., Hoeoeg, J. 0. and Braenden, C. I. (1984). EMBO Journal 3, 1443. Elliott, K. and Whelan, J. (eds) (1979). Ciba Foundation Symposium 72. Elskens, M. T. and Penninckx, M. J. (1986). Archives Internationales de Physiologie et de Biochimie 94, 76. Elskens, M. T. , Penninckx, M. J . , Vandeloise, R. and Vander Donckt, E. (1988a). International Journal of Chemical Kinetics 20, 837. Elskens, M. T., Penninckx, M. J., Vandeloisc, R. and Vander Donckt, E. (1988b). Bulletin de la SociCtt! Chimiaue Belee 97. 397. Elskens, M. T., Jaspers, C. J. and Penninckx, M. J. (1991). Journal of General Microbiology 137, 637. Ermler, U. and Schulz, G. E. (1991). Proteins: Structure Function and Genetics 9, 174. Evans, R . G. (1985). International Journal of Radiation, Oncology, Biology, Physics 11, 1163. Eyer, P. Bnd Podhradsky, D. (1986). Analytical Biochemistry 153, 57. Fahey, R. C. (1977). In “Protein Crosslinking, part A” (R. B. Friedman, ed.), p. 1. Plenum Press, New York. Fahey, R. C. and Newton, G. L. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S . Orrenius, A. Holmgren and B. Mannervik, eds), p. 251. Raven Press, Ncw York. Fahey, R. C. and Newton, G . L. (1986). Methods in Enzymology 143, 85. Fahey, R. C., Brody, S. and Mikoljczyk, S . D . (1975). Journal of Bacteriology 121, 144. Fahey, R. C., Brown, W. C., Adams, W. B. and Worsham, M. B. (1978). Journal of Bacteriology 133, 1126. Fahey, R. C., Bushbacher, R . M. and Newton, G . L. (1987). Journal of Molecular Evolution 25, 81. Fahey, R. C., Newton, G. L., Arrick, B., Overdank-Bogart, T. and Aley, S . B. (1984). Science 224. 70. Y
,
294
M. J. PENNINCKX AND M. T. ELSKFNS
Fairfield, A. S . , Abosch, A . , Ranz, A., Eaton, J. W. and Meshnick, S. R. (1988). Molecular and Biochemical Parasitology 30, 77. Fairlamb, A. H. (1989). Parasitology 99, 93. Fairlamb, A. H. (1990). Biochemical Society Transactions 18, 717. Fairlamb, A. H. and Cerami, A. (1985). Molecular and Biochemical Parasitology 14, 187. Fairlamb, A. H., Blackburn, P., Ulrich, P., Chait, B. T. and Cerami, A. (1985). Science 227, 1485. Fairlamb, A. H., Henderson, G. B. and Cerami, A. (1986). Molecular and Biochemical Parasitology 21, 247. Fairlamb, A. H., Henderson, G . B., Bacchi, C. J. and Cerami, A. (1987). Molecular and Biochemical Parasitology 24, 185. Ficchter, A,, Fuhrmann, G . F. and Kappeli, 0. (1981). Advances in Microbial Physiology 22, 123. Flohe, L. and Gunzler, W. A. (1976). In “Glutathione: Metabolism and Function” (Kroc Foundation Series, vol. 6) (I. M. Arias and W. B. Jacoby, eds), p. 17. Raven Press, New York. Flohe, L., Benohr, H. C., Sies, H., Waller, H . D. and Wendel, A . (eds) (1974). “Glutathione”, Thieme, Stuttgart. Flohe, L., Gunzler, W. A. and Loshen, G. (1980). In “Trace Metal in Health and Disease” (N. Khararash, ed.), p. 263. Raven Press, New York. Fox, G. E., Stakebrandt, E . , Hespell, R. B . , Gibson, J., Maniloff, J., Dyer, T. A,, Wolfe, R. S . , Balch, W. E., Tanner, R. S., Magrum, L. J., Zablen, L. B., Blakemore, R., Gupta, R., Bonen, L., Lewis, B. J., Stahl, D. A , , Luehrsen, K. R., Chen, K. N. and Woese, C. R. (1980). Science 209, 4.57. Freedman, R. B. (1979). FERS Letters 97, 201. Freedman, R . B. and Hillson, D . A. (1980). In “The Enzymology of Post-Translational Modification of Proteins I” (R. B. Freedman and H. C. Hawkins, eds), p. 157. Academic Press, London. Freedman, R. B., Hawkins, H. C., Murant, S . J. and Reid, L. (1988). Biochemical Society Transactions 16, 96. Fridovich, I. (1982). Biochemical Society Transactions 10, 67. Fuchs, J. (1977). Journal of Bacteriology 129, 140. Fuchs, J. A. and Warner, H. R. (1975). Journal of Bacteriology 124, 140. Fuchs, J. A, , Hailer, B. and Tuggle, C. K. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 385. Raven Press, New York. Fukui, S . and Tanaka, A. (1979). Trends in Biochemical Sciences 4, 246. Galiazzo, F., Schlesser, A. and Rotilio, G. (1987). Biochemical and Biophysical Research Communications 147, 1200. Gallori, E., Casalone, E., Nocita, O., Turbanti, L. and Polsinelli, M. (1988). Journal of General Microbiology 134, 3173. Gancedo, C. and Serrano, R. (1989). In “The Yeasts” (A. H. Rose and J. S. Harrison, eds), 2nd cdn, vol. 3, pp. 205-259. Academic Press, London. Gauthier, M. J . , Berge, J. B., Cuany, A , , Breittmayer, V. and Fournier, D. (1988). Pesticide Biochemistry and Physiology 31, 61. Gcnnity, J . M., Bottino, N. R., Zingaro, R. A , , Wheeler, A. E. and Irgolic, K. J. (1985a). Phytochernistry 24, 2817. Gennity, J. M., Bottino, N. R., Zingaro, R. A , , Whceler, A. E. and Irgolic, K. J . (198%). Phytochemistry 24, 2823. Ghisla, S. and Massey, V. (1989). European Journal of Biochemistry 181, 1. Giammanco, G., Buissieres, J., Toucas, M., Brault, G. and Le Minor, L. (1980). Annales de Microbiologie de l’lnstitut Pasteur (Paris) 131, 181. Giddings, T., Wolk, P. and Shomer-Ilan, A. (1981). Journal of Bacteriology 146, 1067. Gillespie, E. (1979). Nature 277, 135. Giovannelli, J. (1987). Methods in Enzymology 143, 419. Glaeser, H., Coblenz, A., Kruczek, R., Ruttke, I., Ebert-Jung, A . and Wolf, K. (1991). Current Genetics 19. 207.
GLUTATHIONE IN MICRO-ORGANISMS
295
Gleason, F. K. and Holmgren, A. (1988). FEMS Microbiology Reviews 4, 271. Goldberger, R. F., Epstein, C. J . and Anfinsen, C. B. (1963). Journalof Biological Chemistry 238, 628. Gorrod, J. W. (1980). Chemical Industry 7, 457. Greer, S. and Perham, R. N. (1986). Biochemistry 25, 2736. Grenson, M. and Hennaut, C. (1971). Journal of Bacteriology 105, 477. Griffith, 0. W. (1987). Methods in Enzymology 143, 366. Griffith, 0. W. and Meister, A . (1979a). Proceedings of the National Academy of Sciences of the United States of America 76, 5606. Griffith, 0. W. and Meister, A. (1979b). Journal of Biological Chemistry 254, 7558. Griffith, 0. W. and Meister, A. (1980). Proceedings of the National Academy of Sciences of the United States of America 77, 3384. Griffith, 0. W., Anderson, M. E. and Meister, A. (1979). Journal of Biological Chemistry 254. 1205. Grill, E., Zenk, M. H. and Winnacker, E . (1985). Science 230, 674. Grill, E., Winnacker, E. L. and Zenk, M. H . (1987). Proceedings of the National Academy of Sciences of the United States of America 84, 439. Gushima, H., Miya, T., Murata, K. and Kimura, A . (1983). Journal ofApplied Biochemistry 5, 210. Halliwell, B. (1991). Drugs 42, 569. Halliwell, B. and Aruoma, 0. I. (1991). FEBS Letters 281, 9. Halliwell, B. and Gutteridge, J. M. (eds) (1989). “Free Radicals in Biology and Medicine”. Clarendon Press, Oxford. Halliwell, B. and Gutteridge, J. M. (1990). Methods in Enzymology 186, I. Harden, A. (1932). In “AlcoholicFermentation”, p. 109. Longmans, Green and Co., London. Harington, C. R. and Mead, T. H . (1935). Biochemical Journal 29, 1602. Hasegawa, M. and Matsubana, I. (1978). Agricultural and Biological Chemistry 42, 385. Hayashi, Y., Nakagawa, C. W., Uyakul, D., Imai, K . , Isobe, M. and Goto, T. (1988). Biochemistry and Cell Biology 66, 288. Hayashi, Y . , Nakagawa, C. W., Mutoh, N., Isabc, M. and Goto, T. (1991). Biochemistry and Cell Biology 69, 115. Heikkila, R. E., Cabbat, F. S. and Cohen, G . (1976). Journal of Biological Chemistry 251, 2182. Henderson, G. B . , Fairlamb, A. H., Ulrich, P. and Cerami, A. (1987). Biochemistry 26,3023. Henderson, G. B., Ulrich, P., Fairlamb, A . H., Rosenberg, I., Pereira, M., Sela, M. and Cerami, A. (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 5374. Henderson, G. B., Yamachuci, M., Novoa, L., Fairlamb, A. H. and Cerami, A. (1990). Biochemistry 29, 3924. Hewick, D. S . (1982). In “Metabolic Basis of Detoxication, Metabolism of Functional Groups’’ (W. B. Jacoby, J. R. Bend and J. Caldwell, eds), p. 151. Academic Press, New York. Hoeoeg, J . O., Holmgren, A , , Silva, C. D., Douglas, K. T. and Seddon. A . P. (1982). FEBS Letters 138, 59. Hoeoeg, J. O., Joernvall, H., Holmgren, A,, Carlquist, M. and Persson, M. (1983). European Journal of Biochemistry 136, 223. Hoeoeg, J. O., von Bahr-Lindstroem, H . , Jocrnvall, H. and Holmgrcn, A. (1986). Genetics 43, 13. Holmgren, A. (1976). Proceedings of the National Academy of Sciences of the United States of America 73, 2275. Holmgren, A. (1979). Journal of Biological Chemistry 254, 3664. Holmgren, A. (1981). Trends in Biochemical Sciences 6, 26. Holmgren, A. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 205. Raven Press, New York. Holmgren, A. (1985). Annual Review of Biochemistry 54, 237. Holmgren, A. (1988). Biochemical Society Transactions 16, 95.
296
M. J . PENNINCKX AND M. T. ELSKENS
Holmgren, A,, Branden, C. I., Jornvall, H. and Sjoberg, B. M. (eds) (1986). “Thioredoxin and Glutaredoxin Systems: Structure and Function”. Raven Press, New York. Hopkins, F. G. (1921). Biochemical Journal 15, 286. Hopper, D. J. and Cooper, R. A. (1971). FEBS Letters 13, 213. Iwami, K., Yasumoto, K. and Fushiki, T. (1978). Agricultural and Biological Chemistry 42,2175. Izuka, M., Murata, K. and Kimura, A. (1988). Agricultural and Biological Chemistry 52,613. Izuka, M., Inorie, Y., Murata, K. and Kimura, A. (1989). Journal of Bacteriology 171,6039. Jackson, P. J., Unkefer, C. J., Dovlen, J. A., Watt, K. and Robinson, N. J. (1987). Proceedings of the National Academy of Sciences of the United States of America 84, 6619. Jacoby, W. B. (1978). Advances in Enzymology 36, 383. Jacoby, W. B. and Habig, W. H. (1980). In “Enzymic Basis of Detoxication” (W. B. Jacoby, J. R. Bend and J. Caldwell, eds), p. 63. Academic Press, New York. Jacoby, W. B., Ketterer, B. and Mannervik, B. (1984). Biochemical Pharmacology 33,2539. Jaspers, C. (1985). Ph.D. Thesis: Free University of Brussels. Jaspers, C. and Penninckx, M. (1981). FEBS Letters 132, 41. Jaspers, C. and Penninckx, M. (1982). Archives Internationales de Physiologie et de Biochimie 90, 198. Jaspers, C. and Penninckx, M. (1984). Biochimie 66, 71. Jaspers, C., Gigot, D. and Penninckx, M. (1985). Phytochemistry 24, 703. Jocelyn, P. C. (1972). “Biochemistry of SH Group”. Academic Press, New York. Jockers-Scherubl, M. C., Schirmer, R . H . and Krauth-Siegel, R. L. (1989). European Journal of Biochemistry 180, 267. Jones, E. W. and Fink, G . R. (1982). In “The Molecular Biology of the Yeast Saccharomyces cerevisiae: Metabolism and Gene Expression” (J. N. Strathen, E. W. Jones and J. R. Broach, eds), pp. 181-299. Cold Spring Harbor Laboratory, New York. Kaback, H. R. (1974). Science 186, 882. Kappus, H. (1985). In “Oxidative Stress’’ (H. Sies, ed.), p. 273. Academic Press, New York. Kappus, H. (1986). Biochemical Pharmacology 35 1. Kappus, H. (1987). Chemistry and Physics of Lipids 45, 105. Karplus, P. A. and Schulz, G . E. (1987). Journal of Molecular Biology 195, 701. Kato, H., Chihara, M., Nishioka, T., Murata, K., Kimura, K. and Oda, J. (1987). Journal of Biochemistry 101, 207. Kato, N., Sakazava, C., Nishizava, T., Tani, Y. and Yamada, H. (1980). Biochimica et Biophysica Acta 611, 323. Kendall, E. C., Mackenzie, B. F. and Mason, H. L. (1929). Journal of Biological Chemistry 84, 657. Kerklaan, P. R., Zoetemelk, C. E. andMohn, G. R. (1985). Biochemical Pharmacology 34,2151. Kistler, M., Summer, K. H. and Eckardt, F. (1986). Mutation Research 173, 117. Kistler, M., Maier, K. and Eckardt, F. (1990). Mutagenesis 5, 39. Kitamoto, K., Yoshizawa, K., Ohsumi, Y. and Anraku, Y. (1988). Journal of Bacteriology 170, 2683. Kosower, E. M. and Kanety-Londner, H. (1976). Journal of the American Chemical Society 98, 3001. Kosower, N. S. and Kosower, E. M. (1978). International Review of Cytology 54, 109. Kosower, E. M., Pazhenchevsky, B. and Hershkowitz, E. (1978). Journal of the American Chemical Society 100, 6516. Kosower, N. S . , Kosower, E. M., Newton, G. L. and Ranney, H. M. (1979). Proceedings of the National Academy of Sciences of the United States of America 76, 3382. Kosower, E. M., Kosower, N. S. and Radkwsky, A. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 243. Raven Press, New York. Krauth-Siegel, R. L., Enders, B., Henderson, G . B., Fairlamb, A. H. and Schirmer, R. H. (1987). European Journal of Biochemistry 164, 123. Kren, B., Parsell, D. and Fuchs, J. A. (1988). Journal of Bacteriology 170, 308. Kumagai, H . , Nakayama, R. and Tochikura, T . (1982). Agricultural and Biological Chemistry 46, 1301.
GLUTATHIONE IN MICRO-ORGANISMS
297
Kumagai, H., Tamaki, H., Koshino, Y., Suzuki, H. and Tochikura, T. (1988). Agricultural and Biological Chemistry 52, 1377. Kumar, K. S., Sancho, A. M. and Weiss, J. F. (1986). International Journal of Radiation, Oncology Biology, Physics 12, 1463. Kumar, S., Ohja, V . , Gangerly, N. K. and Kohti, K. K. (1990). Biochemistry International 20, 539. Kunding, W. (1976). In “The Enzymes of Biological Membranes” (A. Martoni, ed.), p. 31. Wiley, London. Lahti, R. and Suonpaa, M. (1982). Journal of General Microbiology 128, 1023. Larsson, A,, Orrenius, S., Holmgren, A. and Mannervik, B. (eds) (1983). “Functions of Glutathione: Biochemical, Physiological, Toxicologic.al, and Clinical Aspects”. Raven Press, New York. Lau, E. P., Niswander, L., Watson, D. and Fall, R . (1980). Chemosphere 9, 565. Lawrence, R. A. and Burk, R. F. (1976). Biochemical and Biophysical Research Communications 71, 952. Lawrence, R. A. and Burk, R. F. (1978). Journal of Nutrition 108, 211. Levy, H. R. and Christoff, M. (1983). Biochemical Journal 214, 959. Li, L., Seddon, A. P. and Meister, A. (1988). Journal of Biological Chemistry 253, 1799. Llobell, A., Lopez-Ruiz, A,, Peinado, J. and Lopez-Barea, J. (1988). Biochemical Journal 249, 293. Loewen, P. C. (1981). Canadian Journal of Biochemistry 59, 379. Lopez-Barea, J. and Lee, C. Y. (1979). European Journal of Biochemistry 98, 487. Mannervik, B. (1973). Biochemical and Biophysical Research Communications 53, 1151. Mannervik, B. (1982). In “Metabolic Basis of Detoxication” (W. B. Jakoby, J. R. Bend and J. Caldwell, eds), p. 185. Academic Press, New York. Mannervik, B. (1985). In “Advances in Enzymology and Related Areas of Molecular Biology” (A. Meister, ed.), p. 357. Wiley, New York. Mannervik, B. and Axelsson, K. (1980). Biochemical Journal 190, 125. Mannervik, B. and Danielson, U. H. (1988). Critical Reviews in Biochemistry and Molecular Biology 23, 283. Mannervik, B., Guthenberg, C., Jensson, H., Warholm, M. and A h , P. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 75. Raven Press, New York. Mark, D. F. and Richardson, C. C. (1976). Proceedings of the National Academy of Sciences of the United States of America 13, 780. Mark, D. F., Chase, J. W. and Richardson, C. C. (1977). Molecular and General Genetics 155, 145. Mason, R. P. and Josephy, P. D. (1985). In “Toxicity of Nitroaromatic Compounds” (D. E. Rickert, ed.), p. 21. Hemisphere, Washington, DC. Masselot, M. and de Robichon-Szulmajster, H. (1975). Molecular and General Genetics 139, 121. Masselot, M. and Surdin-Kerjan, Y. (1977). Molecular and General Genetics 154, 23. Mata, A. M., Pinto, M. C. and Lopez-Barea, J. (1985a). Molecular and Cellular Biochemistry 67, 65. Mata, A. M., Pinto, M. C. and Lopez-Barea, J . (1985b). Molecularand Cellular Biochemistry 68, 121. Mathews, C . K., Sjoberg, B. M. and Reichard, P. (1987). European Journal of Biochemistry 166, 279. Mehra, R. K., Target, E. B., Gray, W. R. and Winge, D. R. (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 8815. Meister, A. (1974). In “The Enzymes” (P. D. Boyer, ed.), vol. 10, pp. 671-697. Academic Press, New York. Meister, A. (1981a). Current Topics in Cellular Regulation 18, 21. Meister, A. (1981b). Trends in Biochemical Sciences 6, 231. Meister, A. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological,
298
M. J. PENNINCKX AND M . T. ELSKENS
and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 1. Raven Press, New York. Meister, A. (ed.) (1985). Methods in Enzymology 113. Meister, A. (1988). Journal of Biological Chemistry 263, 17205. Meister, A. and Anderson, M. E. (1983). Annual Review of Biochemistry 52, 711. Messenguy, F., Colin, D. and Ten Have, J. P. (1980). European Journal of Biochemistry 108, 439. Meury, J. and Robin, A. (1985). European Journal of Biochemistry 143, 113. Meury, J. and Robin, A. (1990). Archives of Microbiology 154, 475. Meyer, M., Deitrichs, D., Schmidt, B. and Andreesen, J. R. (1991). Journal of Bacteriology 173, 1509. Milbauer, R. and Grossowicz, N. (1965). Journal of General Microbiology 41, 185. Minato, S. (1979). Archives of Biochemistry and Biophysics 192, 235. Mitchell, J. B. (1988). IS1 Atlas of Science: Pharmacology, 155. Mohn, G., de Kniff, P. and Baars, A. (1983). Mutation Research 111, 25. Moncada, C., Repetto, Y . , Aldunate, J., Letelier, M. E. and Morello, A. (1989). Comparative Biochemistry and Physiology 94, 87. Monks, T. J., Anders, M. W., Dekant, W., Stevens, J. L., Lau, S. and van Bladeren, P. J. (1990). Toxicology and Applied Pharmacology 106, 1. Montero, S . , de Arriaga, D. and Soler, J. (1988). Biochimica et Biophysica Acta 952, 56. Montero, S . , de Arriaga, D., Busto, F. and Soler, J. (1990). Archives of Biochemistry and Biophysics 278, 52. Moore, W. R., Anderson, M. E., Meister, A , , Murata, K. andKimura, A. (1989). Proceedings of the National Academy of Sciences of the United States of America 86, 1461. Mooz, E. D. (1979). Biochemical and Biophysical Research Communications 90, 1221. Mooz, E. D. and Meister, A. (1967). Biochemistry 6, 1722. Mooz, E. D . and Wigglesworth, L. (1976). Biochemical and Biophysical Research Communications 68, 1066. Morse, M. L. and Dahl, R. H. (1978). Nature 271, 654. Morzycka, E. and Paszewski, A. (1982). Acta Biochimica Polonica 29, 81. Murata, K . and Kimura, A. (1990). Biotechnology Advances 8, 59. Murata, K., Tani, K., Kato, J. and Chibata, I. (1980). Journal of General Microbiology 120, 545. Murata, K., Tani, K., Kato, J . and Chibata, I. (1981). Agricultural and Biological Chemistry 45, 2131. Murata, K., Inorie, Y., Watanabe, K., Fukuda, Y . , Saikusa, T. and Kimura, A. (1986a). Agricultural and Biological Chemistry 50, 135. Murata, K., Saikusa, T., Watanabe, K., Fukuda, Y., Shimosaka, M., Inorie, Y. and Kimura, A. (1986b). European Journal of Biochemistry 57, 297. Murata, K., Sato, N., Rhee, H. I., Watanabe, K. and Kimura, A. (1987). Agricultural and Biological Chemistry 51, 1901. Murray, H. W., Aley, S. B. and Scott, W. A. (1981). Molecular and Biochemical Parasitology 3, 381. Nagai, S. and Black, S. (1968). Journal of Biological Chemistry 243, 1942. Nakayama, R. (1984). Ph.D. Thesis: Kyoto Univcrsity. Nakayama, R., Kumagai, H. and To Chikura, T. (1984). Applied and Environmental Microbiology 47, 653. Neben, I., Sahm, H. and Kula, M. R. (1980). Biochimica et Biophysica Acta 614, 81. Newton, G. L. and Fahey, R. C. (1990). In “Glutathione: Metabolism and Physiological Functions” (J. Vina, ed.), p. 69. CRC Press, Boca Raton. Newton, G. L. and Javor, B. (1985). Journal of Bacteriology 161, 438. Nikolaev, 0. N., Alver’Yanov, A. A. and Dzhavakhiya, V. G. (1989). Mikologija i Fitopatologija 23, 439. Noda, K., Igato, K., Horikawa, Y. and Fijii, M. (1980). Agriculturaland Biological Chemistry 44, 2419. Ogawa, T., Oka, Y. and Sasaoka, K. (1987). Journal of Food Science 52, 135.
