No title

Advances in

MICROBIAL PHYSI0LOGY

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Advances in

MICROBIAL PHYSIOLOGY edited by

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

J. GARETH MORRIS Department of Botany and Microbiology University College Wales Aberystwyth

Volume 17

1978

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

ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road London NW 1 United States Edition published by ACADEMIC PRESS LTD. 111 Fifth Avenue New York, New York 10003

Copyright 0 1978 by ACADEMIC PRESS INC. (LONDON) LTD.

'

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

Library of Congress Catalog Card Number: 67- 19850 ISBN: 0- 12-0277 17-4

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

Contributors to Volume 17 MARGARET M. ATTWO OD, Department o f Microbiology, University o f Shejjeld, England PATRICK J. BRENNAN, Department of Biochemistry, University College, Dublin, Ireland A. D. BROWN, Department ofBiology, University o f Wollongong, Wollongong, N.S.W. 2500 Australia BRUCE L. A. CARTER, Department o f Genetics, Trinity College, University $Dublin, Dublin 2, Ireland W. HARDER, Department o f Microbiology, The University o f Groningen, The Netherlands DOROTHY M. LOSEL, Department o f Botany, University o f Shefield, Shejjeld SIO 2TN,England A. D. WARTH, C.S.I.R.O. Division $Food Research, North Ryde, N . S . W . 21 I3 Australia

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Contents Molecular Structure of the Bacterial Spore by A . D. WARTH

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I1. Spore Morphology . . . 111. Exosporium and Appendages

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I . Introduction

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VI . VII . VIII . IX . X.

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A . Morphology . . . B.ChemicalComposition . Coats . . . . . . . . A . Morphology . . . . . . B . Chemical Composition and Structure . C. Biosynthesis . . . . . . D . FunctionofSporeCoats . . . . Cortexand GermCellWall . . . . A . Morphology . . . . . . B . ChemicalStructure . . . . . C . Lytic Enzynes . . . . . . D . Biosynthesis . . . . . . Core . . . . . . . . . A . Macromolecular Composition . . B . Low Molecular Weight Compounds . Ionic Composition of Spores . . . . Water Content and Physical State of the Core Mechanisms for the Dehydration of the Core Acknowledgement . . . . . . . . . . . . . References

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Physiology of Fungal Lipids: Selected Topics by PATRICK J. BRENNAN and DOROTHY M . LOSEL I . Introduction . . . . . . . I 1 . Location of Lipid in Fungal Cells . . . 111. Lipids and Fungal Membranes . . . . A . Membranes-General Considerations . B . Phospholipids-TypesandDistribution . C. Phosphoglycerides-PhysiologicalAspects D . Glycolipids . . . . . . . E. Sphingolipids . . . . . . IV . Biosynthesis of Fungal Lipids . . . . A . Phospholipids . . . . . . B . Glycolipids . . . . . . . C . Sphingolipids . . . . . .

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CONTENTS

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. V . Role of Lipids in Fungal Morphogenesis . A . Hormonal and Growth-Regulating Factors B . Lipid Reserves in Morphogenesis . . C. Lipid Reserves and Secondary Metabolites D . Lipid Metabolism in Morphogenesis . . VI . Role of Lipid in Fungus-Host Relationships . A . Fungal Associations with Plant Tissues . B . Fungi Associated with Insects . . . C . Fungi Pathogenic to Man and Animals . D . Discussion . . . . . . . References . . . . . . .

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113 114 121 130 133 140 140 155 159 163 164

Compatible Solutes and Extreme Water Stress in Eukaryotic Microorganisms by A . D. BROWN I . Introduction

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11. RoleofPolyhydric Alcohols

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A . General . . . . . . . B . Specific . . . . . . . . PhysiologyofXerotolerance . . . . A . XerotolerantYeasts . . . . . B . Xerophilic Yeasts . . . . . . C . Xerotolerant Fungi . . . . . D . Halophilic Algae . . . . . . E . IntermediateXerotolerance . . . Regulation of Compatible Solute Accumulation Summary . . . . . . . . Acknowledgements . . . . . . References . . . . . . . .

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181 184 184 188 197 198 212 215 215 223 223 237 239 239

The Yeast Nucleus by BRUCE L. A . CARTER I . Introduction . . . . . . . . . I1 . Nuclear Morphology . . . . . . . 111. Nuclear Division . . . . . . . . IV . Yeast Chromosomes . . . . . . . A . Introduction . . . . . . . . B . Histones . . . . . . . . . C. Sizeofyeast Chromosomes . . . . . D . Localization of Genes o n Chromosomes . . . . . V . Initiation of Nuclear DNA Synthesis . . . . . . . VI . Nuclear DNA Replciation VII . Nuclear Control Over Mitochondrial-DNA Replication . . . . . . VIII . Nuclear DNA Enzymes . A . DNA Polymerases . . . . . . . B . DNA-Dependent RNA Polymerases . . . C. Poly(A) Polymerases . . . . . . .

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244 245 247 249 249 251 254 256 260 266 271 271 271 273 280

CONTENTS

Expression of Nuclear Genes . . . . . . Expression of Yeast Genes in Eschen'chca coli . . . Integration of Growth and Nuclear/Cell Division . SomeTechnical Considerations . . . . . A . Isolation of Yeast Nuclei . . . . . . B. Isolation of a Nucleolar Fraction from Yeast Nuclei . . C . Isolation of Chromatin from Yeast Nuclei D . InhibitionofNuclear Functions . . . . E . DNA Estimation . . . . . . . F. Nuclear Staining . . . . . . . XI11 . Conclusions . . . . . . . . . XIV . Acknowledgements . . . . . . . . References . . . . . . . . . IX . X. XI . XI1 .

Biology. Physiology and Biochemistry of W . HARDER and MARGARET M . A-TTWOOD

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Hyphomicrobia by

I . Introduction . . . . . . . . . . . . I1. BiologyandPhysiologyofHyphomicrobia . . . . . . A . The Genus Hyphomicrobium . . . . . . . . B . Enrichment and Isolation . . . . . . . . . . . . . . . . . . . . . C. Nutrition D . Life Cycles and Pleomorphism . . . . . . . . E . Effect of Environment o n Morphology . . . . . . F. Ecology . . . . . . . . . . . . 111. Biochemistry of Hyphomicrobia . . . . . . . . A . Biochemistry of Growth on Reduced One-Carbon compounds . B . Biochemistry of Growth on Two-Carbon Compounds . . . C. Possible Role of Cytochromes . . . . . . . . D . Biochemical Basis for Restricted Methylotrophy in Hyphomicrobia IV . Conclusion . . . . . . . . . . . . . V . Acknowledgements . . . . . . . . . . . References . . . . . . . . . . . . . Author Index . . . . . . . . . . . . Subject Index . . . . . . . . . . . .

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Molecular Structure of the Bacterial Spore A. D. WARTH C.S.I.R.O. Division of Food Research, North Ryde, N.S. W. Australia 21 13

I. Introduction

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11. SporeMorphology

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111. Exosporium and Appendages . . A. Morphology . . . . . B. ChemicalComposition . . . IV. Coats . . . . . . . A. Morphology . . . . . . B. Chemical Composition and Structure . C. Biosynthesis . . . . . . D. FunctionofSporeCoats . . . V. Cortex and Germ Cell Wall . . . . A. Morphology . . . . . . B. Chemicalstructure . . . . C. LyticEnzymes. . . . . . D. Biosynthesis . . . . . . VI. Core . . . . . . . . A. Macromolecular composition . . B. Low Molecular Weight Compounds . V I I . Ionic Composition of Spores . VIII. Water Content and Physical State of the CoEe IX. Mechanisms for the Dehydration of the Core X. Acknowledgement. . . . . . References . . . . . . .

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I. Introduction

In the differentiation of a bacterial cell into a spore, a number of new morphological structures are formed. The cortex and germ cell wall are specialized adaptations of vegetative structures. Spore coats, 1

2

A. D. WARTH

exosporia and appendages are new structures embodying new classes of microbial products. Their novelty is made possible by the strategy of intracellular synthesis and assembly in the sacrificial sporangial cell. Within the spore cell, or core, unique constituents such as dipicolinic acid, and very different proportions of normal metabolites and electrolytes are found. Some of the intracellular macromolecules have spore-specific modifications, but in the main, a normal complement of enzymes, ribosomes and nucleic acids is present. The mature spore has very well known properties of resistance to heat, radiation, enzymes, disinfectants and other deleterious agents and an absence of endogenous metabolism. In this article, knowledge of the composition and structure of the spore cytoplasm and each of the spore integuments will be reviewed, and the contribution of each component to determining the essential properties of the spore will be considered. Of particular interest is the heat resistant and ametabolic state of spores. This appears to be mainly a consequence of a reduced water content in the core. The final section discusses the possible chemo-mechanical properties of the cortex, and examines models for its role in the dehydration of the core.

11. Spore Morphology

Spores of all species have the same basic structure. For example, the spore of Bacillus cereus (Fig. 1 ) has a central core (c) or protoplast, surrounded in turn by a plasma membrane (pm), germ cell wall (gcw), cortex (cx), coats (cts) and exosporium (ex.).The core, plasma membrane and germ cell wall constitute a condensed cell, which is contained within and is protected by the outer integuments. Much variation between species is found in the complexity of the coats. Even greater variation is found in structures external to the coats. An exosporium as seen in the spore of B . cereus (Fig. 1) is found in only a few species but more elaborate structures, termed appendages, are common among Clostridium spores (Rode, 197 1). Parasporal bodies of various forms are found in some Bacillus species, mostly insect pathogens. The best known of these are the large protein crystals formed by strains of B. thuringiensis. More detailed information on spore morphology is given in reviews of spore formation by Fitz-James and Young (1969)and Murrell(l967) and in a freeze etching study by Holt and Leadbetter (1969).

MOLECULAR STRUCTURE OF THE BACTERIAL SPORE

3

111. Exosporium and Appendages A. MORPHOLOGY

The exosporium of B. cereus consists of an outer layer of hair-like projections ( 2 5 nm thick), an intermediate layer (6 nm) and a basal layer (19 nm). The basal layer has several layers arranged in a hexagonally ordered lattice structure (Gerhardt and Ribi, 1964). Similar structures are found in B . fastidiosus (Holt and Leadbetter, 1969), some strains of B. megaterium (Beaman et al., 1972) and some Clostridium species (Samsonoff et al., 1970; Hoeniger and Headley, 1969). In some other strains and species, slightly different structures are seen (Hodgkiss et al., 1967; Mackey and Morris, 1972). The exosporium may be loosely fitting as in B . cereus or it may be tightly fitting or even integral with the coats. A recent observation of an exosporium in spores of B . subtilis revealed only after partial extraction of the coats, suggests that exosporia may be widespread occurrences but are often obscured by the dense outer coat (Sousa et al., 1976).

