Advances in Microbial Physiology, Volume 13

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Advances in

MICROBIAL PHYSIOLOGY

This Page Intentionally Left Blank

Advances in

MICROBIAL PHYSIOLOGY Edited by A. H . ROSE School of Biological Sciences Bath University England

and

D. W. TEMPEST Laboratoriunz voor Microbiologie Universiteit van Amsterdam Amsterdam- C The Netherlands

VOLUME 13 1976

ACADEMIC PRESS LONDON NEW YORK SAN FRANCISCO A Subsidiary of Harcourt Brace Jovanovich,Publishers

ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road London NWl

United Statea Edition published by ACADEMIC PRESS INC. 11 1 Fifth Avenue New York, New York 10003

Copyright 0 1976 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: 87-19860 ISBN: 0-12-027713-1

PRINTED I N GREAT BRITAIN BY

WILLIAM CLOWES AND SONS LIMITED LONDON, COLCHESTER AND BECCLES

Contributors to Volume 13 W. P. WES, Lehrstuhl fur Mikrobiologie and Botanisdes Iwtitut der Universitat Munchen, Miinchen, West Germany 0. KANDLER,Lehrstuhl fur Mikrobiologie and Botanisches Iwtitut der Universitat Munchen, Munchen, West Germany

R. P. MORTLOOK, Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01002, U.S.A. J. W . PAYNE,Department of Botany, University of Durham, Science Lalioratories, South R o d , Durham D H l 3 L E , England

C. RATTLEDOE, Department of Biochemistry, University of Hull, Kingston upon Hull, HU6 7RX, North Humberside, England

K . H . SUJELEIB-ER, Lehrstuhl fur Mikrobiologie and Botanisches Iwtitut der Universitdit Miinchen, Munchen, West G ' e m n y


Contents Catabolism of Unnatural Carbohydrates by Micro-Organisms ROBERT P. MORTLOCK

I. Introduction 11. Xylitol . A. Fungi . B. Bacteria

.

III. L-Arabitol A.Fungi . B. Bacteria IV. D-ArabhBe A.ul?ngi . B. Bacteria

.

.

2

.

3

.

. .

3 4

.

.

. . .

V. L-Xylose and L - L ~ o s ~ . . A. Pathways of Catabolism B. Origin of the Enzymic Activities

.

.I VI. D - L ~ O S E VII. L - M ~ ~ .o s ~ A. Pathway of Catabolism B. Origin of the Enzymic Activities

.

WII. D-MlOSe

x. D-fiCOSe

.

XI. Conclusions

.

.

. . . .

.

.

XII. Aoknowledgements References

. .

. .

.

IX. D-TagatOSe.

. . . . .

.

. . .

15 16 16 21 21 21 36 36 38 39 41 41 41 43 45 45 47 49 60

viii

CONTENTS

Peptides and Micro-Organisms

J. W. PAYNE I. Introduction. 11. Peptides in the Nutrition of Micro-Organisms A. Introduction B. Historical C. Commercial Peptide Media. D. Strepogenin E. Modes of Utilization

. .

.

,

.

.

111. Roles of Peptidases in Peptide Utilization. , A. Introduction . B. Peptidase Activity and Growth Response to Peptides . C . Location of Peptidases and Mode of Peptide Utilization. D. Functions of Microbial Peptidases . E. Distinction Between Hydrolysis and Transport in Peptide . Utilization

IV. Peptide Transport in Micro-Organisms . A. Modes of Transport . B. Genetic and Environmental Considerations . C. Methodology of Peptide Transport Studies . D. Distinction Between Amino-Acid and Peptide Transport E. Structural Specificities of Peptide Transport Systems . F. Energetics of Peptide Transport. . G. “Binding-Proteins” and “Membrane Vesicles” . H. Regulation of Peptide Transport. .

V. Miscellaneous Relationships Between Peptides and MicroOrganisms . A. Toxin Biosynthesis . B. Toxic Peptides . . C. Concept of “Smugglins” . D. Peptide Antibiotics . E. Peptide Ionophores . F. Conjugated Peptides . . VI. Conclusions References

. .

. . . . .

. .

56 56 56 57 57 58 61

.

63 63 . 6 4 64 68

.

. .

.

. .

. . .

. .

. .

. .

71 72 72 74 74 76 77 94 95 97

.

99 99 99 101 101 103 104

. .

104 104

.

. .

.

ix

CONTNNTS

The Physiology of the Mycobacteria COLIN RATLEDGE

I. Introduction 11. Structure

.

. . . . .

.

A. Cell Wall : Chemical Composition . B. Cell Wall : Electron Microscopy C. Cell Wall : General Conclusions D. Membrane Structures . E. Nuclear Material and Ribosomes . . F. Polyphosphate Granules G. LipidVacuoles 111. Lipids :their Structure and Biosynthesis A. Influence of the Environment on Lipid Formation . B. Straight-Chain Saturated Fatty Acids C. Straight-Chain Unsaturated Fatty Acids D. Branched-Chain Acids E. Mycolic Acids . F. Effect of Isoniazid on Mycolic Acid Biosynthesis . G. Neutral Lipids and Phospholipids H. Glycolipids . . J. Carotenoids . IV. Growth in vivo : Interactions Between Invading Mycobacteria and Host Tissues. . A. Macrophage-Bacteria Interactions a t the Onset of Infection . B. Growth of Mycobacteria in vivo , C. Toxicity of Invading Mycobacteria . D. Cellular Immunity and Delayed Hypersensitivity. E. Other Cell Responses to Mycobacteria and Isolated Fractions F. General Conclusions V. Growth in vitro A. General Observations . B. Nutrition, Nutrients and their Assimilation. . C. Growth Factors and the “Non-Cultivatable” Mycobacteria . VI. Aspects of Metabolism . A. Central Pathways of Metabolism . . B. L-Lactate Oxidase C. Energy Metabolism . D. Nucleic Acids and Protein Biosynthesis References . . Acknowledgements

.

. .

. .

.

. .

.

.

.

.

. . . . . . . .

.

.

.

.

.

.

. .

.

.

. . . . . . . . . .

116 117 118 122 126 127 130 131 133 134 134 136 143 145 147 151 152 154 157 161 161 163 167 170 178 181 183 183 184 197 203 204 205 207 212 216 244

X

UONTENTS

Effect of Endogenous and Exogenous Factors on the Primary Structures of Bacterial Peptidoglycan K. H. SCHLEIFER, W.

P. HAMMES AND 0. KANDLER

. .

I. Introduction.

11. Effects of Endogenous Factors . A. Growthphase . B. Peptidoglycan Structures of Vegetative Cell Walls Compared . . with the Spore Cortex C. Changes in the Peptidoglycan Structure during a Morphological . . Life Cycle D. Genetic Variations oi the Peptidoglycan Structure . .

.

. .

111. Effects of Exogenous Factors . A. Aerobic and Anaerobic Growth . B. Amino-Acid Composition of the Growth Medium . . C. Ionic Environment . D. Osmotic Pressure of the Growth Medium , E. Antibiotics F. Growth-Inhibiting Concentrations of Glycine and D - h i n O Acids .

. .

.

IV. Concluding Remarks V. Acknowledgement. References . Author Index Subject Index

.

.

.

. . . . .

.

.

246 246 246 247 250 255 258 258 260 276 277 279 281 287 288 288 293 311

Catabolism of Unnatural Carbohydrates by Micro-Organisms ROBERT P. MORTLOCK Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01002, U.S.A. I. Introduction

.

11. Xylitol A.Fungi. B. Bacteria

.

.

2

.

. .

3 3

.

4

. . .

16 16 16

111. L-Arabitol A. Fungi. B. Bacteria

. . .

IV. D-Arabinose A. Fungi. B. Bacteria

. . .

V. L-Xyloseand L - L ~ X O S.~ A. Pathways of Catabolism . B. Origin of the Enzymic Activities

.

.

. .

VI. D-LYXOS~ VII. L-Mannose . A. Pathway of Catabolism. . B. Origin of the Enzymic Activities

VIII. D-MlOSe

.

x. D-Fucose . XI. Conclusions

.

XII. Acknowledgements References

.

. .

. . . . . .

.

Ix.D-Tagatose

. . . . . .

.

. 1

21 21 21 36 36 38 39

41 41 41 43

46 46

47

49 60

2

R. P. MORTLOCK

I. Introduction Micro-organisms are noted for their ability to catabolize a wide variety of substrates in order to provide the carbon and energy required for their growth. Many carbohydrate structures are naturally occurring in that they are biologically synthesized and accumulate as potential sources of carbon and energy for growth of micro-organisms. As a result, micro-organisms have evolved regulated, catabolic pathways for degradation of these natural carbohydrates. A large number of carbohydrate structures, however, are not found under natural conditions or are found only rarely and in very small amounts. Micro-organisms could not be expected to have evolved catabolic pathways, specific for the degradation of compounds they have not encountered in their natural environment. Even if certain carbohydrate structures not found in nature today had once been present, micro-organisms would not be expected to retain pathways for substrates which were no longer available. The current concepts of evolution predict that, without positive selection for the presence of a catabolic pathway, mutations should accumulate leading to eventual loss of the pathway. Despite these concepts it has been observed that certain of the chemically synthesized, unnatural carbohydrates will serve as growth substrates for some microorganisms. Stock culture collections include micro-organisms which are capable of using some of the unnatural carbohydrates as sole sources of carbon and energy for growth. The observations that a particular micro-organism possesses the ability to utilize a novel substrate immediately raises the question as to the route of degradation of the substrate and the origin of the structural and regulatory genes of the enzymes involved. Although the most extensive investigations into the metabolism of unnatural carbohydrates occurred in the last decade, much of the work had its origin approximately twenty years ago at the Prairie Regional Laboratories in Saskatoon, Saskatchewan, Canada where investigators were able to synthesize or obtain many carbohydrates of unnatural structures. Dr. P. J. Simpson, working in that laboratory, tested a number of microbial cultures for the ability to ferment rare five-carbon sugars. Of the bacteria available for testing, the most versatile organism with respect to its ability to ferment such suga.rs was Aerobacter aerogenes strain PRL-R3. I n testswith fermentation media, this strain was able t o metabolize all of the eight aldopentose structures (D- andL-ribose, D- and L-arabinose, D- and L-xylose and D- and L-lyxose) and all of the four pentitol structures (ribitol, D- and L-arabitol, and xylitol). This seemed quite remarkable since only three of the aldopentoses @ribose, L-arabinose and D-xylose) and only two of the pentitols (ribitol and

CATABOLISM O F UNNATURAL CARBOHYDRATES BY MICRO-ORGANISMS

3

D-arabitol) were considered to be available as growth substrates in nature. Later investigation by other workers confirmed the versatility of this organism in its ability to utilize these sugars as sources of carbon and energy (Anderson and Wood, 1962a; Mortlock and Wood, 1964a). The possible exception was L-ribose which was not available for testing as a potential growth substrate. It has been recognized in a number of laboratories that studies on the mechanisms by which micro-organisms utilize novel substrates may lead to increased understanding of the events which result in the evolution of the catabolic pathways in micro-organisms. 11. Xylitol

A. FUNGI In 1954, McCorkindale and Edson described a polyol dehydrogenase from rat liver which they called L-iditol dehydrogenase and which could catalyse oxidation of xylitol, sorbitol and L-iditol. Similar enzymic activities have been sometimes referred to as sorbitol dehydrogenases or polyol dehydrogenases since they were often capable of catalysing oxidation of these three polyols in addition to other substrates (Hollman, 1967).Because of the substrate versatility of this type of polyol dehydrogenase, rat liver, which contains a similar enzyme, is able to utilize xylitol at very high rates of metabolism (Woods and Krebs, 1973). Numerous investigators have reported enzymes of a similar nature from strains of fungi which were capable of catalysing oxidation of xylitol. Arcus and Edson reported in 1956 that cell-free extracts of Candida utilis could oxidize mannitol, sorbitol and L-rhamnitol as well as xylitol. An NAD-linked xylitol deliydrogenase was purified 35-fold from C. utilis by Chakravorty et al. (1962) and found to oxidize a variety of polyols. Xylitol was the best substrate tested although the enzyme also possessed significant activity for D-sorbitol, L-iditol, D-altritOl, D-mrtnnitol and ribitol. The reason for the existence in fungi of an enzyme with maximal activity for an uncommon pentitol such as xylitol appears to result from the metabolism of more common substrates through xylitol as an intermediate. The yeast Schwanniomyces occidentalis catalyses inositol through a pathway believed to involve D-glucuronic acid, L-gluconate, L-xylulose, xylitol and D-xylulose as intermediates (Sivak and Hoffman, 1961). A common route for aldopentose degradation in yeast has been shown to involve reduction of the aldopentose by an NADP+-linked reductase to the corresponding pentitol. This is followed by oxidation of the pentitol by an NAD+-linkeddehydrogenase to yield a 2-ketopentose. An enzymic pathway of this nature, catalysing reduction of D-xylose to

4

R. P. MORTLOOK

xylitol and oxidation of xylitol to D-xylulose, has been described for Penicillium chrysogenum, Geotrichumcandidum, Candida utilis, Saccharomyces rouxii and other fungi (Chiang and Knight, 1961, 1966; Moret and Sperti, 1962; Scher and Horecker, 1966; Ingram and Wood, 1966,1966). I n 1960, Chiang and Knight tested 20 strains of yeast or filamentous fungi for their ability to grow in a D-xylose-salts medium. Of the 14 strains which were capable of growth, all possessed the enzymic activities to catalyse reduction of D-xylose t o xylitol and oxidation of xylitol to D- or L-xylulose. Onishi and Suzuki (1969) have reported a procedure for production of xylitol by a strain of Candida guilliermondi. Barnett, in 1968, tested 16 strains of yeasts for their ability to grow using any one of eight different polyols as the sole source of carbon. The polyols tested were erythritol, ribitol, D-arabitol, L-arabitol, xylitol, sorbitol, galactitol and D-mannitol. Six of the strains tested, including one species of Kluyveromyces, one species of Torulopsis, two of Trigonopsis and two species of Debaryomyces, could utilize xylitol as a growth substrate. All of the strains which could grow using a particular polyol could also respire that polyol, but for no strain tested could whole cells respire a substrate on which they could not grow. Growth of the organisms was believed to result from pre-existing enzymic ability and not from selection of mutants. A species of Torulopsis candida was the most versatile of the yeasts tested in being able to use all eight of the polyols tested as growth substrates, including xylitol and L-arabitol. Three separate enzymes, each capable of catalysing oxidation of xylitol, were found in cell-free extracts of T . candida. These enzymes included an NAD-linked dehydrogenase catalysing oxidation of xylitol to D-xylulose, and an NADPlinked enzyme catalysing oxidation of xylitol to D-xylose. In contrast to the data obtained with this Torulopsis species, Candida utilis could not use any of the polyols tested as growth substrates even though cell-free extracts prepared from C . utilis grown on D-xylose did possess enzymic activity for the NAD-linked oxidation of xylitol. For that reason, the lack of ability of C. utilis to grow on xylitol and the other polyols was believed to be due to the inability of the sugars to enter the cells (Barnett, 1968).