GLUTATHIONE IN MICKO-ORGANISMS
299
Ogawa, T., Oka, Y. and Sasaoka, K. (1990). Phytochemistry 29, 303. Ohtake, Y. and Yabuuchi, S. (1991). Yeast 7, 953. Ohtake, Y . , Watanabe, K., Tezuka, H., Ogata, T., Yabuuchi, S . , Murata, K. and Kimura, A. (1989). Journal of Fermentation and Bioengineering 68, 390. Ondarza, R . N., Rendon, J. L. and Ondarza, M. (1983). Journal of Molecular Evolution 19, 371. Ono, B., Naito, K., Shirahige, Y. and Yamamoto, M. (1991). Yeast 7, 843. Ono, B. I., Shirage, Y. I . , Nanjoh, A , , Andou, N., Ohue, H. and Ishino-Arao, Y. (1988). Journal of Bacteriology 170, 5883. Orlowski, M. and Meister, A. (1971). Journal of Biological Chemistry 246, 7095. Osuji, G. 0. (1980). FEBS Letters 110, 192. Overbaugh, J. M. (1985). Journal of Free Radicals and Biological Medicine 1, 187. Overbaugh, J. M. and Fall, R. (1982). FEMS Microbiology Letters 13, 371. Overbaugh, J. M. and Fall, R. (1985). Plant Physiology 77, 437. Overbaugh, J. M., Lau, E. P., Marino, V. A. and Fall, R. (1988). Archives of Biochemistry and Biophysics 261, 227. Pai, E. F. and Schulz, G. E. (1983). Journal of Biological Chem&try 258, 1752. Paszewski, A. and Grabski, J. (1974). Molecular and General Genetics 132, 307. Paszewski, A. and Grabski, J. (1975). Journal of Bacteriology 124, 893. Payne, G. M. and Payne, J. W. (1984). Biochemical Journal 218, 147. Pel, R. and Gottschal, J. C. (1989). FEMS Microbiology Ecology 62, 349. Penketh, P. G. and Klein, R. A. (1986). Molecular and Biochemical Parasitology 20, 111. Penketh, P. G., Kennedy, W. K., Patton, C. L. and Sortorelli, A. (1987). FEBS Letters 221, 427. Penninckx, M. J. and Jaspers, C. J. (1982). Bulletin de l‘fnstitut Pasteur (Paris) 80, 291. Penninckx, M. J. and Jaspers, C. J. (1985). Phytochernistry 24, 1913. Penninckx, M. J., Jaspers, C. J. and Wiame, J . M. (1980). European Journal of Biochemistry 104., 119. Penninckx, M. J., Jaspcrs, C. J . and Lcgrain, M. (1983). Journal of Biological Chemistry 258, 6030. Perham, R. N., Berry, A. and Scrutton, N. S . (1988). Biochemical Society Transactions 16, 84. Perry, A. C., Bhriain, N. N., Brown, N. L. and Rouch, D . A. (1991). Molecular Microbiology 5 , 163. Piccolomini, R., Di Ilio, C., Aceto, A , , Allocati, N., Faraone, A,, Cellini, L., Ravagnan, G. and Federici, G. (1989). Journal of General Microbiology 135, 3119. Pickett, C. B. and Lu, A. Y. (1989). Annual Review of Biochemistry 58, 743. Pinto, M. C., Mata, A. M. and Lopez-Barea, J. (1984). Archives of Biochemistry and Biophysics 228, 1. Pinto, M. C., Mata, A. M. and Lopez-Barea, J. (1985). European Journal of Biochemistry 151, 275. Price, N. W. and Pinhey, K. G . (1929). Journal of Biological Chemistry 84, 321. Prohaska, J. R. (1980). Biochimicu ef Biophysica Acra 611, 87. Pryor, W. A. (1962). “Mechanisms of Sulfur Reactions”. McGraw-Hill, New York. Racker, E. (1951). Journal of Biological Chemistry 190, 685. Ramaswamy, O., Pal, S . , Guha-Mukerjee, S . and Sojory, S . K. (1983). Biochemistry International 7, 307. Reed, D. J. (1986). Biochemical Pharmacology 35, 7. Reed, D. J., Brodie, A. E. and Meredith, M. J. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannevik, eds), p. 39. Raven Press, New York. Reichard, P. (1987). Biochemistry 26, 3245. Reichard, P. and Thelander, L. (1979). Annual Review of Biochemistry 48, 133. Rennenberg, H. (1982). Phytochemistry 21, 2771. Repetto, Y., Letelier, M. E., Aldunate, J. and Morello, A. (1987). Comparative Biochemistry and Physiology 87, 73. ~~
300
M. J. PENNINCKX AND M.
‘r. ELSKENS
Revesz, L. and Malaise, E. P. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 163. Raven Press, New York. Rhee, H. I., Murata, K. and Kimura, A. (1986). Biochemical and Biophysical Research Communications 141, 993. Rhee, H. I . , Sato, N., Murata, K. and Kimura, A. (1988). Agricultural and Biological Chemistry 52, 2243. Richman, P. and Meister, A. (1975). Journal of Biological Chemistry 250, 1422. Rizza, V . and Hu, A. S . L. (1973). Biochemical and Biophysical Research Communications 54, 168. Roberts, R. B., Abelson, P. H., Cowie, D . B., Bolton, E. T. and Britten, R. J. (1963). “Studies of Biosynthesis in Escherichia coli”, p. 424. Carnegie Institute, Washington, DC. Robins, R. and Davies, D . (1981a). Biochemical Journal 194, 63. Robins, R. and Davies, D . (1981b). Phytochemistry 20, 1497. Ropenga, J. S. and Lenfant, M. (1987). Applied Microbiology and Biotechnology 26, 117. Ropenga, J. S . , Yax, P. and Lenfant, M. (1989). Applied Microbiology and Biotechnology 31, 176. Rose, Z . B. and Racker, E . (1962). Journal of Biological Chemistry 237, 3279. Roseman, S . (1969). Journal of General Microbiology 54, 138. Russel, M. and Holmgren, A . (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 990. Sahm, H. (1977). Advances in Biochemical Engineering 6 , 77. Sandberg, V . A, , Kren, B., Fuchs, J. A. and Woodward, C. (1991). Biochemistry 30, 5475. Saxena, M., Mukerji, K. G . and Raj, H . G. (1988). Biochemical Journal 254, 567. Scherens, B., Dubois, E. and Messenguy, F. (1991). Yeast 7 , 185. Schwartz, R., Lucas, M. T., Escalante, L., Vasquez, G . and Sanchez, S . (1988). Journal of General Microbiology 134, 1117. Schutte, H., Flossdorf, J., Sahm, H. and Kula, M. R. (1976). European Journal of Biochemistry 62, 151. Scrutton, N. S . , Berry, A. and Perham, R. N. (1987). Biochemical.iournal245, 875. Sels, A. A. and Brygier, J. (1980). European Journal of Biochemistry 112, 283. Serrano, A,, Rivas, J. and Losada, M. (1984). Journal of Bacteriology 158, 317. Shames, S. L., Fairlamb, A. H., Cerami, A. and Walsh, C. T. (1986). Biochemistry 25,3519. Shames, S . L., Kimmel, B. E., Peoples, 0. P., Agabian, N. and Walsh, C. T. (1988). Biochemistry 27, 5014. Shimuzi, H., Araki, K., Shioya, S. and Suga, K. (1991). Biotechnology and Bioengineering 38, 196. Shishido, T. (1981). Agricultural and Biological Chemistry 45, 2951. Smith, C . V . , Hughes, H., Lauterburg, B. H. and Mitchell, J . R. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S . Orrenius, A. Holmgren and B. Mannervik, eds), p. 125, Raven Press, New York. Smith, J. and Shrift, A. (1978). Comparative Biochemistry and Physiology 63, 39. Soda, K. (1987). Methods in Enzymology 143, 453. Spallholz, J. E. (1987). Medical Hypotheses 23, 253. Stadtman, E. R . and Kornberg, A. (1953). Journal of Biological Chemistry 203, 47. Summer, K. H., Goeggelmann, W. and Greim, H. (1980). Mutation Research 70, 269. Sundquist, R. A. and Fahey, R. C . (1988). Journal of Bacteriology 170, 3459. Sundquist, R. A. and Fahey, R . C. (1989). Journal of Molecular Evolution 29, 429. Suzuki, H., Kumagai, H. and Tochikura, T. (1986). Journal of Bacteriology 168, 1325. Suzuki, H . , Kumagai, H., Echigo, T. and Tochikura, T. (1989). Journal of Bacteriology 171, 5169. Swerdlow, R. D. and Setlow, P. (1983). Journal of Bacteriology 153, 475. Sysoev, 0. V., Govorukhina, N. I. and Gruzman, M. (1990). Prikladnaia Biokhimiia I Mikrobiologiya 26, 456. Szajewski, R . P. and Whitesides, G. M. (1980). Journal of American Chemical Society 102, 2011.
GLUTATHIONE IN MICKO-ORGANISMS
301
Tabor, H. and Tabor, C. W. (1975). Journal of Biological Chemistry 250, 2648. Tabor, H. and Tabor, C. W. (1979). Biochemical and Biophysical Research Communications 41, 232. Tamaki, H. , Kumagai, H . and Tochikura, T. (1989). Journal of Bacteriology 171, 1173. Tate, S. S. and Meister, A. (1978). Proceedings of the National Academy of Sciences of the United States of America 72, 1985. Taylor, M. C., Chapman, C. J., Kelly, J. M., Fairlamb, A. H. and Miles, M. A. (1989). Biochemical Society Transactions 17, 579. Tel-Or, E . , Huflejt, M. and Packer, L. (1985). Biochemical and Biophysical Research Communications 132, 533. Thornalley, P. J. (1990). Biochemical Journal 269, 1. Tomita, K., Ito, M., Yano, T., Kumagai, H. and Tochikura, T. (1988). Agricultural and Biological Chemistry 52, 1159. Tomita, K., Yano, T., Kitagata, T . , Kumagai, H. and Tochikura, T. (1989). Agriculturaland Biological Chemistry 53, 1995. Tomizawa, H. H. (1962). Journal of Biological Chemistry 237, 3393. Tsai, C. S. and Godin, G. R . (1987). International Journal of Biochemistry 19, 337. Uotila, L. and Koivusalo, M. (1974). Journal of Biological Chemistry 249, 7653. Uotila, L. and Koivusalo, M. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 175. Raven Press, New York. van Bladeren, P. J. and van Omen, B. (1991). Pharmacology and Therapeutics 51, 35. van Dijken, J . P., Oostra-Demkes, G. J., Otto, R. and Harder, W. (1976). Archives of Microbiology 111, 77. van Dijken, J. P., Harder, W. and Quayle, J. R. (1981). In “Microbial Growth on C1 Compounds” (H. Dalton, ed.), pp. 199-201, Heyden, London. Van Huffel, C. and Sels, A. A. (1987). Archives Internationales de Physiologie etde Biochimie 95, 96. Verduyn, C., Giuseppin, M. L. F., Scheffers, W. A . andvanDijken, J. P. (1988). Appliedand Environmental Microbiology 54, 2086. Von Sonntag, C. (1987). “The Chemical Basis of Radiation Biology”. Taylor and Francis, London. Vos, R. M. and van Bladeren, P. J. (1990). Chemical and Biological Interactions 75, 241. Wackett, L. P. and Gibson, P. T. (1982). Biochemical Journal 205, 117. Watanabe, K., Murata, K. and Kimura, A. (1986). Agriculturaland Biological Chemistry 50, 1925. Waxman, D. J. (1990). Cancer Research 50, 6449. Wendel, A. (1980). In “Enzymic Basis of Detoxication” (W. B. Jacoby, J. R. Bend and J. Caldwell, eds), p. 333. Academic Press, New York. Wiame, J . M., Grenson,M. andArst,H. N., Jr (1985).Advancesin Microbial Physiology26,2. Wiebers, J. L. and Garner, H. R. (1967). Journal of Biological Chemistry 242, 12. Wiemken, A. (1980). In “Cell Compartmentation and Metabolic Channeling”, p. 225. ElsevierINorth Holland Biomedical Press, Amsterdam. Williams, C. H. (1976). In “The Enzymes” (P. D. Boyer, ed.), vol. 13, pp. 89-132. Academic Press, New York. Williams, C. H. and Arscott, L. D . (1971). Methods in Enzymology 17B, 503. Yamada, Y., Tani, K. and Kamikara, T. (1984). Journal of General Microbiology 130,3275. Yamagata, S. (1980). Journal of Biochemistry 88, 1419. Yan, N. and Meister, A. (1990). Journal of Biological Chemistry 265, 1588. Yawetz, Z . A. and Agosin, M. (1981). Comparative Biochemistry and Physiology 68, 237. Yokota, A., Shigeoka, S . , Onishi, T. and Kitaoka, S. (1988). Plant Physiology 86, 649. Zhang, Y., Hempelmann, E . and Schirmer, R . H. (1987). Biochemical Pharmacology 37,855. Ziegler, D. M., Poulsen, L. L. and York, B. M. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 205. Raven Press, New York.
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Author Index
Numbers in bold refer to the pages on which references are listed at the end of each chapter A
Abarbanel, R. M., 226, 235 Abbas, H. K . , 104, 145 Abdrashitova, S. A., 271, 291 Abe, K . , 99, 134, 139, 204, 212, 221, 228, 235 Abelson, J. N., 25, 41, 59 Abelson, P. H . , 286, 300 Abosch, A , , 272, 294 Abraham, M., 48, 49, 64, 65 Aeeto, A , , 281, 293, 299 Achstetter, T . , 88, 91, 94, 134, 136 Adair, W. L., 91, 135 Adams, D. J . , 111, 115, 116, 117, 121, 122, 125, 126, 130, 135, 142, 145 Adams, L. F., 26, 62 Adams, W. B . , 241, 293 Adar, Y. Y . , 48, 58 Adebodun, F., 126, 127, 136, 137 Agabian, N . , 275, 300 Agosin, M., 272, 282, 301 Aidarkhanov, B. B . , 271, 291 Ainsworth, A. M . , 149, 151, 194 Ainsworth, G. C., 147, 194 Aisaka, K., 242, 272, 291 Akada, R., 99, loo,, 134, 141 Alamashanu, S., 34, 58 Aldunate, J., 251, 283, 298, 299 Aley, S. B . , 242, 272, 293, 298 Alie, M . , 191, 194 Alin, P., 281, 297 Allan, M., 180, 201 Allen, L., 251, 291
Allocati, N., 281, 293, 299 Almar, M. M . , 282, 293 Alver’Yanov, A. A., 272, 298 Ammer, H., 91, 142 Amy, P. S., 229, 231, 235 Anderegg, R . J . , 88, 134 Anders, M. W . , 240, 284, 291, 298 Anderson, A., 285, 292 Anderson, J. B . , 192, 195, 196, 199 Anderson, M. E., 247, 250, 251, 256, 257, 295, 298 Anderson, S. A , , 212, 235 Ando, K., 282, 291 Andou, N., 260, 299 Andreesen, J. R . , 266, 298 Andrews, S. C., 28, 58 Anfinsen, C. B . , 263, 291, 295 Anhalt, L. M . , 8, 25, 43, 61 Anraku, Y . , 94, 142, 260, 296 Ansorge, S . , 264, 291 Anthony, C., 288, 291 Apontoweil, P., 255, 256, 289, 291 Appeltauer, U., 91, 92, 134, 142 Applegate, B . , 5, 63 Arai, S . , 204, 212, 221, 222, 228, 232, 235, 237 Araki, K., 254, 300 Aranachalam, T., 75, 134, 140 Area, P . , 281, 284, 292 Arellano, F., 224, 227, 236, 237 Arias, I . M . , 240, 292 Arita, M., 47, 64 Arni, K., 89, 90, 95, 141 Arnold, E., 92, 142
304
AUTHOR INDEX
Amy, D. C., 204, 212, 222, 229, 230, 231, 235, 236 Aronson, A., 248, 260, 292 Arrick, B., 242, 293 Arscott, L. D . , 274, 301 Arsenault, G. P . , 75, 76, 134, 140 Arst, H . N . Jr 258, 301 Aruoma, 0. I . , 269, 295 Asai, Y., 47, 64 Asgeirsdottir, S. A., 169, 173, 175,177, 183, 200 Ashby, A. M., 103, 143 Ashworth, E. N . , 212, 235 Astorga, A., 23, 60 Au-Young, A., 91, 135 Axelsson, K . , 263, 297 Azuma, Y., 98, 100, 140, 141 B Baars, A., 257, 298 Baba, M., 49, 60, 90, 93, 94, 134 Baba, N., 90, 93, 94, 134 Babel, W., 287, 292 B,abson, J. R . , 280, 292 Bacchi, C. J . , 245, 294 Bacher, A., 11, 12, 22, 27, 29, 65, 66 Badham, E. R . , 183, 194 Baffi, R. A., 88, 134 Bainton, N. J . , 37, 58 Baker, L. S., 229, 235 Balaguer, P., 5, 58 Balandrano, D . , 110, 111, 135 Balch, W. E., 242, 294 Baldwin, T. O., 5 , 7, 8, 11, 14, 16, 17, 18, 23, 25, 26, 27, 28, 30, 31, 33, 34, 39, 40, 41, 43, 45, 48, 52, 58, 59, 61, 62, 63, 65, 66, 67 Ballou, C. E., 90, 92, 93, 139 Balny, C., 12, 61 Banbury, G. H . , 86, 134 Band, P., 120, 135 Bang, S . , 2, 59 Banno, I., 95, 96, 137, 143 Bannuett, F., 161, 199 Barak, E., 283, 292 Barak, M., 35, 41, 59 Barchet, W. R . , 229, 230, 236 Barksdale, A. W . , 74, 75, 76, 77, 134, 137, 140
Barlow, A. J. E., 105, 111, 136 Barnes, D . , 88, 137 Baron, E. S., 263, 292 Baross, J., 49, 59 Barra, D., 281, 293 Barrett, V., 191, 194 Barrow, S. E . , 76, 134 Bartlet, I., 12, 14, 64 Bartnicki-Garcia, S., 98, 143 Bassi, A. S . , 204, 230, 231, 232, 236 Baudoni, R. J . , 98, 134, 137, 138 Bauer, V. J . , 79, 143 Baumann, L., 2, 41, 43, 48, 49, 59, 67 Baumann, P . , 2, 41, 43, 48, 49, 59, 67 Baumstark, A. L . , 13, 59 Beach, D., 160, 194 Beaman, B., 49, 67 Bean, G. A , , 80, 141 Beato, M., 123, 134 Becker, J. M . , 87, 88, 89, 95, 120, 134, 135, 137, 140, 142, 143, 145 Becker, K., 280, 292 Becvar, J. E . , 8, 11, 12, 58, 59, 62 Beechling, J. R . , 151, 194 Beelman, R. B., 190, 201 Bcever, R. E . , 176, 194 Belas, R., 2.5, 41, 59 Bell, A. A., 179, 194 Bell, J., 34-5, 60 Bellofatto, V., 245, 292 Beman, J., 63 Ben Bissat, A , , 289, 292 Benohr, H., 240, 294 Berch, S., 191, 197 Berends, T., 41, 58 Berends, W. P . , 255, 256, 289, 291 Berfold, T., 204, 210, 237 Berge, J. B., 284, 294 Bergemann, J., 201, 202 Bergfeld, R., 187, 197 Bernhagen, J., 160, 195 Berriman, J., 151, 194 Berry, A., 274, 276, 299, 300 Berry, D., 94, 145 Bettiol, M. F . , 78, 138 Betz, R., 87, 88, 95, 134, 135, 144 Bhalerao, U . T., 72, 135, 142 Bhriain, N. N . , 275, 299 Biemann, K., 75, 134 Binniger, D. M., 191, 194
AUTHOR INDEX
Birkinshaw, J. H., 184, 194 Birnbaumer, L., 125, 132, 133, 138 Bironaite, D. A . , 280, 292 Bistis, G. N . , 100, 101, 135, 151, 194 Black, S . , 264, 298 Blackburn, P., 244, 245, 294 Blair, L., 88, 139 Blakemore, R., 242, 294 Blakeslee, A . F . , 81, 135 Blinov, V., 26, 27, 31, 61 Blisset, S. J . , 34, 59 Blobel, G., 88, 145 Blum, L. J . , 5, 61 Boctor, A. M . , 120, 135 Boeke, J . D . , 92, 144 Boettcher, K. J . , 37, 38, 39, 49, 50, 59 Bogacki, I. B., 6, 60 Bognar, A . , 24, 59 Bohley, P., 264, 291 Boivin, R., 34, 59 Bolker, M., 201, 202 Bolton, E. T., 286, 300 Bonen, L . , 242, 294 Bonsignore, A . , 276, 292 Booth, I. R . , 34, 65 Bothner-By, A. A. , 82, 144 Bottema, C. D. K . , 120, 141 Bottino, N. R . , 271, 272, 279, 294 Bourne, H. R . , 94, 136 Boussioux, A.-M., 5, 58 Bouter, S . , 255, 258, 292 Boveris, A . , 269, 292 Bower, L. A., 81, 143 Bowers, B., 91, 143 Boyed, D. H . , 158, 198 Boyer, T. D . , 281, 292 Boylan, M., 6, 16,22,25,26,27,28,30, 31, 34, 41, 42, 58, 59, 63, 64, 66 Boyland, E., 281, 292 Boyle, R., 4, 5, 59 Braenden, C. I . , 268, 293 Brake, A. J 88, 94, 135, 138, 139 Bramley,T. A . , 111, 116,121, 122, 125, 126, 130, 131, 135, 145 Brana, A. F . , 284, 292 Branch, S. K . , 151, 194 Branden, C. I . , 266, 267, 292, 296 Brandhorst, B. P . , 78, 137 Branton, D., 6, 41, 65 Brasier, A., 94, 136
305
Brasier, C. M., 81, 135 Brault, G., 248, 294 Brawley, V., 91, 143 Breittmayer, V., 284, 294 Bremer, W., 169, 195 Brenner, C., 94, 135 Bret, J., 181, 197 Britten, R. J . , 286, 300 Broda, P., 149, 180, 190, 194 Brodie, A. E., 261, 299 Brody, S., 293 Broek, D . , 89, 142 Bromberg, S. K . , 154, 175, 194 Brown, J. P . , 39, 61 Brown, N. L., 275, 299 Brown, R. W., 260, 292 Brown, W. C., 241, 293 Brown, W. M., 26, 61 Broxholme, S. J., 151, 194 Bruinenberg, P. M . , 276, 292 Brunt, S. A., 78, 79, 135 Brus, L. E., 290, 292 Bryant, T., 49, 67 Brygier, J., 273, 300 Bucciarelli, T., 281, 293 Buchanan, B. B., 263, 292 Buck, J. D . , 49, 61 Bucking-Throm, E., 92, 135 Buetow, D. E., 105, 135 Buissieres, J . , 248, 294 Bulawa, C. E., 91, 135 Bull, A. T . , 149, 153, 194 Bu’Lock, J. D . , 70, 71, 82, 83, 84, 135, 179, 194, 195 Bunch, T. A , , 41, 58 Burgeff, H., 81, 135 Burgess, R. R . , 214, 235 Burk, R. F . , 270, 297 Burke, M. J . , 210, 225, 226, 235 Burkholder, A. C., 89,95,135,138,139 Burlage, R . S . , 5, 59, 63 Burlingame, A. L . , 37, 42, 60 Burnett, J. H . , 149, 195 Burshell, A., 115, 119, 120, 124, 135, 136 Burton, G., 264, 293 Bushbacher, R. M., 242, 244, 275, 293 Bussey, H., 88, 135, 136 Busto, F., 276, 298 Butler, R. D . , 148,149,184,185,186,198