FIG. 1. Electron Micrograph of Spore of Bacillus cereus T showing: Core ( c ) ,plasma membrane ( P M ) , germ cell wall (ccw),cortex (cx), coats (CTS) and exosporium (EX).

A. D. WARTH

4

Appendages are loosely fitting structures of very diverse form that are commonly found on spores of Clostridium species. An excellent review is available (Rode, 197 1). Clostridium taeniosporurn spores have fifteen to twenty large ( 4 4 pm) ribbon-like appendages attached through a hook-like structure to the trunk which is continuous with the spore coats. An upper layer 9 nm thick overlies an electron-transparent layer of 3 nm. The main layer is about 100 nm thick and consists of multiple layers of 5 nm spherical subunits (Rode, 197 1). B. C H E M I C A L C O M P O S I T I O N

Chemical analyses are only available for exosporia of B. cereus T and appendages of C1. taeniosporum. Both are voluminous loosely fitting structures which are easily removed from the spore. Sonication was TABLE 1. Composition of the exosporium from Bacillus cereus T and the appendage from Clostridiumtaeniosporum. Exosporium

Appendage

%ofdry weight

Protein ( 17 amino acids) Neutral lipid Phospholipid Total P Neutral carbohydrate (as glucose) Glucose Glucosamine Rhamnose Dipicolinic acid Muramic Acid Diaminopimelic acid Ribose ~

~~

52.1 12.5 5.5 1.8 9.1 3.8 6.4 0.2

ND ND 0.7

79.7

10.4 3.7 4.9

+

ND ND ND

~~

N D indicates that none was detected. Data for B . cereus T from Matz et al. (19701, and for C . taemosporum tram Yolton etal. (1972).

used for isolation of appendages (Yolton et al., 1972) and passage through a needle valve under high pressure for exosporium (Gerhardt and Ribi, 1964). Estimates of the amount of exosporium range from 2% (Matz et al., 1970) to 10% (Gerhardt et al., 1972) of the spore dry weight.

MOLECULAR STRUCTURE OF THE BACTERIAL SPORE

5

In some aspects of their composition, the two preparations were similar (Table 1). In both, protein was the major component and both contained significant amounts of carbohydrate made up of glucose, rhamnose and glucosamine residues. Lipid composition was not reported for appendages but evidently it is much less than the 18% TABLE 2. Amino acid composition of exosporium appendages a n d coats. Amino Acid

Clostridiurn taeniosporum

Bacillus cereus Exosporium'

Coatb

AppendageC

CoatC

Moles/100 moles Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Pheny lalanine Lysine Histidine Arginine Cystine

+

13.5 6.8 6.2 12.0 4.2 6.8 5.5 6.8 0.9 5.8 7.5 6.2 4.5 3.1 2.1 6.5 1.2

11.3 7.2 6.1 8.2 4.9 11.3 7.8 6.8 5.1 7.2 4.7 5.4 4.5 2.5 3.8 3.4

14.9 9.8 6.5 7.5 6.8 11.9 5.6 7.6 0.6 5.4 4.1 4.6 3.0 5.0 0.6 2.8 2.7

13.8 4.6 5.2 10.4 4.1 10.6 5.7 5.0 2.0 4.4 6.0 4.6 3.6 9.7 1.7 4.9 3.6

References: a Matz et al. (1970); bAronson and Fitz-James (1968); 'Yolton et al. ( 1 9 7 2 ) .

found in exosporium. The amino-acid compositions of the exosporium and appendages are similar and resemble that of the spore coats (Table 2). Contamination with cell-wall material and ribonucleic acids was low, as shown by the small amounts of muramic acid, diaminopimelic acid and ribose detected. O n the other hand, some caution may be necessary with lipid analyses as lipid may be absorbed from the sporulation medium which contains lysed sporangia. The phospholipid in B. cereus T exosporium was almost entirely disphosphatidylglycerol (Matz et al., 1970). In other studies using whole spores of B. cereus T (Lang and Lundgren, 1970) and B . megaterium (Bertsch et al., 1969), diphosphatidylglycerol was found in a readily extractable form and could have originated in the exosporium. Exosporium lipid

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A. D. WARTH

was not significantly different from whole spore lipid in its fatty acid composition. Straight chain n-C,, and n-C,, fatty acids predominated, and branched-chain fatty acids were present in much lower amounts than is common among Bacillus species (Kaneda, 1967)or was found in vegetative cell membranes of B . cereus T (Beaman et al., 1974).Treatment of exosporia with phenol plus acetic acid, or with sodium dodecyl sulphate (SDS), solubilized components probably from the basal layer. The SDS extract contained 15%of the exosporium protein and consisted of spherical particles 11-44 nm in diameter, which on dialysis, spontaneously re-aggregated into sheets having a hexagonal lattice structure similar to that of the basal layer (Beaman et al., 197 1). This propensity for self assembly is consistent with the formation of the exosporium in the cytoplasm of the mother cell, apparently unaided by pre-existing cytoplasmic structures (Ohye and Murrell, 1973). Relative to exosporia, re-aggregated exosporia were enriched in lipid and contained 39%protein, 33%lipid and 12% carbohydrate. A component chemically related to the complex carbohydrate of the exosporia and appendage may be a common feature in spores, despite the difficulty with some species of recognizing exosporia in electron micrographs. Spores of a number of Bacillus species contained glucosamine in excess of the stoicheiometry required for peptidoglycan (Murrell and Warth, 1965) and a carbohydrate content of 1 to 5% is typical of spores (see Murrell, 1969). In spores ofB. subtilis and B . cereus, rhamnose, glucose and minor saccharides were present (Warth et al., 1963). The carbohydrate components could be associated with the delicate nap seen on exosporia or they may be present in the capsular material which very commonly engulfs spores. Walker (1969) looked for the location of carbohydrate in spores of B . cereus by oxidation of thin sections with periodate and staining with silver. Unexpectedly this procedure did not stain the exosporium, but did stain the developing cortex. However, the structure established for spore cortex peptidoglycan (Warth and Strominger, 1972) does not have periodate-sensitive bonds in the glycan chains, and other saccharides were not detected in cortex preparations. N o direct evidence exists as to the function of exosporia and appendages. Tipper and Gauthier (1972) suggested a function for the exosporium in controlling assembly of coat subunits during spore formation. Exosporia and appendages do not appear to be significant permeability barriers. Openings are present in appendages (Rode, 197 1) and in exosporia of Cl. pasteurianum (Mackey and Morris, 1972) and B. megaterium (Beaman et al., 1972). Solute uptake studies on B .

MOLECULAR STRUCTURE OF THE BACTERIAL SPORE

7

cereus did not distinguish an effect attributable to limited permeability of the exosporium (Gerhardt et al., 1972). Together with capsular material, and cell wall layers that in some species persist after sporangial lysis, exosporia and appendages usually form the outermost layer of the spore. As such they determine superficial properties such as adhesion, and antigenicity, which are of obvious ecological importance. I t is perhaps relevant that discrete exosporia appear to be very common in toxic o r pa thogenic species. IV. Coats A.

MORPHOLOGY

Spore coats show an interesting variety both in appearance and in complexity. Some examples are shown in Fig. 2. Three main types of layer can usually be distinguished in thin sections. The most distinctive is the middle layer which shows a very characteristic laminar pattern. This pattern is well developed in the spore coat of B . cougulans which has about seven lamellae spaced 5-7 nm apart. Beneath the laminated coat layer is a region of poorly structured material sometimes referred to as undercoat. Other less consolidated material, including possible remnants of the mother cell cytoplasm and the forespore membrane, may constitute the inner boundary of the coats. Outer coats particularly, vary in complexity between species. At one extreme, some species have heavily ridged and ornate coats (Bradley and Franklin, 1958; Murphy and Campbell, 1969).Thin sections show these to be of complex morphology (Fig. 2d; Holbert, 1960; Leadbetter and Holt, 1968). The spore of B . coagulans (Fig. 2b) has a simpler, thick, heavily staining layer, while that of B . cereus appears to lack an outer coat (Fig. 2a). Structures equivalent to the exosporium are integral with the coats in some species (Leadbetter and Holt, 1968; Beaman et ul., 1972). Freeze etching also reveals interesting details of coat structure (Holt and Leadbetter, 1969) but correlation with features seen in stained sections is not straightforward. A characteristic array of parallel fibrils, about 5 nm in diameter, is generally present and these probably correspond to a laminated layer. Often the fibrils wrap around much of the spore, but in spores of B . cereus they are present as smaller patches or domains. Underlying the fibrillar layer is a pitted layer. During sporulation, coats are formed in the mother cell cytoplasm and not on the cell or forespore membranes (Ohye and Murrell, 1973).The

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D. WARTH

morphogenesis of the spore coat and its morphological and chemical structure have been comprehensively reviewed recently by Aronson and Fitz-James (1976). B. CHEMICAL COMPOSITION A N D STRUCTURE

Spore coats consist very largely of structural protein. Smaller amounts of complex carbohydrate and lipid are generally also found and in some species quite large amounts of phosphorus. Coats can be prepared by mechanical disruption of spores, followed by extensive washing with buffers to remove soluble cytoplasmic components, and digestion with lysozyme to eliminate the cortex and germ cell wall. Such preparations comprise 30 to 60% of the spore dry weight and 40 to 80% of the spore protein. Electron microscopy shows the presence of the major morphological structures of the spore coats including the laminated inner coat, the more diverse and complex outer layers and, where present, the exosporium. Soluble, finely dispersed and protease-sensitive material can be lost. Cytoplasmic membranes are usually eliminated during the washing procedure, but the fate of the outer forespore membrane and the poorly structured material often seen between the cortex and the inner coats is not clear. In general, electron microscopy of thin sections does not give a clear indication of the chemical integrity of spore coats. Large proportions of the weight of the coats can often be extracted with little change in the appearance of the different coat layers and the presence of capsular material and close fitting exosporia is difficult to detect. Spore-coat components can therefore be lost if, during cleaning of the spores or spore coats, treatments with detergents, proteases, alkali or sonication are used, even though these treatments may not affect the viability, heat resistance and refractility of spores. Spore coats are substantially resistant to proteolytic enzymes and to a wide variety of chemical reagents. Part of the coat structure normally remains insoluble after all treatments short of severe hydrolysis or oxidation. The most useful agents for extracting coat components have been disulphide bond-breaking reducing agents, alkali, sodium dodecyl sulphate and urea. Despite the often striking differences in the morphological structure of the different spore coat layers, there appears to be remarkable uniformity in the extractable protein components of each layer. Unlike most other species, the spores of B . cereu5 T and B . megaterium KM have coats which can be almost entirely

MOLECULAR STRUCTURE OF THE BACTERIAL SPORE

9

FIG. 2. Electron micrograph showing structure of spore coats (a) Bacillus cereus T ib)

Bacillus coagulans ( c )Bacillus stearothermophilus id) Bacillus apiarius.