B. BACTERIA 1. Enzyme Activities There have been a number of reports in the literature of bacterial dehydrogenases which catalyse oxidation of xylitol, in addition to other and more naturally occurring substrates. I n 1956, Arcus and Edson found several enzymes in Acetobacter suboxydans which could oxidize xylitol. One enzyme was a pEbrtiCdate, cytochrome-linked D-mannitol

OATABOLISM O F UNNATURAL CARBOHYDRATES BY MICRO-ORGANISMS

5

dehydrogenase which would oxidize erythritol, ribitol, D-arabitol, D-mannitol and sorbitol rapidly, and xylitol, allitol and D-talitol slowly. Cell-free extracts also contained a soluble NAD+-linked,polyol dehydrogenase having its best catalytic activity on xylitol, mannitol and sorbitol. Growth of the organism on xylitol was not reported. Shaw, in 1956, studied an NAD+-linkedpolyol dehydrogenase from a bacterium in the genus Pseudomonas. Growth on sorbitol or dulcitol induced formation of a galactitol dehydrogenase which oxidized dulcitol to D-tagatose. Fully hydroxylated polyols containing an L-threo configuration adjacent to a primary alcohol group were attacked by the enzyme. In addition to dulcitol, sorbitol was oxidized to D-fructose, L-iditol to L-sorbose, L-arabitol to L-ribulose, and xylitol to xylulose. Cells grown on dulcitol also contained a D-iditol dehydrogenase which would oxidize D-iditol, D-gulitol, D-talitol to D-allulose, and xylitol to L-xylulose. Whether or not the organism was capable of growth utilizing xylitol was not reported. In 1968, Yamanaka and Sakai reported that Pseudomonas jlwrescens grown on sorbitol or mannitol, Pseudomonas coronafaciens grown on mannitol, Sarcina marginata grown on sorbitol and Sarcina aurantiaca grown on mannitol all possessed activity for oxidation of xylitol in addition to other substrates. The ability ofthese organisms to use xylitol as a growth substrate was not tested. Kersters et al. (1966) isolated a number of polyol dehydrogenases with differing substrate specificities from Gluconobacter oxydans. They found an NADP+-linkedxylitol dehydrogenasespecificfor xylitol as a substrate and capable of catalysing oxidation of xylitol to L-xylulose. The cells also contained an NAD+-linked D-erythro dehydrogenase which would oxidize ribitol to D-ribulose, xylitol to D-XyhdOSe, L-arabitol to both L-xylulose and L-ribulose, L-mannitol to L-fructose, and L-glucitol to D-sorbose.A third enzymeidentifiedwasanNAD+-linkedD-xylodehydrogenase which would oxidize D-glucitol to D-fructose. With the activity of this latter enzyme on D-glucitol at loo%, other activities were: xylitol to D-xylulose (21?40),rAditol to L-sorbose (la%), and D-mannitol to Dfructose (2%).Other dehydrogenaseactivities includedanNADP+-linked D-lyxo dehydrogenase catalysing oxidation of D-mannitol to D-fructose (loo%), D-glucitolto L-sorbose (la%),and D-arabitolto D-xyldose (2%). Two different NADP-linked gluconate dehydrogenases and one or more L-erythro dehydrogenases were also found attached to the cytoplasmic membrane. The latter enzymes could oxidize L-threitol, erytfitol, D-arabitol, ribitol, mannitol, D-glucitol, allitol, and D-altritol. Even though the organism was capable of synthesizing at least three separate cytoplasmic enzymes which could catalyse oxidation of xylitol, growing cells could not oxidize xylitol as a substrate. Likewise, growing

6

R . P. MORTLOCR

cells could not oxidize L-arabitol, L-mannitol or L-rhamnitol. The lack of ability to grow using any of these three carbohydrates could result from an inability t o transport the sugars through the cell membranes or from the toxicity of products formed by oxidation of the s u p r s .

2. Growth of Bacillus In 1964, Horwitz and Kaplan reported growth of Bacillus subtilis using xylitol as the carbon and energy source. Cells of B. suhtilis grown on D-sorbitol contained an inducible sorbitol dehydrogenase which would catalyse oxidation of a number of polyols. I n addition to sorbitol, xylitol, L-iditol, allitol and ribitol were active as substrates (Horwitz, 1966). Xylitol was 25% more active as a substrate than was sorbitol but, in spite of this, the organism could not utilize xylitol as an only carbon source for growth. Apparently, xylitol did not serve as an inducer for the enzyme, and incubation of cells with xylitol did not result in synthesis of sufficientquantities of the enzyme t o permit growth. Horwitz and Kaplan (1964) found that the organism could grow using xylitol as a substrate, providing a small amount of sorbitol was also present in the medium to cause induction of the dehydrogenase. It could now be predicted that xylitol should serve as a selective substrate for the isolation of mutants of B. subtilis which would be constitutive for the enzyme and therefore capable of utilizing xylitol as the sole carbon source for growth. The authors did not report whether or not they had attempted t o isolate xylitol-positive mutants.

3. Growth of Azotobacter In 1961, Marcus and Marr reported growth of Azotobacter agilis with xylitol as the substrate. This organism was found t o be capable of synthesizing two separate polyol dehydrogenases. One was an L-iditol dehydrogenase which was induced by polyols with a D-xylo configuration such as sorbitol, L-iditol, xylitol, and ribitol with relative rates of activity of 100, 102, and 9%, respectively. The other enzyme was a D-mannitol dehydrogenase oxidizing D-mannitol, D-arabitol, D-rhamnitol and perietol. D-Ambit01 was also one of the inducers of this enzyme. The organism was capable of growth on D-mannitol, sorbitol, D-arabitol, erythritol, glycerol, D-fructose, L-sorbose, sucrose and D-glucose, hut not galactitol, ribitol, L-arabitol, D-mannose, D-ribose, or lactose. The product of the oxidation of both xylitol by the L-iditol dehydrogenase and D-arabitol by D-mannitol dehydrogenase was D-XyhdOSe. Since D-arabitol is a naturally occurring carbohydrate, the organism presumably possessed the required enzymic activities for the further degradation of D-xylulose. Thus, the ability of xylitol to act as both

CATABOLISM OF UNNATURAL CARBOHYDRATES BY MICRO-ORGANISMS

7

inducer and substrate for the L-iditol dehydrogenase permitted the organism to utilize xylitol as a growth substrate.

4 . Growth of Klebsiella (Aerobacter) Many of the organisms previously called Aerobacter would now be classified in the genus Klebsiella. I n referring to previous work, the generic name actually used by the investigators a t the time will be repeated. As mentioned previously, F. J. Simpson observed fermentation of all four pentitols, including xylitol and L-arabitol, by Aerobacter aerogenes strain PRL-R3. Later investigation in the laboratory of W. A. Wood confirmed the versatility of this strain and showed that it was able to utilize all of the four pentitols a,s sole sources of carbon and energy for growth (Mortlock and Wood, 1964a). When cells which had been grown with D-glucose as the substrate were transferred to a medium with either of the two common pentitols, ribitol or n-arabitol, as sole carbon sources, growth followed a lag period of several hours and was complete within one day. However, as shown in Table 1, when xylitol was used as the growth substrate in similar experiments, a lag period of a t least three days was normally required before growth could be observed. Transfer TABLE1. Growth of Aerobacter aerogenes P R L - R 3 o n various pentoses and pentitols Substrate used for growth of inoculum ~

Growth substratc D-G~UCOSC D -XylOSO

L-Xylosc D -Arabinose L-Arabinose D-Riboso D -LyXOSe D -Ambit01 L-Arabitol Ribitol Xylitol

D-

Glucose

0*5+ 0.5 27 2 0.5

1 4 0.5

2 1 4

D-

L-

Arabinose Arabitol

D-

L-

Lyxose

Xylose

0.5

0.5

0.5

0.5 25 1

0.5 22 4 1

0.5 27 2 1 1

0.5

1 4

0.6 2 1 4

1 4 1 1 1 4

1 1

2 1 4

0.5 0-5

2 1.6 1 1

Xylitol

0.5 0.5 5 1 1 1

4 0.5 1

4 1 1

1 4

1 2

Incubation was carried out in screw-capped tubes containing 5.5 ml of salts solution supplernentcd with 0.6% (w/v) of the indicated carbohydrate. Tho inoculum consisted of 2 x 106 cells per ml, and was prepared from a culture grown on the indicated carbohydrate, harvested by centrifugation, and washed twice with sterile salts solution. The tubes were slanted in test-tube racks and shaken at 26°C. Turbidity determinations were made at time intervals, and were corrected for carbohydrate blanks. Values indicate the number of days required to reach a, turbidity reading of 1.0. From Mortlock and Wood (19648).

8

R. P. NORTLOCK

of xylitol-grown cells to a second xylitol-salts medium resulted in elimination of this lag period and completion of growth in only two days (Table 1). Cells which had been isolated on xylitol retained the ability to grow on xylitol without the original long lag period even after transfer through a glucose-saltsmedium. The latter observation indicated that the original lag period had been required for selection of mutants which had acquired the ability to utilize xylitol as a growth substrate. When wildtype cells were transferred to a xylitol-containing medium, the culture did not possess the enzymic activities required for catabolism of xylitol until after several days’ incubation and not until growth of the culture was f i s t detected. I n contrast, incubation of wild-type cells with the naturally occurring pentitols, ribitol or D-arabitol, lead to induction of the enzymic activities for catabolism of the particular pentitol being tested within several hours (Mortlock and Wood, 1964b). (a) Pathway of catabolism. The pathways of catabolism of ribitol and Darabitol have been studied in different laboratories using several strains of Aerobacter aerogenes (Fromm, 1958; Nordie and Fromm, 1959; Wood et al., 1961; Fromni and Nelson, 1962; Fromm and Bietz, 1966; Lin, 1961; Hully et al., 1962). Ribitol has been shown to be oxidized by an NAD-linked dehydrogenase to the corresponding ketopentose, Dribulose (Fromm, 1958; Wood et al., 1961; Hully et al., 1962). A Dribulokinase then catalysed phosphorylation at the five-carbon position t o yield D-ribulose 6-phosphate as shown in Fig. la. I n a similar manner, as shown in Fig. lc, D-arabitol was oxidized to D-xylulose and then phosphorylated to yield D-xylulose 5-phosphate (Wood et al., 1961; Lin, 1961). Epimerization reactions and transketolase and transaldolase re-arrangements then converted these pentulose phosphates into intermediates of the hexose diphosphate catabolic pathway. Fossitt et al. in 1964 reported the pathway for degradation of xylitol by A . aerogenes PRL-R3 to comprise oxidation of xylitol to D-xylulose catalysed by an NAD+-linked dehydrogenase, followed by phosphorylation to yield D-xylulose 5-phosphate as illustrated in Fig. Id. Since the aldopentose D-xylose was metabolized by an inducible enzyme pathway involving isomerization to D-xylulose (Fig. lb), D-xylulose was a known intermediate in at least two existing catabolic pathways (Altermatt et al., 1966; Simpson and Bhuyan, 1962).Thus, discounting possible problems in permeability, the mutational event required to permit growth on xylitol appeared to result in the acquisition of one new enzymic activity which could catalyse oxidation of xylitol to D-xylulose (Fossitt et al., 1964; Mortlock and Wood, 1964b).

(b). Origin of dehydrogenase activity. Several workers have reported the presence of high levels of ribitol dehydrogenase activity in cell-free

9

CATABOLISM OB UNNATURBL CARBOHYDRATES B Y MICRO-ORQANISYS

CHzOH

(a)

I H-C-OH I H-C-OH I H-C-OH I

CH,OH

)I Ribilol dchydiogcnaae

,

NADf

-

C=O

I I H-C-OH I

I>-Ilibulokinas~.

H-C-OH

CH,OH

I I

C=O

H-C-OH

A l'P, M g 2 +

I I

H-C-OH CHzOPOjHz

CHzOH

CHzOH

Ribitol

D-Ribulose

D-Ribulose 5-phosphate

/

D -Ribulose 5- pbephnte 3- epimerase

CHZOH

I c=o I

IEO-C-H

I

H-C-OH

I

CHzOPOJH2 D-XYhlOSe 5-phosphate

CHO

(b)

CH,OH

I H-C-OH I HO&H I H-C-OH I

n-xylose isoineraac

,

*ugz+

I

I

(d) HO-C-H

I 1

H-C-OH CH,OH Xylitol

D-Xylulose6-phosphate

CHZOH

CHZOH

c=o

c=o

I

n-Arabitol ddydroornase

4 - 2

NADf

CHZOH

I H-c-oH

I I HO--CH I H-&OH I

CHzOPOjHz

D-XylulOSe

I

I

A Y'P, Mgz+

I

I

CHzOH

HO-C-H

D-X'Ululokinaael

CHZOH

D-Xylose

(0)

110-C-H

CH20H

c=o

H-C-OH

CH2OH

H0-C-H

I c-0 I

I I

HO-C-€1

I

~-X&dokin~e

A TP,bf@+

I

HO-C-H

I

CH,OH

CHZOH

I

I

c=o I I HO-C-H F HO-C-H 7A T P , Mg2+ NADt I I H-SOH H-C-OH I I Ribit01 dehydrogeanse

D-Xylulokinase (1 or 2)

CHZOH

D-XYlulOm

CHzOP,OIHS

D-Xylulose6-phosphate

FIQ.1. Pathways for degradation of (a) ribitol, (b) D-XylOBe, (d) xylitol by Aerobacter aer0gene.s strain PRL-R3.