306
AUTHOR INDFX
Butt, T. R., 110, 125, 130, 137 Buttner, M. P., 229, 231, 235 Bycroft, B. W., 37, 58 Byers, D. M., 19, 22, 24, 28, 41, 42, 59, 64, 67 C
Cabbat. F. S., 279, 295 Cabib, E., 91, 92, 143, 144 Cabib. J., 91, 135 Caccuri, A . M., 293 Cain, R. B., 180, 191, 195 Caldwell, D. R . , 204, 222, 236 Caldwell, G. A., 88, 89, 140, 145 Callahan, S. M., 45 Camarero, V. C. P., 60 Cambillau, C., 268, 293 Campbell., A. K., 5, 59, 66 Cano, L. E., 107, 115, 117, 124, 129, 131, 143 Cano, R. J., 101, 144 Cao, J.-G., 37, 42, 59 Cao, Y . , 49, 67 Capage, D. A., 83, 144 Capek, A . , 105, 110, 111, 135 Caplan, S.. 88, 135 Caple, G . , 231. 237 Cappelaro, C., 90, 138 Carattoli, A , , 184, 198 Carey, L. M., 19, 59 Carlile, M. J., 73, 74, 135 Carlquist, M., 267, 295 Carlson, G. L., 75, 79, 141, 143 Carlstedt-Duke. J., 124, 145 Carmi, 0. A., 34, 59 Caroll, P. J., 290, 292 Caron, M. G . , 127, 139 Carr, S. A., 88, 135 Carter, B. L. A., 88, 137 Cartledge, J. L., 81, 135 Casalone, E., 273, 282, 283, 292, 294 Casas-Campillo, C., 110, 111, 135 Casperson, G . F., 94, 136 Casselton, L. A , , 157, 158, 159, 160, 191, 194, 195, 196, 197, 198, 201, 202 Castle, A . J . , 192, 195, 199 Catanzaro, A , , 128, 136 Catt, K. J., 127, 140 Cedergren, R., 43, 61
Cejek, E., 95, 136 Cellini. L,. , 281, 293, 299 Cenas, N. K . , 280, 292 Cerami, A , , 244,245,272,275,280,294, 295, 300 Cerda-Olmedo, E., 84, 137 Cha, M., 112, 121, 126, 140 Chadha, R. K., 76, 141 Chait, B. T., 244, 245, 294 Chakrabarty, A . M., 39, 59 Chalifour, F.-P., 34, 58 Challcn, H. P., 192, 195 Chambers, J. A . A , , 184, 199 Chambon, P., 123, 124, 140, 145 Champe, S. P., 103, 136, 140, 151, 195 Chan, K., 111, 136 Chance, B., 269, 292 Chandramohan, D., 49, 65 Chang, A , , 103, 136 Chang, C. N., 88, 144 Chang, H. S., 81, 136 Chang, S. T., 190, 195 Chang-Chien, M., 204, 222, 236 Chanter, D. O., 150, 195 Chapman, C. J., 275, 301 Chapman, D., 231, 237 Chase, J. W., 267, 268, 297 Chasseaud, L. F., 281, 282, 292 Chater, K., 34, 66 Chattaway, F. W., 105, 111, 136 Chen, E. Y., 88, 144 Chen, K. N., 242, 294 Chen, L. H., 7, 16, 17, 58, 59 Chen, P.-F., 44, 59 Cheng, H., 248, 260, 292 Cheng, T. M., 178, 196 Cherapak, C. N., 126, 137 Cherest, H., 260, 262, 292 Chessin, H., 207, 237 Chiba, S., 282, 291 Chibata, I., 255, 286, 298 Chihara, M., 250, 296 Chimera, J. A , , 34, 66 Chlabra, S. R., 37, 58 Chlumsky, L. J., 16, 17, 58 Cho, K.-W., 7, 13, 14, 25, 26, 43, 46, 47, 59, 60, 67 Choi, S. H., 40, 59 Chou, P. Y., 226, 235 Christoff, M., 277, 297
307
ACJTWOK INIIFX
Christophervon, R. I., 280, 292 Chung, Y. C., 274, 292 Chvatchko, Y., 89, 136 Clark, A. J., 256, 292 Clark, K. L., 90, 136 Claus. R., 132, 136 Clay, F. J., 92, 140 Claydon, N., 180, 201 Clcland, R. E., 187, 195 Clemons,K.V., 110,111, 115,117,118, 119. 124, 129, 130, 131, 136, 143, 144 Cline, T. W., 13, 14, 17, 58, 59 Close, T. J . , 5 , 34, 65 Clutterbuck, A. J . , 179, 195 Coblenz, A , , 255, 258, 290, 294 Cochrum, L., 26, 43, 59, 62 Coffey, J . J., 35, 59 Cohcn, A., 5, 65 Cohen, E., 284, 292 Cohen, F. E., 226, 235 Cohen, G. M., 240, 279, 292, 295 Cohn, D. H., 25, 26, 41, 58, 59, 61 Cole, G. T., 176, 195 Colepicolo. P., 7, 13, 14, 43, 46, 47, 59, 60 Colin, D., 254, 259, 298 Colowick, S., 240, 292 Colwell, R. R., 2, 37, 48, 49, 50, 61,63, 64 Constantinidou, H. A , , 229, 235, 236 Contcr, A , , 271, 293 Cooper, R. A , , 285, 286, 292, 296 Cooper-Palomar, J. L , 77, 136 Cormier, M. J., 8, 60 Corotto, L. V., 204, 211, 212, 213, 214, 220,221,225,226,227,228,233,235, 237 Costa, S . , 34-5, 60 Coulet, P. R., 5 , 61 Coulson, C. J., 110, 111, 129, 139 Countryman, C., 27, 40, 45, 59 Cowden, W. B., 280, 292 Cowie, D. B., 286, 300 Cox, J., 186, 195 Cox, R. B., 289, 292 Crabb, J,. W., 88, 135 Cragg, L., 30, 63 Craig, G., 78, 138, 185, 186, 195 Creighton, T. E., 263, 292 Cresnar, B., 123, 138
Cristenscn, M., 231, 237 Crook, E. M., 240, 292 Cross, F., 87, 90. 92, 136 Cross, G. A , , 245, 292 Croutc, F., 271, 293 Csonka, L. N., 256, 292 Cuany, A , , 284, 294 D
Dadok, J., 82, 144 Dahl, M., 161, 199 Dahl, R. H., 256, 298 Dahlbeck, D., 21 I , 212, 229, 236 Dameron, C. T., 290, 292 Daniclson, U. H., 281, 297 Danilov, V. S . , 13, 62 Danson, M. J . , 275, 293 Daubner, S. C., 7,23,25,27,31,59,60 Davey, J . , 96, 136 Davies, D., 253, 259 Davis, J., 49, 60 Davis, N. G., 90, 136 Day, P. R., 149, 158, 195 Dayhoff, M. O., 214, 237 Dayoff, M. D., 127, 138 Dean, R. T., 269, 293 Deen, R. A , , 199 dc Arriaga, D., 274, 276, 298 De Flora, A , , 276, 292 dc Haan, A., 192, 196 Deininger, C. A , , 209, 211, 212, 220, 222, 223, 226, 229, 233, 235, 237 de Jonckhheere, J . F., 282, 293 Dekant, W., 240, 284, 291, 298 dc Kniff, P., 257, 298 Delong, E. F., 25, 42, 60 DeLuca, M., 11, 24, 28, 62 Dempsey, G. P., 176, 194 Denycr, S., 5 , 34, 62, 66 Deretic, V., 39, 60 dc Rey-Pahlade, J., 240, 293 de Robichon-Szulmajster, H., 260, 262, 292, 297 De Rousset-Hall, A., 186, 196 Dcsmarchelier, P. M., 49, 50, 60 deSmet, G.. 231, 236 Devereux, J., 214, 235 Dcvine, J. H., 16, 17,25,26,27.30,31, 33, 40, 43, 45, 48, 58, 60, 66
308
AUTHOR INDEX
DeVries, A. L., 223, 236 deVries, 0. M. H., 154, 161, 162, 165, 166,169,173,175,176,177,179, 183, 185, 195, 200 de Vries, S. C., 167, 195 Dhephlagnon, S., 16, 34, 58 Di Illio, C . , 273,281,282,292,293,299 DiCera, E., 223, 236 Dick, S . , 181, 197 Dickinson, K., 111, 116, 125, 126, 130, 145 Dierickx, P. J., 281, 282, 293 Dietrichs, D., 266, 298 Dietzel, C., 95, 136 Dignard, D., 88, 136 Digrazia, P. M., 5, 63 Dikshit, R., 39, 60 Dilmore, L., 49, 60 Dingernanse, M. A., 191, 198 Dion, P., 34, 58 Distel, D. L., 51, 62 Dixon, R. K., 191, 194 Dmochowska, A., 88, 136 Do, Y. S . , 115, 119, 120, 124, 135, 136, 137 Doel, S., 88, 137 Dohlrnan, H. G., 127, 139 Dohn, D. R., 284, 291 Dolan, K. M., 40, 48, 60 Donchenko, A. P., 26, 27, 31, 62 Dons, H., 161, 201 Dons, J. J. M., 166, 167, 169, 179, 195, 200 Douglas, K. T., 268, 285, 293, 295 Douville, E., 34-5, 60 Douzou, P., 12, 62 Dovlen, J. A , , 290, 296 Drinkard, L. C., 81, 136 Driver, C. H., 101, 136 Drotar, A., 264, 293 Drurnmond, B. J., 191, 198 Drutz, D. J., 107, 110, 113, 115, 116, 117, 118,125,128, 130, 131, 136, 137, 142 Dry, M. A., 24, 49, 65 Dubois, E., 253, 266, 293, 300 Dudani, A. K., 287, 293 Duddles, N. D., 181, 197 Dudley, K., 180, 201 Dulic, V., 89, 136
Dunbar, P., 5, 63 Dunlap, P. V., 4, 5, 6, 36, 38, 39, 40, 44,45,46,47,49,53,59,60,61,62,63 Dunn, D. K., 6, 60 Duntze, W., 87,88,92,95,135,136,144 Dupouy, D., 271, 293 Duran, A , , 91, 92, 143, 144 Durand, R., 181, 183, 195, 197, 199 Durrant, A. J., 180, 191, 195 Dutsch, G. A., 186, 195 Dyer, T. A , , 242, 294 Dzhavakhiya, V. G., 272, 298 E
Eaton, J. W., 272, 294 Eberhard, A , , 13,35,36, 37,38, 39,41, 42, 43, 60, 62, 64 Eberhard, C., 37, 38, 42, 60 Eberle, J., 184, 199 Ebert-Jung, A., 255, 258, 290, 294 Echigo, T., 251, 300 Eckardt, F., 255, 257, 258, 296 Eckstein, J., 7, 13, 14, 60, 63 Economou, A , , 157, 158, 159, 195, 196 Eddy, A. A , , 259, 293 Edelstein, L., 150, 195 Edgington, L. V., 277, 283, 292, 293 Edwards, J. A , , 75, 135, 136, 137 Egel, R., 96, 97, 136, 137, 139 Eger-Hummel, G., 181, 195 Egerton, M., 89, 136 Egidy, G., 111, 126, 130, 142 Egli, T., 289, 293 Egyud, L. G., 287, 293 Eichler, F., 260, 262, 292 Eilam, Y., 93, 94, 142 Eilers, F. I., 185, 186, 195 Eisenthal, R., 275, 293 Eklund, H., 267, 268, 292, 293 Elfarra, A. A , , 284, 291 Elguindni, I., 89, 136 Elhai, J., 5, 34, 60, 66 Elhiti, M. M. Y., 148, 149, 184, 185, 186, 198 Eliaou, J.-F., 5, 58 Eliasson, R., 28, 61 Elliot, T. J., 191, 192, 195 Elliott, C. G., 80, 81, 136, 137 Elliott, K., 240, 293
AUTHOR INDEX
Ellis, E . A., 76, 78, 141 Ellison, A., 34, 62 Elskens, M. T., 253,257,260,261,262, 277, 278, 293 El-Zayat, A. A. E., 103, 136, 140, 151, 195 Emori, Y., 204, 212, 221, 228, 235 Emter, O., 88, 136 Enders, B., 275, 296 Engebrecht, J., 5,26,27, 29,30, 34,37, 38, 39, 40, 42, 45, 60, 61, 66 Engelberg, D., 133, 136 Epstein, C. J., 263, 295 Eriotou-Bargiota, E . , 88, 145 Ermler, U., 275, 293 Ero, L., 77, 141 Erwin, D. C., 81, 143 Escalante, L., 254, 300 Escher, A., 17, 34, 35, 61 Esser, K., 156, 158, 170, 171, 195, 199 Esteve, C., 49, 65 Esumi, Y., 95, 96, 143 Evans, E. A., 88, 145 Evans, J. F., 31, 64 Evans, R. G., 279, 293 Ewenson, A , , 88, 145 Eyer, P., 244, 293 Eymers, J. G., 6, 61 F Fahey, R. C., 241, 242, 244, 263, 264, 271, 274, 275, 293, 298, 300 Fairfield, A. S., 272, 294 Fairlamb, A. H., 242, 244,, 245, 272, 275, 280, 292, 294, 295, 296, 300, 301 Falkow, S., 39, 64 Fall, R., 210, 221, 222, 223, 227, 229, 235,236,242,264,271,281,282,293, 297, 299 Faraone, A., 281, 293, 299 Farghaly, A., 47, 61 Farmer, J. J., 2, 5, 48, 61 Fasman, G. D., 226, 235 Fawell, S. E . , 112, 121, 126, 127, 136, 140 Federici, G., 273, 281, 282, 292, 293, 299 Fegeler, K., 111, 141
309
Feldman, D., 107, 110, 111, 112, 113, 115,116, 117,118,119,120, 129,130, 131, 135, 136, 137, 140, 141, 143, 144 Feldman, K. A., 49, 61 Felton, F. G., 105, 141 Ferri, S. R., 8, 19, 25, 26, 27, 42, 52, 61, 64, 66 Fevre, M., 181, 197 Fiechter, A , , 254-5, 289, 293, 294 Field, J., 89, 142 Fields, S., 87, 137 Fijii, M., 246, 298 Fincham, J. R. S., 149, 195 Findlay, J. B., 28, 58 Findlay, W. P. K., 184, 194 Fink, G. R., 92, 143, 144, 254, 260, 296 Finney, D. J., 208, 235 Fitzgerald, J. M., 49, 61 Fitzgerald, K. J., 34, 63 Flawia, M. M., 126, 137 Flegcl, T. W., 99, 137 Flegg, P. B., 190, 191, 195 Fletcher, N. H., 204, 206, 207, 226, 235 Fletterick, R. J., 226, 235 FlohB, L., 240, 263, 270, 294 Flossdorf, J., 289, 300 Floyd, A. J., 151, 194 Fontecave, M., 28, 61 Foran, D. R., 26, 61 Ford, S., 92, 137 Foster, L. M., 165, 199 Fournier, D., 284, 294 Fox, G. E., 242, 294 Frackman, S., 8, 25, 43, 61 Franks, F., 204, 206, 207, 226, 235 Frederick, R., 224, 233, 236 Freedman, R. B., 244, 263, 264, 294 Fresh, R. W., 231, 237 Frey,C. L., 110,115,116,117,125,130, 137, 142 Fridovitch, I., 269, 294 Fried, J. H., 75, 136 Fried, J. N., 75, 136 Friedman, R. B., 240, 292 Friedmann, K. L., 96, 137 Friedrich, W. F., 38, 39, 44, 61 Friend, J., 103, 143 Froeliger, E., 191, 198 Fry, S. C., 179, 195
3 10
AUTHOR INDEX
Fuchs, J. A., 242, 255, 256, 265, 267, 268, 269, 294, 296, 300 Fuerst, J. A., 48, 65 Fuhrmann, G. F., 254-5, 294 Fujii, S., 23, 63 Fujii, T., 190, 196 Fujimura. H., 95, 96, 137, 143, 145 Fujino, M., 95, 98, 99, 137, 139, 143 Fujino, Y., 181. 197 Fujiyama, A., 89, 137 Fukasawa, S., 49, 61 Fukuda, Y., 285, 298 Fukui, S., 94,98, 99, 100, 135, 138, 139, 140, 141, 144, 288, 294 Fukui, Y., 96, 137 Fuller, M. S., 71, 142 Fuller, R. S., 88, 137 Furukawa, S., 183, 197 Furuya, M., 183, 195 Fushiki, T., 251, 296
Giddings,T. H., 210,221,222,236,260, 294 Gics, D., 221, 223, 236 Gigot, D., 245, 248, 253, 254, 259, 296 Gill, S. J., 223, 236 Gillespie, E., 287, 294 Gillissen, B. 207, 202 Giovannelli, J., 260, 294 Giroux, S., 43, 61 Gitelson, I. I., 25, 26, 27, 31, 61 Giuseppin, M. L. F., 273, 301 Glacscr, H., 255, 258, 290, 294 Glass, N. L., 160, 197, 202 Gleason, F. K., 266, 295 Gledhill, L. 37, 58 Glen, B., 81, 136 Go, S. J., 192, 196 Godin, G. R., 275, 301 Goegglemann, W., 284, 300 Gold, M. H., 149, 178, 180, 190, 191, 194, 196 G Goldberg, I., 289, 292 Goldberger, R. F., 263, 295 Galarza, A.. 110, 111, 135 Gooday, G. W., 70, 71, 76, 81, 82, 83, Galiazzo, F., 273, 294 84, 86, 90, 137, 139, 151, 186, 187, Gall, A. M., 80, 137 190, 196 Gallori, E., 283, 294 Goodenough, P. W., 180, 200 Galun, E., 184, 195 Goodey, A. R. , 88, 137 Galvagno, M. A , , 111, 126, 130, 142 Goodman, L. E., 89, 137 Galyan, E. L., 204, 222, 231, 236, 237 Gordon, A . S., 7, 63 Gambello, M. J . , 40, 61 Gordon, S. A , , 183, 198 Gambiel, A., 284, 292 Gorrod, J. W., 277, 295 Gancedo, C., 262. 294 Goto, T., 290, 295 Gangerly, N . K., 250. 251, 297 Gottgens, B., 160, 198, 201, 202 Garcia, P., 292 Gottschal, J . C., 264, 299 Garner, A, , 269, 293 Govind, N. S., 84, 137 Garncr, H. R., 260, 301 Govindarajan, A. G., 210, 221, 222, Garnier, J., 226, 235 223, 224, 227, 233, 235, 236 Gast, R., 13, 61 Govorukhina, N. I., 281, 300 Gaubatz, S., 160, 197, 201, 202 Gow, N. A. R . , 76, 137 Gauthier, M. J., 284, 294 Grabski, J., 260, 299 Gautier, S. M., 5, 61 Graham, A . F., 6,22,25,26,27,28,29, Gennity, J. M., 271, 272, 279, 294 30, 31, 34, 41, 42, 58, 64, 67 Georgopoulos, S. G., 64, 101, 135 Granot, D., 172, 197 Ghisla, S., 7,13,14,60,63,274,276,294 Grant, G. G., 5, 8, 9, 64 Giammanco, G., 248, 294 Gray, K. M., 25, 39, 61 Giasson, L., 159, 160, 196, 200201, 202 Gray, W. R., 245, 290, 297 Gibson, J., 242, 294 Greaves, A. M., 75, 141 Gibson, P. T., 284, 301 Green, D. M., 88, 137 Gibson, Q. €I., 11, 12, 14, 61, 62 Green, J. M., 75, 135
AIJTHOR I N D E X
Green, R., 88, 135 Green, R. L., 204, 212, 221, 225, 228, 231, 233, 235, 237 Green, S., 123, 124, 145 Grecnberg, E. P., 4, 5 , 25, 37, 38, 39, 40, 42, 44, 45, 49, 50, 59, 60, 61, 62, 64, 65, 66 Greenfield, N. J., 126, 127, 136, 137 Greer, D. L., 129, 143 Greer, S., 275, 295 Gregoridas, A. 201, 202 Gregory, P. H., 151, 196 Greim, H., 284, 300 Grenson, M., 253, 258, 259, 293, 295, 301 Gribskov, M., 214, 235 Griffith, 0. W., 247, 250, 251, 261, 295 Grill, E., 245, 290, 295 Grogan, D. W., 46, 61 Groner, B., 78, 137 Grossbard, M. L,., 37, 42, 61 Grossman, A , , 120, 135, 137 Grossowicz, N . , 248, 298 Grover, S., 131, 137 Gruen, H. E., 151, 154, 185, 186, 196, 197, 200 Gruzman, M., 281, 300 Guerrero, M. A , , 46, 61 Guest, J. R., 28, 58 Guha-Mukerjee, S., 287, 299 Guijarro, J., 34, 66 Gull, K., 185, 186, 195 Gunsalus-Miguel, A., 17, 61 Gunzlcr, W. A , , 263, 270, 294 Guo, L. Y., 81, 137 Gupta, R., 242, 294 Gupta, S. C., 5 , 6, 41, 46, 50, 61, 64 Gushima, H., 250, 295 Gustafson, G. D., 34, 63 Gustafsson, J.-A., 124, 145 Guthenberg, C., 281, 297 Guttcridgc, J. M., 269, 270, 295 Guttman, S. M., 80, 138 Gwynne, D. I., 78, 138 H Haagen-Smit, A. G., 74, 142 Haas, J. E., 230, 237 Habig, W. H., 282, 296
31 1
Hada, H. S., 42, 61 Hadar, R., 34, 37, 42, 58 Hacberli, P., 214, 235 Haffner, L., 185, 196 Hagag, N . , 44, 59 Hagemeier, H. H., 111 , 141 Hagen. D. C., 95, 138 Haggard-Ljungquist, E., 28, 66 Hall, S., 275, 293 Haller, B., 255, 256, 265, 267, 268, 294 Halliwell, B., 269, 270, 279, 295 Hamada, Y., 190, 196 Hamlett, N. V., 50, 65 Hammond, J . B. W., 186, 196, 200 Hanifa Moursi, S. A , , 105, 138 Hanson, F. E., 35, 36, 63 Hanson, H., 264, 291 Harden, A., 285, 295 Harder, W., 288, 289, 293, 301 Harding, K. E., 72, 138, 142 Hardisson, C., 281, 284, 292 Harington, C. R., 240, 295 Harley, J. L., 191, 196 Harmsen, M. C., 192, 196 Harris. R. E., 128, 138 Harrison, P. M. 28, 58 Harrison, T. L., 83, 144 Hartig, A , , 89, 138 Hartwell, I,. H., 87, 89, 90, 92, 94, 95, 135, 136, 138, 139 Harvey, D., 120, 137 Harvey, E. N., 47, 49, 61 Harvey, R. P., 128, 138 Hasegawa, M., 245, 295 Hasegawa, S., 105, 123, 144 Hasegawa, Y., 212, 235 Hasek, J., 94, 138 Hasel, K. W., 31, 64 Hasclbcck, A., 92, 142 Hasting, J. W., 2, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 17, 24, 35, 36, 37, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 58, 59, 60, 61, 63, 64, 65, 66, 67 Hataro, T., 98, 141 Hatomo, T., 98, 141 Hauser, K., 90, 91, 138 Havrankova, J., 131, 143 Hawkins, H. C., 264, 294 Hayashi, H., 181, 198 Hayashi, Y., 290, 295
312
AUTHOR INDEX
Hayes, W. A . , 190, 191, 195, 196 Haygood, M. G . , 25,26, 45,46, 49, 51, 52, 61, 64, 66 Haylock, R. W., 158, 159, 196 Heckel, R. C . , 25, 26, 27, 30, 31, 33, 40, 43, 48, 58 Hegnauer, H., 179, 198 Heijne, G. V., 213, 235 Hei,kkila, R. E., 279, 295 Hempelmann, E., 280, 301 Henderson, G. B., 244, 245, 272, 275, 280, 292, 294, 295, 296 Hendrix, J. W., 80, 138 Henikoff, S . , 39, 62 Hennaut, C., 259, 295 Henning, D., 88, 136 Henry, J. P., 6, 47, 62 Herman, C. A , , 78, 138 Herman, R. P., 78, 138 Herndon, M. E., 16, 67 Herring, P. J . , 51, 62 Hershkowitz, E., 241, 263, 296 Herskowitz, I., 87, 88, 89, 90, 94, 95, 132,137, 139,140, 141,142, 161, 172, 197, 199 Hespell, R. B., 242, 294 Hewick, D. S., 278, 295 Hickman-Brenner, F. W., 2, 5, 48, 61 Hicks, J., 160, 196 Higgins, C. F . , 33, 34, 62, 65 Higuchi, T . , 180, 197 Hill, P. J. 37, 58 Hillson, D. A . , 244, 294 Hilmen, M., 25, 41, 59 Hintz, W. E. A., 192, 196 Hiraga, K., 94, 138 Hirano, S. S . , 204, 229, 235, 236 Hirata, A . , 98, 138 Hirooka, T., 34, 62 Hirst, M. A . , 120, 138 Hisatomi, T., 87, 90, 95, 96, 143, 145 Hishinuma, F . , 105, 123, 144 Hitchcock,C. A . , 115,116,117,130,142 Hitzman, R. A , , 88, 144 Hoeoeg, J. O., 267, 268, 269, 293, 295 Hoffman, R . M . , 163, 196 Hofmann, K. H., 287, 292 Hoge, J. H. C., 161, 165, 16G7, 185, 195, 196, 201 Hollis, M., 124, 145