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A. D. WARTH

solubilized under mild conditions. Extraction of B. cereus T spore coats (Aronson and Fitz-James, 1968) or spores (Aronson and Horn, 1972) with dithioerythritol at pH 10.3 dissolved 82% of the coat protein with the concommitant disappearance of the inner or undercoat but not of the outer laminated layer. The extract contained only protein, which except for a lower cystine content closely resembled total coats in its amino-acid composition (Tables 2, 3). Further extraction in the presence of sodium dodecyl sulphate solubilized most of the remaining coat protein together with some polysaccharide, and eliminated the main structural features of the coats visible in the electron microscope. The residue, comprising less than 5% of the total protein, was mainly exosporium and contained lipid, protein and a small amount of carbohydrate. Both the dithioerythritol and sodium dodecyl sulphate extracts appeared to consist principally of the same polypeptide of about 12,000 molecular weight (Aronson and Horn, 1972). Apart from a small amount of high molecular-weight material which was attributed to aggregation, both extracts showed a single peak on gel electrophoresis, gel exclusion chromatography and sucrose gradient centrifugation. After dansylation and digestion of the extract with keratinase, three dansyl peptides were isolated. Their composition was consistent with a common amino terminal sequence for the polypeptide of: NH,-Ser-Gly-(Glu, Thr), in which the terminal serine residue was sometimes absent. Coat protein extracted from whole spores had mainly amino- terminal serine, whereas coat extracts, which presumably had suffered more exposure to peptidases, yielded more amino terminal glycine. The major extractable protein appears to be very similar in different species. With spores of B . subtilis, 85% of the coat was solubilized with sodium dodecyl sulphate and dithiothreitol and the major protein component had a molecular weight of 14,000. Serine was the major amino-terminal residue (Mitani and Kadota, 1976). Sodium dodecyl sulphate extracts of B . thiaminolyticus spore coats contained 53% of the protein and showed a single band of about 15,000 molecular weight on gel electrophoresis (Watabe et al., 1975). Urea plus mercaptoethanol extracts of spores of four Bacillus species and C1. bzfrmentans behaved identically on gel electrophoresis, showing a single major band. Their amino-acid compositions were very similar to each other and to the extract of B. cereus T spores (Table 3) and each showed partial antigenic homology with the crystal protein from sporangia of B . thuringzensis (Somerville et al., 1970). Similar results were obtained for a different

TABLE 3. Amino Acid Composition of Spore Coats and Crystal Proteins (For abbreviations see foot of table.) Bacillus thuringiensis alesti' berliner'

Bacillus cereu5 T a

Bacillus

Bacillus subtilis'

Banllus subtilis'

Bacillus subtilis"

Five species'

cereus

Fraction extracted

coats

coats

coats

coats

coats

spores

Extractant

50mMDTE 50mMME PH 10.5 PH 10.2

-

50 mM DTT 50 mM DTT 8 M urea, 1.6 M ME pH 10.5 pH 10.0 pH 8.5 -

Species

-

crystal

crystal

coats

8 M urea, pH 8.0

1.6M ME

z 0 E 0

;

Moles/100 moles

3

cn Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine 4 Cystine Tryptophan

11.3 6.8 5.8 7.9 5.3 11.6 8.3 7.0

-

5.4 7.9 5.0 5.3 4.3 2.5 3.8 1.2

-

13.1 6.7 6.2 10.4 5.3 10.1 8.4 7.9

1 .o

4.7 8.6 2.0 4.9 6.7 1.6 2.1 1.8

-

6.9 4.6 7.6 6.0 2.8 21.1 7.0 4.4

-

3.2 3.6 11.2 6.2 6.6 3.0 4.0

-

9.7 9.5 6.6 6.8 6.9 11.5 8.4 7.5

-

5.5 7.5 1.5 6.8 4.0 2.1 4.5 0.5

-

12.2 5.0 7.3 6.8 3.6 14.3 8.7 5.2 1.7 4.3 5.6 7.1 4.6 6.1

2.0 4.9 0.8 -

10.3-10.9 6.1-10.5 5.4-8.7 6.6-10.9 4.8-7.6 10.6-13.3 7.&12.0 7.4-8.0

-

10.6

9.1 6.8 8.5 6.4 11.6 9.3 7.7

12.4 6.3 7.3 12.0 4.8 7.7 5.4 8.1

-

-

5.8-8.2 6.8-8.8 1.2-3.2 3.6-5.5 3.5-5.2 1.6-2.1 1.4-3.8

6.6

6.0

7.8 2.1 4.7 4.1 1.9 2.7

8.3 4.8 5.2 3.2 2.2 6.4

-

-

-

11.2 6.2 6.1

12.7 4.2 8.8 7.2 6.7 1.9 5.0 8.3 3.3 4.1 4.6 2.2 5.0 0.9 1.4

-I p -I C p

m

$ -I

I

7 m

? v)

p

-o

Abbreviations: ME, 2 mercaptoethanol; DTE, dithioerythritol; DTT, dithiothreitol. References: "Aronson and Fin-James (1968); bSomerville and Pockett (1975); 'Spudich and Kornberg (1968); "Mitani and Kadota (1976); CSomervilleetal., (1970);fLecadetetal., (1972). a Data are given as moles of amino acid per 100 moles total amino acid recovered. Tryptophan, methionine and cystine analyses are often not reported. -..

12

A. D. WARTH

strain of B. thuringiensis by Lecadet et al. (1972). Some differences in amino-acid composition are apparent (Table 3) and in distinction to B. cereus T spore coat protein, phenylalanine was the principal amino-terminal amino acid of the extracted protein, although aminoterminal serine and methionine were found in intact coats. Lysine appeared to be the carboxyl-terminal residue. Peptide maps of keratinase digests of protein from spore coats of B. cereus T, B. subtilis 168 and B. megaterium KM gave very similar patterns of about twelve peptides (Aronson and Fitz-James, 1975) and coat proteins from B . cereus T and B . megaterium were interchangeable in in uitro reconstitution of spore coat layers (Aronson and Fitz-James, 197 1). A considerable amount of evidence has been obtained to indicate a close relationship between the protein of the parasporal crystal formed by strains of B. thuringiensis and the extractable protein of the spore coats. Amino-acid compositions are similar but not identical (Table 3), identical bands are obtained on gel electrophoresis, considerable antigenic homology exists, and both proteins are similar in the conditions required for solubilization and in their tendencies to reaggregate (Delafield et al., 1968; Somerville et al., 1968, 1970; Lecadet et al., 1972). Each contains the same amino and carboxyl terminal amino acids (Lecadet et al., 1972). Maps of the tryptic peptides from performic acid-oxidized protein of crystal and spore extract also were identical (Somerville et al., 1970). Spore extracts of several Bacillus species and acrystaliferous strains were all toxic to larvae of Lepidoptera species. Purification of the toxin from B . cereus strain 64a gave a protein of molecular weight approximately 32,000 very similar to B . thuringzensis crystal protein in composition (Table 4) but with a much lower specific toxic activity. Exosporium protein from B. cereus T also has similarities to this coat protein fraction, both in composition (Tables 2, 3) and in its tendency to re-aggregate (Beaman et al., 197 1). Morphologically the parasporal crystal seems to be formed on the developing exosporium (Somerville and James, 1970; Somerville, 197 1) and the crystal-specific antibody reacted with the inner layers of the spore coat and exosporium (Short et al., 1974) The foregoing work was based upon extraction of spores or coats with urea or sodium dodecyl sulphate in the presence of reducing agents. Kondo and Foster (1967) extracted spore coats of several Bacillus species with alkali. Mild sonication then dispersed a “paracrystal” fraction leaving a resistant residue of from 50 to 70% of the coat weight. Analyses o f these fractions from B . megaterium showed

TABLE 4. Amino acid composition of alkali-soluble proteins from spore coats

Amino acid

Species -.

Bacillus megatenurn

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine 4 Cystine Tryptophan Ornithine

Alkali Extract'

Paracrystal fraction"

9.8 4.0 5.1 7.5 6.5 12.1 5.6 4.0 1.1 3.2 5.1 11.6 4.7 7.5 4.8 6.2 1.3

15.0 2.9 6.6 8.3 1.2 16.0 4.7 2.3 1.o 2.0 0.3 2.1 6.7 2.5 2.4 5.6 8.2 -

-

-

Residue" 11.0 5.1 6.1 10.0 4.8 6.2 4.7 3.8 3.5 5.8 0 4.9 5.4 18.4 3.1 4.5 1.9 -

Bacillus coagulans

Bacillus

Clostridium

cereus

sporogenes

Bacillus subtilis

Alkali extractb

Alkali extractb

Alkali extract

Alkali extractC

8.2 6.3 5.6 12.6 8.1 6.1 7.1 1.5 5.3 7.2 7.1 4.3 8.8 5.3 4.2 0 2.1

11.6 7.7 3.7 12.7 9.7 9.3 7 .O 1.5 5.5 8.0 3.5 4.2 6.3 2.6 5.4 0.3 0.9

10.0 7.1 5.3 11.1 10.2 8.7 8.1 1.8 4.5 9.3 4.0 5.0 7.6 3.6 3.1 0

8.1 3.4 4.3 8.1

0

References: a Kondo and Foster (1967).Coats were extracted with 0.06 N NaOH at 5 O O C . The paracrystalline fraction was solubilized by sonication. b Gould et al. (1970). Coats were treated with 7 M urea and 10%mercaptoethanol pH 2.8 before extraction with 0.1 N NaOH at 4°C. cWood (1972). Extracted as in Gould eta!. (1970).