(0) D-arabitol

and

10

R. P. MORTLOCK

extracts prepared from cells grown with xylitol as the carbon source (Mortlock and Wood, 1964a; Fossitt et al., 1964). I n 1964,afterastudy of the physical and immunological properties of the pentitol dehydrogenases and pentulokinase of the PRL-R3 strain, Mortlock et al. showed that oxidation of xylitol was catalysed by ribitol dehydrogenase. The ribitol dehydrogenase purified from xylitol-grown cells appeared to be identical with the dehydrogenase purified from ribitol-grown cells, with respect to the properties listed in Table 2. Furthermore, the ribitol and xylitol dehydrogenase activities could not be separated by purification involving electrophoresis, chromatography on DEAE-cellulose or sucrose-gradient centrifugation (Mortlock et al., 1965). Antiserum prepared using purified ribitol dehydrogenase as antigen inhibited the enzymic activity of ribitol dehydrogenase from ribitol-grown cells. Such antiserum was also effective in inhibiting both the ribitol and xylitol dehydrogenase activities from xylitol-grown cells. Therefore, the xylitolpositive mutants did not possess a unique or specific xylitol dehydrogenase, but, during growth on xylitol, the ribitol dehydrogenase of the ribitol catabolic pathway was catalysing oxidation of xylitol t o the corresponding ketopentose, D-xylulose. TABLE2. Comparison of ribitol dehydrogenases from different sources Source of dehydrogenase Inducible (ribitolcontaining culture)

Constitutive (xylitolcontaining culture)

Constitutive (L-arabitolcontaining culture)

2.6 x 10-3 M 2.9 x lo-' M 2.9 x lo-' M

3.5 x 10-3 M 2.5 x lo-' M 2.7 x lo-' M

2 4 x 10-3 M 4.0 x lo-' M 4.2 x lo-' M

100 35 34

100 39 23

100 46 44

4.5-4-8 6.0

5.0 6.8-6.2

5.0 -

0.4-0.41 0.40 30,000-32,000

0.39 0.40 32,000

___.-

K , valuo, Ribitol K , value, L-Arabitol K , value, Xylitol Ratio of activity at Vmax

Ribitol L-Arabitol Xylitol Heat sensitivity s20, w

Gel electrophoresis Protein (R,) Activity (R,) Specific activity

0.40 0.40 32,000

Specific activity is expressed in units of enzyme per mg of protein. One unit of enzymic activity catalysed an absorbancy change of 1.0 per min. From Mortlock et al. (1966).

CATABOLISM OF UNNATURAL CARBOHYDRATES BY MICRO-ORQANISMS

11

Since the wild-type cells already possessed the ability to synthesize an cnzyme capable of catalysing oxidation of xylitol, the question arose as to the nature of the mutation which was required to permit growth on xylitol. As an enzyme evolved for the ribitol catabolic pathway, ribitol dehydrogenase was regulated for the catabolism of ribitol and induced when cells were incubated in the presence of ribitol. The activity of the enzyme with xylitol as a substrate was relatively low as compared with ribitol (Table 2)) and the amount of enzyme present in un-induced cells was not sufficient to permit detectable growth with xylitol as the substrate. The situation was similar to that previously mentioned for Bacillus subtilis, in that xylitol would serve as a substrate but not an inducer for the enzyme. The ability to utilize xylitol as a growth substrate was found only in mutants which were constitutive for ribitol dehydrogenase and therefore able to synthesize high levels of the enzyme in the absence of the normal inducer (Mortlock et al., 1964). Thus, the ability of the organism to grow with xylitol as the sole carbon and energy source resulted from both the gratuitous ability of a pre-existing enzyme to catalyse the key reaction in conversion of the novel substrate to an intermediate which could then be further degraded by the existing enzymic pathways of the cell, and the ease of selection of mutants constitutive for that enzyme. Ribitol dehydrogenase was purified and crystallized from ribitolgrown cells and also from a constitutive mutant which had been isolated by growth on xylitol (Mortlock et al., 196513).The enzymes from either source showed similar physical and immunological properties (Table 2). Each dehydrogenase migrated in polyacrylamide gel with an identical single protein band. The enzyme was capable of catalysing the NAD+linked oxidation of ribitol to D-ribulose, xylitol to D-xylulose, and in addition, L-arabitol to L-xylulose. Constitutive synthesis of the enzyme was repressed by growth on D-glucose but not by growth on casein hydrolysate. The maintenance of constitutive mutants on slants of casein hydrolysate, peptone or nutrient agar led to the selection of revertants which were again inducible for the enzyme and incapable of growth on xylitol. A similar mechanism permitting growth on xylitol was reported by Lerner et al. in 1964 for Aerobacter aerogenes strain 1033. Mutants of this strain which were capable of growth on xylitol were also constitutive for ribitol dehydrogenase. A mutant which had been directly isolated for constitutive synthesis of ribitol dehydrogenase without the use of xylitol was capable of growth on xylitol without requiring further mutation, and a mutant which had lost the ability to synthesize ribitol dehydrogenase simultaneously lost the ability to grow on both ribitol and xylitol (Lerner et al., 1964). The same mechanism for mutation to growth

12

R . P. MORTLOCK

on xylitol has now been reported for Klebsiella aerogenes W-70, a strain which can be subjected to genetic analysis by transduction (MacPhee et al., 1969).I n this strain, mutations which permit constitutive synthesis of both enzymes of the ribitol catabolic pathway (ribitol dehydrogenase and D-ribdokinase) and also permit growth on xylitol have been mapped adjacent to, but distinct from, the dehydrogenase and kinase structural genes (Charnetzky and Mortlock, 1970). These genetic loci are postulated to represent the regulatory genes of the ribitol catabolic pathway. (c). Origin of kinase activity. In A . aerogenes PRL-R3, the two enzymes of the D-xylose catabolic pathway, namely D-xylose isomerase and Dxylulokinase, were found to be co-ordinately induced (Fig. lb, p. 9). D-Xylose was the apparent inducer, since D-xylose would induce normal levels of kinase in isomerase-deficient mutants (Wilson and Mortlock, 1973). The two enzymes involved in D-arabitol catabolism, D-arabitol dehydrogenase and a separate D-xylulokinase, have been shown to be co-ordinately controlled with D-arabitolas the apparent inducer (Fig. lc). The two D-xylulokhases had similar activity for D-xylulose but could be differentiated immunologically and also by the cold sensitivity of the b a s e of the D-arabitol pathway (Wilson and Mortlock, 1973). The presence of either D-arabitol dehydrogenase or D-xylose isomerase in cell-free extracts prepared from cells grown on xylitol could be used as an indication of which of the two D-xylulokinases was being employed to catalyse phosphorylation of the D-xylulose obtained from the oxidation of xylitol. Cells of strain PRL-R3 grown on xylitol (constitutive for ribitol dehydrogenase)possessed in addition to ribitol dehydrogenase and the co-ordinately controlled D-ribulokinase,low levels of D-arabitol dehydrogenase and D-xylulokinase, indicating the use of D-xylulokinase of the D-arabitol pathway (Mortlock and Wood, 1964a; Fossitt et al., 1964). The reason for the slight induction of the D-arabitol catabolic pathway by xylitol has not been determined. Either some of the Dxylulose formed by oxidation of xylitol might be converted to D-arabitol resulting in induction of the enzymes of the D-arabitol pathway, or xylitol itself might act as a weak inducer of the D-arabitol pathway enzymes. Occasionally, low levels of D-xylose isomerase were also detected. I n this latter case, the isomerase might have been induced along with the kinase of the D-xylose pathway by conversion of some D-xylulose t o D-xylose (Wilson and Mortlock, 1973). Such data indicated that either of the two D-xylulokinases could be used for catabolism of xylitol. When mutant strains, which were lacking either the D-xylulokinase of the D-arabitol pathway or the D-xylulokinase of the D-xylose pathway, were used in an attempt to select

UATABOLISM OF UNNATURAL CARBOHYDRATES BY MCRO-ORGANISMS

13

mutants capable of growth on xylitol, xylitol-positive strains (ribitol dehydrogenase constitutive) could be obtained. However, when a parent strain deficient in both of these kinases was used in similar experiments, mutants capable of growth on xylitol could not be isolated. This also suggested that either kinase could function for growth on xylitol (Wilson and Mortlock, 1973). Genetic data obtained by means of transduction with K . aerogenes W-70 have shown a similar situation to exist with respect to the origin of the b a s e activities of the xylitol catabolic pathway (Charnetzky and Mortlock, 1970, 1972). (d). Origin of transport activity. As mentioned previously, Lin and his coworkers established the mechanism of growth of A . aerogenes strain 1033 on xylitol as involving a mutation for constitutive synthesis of ribitol dehydrogenase (Lerner et al., 1964). The mutant, termed XI, originally possessed a doubling time of 270 minutes on xylitol. After treatment with N-methyl-N-nitro-N-nitroso guanidine and cultivation of X I on xylitol, strain X2 was obtained which doubled on xylitol every 110 min (Wu et al., 1968). This new mutant possessed an altered ribitol dehydrogenase with increased activity for oxidation of xylitol. Cell-free extracts from strain X2 showed a specific activity of 0.164 units per milligram of protein for xylitol oxidation, as compared with 0.066 for the X1 strain. No change was observed, however, in the K , value of the enzyme for ribitol or xylitol. Additional cultivation on xylitol a t a low concentration of substrate (0.05%) led to the isolation of strain X3, with a doubling time of 55 min. The ribitol dehydrogenase of strain X3 was similar to that of strain X2, but strain X3 possessed a constitutive active transport system for xylitol. This transport system appeared to be that normally employed for transport of D-arabitol and induced by D-arabitol in the parent strains. It is possible that, in the parent X1 or X2 strains where this transport system was inducible, enough D-xylulose was converted to D-arabitol to cause a limited induction of this transport system, or perhaps xylitol itself might have acted as a weak inducer of the system. Mutation to constitutive synthesis of the transport system increased the uptake of xylitol from the medium and thus permitted an increased growth rate on xylitol (Table 3). (e). Evolutionary studies. The possible advantages of using xylitol as a substrate to study mutations which lead to the establishment of a new catabolic pathway in bacteria has been discussed by a number of authors (Lerner et al., 1964;Wu et al., 1968;Mortlock and Wood, 1971;Hegeman and Rosenberg, 1970; Hartley et al., 1972; Clarke, 1974).Since the initial mutation which permits growth (the constitutive synthesis of ribitol dehydrogenase) results in relatively slow growth with xylitol as the carbon source, continued cultivation of cells on xylitol can lead to the

14

R . P. MORTLOCK

TABLE3. Origin of enzyme activities used for xylitol degradation ~~~

~~

Reaction

Enzyme

Enzyme source

Regulation of the enzyme during growth on xylitol

Oxidation of xylitol to D -xylulose

Ribitol dehydrogenase

Ribitol pathway

Requires a mutation to constitutive synthesis

Phosphorylation of D-xylulose to ~-xylulose 5-phosphate

D-Xylulokinase 1 or 2

D-Arabitol or D-XylOse pathway

Induced by incubation with, or metabolism of, xylitol

Transport of xylitol through cell membrane

~-Arabitol transport system

n-Arabitol pathway

Induced but will result in an increase in growth rate when constitutive

selection of additional mutations which establish faster growth rates due to more efficient catabolism of the pentitol. Lin and his coworkers (Lerner et al., 1964), after treating a xylitolpositive strain of A . aerogenes strain 1033 with a mutagen, isolated a mutant which was capable of more rapid growth on xylitol. The new mutation was apparently located in the dehydrogenase structural gene, and the altered dehydrogeiiase possessed high activity for oxidation of xylitol. As mentioned previously, the doubling time on xylitol of this new mutant was 110 min as compared with 270 min for its parent strain. The specific activity of the enzyme for xylitol had changed from 0.066 units per milligram of protein to 0.164 units per milligram of protein. However, no change in the specific activity for ribitol was observed, Chemostats have been used in attcmpts to isolate mutants with increased growth rates on xylitol. Mortlock and Wood (1971)reported that cultivation of a xylitol-positive strain of A . aerogenes PRL-R3 in a chemostat with xylitol as the only carbon and energy source resulted in the selection of a mutant whose growth rate on xylitol had increased from 0.26 to 0.5 generations per h. No change could be detected in the activity of the purified enzyme for either xylitol or ribitol, but the constitutive dehydrogenase level found in crude cell-free extracts had increased from 7.5 to 31 units per milligram of protein. Since the activity of the enzyme for xylitol as a substrate was low as compared with the activity for ribitol, the increase in amount of enzyme apparently permitted a more rapid rate of oxidation of xylitol and an increase in the growth rate with xylitol a s a substrate.