Holly, J. A . , 89, 91, 95, 138, 139, 140 Holmgren, A . , 240, 266, 267, 268, 269, 293, 295, 296, 297, 300 Holt, S., 248, 260, 292 Holzman, T. F . , 11, 41, 58, 62 Honma, M., 282, 291 Hood, M., 49, 60 Hopkins, F. G . , 240, 296 Hoppen, H. O . , 132, 136 Hopper, D . J . , 286, 296 Horgen, P. A . , 78, 138, 143, 192, 195, 196, 199 Horikawa, Y . , 246, 298 Horikoshi, T., 181, 183, 197 Horner, J., 187, 198 Horton, J. S., 78, 138, 173, 174, 196, 198 Houwink, A. L . , 187, 199 Howald, I., 89, 136 Hruska, K. S . , 26, 4,3, 59, 62 Hu, A. S. L., 286, 300 Hubscher, U., 186, 198 Hudnik-Plevnik, T., 123, 138 Huflejt, M., 271, 301 Hughes, H., 277, 300 Huisman, J. G . , 81, 86, 140 Hulton, C. S. J., 33, 34, 62, 65 Humbert, M . , 5, 58 Hund, N. H., 280, 292 Hunsley, D., 186, 196 Hunt, L. T., 127, 138 Hunzicker-Dunn, M., 125, 138 Huppert, M., 107, 113, 115, 118, 128, 131, 136, 142 Hurlbert, R. E., 274, 292 Huxley, J. S . , 69, 138 Huynh, T. V . , 229, 236 Hwan-Shum, J. J . , 90, 136 Hynes, N., 78, 137 I Igato, K . , 246, 298 Iglewski, B. H., 40, 61 Ikeda, Y., 104, 139, 181, 197 Illarionov, B. A . , 25, 26, 27,31, 42,43, 62 Ilott, T. W., 103, 138 Ilyaletdinov, A. N . , 271, 291 Imai, K., 290, 295
313
AUTHOR INDEX
Inaba, H., 47, 67 Inaba, T., 104, 138 Ingold, C. T . , 151, 196 Ingolia, T. D . , 34, 63 Ingram, D. S . , 103, 138, 143 Inorie, Y., 281, 285, 296, 298 Insley, M. Y., 95, 140 Irgolic, K. J., 271, 272, 279, 294 Irvin, R. T . , 78, 138 Ishibashi, Y . , 98, 138 Ishikawa, T., 15&9, 172, 174, 177, 178, 181, 184, 196, 199, 200 Ishino-Arao, Y., 260, 299 Islam, M. S . , 101, 138 Ismailov, A. D . , 13, 62 Isobe, M., 2,90, 295 Isogai, A., 98, 99, 137, 138, 139, 143 Israel, A . , 25, 42, 60 Iten, W., 190, 196 Ito, M., 251, 301 Itoh, H., 132, 133, 139 Iwadare, T., 75, 136 Iwami, K., 251, 296 Iwanochko, M., 78, 138 Iyengar, R., 132, 133, 138 Izuka, M., 255, 281, 296
J Jablonski, E., 11, 24, 28, 62 Jackson, C. L., 87, 90, 92, 94, 136, 138 Jackson, P. J., 290, 296 Jacoby, W. B . , 240, 281, 282, 292, 296 James, C . J., 46, 63 Janssens, P. M. W., 131, 138 Jaspers, C. J., 240, 244, 245, 248, 251, 252,253,254,257,259,260,261,262, 273, 282, 28,&7, 293, 296, 299 Jassim, S. A. A , , 34, 62 Javor, B . , 242, 298 Jelinek, B. G . , 81, 144 Jenness, D. D . , 89, 138, 139 Jennings, D . H . , 150, 151, 155, 196 Jensson, H., 281, 297 Jeong, Y. K., 100, 141 Jin, J. R . , 123, 124, 145 Jirjis, R., 18.5, 198 Jocelyn, P. C., 263, 296 Jockers-Scherubl, M. C., 245, 296 Jockusch, H., 84, 145
Joernvall, H., 267, 268, 269, 293, 295 Johnson, F. H . , 8, 66 Johnston, T. C . , 14, 17, 26, 43, 58, 59, 62, 67 Johnstone, K., 103, 143 Jones, B. E., 81, 82, 83, 84, 90, 135, 137, 139 Jones, E. W., 254, 260, 296 Jones, H,. E., 130, 139 Jordan, F., 126, 127, 136, 137 Jornvall, H . , 28, 66, 266, 296 Josephy, P. D . , 278, 297 Jowett, T., 131, 135 Julius, D., 88, 139 K Kaback, H. R., 286, 296 Kado, C. I., 5, 34, 61, 62, 66 Kadota, T., 98, 141 Kahmann, R., 161, 199, 201, 202 Kai, J., 99, 135 Kaidoh, T., 6, 66 Kaji, M., 100, 141 Kamada, T., 174, 181, 185, 187, 189, 190, 196, 200 Kamikara, T . , 255, 301 Kamiya, Y . , 99, 139 Kamoun, S . , 5, 62 Kanaya, K., 90, 93, 94, 135 Kanda, T., 172, 174, 196 Kanety-Londner, H., 278, 296 Kaplan, H,. B . , 37, 38, 39, 40, 45, 62 Kapoor, S . , 26, 27, 29, 30, 67 Kappeli, O., 2 5 4 5 , 294 Kappus, H., 269, 270, 278, 296 Karesch, H., 186, 199 Karg, H., 132, 136 Karginova, V. A., 25, 26, 27, 31, 61 Karl, D. M . , 6, 62 Karp, M., 34, 62 Karplus, P. A , , 275, 296 Kasai, R., 176, 195 Kasai, S . , 23, 33, 63 Kassir, Y., 172, 197 Katagiri, M., 6, 66 Katan, J., 284, 292 Katayose, Y . , 178, 199 Kato, H., 250, 296
314
AUTkIOR INDEX
Kato, J., 255, 286, 298 Kato, N., 289, 296 Katsuda, H., 189, 196 Katzenellenbogen, B. S., 117, 139 Katzenellenbogen, J. A , , 117, 139 Katznelson, R., 35, 42, 63 Kawai, G., 104, 139, 180, 181, 197 Kawate, S., 212, 235 Kaziro, Y., 96, 132, 133, 137 Keen, J. W., 28, 58 Keen, W. A., 83, 144 Kelly, J. M., 275, 301 Kelman, A, , 204, 235 Kelsey, K., 92, 140 Kempner, E. S., 35, 36, 63 Kendall, E. C., 240, 296 Kendrick, B., 191, 197 Kennedy, W. K., 272, 299 Kenyon, G. L., 37, 42, 60 Kerklaan, P. R. M., 255,258,284,292, 296 Kenvin, J. K., 181, 197 Kenvin, J. L., 80, 139 Kessel, S. R., 275, 293 Ketterer, B., 281, 282, 296 Kiltz, H. H., 87, 144 Kim, S. H., 89, 143 Kimmel, B. E., 275, 300 Kimura, A., 249,250,254,255,256,257, 281,285,286,288,289,295,296,298, 299, 300, 301 King, J. M. H., 5, 63 King, K., 127, 139 Kinsman,O. S., 110, 111,116, 121, 122, 125, 126, 129, 130, 135, 139, 145 Kirchner, G., 34, 63 Kirschke, H., 264, 291 Kistler, M., 255, 257, 258, 296 Kitada, C., 95, 98, 99, 137, 139, 143 Kitagata, T., 246, 301 Kitamoto, K., 260, 296 Kitamoto, Y., 154, 181, 183, 197 Kitaoka, S., 271, 301 Klapper, B. F., 74, 139 Klapper, M. H., 74, 139 Klar, A. J. S., 160, 196 Klein, H., 120, 137 Klein, R. A., 272, 299 Klis, F., 90, 91, 145 Knochelmann, K., 46, 63
KO, W. H., 80, 81, 136, 137, 139 Kobayashi, H., 49, 61 Kocak, R., 231, 232, 236 Kohli, K. K., 250, 251, 297 Koivusalo, M., 288, 289, 301 Kole, H. K., 121, 127, 139 Kolibachuk, D., 39, 66 Koltin, Y., 149, 1.57, 165, 171, 197 Koncz, C., 17, 34, 35, 63, 65 Koncz-Kalman, Z., 34, 63 Konopka, J. B., 87, 90, 92, 136 Konyecsni, W. M., 39, 60 Kooistra, W. H. C. F., 162, 179, 195 Kopecky, K., 43, 66 Korber, H., 34, 63 Kornberg, A., 244, 300 Kornegay, J. R., 191, 194 Kortan, A . R . , 290, 292 Koshino, Y., 282, 297 Kosower, E. M., 241,242,244,247,255, 263, 278, 296 Kosower, N. S., 241,242,244,247,255, 263, 278, 296 Kossler, R., 47, 63 Kou, Y. S., SO, 63 Kozasa, T., 132, 133, 139 Kozloff, L. M., 222, 224, 227, 236, 237 Kram, A , , 192, 196 Krauth-Siegel, R. L., 245, 275, 296 Krebs, H. A , , 277, 293 Kreger, D. R., 1.54, 176, 200 Kren, B., 267, 269, 296, 300 Kricka, L. J., 5, 63 Krieger, C. 27, 65 Krongelb, G. S., 172, 174, 198 Kronstad, J. W., 91, 95, 139, 161, 197 Krook, M., 28, 66 Kruczek, R., 255, 258, 290, 294 Kuchler, K., 89, 139 Kues, U., 160, 197, 201, 202 Kuhad, R. C., 178, 197 Kuhn, J., 5, 34, 48, 58, 59, 67 Kuipers, A. G. J., 191, 192, 197 Kula, M. R., 289, 298, 300 Kulys, 280, 292 Kumagai, H., 246, 249, 251, 254, 255, 282, 296, 297, 298, 300, 301 Kumar, K. S., 279, 297 Kumar, S., 250, 251, 297 Kumeno, K., 232, 237
AUTHOR INDEX
Kunding, W., 259, 297 Kundu, B . , 88, 135, 143 Kunisawa, R., 88, 137, 139 Kuntz, I. D . , 226, 235 Kuo, A., 46, 47, 60, 63 Kurata, S., 105, 123, 144 Kurfurst, M., 5, 6, 46, 62 Kurita, R., 181, 196 Kurjan, J., 88, 91, 95, 132, 133, 135, 136, 139 Kurz, W., 120, 137 Kusaka, I., 99, 135 Kutschera, U., 187, 197 L
Labarere, J . , 162, 169, 199 Lahm, M.-V., 27, 29, 65 Lahti, R., 244, 297 Lahue, E., 221, 223, 236 Langerman, N., 7, 63 Langger, J . , 264, 291 Langhans, V. E., 231, 236 Langridge, W. H. R., 34, 35, 61, 63 Larimer, F., 5, 59, 62 Larsson, A . , 240, 297 Lash, L., 284, 291 Lasure, L. L., 75, 135 Lau, E. P . , 242, 281, 282, 297, 299 Lau, S . , 240, 298 Lauter, E. R., 184, 199 Lauter, F. R . , 184, 199 Lauterburg, B. H., 277, 300 Lawless, R. L. Jr 229, 236 Lawlor, E., 34, 66 Lawrence, R. A . , 270, 297 Laybourn, P., 94, 135 Layton, R. G . , 231, 237 Lazarow, A., 240, 292 Le, P. H . , 76, 140 Leatham, G. F., 151, 179, 197 Lee, C. J . , 81, 139 Lee, C. Y., 22, 25, 26, 27, 31, 43, 63, 276, 277, 297 Lee, J . , 5, 6, 7 , 8 , 11, 12, 13, 14, 17, 18, 22, 23, 25, 27, 31, 34, 35, 49, 50, 60, 61, 62, 63, 64, 65, 67 Lee, K.-H., 49, 63 Lee, P.-Y., 49, 67 Lefkowitz, R. J., 127, 139
315
Legocki, M., 34, 63 Legocki, R. P., 34, 63, 64 Legrain, M., 2867, 299 Leisman, G., 23, 60 Leith, W. H., 83, 90, 137 Lemke, P. A . , 1.57, 191, 192, 194, 197 Le Minor, L., 248, 294 Lenard, J., 112, 121, 126, 127, 136,137, 139, 140 Lenfant, M., 283, 300 Leonard, T. J . , 104, 143, 171, 179, 181, 197, 198, 199 Leong, S. A , , 161, 197 LePeuch, C., 12, 61 LeRoith, D., 106, 121, 131, 139, 143 Leslie, J. F., 171, 197 Lesniak, M. A . , 106, 121, 131, 139, 143 Letelier, M. E . , 251, 283, 298, 299 Leupold, U . , 96, 97, 139 Levedahl, B. H . , 105, 135 Levi, J. D . , 87, 139 Levin, Z . , 204, 210, 212, 237 Levisohn, R., 46, 63 Levitzki, A . , 93,94, 125, 133, 136, 139, 142 Levy, H. R., 277, 297 Lewis, B . J . , 242, 294 Lewis, K. E., 101, 142 Li, L., 248, 297 Liao, H., 94, 139 Lin, J.-W., 17, 25, 26, 27, 30, 31, 33, 40, 43, 48, 58 Lindemann, J., 229, 231, 236, 237 Lindgren, P. B . , 224, 233, 236 Lindow, S. E . , 204, 210, 211, 212, 221, 222,223,224,227,229,230,231,233, 235, 236 Lingle, W. L., 34, 65 Lipke, P. N . , 88, 90, 91, 92, 93, 134, 135, 139, 144 Lisa, T. A , , 45, 60 Little, P. F. R., 160, 195, 198 Liu, M., 187, 198 Liu, Y., 100, 140 Llobell, A . , 276, 277, 297 Lloyd, D., 46, 63 Loewen, P. C . , 244, 297 Loftus, M. G . , 165, 199 Long, H., 105, 141 Longin, T. L., 38, 60
316
AUTHOR INDEX
Loose, D. S., 107, 110, 112, 113, 115, 116,117, 119, 120, 124, 129, 130, 131, 136, 140, 144 Lopez-Barea, J., 276, 277, 297, 299 Lopez-Ruiz, A., 276, 277, 297 Losada, M., 274, 275, 276, 300 Loshen, G., 270, 294 Losick, R., 34, 66 Loumaye, E., 127, 140 Lu, A. Y., 281, 299 Lu, B. C., 149, 171, 181, 197, 200 Lucas, M. T., 254, 300 Luchini, M. M,., 78, 138 Luehsen, K. R., 242, 294 Lugovoy, J. U. M., 88, 144 Lummen, P., 42, 46, 63 Lundberg, K. S. 45, 60 Lute, M., 222, 224, 236 Lydan, M. A., 101, 142 M Mabey 65 MacDonnell 2, 48, 63 Macheroux, P., 7, 63 Machino, G., 184, 198 Machlis, L., 70,71,72,73,74, 135, 140, 141 MacKay, V. L., 87, 89, 90, 91, 94, 95, 135,136,138,139,140,144
Mackenzie, B. F., 240, 296 MacKenzie, R. E., 7, 63 MacLellan, A., 183, 199 MacLeod 47, 63 Madelin, M. F., 154, 197 Magrum, L. J., 242, 294 Maher, E. A., 204, 235 Maier, K., 257, 258, 296 Makemson, J. C., 4, 5, 6, 7, 45,46,49, 50, 61, 62, 64 Maki, L. R., 204, 222, 231, 236, 237 Makiguchi, N., 47, 64 Malaise, E. P., 242, 300 Mallo, G., 111, 126, 142 Malnic, G. F., 8, 62 Manachere, G., 181, 197 Mancini, J. A., 25, 27, 42, 64, 66 Maniloff, J., 242, 294 Mannervik, B., 240, 263, 276,281, 282, 283, 296, 297
Manney, T. R., 87,92,95,135,136,140 Marchant, R., 149, 153, 154, 163, 165, 176, 179, 185, 188, 197, 200 Marcus, S., 88, 89, 95, 135, 140 Margartis, A., 204, 230, 231, 232, 236 Margulis, L., 105, 140 Marino, R., 192, 197 Marino, V. A., 282, 299 Mark, D. F., 266, 267, 268, 297 Marquardt, L., 264, 291 Marsh, L., 87, 89, 90, 132, 140 Martin, M. O., 26, 27, 30, 37, 41, 42, 64, 66 Martin, T., 161, 199 Martini, N., 34, 63 Maruyama, H., 99, 141 Mason, H. L., 240, 296 Mason, J. C., 149, 180, 190, 194 Mason, R. P., 278, 297 Massa, J., 6, 11, 62 Masselot, M., 260, 297 Massey, V., 274, 276, 294 Mata, A. M., 276, 297, 299 Matcham, S. E., 180, 201 Matheson, I., 5, 7, 8, 14, 23, 63 Mathews, B. W., 18, 66 Mathews, C. K., 266, 297 Matile, P., 190, 196 Matsubana, I., 245, 295 Matsui, K., 23, 33, 63 Matthews, T. R., 185, 197 Mayer, M. P., 89, 143 Mayerhofer, R., 34, 63 Mayfield, J. E., 192, 197 Mazur, P., 103, 140 McBath, P., 37, 38, 60 McCaffrey, G., 92, 95, 138, 140 McCosker, J., 49, 65 McCullough, J., 95, 140 McDonnell, D. P., 110, 125, 130, 137 McFall-Ngai, M. J., 4, 5, 46, 49, 50,60, 63, 65, 66 McFarland, B. L., 231, 236 McGee, Z. A., 131, 137 McGrath, J. P., 89, 140 McGuire, W. L., 107, 113, 128, 136 McIlvaine 7, 63 McKenzie, M. A., 112, 121, 126, 127, 136, 137, 140 McKown, B. A., 107, 141
AUTHOR I N D t X
McMorris, T. C., 75, 76, 77, 79, 135, 137, 140, 141, 142, 143, 144, 145 Mead, T. H., 240, 295 Mechler, B., 88, 136 Meck, R., 251, 291 Mehra, R. K., 245, 290, 292, 297 Meighen, E. A . , 5 , 6 , 7 , 8,9, 10, 11, 12, 14, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 33, 34-5, 37, 41, 42, 43, 52, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 Meinhardt, F., 171, 195 Meister, A., 244,246,247,248,249,250, 251,256,257,258,275,295,297,298, 299, 300, 301 Mekalanos, J. J., 39, 64 Mellon, F. M., 160, 195, 198 Mendez, C., 34, 66 Menzies, G. S., 111, 116, 121, 122, 125, 126, 130, 131, 135, 145 Meredith, M. J., 261, 299 Merryweather, J., 94, 135 Mertvetsov, M. P., 25, 26, 27, 31, 62 Meshnick, S. R., 272, 294 Mesland, D. A. M., 86, 140 Messenguy, F., 254, 259, 266, 298, 300 Metzenberg, R. L., 160, 197, 202 Metzger, D., 124, 140 Meury, J., 260, 298 Meyer, H. E., 88, 135 Meyer, M., 266, 298 Meyer, M. D., 75, 79, 141, 143 Meyers, H. V., 103, 140 Michaelis, S., 87, 89, 94, 141, 142 Michaliszyn, G. A., 6, 11, 24, 28, 41, 60, 64 Michalski, C. J., 78, 141 Michelson, A. M., 6, 47, 62 Middleton, A. J., 37, 64 Mikoljczyk, S. D., 293 Milbauer, R., 248, 298 Mileham, A. J., 25, 26, 41, 58, 59 Miles, M. A , , 275, 301 Miles, P. G., 173, 198, 199 Milgrim, C., 159, 196 Miller, D., 88, 145 Miller, E., 49, 65 Miller, J. F., 39, 64 Miller, M. L., 82, 141
317
Miller, R. E., 156, 198 Miller, S. C., 115, 119, 120, 124, 135, 137, 141 Mills, J. S., 75, 136 Mimura, N., 6, 35, 36, 67 Minato, S., 246, 251, 298 Minomi, K., 99, 135 Mirocha, C. J., 104, 138, 142, 145 Mishra, P. K., 82, 144 Misra, T. K., 39, 60 Mitchell, A. P., 172, 197 Mitchell, J. B., 246, 298 Mitchell, J . R., 277, 300 Miura, R., 23, 62 Miya, T., 250, 295 Miyajima, A., 89, 90, 95, 141 Miyakawa, T., 94,98,99, 100, 135, 138, 140, 141, 144 Miyamoto, C., 26,27,28,29,30,31,34, 41, 42, 58, 59, 63, 64, 67 Mizuno, H., 225, 226, 236 Mizutani, J., 282, 291 Mohn, G. R., 255, 257, 258, 284, 292, 296, 298 Mohr, J. A., 105, 107, 141 Mol, P. C., 91, 92, 143, 144, 188, 189, 197 Moncada, C., 283, 298 Monks, T. J., 240, 298 Montero, S., 274, 276, 298 Mooibroek, H., 191, 192, 197 Moon, S. S., 76, 141 Moore, D., 148,149,178,184,185,186, 187, 197, 198, 199 Moore, S. A., 90, 95, 141 Moore, W. R., 256, 257, 298 Mooz, E. D., 246, 248, 250, 258, 298 Mordecai, D., 117, 139 Morelli, G., 184, 198 Morello, A., 251, 283, 298, 299 Morin, R. Y., 2, 49, 58, 62, 65 Morise, H., 8, 66 Morita, R. Y., 49, 59 Morse, M. L., 256, 298 Morzycka, E., 260, 298 Mrsa, V., 90, 138 Muchmore, H. G., 105, 107, 141 Mueller, G. M., 209,211,212,220,222, 223, 224, 226, 229, 233, 235, 236 Mueller, N., 45, 60
318
AUTHOR INDEX
Mukerji, K. G., 284, 300 Mulder, G. H., 162, 167, 169, 177, 183, 184, 195, 198, 200 Muller, F., 5, 7, 11, 12, 14, 22, 23, 63, 65, 67 Muller, H., 88, 136 Miiller-Breitkreutz, K., 48, 64 Mullins, J. T., 74, 76, 77, 78, 141, 144 Munoz-Rivas, A , , 191, 198 Murant, S. J., 264, 294 Murao, S., 181, 198 Murat, M., 271, 293 Murata, K., 249,250,254,255,256,257, 281,285,286,288,289,295,296,298, 299, 300, 301 Murphy, K. P., 223, 236 Murray, H. W., 272, 298 Musafia, B., 34, 58 Musgrave, A , , 77, 141 Mutasa, E. S., 160, 195, 197, 198, 201, 202 Muthukumar, G., 121, 127, 139 Mutoh, N., 290, 295 Mynbaeva, B. N., 271, 291 N Nabi, I. R., 8, 20, 22, 65 Nadeau, T. L., 50, 51, 67 Nadler, T., 212, 237 Nagai, S., 264, 298 Naider, F., 87, 88,89,95, 120,135,137, 140, 141, 142, 143, 145 Nair, G. B., 49, 64, 65 Nair, N. G., 191, 196 Naito, K . , 261, 262, 299 Najarian, R., 94, 135 Nakafuku, M., 132, 133, 139 Nakagawa, C. H., 78, 138 Nakagawa, C. W., 290, 295 Nakagawa, Y., 285, 293 Nakahama, N., 232, 237 Nakamura, T. ,6, 23, 33, 35, 36, 63, 67 Nakanishi, K., 103, 140 Nakaya, T., 23, 63 Nakayama, N., 89, 90, 95, 141 Nakayama, R., 249, 250,251, 254,255, 296, 298 Nandakumar, R., 49, 65 Nanjoh, A., 260, 299
Nasmyth, K. A , , 160, 198 Natarajan, R., 49, 64, 65 Nawrath, C., 184, 198 Ne’eman, Z., 6, 41, 65 Nealson, K. H., 2, 4, 5, 6, 7, 8, 9, 17, 25, 26, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 59, 60, 61, 62, 63, 64, 65, 66, 67 Nearhos, S. P., 48, 65 Neben, I., 289, 298 Needleman, S. B., 214, 236 Neilson, O., 96, 136 Neiman, A. M., 87, 89, 90, 132, 140 Nelson, G. E., 81, 136 Nelson, M. A , , 184, 198 Nelson, R . R., 104, 141 Ncmccck-Marshall, M., 223, 227, 236 Nes, W. D., 80, 141 Newton, G. L., 241, 242, 244, 275, 293, 296, 298 Ng, K. M. E., 75, 141 Nicholas, J.-C., 5, 58 Nichols, R., 186, 196 Nicoli, M. Z., 8, 14,, 16, 17,58,61,63, 64, 65 Niederpruem, D. J., 154, 163, 184, 185, 186, 195, 197, 198, 200 Nielsen, O., 96, 97, 139 Niest, D. K., 90, 136 Nieuwenhuis, D., 77, 141 Nikolaev, 0. N., 272, 298 Nilsson, E., 28, 66 Nilsson, O., 17, 34, 65 Nishihara, M., 99, 141 Nishioka, T., 250, 296 Nishizava, T., 289, 296 Nissen, V., 34, 64 Niswander, L., 242, 281, 297 Nocita, O., 283, 294 Noda, K., 246, 298 Nolting, S., 111, 141 Nordheim, E. V., 204, 236 Novotny, C. P., 159,160,191,196,198, 200, 201, 202 Novova, L., 245, 295 Nozawa, Y., 176, 195 Nurse, P., 160, 194 Nussbaum, A , , 5, 65 Nutting, W. H., 71, 72, 140, 141 Nylhlen, L. E., 179, 198
3 19
AUTl1OR INDFX