8.2 8.3 4.4 3.4 1.1 3.2 4.1 12.9 2.8 14.6 4.4 8.9

-

3 rn

k 50

cn -I

W

i c W

rn

$ -I

I m

rn

E D cn I-

T

0 2 m

14

A. D. WARTH

some marked differences in amino-acid composition (Table 4). The paracrystal fraction clearly resembles sodium dodecyl sulphate and dithioerythritol extracts in composition and properties. It has high glycine, cystine and aspartic acid contents. Its colloidal suspension was cleared by sodium dodecyl sulphate to give a heterogeneous solution of high molecular-weight components which dissociated into small units in the presence of mercaptoethanol. Partial reduction of the paracrystalline fraction led to aggregation and precipitation. The alkali extract had quite a different amino-acid composition from the paracrystalline fraction, having a high tyrosine and low cystine content (Table 4). Other workers (Delafield et al., 1968; Gould et al., 1970; Wood, 1972; Somerville and Pockett, 1975) have examined alkali extracts of whole spores, often after prior treatment with urea and mercapto-ethanol. Extracts from a number of species showed a general similarity in amino acid composition to the alkali soluble fraction of B . megaterium coats (Table 4) and comprised from 1 to 6%of the weight of the spores. The protein from B. subtilis behaved as a single species on ion-exchange chromatography and gel filtration. Both very large and small molecular weight components were present but each appeared identical by immunological criteria. Treatment with sodium dodecyl sulphate, mercapto-ethanol and urea at 100°C effectively disaggregated the higher molecular weight components, and after sodium dodecyl sulphate gel electrophoresis only two bands were seen, corresponding to molecular weights of 10,000 and 56,000 (Wood, 1972). The resistant residue of B . megaterium spore coats after alkali treatment and sonication had a distinctive amino acid composition, high in lysine, aspartic acid and glutamic acid but deficient in cystine (Table 4). Most of the phosphorus of the coats was present in this fraction (Kondo and Foster, 1967). Although in the case of spores of B . cereus, some B . megaterium strains and some Clostridium species, a major part of the coat protein can be solubilized, this is not typical, and with spores of many other species a substantial resistant residue remains. Possibly this is correlated with the presence of coat layers external to the laminated coat, including possible equivalents of the exosporium. Minor coat components such as lipid, complex carbohydrate, hexosamine and phosphate (Warth et al., 1963) are reminiscent ofexosporia (Table 1) and the superficial naplike morphological structure in some species is also similar (Holt and Leadbetter, 1969). The phosphorus content of spore coats differs considerably between species (Murrell, 1969). However, some of the higher values may have resulted from

MOLECULAR STRUCTURE OF THE BACTERIAL SPORE

15

contamination of the coat preparation with inorganic phosphate precipitates. Serine phosphate linkages have been demonstrated in B . subtilis spore coats (Sano et al., 1975; Kondo et al., 1975) and galactosamine phosphate in those ofB. megaterium (Kondo et al., 1975). Earlier reports of a phosphomuramic acid polymer appear to be incorrect. Other unusual chemical features of spore coats include taurine and a very high glutamic acid content in the ridged coats of B. breuis 636 spores (Warth et al., 1963). Contrary to a previous report, bacitracin does not appear to be a component of B. lichen$ormis spore coats (Marschke and Bernlohr, 1970). The reported presence of t--(aspartyl)lysine links in the spore coats of B. sphaerzcus (Tipper and Gauthier, 1972) points to an unusual type of cross-link between peptide chains in this species. Other types of cross link such as the t.-(y-glutamyl)-lysine found in keratins from the medulla (Pisano et al., 1968) have not yet been reported in spore coats. Spore coats have a number of similarities with the keratins, including X-ray diffraction patterns (Kadota et al., 19651, extraction properties and resistance to enzymes. Both are complex morphological structures containing a number of structural proteins including some rich in cystine. A number of specific differences and similarities are evident but a detailed comparison does not seem warranted at present as knowledge of coat structure is still fragmentary and keratins consist of several groups of' proteins with diverse properties and compositions (Bradbury, 1973). Compared with the efforts expended on the study of structural proteins from higher vertebrates, spore coat chemistry has so far received trivial attention. Progress had been made, especially in the B. cereus system, to a point where questions can be more clearly stated and the major practical problems are apparent. In many ways, the properties and the problems such as solubilization techniques, tendencies to aggregate, heterogeneity and complex morphology are similar to those of the keratins but hopefully will prove somewhat less complex. In particular, the use of stable alkylated or oxidized derivatives of the reduced proteins has proved essential in the keratin studies but has been little used in spore coat investigations and may help resolve questions of polydispersity of molecular weight and heterogeneity. Many of the spore-coat studies have not described precautions taken to prevent oxidation of thiol groups during manipulation of reduced protein, nor has the extent of reduction or denaturation achieved been apparent in some cases. These conditions together with the need sometimes to work near the lower limit of p H value for solubility, are conducive to aggregation and molecular weight heterogeneity. Despite these diffi-

16

A. D. WARTH

culties it is clear that a degree of uniformity exists in the major polypeptides within and, to a lesser extent, between species. Whether there exist a few small polypeptides o r families of closely related polypeptides, will require genetic analysis o r sequencing studies lor resolution. Investigation of the chemical linkages and polypeptides in the resistant fraction is understandably more difficult and may require much work to develop suitable chemical o r enzymatic techniques. Unfortunately this resistant fraction is often a major part of the coat. The major challenge lies in describing the processes involved in the assembly of polypeptides into the morphological structures of the spore coats. C. BIOSYNTHESIS

Construction of spore coats is a major activity of the mother cell during spore formation. Two aspects of coat formation can be distinguished; first, biosynthesis of coat polypeptides, and second, aggregation, modifications, additions and re-arrangements involved in the assembly of the morphological structures. Spore-coat structures first appear in electron micrographs after the forespore has contracted and concommitant with cortex formation and the beginning of refractility (e.g. see Fig. 4). Completion of coat formation is a very late event, occurring at the same time as full refractility and heat resistance (Fitz-James and Young, 1969). Different coat components may be synthesized at different times. In B. subtilis, incorporation of phenylalanine into coat precursors was a late event, occurring maximally at the time of appearance of the morphological structures, but the fraction soluble in sodium dodecylsulphate with dithiothreitol was labelled slightly earlier than the resistant fraction (Spudich and Kornberg, 1969). Serine-phosphate linkages were also synthesized late in spore formation (Sano et al., 1975). In contrast, the alkali-soluble coat protein was synthesized at a uniform rate from very early in spore formation (Wood, 1972). In B . cereus, unlike B . subtilis, incorporation of amino acids was continuous from an early stage, suggesting the accumulation of precursor proteins (Aronson and Fitz-James, 1968; Aronson and Horn, 1969). Cystine incorporation, however, was much greater late in spore formation Winter, 1959; Aronson and Fitz-James, 1968). Some cystine appears to be incorporated directly into outer coats of B. cereus by disulphide interchange reaction with coat protein thiols o r disulphides. Reduction of coats released free cystine, predominantly from the sodium

MOLECULAR STRUCTURE OF THE BACTERIAL SPORE

17

dodecyl sulphate-soluble outer coat fraction. About one mole per mole of coat polypeptide was released (Cheng et al., 1973). Furthermore, the in vitro reconstitution of spore coats as judged by antigenic (Horn et al., 1973) or morphological criteria (Aronson and Fitz-James, 197 l ) , was facilitated by cystine. On the other hand Setlow and Kornberg (1969) could not show Cys-S-protein links in B . megaterum spore coats. Much of the cystine for coat formation comes from a pool of' reduced glutathione. Limitation of cysteine availability results in lysozyme sensitive spores, presumably defective in outer coat formation. Achievement of this cysteine deprivation required the use of mutants defective in both cysteine synthesis and glutathione reductase (Cheng et al., 1973). Since, in B. cerem T, the major polypeptide ofboth the inner and outer coats appears to be the same (Aronson and Horn, 19721, the major difference in their morphological structure must be caused by packing and conformational differences, perhaps mediated through disulphide-sulphydryl interchanges between polypeptides and some cysteine. Minor components may also be important. The presence of minor proteins up to 20% of the total coat protein has not been excluded, but the occurrence of very cystine-rich proteins such as are found in keratins has not been reported. D . FUNCTION O F S P O R E COATS

Coats have no significant role in the heat, and ultraviolet radiation resistance mechanisms of the spore. Disruption of coat structure by mutation (Cassier and Ryter, 197 1 ; Aronson and Fitz-James, 19751, inhibition of synthesis (Fitz-James and Young, 1969) or extraction of coat proteins (Gould et al., 1970; Somerville et al., 1970; Aronson and Fitz-James, 1971; Wood, 1972; Vary, 1973) generally gives spores which are heat and radiation resistant and which retain their calcium and dipicolinic acid (DPA) contents and their refractility. Such spores are, however, made sensitive to lysozyme and octanol and may show differences in their response to germinants. These latter properties suggest a role for the spore coat as a protective permeability barrier preventing access of lysozyme to the sensitive cortical peptidoglycan. In conjunction with the exosporium, the coats doubtless function to protect the interior parts of the spore from a wide range of deleterious substances, particularly surfactants and enzymes. Their own extreme resistance to enzymes and other agents may well be due to the complementary resistance of the various components, hence their morpho-

18

A. D. WARTH

logical and chemical complexity. Much of their stability appears to result horn hydrophobic interactions and covalent cross-linking. Coats also provide obvious mechanical protection and, together with other superficial layers, determine the physical properties of the spore surface which affects binding and dispersal from surfaces. Spores have quite unusual surface behaviour as exemplified by their tendencies to form films on glasslair surfaces and their hydrophobic behaviour in two phase systems (Sacks and Alderton, 1961). V. Cortex and Germ Cell Wall A. MORPHOLOGY

The cortex in mature spores appears as a featureless, electrontransparent zone between the core and the coats. When the spore germinates, the cortex loses its refractiveness to staining and a fibrous network can be seen. At its inner surface is a more dense layer which develops into the cell wall of the emergent cell while the cortex lyses. The inner layer has been termed cortical membrane, primordial cell wall and germ cell wall. O n disruption of the spore and in preparations of the spore integuments, the cortex swells greatly, revealing a fibrous network which is now readily stained by heavy metals. Under conditions where the cortex is swollen, its inner surface and the germ cell wall become folded; see figures in Warth et al., (1963);Murrell and Warth (1965) and Fitz-James and Young (1969). The folding appears to result from anisotropic swelling of the cortex. Swelling occurs along the radial axis but no extension of the surface dimensions is evident. Thus, in a fragment of a spherical shell, expansion is directed inwards with distortion of the inner surface. B. CHEMICAL STRUCTURE

Treatment of disrupted spores with lysozyme dissolves the cortex and usually the germ cell wall. Structural determination of the digestion products from B. subtilis spores indicated the structure shown in Fig. 3 (Warth and Strominger, 1972). Essentially identical results were obtained with spores of B . sphaericus (Tipper and Gauthier, 1972), B. cereus, B. megaterium, B . stearothevnophilus and Cl. sporogenes (Warth and Strominger, 19721, and spores of eight other Bacillus species (A. D. Warth, unpublished observations) appear also to have the same struc-

35% r

47%

A

V

18%

A

f

I /

h

1

in

0, H

0

NHAc H-C-CH,

co

3.