15

Hartlcy et ul. (1 972) reported extensive chemostat experiments using the xylitol-positive mutant of A . aeroyenes strain 1033. With xylitol as the selective substrate, a series of mutants with increasing growth rates for xylitol were obtained. No changes in the apparent K , value of the dehydrogenase for ribitol or xylitol were observed with these mutant strains, but increases in the amount of enzyme (specific activity per milligram of protein) were observed. One strain isolated from the chemostat produced 13-15 times more enzyme than the parent strain, with ribitol dehydrogenases now accounting for 18% of the total protein of cell-free extracts. The strain carrying this elevated dehydrogenase level was found to be unstable in that it segregated to yield clones with lowered enzyme levels. This latter fact, plus the inability to isolate ribitol dehydrogenase-negative mutants from this strain, led the authors to conclude that the high levels of dehydrogenaso observed resulted from multiple copies of the deliydrogenase structural gene (Hartley et al., 197 2 ) . Similar results were obtained using cultures that were treated with ultraviolet radiation. Since these extensive chemostat experiments did not select for a mutant possessing an altered dehydrogenase with increased activity on xylitol, it seems likely that the mutant obtained by Lcriier et al. (1964),after treatment with nitrosoguanidine, resulted from more than a single step amino-acid substitution (B. S. Hartley, personal communication). 111. L-Arabitol A. FUNGI L-Arabitol is believed to be rare in nature although it is present in the urine of persons suffering from the metabolic disease, pentosuria (Touster and Harwell, 1958), and an L-arabitol-oxidizing enzyme has been purified from sheep seminal vesicles (Horecker et al., 1967). Certain fungi are capable of metabolizing L-arabitol through ~-xylulose, xylitol, and D-xylulose as intermediates (Chiang and Knight, 1967). L-Xylulose is also an intermediate in the metabolism of glucuronic acid (Stirpe and Comporti, 1965; Stirpe et al., 1965). Chiang and Knight (1961, 1966) reported two independent NAD+linked enzymes in Penicillium chrysogenum which were each capable of catalysing oxidation of L-arabitol. One enzyme produced L-ribulose and the other L-xylulose as the oxidation product. Scher and Horecker (1966) found an NAD+-linked enzyme from Candida utilis which catalysed oxidation of xylitol to D-xylulose but also possessed higher activity for oxidation of L-arabitol. The product of L-arabitol oxidation was presumably L-arabinose.

16

a.P. MORTLOCK

Martinez de Drets and Arius (1970),while studying the induction of a D-arabitol dehydrogenase and a D-sorbitol dehydrogenase in Rhizobiurn rneliloti, observed growth of these cells withL-arabitol as a substrate. The L-arabitol-grown cells did not possess either of the two dehydrogenase activities under study, and the pathway of L-arabitol utilization was not investigated. Of the yeast strains tested by Barnett for growth on polyols, two strains of Torulopsis and two strains of Debaryornyces were able to grow with L-arabitol as the only carbon source (1968).Whole cells of T . candida could oxidize L-arabitol much faster when grown on sorbitol than on other substrates, but cell-free extracts could not catalyse oxidation of either sorbitol or L-arabitol. Extracts prepared from sorbitol-grown cells did reduce L-arabinose in the presence of NADPH, but L-arabitol was not identified as the product of reduction. Since a common pathway of L-arabinose catabolism in fungi involves reduction of L-arabinose to L-arabitol, and oxidation of L-arabitol to a 2-ketopentoseYit would appear that such strains of fungi possessed the enzymic capability for catabolism of L-arabitol, providing they were capable of transporting L-arabitol into the cells.

B. BACTERIA 1. Enzymic Activities Shaw, in 1956, showed a pseudomonad to be abIe to synthesize a galactitol dehydrogenasewhich could also catalyse oxidation of L-arabitol to L-ribulose. Kersters et al. (1965)reported a D-erythro dehydrogenase from Gluconobacter which could oxidize L-arabitol to either L-ribulose or L-xylulose, but L-arabitol was not oxidized by growing cells. Jakoby and Fredericks in 1961, and Jakoby in 1966,reported the partial purification of an enzyme from Aerobacter aerogenes strain ATCC 13797 which had been grown with erythritol as the sole carbon source. I n addition to erythritol (100% activity), the enzyme also oxidized ribitol (27% activity) and L-arabitol (16% activity). The product of L-arabitol oxidation was presumably L-ribulose.

[2. Growth of Aerobacter Simpson first observed the fermentation of L-arabitol by Aerobacter aerogenes strain PRL-R3 and the ability of this strain to grow using L-arabitol as a sole carbon source was later confirmed by Mortlock and Wood (1964a).As shown in Table 1 (p. 7), after inoculation of wild-type cells into an L-arabitol-salts medium, a lag period of from one to two days was required before growth was first detected. Enzymic activities for oxidation of L-arabitol and phosphorylation of L-xylulose could be detected in cell-free extracts prepared from cells growing on L-arabitol (Mortlock and Wood, 19648, b). Once growth on L-arabitol had been


17

completed, transfer to a second L-arabitol-containing medium resulted in a lag phase of only several hours, and growth was completed within 24 h. These observations suggested that an L-arabitol-positive mutant had been selected from the original population of cells. In addition to the relatively low levels of L-arabitol dehydrogenase activity observed in cells grown on L-arabitol, high levels of ribitol dehydrogenase activity were also found.

(a). Pathway of catabolism. Fossitt et al. (1964)also showed that cell-free extracts prepared from A . aerogenes strain PRL-R3, which had been grown with L-arabitol as the only carbon and energy source, contained enzymic activity for oxidation of L-arabitol, D-arabitol and ribitol. The product of L-arabitol oxidation by an NAD+-linked dehydrogenase was identified as L-xylulose, and L-xylulose was reduced to L-arabitol in the presence of the same enzyme and NADH. Anderson and Wood (1962a) had previously shown this organism to be capable of the synthesis of an L-xylulokinase which would phosphorylate L-xylulose to L-xylulose 5-phosphate, and this kinase activity was detected in extracts of Larabitol grown cells (Fossitt et al., 1964). Anderson and Wood (1962a) had also shown that L-xylulose 5-phosphate could then be converted to L-ribulose 5-phosphate by an L-xylulose 5-phosphate 3-epimerase, and this could be epimerized to D-xylulose &phosphate by the action of an L-ribulose &phosphate 4-epimerase. This last epimerase was also required during catabolism of the naturally occurring aldopentose, L-arabinose (Fig. 2a). Fossitt et al. (1964)were able to demonstrate both of these epimerase activities in extracts of cells grown on L-arabitol, and showed conversion of L-xylulose 5-phosphate to D-xylulose 5-phosphate. In this manner, the pathway for L-arabitol degradation was shown to involve oxidation of L-arabitol to L-xylulose, phosphorylation to Lxylulose 5-phosphate, and two epimerization reactions t o yield Dxylulose &phosphate (Fig. 2b). (b). Origin of the dehydrogenase activity. As mentioned previously, cells grown on L-arabitol contained high activity for oxidation of ribitol. Activity was also observed in the same cell-free extracts for Dribulokinase, an enzyme known to be co-ordinatelyregulated with ribitol dehydrogenase and normally used for catabolism of ribitol (Mortlock and Wood, 1964a).With elucidation of the mechanism of growth on xylitol, involving oxidation of xylitol to D-xylulose catalysed by constitutively synthesized ribitol dehydrogenase, a similar mechanism was suspected for growth on L-arabitol. Examination confirmed that L-arabitol-positive mutants were constitutive for both enzymes of the ribitol pathway (Mortlock et al., 196513). Purified ribitol dehydrogenase was found to catalyse oxidation of L-arabitol to L-xylulose,and no separate or specific L-arabitol dehydrogenase could be identified in cells grown on L-arabitol. The enzyme, crystallized from extracts of a constitutive mutant which

CH,OH

I

H ~ c - 0 ~I-drabinose (a)

I

HO-C-H

I

HO-('-H

I

CH,OH L-Arnbinose

. isomerase

C=O

HO--C--HI

1

- V I l ~ +

CH,OH

CH,OH

I

I

6 -Rihuloro

C=O HO-C-HI

5 - phosphate 4 - rpimrrnsr

, A T P ,Mgz+

.

I

HO-C-H

30-C-H

CH,OH ~-Ribulose

C=O

I I

HO-C-H H-C-OH

I

I

I

I

CH,OPO,H, ~-Ribulosc5-l~liosphate

CH,OPO,H, D-SylUlOSC

5-phosphate

R

P

9

a

(a) L-arabinose, and (b) L-arabitol by

A eiobucter uerogenes strain PRL-R3.

R. P. MORTLOCK

*I

L-Ribulow

5 - phosphuits 4- ppimcrase

FIG.2. Pathways for degradation of

18

CHO

0

w


19

had been isolated on L-arabitol, was identical in its physical and immunological properties to the enzyme purified from wild-type cells which had been induced with ribitol (Table 2, p. 10). When wild-type cells were incubated with L-arabitol as the only carbon source, in order to select for L-arabitol-positivc mutants, examination of the culture after eight generations of growth showed 41% of the cells to be constitutive for ribitol dehydrogenase. After serial transfer on L-arabitol, the proportion of constitutive mutants continued to increase. Mutants which were constitutive for ribitol dehydrogenase were found to be capable of growth on L-arabitol without any requirement for further mutation even though such constitutive mutants had been selected on xylitol and not previously exposed to L-arabitol. (c). Origin of the kinase activity. I n addition to L-arabitol, L-xylose and L-lyxose are also metabolized through L-xylulose as an intermediate. In metabolism of the two aldopentoses, L-xylulose is converted to L-xylulose 5-phosphate. Extracts of cells grown on each of the three sugars have been shown to contain L-xylululokinase activity (Anderson and Wood, 1962a; Mortlock and Wood, 196413). When a ribitol dehydrogenaseconstitutive mutant, which had been isolated on a xylitol medium and not previously exposed to L-arabitol, was incubated with ~-arabitolas the carbon source, growth occurred after a lag period of from five to seven h (LeBlanc and Mortlock, 1973). Once cells possessed the ability to oxidize L-arabitol to L-xylulose, further mutations did not appear to be required for growth. Significant amounts of L-xylulokinase were induced aftcr six h incubation of the ribitol dchydrogenase-constitutive mutant with L-arabitol. I n contrast the parent strain, which was inducible for the dehydrogenase, could be incubated with L-arabitol for 24 h without detectable growth or induction of L-xylulokinase activity. It is apparent that synthesis of L-xylulosc, or some intermediate derived from Lxylulose, resulted in induction of the kinase and epimerase activities required for further metabolism of the substrate. Klebsiella aerogenes strain W-70 has also been shown to be capable of growth on xylitol using constitutively synthesized ribitol dehydrogenase to catalyse oxidation of xylitol to D-xylulose (Charnetzky and Mortlock, 1970). The ribitol dehydrogenase from this strain is also capable of oxidizing L-arabitol to L-xylulose, and mutants constitutive for the enzyme (isolated on xylitol) will produce L-xylulose when incubated with L-arabitol. However, this organism appears to be incapable of the further metabolism of L-xylulose, and mutants could not be selected capable of growing with L-arabitol as the only carbon and energy source (Charnetzky and Mortlock, 1974a, b, c). It appears likely at this time that K . aerogenes strain W-70 is unable t o catalyse conversion of L-xylulose to r,-ribulose 5-phospha te,

20

R. P. MORTLOOK

(d). Origin of the epimerase activities. The 3-epimerase which catalyses conversion of L-xylulose 5-phosphate to L-ribulose &phosphate is apparently induced in A . aerogenes strain PRL-R3 following the appearance of L-xylulose in the cells. The 4-epimerase, which converts Lribulose 5-phosphate to D-XylUlOSe 5-phosphate, is also required for catabolism of the common aldopentose, L-arabinose (Fig. 2, p. 18). Metabolism of L-arabinose and the genetic determinants of the Larabinose pathway in Escherichia coli B/r have been carefully studied by Englesberg and other workers (Patrick and Lee, 1968; Englesberg et al., 1965; Englesberg, 1971). The structural genes for three enzymes, L-arabinose isomerase, L-ribulokinase and L-ribulose 5-phosphate 4-epimerase, are co-ordinately controlled with L-arabinose as the apparent inducer. The structural genes for these three enzymes are part of a single operon responding to a positive rather than a negative control system. A similar metabolic pathway has been found for catabolism of L-arabinose by A . aerogenes PRL-R3 (Simpson et al., 1968; Simpson and Wood, 1958; Wolin et al., 1968). LeBlanc and Mortlock (1973)found that mutants of A . aerogenes PRL-R3, which were selected as constitutive for one of these three enzymes were also constitutive for the other two enzymes indicating that the three enzymes were under co-ordinate control in Aerobacter as well as Escherichia. The Aerobacter pathway also appears similar to that of E . coli in that L-arabinose is the apparent inducer of the three enymes (LeBlanc and Mortlock, 1973).The L-ribulose &phosphate 4-epimerase from the PRL-R3 strain has been purified and characterized by Deupree and Wood (1970,1972). With the 3-epimeraseunder common regulation with the other enzymes of the L-arabinose catabolic pathway, the question arose as to the origin of the 3-epimeraseactivity found in L-arabitol-growncells since such cells did not contain detectable activity for the other enzymes of the Larabinose catabolic pathway, Investigation showed that an L-arabinose-negativemutant, which was deficient in the 3-epimerase normally induced by L-arabinose, was still capable of growth on L-arabitol providing that it was also constitutive for ribitol dehydrogenase. Extracts prepared from cells grown on L-arabitol possessed 3-epimerase activity but did not possess L-arabinose isomerase activity. These data suggested the possibility of the existence of two separate L-ribulose 5-phosphate 3-epimerases, one which was regulated for the L-arabinose pathway and a second which was involved in Lxylulose catabolism. I n studying the L-arabinose-negative (epimerase deficient) mutant, it was found that revertants which were capable of growth on L-arabinose occurred at high frequency. Isolation and assay of several such revertants showed them to be constitutive for synthesis of L-ribulose &phosphate 3-epimerase activity but not for L-arabinose


21

isomerase or L-ribulokinase activity (LeBlanc and Mortlock, 1973). Such revertants had apparently overcome the deficiency in the Larabinose pathway by a mutation for constitutive synthesis of the 3-epimerase involved in L-xylulose catabolism. Such mutants were not constitutive for L-xylulokinase activity. The data indicate that a single mutation ,which results in constitutive synthesis of the enzymes of the ribitol catabolic pathway, permits growth of A . aerogenes PRL-R3 on L-arabitol. Oxidation of L-arabitol to Lxylulose by ribitol dehydrogenase results in induction of other enzymic activities required for further degradation. It should be mentioned that, occasionally, mutants are isolated for growth on L-arabitol which are not constitutive for ribitol dehydrogenase. The mechanism of this mutation and the pathway of L-arabitol degradation by such mutants is unknown at this time.