0 Oakey, R. E . , 115, 116, 117, 130, 142 Obata, H., 212, 235 O’Brien, C. H . , 49, 65 Oda, J., 250, 296 Odani, S., 23, 63 O’Day, D. H . , 101, 142 Odds, F. C . , 110, 129, 142 Odell, W. D., 131, 137 Odier, E., 149, 180, 190, 198 Ogata, T., 254, 299 Ogawa, T., 246, 299 Ogden, R. C . , 25, 41, 59 Ohja, V., 250, 251, 297 Ohki, O., 100, 140 Ohsumi, Y., 90, 93, 94, 135, 142, 260, 296 Ohtake, Y., 250, 254, 299 Ohue, H. 260, 299 Oka, Y., 94, 100, 141, 246, 299 O’Kane, D. J . , 5 , 7, 11, 12, 14, 17, 22, 23, 25, 27, 31, 34, 35, 61, 63, 65, 67 Okubo, Y . , 98, 141 Olive, L. S . , 101, 135 Oliver, J. D., 24, 49, 65 Olivcr, R. P., 191, 198 Olson, L. W . , 74, 142 Olsson, O., 17, 34, 35, 63, 65 Ondarza, M . , 274, 299 Ondarza, R. N., 274, 299 Onishi, M., 181, 197 Onishi, T., 271, 301 Ono, B., 260, 261, 262, 299 Oostra-Demkes, G. J . , 289, 301 Oppenheimer, N. J . , 37, 42, 60 Orlean, P., 91, 92, 142 Orlowski, M., 249, 299 Orndorff, S. A . , 49, 65 Orr, W. C., 78, 144 Orreenius, S . , 240, 297 Orser, C. S . , 204, 211, 212, 236, 237 Ortigosa, M . , 49, 65 Osagie, A . U . , 84, 135 Osguthorpe, D. J . , 226, 235 Osmond, B. C., 91, 135 Osuji, G. O . , 258, 299 Osumi, M., 49, 61, 90, 93, 94, 135 Othman, Y. H. S., 115, 116, 117, 130, 142
Otto, R., 289, 301 Overbaugh, J. M . , 271, 274, 282, 299 Overdank-Bogart, T., 242, 293 P
Packer, L., 271, 301 Padan, E., 93, 94. 142 Pai, E. F . , 275, 299 Pal, S . , 287, 299 Pall, M. L . , 177, 198 Palmer, L. M . , 2, 50, 65 Panopoulos, N. J . , 211, 212, 221, 223, 23 1, 236 Paquatte, O., 8, 65 Parag, Y., 158, 198 Park, S. F . , 34, 65 Parker, M. L . , 95, 140 Parody-Morreale, A . , 223, 236 Parsell, D., 267, 269, 296 Passeron, S., 111, 126, 130, 142 Paszewski, A , , 260, 298, 299 Patterson, G. W . , 80, 141 Patton, C. L., 272, 299 Paveto, C., 111, 126, 130, 142 Payne, G. M . , 251, 259, 299 Payne, J. W., 251, 259, 299 Pazhcnchcvsky, B., 241, 263, 296 Pearson, G. R . , 79, 143 Peinado, J., 276, 277, 297 Pekkala, D., 79, 142 Pel, R., 264, 299 Pelletier, J., 16, 34, 58, 59 Penketh, P. G . , 272, 299 Penninickx, M. J . , 240, 244, 245, 248, 251,252,253,254,257,259,260,261, 262, 273, 277, 278, 282, 2867, 293, 296, 299 Peoples, 0. P., 275, 300 Pereira, M., 280, 295 Perham, R. N., 274,275, 276, 295, 299, 300 Perkins, J. H., 173, 181, 183, 198 Perlman, R., 93, 94, 133, 136, 142 Pcron, C. M . , 89, 137 Perry, A . C., 275, 299 Perry, C., 180, 201 Persson, M., 267, 295 Pertscva, M. N., 131, 142 Pfyffer, G. E . , 186, 198
320
AUTHOR INDEX
Phelps, P., 210, 221, 222, 236 Phillips, L. E., 179, 197, 198 Phu, H. L., 80, 141 Piccolomini, R., 281, 293, 299 Pickett, C. B., 281, 283, 299 Piggott, J. R., 88, 137 Pinhey, K. G., 240, 299 Pinto, M. C., 276, 297, 299 Pitblado, K., 110, 111, 129, 139 Planel, H., 271, 293 Plasenicia, J., 104, 142 Platt, T., 35, 36, 41, 64 Plattner, J. J., 72, 135, 142 Plempel, M., 86, 142 Podhradsky, D., 244, 293 Poinar, G. O., 43, 46, 47, 49, 60, 65 Polsinelli, M., 273, 282, 283, 292, 294 Pommerville, J., 71, 72, 73, 74, 138, 142 Ponopoulos, N. J., 224, 233, 236 Popplestone, C. R., 76, 142 Potrikus, C. J., 5 , 6, 37, 42, 46, 50, 62, 65 Poulsen, L. L., 263, 270, 301 Poulter, C. D., 89, 143 Pouwels, P. H., 191, 198 Powell, B. L., 113, 115, 116, 117, 128, 130, 142 Powell, K. A., 151, 194 Powell, M. J., 77, 136 Powers, D. A., 52, 58 Powers, S., 89, 142 Prasad, R., 287, 293 Prasher, D. C., 7, 23, 25, 27, 31, 65 Presswood, R. P., 12, 62 Preus, M. W., 76, 140, 142 Pribnow, D., 191, 194 Price, B. R., 92, 143 Price, N. W., 240, 299 Pringle, J. R., 92, 137, 145 Prochoda, M., 210, 221, 222, 236 Prohaska, J. R., 270, 299 Prosser, J. I., 190, 196 Protopopova, M. V., 25,, 26,27,31,42, 62 Pryke, J. A , , 151, 194 Pryor, W. A., 244, 299 Pujalte, J.-J., 49, 65 Pukkila, P. J., 170, 191, 194, 201 Punt, P. J., 191, 192, 197, 198
Q Quayle, J. R., 288, 289, 292, 301
R Racker, E., 240,284,288,292,299,300 Radford, A., 149, 195 Radkswsky, A , , 241, 296 Raina, 65 Raj, H. G., 284, 300 Ralinson, C. J., 103, 138 Rama, R. A , , 101, 142 Ramaiah, N., 49, 65 Ramaswamy, O., 287, 299 Ramesh, A., 49, 65 Ranes, M., 34, 65 Ranney, H. M., 241, 296 Ranz, A., 272, 294 Rao, B. G., 192, 195 Rao, P., 103, 136 Rao, P. S., 186, 198 Raper, C. A , , 149, 156, 157, 158, 161, 173, 174, 196, 198 Raper, J. R., 70,142,156,158,163,171, 172, 173, 174, 196, 197, 198 Raper, J. T., 74, 142 Rapoport, H., 71,72,135,140,141,142 Raptis, S., 20, 66 Rast, D. M., 154,179,186,195,198,199 Raths, S. K., 88, 89, 136, 142, 143 Raudaskoski, M., 149, 153, 171, 181, 183, 185, 198, 199, 201 Rausch, S. K., 26, 41, 58, 59 Rauschel, F. M., 14, 65 Ravagnan, G., 281, 293, 299 Ray, J. M., 40, 45, 60 Rayner, A. D,. M., 151, 194 Read, D. J., 191, 199 Redei, G. P., 34, 63 Reed, D. J., 261, 270, 277, 278, 280, 292, 299 Rees, C. E. D., 37, 58 Reese, C. P., 41, 61 Reese, R. N., 290, 292 Refai, M., 105, 138 Regensburg, B. A., 154, 176, 200 Reichard, P., 28, 60, 66, 266, 297, 299 Reichelt, J. L., 49, 50, 60, 65 Reid, I. D., 98, 142, 143
32 1
AUTHOR INDEX
Reid, L., 264, 294 Reijnders, A. F. M., 148, 185, 199 Reinhertz, A., 46, 67 Remington, S. J., 18, 66 Rendon, J. L., 274, 299 Rennenberg, H., 240, 247, 299 Repetto, Y., 251, 283, 298, 299 Restrepo, A., 107, 115, 117, 124, 129, 131, 140, 143 Revesz, L., 242, 300 Rhee, H. I., 285, 289, 298, 300 Ribas, J. C., 92, 143 Richardson, C. C., 266, 267, 268, 297 Richardson, W., 160, 197, 201, 202 Richartz, G., 183, 199 Richman, P., 249, 300 Richter, G., 27, 29, 65 Rickards, R. W., 81, 143 Rico, M., 284, 292 Riehl, R. M., 76, 78, 79, 135, 143 Riendeau, D., 18, 65 Riezman, H., 89, 136, 143 Riley, W., 6, 11, 62 Rine, J., 89, 143 Rivas, J., 274, 275, 276, 300 Rizza, V., 286, 300 Robbins, P. W., 91, 135 Robert, J. C., 154, 181, 183, 197, 199 Roberts, C., 131, 139 Roberts, D. M., 24, 49, 65 Roberts, J. L., 34, 63 Roberts, R. B., 286, 300 Robin, A., 260, 298 Robins, R., 253, 259, 300 Robinson, N. J., 290, 296 Robson, B., 226, 235 Rocco, J., 88, 135 Rodriguez, A., 6, 8, 18, 19, 20, 21, 22, 31, 59, 65, 66, 67 Roelofsen, P. A., 187, 199 Rogowsky, P. M., 5, 34,62, 66 Roland, J. C., 187, 199 Roman, M., 49, 65 Romano, N., 184, 198 Romo, D., 72, 142 Roncero, C., 92, 143 Ropenga, J. S., 283, 288, 300 Ropes, I., 94, 138 Rose, M. D., 92, 143 Rose, Z. B., 288, 300
Roseman, S., 259, 300 Rosen, 0. M., 121, 126, 131, 143 Rosenberg, I., 280, 295 Rosenburg, S., 89, 143 Rosenzweig, J. L., 131, 143 Rosin, I. V., 178, 197 Ross, I. K., 165, 179, 183, 199 Rosson, R. A., 35, 37, 41, 66 Roth, J., 106, 121, 131, 139, 143 Rothlisburger, U., 27, 29, 65 Rotilio, G., 273, 294 Rouch, D. A., 275, 299 Rouwendal, G. J. A , , 169, 195 Royce, D. J., 190, 201 Royer, J. C., 192, 199 Rozek, C. E., 78, 143 Ruby, E. G., 4, 7, 37, 38, 39, 44, 49, 50, 58, 63, 65 Rucker, E. B., 26, 43, 59, 62 Ruffner, H. P., 186, 199 Ruggles, J. A., 223, 227, 236 Ruiters, M. H. J., 1,51, 153, 167, 168, 171, 177, 181, 184, 199, 201 Rusmin, S., 104, 143, 181, 199 Russel, M., 267, 269, 300 Russo, V. E. A., 184, 198, 199 Ruttke, I., 255, 258, 290, 294 Ryley, J. F., 129, 143
S Saari, G. C., 89, 95, 138, 140 Sagan, D., 105, 140 Sahm, H., 288, 289, 298, 300 Saikusa, T., 285, 298 Sakagami, Y., 98,99,137,138,139,143 Sakai, N., 212, 235 Sakamoto, M. 95, 96, 143 Sakata, K., 81, 143 Sakazava, C., 289, 296 Saksena, K. N., 192, 1W Sakurai, A., 95, 96, 137, 139, 143 Sakurai, S., 99, 139 Salazar, M. E., 107, 115, 117, 124, 129, 131, 143 Saleh, F., 171, 195 Salmond, G. P. C., 37, 58 Salmond, W., 75, 136 Salonen, M., 183, 198 Salvado, J. C., 162, 169, 199
322
AUTHOR INDEX
Sanchez, S., 254, 300 Sancho, A. M . , 279, 297 Sandberg, G., 34, 64 Sandberg, V. A., 267, 300 Sandlerman, N., 204, 210, 237 Sandmann, C. 201, 202 Sands, D. C., 231, 236 Sanseverino, J., 5, 63 Santa Anna, A. S., 89, 142 Santamaria, R., 34, 66 Sasaoka, K., 246, 299 Sato, N . , 285, 289, 298, 300 Satoh, T., 132, 133, 139 Saxena, M . , 284, 300 Sayler, G. S., 5, 59, 63 Sburlati, A., 91, 135 Scard, P. R. , 151, 194 Schafer, W., 89, 143, 161, 199 Schar, G., 110, 111, 115, 117, 118, 119, 124, 130, 131, 136, 143, 144 Scharen, A. L . , 231, 236 Schauer, A. T., 5, 34, 66 Scheer, J., 192, 196 Scheffer, R., 77, 141 Scheffers, W. A , , 273, 276, 292, 901 Schekman, R., 88, 91, 139, 143 Schell, J . , 34, 35, 62, 64 Scherens, B . , 266, 300 Schineller, J . B . , 37, 38, 60 Schipper, M. A. A., 81, 86, 143 Schirmer, R. H . , 245, 275, 296 Schlesser, A., 273, 294 Schlessinger, R., 161, 199 Schmetterer, G., 34, 66 Schmidhause,r, T. J., 184, 199 Schmidt, B., 266, 298 Schmidt, T. M . , 43, 66 Schnell, R. C., 204, 229, 231, 235, 236, 237 Schofield, M. A., 222, 236 Schopfer, P., 187, 197 Schow, S. R . , 76, 140 Schroeer, B . , 201, 202 Schuler, M., 192, 197 Schultz, D. W., 5, 66 Schulz, B., 161, 199 Schulz, G. E., 275, 293, 296, 299 Schuren, F. H. J., 162, 169, 175, 177, 183, 199, 200
Schurman,D. J.,110,112,113,115,116, 119, 130, 140 Schiitte, H . , 289, 300 Schutz, G., 78, 137 Schuurs, T. A., 192, 196 Schwalb, M. N . , 154,173,174,175,178, 179, 185, 194, 199 Schwartz, R . , 254, 300 Schwartz, R. M., 214, 237 Schwarz, D. R . , 240, 292 Schwiager, H., 92, 142 Scirmer, R. H . , 280, 292, 301 Scott, W. A., 272, 298 Scrutton, N. S . , 274, 276, 299, 300 Seddon, A. P . , 248, 268,285, 293,295, 297 Seeburg, P. H., 88, 144 Segel, L. A., 150, 195 Sela, M., 280, 295 Seliger, H. H . , 51, 66 Sels, A. A., 273, 300, 301 Serrano, A., 274, 275, 276, 300 Serrano, R . , 262, 294 Seshadri, R., 75, 76, 135, 140 Setlow, P . , 251, 274, 300 Settles, L. G . , 34, 66 Sewall, T. L. 199 Shadel, G. S . , 25,26, 27,30,31, 33,39, 40, 45, 43,, 48, 58, 60, 65 Shamansky, L., 41, 58 Shames, S. L . , 245, 275, 300 Sharp, P. M . , 33, 62 Shaw, J. A., 91, 92, 143, 144 Shaw, J. J., 5, 34, 66 Shen, C. Y., 81, 143 Shenbagamurthi, P., 88, 135, 142, 143 Sherwood, W. A , , 80, 143 Shigeoka, S., 271, 301 Shilo, M., 24, 49, 66, 67 Shiloach, J . , 106, 121, 131, 139, 143 Shimomura, O . , 8, 66 Shimosaka, M., 285, 298 Shimuzi, H . , 254, 300 Shioya, S . , 254, 300 Shipley, D., 28, 58 Shirahige, Y . , 260, 261, 262, 299 Shishido, K . , 178, 199 Shishido, T., 282, 300 Shomer-Ilan, A., 260, 294 Showalter, R., 26,27,30,41,42,64,66
AUTHOR INDEX
Shrift, A , , 242, 249, 273, 300 Shu, I . M., 81, 136 Siddiq, A . A , , 103, 143 Sies, H., 240, 269, 292, 294 Sietsma, J . H., 154, 163, 167, 171, 191, 192, 197, 199, 200 Silva, C. D . , 268, 295 Silvcr, J. C . , 78, 79, 135, 138, 142, 143 Silverman, M . , 5,2.5, 26, 27, 29, 30, 34, 37, 39, 38, 40, 41, 42, 45, 58, 60, 61, 64, 66, 67 Silverman, S. J., 91, 143 Simchcn, G., 93, 94, 142, 172, 197 Simek, A , , 105, 110, 111, 135 Simon, M., 5, 25, 26,29, 34, 41, 58,59, 61 Simon, R. D., 24, 66 Simpson, L. M , . 24, 49, 65 Sims, P. F. G., 149, 180, 190, 194 Singer, B., 89, 136, 143 Singh, A , , 88, 144 Singleton, R. J . , 49, 66 Sipiczki, M., 97, 139 Sippel, A . E . , 78, 137 Sizcmore, R., 49, 60, 65 Sjoberg, B . M . , 266, 296, 297 Sjoeberg, B . M . , 268, 293 Skerman, T. M . , 49, 66 Skowronski, R., 115, 116, 117, 130, 144 Skrzynia, C., 191, 194 Slater, E., 91, 135 Slessor, K. N., 5, 8, 9, 64 Slock, J., 39, 66 Smith, C. V . , 277, 300 Smith, E. B . . 37, 64 Smith, J ,242, 249, 273, 3M Smith, R. R., 78, 82, 138, 144 S n k k , S .F, ~, 1% ,1996 Smith, T., 5, 66 Smithies, O . , 214, 235 Snider, J. R . , 231, 237 Sobel, J . D . , 129, 144 Sobolev, A . Y. V., 13, 62 Soda, K., 260, 261, 300 Soderberg, B. O . , 267, 292 Sojory, S. K . , 287, 299 Soler, J., 274, 276, 298 Soli, G., 47, 66 Soly, R. R . , 20, 23, 2.5, 26, 27, 31, 42, 52, 61, 64, 66
323
Sommer, T., 184, 199 Sonenberg, N . , 16, 34, 58 Sonnenberg, A . S. M., 192, 199 Sortorelli, A . , 272, 299 Southworth, M. W., 2I2, 222, 224,226, 237 Spallholz, J. E . , 243, 300 Spatrik, P., 89, 138 Specht, C. A , , 159, 160, 191, 196, 198, 200, 201, 202 Spencer, D. M., 190, 191, 195 Sprague, G. F., 87, 88, 90, 92, 95, 136, 138, 139, 140, 144 Springer, J., 161, 162, 1 6 6 7 , 169, 172, 173, 174, 175, 183, 184, 195, 196,199, 200 Spudlich, J., 8, 62 Spyrou, G., 28, 66 Srivastava, L. K . , 287, 293 Stadtman, E . , 240, 244, 292, 300 Stahl, D . A , , 242, 294 Stahl, U., 170, 171, 199 Stahmann, M. A , , 179, 197 Stakebrandt, E., 242, 294 Stambcrg, J., 157, 197 Stankis, M. M . , 159, 160, 200. 201, 202 Stanley, P. E . , 5, 66 Staskawicz, B. J . , 211, 212, 224, 229, 233, 236 Stathis, P. A , , 11.5, 119, 120, 124, 135, 136, 137, 141 Stead, P. 37, 58 Steden, M., 95, 144 Steigerwald, M. L . , 290, 292 Steinhaucr, D., 25, 42, 60 Stemmler, J , 37, 42, 61 Sterne, R. E., 88, 89, 137, 139 Stevens,D . A . ,1% ,\a7 ,\\a ,1\-5,\1\, 115,117, 118,119, 124,129,130,131, 136, 140, 143, 144 Stcvcns, J. Id., 240, 298 Stewart, G. S. A. B., 5 , 34, 37, 58, 59, 62, 64, 66 Stirling, D. A . , 34, 65 Stotzler, D., 87, 144 Stover, E. P . , 107, 110, 111, 113, 115, 117,118,119,120,124,129,130,131, 136, 137, 140, 143, 144 Stranick, S. J., 38, 60 Strathern, J. N . , 160, 196
324
AUTHOR INDEX
Stratmann, R., 160, 195 Strazdis, J. R., 144 Strehler, B. L., 8, 60 Streiblova, E., 94, 138 Strickland, J. B., 72, 138, 142 Stringer, M. A , , 199 Strobl, G . , 22, 67 Stubmiller, L. M., 76, 141 Stussi, H., 179, 198, 199 Su, H. J., 81, 139 Suarez, J. E., 281, 284, 292 Suga, K., 254, 300 Sugihara, J., 17, 31, 58, 66 Suissa, M., 34, 58 Sukhova, N. M., 280, 292 Sullivan, W. P., 79. 143 Summer, K. H., 255,257,284, 296,300 Summerbell, R. C., 192, 199 Sun, S. H., 107, 113, 115, 118, 128, 136, 142 Sundeen, J., 75, 135, 136 Sundquist, R. A , , 264,271,274,275,300 Suonpaa, M., 244, 297 Surdin-Kerjan, Y., 260, 297 Suslow, T. V., 231, 236, 237 Sutter, R. P., 81, 82, 83, 136, 141, 144 Suzuki, A., 98, 99, 137, 138, 139, 143, 181, 183, 197 Suzuki, H., 251, 282, 297, 300 Suzuki, K., 6, 66 Svobodova. J., 94, 138 Swamy, S., 158-9, 178, 199 Swanson, R. F., 18, 66 Swartzman, E., 22, 26, 27, 29, 30, 41, 42, 59, 67 Swerdlow. R. D., 251, 274, 300 Sysoev, 0. V., 281, 288, 300 Szajewski, R. P., 263, 301 Szalay, A., 17, 34, 35, 61, 63, 64, 65 Szent-Gyorgy, A., 287, 293 Szittner, R., 8, 22, 25, 26, 17, 31, 33, 42, 43, 52, 61, 63, 64
T Tabor, C. W., 244, 245, 301 Tabor, H., 244, 301 Tachikawa, T., 94, 100, 140, 141, 144 Taguchi, N., 94, 138 Tahara, N., 94, 138
Tahara, S., 282, 291 Takagi, Y., 178, 199 Takahashi, N., 95, 96, 143 Takashi, N., 99, 139 Takashi, S., 176, 195 Takemaru, T., 174, 181, 185, 187, 189, 190, 196, 200 Takimoto, A., 6, 35, 36, 67 Tamaki, H., 282, 297, 301 Tamanoi, F., 89, 137 Tamura, S., 98, 99, 137, 139, 143 Tamwa, S., 99, 139 Tan, K. K., 181, 183, 200 Tanaka, A., 288, 294 Tanaka, H., 95, 96, 143 Tanaka, K., 98, 138 Tanaka, S., 105, 123, 144 Tang, J. S., 49, 67 Tani, K., 255, 286, 298, 301 Tani, Y., 289, 296 Tanner, R. S., 242, 294 Tanner, W., 90, 91, 92, 138, 142, 145 Target, E. B., 245, 290, 297 Tarui, N., 181, 198 Tatchcll, K., 160, 198 Tate, S. S., 251, 301 Tatem, B. A , , 105, 141 Tavernicr, J. E., 264, 293 Taylor, A., 90, 92, 93, 139 Taylor, M. C., 275, 301 Taylor, S., 260, 195 Tcbo, B. M., 49, 62, 65 Tel-Or, E., 271, 301 Ten Have, J. P., 254, 259, 298 Tereda, O., 242, 272, 291 TCrouanne, B., 5, 58 Terrance, K., 88, 90, 135, 144 Tester, P., 49, 58 Tezuka, H . , 254, 299 Thelander, L., 266, 299 Thielke, C., 185, 196 Thomas, D. S., 74, 77, 78, 79, 144 Thomas, D. Y., 88, 136 Thomas, G., 49, 64 Thomas, S. M., 269, 293 Thompson, R. B., 14, 26, 62 Thornalley, P. J., 285, 287, 301 Thorner, J., 87,88,89,94,95,127,136, 137, 139, 140, 144 Thornley, J. H. M., 150, 195
325
AUTHOR INDEX
Thurston, C. F., 180, 201 Timberlakc, W. E., 78, 143, 144, 161, 170, 198, 199, 200 Tkacz, J. S., 90, 144 Tobler, H., 186, 199 Tochikura, T., 246, 249, 251, 254, 255, 282, 296, 297, 298, 300, 301 Toft, D. O., 75, 76, 79, 141, 143 Tokes, L. G . , 120, 136, 137, 141 Tokuyama, T., 212, 235 Tolner, B., 192, 196 Tomita, K., 246, 251, 301 Tomizawa, H., 263, 301 Torres, H. N., 126, 137 Toucas, M., 248, 294 Townsley, J. D., 105, 111, 136 Treat, M. L., 7,23,25,27,31,41,58,59 Trinci, A. P. J., 149, 153, 194, 200 Trudel, S., 16, 34, 58 Trueblood, C. E., 89, 143 Trueheart, J., 92, 144 Tsai, C. S., 275, 301 Tsao, P. H., 81, 143 Tsuchiya, E., 94, 98, 99, 100, 138, 139, 140, 141, 144 Tsuchiya, T., 6, 66 TsusuC, Y. M., 181, 183, 200 Tu, S.-C., 8, 12, 13, 15, 16, 25, 26, 27, 43, 44, 59, 61, 64, 66, 67 Tuggle, C. K., 255, 256, 265, 267, 268, 294 Turbanti, L., 283, 294 Turner, E. M., 180, 186, 200 Turner, M. A , , 224, 227, 236, 237 Tymon, A. M., 160, 195, 197, 198, 201, 202
U Ulitzur, S., 5, 6, 8, 24, 34, 37, 41. 42, 43, 46, 48, 50, 58, 59, 61, 63, 64, 67 Ullrich, R. C., 159, 160, 191, 196, 198, 200, 201, 202 Ulrich, P., 244, 245, 272, 275, 280, 294, 295 Umezawa, T., 180, 197 Unkefer, C. J., 290, 296 Uno, I., 15&9, 174, 177, 178, 181, 184, 199, 200 Unrau, A. M., 76, 142
Uotila, L., 288, 289, 301 Upper, C. D., 204, 212, 222, 229, 230, 231, 235, 236 Urban, M., 201, 202 Uwajima, T., 242, 272, 291 Uyakul, D., 290, 295 V
Valdivieso, M. H., 91, 92, 143, 144 Vali, G., 204, 208, 209, 231, 237 Valli, K., 149, 180, 190, 196 van Bladercn, P. J., 240, 281, 283, 298, 30 1 Vandegrift, V., 26, 43, 62 Vandekerckhovc, J., 212, 222, 226,237 Vandeloise, R., 278, 293 van den Endc, H., 71, 81, 83, 86, 140, 144 van den Hondel, C. A. M. J. J., 191,198 Vander Donckt, E., 278, 293 van der Valk, P., 149, 153, 154, 163, 179, 185, 197, 200 van Dijken, J. P., 273, 276, 288, 289, 292, 293, 301 VanGcmert, H., 231, 232, 236 van Griensven, L. J. L. D., 192,196,199 Van Huffel, C., 273, 301 van Montagu, M., 212, 222, 226, 237 van Ommen, B., 281, 283, 301 VanRiet, D., 39, 66 Van Schouwenberg, K. L., 6, 61 Varshavsky, A., 89, 140 Vasqucz, G., 254, 300 Vauras, R.,149, 153, 183, 185, 198 Vcenhuis, M., 289, 293 Venugopalan, V. K., 49, 65 Verduyn, C., 273, 301 Vcrmuelen, C. A., 188, 189, 197 Verrinder-Gibbins, A. M., 171,181,200 Vervoort, J., 5, 7, 11, 12, 14,22,23,63, 65, 67 Vim, B., 187, 199 Vigfusson, N. V., 101, 144 Viitanen, H., 153, 183, 198 Villar, C. J., 284, 292 Visser, J.. 5, 7, 14, 23, 63 Vissers, S., 253, 293 Volk, R., 27, 29, 65 von Bahr-Lindstroem, H., 267,269,295