L-Ala

5-

c-c i

I

II

H O Muramic d-lactam

0

J. L-Ala

D-Glu J.V IIl-Dpill

3. D-Ala FIG. 3. Repeating units of peptidoglycan from spores of Bacillus subtilzs. The relative frequency of each unit is shown as a pri~cc~ntage. The sequence ofthe units is not random. Muramic lactam units tend to alternate with the other units. O n average 19% ol tliv tetrapeptides were linked through their D-alanine carboxyl group to the €-amino group of diaminopimelic acid ( D i m )ol'another-peptide side chain (from Warth and Strominger, 1972).

-I

I

m

20

A. D. WARTH

ture. The structure is related to type I peptidoglycan (Ghuysen, 1968) which is common in vegetative cell walls, but some modifications unique to spores are present. The most striking of these is that 45-60% of the muramic acid residues in the glycan chains lack both a peptide and an N-acetyl substituent and instead form an internal amide muramic lactam”. In a further 18% of the muramyl residues, the peptide side chain is curtailed to a single L-alanine residue. Compared with vegetative cell-wall peptidoglycans, the degree of cross-linking is very low. Only one peptide in five initiates a cross-link and only 35% of the muramyl residues bear a peptide, thus giving about one peptide crosslink to every sixteen residues in the glycan chain. End-group determinations indicate an average glycan chain length of 80 to 100 saccharides which is longer than is common in vegetative cells. Other polymers found in the cell wall, such as teichoic acid, have not been detected in spores (Chin et al., 1968; Warth and Strominger, 1972). Perhaps the most significant structural feature is the uniformity of structure of the spore peptidoglycan compared with the species variability of cell walls. Walls of B . subtilzs differed from the spore cortex in having amidated diaminopimelic acid residues and in lacking the carboxyl terminal D-alanine from the peptide (Warth and Strominger, 1971). In B . sphaericus, the change in structure is even more radical, with the diaminopimelic acid in the spores being replaced by lysine in the cell wall and a D-isoasparaginyl residue being incorporated in the peptide cross link (Hungerer and Tipper, 1969). I t is likely that other variations in peptidoglycan structure exist in the vegetative cell walls of spore-forming species, but the structure of the cortex does not appear to vary. This conservation and specificity of structure must imply an important function for the cortex, common perhaps to all species. Although the sequence along the glycan chain of the various muramic acid substituents is not known, the relative yields of the products of lysozyme digestion suggest that a degree of regularity exists, with lactams alternating with peptide or alanine substituents (Warth and Strominger, 1972).This conclusion has been confirmed by a study of the kinetics of formation of lysozyme digestion products and by a non-enzymic degradation procedure employing alkaline hydrolysis and nitrous acid, which specifically breaks the glycan chain at muramic lactam residues (A. D. Warth, unpublished results). Direct analyses of purified germ cell walls have not been reported. A number of observations suggest that it is a rudimentary form of cell (6

MOLECULAR STRUCTURE OF THE BACTERIAL SPORE

21

wall lacking some of the distinctive features of the vegetative cell wall, but it probably does not have the unique structural features of cortical peptidoglycan. Germ cell wall is formed earlier than the cortex and the morphology of their formation strongly suggests that the germ cell wall is synthesized by the forespore cytoplasm and membrane, whilst the cortex is synthesized by the mother-cell cytoplasm and outer forespore membrane. Bacillus sphaericus spores contain peptide side chains of both cell and spore types (Tipper and Gauthier, 1972).During spore formation, diaminopimelate ligase, a n enzyme specifically required for synthesis of spore-type peptides, was found only in the mother cell (Tipper and Linnett, 1976). I t was formed after most of the germ cell wall had been laid down (Holt et al., 1975), whereas L-lysine ligase which is required for cell wall type peptides was present at highest specific activity in the forespore. I n B . cereus, the germ cell wall shares the vegetative cell wall property of lysozyme resistance, which is probably occasioned by the absence of N-acetyl substituents from some glucosamine residues in both cells walls (Araki et al., 197 1 ) and spores (Warth, 1968). O n the other hand, amidated peptides characteristic of B. subtilis walls were not present in spore peptidoglycan (Warth and Strominger, 1972). Teichoic acid and other major cell-wall polymers have not been detected chemically in spores of B . subtilis (Chin et al., 1968; Warth and Strominger, 1972) o r other species (Warth, 1965, 1968). Electron micrographs show the germ cell wall as a simple layer adjoining the cortex and d o not suggest the presence of any of the more complex features of cell walls of some species, although cell-wall antigens were detected in the germ cell wall of B . cereus (Walker, 1970). A characteristic cell-wall layer of protein subunits in B . polymyxa was formed immediately after germination (Murray et al., 1970). Cross linking of peptides appears to be greater in the germ cell wall than in the cortex, as is suggested by its less expanded appearance. Peptides from cell walls of germinated B . megaterium were more cross-linked than total spore peptidoglycan (Cleveland and Gilvarg, 1975). The lysozyme-resistant fraction of B . cereus and B . subtilis spore peptidoglycan was mainly germ cell wall and had more peptides and peptide cross-links than cortical peptidoglycan (Warth, 1968, and unpublished results). Muramic lactam appears to be confined largely o r entirely to the cortex. It is formed late during spore formation at the same time as dipicolinic acid (Wickus et al., 1972; Imae and Strominger, 1976a, b). Peptidoglycans containing muramic lactam residues can be solubilized after mild alkaline hydrolysis by treatment with nitrous acid which

22

A. D. WARTH

breaks the glycan chains specifically at the muramic lactam residue (A. D. Warth, unpublished results). This method removed the cortex from B . cereus and B. subtilis spores leaving the germ cell wall apparently unaltered. C . LYTIC ENZYMES

In addition to peptidoglycan, the cortex and germ cell wall contain a number of degradative enzymes. On germination or even simply on disruption of the spore, the enzymes become active. The cortex structure is more or less completely solubilized whereas the germ cell wall is stretched by the swelling cell but persists and becomes the cell wall of the young cell. In disrupted spores both the cortex and the germ cell wall often autolyse but, in some species, germ cell wall may persist. The principal lytic activities present in B . subtilis, B . cereus and B . megaterium spores are endo-N-acetylglucosaminidase, which hydrolyses glycosidic links in the glycan chain of the peptidoglycan, and N-acetylmuramyl- L-alanine amidase which cleaves peptide side chains from the glycan chains (Warth, 1972; Hsieh and Vary, 1975). Each of these enzymes has only a limited action during germination or autolysis, cleaving only a few of the glycosidic and amide bonds present. The products are large peptidoglycan fragments, which in B . megaterium had a molecular weight of 15,300 and appeared relatively monodiserse (Record and Grinstead, 1954) and a few small peptides. Spores of B . cereus also contain D-alanine carboxypeptidase and N-acetylglucosamine deacetylase activities (Warth, 1972). These two enzymes are not themselves lytic but could modify the germ cell-wall structure in such a way that it approaches that of the vegetative cell wall and may modulate the action of the lytic enzymes. For example, removal of acetyl groups from N-acetylglucosamine residues makes the peptidoglycan resistant to lysozyme (Araki et al., 1971). In B. cereus, the lytic enzymes are readily extractable (Strange and Dark, 1957) but, in most other species, they are bound to spore structures. The spore lytic enzymes have not been separated and the particular properties and function of each enzyme determined. All studies using spore lytic enzyme have used this mixture of enzymes, and the relative participation of each will depend upon the conditions of pH value, cation concentration and substrate used. Great caution is therefore necessary in comparing work from different laboratories or involving quantitative measurements of activity.

MOLECULAR STRUCTURE OF THE BACTERIAL SPORE

23

Two major hnctions for the spore lytic enzymes can be envisaged, namely lysis of the cortex and decrease in the rigidity of the germ cell wall to facilitate swelling of the young cell. In addition, the normal complement of cell-wall enzymes necessary for growth may be present. N-Acetylglucosaminidase appears to be the main enzyme associated with cortex lysis. Its action would be essential to break the long glycan chains of the cortical peptidoglycan to the size of' the fragments produced. A spore enzyme preparation from B . cereus lysed isolated cortices with the formation of reducing groups but not of amino groups (Gould and King, 1969). The optimum pH value of 5 to 6 found for autolysis in B. subtilis and B. coagulans (Warth, 1965, 1972) is typical of bacterial cell-wall N-acetylglucosaminidases (Berkeley et al., 1973). Lytic activity in a B. cereus spore extract was stimulated by Co2+ at an optimal pH of 7.8 (Strange and Dark, 1957). This suggests the presence of the muramyl- L-alanine amidase with like properties which was purified from autolysing sporangia of B. thuringiensisby Kingan and Ensign ( 1968). A proper comparison of the spore enzymes with those from vegetative cell walls will require a more thorough characterization of the individual spore enzymes. Both amidase (Herbold and Glaser, 1975) and N-acetylglucosaminidase (Berkeley et al., 1973)have been purified from B. subtilis walls. A mutant lacking N-acetylglucosaminidase apparently was capable of both sporulation and germination (Ortiz, 1974) but a mutant with a temperature-sensitive amidase (Fan and Beckman, 1973) has not yet been tested for spore related functions. D. B I O S Y N T H E S I S

The existence of differences in structure between spore and vegetative peptidoglycans has made a study of peptidoglycan synthesis during spore formation an attractive system for gaining insight into control mechanisms operating during the cellular differentiation process. Interesting questions arise as to the respective roles of the forespore and forespore membrane on the one hand, and the mother cell and the outer forespore membrane on the other. Vinter (1963) showed that peptidoglycan synthesis as indicated by diaminopimelic acid incorporation was maximal at two periods during spore formation of B. cereus. The first period corresponded to the formation of the germ cell wall and the second was co-incident with cortex synthesis and the development of refractility. In B. megaterium,