IV. D-Arabhose

A. FUNGI The aldopentose D-arabinose can be found infrequently in nature as a constituent of certain plants and bacteria (Schaffer, 1972). Enzymes which catalyse catabolism of D-arabinose are apparently quite rare in fungi (Lewisand Smith, 1967). None of the yeast strains tested by Barnett (1968) could utilize D-arabinose as a growth substrate but, on the other hand, only two of those strains could utilize the very common aldopentose, L-arabinose, as a carbon source for growth. Wickerham and Burton (1948) tested over 70 strains of Hansenula anomala for the ability to use D-arabinose and found all strains tested to be negative in that respect. Most strains could not use L-arabinose either.

B.BACTERIA 1. Growth of Pseudomonas Schacker et al. (1969) and Mobley et al. (1970) isolated L-fructose dehydrogenases from pork and sheep liver, respectively. The enzyme from either source oxidized both L-fructose and D-arabinoseas substrates. For the sheep-liver enzyme, the maximum rates of activity were approximately equal for both substrates at pH 10.4, but L-fructose had only about 70% as much activity as D-arabinose at pH 8.5. The apparent K , value was found to be lower for L-fructose in each case. The values reported were 1.5 mM for L-fructose and 7.2 mM for D-arabinose for the enzyme from sheep liver, and 0.32 mM for L-fructose and 2.1 mM for D-arabinose for the enzyme from pork liver. An enzyme capable of catalysing oxidation of D-arabinose has also

22

R . P. MORTLOCK

been found in pseudomonads (Hu and Cline, 1964). I n 1965, Clinc and Hu purified three separate sugar dchydrogenases from a pseudomonad one of which they termed a D-arabinose dehydrogenase. These same workers later studied a mutant from a different strain of Pseudomonas which had a constitutive production of a D-galactose dehydrogenase and also possessed high levels of two other dehydrogenases, a D-arabinoee dehydrogenase and a D-aldopyranose dehydrogenase. The D-arabinose dehydrogenase was purified and found to be specific for sugars with the L-galactose configuration with either NAD+ or NADP+ serving equally as well as electron acceptors. The relative rates of activity with different sugars were: D-arabinose, loo%, and L-galactose, 27%. Activity was not detected with D-galactose, L-arabinose, D-glucose, D-xylose, L-xylose, D-mannose, L-mannose, D-lyxose, L-lyxose, D-ribose or D-fructose. The apparent K , value for D-arabinose was 8.2 x &I, which was lower than the value obtained with the enzyme from liver described previously. In 1956 Doudoroff et al. reported the isolation of mutants of Pseudomonas saccharophila which could utilize D-arabinose as a sole carbon source for growth. When wild-type cells were transferred to a medium with D-arabinose as the substrate, a lag phase of from two to three days was observed before growth occurred. The D-arabinose-positive mutants were isolated on agar containing D-arabinose as the carbon source, and used to determine the pathway of D-arabinose catabolism. Cells grown on D-arabinose contained activity for oxidation of D-arabinose (Palleroni and Doudoroff, 1957), but no isomerase or lrinase activity could be observed for D-arabinose as a substrate. An enzyme was found in cell-free extracts which would catalyse NAD+-linked oxidation of D-arabinose to D-arabonolactone. The lactone was then hydrolysed to yield D-arabonic acid which was next converted to 2-keto 3-deoxy-~-arabonic acid. The latter compound was cleaved to pyruvate and glycolic acid with an accompanying reduction of NAD+. Oxidation of the compound was required in order for cleavage to occur since pyruvate was not formed in the absence of NAD+. This pathway for D-arabinose catabolism by Pseudomonas is illustrated in Fig. 3a. Glycolic acid was actually observed to accumulate in cultures growing with D-arabinose as the carbon source (Palleroni and Doudoroff, 1957). The exact mechanism or nature of the mutation which permitted metabolism of D-arabinose by this pathway was not determined.

2. Growth of Propionibacterium Volk, in 1959, observed D-arabinose 5-phosphate occurring as an intermediate in the catabolism of L-arabinose and other pentoses by Propionibacterium pentosaceum. He purified a phosphoarabinoisonierase from L-arabinose-grown cells, and showed the enzyme to be capable of


23

isomerizing D-ribulose 5-phosphate to D-arabinose &phosphate (Volk, 1960). The enzyme was inactive on L-arabinose, D-arabinose, D-xylose, D-ribose, D-ribose 5-phosphate or D-glucose 6-phosphate. Although this particular strain of P. pentosaceum could catalyse isomerization of D-arabinose 5-phosphate it could not use free D-arabinose as a substrate. Volk later studied another strain of P. pentosaceum, strain El4, which can utilize D-arabinose as a sole carbon source for growth (Volk, 1962). Cells of this strain which had been grown on D-arabinose possessed a n isomerase similar to that found in the fist strain, and also contained a kinase for phosphorylation of D-arabinose to D-arabinose 5-phosphate. The organism did not possess an enzyme for isomerization of nonphosphorylated D-arabinose. D-Ribose, but not D-glucose, D-galactose, D-mannose,D-fructose, D-xylose or L-arabinose, also served as a substrate for the kinase. Therefore, the data indicated that the route of degradation of D-arabhose by this strain of P. pentosaceum involved phosphorylation t o the 5-phosphate followed by isomerization t o ribulose &phosphate, (Fig. 3b, p. 24).

3. Growth of Klebsiella (Aerobacter) (a). Pathway of catabolism. I n the early 19509, Simpson observed fermentation of D-arabinose by A . aerogenes strain PRL-R3. Studies with 14Clabelled sugars by Neish and Simpson (1954) indicated that D- and L-arabinose were both degraded by similar types of metabolic pathways. The route for L-arabinose catabolism by this organism involved isomerization to a 2-ketopentose (L-ribulose) and phosphorylation t o the ketopentose 5-phosphate as shown in Fig. 2a (p. 18); Simpson and Wood, 1958; Simpson et al., 1958; Wolin et al., 1958). Mortlock and Wood (1964a) confirmed the ability of this organism to utilize D-arabinose as a growth substrate. A lag period, presumably for selection of mutants, was required before growth was initiated (Table 1, p. 7). When a starved inoculum of glucose-grown wild-type cells was incubated in a growth medium with D-arabinose as the only carbon source, growth could not be detected for a period of about 15 h. An increase in cell mass was observed after about 17 h of incubation at 30"C, under aerobic conditions, and the viable cell count began to increase after about 25 to 35 h. Extracts prepared from cells grown with D-arabinose as a substrate contained enzymic activityfor jsomerization of D-arabinose to D-ribdose, and low but detectable activity was also observed for isomerization of L-xylose to L-xylulose. Cells which had been grown on L-xylose (L-xylosepositive mutants) contained higher activity for isomerization of L-xylose but also possessed proportionately higher activity for isomerization of D-arabinose. Cells grown on D-arabhose were also found to contain both

/p

CHO

I

HO-C-H (a)

I I

H-C-OH

D-A,abinose

I

CH,OH

CH,OH

HO-4-H

CH,OH

D-Arabinose

CH,OH D- Arabonic acid

CHO

I

I HA-OH I

I

D-ArabonoY- lactone

CHO

H-C---OH

D-drabonic

Ozidatim

0

acid

I

D-Arabinose

(b)

C-OH

c=o I

dchydrogenaae NAD+

H-C-OH

I

I

I I H-C-OH I H--C-OH I

CH,OH

HO-C-H D-Arabinokinast-

+-

ATP

Phosphwrabino isomerase

.

CHZOPOjHz

D-Arabinose 5-phosphate

I

c=o I H-C-OH

I

H-C-OH

I

CH,OPO,H, D-Hibldosr 5-phosphate

H-C-OH

CH,

CHZOH

Pyruvic acid

Glycolic acid

I

‘d

CH20H 2-Keto 3-deoxpD-nrabonic acid

w

8

80

2

CH,OH

CHO

(c)

I HO-C-H I H-C-OH I

I c=o I

~-Fucose isomerase

H-C-OH

D-Arabinose

H-C-OH

isomernee

CH,0P03Hz

C=O

~

MIL?+

I H-C-OH I H-C -OH I

E

0 4

u-Ribnlose 5-phosphate

I

~

zrn

CH,OPO3H2

CH,OH ~-Furose

k0

I I

D-Ribulose

CHO

CH,OH

H-C-OH CH,OH

1)- hrabinose

(d)

D- Ribulokinase

F H-C-OH A T P , MgZf

I

I

CH,OH

I HO-C-H I H-C-OH I H-C-OH I

I I

c=o

H-C-OH

I

Mn2+

CH,OH

1.-Fuculokinaee

F A T P , Mg2+

H--C

I c=o I

i-

OH

-

Fuculose 1L-phosphate a~o~ase

.

H--C-OH

I

~

CHZOPO3Hz

I c=o I

CHO

+I

CHZOH

CH,OH


u-Ribulose

d

s

W

CHzOH

CH,OH

k P

Dihydroxyacetone Glycolaldehyde phosphate

CH,OH

@

U

s ki

I c=o

e!

L-Ribulokiriase

2

A T P , Mg2+

I

Fi0

I CH,OPO3HZ

8m

H-C-OH


s

!a

P

5

FIG.3. Pathways for degradation of D-arabinosc by bacteria. ( a ) Pathway for degradation of D-arabinose by P s e u d o m o n a s sp. ( b ) Pathway for degradation of D-arabinose by P r o p i o n i b a c t e r i u m sp. (c) Pathway for degradation of D-arabinose by Aerobacter rn a e r o g e n e a s t r a i n PRL-R3 and Klebsiella aerogenes strain W70. (d)Pathways for degradation of ~-arabinose by E s c h e r i c h i a coli strain t.3 cn K-12.

26

R. P. MORTLOOK

ribitol dehydrogenase and D-ribulokinase activities which were equivalent to the levels of activity normally observed in ribitol-grown cells (Mortlock and Wood, 1964a). Incubation of wild-type cells with Darabinose did not lead to induction of the enzymes of the ribitol pathway, but the metabolism of n-arabinose by D-arabinose-positive mutant cells resulted in rapid induction of ribitol dehydrogenase and n-ribulokinase (Mortlock and Wood, 1964b). The ribulokinase present in D-arabinose-grown cells was later shown to be identical in its physical and immunological properties with the kinaseof theribitol catabolicpathway (Mortlocketal., 1965a).The product of the phosphorylation of D-ribdose by this kinase had been previously shown to be D-ribdose 5-phosphate (Wood et al., 1961). Thus, the pathway for degradation of D-arabinose by D-arabinose-positive mutants appeared to consist of isomerization of D-arabinose to D-ribulosefollowed by phosphorylation to D-ribulose &phosphate as shown in Fig. 3c, p. 25. (b).Origin of isomeraseactivity. With the elucidation of the mechanism of the mutation which permitted growth on xylitol (constitutive synthesis of a n enzyme normally used for a naturally occurring carbohydrate), a similar mechanism could also be suspected for growth on D-arabinose. Investigation of mutants of A . aerogenes strain PRL-R3 which were capable of growth on D-arabinose confirmed the suspicion that they were constitutive for the isomerase activity. Cell-free extracts prepared from the constitutive mutants grown on peptone contained isomerase activity for the methylpentose L-fucose, as well as Dmabinose. L-Pucose is a naturally occurring carbohydrate (Schaffer, 1970), metabolized by coliforms by a pathway involving its isomerization to L-fuculose (Green and Cohen, 1956; Hotta and Kurokawa, 1973; Messer and Kerry, 1973). The activity for L-fucoseisomerization was several times higher than that for D-arabinose indicating that the enzyme observed might be the natural isomerase of the catabolic pathway for L-fucose. Wild-type cells were found to be capable of growth on L-fucose without the requirement of mutation, and L-fucose was found to induce isomerase activities in wildtype cells similar to those activities observed in the constitutive mutants. Therefore, growth on D-arabinose was postulated as resulting from a mutation to constitutive synthesis of an isomerase which was normally induced for degradation of L-fucose (Camyre and Mortlock, 1965). Very low levels of constitutive L-xylose isomerase activity were also observed in these mutants (Camyre and Mortlock, 1965). Later investigation confirmed this mechanism for growth on Darabinose. Isomerase activities for L-fucose, D-arabinose and L-xylose were induced in the wild type, PRL-R3 strain upon incubation of cells withL-fucose (OliverandMortlock, 1971a).Activityfor bothL-fucose and

OATABOLISM OF UNNATURAL CARBOHYDRATES BY MICRO-ORGANISMS

27

D-arabinose isomerization was detectable after two h incubation of wild-type cells with L-fucose, and the ratio of D-arabinose to L-fucose activity remained constant during growth of the cells (Oliver and Mortlock, 1971b). A D-arabinose-positive mutant was constitutive for these same isomerase activities, and the mutant continued to synthesize high isomerase activities for over 30 generations of growth on a casein hydrolysate medium. Longer cultivation led to selection of revertants which were inducible for the isomerase and incapable of growth on D-arabinose. By the use of mutagenic treatment, a D-arabinose-negative mutant was selected from a D-arabinose-positiveculture. Upon examination, the mutant was found to have lost the ability to isomerize L-fucose to Lfucolose and to grow with L-fucose as a substrate. Revertants of this mutant, which were selected for growth on L-fucose, regained constitutive synthesis of both of these isomerase activities and also regained the ability to grow on D-arabinose (Oliver and Mortlock, 1971a). Another mutant was selected from the wild-type, PRL-R3, strain which had lost its ability to grow on L-fucose as a carbon source but was still capable of inducing L-fucose isomerase when incubated in the presence of L-fucose. Although this strain had not been previously exposed to D-arabinose, it was now capable of growth on D-arabinose, providing a small amount of L-fucose was present in the medium to permit continuous induction of the required isomerase activity. Since the growth rate of this strain on D-arabinose under these conditions was comparable with that of the D-arabinose-positive mutants, the mutation establishinggrowth on D-arabhose apparently only affected regulation of the enzyme and not its catalytic activity. The isomerase was purified to homogeneity from three different sources, including the wild-type, PRL-R3, strain induced with L-fucose,a constitutive mutant isolated on D-arabinose,and a constitutive mutant isolated on L-xylose. The purified enzyme gave a single band following disc-gel electrophoresis and a single peak upon column chromatography or ultracentrifugation (Oliver and Mortlock, 1971b). The ratios of the activities for D-arabinose isomerization and L-fucose isomerization remained constant throughout all of the purifications. It is interesting to note that dithiothreitol was found to act as a competitive inhibitor for both substrates of the enzyme (Oliver and Mortlock, 1969). The apparent K , value of this isomerase was 56 mM for L-fucose and 140 mM for D-arabinose, and the ratio of D-arabinose to L-fucose activity under normal assay conditions was between 0.6 and 0.7. Izumori and Yamanaka (1974)purified and crystallized a D-arabinose isomerase from a strain of A . aerogenes. The strain, A . aerogenes M-7, was isolated from sea water. The enzyme was purified over 27-fold by a