326
AUTHOR INDEX
Vonnegut, B., 207, 237 Von Sonntag, C . , 269, 301 Vos, R. M . , 283, 301 Vroman, B. T . , 79, 143
w Wackett, L. P., 284, 301 Waddle, J. J . , 17, 58, 67 Waelsh, H . , 240. 292 Wagner, J. C., 88, 145 Walker, D. C., 179, 195 Walker, N., 94, 136 Wall, L. A . , 6, 18, 20, 21, 22, 31, 42, 58, 65, 66, 67 Wallace, J. C., 39, 62 Waller, H. D . , 240, 294 Walsh, C. T . , 245, 275, 300 Walton, D. S . , 50, 64 Wampler, J. E . , 34, 65 Wang, Y . , 14, 62 Warholm, M . , 281, 297 Wariishi, H., 149, 180, 190, 196 Warner, H. R . , 242, 255, 256, 294 Warren, C. O . , 76, 141 Warren, G. J . , 204, 208, 210, 211, 212, 213,214,220,221, 222,223,224,225, 226,227, 228,229, 231,233,235,236, 237 Washino, R. K., 80, 139 Watabe, S.,204,212,221,222,228,235, 237 Watanabe, H., 6, 35, 37, 42, 47, 67 Watanabe, J., 232, 237 Watanabe, K., 249, 250, 254, 285, 289, 298, 299, 301 Watanabe, M., 204, 212, 221,222, 224, 226, 228, 232, 235, 237 Waters, M. G., 88, 145 Watson, D., 94, 145, 242, 281, 297 Watson, H. E. E., 88, 137 Watt, K., 290, 296 Watzele, G., 91, 92, 145 Watzele, M., 90, 91, 142, 145 Waxman, D. J . , 281, 301 Weaver, L. H . , 18, 66 Webb, R. A , , 184, 194 Webster, J. G . H., 148, 149, 200 Webster, N. J . G., 123, 124, 145 Wei, S. L., SO, 67
Weihe, G. R., 75, 76, 140, 145 Weilguny, D . , 96, 136 Weiping, X., 104, 145 Weiss, J. F . , 279, 297 Welch, S. K . , 95, 140 Welches, W. R . , 16, 67 Wendel, A , , 240, 270, 294, 301 Werkman, T. B . A , , 84, 145 Wessels, J. G . H., 149, 151, 153, 154, 155, 161, 162, 163, 164, 165, 166-7, 168,169,171,172, 173,174,175,176, 177,179,181,183, 184,185,187,188, 189, 191, 192, 195, 196, 197, 198,199, 200, 201 West, P. A . , 37, 42, 49, 50, 61, 67 Westaway, D . , 224, 236 Wheeler, A . E., 271, 272, 279, 294 Wheeler, H. E., 101, 136 Wheeler, M. H . , 179, 194 Whelan, J., 240, 293 Whitaker, J. P . , 81, 83, 144 White, G. F . , 230, 237 White, J. H . , 124, 140 White, R. H . , 76, 145 White, R. M . , 77, 140 White, V. K . , 24, 49, 65 Whitesides, G. M . , 263, 301 Wiame, J. M . , 244, 251, 252, 253, 258, 259, 293, 299, 301 Widrig, C. A , , 37, 38, 39, 60, 62 Wiebers, J . L . , 260, 301 Wiederanders, B . , 264, 291 Wiemken, A . , 259, 301 Wiggiesworth, L., 248, 251, 298 Wigler, M., 89, 142 Wilkinson, L. E . , 92, 145 William, M. W., 71, 140 Williams, C. H . , 274, 275, 301 Williams, D. L . , 275, 293 Williams, P., 37, 58 Williams, R. J . , 111, 116, 121, 122, 125, 126, 130, 135, 145 Williamson, I . P . , 81, 82, 83, 84, 139 Willoughby, K. J . , 204, 236 Wilson, M. K . , 37, 58 Wilson, T., 7, 13, 14, 60 Wimpee, C. F . , 50, 51, 67 Windels, C. E., 104, 145 Wing, S., 11, 24, 28, 64 Winge, D. R . , 245, 290, 292, 297
327
AUTHOR INDEX
Winkler, U. K., 42, 46, 48, 63, 64 Winnacker, E., 245, 290, 295 Winskill, N., 82, 83, 84, 135 Wittig, R., 88, 135 Woerpel, M. D., 231, 237 Woese, C. R., 48, 66, 242, 294 Wojcicchowecz, D., 91, 139 Wolber, P. K . , 204, 208, 209, 210, 211. 212,213,214,220,221,222,223,224, 225,226,227,228,229,231,233,235, 236, 237 Wolf, D. H., 88, 135, 145 Wolf, D. U., 88, 136 Wolf, J . C., 104, 145 Wolf, K., 255, 258, 290, 294 Wolfe, C. J., 51, 67 Wolfe, R. S., 242, 294 Wolk, C. P.. 5, 34, 60, 66 Wolk, P., 260, 294 Wong, W. M., 154, 185, 196, 200 Wood, D. A . , 180, 185, 186, 190, 191, 195, 200, 201 Woodward, R . , 7, 23, 25, 27, 31, 65 Woodward, C., 267, 300 Woolkalis, M., 2, 58 Worsham, M. B., 241, 293 Wright, A. P. H., 124, 145 w u , c . - w . , 44, 59 WU, F. Y.-H., 44, 59 Wucst, P. J., 190, 201 Wunsch, C. D., 214, 236 Wurtz, T., 84, 145
Yamamoto, M., 96, 124, 137, 261, 262, 299 Yamashita, I., 98, 99, 134 Yamomoto, K. R., 123, 145 Yan, N., 250, 301 Yanagishima, N . , 87, 90, 95, 96, 137, 143, 145 Yang, C. C., 89, 143 Yang, H., 159, 160, 200, 201, 202 Yang, Y., 49, 67 Yankofsky, S. A , , 204, 210, 212, 237 Yano, T., 246, 251, 301 Yanofsky, C., 184, 199 Yarus, M., 5 , 66 Yasataka, T., 100, 141 Yashar, B . M., 170, 201 Yashphe, J., 42, 44, 67 Yasumoto, K., 251, 296 Yawetz, Z. A , , 272, 282, 301 Yax, P., 283, 300 Yetinson, T., 49, 66, 67 Yli-Mattila, T., 153, 171, 179, 181, 183, 184, 199, 201 Yokota, A . , 271, 301 Yokoyama, T., 176, 195 York, B . M., 263, 270, 301 Yoshida, K., 87, 90, 95, 96, 145 Yoshida, M., 98, 139, 143 Yoshitome, H., 212, 235 Yoshizawa, K., 260, 296 Young, R., 39, 50, 66, 67 Yunis, A . , 251, 291
X
Zablen, L. B . , 242, 294 Zantinge, B . , 161, 167, 196, 201 Zarkadas, C., 20, 66 Zawodny, P. D., 83, 144 Zenk, M. H., 245, 290, 295 Zhang, Y., 280, 301 Zhao, J., 204, 212, 237 Zickler, H . , 101, 145 Ziegler, D. M., 263, 270, 301 Ziegler, M. M., 5 , 7, 11, 14, 16, 17, 28, 41, 52, 58 Zingaro, R. A . , 271, 272, 279, 294 Zoetemelk, C. E. M.,255,258,284,292, 296
Xi, L., 15, 16, 26, 43, 67 Xin, X., 15, 16, 27, 67 Xue, C. B., 88, 89, 95, 140, 145 Y
Yabuuchi, S . , 250, 254, 299 Yamachuchi, M., 245, 295 Yamada, H., 289, 296 Yamada, Y., 255, 301 Yamagata, S., 260, 301 Yamaguchi, M., 184, 200
Z
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Subject Index
A
a-agglutinin, 90, 91 a-cells of Sacch. cerevisiae, sex hormones and, 87-96 passim a-factor, Sacch. cerevisiae, 87-96 passim, 102, 132 amino-acid sequence, 87 receptor, 90 signal transduction and, 132 a-factorase, Sacch. cerevisiae, 95 A mating-type gene, 158 dikaryon formation and the, 163-5 of U . maydis, 160-1 A u mating-type gene of S. commune, 159-60 0-Acetyl transferase (OAS) sulphydrylase, 260, 261, 262 0-Acetylhomoserine (OAH) sulphydrylase, 260, 261, 262 Achyla spp. ambisexualis, 75, 76, 78 heterosexualis, 75, 79 sex hormones in, 74-80 Aeon mutation, 157, 158, 159, 161, 165, 167, 172 Acyl carrier proteins, 19 Acyl group, transfer between synthetase and reductase, 21-2 Acyl-AMP, formation, 20 Acylation of synthetase, 20-1 Acyltransferase subunit (t; LuxD) of fatty-acid reductase complex, 18-20 amino-acid sequence comparisons with other lux proteins, 53 gene, see L u x D
Adenosylhomocysteinase, 261 S-Adenosylmethionine demethylase, 261 S-Adenosylmethionine synthetase, 261 Adenylate cyclase C. albicans, mammalian hormones affecting, 125 Sacch. cerevisiae sexual reproduction and, 94 V. fischeri luminescence and, 44 p2-Adrenergic receptor expresses in Sacch. cerevisiae, human, 127 Aerobic bacteria glutathione in, 241, 242 Gram-negative, bioluminescent, 2 Agaricus spp. bisporus fruiting in, 148, 156, 179, 180, 181, 185, 186, 188, 190 genetic manipulation, 192 sex hormones in, 104 bitorquis, fruiting in, 156, 190 Agglutinin, fungal sexual, 9@1 Agriculture, bacterial ice nucleation as a problem in, 230-1 Agrocybe aegerita haploid fruiting in, 171 protein patterns in vegetative monokaryons and dikaryons of, 162 Aldehyde(s) in bioluminescence, 8-9, 18-22 biosynthesis, 18-22 requirement for, &9, 9, 10, 11 as glutathione S-transferase substrates, 282 Aldehyde dehydrogenase, V. harveyi, 24
330
SUBJECT INDEX
Allomyces macrogynus, sex hormones in, 7 1 4 a-agglutinin, Sacch. cerevisiae, 9 6 1 a-cells of Sacch. cerevisiae, sex hormones and, 87-96 passim a-factor, Sacch. cerevisiae, 87-96 passim, 102, 132 amino-acid sequence of, 87 gonadotrophin-releasing hormone (GnRHILHRH) and the, homology between, 127 oestradiol-binding protein in pancreas and effects of, 120 receptor, 89 signal transduction and, 132 a-mating-type gene, 158 a1 hormone of P. sylvaticum, 81 a2 hormone of P. sylvaticum, 81 Alteromonas hanedia, bioluminescence, 2, 50, 51 Amino-acid transport, yglutamyltranspeptidase and, 25840 Ammonium ions as inhibitors of pileus expansion and sporulation, 187 cAMP (cyclic AMP) C. albicans, mammalian hormones affecting levels of, 126 fruiting in fungi and, 177-9 lux gene expression in luminescent bacteria and the role of, 43-5 Sacch. cerevisiae sexual reproduction and, 94 CAMPreceptor protein (CRP), lux gene expression and the role of, 43-5 cAMP receptor protein-binding site (CRP-binding site), luminescence and, 30, 45 Anaerobic bacteria, Gram-negative, bioluminescent, 2 5-a-Androst-16-en-3a-ol metabolite of T. melanosporurn, 132 Antheridia branches antheridiol-induced chemotropism. 76 antheridiol-induced formation, 76 differentiation, antheridiol-induced, 76 Antheridiol, 74, 76-80, 102
activity, 7 6 8 0 , 102 receptor, 79 structure, 75 Anthranilic acid as fruiting-inducing substance in F. arcularius, 181 Antibacterial antibiotics from fungi, glutathione and, structural similarities, 243 Antimalarial drugs interfering with glutathione metabolism, 280 Anti-oestrogen effects on C. immitis, 108 Anti-oxidant defense system in microorganisms, 242-3, 269-74 A p f mutation, 252, 259 Argp- mutation, 252, 259 Arsenite metabolization, 271 Arthroderma spp. benhamiae disease caused by, 130 mammalian sex hormones affecting, 111 , 130 mammalian sex hormones with binding sites in, 115, 119 incurvatum, sex hormones in, 101 Ascocarps, 148 Ascomycotina, fruit bodics of, 148 Ascorbate pcroxidase in cyanobacteria, 27 1 Ascosporogcnous yeasts, sex hormones in, 8&97 Aspartatc residue (position 113) of luciferasc a subunit, 17 Aspergillus spp. Jlavus, glutathione-related processes, 284 fumigatus, mammalian hormones affecting, 106 nidulans glutathione-related processes, 260 psi factors, 103, 104 spore rodlet-deficient mutant, 17G7 oryzae, glutathione-related processes, 245-6, 251 AT contcnt of lux gcnc upstream DNA, 30, 30-1 ATP rcquirements in bioluminescence, 6 Auto-induction of lux gene expression, 3543
331
SUBJECT INDEX
Auxins, hyphal walls and the effects of, 187 A x mutation, 157, 158, 159, 161, 163, 165, 167 A y mutation, 157, 158 25-Azacholesterol, 80 Azospirillurn brasilense, captanresistant mutant. 283 B
B mating-type gene, 158 dikaryon formation and the, 163-5 of U. maydis, 16&1 Bacillus spp . cereus, glutathione-related processes, 248, 260 coagulans, glutathione-related processes, 284 megatheriurn, glutathione-related processes, 274 natto, glutathione-related processes, 246 Bacteria, see also individual species aerobic and anaerobic, see Aerobic bacteria; Anaerobic bacteria bioluminescent, see Bioluminescence ice nucleation in, see Ice nucleation BAR1 gene, 91, 95 Basidiosporogenous yeasts, sex hormones in, 98-100 Basiodiocarps (basidiomes), 148 Basiodiomycotina/basiodiomycetes fruiting in, 148, 149 in industrial mycology, 190, 191 Bcon mutation, 157, 158, 159, 161, 163, 165, 167, 172 Beneckea alginolytica, glutathionerelated processes, 264 P-mating-type gene, 158 Beta,-Adrenergie receptor expresses in Sacch. cerevisiae, human, 127 BIND assay, 233, 234 Bioluminescence, 1-67 applications, 5 control, 3 5 4 8 molecular biology, 5, 24-35 reaction giving, 11-14 intermediates in, 11-14
species of bacteria with, 2 4 , 43, 48-9, see also individual species ecology, 2, 48-52 evolution, 48-57 identification, 49-52 Biotcchnology, fungi in, 190-2 Bis-y-glutamylcystcine redwtase, 275 N'N8-Bis(glutathiony1)spermidine in trypanosomatids, 245 Blakeslea trispora, sex hormones in, 81, 82, 8 3 4 Boletus edulis, cultivation, 191 Botyris cinerea, captan-resistant mutant, 283 Brackets, fruiting in, see Fruiting Use mutation, 174 Bug's ear fruiting body morphology, 174 Buthionine (S,R)-sulphoxime, glutathione biosynthesis inhibited by, 250 Bx mutation, 157, 158, 159 By mutation, 157, 158
c Cadmium detoxification, 290 Cadystins, 290 Calmodulin-binding proteins in Rh. toruloides, 100 Candida spp., 109-10, 111-12, 112-17, 124, 129-30 albicans, 109-10, 11 1-12, 112-17, 129-30 disease caused by (candidosis), 129-30 glutathione-related processes, 255 mammalian hormones affecting, 1 24, 106, 109-10, 111-12, 112-17, 124, 124-5, 129-30 mammalian hormones with binding sites in, 112-20, 121 boidinii, glutathione-related proeesses/methanol dissimilation, 288, 289 budding yeast and mycelial growth phases of, 110 glabrata (Torulopsis glabrata) glutathione-related processes, 245, 290 heavy-metal detoxification, 290
332
SUBJECT INDFX
Candida spp., (con?) mammalian hormones affecting other (non-C. albicans), 106 mammalian hormones binding sites in other (non-C. albicans), 114, 116 tropicalis, glutathione-related processes, 255 utilis, glutathione-related processes, 258, 276 Cantherellus cibarius, cultivation, 191 Captan resistance, glutathione metabolism and, 283 Carbon dioxide, fruiting and effects of, 1814 Carboxypeptidase Y, Sacch. cerevisiae, 88 0-Carotene, trisporic acid formation and, 82, 83, 84 Cascade hybridization, 161-2 Catalase in antioxidant defense, 272 Catecholamines, fungal responses to, 127-8 Cathepsin B-like protease, Sacch. cerevisiae, 88 Cellulase, Achyla spp., antheridiol effects, 77-8 Cephalosporium spp., glutathione and antibiotics from, structural similarities, 243 Cerebrosides, S. commune, as sex hormones or as fruiting-inducing substances, 104, 181 Chemical sensitivity, glutathione in the modulation of, 277-80 Chitin Clostridium spp. and fermentation of, 264 hyphal wall, 187, 188, 189 Sacch. cerevisiae sexual reproduction and formation of, 91 Chitin synthases, 91-2 Chlamydomonas reinhardtii, glutathione-related processes, 27 1 Chorionic gonadotrophin, human, C. albicans binding sites for, 121, 122, 125, 126 CHSl gene and Chsl product, 91 CHSl gene and Chs3 product, 91, 91-2 CHS2 gene and Chs2 product, 91, 92
Chytridiomycetes, sex hormones in, 714 Cladosporium cladosporioides extracts, fruiting induced by, 181 Clostridium spp., glutathione-related processes, 264 COz, fruiting and effects of, 1814 Coccidioides immitis, 107-8, 118, 128 disease caused by (coccidioidomycosis), 107, 128 growth phases, 109 mammalian hormones affecting, 106, 107-8, 128 mammalian hormones with binding sites in, 115, 118 Coenzyme A, glutathione metabolism and, 244 Conjugation, glutathione, 2 8 1 4 Conjugation tube of shmoo, formation, 92-3 Coprinus spp. cinereus (lagopus), fruiting in, 148-9, 154, 156, 157-9, 160, 165, 170, 17&1, 174, 181, 184, 185, 186, 187-8, 188-9 congregatus, fruiting in, 148,154,179, 181, 183, 184 radiatus, fruiting in, 185 Corticosteroid-binding proteins, C. albicans, 113, 130 Corticosterone Candida albicans binding sites for, 112, 113, 114, 130 Candida spp. other than C. albicans with binding sites for, 114 dermatophyte binding sites for, 118, 119 11p-Cortisol, C. albicans binding sites for, 113 Corynebacterium glutamicum, glutathione-related processes, 245 Crithidiafasciculata, glutathione-related processes, 272 Crop plants, bacterial ice nucleation as a problem with, 23Gl CRP and CRP-binding site, see AMP CTT y-lyase (y-cystathionase), 261, 262 Cunninghamella elegans, glutathione and the transferase system in, 284
333
SURJECI' INDEX
Cya gene, V . fischeri luminescence and, 44 Cyanobacteria, hydropcroxide scavenging in, 271 Cyclic AMP, see AMP P-Cystathionase, 261 y-Cystathionase (C7T y-lyase), 261,262 y-Cystathionine synthase, 261 p-Cystathionine synthase, 261 Cysteine residue of luciferase a-subunit, position, 106 16 of synthetase of fatty-acid reductase complex (position 364), 2&1 Cysteinylglycine dipeptidasc, 248, 261 Cytochrome system, luciferase as alternative electron carrier to, 46 Cytoplasm, active, fruiting and the redistribution/movement/ translocation of, 151-3
D Dehydroepiandosterone effects on T. mentagrophytes, 111 Dehydro-oogoniol and 24(28)-Dehydrooogoniol-1, 76 23-Deoxyantheridiol, 74 Deoxycorticosterone effects on T. mentagrophytes, 111 Dermatophytes, 11&11, 118-19, 130-1 disease caused by (dermatophytosis), 130-1 mammalian hormones affecting, 106, 11&11,13&1 mammalian hormones with binding sites in, 118-19 Deuterium oxide, ice nucleation and effects of substituting water with, 2234 Diamide, glutathione oxidized by, 278 Dictyuchus spp., sex hormones, 80 Diethyldithiocarbamate metabolism, 278-9 Diethylstilboestrol P. brasiliensis and effects of, 107 P . brasiliensis binding sites for, 117 Dihydro-4a-peroxy-FMN in bioluminescent reaction: 12, 13
Dihydrotestosterone C. immitis binding sites for, 118 dermatophytes and effects of, 111 4-Dihydrotrisporic acid, trisporic acid formation and, 82, 84 4-Dihydrotrisporol, trisporic acid formation and, 82, 83 5,8-Dihydroxylinoleic acid (psi C) in A . nidulans, 103, 104 8-Dihydroxylinoleic acid (psi B) in A . nidulans, 103 Dikaryon, 163-70 formation, 158, 163-5 biochemical changes during, 163-5 RNA and protein regulation in, during fruiting, 165-70 Dimethyldithiocarbamate metabolism, 27&9 Dipeptidyl aminopcptidase, S . cerevisiae, 88 Disulphidc(s), in microorganisms, mixed, 244, see also Glutathione disulphide; Thiol-disulphide exchanges Disulphide isomerase (PDI), 263, 266 Dithionitc assay for luciferases, 10-1 1 DNA-binding domain of oestrogenreceptor proteins in fungi, 123-4 DNA-mediated transformation systems for fruiting basidiomycetes, 191 Drug toxicity, glutathione protecting against, 277-80 Dunaliella primoleeta, glutathionerelated processes, 271 E Ebselen as an antioxidant in disease therapy, 279-80 Electron carrier, luciferase as alternative, to cytochrome system, 46 Electron transfer in E. coli by thioredoxin and glutaredoxin system, 264, 2 6 6 9 Eln mutation, 174 Energy requirements of bioluminescence, 6-7 Entamoeba histolytica, glutathione not produced in, 242