24

A. D. WARTH

Pitel and Gilvarg (197 1 ) failed to find diaminopimelate incorporation until engulfment of the forespore was complete. Subsequent incorporation was not clearly separated into two maxima. Muramic lactam synthesis in B. cereus T and B. megaterium occurred only during the later phase (Wickus et al., 1972 ) . Penicillin-binding capacity, taken as a measure of transpeptidase activity and possibly other reactions involved in biosynthesis and crosslinking of peptidoglycan, also showed maxima during these two periods (Lawrence et al., 1971). Mutants blocked at different stages of'spore formation failed to express either the second or both maxima in binding (Rogolsky et al., 1973). Anwar et al. ( 1974) separated five binding components from B . subtilis cells, but found no new components or changes in the relative proportions during spore formation. Possible pathways for the formation of muramic lactam and the N acetylmuramyl- L-alanine units in the cortex have been discussed (Warth, 1968; Tipper and Gauthier, 1972). So far, neither muramic lactam synthesis nor any of the postulated enzymic activities have been reported in cell free preparations. Sporulating cells of B . sphaericus (Guinand et al., 1974) and of B . subtilis (Guinand et al., 1976) contained a particulate y-glutamyl diaminopimelate peptidase. Bacillus subtilis also had muramyl-L-alanine amidase activity. The relevance of these enzymes to cortex formation is not known. Other functions of these enzymes could be sporangial lysis or post-germinative modification of the cell wall. Tipper and his colleagues have studied the activity of the enzymes involved in synthesis of the UDP-N-acetyl muramyl peptide precursors of peptidoglycan in B . sphaericus. Most of the enzymes are common to both vegetative cell and spore peptidoglycan, and were synthesized during two periods preceding the two peaks of biosynthetic activity. Diaminopimelate-ligase is specifically required for addition of diaminopimelic acid to the precursor and its activity does not appear until just before cortex synthesis (Tipper and Pratt, 1970) and is confined to the mother cell (Tipper and Linnett, 1976) thus providing strong evidence for synthesis of the cortex by the outer forespore membrane under the control of the mother cell cytoplasm. This is to be expected since, at this stage, the forespore cytoplasm is condensed and is unlikely to be active metabolically and the outer forespore membrane is proximal to the cortex. The lysine-adding enzyme is present throughout all stages of spore formation. It is found also, along with the other enzymes necessary for formation of vegetative type pre-

MOLECULAR STRUCTURE

OF THE BACTERIAL SPORE

25

cursor, at a relatively high specific activity in the mature spore. It seems probable that these enzymes participate in synthesis of the germ cell wall and later, after germination, of new vegetative cell wall. VI. Core A.

MACROMOLECULAR COMPOSITION

In terms of its macromolecular constitutents, the core is a relatively normal cell. Many of the enzymic activities of vegetative cells are found in spore extracts. Most spore enzymes which have been studied had very similar properties to their vegetative counterparts, and it is probable that both are specified by common genes (Kornberg et a/., 1968; Sadoff, 1969).Adenylate kinase (Spudich and Kornberg, 1969)deoxyribonucleic acid polymerases (Falaschi and Kornberg, 1966; Terano et al., 19751, inorganic pyrophosphatase (Tono and Kornberg 1967a, b), lysyl- tRNA synthetase (Steinberg, 1974) and ribonucleic acid polymerase core enzyme (Ben-Ze’ev et al., 1975) from cells and spores have been studied in detail and no important differences in kinetic or molecular properties have been found. Other enzymes, such as aldolase (Sadoff, 1969), glucose 6-phosphate dehydrogenase (Ujita and Kimura, 1975)and purine nucleoside phosphorylase (Gilpin and Sadoff, 197 11, are similar but have significant differences in properties suggesting specific modification of the spore form. In some cases, spore enzymes have been modified by serine protease action either in vivo or during extraction; in others, the nature of the modifications are unknown. Ribosomes from B. subtilis (Bishop et al., 1969) and B . megaterium (Chambon et al., 1968) spores are similar to vegetative ribosomes in physical properties and protein-synthesizing activity but, in B. cereus spores, some ribosomal proteins were missing, causing defective subunits and poor synthetic activity (Kobayashi, 1973). Hybridization studies with B. subtilis DNA (Bishop and Doi, 1968; Edge11 et al., 1975) indicate that both spore and vegetative rRNAs are transcribed from the same genes. Messenger-RNA competitive with vegetative and sporulation messenger has also been found in spores (Jeng and Doi, 1974). The tRNA complement of spores is similar to that of vegetative cells. Several new types have been detected in sporulating cells and some of these were also found in spores (Lazzarini, 1966;Jeng and Doi, 1975). DNA from spores of B. megaterium (Chambon et al., 19681, B . subtilis (Sakakibara et al., 1969)and B . cereus (Tsuji et al., 1975)had properties

26

A. D. WARTH

not significantly different from vegetative DNA. Unlike spore protein and RNA, which are largely synthesized during spore formation, DNA is incorporated into the spore by partition of the parent cell DNA. Bacillus subtilis contained a single complete genome (Callister and Wake, 1976), but other species may have multiple copies (Fitz-James and Young, 1959).The properties of spore and cell nucleic acids have been reviewed by Doi (1969). The lipid composition of spores is very similar to that of vegetative cells. The major phospholipids of both cells and spores of B . polymyxa (Matches et al., 19641, B. megaterium (Bertsch et al., 1969)and B . cereus (Lang and Lundgren, 1970) were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. Bacillus megaterium also has a glycosaminylphosphatidylglycerol. Disruption or hydrolysis of spores is necessary for complete extraction of lipid. The readily extractable fraction in B. cereus and B. megaterium had a high diphosphatidylglycerol content and probably came from the exosporium and other peripheral structures. Lipid from Bacillus species is characterized by a very high proportion of branched-chain fatty acids (Kaneda, 1967). In B. megaterium, CI5branched-chain isomers comprised 70%of the total fatty acids. Spores contained relatively more of the C,, branched-chain isomers than cells but the proportion depended upon the amino-acid composition of the sporulation medium. Phospholipids accounted for two-thirds of the fatty acids of cells but only one third of those of the spore. (Scandella and Kornberg, 1969). Bacillus thuringzensis spore lipid contained more is0 and less anteiso isomers than cell lipid, whereas straight-chain fatty acids remained constant at 10 to 11% of the total (Bulla et al., 1975). Bacillus spores have quite low lipid contents, but the data are not reliable (Murrell, 1969). Clostridial spores on the other hand contained 13 to 38% lipids. In two species of thermophilic clostridia, the lipid content was 13.5%and 16.3%and was nearly all firmly bound in the spore. Normal saturated and monounsaturated fatty acids from C,, to CLB, and a hydroxystearic acid were the main components (Pheil and Ordal, 1966). Setlow (1974, 1975a, b) has recently discovered a group ofbasic, low molecular-weight proteins present in the core of B. megaterium spores. These proteins may constitute as much as 30 to 50% of the protein in the core. Their main function is as a reserve material for germination as they are very sensitive to proteolytic enzymes and are rapidly degraded on germination. Amino-acid analysis show a very high proportion of polar amino acids, and cystine and tryptophan were absent.

MOLECULAR STRUCTURE OF THE BACTERIAL SPORE

27

Most of the glutamic and aspartic acid residues must be amidated as the isoelectric points were high (pH 9.8). N o enzymic function has been ascribed to these proteins but in vitro they complexed with DNA, raising its melting temperature. Other storage polymers have not been conclusively demonstrated in mature spores. B. megaterium accumulated /3-hydroxybutyrate (Slepecky and Law, 1961) at the end of exponential growth and C. butyricum formed an intracellular polysaccharide (Bergcre et al., 1975), but these reserves are utilized during spore formation. B.

LOW MOLECULAR WEIGHT C O M P O U N D S

In contrast to the unexceptional nature of the macromolecules of the spore core, the composition of small molecules in the spore differs dramatically fi-om that of vegetative cells. Localization of small molecules presents special problems in a structure with the morphological complexity but small size of a bacterial spore. The ionic composition of the spore is dominated by dipicolinate anions and calcium cations. After many years of uncertainty it now seems clear that the dipicolinate and much of the calcium are located in the core and not the cortex. Leanz and Gilvarg (1973) studied the attenuation of /3 particles emitted from tritium labelled dipicolinate and labelled components in the coats, cortex and core. A central location, in the core, was clearly indicated. Some excellent ultraviolet photomicrographs of sporulating B. subtilis (Wyckoff and Ter Louw, 193 1) also provide convincing evidence for a core or core plus cortex location. Arguments in support of a cortical location have been based on the similar time of syntheses of dipicolinate and the cortex, the large cortical space, and the dependance of dipicolinate retention on the cortex. A cortical location seems most unlikely, however, as the cortex shows no significant capacity to bind dipicolinic acid in vitro. In coat-deficient spores, the cortex is accessible to lysozyme but the spores retain dipicolinic acid. The coats may even be extracted (Aronson and Fitz-James, 1971) leaving no visible permeability barrier outside the cortex, and yet the spores still retain dipicolinic acid. Calcium, as the dominant cation, must be associated with at least some of the dipicolinate. In several species, the calcium and dipicolinic acid are present in nearly equivalent amounts (Murrell and Warth, 1965) but significant departures from 1 : 1 stoicheiometry implies that not all the dipicolinic acid or the calcium need be associated as the 1 : 1 chelate. Attempts to locate cal-