28

R. P. MORTLOCK

technique involving precipitation by polyethylene glycol. The enzyme has a molecular weight of approximately 2.5 x lo5 daltons as determined by Sephadex G-200 gel filtration, and has catalytic activity for isomerization of L-fucose ( K , 57 mM) as well as D-arabinose ( K , 160 mM). After continued transfer on media containing D-arabjnose as the only carbon source, a, new mutant was selected from the D-arabinose-positive constitutive strain (strain 502) which possessed a larger colony size when grown on D-arabinose-saltsagar. This new mutant possessed an isomerase with an altered ratio of D-arabinose to L-fucose activity. The new ratio of 1.4 was maintained through purification of the altered isomerase from this mutant (strain 531). Although the mutant strain (531)was constitutive for isomerase activity, it was possible to isolate a revertant which was again inducible for the isomerase activity. Since the isomerase was now inducible, this new mutant had lost the ability to utilize D-arabinose as a growth substrate, but incubation with L-fucose led to induction of the isomerase which possessed the altered activity ratio. These data were taken as indicating that the alteration observed in the activity ratio was indeed the result of a mutation in the L-fucose isomerase structural gene (Oliver and Mortlock, 1971b). The new apparent K , values were 35 mM for L-fucose and 98 mM for D-arabinose. These results with D-arabinose were similar to those which had been obtained by Lin and his coworkers with A . aerogenes strain 1033 where xylitol was the substrate. These workers had obtained a mutant constitutive for ribitol dehydrogenase, and thus able to grow slowly using xylitol as a growth substrate. They later isolated a “fitter” mutant with an alteration in the dehydrogenase structural gene which resulted in increased activity for oxidation of xylitol and a more rapid growth rate for that pentitol (Wu et al., 1968). (c). Origin of kinase activity. Mortlock and Wood (1964a) demonstrated that cell-free extracts of A . aerogenes strain PRL-R3 grown on Darabinose contained activities for ribitol dehydrogenase and D-ribulokinase which were comparable to those activities obtained from cells grown and induced on ribitol. After purification of the enzymes and characterization of their physical and immunological properties, it was shown that the dehydrogenase and kinase synthesized in ribitol-grown cells were identical with the enzymes from D-arabinose-grown cells (Mortlockand Wood, 1964b).D-Arabinose itself did not act as an inducer of the enzyme in wild-type cells, but in cells possessing L-fucose isomerase incubation with D-arabinose resulted in the induction of the enzymes of the ribitol pathway. It was later shown by Bisson and Mortlock (1968) and Bisson et al. (1968)that the apparent inducer of the ribitol pathway

CATABOLISM OF UNNATURAL CARBOHYDRATES BY MICRO-ORQANISMS

29

was the intermediate D-ribulose, rather than the initial substrate, ribitol. When cells were introduced into a medium containing ribitol, the basal levels of ribitol dehydrogenase present catalysed oxidation of ribitol to D-ribulose, resulting in induction of both enzymes of the ribitol pathway (Fig. l a ; p. 9). When cells containing L-fucose isomerase were incubated with D-arabinose, isomerization of D-arabinose to Dribulose also resulted in induction of both enzymes of the ribitol pathway. Once induced, the kinase could then catalyse phosphorylation of Dribulose a t the five-carbon position (Oliver and Mortlock, 1971a; Mortlock and Wood, 1971). Mutants of A . aerogenes PRL-R3, unable to utilize ribitol because of a deficiency in D-ribdokinase activity, were also unable to grow using D-arabinose even though they were constitutive for L-fucose isomerase. Similar data for regulation of the ribitol pathway and mechanism of growth of D-arabinose have been obtained for Klebsiella aerogenes strain W-70 (Charnetzky and Mortlock, 1970). (d). Origin of D-arabinosetransport activity. Cellsof the wild-type PRL-R3 strain could be shown to possess increased activity for uptake of I4CD-arabinose after induction with L-fucose. This activity was found to be constitutive in mutants which had been selected as constitutive for L-fucose isomerase and was maintained after transfer of the constitutive strain to a medium with casein hydrolysate as the only carbon source (Oliver and Mortlock, 1971). Although not conclusive, such data suggested that a transport system for L-fucose could also be utilized for D -arabinose. Therefore, the pathway for catabolism of D-arabinose in D-arabinosepositive mutants of A . aerogenes PRL-R3 is catalysed by enzymes borrowed from two pathways which evolved for degradation of more common sugars. The existence of spontaneous mutants constitutive for the enzymes of the L-fucose catabolic pathway permits utilization of Lfucose isomerase to catalyse conversion of D-arabinose to D-ribulose. Accumulation of D-ribulose results in induction of the enzymes of the ribitol catabolic pathway with the kinase of this latter pathway catalysing phosphorylation of D-ribulose. The D-ribulose 5-phosphate is further metabolized by epimerization to D-xylulose 5-phosphate and transketolase and transaldolase re-arrangements (Mortlock and Wood, 1971).

4. Growth of Escherichia coli (a). Escherichia colistrains Band BIR. I n 1951,Cohen et al. reported that enzyme preparations from Escherichia coli degrade 6-phosphogluconate with production of D-arabinose and other pentoses. Cells adapted to D-arabinose were found to contain a specific D-arabinokinase. Later, in 1963, Cohen reported the isolation of ti mutant of E. coli capable of

30

R. P. MORTLCOK

growth on D-arabinose. The parent E . coli B strain did not show appreciable growth on either D-arabinose or D-ribose while the D-arabinosepositive mutant, strain Ba15, was capable of growth on D-arabinose but not D-ribose. Another mutant strain (Brl) was selected from the parent E . coli B strain by its ability to grow on D-ribose.AlthoughthisD-ribosepositive strain had not been exposed to D-arabinose, it was found to grow equally well on D-arabinose or D-ribose. Cell-free extracts of the Ba15 strain, which had been prepared from cells grown on D-arabinose, contained enzymic activity for isomerization of D-arabinose to D-ribulose and also contained a kinase for phosphorylation of D-ribulose.The isomerase was inactive on L-arabinose D-lyxose, D-xylose and D-ribose but it was active for the isomerization of L-fucose in addition to D-arabinose. Although the parent strain B was not capable of growth on D-arabinose, incubation of cells with D-arabinoseled to induction of isomerase activity in strain B as well as in the D-arabinose-positivemutant strain Ba15. The author concluded that inability of E . coli B to grow on D-arabinose did not result from incapacity to isomerize D-arabinose but resulted from the lack of ability to phosphorylate D-ribulose. I n 1956, Green and Cohen reported that the same D-arabinose-positive strain, Ba15, was capable of growth on both D-arabinose and L-fucose. Cells adapted to either L-fucose or D-arabinosepossessed enzymic activity for isomeration of L-fucose to L-fuculoseand isomerization of D-arabinose t o D-ribulose. The activity of L-fucose isomerization was always found t o be two and a half times higher than that for D-arabinose isomerization and, for this reason, the authors suggested that both isomerase activities were catalysed by the same enzyme. The estimated K , value for L-fucose was 17 mM and the K , for D-arabinose was 3.5 mM. The enzyme was also active for L-galactose and D-altrose as substrates. Green and Cohen postulated a pathway for degradation of D-arabinose involving isomerization of D-arabinose to D-ribulose, catalysed by L-fucose isomerase, followed by phosphorylation of the D-ribulose. They also reported that their strain of E . coli B, which was originally incapable of growth on D-arabinose, was now able to utilize D-arabinose as a growth substrate and suggested that the new observed growth was due to “mutative variation’’ during years of culture passage. In this latter paper they stated that this E . coli B strain was not capable of growth on L-fucose. In 1966, Lin and Cohen reported that the kinase that phosphorylates D-ribdose during degradation of D-arabinose catalysed phosphorylation of D-ribulose at the C-5 position to yield D-ribulose lj-phosphate, while phosphorylating L-fucose at the one-carbon position to give L-fucose 1-phosphate. The authors reported that, when D-ribulose was used as a substrate, both D-ribulose 5-phosphate and small amounts of D-ribulose

CATABOLISM OF UNNATURAL CARBOHYDRATES B Y MICRO-ORGANISMS

31

1-phosphate were products of the reaction. The possibility of contaminating enzymes in their enzyme preparation could, however, not be eliminated. Recently, Boulter and Gielow (1973) purified the enzyme responsible for isomerization of D-arabinose from D-arabinose-induced cultures of E . coli B/r. Their strain was reported to be capable of growing on Darabinose without requiring a mutation, but it was not capable of growth on L-fucose. The isomerase had activity for L-fucose as well as Darabinose and the apparent R, values were 170 mM for D-arabinose and 42 mM for L-fucose. The enzyme was very similar in its properties to an isomerase purified by the same authors from E. coli strain K-12 which had been induced by L-fucose, and to an L-fucose isomerase previously purified from A . aerogenes PRL-R3 (Oliver and Mortlock, 1971b). (b). Escherichia coli strain K-12. Le Blanc and Mortlock (1971a, b) became interested in mutants of E . coli K-12 which had gained the ability to grow using D-arabinoseas a substrate. It could be postulated that such mutants utilized L-fucose isomerase to catalyse isomerization of Darabinose to D-ribulose, since this had been found to be true for A . aerogenes and other strains of E . coli. However, since E. coli K-12 could not utilize ribitol as a growth substrate, it presumably was lacking the enzymes of the ribitol catabolic pathway and would have to employ some other kinase for phosphorylation of D-ribulose. Various strains of E . coli were known to be capable of synthesizing several kinases which could catalyse the phosphorylation of D-ribulose. One such kinase was the L-ribulokinase of the L-arabinose catabolic pathway which was known to possess activity for phosphorylation of D-ribulose as well as L-ribulose (Leeand Bendet, 1967).Another possibility was the L-fuculokinaseofthe L-fucose catabolic pathway. The pathway of L-fucose catabolism, in both E. coli and A . aerogenes involves isomerization of L-fucose to L-fuculose, catalysed by L-fucose isomerase,followed by phosphorylation of the L-fucose at the one-carbon position. The latter reaction is catalysed by L-fuculokinase. The Lfuculose 1-phosphate is then cleaved by an aldolase to yield L-lactaldehyde and dihydroxyacetone phosphate (Green and Cohen, 1966; Eagon, 1961; Heath and Ghalambar, 1962; Ghalambar and Heath, 1962). Heath and Ghalambar (1962) purified L-fuculokinase from E . coli and showed the enzyme to be active for phosphorylation of D-ribulose with 38% of the activity shown for L-fuculoseas a substrate. The same workers also showed that L-fuculose 1-phosphate aldolase was active for cleavage of D-ribulose I-phosphate as well as L-fuculose 1-phosphate. LeBlanc and Mortlock (1971b), working with E . coli K-12, found the wild-type organism to possess an inducible pathway for catabolism of L-fucose,butit was not capableof growthusing D-arabinoseas a substrate.

32

R. P. MORTLOCK

D-Arabinose-positive mutants could be selected following at least five days incubation of the wild-type strain with D-arabinose as the sole carbon source. One such isolate (strain 1102)was selected for study and was found to be capable of exponential growth on D-arabinose at the rate of 0.57 generations per h. Cell-free extracts prepared from cells grown on D-arabinose showed the presence of the enzymes of the L-fucose catabolic pathway. The mutation which permitted growth on D-arabinose as a substrate allowed induction of the L-fucose pathway enzymes when cells were incubated in the presence of D-arabinose. Thus, the mutation was regulatory in that it permitted D-arabinose, or a metabolite derived from D-arabinose, to act as an inducer for enzymes of the L-fucose pathway. Cell-free extracts prepared from the D-arabinose-negative parent strain that had been induced with L-fucose possessed isomerase activity for L-fucose and D-arabinose, kinase activity for both L-fuculose and Dribulose, and aldolase activity for L-fuculose 1-phosphate and D-ribulose 1-phosphate. Incubation of the parent K-12 strain with D-arabinose did not lead to induction of these activities. When the D-arabinose-positive mutant was used, however, incubation with either L-fucoseor D-arabinose led to induction of identical isomerase, kinase and aldolase activities. The kinase activity was partially purified from cells grown on D-arabinose, and the ratio of activity for L-fuculose as compared to D-ribdose did not change over the coursc of the purification (LeBlanc and Mortlock, 1971a). D-Ribulosewas phosphorylated at 55% the rate of L-fuculose with apparent K , values of 0.19 mM for L-fuculoseand 0.73 mM for D-ribulose. The higher K , value for D-ribulose, and the greater relative activity for L-fuculose plus induction of the enzyme by L-fucose in the wild-type cells, indicated that L-fuculose was the natural substrate for the enzyme. The product of the phosphorylation of D-ribulose by the enzyme waa isolated and identified as D-ribdose 1-phosphate,and the products of the activity of the aldolase on D-ribulose 1-phosphate were identified as dihydroxyacetone phosphate and glycolaldehyde,in agreement with the observations of Ghalambar and Heath (1962). Glycolate, which was the product of glycolaldehyde oxidation, accumulated in the medium of cells growing on D-arabinose until such time as the D-arabinose was exhausted from the medium. The D-arabinose-negative parent strain was capable of growth on D-arabinose at a linear rather than exponential growth rate, providing the cells had been pre-induced on L-fucose (LeBlancand Mortlock, 197lb). In this case, as would be predicted, the growth rate decreased by approximately half at each doubling of cell mass due to dilution of the preinduced enzymes by growth in the absence of L-fucose. Since cells which were programmed in this manner with the enzymes of the L-fucose catabolic pathway initially possessed growth rates on D-arabinose