334
SUBJEC T INDEX
Enterobacteriaceae, bioluminescent, 2 Enterobacterial repetitive intergenic consensus (ERIC) sequence, 334 Environment fruiting in higher fungi and effects of, 18W ice nucleation in bacteria and its significance for the, 230-1 Epoxides as glutathione S-transferase substrates, 282 ERIC (enterobactcrial repetitive intergenic consensus) sequence, 334 Erwinia spp. ananas, ice nucleation gene and protein product in, 212, 220, see also specific gene herbicola, ice nucleation in, 209 genes and proteins, 212, 220, 221, 233, see also specific genes Escherichia coli glutathione-related processes, 242, 249, 251, 254, 255,256,258,264, 265, 26G9, 274, 275, 276, 284, 285, 286, 287, 289 HtrP gene, 29 ice nucleation phenotype transferred to, 211, 222, 224 iron transport and regulation mutants, bioluminescence studies in, 45 N-Ethylmaleimide hormone binding in C. albicans and effects of, 116 ice nucleation in bacteria and effects of, 222 Euglena gracilis, glutathione-related processes, 271 Euprymna scolopes, light organs of, 38-9. 50 Evolution of bioluminescence in bacteria, 48-57 of glutathione metabolism, 242 of ice-nucleation genes in bacteria, 228-30 of mammalian hormone-like molecules and receptor-effector systems in fungi, 131-2 Exp mutation, 174
F Farming, bacterial ice nucleation as a problem in, 230-1 Farncsylatcd fungal sex hormones, 89, 102 Fatty-acid reductase complex, 18-22 in bioluminescence, 18-22 components/subunits, 18-22 see also individual components amino-acid sequence comparisons between various, 5 3 4 identification, 22 luciferase and, direct interaction, 22 Fatty-acid reductase subunit (r; LuxC), 21-2 amino-acid sequence comparisons with other lux proteins, 53 gene, see LuxC Fb+ alleles, haploid fruiting and the, 171 FBF gene andfbf mutation, 172-3, 173, 175 Fi+ alleles, haploid fruiting and the, 171 Fibrillar architecture of hyphal walls, 187 Fis' allele, 174 Fish, monocentrid, light organs of, 38 Flammulina spp. velutipes, fruiting, 154, 185, 190 vetupilis, glutathione-related processes, 246 Flavin (s) bioluminescent reaction in bacteria and its specificity for, 7-8 fruiting in fungi a n d t h e role of, 183 Flavin analogues as antimalarial drugs, 280 Flavin mononucleotide (FMN) and reduced form (FMNHJ, see FMN Flavolus arcularius, anthranilic acid as fruiting-inducing substance in, 181 Flavoprotein, non-fluorescent, of Photobacterium, 2 3 4 FMN and FMNH2 in bioluminescence, 6 , 7 , see also Dihydro-4a-peroxyFMN; -4a-Peroxy-FMN assays using, 9
335
SURJFCT INDEX
FNR (fumarate nitratc reductase), lux gene regulation and, 48 Food(s) freeze processing of, bacterial ice nuclei in, 232 fungi as, 190, 190-1 Salmonella spp., assay in, 233 Formaldehyde dehydrogenase, 288-9 S-Formylglutathione hydrolase, 289 Fosfomycin-resistant E. coli, 284 Fre gene, 28 Free-encrgy relationships governing initiation of ice crystallization, 205 Freeze processing of foods, bacterial ice nuclei in, 232 Frost damage to plants, bacterial ice nucleation causing, 230-1, 230-1 FRTl control element, 1 7 3 4 Fruit bodies (fruiting bodies), 148-55, 185-90 emergence, 148-55 rapid expansion of, 185-90 Fruiting in higher fungi (brackets; mushrooms; toadstools), 147-201 biotechnology and, 19&2 environmental control of, 180-4 genes controlling, 155-75 accessory, 17C-5 mating-type, 155-70 molecular and biochemical indices of, 175-80 R N A and protein regulation in dikaryon during, 165-70 Fucosterol, sex hormones derived from, 76 Fumarate nitrate reductase, lux gene regulation and, 48 Fungi, see also individual species antibiotics from, glutathione and, structural similarities, 243 as food, 190, 1 9 6 1 higher, fruiting in, see Fruiting sex hormones and, see Sex hormones Fur gene mutants of E. coli, bioluminescence studies in, 45 Fur-like protein, 46 FUSl gene, 92 FUS3 gene, 132
Fusarium spp. glutathione and the transferase system in, 284 sex hormones in, 104 G
G-proteins C. albicans, 125 Sacch. cerevisiae, 133 and mammalian G-protein, comparisons, 132 Gap- mutant, 252, 259 GdhA gene, Sacch. cerevisiae, 252 GdhCR gene, Sacch. cerevisiae, 252,253 Gibberella zea, sex hormones, 104 Globulin, corticosteroid-binding, C. albicans, 113 Glucagon effects on C. albicans, 111 on N . crassa, 126 P-Glucan, chitin and, links between, in hyphal walls, 188, 189 p-(1 + 3)-p-(1 + 6)-Glucan, degradation, fruiting and, 154, 163 R-Glucan, degradation, fruiting and, 154, 155, 188 R-Glucanase, 155, 163 Glucocorticoid C. albicans susceptibility associated with use of, 130 receptors (mammalian) C. albicans corticosteroid-binding protein and, 113 Sacch. cerevisiae expression of, 124 Gluconate metabolism in Pseudomonas spp., 286 Glucose-6-phosphate dehydrogenase, glutathione reductase and, interactions, 276-7 Glutacillin, 243 Glutamate dehydrogenase genes, see G d h A ; GdhC NADH-dependent, fruiting and, 186 NADPH-dependent, fruit body expansion and, 186 Glutamine analogues, glutathione degradation inhibited by, 251
336
SUBJECT INDEX
G1utamine:fructose-&phosphate aminotransferase, Sacch. cerevisiae, 92 y-Glutamyl cycle, 247-9, 252-5, 259 regulation, 252-5 y-Glutamylcyclotransferase, 248 y-Glutamylcysteine synthetase, 249-50, 261 gene and deficient mutant, see GshA y-Glutamyltranspeptidase, 248, 250-5, 2 5 8 4 2 , 261 physiological roles, 258-62 Glutaredoxin system, 26&9 Glutathione, 239-301 conjugation, 2 8 1 4 glutathione disulphide and, interconversion, 262-80 metabolism, 247-90 general outlines, 247-60 mutants defective in, 255-8 occurrence and distribution of, and related compounds, 241-7 as sulphur source, mobilization of, 260-2 Glutathione disulphide and glutathione. interconversion, 262-80 Glutathione peroxidase, 262, 269-74 Glutathione redox cycle, 274-80 Glutathione reductase cycle, 274-7 Glutathione synthetase, 249-50, 250, 26 1 mutant deficient in (gshB-), 244 Glutathione thiol transferase, 264 Glutathione S-transferases, 2 8 1 4 Glutathione transhydrogenases, 262-6 Glutathionylspermidine, 244-5 N'-Glutathionylspermidine, in trypanosomes and Leishmania spp., 245 Glyceraldehyde-3-phosphate dehydrogenase (GPD) gcne, A . bisporus genetic manipulation and use of the, 192 Glycogen synthase, N . crassa, insulin effects on, 126-7 Glyoxylase pathway, 284-8 Gonadotrophin, human chorionic, C. albicans binding sites for, 121, 122, 125, 126
Gonadotrophin-releasing hormone and Succh. cerevisiae a-factor, homology between, 127 Gor genes, 275 G P D gene, A. bisporus genetic manipulation and use of the, 192 Gram-negative bacteria, bioluminescent, 2 G s h A gene, 249-50, 252 mutant (gsh-), 244, 253, 255, 256, 257 product, see y-Glutamylcysteine synthetase G s h B gene mutant (gshB-), 244, 255, 256 product, see Glutathione synthetase Guanine nucleotide-binding proteins, see G-proteins H h+(P-ccll) mating type of Schiz. pombe, sex hormones and the, 96 h- (M-cell) mating type of Schiz. p o m b e , sex hormones and the, 96 Halobacterium salinarurn, bis-yglutamylcysteine reductase, 275 Hunsenula polymorpha antioxidant defense in, 273 methanol dissimilation, 288 Hap-5 allcle, haploid fruiting and the, 171 Hap-6 allclc, haploid fruiting and the, 171 Haploid fruiting genes, 170-5 Hawaiian squid, light organs of, 38-9 Heat shock proteins of Achyla spp., antheridiol effects, 79 Heavy metals, detoxification, 289-90 Heterogenic incompatibility, 158 Heterokaryon, formation, 155-9 Heterothallism primary, 148 secondary, 148 Histidine residue (site unknown) of luciferase a subunit, 16 Homocysteine methyltransferase, 261 Homocysteine synthase (OAH sulphhydrylase), 260, 261, 262 Homogenic incompatibility, 156, 158
337
SUBJECT INDFX
Homokaryotic (haploid) fruiting, genes involved in, 170-5 Homoserine acetyltransferase, 261 Homothallism, primary, 148 Hormone(s), sex, 69-145 fungal, 7C-145 mammalian, 105-32 binding sites in fungi for, 112-23 biochemical responses of fungi to, 123-8 in vitro growth and morphogenesis of fungi affected by, 105-12 pathogenesis of fungi and, 128-32 Hormone-binding proteins in fungi, 112-23 HPT gene, L. laccata transformation and the, 191 HtpR, lux gene regulation and, 48 HtrP gene (of E. coli), 29 Hydrogen peroxides, disposal, 269-74 Hydrophobins, 151, 162, 169, 175-7 genes, 162, 169, 173, see also specific genes N-(p-Hydroxbutyryl) homoserine lactone as a luminescence autoinduccr, 37, 42 Hygromycin phosphotransferase gene, L. laccutu transformation and the, 191 Hymenomycetes, fruiting, 148 Hyp hae , 1 87-90 growth in Achyla spp., antheridiol effects, 76 mass flow through, 151 wall, expansion, 187-90 wall-bound hydrophobic proteins shielding, 151 I Ice nucleation (by bacteria), 203-37 activity, measurement, 208-9 applications, 231-2 environmental significance, 230-1 genes, 211-12, 233, see also specific genes evolution, 228-30 as reporters for linked events, 233 heterogeneous, 204, 205, 206-8 by coherent templates, 20-
homogenous, 204 physical basis of, 204-1 1 proteins, 21 1-30 biochemistry and immunology, 22 1-5 domain structure, 212-21 sequence, 212-21 structural models, 225-7 secondary, 204, 204-5 temperatures, 209-1 1, 224, 225 Ice-nucleation diagnostic assay, bacterial (BIND assay), 233, 234 IceC gene, 212 IceE gene and its protein product, 212, 215-19. 220 I M E gene of Sacch. cerevisiae, 172 InaA gene and its protein product, 212 InuC gene, 212 InaW gene and its protein product, 212, 222-3 InaX gene and its protein product, 212 InuY gene, 212 InaZ gene and its protein product, 212, 215-19, 233 Insulin C. albicans and effects of, 11 1 N. crussu and effects of, 112, 1 2 6 7 N . crassa binding sites for, 112 Ionizing radiation, glutathione protecting against effects of, 242, 25&7,277-80 Iron, lux gene regulation and the role of, 45-6 Irradiation, glutathione protecting against effects of, 242, 2 5 6 7 , 277-80 Isopenieillin N and glutathione, structural similarities, 243 Isoprenoids as fungal sex hormones, 7 1-86 Issatchenku orierztalis, glutathione transferase activity in, 282 K Ketoconazole, C. ulbicans eorticosterone binding and effects of, 113, 130 KEXl gene, 88 KEX2 gene, 88
338
SUBJECT INDEX
Kinase, protein, CAMP-dependent, fruiting and, 178 Kloeckera spp., S-formylglutathione hydrolase, 289 Kryptophanaron alfredi symbiont, lux genes, 25 L
Laccaria laccata, transformation, 191 Laccase, 162, 179-80 p-Lactam form of glutathione, 243 S-D-Lactoylglutathione, therapeutic uses and production, 287-8 Lagenidium giganteum, sex hormones, 80 LasR, luxR and, homology, 40 Leishmania spp., glutathione-related processes, 245, 275 Lentinus edodes, fruiting in, 151, 178, 179-80, 190 LexA protein, lux gene expression and, 47-8 Light effects, on fruiting, 1 8 1 4 emission, in bioluminescent bacteria, 6, 6 7 Lignin degradation by fungi, industrial application, 190 Lipids bacterial ice nucleation activity and, 222 peroxidation, 271 glutathione S-transferase protecting against effects of, 282 Lipoxygenase activity in Achyla spp., antheridiol effects, 78 Luciferase(s), 9-18 active-site residues, 1G18 aldehyde specificity, 8-9 assays, 9-14 coupled, 11 dithionite, 1&11 standard, 9-10 electron-carrier function, 46 fatty-acid reductase complex and, direct interaction, 22 flavin substrate specificity, 7-8 genes, duplication, 15, 54-7, see also specific genes
light emission and, 7 mutations, 16-18 reaction involving, 11-14 structurc, 14-18, see also specific subunits (below) primary (=amino acid sequence), 14-18, 5 2 4 quaternary, 14 Luciferase, a subunit (LuxA protein) active sites, 16-17 amino-acid sequence, 14-15, 16 high conservation, 16 amino-acid sequence compared with other lux proteins, 52, 54-7 gene, see L u x A Luciferase, p subunit (LuxB protein) active sites, 17 amino-acid sequence, 14-15, 16 amino-acid sequence compared with other lux proteins, 52, 54-7 gene, see L u x B Lumazine protein (of Photobacterium SPP.), 7, 22-3 in bioluminescent reaction, 13 gene (lump), function/properties/ location, 27, 3CL1 Luminescence, see Bioluminescence L u m p gene (lumazine protein), function/properties/location, 27, 3&1 Luteinizing hormone C. albicans and effects of, 111, 124, 125 C. albicans binding sites for, 121-2 Luteinizing hormone-releasing hormone and Sacch. cerevisiae afactor, homology between, 127 L u x (genes), 5, 24-48 DNA downstream from, 26-9 DNA upstream from, 29-31 duplication, 15, 54-7, see also specific genes expression, 31-48 auto-induced, 3 5 4 3 CAMP-rcgulated, 43-5 differential, 3 1 4 iron-regulated, 45-6 in non-derivative organisms, 34-5 osmolarity-regulated, 47 oxygen-regulated, 46
339
SUBJECT INDEX
physiological and genetic control, 3548 organization, 24-31 Lux (proteins), protcins related to/ accessory, 2 2 4 , see also specific proteins LuxA gene, 1 5 , 24-5, 26 expression, 31 in non-derivative species, 34-5 probes containing and/or probing for, 50, 51 LuxA protein (LuxA protein), see Luciferase, a subunit LuxB gene, 15, 24-5, 26 expression, 31 in non-derivative species, 3 4 4 LuxB protein, see Luciferasc, subunit LuxC gene (for fatty-acid reductase subunit (r)), 24-5, 26 DNA upstream from, 30 expression, 31 LuxC protein, see Fatty-acid reductase subunit LuxD genc (for acyltransferase subunit (t) of fatty acid reductase complex) gene, 24-5, 26 expression, 31 LuxD protein, see Acyltransferase subunit LuxE gene (of synthetasc subunit of fatty-acid reductase complex), 24-5, 26 expression, 31, 33 stem-loop structures, 32 LuxE protein, see Synthetase LuxF gene, 24, 25-6, 27 function/properties/location, 27, 43 LuxF protein, amino-acid sequence, lux proteins with sequences related to, 53, 54-7 LuxG, amino-acid sequence comparisons with other lux proteins, 54 LuxG gene, 2 6 9 function/properties/location, 2 6 9 LuxH gene, 29 functiodpropertiedlocation, 27, 29 Luxl gene (auto-inducer synthase), 27, 29-30, 39 function/propertiesfocation, 27, 29-30
LuxN gene, 43 LuxR gene, 27, 29-30, 40, 41 function/properties/location, 27, 29-30 as member of superfamily of transcriptional regulators, 40 probes containing and/or probing for, 50 LuxR protein, 3 9 4 0 , 45 LuxR protein-binding site (lux rcgulon operator), 40, 48 LuxR* gene, 27, 30 function/properties/location, 27, 30 LuxY gene (yellow fluorescence protein), function/properties/ location, 27, 31 Lysp- mutation, 252, 259 M M-cells of Schiz. pombe, sex hormones and the, 96, 97 M-factor, Schiz. pombe, 96, 97 Malaria, glutathione metabolismaffecting drugs in, 280 Malonic dialdehyde accumulation, 271 Mammalian sex hormones, fungal interactions with, see Sex hormones Mannitol, fruit-body expansion and, 186 Marine environments, luminous bacteria in, 49, 50 Mating type, yeast, 8 6 9 7 , 172 sex hormones and the, 8 6 9 7 Mating-type genes, 155-70 fruiting controlled by, 155-70 as master regulators, 155-9 molecular structure, 159-61 Messenger RNA, see RNA Metals, heavy, detoxification, 289-90 Meteorological significance of bacterial ice nucleation, 231 Methanol dissimilation, 288-9 L-Methionine (S)-sulphoxime, glutathione biosynthesis inhibited by, 250 Methylglyoxal metabolism, 289 Methylglyoxal synthase, 285-6 Methylmercaptan release from S. commune, 184
340
SUBJECT INDbX
Methylobacterium organophilurn, glutathione S-transfcrase in, 281 Methyltrienolone, C. irnmitis binding sites for, 118 MFal genes, 88 M F d genes, 88 Microfibrils, hyphal walls, 187 Microsporum canis disease caused by, 130 mammalian hormones affecting, 11 1, 115, 130 Mitochondria1 genome of A . bisporus, structural studies, 192 Modifier mutations, 161 Monocentris japonicus, light organs of, 38 Monokaryotic (haploid) fruiting, genes involved in, 17@5 Monophenol oxidase, fruiting and, 179 Mucor spp. flavus, 86 glutathione peroxidase and antioxidant defense in, 272 japonicus, glutathione transferase activity in, 282 mucedo, 81, 82 sex hormones in, 81, 82, 86 Mushrooms, fruiting in, see Fruiting Mycelium, secondary (heterokaryon), formation, 155-9 Mycobacterium smegmatis, glut at hion e degradation in, 250 Mycotoxin, ocstrogenic, zearelenone as an, 104
N NADH-dependent glutamate dehydrogenase, fruiting and, 186 NADH-dependent glutathione reductase, 274, 275 NADPH, acylreductase intermediate reduced by, 22 NADPH-dependent glutamate dehydrogenase, fruit-body expansion and, 186 NADPH-dependent glutathione reductase, 274, 275, 276-7 NAD(P)H:FMN oxidoreductases, 24 Nafoxidine effects on C. irnmitis, 108
NeuroJpora crassa, 112, 126-7 glutathione-related processes, 260 light effects on gene expression in, 184 mammalian sex hormones affecting, 106, 112, 126-7 mammalian sex hormones with binding sites in, 121 mating-type genes, 160 sex hormones in, 100-1 spore rodlet-deficient mutant, 176 Nitrofurans, glutathione reductase inhibited by, 280 Nitrosoureas, glutathione reduetase inhibited by, 280 Nostoc muschorum, hydro peroxide scavenging in, 271 Nuclear migration, fruiting and, 158 0