28

A. D. WARTH

cium by electron probe X-ray micro-analysis (Scherrer and Gerhardt, 1972) and by micro-incineration (Knaysi, 1965) showed calcium throughout the spore. Calcium was concentrated in the core, but significant amounts could well be present also in the cortex and coats. Indirect evidence for the location of calcium dipicolinate in the core comes from the peculiar property of spores, discovered by Robinow (1953), of exploding when treated with strong acid. Electron microscopy (Robinow, 1953; Fitz-James and Young, 1969) shows the core contents extruded through a break in the membrane, cortex and usually the coats. Only strong acids which have a soluble calcium salt will cause the reaction and only spores containing calcium dipicolinate will explode (A. D. Warth, unpublished results). I t would appear that the acid entering the core dissociates calcium dipicolinate forming a high concentration of calcium ions. If the membrane or cortex retains a low permeability to calcium ions, a transient osmotic force would be generated. If the calcium dipicolinate were located in the cortex the coats, but not the membrane, and cortex would be disrupted . The content of free amino acids was similar in spores and cells, but in spores consisted very largely of glutamic acid, arginine and lysine with very low levels of some of the other amino acids (Pfennig, 1957; Lee and Ordal, 1963; Nelson and Kornberg, 1970a). Spermidine was the predominant polyamine in B. megaterium (Setlow, 1974). Lesser amounts of putrescine and spermine were sometimes found. The total ribonucleotide pool in B. megaterium spores was somewhat less than in cells, and consisted mainly of the monophosphates and some diphosphates. Some high-energy compounds such as ribonucleotide triphosphates, reduced nicotinamide nucleotides and sugar phosphates were present at very low levels (Setlow and Kornberg, 1970; Setlow, 1973). On the other hand, a relatively large amount of 3-phosphoglyceric acid was present in B . megaterium and other Bacillus species. This was utilized during the first few minutes of germination to generate and maintain ATP levels (Nelson and Kornberg, 1970b; Setlow, 1975~).In addition to high levels of dipicolinic, glutamic and phosphoglyceric acids, B. subtilis but not B . cereus or B. megaterium spores contained a large amount of sulpholactic acid (Bonsen et al., 1969; Wood, 1971).Among the 14 strains of Bacillus listed inTable 6, sulpholactic acid was present in seven including B. subtilis, B. licheniformis and B . brewis.

MOLECULAR STRUCTURE OF THE BACTERIAL SPORE

29

The unique nature of the molecular environment of the vital macromolecules in the spore core is strikingly evident from the quantitative composition of the soluble fraction of spores (Table 5 ) . Protein and nucleic acids comprise only 50 to 60% of the dry weight, the remainder being principally dipicolinate, and other low molecular-weight anions and cations. The amount of enzymic protein is probably even less than that shown, at least in B . megaterium, as 30 to 50% of the protein is a basic low molecular-weight fraction that serves as a storage polymer and is probably of some importance itself in the physical structure of the core (Setlow, 1975b). Evidently the enzymes and other sensitive macromolecules are in a solvent containing a very high concentration of electrolytes. The properties of the solvent phase and its effect upon the activity and heat stability of the macromolecules depends very critically on the amount of water present. Evidence relating to the water content and physical state of the core is discussed later. VII. Ionic Composition of Spores

Divalent inorganic cations are of particular importance in the ionic composition of spores and have major effects on spore formation, heat resistance, and dormancy. Compared with vegetative bacteria, spores have very high calcium and manganese contents but often contain less TABLE 5. Composition of the Soluble Fraction of Spores Component

Bacillus megateriuma

Bacillus subtilis a

Bacillus subtilisb

% of spore dry weight

Dipicolinic acid Phosphoglyceric acid Sulpholactic acid Glutamic acid Arginine Ribonucleic acid Deoxyribonucleic acid Protein Inorganic cations

9.8 0.9 0 0.4 0.2 5.2 0.9 14.8 2.9

8.8 0.8

10 1

1.3 0.5 1.4

3-6

3.3 0.6 8.8 2.6

1

-

1

4 8 3.6

'Unpublished data of A. D. Warth Bacillus megaterium QM B 155 1 , Bacillus subtilis Porton strain. 'Data from Nelson et al.. (19691.'

A. D. WARTH

30

TABLE 6. Content of Ions in Spores

Bacillus cereus

Bacillus subtilis

Bacillus

SPP.

,u equiv./gm dry weight

Calcium Magnesium Manganese Potassium Sodium Polyamines Lysine Histidine Arginine Peptidoglycan" Total cations

1893 300 56 54 20 22 3 14 126 210 48 3043

1832 173 54 140 30 16 287 129 164 77 2902

780-1900 93-526 48-253 40-640 10-70 10-32 200-400 50-220 130-270 28-8 1 2050-3600

Dipicolinic acid Phosphate estersb Peptidoglycan" Sulpholactic acid Glutamic acid' Aspartic acid Amide

1890 3 16 195 < 10 590 500 -450

1513 293 317 i

1 .

NH-CH,OCOCH, -

1 71 0

“CIS-Sphingosine, sphing-4-enine, ~-erythrol,3-dihydroxy-2-amino-trans-4-octadecene, CH,-(CH,),,-CH=CH-CH-CH-CH,OH; C,,-Dihydro-

I

I

cn

rn

O H NH, r sphingosine, sphinganine, o-erythm-1,3-dih~droxy-2-amino-octadecane; C,,-Phytosphingosine, 4-~-hydroxysphinganine,o-ribo-1,3,4-trihydroxy-Z- rn 0 amino-octadecane. u b Ceramide, cerebrin, cer, a N-acyl derivative of a sphingosine-type base.

4 +

z

0

cn

82

P. J. BRENNAN AND D. M. LOSEL

Hemming, 1972). In Sacch. cerevisiae, the polyprenolmannose acceptor consists of a family of dolichols with from 14 to 18 isoprene units (Jung and Tanner, 19 7 31. Letoublon et al. ( 1973) characterized the particulate mannosyl transferase enzyme from A . niger responsible for transfer of mannose from GDP-mannose to the polyprenol phosphate. The ensuing polyprenol-phosphate mannose is apparently involved in the biosynthesis of aspergillus mannan (Barr and Hemming, 1972); that these mannan units are attached to protein can be inferred from the results of Letoublon and Got (1974). Similar results were obtained with the yeasts, Hansenula holstii and Sacch. cereuisiae (Bretthauer et al., 1973; Babczinski and Tanner, 1973). In addition, it was shown that the products of mannose transfer from polyprenol phosphate were mostly glycopeptides with mannose linked to serine or threonine (Bretthauer and Tray, 1974; Bretthauer and Wu, 1975; Sharma et al., 19741, although there was evidence for linkage to other amino-acids. It appears that these mannosyl- 0-serine (threonine) linkages are part of the cell-wall mannan-protein complex of yeasts. Sharma et al. (1974) and Lehle and Tanner (1974) made the important distinction that the dolicholmonophosphate is involved only in transfer to an appropriate amino acid of the yeast mannan-protein. N o lipid intermediate takes part in mannosyl-transfer reactions to mannosyl groups, in which case GDP-mannose is used directly. Therefore, the sequence depicted in Fig. 2 A probably applies for biosynthesis of yeast and aspergillus mannan-protein. Moreover, from several lines of evidence reported by Gold and Hahn ( 1976), it appears that a mannosylphosphorylpolyisoprenol is an obligatory intermediate in the in uiuo mannosylation of particulate protein in N. crassa. Recently, Lehle and Tanner (1975) reported that incubation of a membrane fraction from Sacch. cerevisiae with UDP[14C]-N-acetylglucosamine catalysed transfer of N-acetylglucosamine to endogenous lipid as well as to a methanol-insoluble polymer. The lipid fraction was subdivided into three components by thin-layer chromatography. Two were identified as dolicho1pyrophosphate-Nacetylglucosamine and dolicholpyrophosphate-di-N-acetylchitobiose. Radioactivity was also transferred to a lipid containing two mannose residues and a di-N-acetylchitobiose (i.e. a tetrasaccharide). In view of evidence (Lennarz, 1975) for pre-assembly of oligosaccharide chains of certain animal glycoproteins on a polyprenol carrier prior to their transfer to the nascent polypeptide, it seems that

nGD P- Man

A # Mannosyl- 0 serine-peptide

Polypeptide with free hydroxyl group of L-serine (threonine)

N-acetylglucosaminylasparaginyl-peptide

Polypeptide with free amino group of L-asparagine

NH,

+

dolicholypyrophosphate

I

(GlcNAc),-(Man), Polypeptide with free amino group of L-asparagine

-c

Mannan-glycoprotein of cell walls

Mannan-glycoprotein of cell walls

nGDP-Man N H -( GlcN Ac)2 - Man2

di-mannosyl-di-N-acrtylchitobiosylasparaginyl peptide

4

c

NH -( GlcNAc),(Man)n+2

Mannan glycoprotein of cell walls

u !?

8

r rn

9 mi

0

g

n

FIG. 2. Postulated biosynthesis of yeast mannan demonstrating the involvement of polyprenol and nucleotide sugars Yeast mannan is a covalently linked polysaccharide-protein complex. Some of the mannose is attached to the polypeptide chain as short oligosaccharides, glycosidically linked to serine and threonine (Ballou, 1974). Biosynthesis of these segments is represented in A. However, the majority of mannose is attached as polysaccharide chains with perhaps 150 o r more mannose units linked via N-acetylo-glucosamine to asparagine (Sentandreu and Northcote, 1968). Possible routes for biosynthesis of these segments are demonstrated in B and C. Ballou (1974)pointed out that little is known about this type of linkage because the attachment of mannose to glucosamine and the number of glucosamine units at the linkage point are uncertain.

i/j

84

P.-J.

BRENNAN AND D. M. LOSEL

Tanner’s results point to a similar phenomenon existing in eukaryotic micro - organisms. Ballou ( 19741, in discussing yeast mannans and their biosynthesis, envisaged a mechanism in which protein or short polypeptides are assembled; manno-oligosaccharides are then built on the serine and threonine units, and the longer polysaccharide chains are formed by addition first of N-acetylglucosamine to asparagine followed by stepwise addition of mannose units. From Tanner’s results, it appears that part of the longer chains are preformed on the carrier polyprenol before donation to the asparagine residue. Since it is not yet clear how many glucosamine units are at the linkage point, the relative importance of the two procedures for glycosylation is not known. Schemes by which biosynthetic and structural observations on yeast mannan can be correlated are summarized in Fig. 2. Letoublon and Got (1974) suggested that polyprenolphosphomannose is the form in which active mannose is transported across the plasma membrane for cell-wall biosynthesis. A difficulty in the Roseman (1974) hypothesis of cellular adhesion, extended to slime moulds in Section E (p. 98), is to explain how the sugar nucleotides can pass through the permeability barrier of the cell membrane into the extracellular area. However, if the active sugar is lipid-linked, then it should readily diffuse through the plasma membrane. E.