33

equivalent to those obtained with D-arabinose-positive mutants, alterations in the catalytic activity of the enzymes were not required to establish growth on D-arabinose. One of the D-arabinose-positive strains was subjected to mutagenic treatment, and L-fucose-negative mutants were selected. All mutants which were L-fucose-negative had also lost the ability to grow using D-arabinose as a substrate. Included among these mutants was at least one kinase-negative mutant and one aldolase-negative mutant, showing the requirement for activity of all of these enzymes of the L-fucose catabolic pathway to permit growth on D-arabinose. Data such as these have shownthepathway of D-aritbinosedegradation, in D-arabinose-positive mutants of E. coli strain K-12, to consist of isomerization of D-arabinoseto D-ribulose,phosphorylation to D-ribulose 1-phosphate, and cleavage to dihydroxyacetone phosphate and glycoaldehyde (Fig. 3d, p. 25). Even though this strain lacked the ribitol catabolic pathway of A . aerogenes PRL-R3, the activity of the Lfuculokinase for D-ribulose permitted further degradation of this 2-ketopentose intermediate. As mentioned previously, the L-ribulokinase of the L-arabinose catabolic pathway has been shown to possess activity for phosphorylation of D-ribulose. LeBlanc and Mortlock (1972) found that a ribitol-negative strain of A . aerogenes PRL-R3, deficient in D-ribulokinase activity, possessed a high rate of reversion to growth on ribitol. Upon analysis it was found that some of these revertants were constitutive for the Larabinose pathway enzymes and others were constitutive for the L-fucose pathway enzymes. In studying the L-arabinose-constitutivemutants, L-ribulokinase was shown to be replacing the b a s e deficiency for phosphorylation of D-ribulose. These D-ribulokinase-negative L-ribulokinase-constitutive mutants were able to grow on ribitol a t about one half the rate of the wild-type strain. In the other revertants which were constitutive for the L-fucose pathway, the L-fuculokinase was apparently catalysing phosphorylation of D-ribulose. By analogy with E . coli strain K-12, the product of phosphorylation in this latter reaction would be predicted to be D-ribulose 1-phosphate which might then be cleaved by the aldolase of the L-fucose pathway to yield dihydroxyacetone phosphate and glycolaldehyde.Glycolaldehyde could be oxidized to glycolic acid which had been found to accumulate in the medium during growthof E . coli K-12 on D-arabinose. In confirmationofthis assumption, when cells of the D-ribulokinase-deficientmutants of A . aerogenes strain PRL-R3 were grown on L-fucose and then transferred to either a ribitolcontaining or D-arabinose-containing medium, glycolic acid was found to accumulate in the medium (LeBlanc and Mortlock, 1972). It was predicted that E . coli K-12 mutants which were capable of

34

R, P. MORTLOCK

growth on D-arabinose might show an increased growth rate with this substrate if they could catalyse phosphorylation of D-ribulose at the five-carbon position and utilize the carbohydrate by this more direct metabolic route. Two separate methods were used to establish Lribulokinase activity in D-arabinose-positive mutants of E . coli K-12. The first method involved selection of a mutant which was negative for L-arabinose isomerase activity. Using this latter strain, incubation with a low concentration of L-arabinose (0*005%),in the medium resulted in continuous induction of L-ribulokinase. The second method required the isolation of a mutant constitutive for L-ribulokinase activity. I n both cases, the presence of L-ribulokinase activity in the cells resulted in an increase in the growth rate on D-arabinose and a decrease in the amount of glycolic acid accumulated in the medium (Fig. 3d, p. 25; LeBlanc and Mortlock, 1972). An interesting question which can be raised is why E . coli K-12 should mutate to growth on D-arabhose by a regulatory alteration permitting D-arabhose (or a metabolite derived from D-arabinose)to act as inducer of the L-fucose enzymes, while A . aerogenes PRL-R3 mutates to constitutive synthesis of enzymes on the pathway. These events could simply reflect a differencein mutation rates for the respective mutations in the regulatory genes, or possibly might result from basic metabolic differences between the two strains. If a mutation should occur to permit a new carbohydrate (i.e. D-arabinose) to act as a new inducer for a pathway, it could be postulated that the efficiency of the compound for induction would be poor. Relatively high intracellular concentrations of the new inducer might be required for full induction of the pathway to occur. The natural D-ribulokinase of the A . aerogenes strain might prevent accumulation of such a new inducer to the required levels by rapidly phosphorylating at the 5-carbon position the D-ribulose resulting from D-arabinose isomerization. This could result in the observed requirement for constitutive synthesis of the enzymes of the L-fucose pathway. On the other hand, lack of an efficientD-ribulokinase in E . coli K-12 could permit some accumulation of such intermediates of D-arabinose metabolism as D-ribulose and D-ribulose I-phosphate which possess structural similarity to L-fuculose and L-fuculose I-phosphate. The actual inducer of enzymes of the L-fucose pathway in wild-type Aerobacter or Escherichia has not been identified at this time. (c). Escherichia, coli strains from natural sources. I n 1957, Tecce and coworkers reported that cells of E . coli strain 30 would utilize L-rhamnose, D-xylose,L-arabinose or D-ribose, but not D-arabinose, as carbon sources for growth. If cells were first grown on L-rhamnose, however, they were then able to metabolize D-arabinose. The following year, Di Girolamo

CATABOLISM OB UNNATURAL CARBOHYDRATES BY MICRO-ORGANISMS

35

et al. (1 958) isolated over one hundred strains of E . coli from human faeces and tested their ability to utilize L-fucose, L-rhamnose and D-arabinose as growth substrates. The strains were tested on agar containing a single sugar as the sole carbon and energy course. After 48 h incubation a t 37"C, 78 strains were able t o grow on both L-fucose and L-rhamnose but none of these 78 strains could grow on D-arabinose. Eight strains grew on L-rhamnose only, four strains grew on L-fucose only, 16 grew on all three sugars including D-arabinose, and four grew on none of the sugars tested. Thus 15% of the E . coli strains were apparently able to use D-arabinose as a growth substrate immediately following their isolation from nature. Some plates which showed no growth on D-arabinose after 48 h had colonies present after seven days' incubation. Five D-arabinose-positive mutants were isolated from such plates. Twenty-six strains which were D-arabinose-negative were tested for their ability to grow using D-arabinose after they had been previously induced with small amounts of L-rhamnose or L-fucose. All of the strains which were L-fucose-positivewere able to utilize D-arabinose when incubated on D-arabinose in the presence of L-fucose. Some of these strains could also utilize D-arabinose if they were incubated with D-arabinose in the presence of L-rhamnose, but none of the L-fucose-negative strains could use D-arabinose under any conditions. Resting cells of L-fucosepositive strains could oxidize D-arabinoseproviding they had been grown on either L-fucose or L-rhamnose. Since cells grown on L-rhamnose possessed D-arabinose isomerase activity, with the exception of the fucose-negative strains, and since cells grown on D-arabinose did not possess L-rhamnose isomerase activity, the authors concluded that the observed activity for isomerization of D-arabinose was caused by L-fucose isomerase. They postulated that D-arabinose was utilized by enzymes of the L-fucose catabolic pathway and the enzymes of this pathway could be induced by growth on either L-fucose or L-rhamnose. It is of importance t o note that no strains were observed to be D-arabinose-positiveand L-fucose-negative. For all of the coliforms whose growth on D-arabinose has been studied, L-fucose isomerase apparently catalyses the initial reaction of Darabinose catabolism, namely isomerization of D-arabinose to D-ribdose. With A . aerogenes PRL-R3 and Klebsiella aerogenes W-70, phosphorylation of D-ribdose to the &phosphate is catalysed by the D-ribulokinase of the ribitol pathway. For E . c01i K-12 and perhaps the E . coli strains studied by Tecce et al. (1967), all of the enzymes of the L-fucose catabolic pathway are employed with L-fuculokinase catalysing phosphorylation of D-ribulose to the l-phosphate. The pathway of catabolism of Darabinose in E . coli strains B and B/r is still not clearly elucidated. Gohen (1953) first reported the strain of E . coli B under study in his

36

R. P. MORTLOCK

laboratory to be D-arabinose-negative, but later reported the strain t o be D-arabinose-positive and L-fucose-negative. Boulter and Gielow (1973) reported their strain of E . coli B/r to be D-arabinose-positive and L-fucosenegative, and several strains of E . coli B and B/r examined in this laboratory confirm this observation. We have recently found, however, that continued cultivation of a D-arabinose-positive mutant of K . aerogenes W-70 on D-arabinose leads to selection of L-fucose-negative mutants. With this strain of K . aerogenes, D-arabinoseis metabolized to some extent by both the &phosphate and l-phosphate metabolic routes. Elimination of the L-fuculose l-phosphate aldolase activity can be postulated to permit all of the D-ribulose obtained by the isomerization of D-arabinose to be degraded by the more efficient 6-phosphate route (S. L. Rosenberg, E. J. St. Martin and R. P. Mortlock, unpublished observations).

V. L-Xylose and L-Lyxose A. PATHWAYS OF CATABOLISM F. J. Simpson (personal communication) made the original observations on fermentation of both L-xylose and L-lyxose by A . aerogenes strain P R L R 3 . Some years later Anderson and Wood (1960) began studying the metabolism of L-xylose by this organism and found a cobalt-activated isomerase which catalysed isomerization of L-xylose to L-xylulose (Fig. 4). Two additional enzymes involved in L-xylose catabolism were also reported. One was an L-xylulokinase catalysing phosphorylation of L-xylulose to L-xylulose 5-phosphate. The other enzyme was an epimerase which catalysed epimerization of L-xylulose 5phosphate at the three-carbon position to form L-ribulose &phosphate (Anderson and Wood, 1960, 1962a, b). The pathway of L-xylose degradation, therefore, was shown to involve isomerization of L-xylose t o L-xylulose,phosphorylation of L-xylulose to yield L-xylulose&phosphate, and two separate epimerizations t o yield eventually D-xylulose 6phosphate. This pathway is illustrated in Fig. 4. The L-xylulokinase was purified by Anderson and Wood (1962a) and found to be specific for L-xylulose as a substrate with an apparent K , value for L-xylulose of 0.4 mM. They also found that cells which were grown on L-xylose possessed activity for isomerization of L-lyxose to L-xylulose. Therefore, cells which had been grown on L-xylose contained enzyme activities which could catalyse degradation of L-lyxose (Fig. 4). Mortlock and Wood (1964a), in their studies of growth of this strain of A . aerogenes on a variety of pentoses and pentitols, found that incubation of wild-type cells in a medium containing L-xylose as the substrate resulted in a lag period of from 25 to 35 days before growth was first observed. After growth had occurred, transfer of cells to a, new L-xylose-

X

t

X

o

x

2

I -0 I

8 x I I

x

U-V -V--u

27

x

I

0

$3 dfa

.B 0.2 A

2

1

x f

o x

6

a

X

I I -0-v-v I

x

CATABOLISM OF UNNATURAL CARBOHYDRATES B Y MICRO-ORGANISMS

I

I

o x X 0--u

37

38

R. P. MORTLOUK

containing medium resulted in completion of growth within two days. Cells which were cloned on L-xylose agar were found to be capable of growth on an L-xylose-containing liquid medium within a two day period, indicating that L-xylose-positivemutants had been selected from the original population. Cells grown on L-xylose were found to contain very high levels of D-arabinose isomerase activity, in addition to the activities reported by Anderson and Wood (1962b). In comparablegrowth experiments with L-lyxoseas a growth substrate, Mortlock and Wood (1964a)found that mutants capable of growth on L-lyxose could be selected from a wild-type population in two days. Unfortunately, L-lyxose was not available in sufficient quantity to permit assay of the enzymic activities of cell-free extracts. Nevertheless, the isomerase activity reported by Anderson and Wood (1962b),which could catalyse isomerization of L-lyxose to L-xylulose, suggested the pathway illustrated in Fig. 4.

B. ORIGIN OF

THE

ENZYMIU ACTIVITIES

The high levels of activity for isomerization of D-arabinose which had been found in L-xylose-grown cells indicated the possibility that the L-xylose and D-arabinose isomerization activities observed might be catalysed by a common enzyme. Upon examination, it was found that both the D-arabinose isomerase and the L-xylose isomerase activities present in the L-xylose-positive mutants were constitutive, and maintained during growth on a medium containing casein hydrolysate in the absence of either L-xylose or D-arabinose (Camyre and Mortlock, 1965; Oliver and Mortlock, 1971a).After identification of the isomerase activity of D-arabinose-grown cells as L-fucose isomerize, it was suspected that the L-xylose isomerase activity might also be catalysed by L-fucose isomerase. This concept was supported by Camyre and Mortlock (1965), who showed that the wild-type cells which had been induced with L-fucose and the isomerase-constitutive mutants isolated on either D-arabinose or L-xylose, all contained equal ratios of activity for isomerization of L-fucose, D-arabinose and L-xylose. The enzyme was eventually purified to homogeneity and indeed found to be active for isomerization of all three aldopentoses even though L-xylose was isomerized at very low activitie8 (Olivera.ndMortlock,1971b).The apparent K , value for L-xylose was calculated at approximately 0.4 M, although L-xylose actually became inhibitory at concentrations over 0.26: M. The extrapolated VmaXvalue for L-xylose represented only 0.83% of the value for L-fucose or D-arabinose, but cells grown on L-xylose compensated for this poor activity by producing high constitutive levels of the enzyme. Crude extracts of L-xylose-growncells had ten times higher


39

isomerase activity than crude extracts of D-arabinose-grown cells (Mortlock and Wood, 1964a). Studies on the nature of the mutation permitting growth on L-lyxose have been hampered by the lack of sufficient quantity of pure L-xylose. As mentioned previously, Anderson and Wood (1962b) detected both L-xylose and L-lyxose isomerization activities in extracts prepared from L-xylose-grown cells. Oliver and Mortlock (197lb) reported that cells grown on L-lyxose were constitutive for L-fucose isomerase, and such data as these suggested that constitutive L-fucose isomerase catalysed isomerization of L-lyxose in cells growing on L-lyxose, and a mutation in the regulation of the L-fucose pathway permitted growth on L-lyxose in a manner similar to that found for growth on L-xylose and D-arabinose. I n spite of this hypothesis, Oliver and Mortlock (197 1b) could not detect any activity for isomerization of L-lyxose to L-xylulose using a highly purified L-fucoseisomerase preparation. At this moment the origin of the L-lyxose isomerase activity is still obscure. As was mentioned previously in the discussion of L-arabitol degradation by this strain of A . aerogenes, formation of L-xylulose within the cells resulted in the induction of L-xylulokinase and probably L-xylulose 6-phosphate 3-epimerase activity (LeBlanc and Mortlock, 1973). Isomerization of L-xylose to L-xylulose should also result in induction of the required enzymic activities to convert the L-xylulose to D-xylulose 5-phosphate. Since mutants can be selected from the wild-type population which are capable of growth on D-arabinose only after several days incubation in a D-arabinose-containing medium, the requirement for several weeks incubation to select mutants for growth on L-xylose suggested that some additional mutation was required for growth on this latter substrate. When isomerase-constitutive mutants which had been previously selected on D-arabinose were transferred to an L-xylose medium, the lag period required before growth was detected was shortened to about seven days (Oliver, 1969).Although further data are not available a t this time, one possible explanation for these results would be the requirement for some additional mutation to establish transport activity for L-xylose.