Oestradiol Candida albicans binding sites for, 114, 116-17 Cundida albicans and effects of, 110, 114, 125 Candida spp. other than C. albicans with binding sites for, 116 Cundida spp. other than C. albicans and effects of, 114 Coccidioides irnmitis and effects of, 107-8, 128 Coccidioides irnmitis binding sites for, 118 P. brasiliensis and effects of, 107, 114, 124, 129 P. brasiliensis binding sites for, 117 pancreatic protein binding, 120 Sacch. cerevisiae and effects of, 105, 115, 123,124 Sacch. cerevisiae binding sites for, 119-20 Oestriol C. albicans and effects of, 110 P. brasiliensis binding sites for, 117 Ocstrogen(s), see also specific oestrogens and Anti-oestrogens P. brasiliensis and effects of, 107 zcarelenone acting as an, 104, 117 Oestrogen-receptor proteins of fungi, characteristics, 1 2 3 4
SUBIECT INDEX
Oestrone, P. brasiliensis binding sites for, 117 Oogoniol, 7 5 4 , 102 structure, 75 synthesis and release, 77, 102 Oogoniol-1 , 76 Oomycctes, sex hormones in, 74-81 Open-reading framcs, mating-type genes and, 160, 161 Ophthalmic acid in U . pinnatifida, 246 ORFs (open-reading frames), matingtype genes and, 160, 161 Osmolarity, lux gene expression regulated by, 47 Osmotica, fruit-body expansion dependent on, 186 Oxidases, phenol, fruiting and, 179-80 Oxidative stress glutathione reductase cycle during, 280 luminescence system with role in, 46-7 Oxidoreductases, NAD(P)H:FMN, 24 N-( p-Oxohexanoyl) homoserine lactone as a luminescence auto-inducer, 37 Oxygen luminescence dependent on, 4, 5 , 46 in Photobacterium spp., 4, 5, 46 toxicity in micro-organisms, preventive mechanisms, 242-3, 269-74 5-0xyprolinase, 248 P
P-cells of Schiz. p o m b e , sex hormones and the, 96, 97 P-factor, Schiz. p o m b e , 96, 97 P A B l gene, mating-type gene locations in relation to, 159, 160 Pancreatic oestradiol-binding protein, 120 Paracoccidioides brasiliensis, 107, 117, 124, 128-9 disease caused by (paracoccidioidomycosis), 128-9 growth phases, 108 mammalian hormones affecting, 1 28-9, 106, 107, 124
341
mammalian hormones with binding sites in, 114, 117 Parisin, 74 Pat1 mutants of Schiz. p o m b e , 97 PDI protein (disulphide isomerase), 263, 266 Penicillium spp. antibiotics from, structural similarities with glutathione, 243 chrysogenurn, glutathione-related processes, 254 oxalicum, glutathione-rclated processes, 246 Peptides as sex hormones in yeast, 86100 Peroxidation, lipid, see Lipid Peroxides, disposal, 269-74 Peroxyflavin hemiacetal in bioluminescent reaction, 12 4a-Peroxy-FMN intermediates in bioluminescent reaction, 12, 13, 14 Phanerochaete chrysosporum CAMP and fruiting control in, 178 lignin degradation by, 190 Phenol oxidases, fruiting and, 179-80 2-Phenylethanol, bacterial ice nucleation and effects of, 222 Pheromones aldehyde, unsaturated, 9 bacterial, autoinducers as, 37 fungal, 7G104, 132 insect, 9 Phosphoglucomutase, fruit body, 185 Photobacterium spp. leiognathi, 2, 23 lux genes and their regulation, 25, 25-6, 30, 31, 33, 42-3, 46, 47 lux protein sequence comparisons with other species, 52-7 passim lumazine protein, see Lumazine protein non-fluorescent flavoprotein, 2 3 4 oxygen dependent-luminescence, 4, 5 , 46 phosphoreum, 2, 4 acyltransferase subunit of fatty-acid reductase complex, 19 luciferasc assay, 12
342
SUBJECT INDEX
Photobacterium spp., phosphoreum, (cont) lux genes and thcir regulation, 25, 2 5 4 , 30-1, 31, 42, 46, 47 lux protein sequence comparisons with other species, 52-7 passim synthetase subunit of fatty-acid reductase complex, 20-1 tetradecanal isolated from, 8 Photosynthetic bacteria, anti-oxidant defense systems, 271 Phototrophic organisms, glutathione metabolism, 242 Phycomyces spp. blakesleeanus, sex hormones in, 76, 81, 82, 83 hyphal wall expansion, 187 Phytochelatins, 290 Phytophthora spp. cactorum, 80 parasitica, 81 sex hormones, 80, 81 Pileus, stipes elongation and the, 186 Plants, crop, bacterial ice nuclcation as a problcm with, 23@1 Plasmodium spp. berghei, antioxidant defense systcm, 272 falciparum, antioxidant defense system, 272, 280 Pleurotus ostreatus, commercial use, 190 Polyporus ciliatus, fruiting in, 171 E’orphyridiurn cruentum, glutathionerelated processes, 271, 272 Potassium-ion transport, glutathione involvement in, 260 Precocious sexual inducer, A . nidulans, 103 Prednisolone, C. albicans binding sites for, 113 Pregnanediol effects on C. ulbicuns, 110 Pregnanetriol effects on C. albicans, 110 Prepro-a-factor, 88 Primordia, fruit body, light-induced, 181-3 Progesterone A . benhamiae binding sites for, 115, 119 Candida albicuns binding sites for, 112, 113
Coccioides immitis and effects of, 108, 128 Coccioides immitis binding sites for, 118 M . canis binding sites for, 115, 119 P. brasiliensis and effccts of, 107 P. brasiliensis binding sites for, 117 T. mentagrophytes binding sites for, 115, 118 T. mentagrophytes and effects of, 111 T. rubrum binding sites for, 115, 119 Progestins C. immitis binding sites for, 118 dermatophyte-binding sites for, 115 Promcgestone (RS020) Candida albicans binding sites for, 113 Coccidioides irnmitis binding sites for, 115 T. mentagrophytes and effects of, 111 Protein(s), see also specific proteins in higher fungi, 161-3, 165-70 during fruiting, in dikaryon, regulation, 165-70 during vegetativc growth, regulation of, 161-3 hydrophobic, 151 hormonal (and polypeptide hormones), mammalian, fungi affected by, 106, 111-12, 121-3, 125-7 hormone-binding, in fungi, 112-23 ice-nucleation, in bacteria, see Ice nucleation Protein kinase, CAMP-dcpendcnt, fruiting and, 178 Protein tyrosine kinase activity of insulin-binding proteins in N . crassa, 121 Proteus mirabilis, glutathione-related processes, 2 55,249,250,251,282 Protozoa, anti-oxidant defense system, 272 pSc1 protein, 169, 177 pSc3 protein, 169, 176, 177 pSc4 protein, 169, 177 Pseudornonas spp. aeruginosa glutathione reductase in, 275 glutathione S-transferase in, 281 lasR of, luxR homology with, 40
343
SUBJECI INDEX
fluorescens, ice-nucleation gene in, 212, see also specific gene putida glutathione peroxidase activity, 248, 271 glyoxylase pathway in, 28.5, 287-8 syringae, ice nucleation in, 209, 210 applications, 232 genes for, 212,233, see also specific genes viriflava, ice nucleation gene, 212, see also specific gene otherhon-specified species, glyoxylase pathway in, 286 Psi factors, A. nidulans, 103, 104 Pyrenopeziza brassicae, sexual factors, 1034 Pyricularia oryzae, anti-oxidant defense in, 272 Pythium spp. sex hormones, 80, 8Gl sylvaticurn, 80-1
Rhodosporidium toruloides, sex hormones in, 87, 99-100 Rhodotorucine A, 1 02, 99-100 amino-acid sequence, 87 Riboflavin as luciferase substrate, 7 Ribonucleotide reductase, 267,268,269 Ribosomal RNA, fruiting and detection of, 151-3 R M E gene of Sacch. cerevisiae, 172 RNA (in general), regulation, in higher fungi during fruiting, in dikaryon, 165-70 during vegetative growth, 161-3 mRNA (messenger RNA) synthesis in Achyla spp., antheridiol effects, 78 in lux genes of luminescent bacteria, 31 in S. commune during fruiting, 16670, 175, 176 rRNA (ribosomal RNA), fruiting and detection of. 151-3 S
R R1881, C. immitis binding sites for, 118 R5020, see Promegestone Radioprotective effects of glutathione, 242, 2 5 6 7 , 277-80 R A M I D P R I , 89 RAS proteins in C. albicans, and their genes, 133 in Sacch. cerevisiae, farnesylation, 89 Reductase(s) bis-y-glutamylcysteine, 275 fatty-acid, see Fatty-acid reductase fumarate nitrate, lux gene regulation and, 48 glutathione, 274-7 NAD(P)H:FMN oxido-, 24 ribonucleotide, 267, 268, 269 trypanothione, see Trypanothione reductase p-Resorcyclic acid as a fungal sex hormone, 104 Retinal, trisporic acid formation and, 82, 83 R H A I I R H A U R H A 3 , 99 Rho-dependent terminator, Vibrio spp. lux gcnes, 29
Saccharomyces spp., 8 6 9 6 , 119-20, 1 2 3 4 , 127, 132-3 cerevisiae, 8 6 9 5 , 106, 119-20, 123-4, 127 glutathione-related processes, 242, 244,248,249-50,252-3,254,255, 257, 258, 259, 26G2, 273, 275, 280, 282, 285, 286289 mammalian hormones affecting, 105, 106, 123-4, 127-8, 133 mammalian hormones with binding sites in, 115, 119-20, 121 exiguus, 95, 96 kluyveri, 95, 96 mating-type control of sporulation in, 86-96, 172 sex hormones in, 8 6 9 6 , 132 Saccharomycopsis lipolytica, glutathione-related processes, 260 Salmonella spp. assay in food, 233 typhimurium, glutathione-related processes, 258, 284 Salt concentration, lux gene expression regulated by, 47
344
SUBJECT INDEX
SAM dcmethylase, 261 SAM synthetasc, 261 Saprolegnia ferex, sex hormones, 80 Scl gene, 162, 169, 173, 175 protein product (pscl), 169, 177 Sc.? gene, 162, 167, 169, 173, 175 protein product (pSc3), 169, 176, 177 Sc4 gene, 162, 169, 173, 175 protein product (pSc4), 169, 177 Sc7gene, 169 Sc14 gene, 169 Schizophyllum commune fruiting in, 148-9, 151-3, 154, 155, 156, 157-9, 15940, 163, 165-8, 171, 172-5, 175-6, 179, 180, 181, 183, 184, 185 scx hormones, 104 Schizosaccharomyces p o m b e , 9 6 7 glutathione-relatcd processes, 245, 258, 290 heavy-metal detoxification, 290 sex hormones in, 9 6 7 Sea water (marine environments), luminous bacteria in, 49, 50 Selenium-containing organic compounds in anti-oxidant defense, 278-9 Selenium-dependent glutathione pcroxidase, 262, 270 Septa1 dissolution, fruiting and, 158 Serine, acetylation, 260, 261 Serine acetyltransferase, 261 Serine residue of acyltransferase as acylation site (position 70), 19, 20 of ice-nucleation proteins, 228 of luciferase u subunit (position 227), 17 Serpula lacrymans, water flow in, 151 Sex hormones, 69-145 fungal (endogenous hormones; pheromones), 7C104, 132 mammalian, fungi affccted by, 105-32, 133 binding characteristics, 112-23 biochemical responses of, 123-8 in vitro growth and morphogenesis of, 105-12 Sexual factors, P . brassicae, 1 0 3 4
Sexual morphogens, fungal hormones as, 103-4 Sexual spores, fruit bodies for dissemination of, see Fruit bodies; Fruiting SF, P. hrassicae, 1 0 3 4 Shewanella hanedai, bioluminescence, 2, 50, 51 Shmoos, formation/development and behaviour, regulation, 89-90,90, 91, 92-3, 93 Sigma factors, L u x R gene as member of superfamily of, 40 Signal transduction in Sacch. cerevisiae following interactions with u/a mating factors or mammalian hormones, 132-3 Sirenin, 7 1 4 , 102 structure, 73 Sirobasidiurn magnum, sex hormones in, 99 Slime moulds, sex hormones in, 101 Snow-making with ice-nucleating bacteria, 231-2 Sodium chloride, lux gene expression regulated by, 47 Somatostatin as a coligand with pancreatic ocstradiol-binding protein, 120 Spermidine glutathione metabolism and, 244-5 Sphingolipids, S. commune, as fruitinginducing substances, 181 Spores, sexual, fruit bodies for dissemination of, see Fruit bodies; Fruiting Squid, light organs of, 38-9, 50 STE2 gene, 89 S T E 3 gene, 90 STE7 gene, 132 S T E l l gene, 132 STEl2 gene, 132 STE13 gene, 88 Stem-loop structures in lux genes, 31-3 Steroid hormones, mammalian, 105-1 1, 112-20, see also specific hormones fungal binding sites for, 112-20 fungi affected by, 105-11, 112-20 biochemical responses of, 123-5
345
SUBJECT INDEX
in vitro growth and morphogenesis of, 105-1 1 Sterols, as fungal sex hormones, 80 Stilboestrol, P . brasiliensis and effects of, 107 Stipes of fruit bodies, elongation, 185-6, 187-8 Streptococcus faecalis, glutathionerelated processes, 244 Stress, oxidative, see Oxidative stress Sulphate esters as glutathione Stransferase substrates, 282 Sulphides in bacteria, 241, see also Disulphides Sulphur source, glutathione mobilization as, 2 6 C 2 Sulphydryl blocking/modifying agent hormone binding in C. albicans and effects of an, 116 ice nucleation in bacteria and effects of, 222 Superoxide dismutase in anti-oxidant defense, 272 Synechococcus spp., hydroperoxide scavenging in, 271 Synthase(s) auto-inducer, 38 gene, in luminescent bacteria, see LUXZ p-cystathionine, 261 chitin, Sacch. cerevisiae, 91-2 y-cystathionine, 261 glycogen, N . crassa, insulin effects on, 1267 homocysteine ( O A H sulphydrylase), 260, 261, 262 methylglyoxal, 2 8 5 4 Synthetase(s) of fatty-acid reductase complex (IuxE) acylation of, 20-1 amino-acid sequence comparisons with other lux proteins, 53, 54 gene, see LuxE y-glutamylcysteine, see yGlutamylcysteine synthetase glutathione, see Glutathione synthetase SAM (S-adenosylmethionine), 261
T Tamoxifen effects on C. immitis, 108 Temperature fruiting and effects of, 1 8 1 4 ice nucleation and effects of, 209-1 1, 224, 225 Testosterone C. irnmitis affected by, 108 C. irnmitis binding sites for, 115, 118 Tetradecanal as natural aldehyde in bioluminescence, 8 Tetradecanoyl derivatives as acyl substrates for acyltransferases, 19 Tetrahymena thermophila, glutathione transferase in, 282 Tetramethylthiuram disulphide (thiram), glutathione metabolism and effects of, 27&9, 280 Thin (thn) mutation in S. commune, 173 Thiol-disulphide exchanges, 2 6 3 4 Thioredoxin system, 2 6 6 9 Thiram, glutathione metabolism and effects of, 278-9, 280 T H N gene and thn (thin) mutation in S. commune, 173, 175 Toadstools, fruiting in, see Fruiting TonB gene 1:iutants of E .coli, bioluminescence studies in, 45 (+)-Torreyol, hydrophobicity of, fruiting and the, 151 Torulopsis glabrata, see Candida spp. Transcriptional regulators, superfamily of, luxR as member of, 40 Transferase(s) y-glutamylcyclo-, 248 glutathione S-, 281-4 glutathione thiol, 264 homocysteine methyl-, 261 homoserine acetyl-, 261 hygromycin phospho-, L. laccata transformation and the gene for, 191 serine acetyl-, 261 as subunit (t) of fatty-acid reductase complex, see Acyltransferase Transformation systems, DNAmediated, for fruiting basidiomycctes, 191
346
SUBJECT INDEX
Tremella spp., 98-9 brasiliensis, 98 mesenterica, 87 sex hormones in, 87, 98-9 Tremerogen A-10, 98, 102 amino-acid sequence, 87 Tremerogen a-13, 98, 102 amino-acid sequence, 87 Tremerogen A-9291-1, 98 Trichoderma spp., sex hormones, 80, 81 Tricholoma spp. matsutake, cultivation, 191 shimeji, glutathione degradation in, 250 Trichophyton spp. mentagrophytes disease caused by, 130 mammalian hormones affecting, 11G-11, 115, 130 rubrum disease caused by, 130 mammalian hormones affecting, 111, 115, 130 Trisporic acids, 8 1 4 , 102 Trisporol, trisporie acid formation and, 82, 84 TrxA mutation, 268 TrxB mutation, 268 Trypanosoma spp. (and trypanosomatids) drugs acting against, 283 glutathione-related processes, 245, 251, 272, 275, 282, 283 Trypanothione, trypanosomatid, 245 Trypanothione reductase in H . salinarum, 275 in trypanosomatids, 245, 280 inhibitors of, as antimalarial drugs, 280 Tuber spp. magnaturn, cultivation, 191 melanosporum 5-a-androst-16-en-3cI-olmetabolite of, 132 cultivation, 191 Tyrosinase, fruiting and, 179 Tyrosine kinase activity of insulinbinding proteins in N . crassa, 121
U UDP-N-acetylglucosamine, Sacch. cerevisiae, 92 Undaria pinnatifida, ophthalmic acid, 246 Ustilago maydis, mating-type genes, 16G1
v Vegetative growth of higher fungi, RNA and protein regulation during, 161-3 Vibrio spp. bioluminescent strains, 2, 43, 49 identification and ecology of, 50,51 cholerae, 2, 49, 50 jischeri, 3 9 4 0 assay of luciferase, 13 lux genes, amino-acid sequence comparisons with other species, 52-7 passim lux genes, DNA downstream from, 29, 30 lux genes, DNA upstream from, 30 lux genes, expression, 36, 3 7 4 0 , 43-5, 45-6, 4G7, 47, 47-8 yellow fluorescence protein, see Yellow fluorescence protcin harveyi, 4&2 active-site residues in luciferase, 16-17 aldehyde biosynthesis and the transferase subunit in, 19 a and p luciferase subunit sequence, 14-15 assay of luciferase, 10, 12 flavin as substrate for luciferase in, 7 lux genes, amino-acid sequence comparisons with other species, 52-7 passim lux genes, DNA downstream from, 29 lux genes, DNA upstream from, 30 lux genes, expression, 31, 36, 4G2, 43-5, 46 lux-related proteins, 24 otherhinor references, 4, 8 logei, 2, 50
347
SUBJECT INDEX
orientalis, 2, 51 splendidus, 2, 51 vulnificus, 2, 49, 51 Vibrionaceae, bioluminescent, 2 Volvariella volvacea, commercial use, 190
auto-induced, 43 oxygen induced, 46 lux gene organization, 29 lux protein sequence comparisons with other species, 52-7 passim Y
W
Water fruiting in fungi and the transport of, 151, 177, 186 supercooled crystallization by bacteria of ice from, see Ice nucleation metastability, 205
x Xunthornonas curnpestris, ice nucleation gene, 212, see also specific gene Xenorhabdus lurninescens aldehyde specificity, 8 bioluminescence (in general), 2 lux gene expression, 3 3 4 , 43, 46
YCL313 gene, 266 Yeasts, sex hormones in, 86100, 132-3, see also individual species Yellow fluorescence protein (of V . fischeri), 7, 23 in bioluminescent reaction, 13 gene (luxY), functionlpropcrticsl location, 27, 31 1
Zearelenone as a fungal sex hormone, 104, 117 Zygomycctes, sex hormones in, 8 1 4 Zygophore, 8 4 6 formation, induction, 81, 84 Zygophotropism, 8 4 6
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