SPHINGOLIPIDS

Long-chain sphingosine-type bases are found in fungal extracts in the form of glycophosphosphingolipids, phosphosphingolipids, glycosylceramides, ceramides, acylated long-chain bases or sometimes in the free form. A small amount of an anhydrocerebrin has been obtained from baker’s yeast without the use of hydrolytic procedures (Table 5, p. 80). Whether it occurs in nature or is an artifact of isolation is not known. Previously we (Brennan et al., 1975) suggested that the bulk of fungal sphingolipids will prove to be glycosphingolipids of the type found in higher plants or animals. In animals tissues, the sugar residues are in direct glycosidic conjugation with the primary hydroxyl group of the N-acetylated sphingosine-type base (ceramide). In higher plants, the direct glycosidic bond is seen only in the simple monoglycosylceramides (cerebrosides).The remainder of plant glycosphingolipids have a phospho-inositol bridging the ceramide and

PHYSIOLOGY OF FUNGAL LIPIDS: SELECTED TOPICS

85

glycosyl moieties. Both types of glycosphingolipids have recently been found in fungi and have often been given the generic name of mycosphingolipids. 1. Free Long- Chain Bases and Ceramides

There have been several isolated reports of the presence of free bases in fungi. For instance, in Hansenula cferri, about 3% of’ the phytosphingosine occurs in the free form; the remainder is present as the tetra-acetylphytosphingosine. Probably the best authenticated report is that from Kimura et al. (1974; Table 5 , p. 80). They isolated substantial quantities (60 mg from 120 g of dried cells; 2-3% of total lipid) of a ninhydrin-positive substance from Candida intermedia grown on glucose. The compound corresponded to authentic C,,-phytosphingosine, particularly with regard to the infrared and nuclear magnetic resonance spectra, and the production of serine after periodate oxidation and hydrolysis. However, gas-liquid chromatography-mass spectroscopy of this material showed that 10%of it was the C,,-phytosphingosine. I t is noteworthy that this yeast was devoid of free ceramides of tetra-acetylphytosphingosine. Hence it appears that the organism may be deficient in the enzymes responsible for the acetylation of phytosphingosine. Until recently, it was considered that the major class of fungal sphingolipids were free ceramides (cerebrins). Table 5 (p. 80) list the fungi from which these have been isolated, and their probable structures. In some of these cases, the ceramides comprise a very large proportion of the total cell weight; in several of the moulds, mushrooms, phycomycetes and Fungi Imperfecti, they account for 0.2-0.3% of the dry tissue. In Amanita muscaria, ceramides may amount to about 3% of the dry sporophore. On the question of the distribution, total levels, and structures of fungal ceramides, the reports of Weiss and colleagues (1972, 1973) are the most comprehensive. The unprecedented abundance of ceramides in fungal extracts led to the suggestion (Brennan et al., 1975) that they might result from alkaline degradation of complex glycosphingolipids; most of the authors listed in Table 5 (p. 80) used an alkali-stable lipid fraction as a source of ceramides. I t was conceivable that treatment of lipid extracts with alkali cleaved the phosphodiester bridge of glycophosphosphingolipids resulting in the release of some free ceramides. In fact, Steiner et al. (1969) demonstrated degradation of mannosyldi-inositoldiphosphorylceramide(CerP,I,M)

TABLE 6. Complex sphingolipids of fungi Probable structure

Trivial name and abbreviation

Composition of ceramide

Source

Reference

0

II

Cer-( l+j-O-P-O-inositol

Inositolphosphorylceramide (CerPII

I

0-

Phytosphingosine and hydmxy Cz6: tatty acid

Saccharomyces cerevisiae

Smith and Lester (19741

P

0

II

L

Cer-( 1-4-O-P-O-inositol

Inositolphosphorylceramide ( CerPl]

I 0-

Phytosphingosine and C z 6 : tatty ac1d

Saccharomyces cerevisiae

Smith and Lester (19741

2

II

Inositolphosphorylceramide ( Cer PI I

Cer-( l-W-P-O-inositol

I 0-

Phytosphingosine and Saccharomyces dihydroxy Cz6: fatty acid cerevisiae

Smith and Lester (19741

.

Cer-( I+I-O-P-O-inositol

.

I

Inositolphosphorylcerarnide (CerPI)'

Not determined

Neuro$ora crassa Lester e l al. (19741

Inositolphosphorylceramide ( Cer PI Ib

Not determined

Aspergillus n i p

Di-inositoldiphosphorylteramide (CerP~Ipl

Phytosphingosine and hydroxy Cz*: tatty acid

Neurospora crassa Lester et al. ( 19741

00

II

Cer-( l+l-O-P-O-inositol

I

0-

II

Cer-0-P-0

I

Hackett and Brennan(l977)

0

II

-inos-0-P-0

O-Na+

I

> 2 0

P T

0

0

rn 2 2

P

0

I1

33

-inos

0-Na'

0

0

0-

0-

II II Cer-0-P-0-inos-(mann)-O-P-O-inos I 1

Mannosyldi-inositoldiphosphoryl- Not determined' ceramide (CerP,I,M)

Saccharomyces cereuisiae

Steiner et al. ( 1969); Steiner and Lester (1972)

Mannosylinositolphosphorylceramide (CerPIM)

C,*- and Cz0Phytosphingosine. Mixture of 2-hydroxyand non-hydroxysaturated and unsaturated fatty acids

Saccharomy ces cereuisiae

Wagner and andZofcsik (1966a. b)

Mannosylinositolphosphorylceramide (CerPIM)

C I8 -Dihydrosphingosine, 2-hydroxy- and nonhydroxy C,, fatty acids

Saccharomyces cereuisiae

Mannosylinositolphosphoryceramide (CerPIM)

C 1 8-Sphingosine, 2-hydroxy C,,-C,, fatty acids

Agaricus bisporus

0

II

Cer-0-P-0-inos-mann

I

0

W

< 0,

and Candida utilis

0

II

Cer-0-P-0-inos-mann

I

0Cer- 1'-phosphory- l-inos-(Ztl)-a-O-mann

GalactosylmannosylinositolNot determined phosphorylceramide (CerPIMGal)

Aspergtllus niger

P. J. Brennan (unpublished results)

Brennan and Roe (19751 Roe (1976)

v)

-I

? 0 v)

0

II

Cer-0-P-0 -inos- (rnann-gaI-glcId

1

0-

Glucosylgalactosylmannosylinositolphosphorylceramide (CerPIMGalClc)

Not determined

Aspergillus niger

Roe (1976)

TABLE 6.-cont. Probable structure

Trivial name and abbreviation

03 03

Composition of ceramide

Source

Rehence

0

I

Cer-0-P-0-inos-(mann,-gal,)

Trigalactosyldimannosylinositolphosphorylceramide (CerPIM,Gal,)

Not determined

Aspergillus nzger

Cer-(l’)phosphoryl-(l)inos(6cl)a-o-GlcUA Fucosyltrigalactosylglucuronosyl2 inositolphosphorylceramide (CerPI(GlcUA)Gal,Fuc)

Not determined

Agaricus bisporus

Large variety ofphytosphingosines. Mostly 2-hydroxy C , 6 : ,fatty acid Mostly C,,-sphingosine and C , , tatty acid Mostly C,, phytosphingosine and 2-hydroxy C,, fatty acid Mostly phytosp hingosines. In Amanita muscaria the major bases are o f n 22 : O and i 21 : O types. In Amanita rubescens they are mostly i 19 : 0 and i 20 :O. In Agaricus bisporus the principal bases are of the n 18 :0 and i 2 1 : 0 variety. In all sources 2-hydroxy fatty acids constitute 40-60% of the total fatty acids

Phycomyces blakesleeanus

Weiss et al. (1973)

Hansenula cferri Fusarium lint

Kaufmann et al. (1971) Weiss et al. (1973)

I 0-

t

Byme and Brcnnan (1976) (unpublished results) P. J. Brennan and J. A. Hackett, (unpublished

1

a - o - G a l ( 2 cl)a-D-GaUZ+ I ) a - o - G a l ( 2 c l)a-L-Fucc C e r-(l’tl)gl u c o s e

Glucosylceramide (Cer-Glc)

Cer4 1 ’+ 1)glucose

Glucosylceramide (Cer-Glc)

Cer-(l’+-l)glucose

Glucosylceramide (Cer-Glc)

...

,

Cer-( 1’+- 1)glucose

Glucosylceramide (Cer-Glc)

z Z

B

z B

z

P

5 r

Amanita muscaria Weiss and Stiller Amanita rubescens (1972) Agaricus bisporusf Prostenik and Clttucybe /abuscuns@ C o s ~ \ . l C( 1974)

rn

r

Cer-( 1‘C1)-galactose

Galactosylceramide (Cer-Gal)

Cer-(glucose-galactose-galactosegalactose)

Monoglucosyltrigalactosyl ceramide (Cer-(glc-gal,))

Mostly C,,-, C,,-dihydro- Saccharomyces cereuisiae sphingosine, C l x sphingosine, and 2Candida utilis hydioxy Ci6: tdtty acid Cis-sphingosine. C,,-di- Aspergillus nigerf hydrosphingosine and 2hvdroxv-Clu I fattv .” . , acid. Phytos;hiiigosine and 2- Neurospora crassa hydroxy C,,: fatty acid

Wagner and Zofcsik ( 1 966a) Wagner and Zofcsik (1966hi Wagner and Fiegert, (1969) -u

Lester etal. (1974)

5

“Neurospora c~assaseems to contain three CerPIs. bAspergillus niger seems to contain two CerPIs. ‘Tyorinoja et al. (1974)seem to have also isolated this sphingolipid from Saccharomyces cereviszae. If so, the major long-chain base is C,,-phytosphingosine and, from a previous publication (Nurminen and Suomalainen, 197 11, the principal fatty acids are 2-hydroxy-C,,: and non-hydroxy C,,: o. *Tentative structures. ‘This is a generalized structure. Two spingolipid preparations were examined, each containing at least two glycophosphosphingolipids. These differed in the presence or absence of o-glucuronic acid and L-fucose. ’P. J. Brennan and J. Madden (unpublished results) also found g1ucos;lceramide in Agaricus bisporus and Aspergillus niger. N o galactosylceramide was found in either sperics. B O f the two glucocei-ebi-osides obtained from Clitocybe tabescens, one is a glucosylceramide containing some 2-hydroxystearic acid, but with a predominance 01 heptadecanoic and decanoic acids. The other cerebrosides is a N-acilphytosphingosylglucosidecontaining mostly 2-hydroxystearic acid. There is tenuous evidence for a P-glycosidic link in these cerebrosides.

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