M. D-LyXOSe The PRL-R3 strain of A . aerogenes was also reported by F. J. Simpson (personal communication) to be able to ferment D-lyxose, in addition t o the other pentoses. Mortlock and Wood (1964a) found that, after inoculation of a wild-type population of cells into a D-lyxose-containing salts medium, a lag of about four days was required before growth of the culture was apparent. Transfer of an inoculum into a second D-lyXOSe-

40

R. P. MORTLOCK

containing medium resulted in growth being completed within one day, suggesting that a mutant had been selected from the original population (Table 1, p. 7). Cells which had been grown on D-lyxose were assayed for enzymic activities, and were found to contain isomerase activity for both D-lyxose and D-xylose. The product of isomerization in each case was D-xylulose. D-Xylulokinase activity was also present in cell-free extracts as was D-arabitol dehydrogenase activity. When the D-lyxose-negative wild-type cells were incubated in a medium with D-lyxose as the carbon source, growth was not detectable until after 83 h of aerobic incubation a t 30°C. This first measurable increase in cell mass corresponded with the disappearance of D-lyxose from the medium and the first appearance of isomerase, D-xylulokinase, and D-arabitol dehydrogenase activities in cell-free extracts (Mortlock and Wood, 196413). The lag observed in the appearance of these enzymic activities indicated that D-lyxose itself did not serve as inducer for these enzymes in the wild-type strain. Allison and Anderson (1964) confirmed the suspected pathway of catabolism of D-lyxose as consisting of isomerization of D-lyxose to D-xylulose followed by phosphorylation to yield D-xylulose 5-phosphate. The latter compound was known to be a naturally occurring intermediate in degradation of the other five-carbon sugars. The enzyme catalysing isomemzation of D-lyxose was purified 130-fold from D-lyxose-grown cells of A . aerogenes PRL-R3 by Anderson and Allison (1965). This isomerase possessed activity for both D-lyxose and D-mannose as substrates, but it was not active on D-glucose, D-ribose, D-arabinose, L-arabinose or L-xylose. Palleroni and Doudoroff, in 1956, had reported a mannose isomerase from Pseudomonas saccharophila which was capable of catalysing isomerization of D-mannose to D-fructose, D-lyxose to D-xylulose, and D-rhamnose to D-rhamnulose. With the enzyme from A . aerogenes, however, the V,,, value was 2.5 times greater for D-lyXOSe than for D-mannose and the K , value for D-lyxosewas 36 mM as compared with 10 mM for D-mannose. Since D-lyxose was a better substrate than D-mannose, and since cells grown on D-mannose did not possess D-lyXOSe isomerase activity, the enzyme purified from D-lyxose-grown cells of A . aerogenes by Anderson and Allison (1965) was not a true D-mannOSe isomerase. The enzyme was apparently induced only in D-lyxose-positive mutants by the presence of D-lyxose in the growth medium. The genetic origin of this enzymic activity is still unknown, and further investigation will be required to determine if this is a naturally occurring enzyme for an unknown substrate which happens to possess activity for D-lyxose or if it is an enzyme whose structural gene has been modified to permit catalytic activity with D-lyxose as a substrate. Aerobacter aerogenes PRL-R3 has been shown to synthesize two separate D-xylulokineses. One of the kinases is co-ordinately controlled

CATABOLISM OB UNNATURAL CARBOHYDRATES BY MICRO-ORGANISMS

41

with D-arabitol dehydrogenase and induced by D-arabitol, and the other is induced by D-xylose (Wilson and Mortlock, 1973). The presence of D-arabitol dehydrogenase activity and D-xylose isomerase activity in cells metabolizing D-lyxose indicated that both of these kinases were partially induced in these cells and could have been catalysing phosphorylation of the D-xylulose derived from isomerization of D-lyxose.

VII. L-Mannose A. PATHWAY OF CATABOLISM Mayo and Anderson, 1968, reported that wild-type A . aerogenes strain PRL-R3 could grow slowly with a generation time of 33 h when utilizing L-mannose as a substrate. These authors were able to select a mutant which was capable of more rapid growth on L-mannose and possessed a generation time on L-mannose-salts medium of 2.5 h. Cells which had been grown on L-mannose had the ability to oxidize L-mannose, but this ability was not found in glucose-grown cells indicating that the enzymic activity for L-mannose catabolism was either inducible by L-mannose or subject to catabolite repression by D-glucose. Crude cell-free extracts of L-mannose-growncells contained a cobalt-activated L-mannoseisomerase a t specific activities of from 0.2 to 0-24 mM per min per mg of protein. The product of L-mannose isomerization was identified as L-fructose, and the mutant which had been isolated for rapid growth on L-mannose would also grow on L-fructose without the requirement of an additional mutation. The L-fructose which was produced by isomerization of L-mannose was found to be phosphorylated with ATP to yield L-fructose l-phosphate. The phosphorylated compound was cleaved by a n aldolase to yield dihydroxyacetone phosphate and L-glyceraldehyde (Mayo and Anderson, 1968). This pathway, which is illustrated in Fig, 6 , involves isomerization of the substrate to 2-keto sugar, phosphorylation t o a ketohexose l-phosphate and cleavage by a n aldolase, and is similar to the pathways for degradation of the 6-deoxyhexoses (methyl pentoses), L-fucose and L-rhamnose by coliform bacteria.

B. ORIGIN OF

THE

ENZYMIC ACTIVITIES

The following year, Mayo and Anderson (1969)reported that wild-type cells which had been grown on L-rhamnose contained enzymic activities necessary for oxidation of L-mannose. The isomerase, kinase and aldolase activities of the L-rhamnose catabolic pathway were induced in both wild-type cells and the L-mannose-positive mutant by incubation with either L-rhamnose or L-mannose. L-Mannoseappeared to act gratuitously as both an inducer and a substrate of the L-rhamnose pathway enzymes, and incubation of the wild-type strain with L-mannose resulted in synthesis of the required enzymic activity (the L-rhamnose catabolic

1I

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4

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4

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I H-C-OH I HO&H I

-

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0

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I I

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CH,OH L-Fructose 1-phosphate

(

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A W e

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Dihydroxyacetone phosphate

\ H

R0 4

I

HOG-H

I

CH,OH L-GI yceraldehyde

FIG.6. Pathway for L-mannose degradation by Aerobucter uerogenea strain PRL-R3. From Mayo and Anderson (1968).

OATABOLISM OF UNNATURaL CARBOHYDRATES BY MICRO-ORGANISMS

43

pathway) to convert L-mannose to dihydroxyacetone phosphate and L-glyceraldehyde. In confirmation of this concept, a mutant which had been derived from the L-mannose-positive strain, but which had lost its isomerase activity, failed to grow on either L-rhamnose and L-mannose. Even though wild-type cells which had been pre-induced with Lrhamnose contained the required enzymic activities to catabolize L-mannose, degradation of L-mannose apparently resulted in products which were toxic to cellular growth. The actual mutation which had permitted the more rapid growth rate on L-mannose had allowed the cells to overcome this toxicity. The metabolite responsible for the toxicity was not identified, although L-glyceraldehyde was suggested as a likely candidate (Mttyo and Anderson, 1969).By analogy with this mechanism, it would be interesting to test the enzymes of the L-fucose catabolic pathway for the ability to degrade substrates such as L-galactose or D-altrose.

VIII. D-Allose I n 1956, Altermatt et al. reported that I4C-labelled D-allose was utilized by A . aerogenes PRL-R3 with a metabolic route which was similar to that used for catabolism of D-glucose.Such a route presumably involved fructose 1,6-diphosphate as a metabolic intermediate. Later, in 1963, Gibbins and Simpson reported that cell-free extracts prepared from D-allose-grown cells contained a kinase which was capable of phosphorylating D-allose to yield D-allose 6-phosphate. The kinase was purified 28fold and was found to have an apparent K , value of 0.98 mM for D-allose. Lower activity was also found for phosphorylation of Dglucose, D-ribose and D-galactose.Cell-free extracts contained an enzyme capable of catalysing isomerization of D-allose 6-phosphate to allulose 6-phosphate (Gibbinsand Simpson, 1964a,b). Another enzyme catalysed an epimerization of the latter compound at the 3-carbon position to form D-fructose 6-phosphate (Fig. 6). Because of similarities in structure, it was suggested that this epimerase might be the same as that which normally functioned for epimerization of D-ribulose &phosphate to D-xylulose &phosphate. Since D-allokinase activity and the D-allOSe 6-phosphate activity were low or absent in D-glucose- or D-ribose-grown cells, and because cells grown on D-glucose showed a lag period before initiating oxidation of n-allose, the enzyme activities required for catabolism of D-allosewere believed to be inducible. It is also possible that the enzymes required for D-allose utilization were subjected to a form of catabolite repression when cells were grown on such naturally occurring substrates as D-glucose or D-ribose. The genetic origin of the enzymes which were used for D-allose catabolism or their normal role in the metabolism of cells is unknown.

CHO

I H-C-OH I H-C-OH I H-0-OH I H-C-OH

I

CH,OH u-Allose

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CH,OH

I

I C=O I

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I

H-C-OH

sBucse

-

-4TP

I

H-C-OH

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CHZOPOIH,

D-Allose 6-pliosphatc

leomerase

H-C-OH

I

H-C-OH

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CH,OPO,H, D-Allulose 6- phosphate

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D-Fructosc 6- phosphate

FIG.6. Pathway for degradation O f D-allOSe by Aerobacter aerogenes.


45

IX. D-Tagatose Recently, Bissett and Anderson (1973) have described a catabolic pathway which involved phosphate derivatives of the rather uncommon hexose, n-tagatose, as metabolic intermediates. In Staphylococcus aureus NCTC 8511, lactose was reported to be metabolized by a pathway involvingphosphorylation, with phosphoenolypyruvate as the phosphate donor, and then cleavage of the phosphorylated product by a phospho/3-galactosidase to yield glucose and galactose 6-phosphate. Bissett and Anderson (1973)found the pathway of galactose 6-phosphate degradation to involve isomerization to yield D-tagatose 6-phosphate, followed by phosphorylation to D-tagatose 1,g-diphosphate. The diphosphate was cleaved by an aldolase to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Since the isomerase, kinase, and aldolase were all induced by growth on either lactose or galactose, these investigators suggested that galactose might also be degraded by the same route following its phosphorylation by phosphoenolpyruvate. The same authors have recently reported a similar pathway in some of the group-N streptococci. The four group-N Streptococcus strains tested possessed the tagatose 6-phosphate pathway in addition to the normal D-galactose l-phosphate 3 D-glucose l-phosphate route for galactose degradation. The tagatose 6-phosphate pathway was not found in E . coli B/r or A . aerogenes PRL-R3 (Bissett and Anderson, 1974).

X. D-Fucose Cline and Hu (1965a,b), while studying the sugar dehydrogenases of a mutant of a Pseudomonas species, found two separate enzymes which were each capable of catalysing oxidation of D-fucose. Each of the enzymes was purified and studied in order to determine its various substrate specificities. One enzyme was found to be an NADP+-linked D-galactose dehydrogenasewhich would oxidize D-galactoseto galactonolactone. The relative rates of activity with other substrates were found to be Dgalactose (100%)) L-arabinose (44%), D-fucose (156%) and 2-deoxy-~galactose (90%).The activity with D-fUCOSewas actually 50% higher than the activity with the naturally occurring substrate D-galactose. The other enzyme possessing activity on D-fucose was a D-aldose dehydrogenase which was capable of oxidizing a variety of D-aldopyranoses to the corresponding aldonolactones. Among the substrates oxidized by the NAD+-linked enzyme were D-galactose (loo%), L-arabinose (1IS%), D-glucose (103%)) D-XylOse (102%), D-fUCOSe (6O%), D-guinovose ; 67%), and 2-deoxy-~-galactose(470/,). Nsihizuka (6-deoxy-~-glucose and Hayaishi (1962) detected an enzyme in Pseudomonas gravolens

46

R . P. MORTLOUK

which could catalyse oxidation of D-fucose in addition to other substrates. Dahms and Anderson (1972a) isolated a pseudomonad which was capable of growth with D-fucose as the sole carbon source. The organism grew well on either D-fucose, D-glucose, L-arabinose or D-galactose with a generation time in each case of about three hours. These authors also identified and purified from this organism two separate enzymes which were each capable of oxidizing D-fucose. One of these enzymes was a Daldohexose dehydrogenase similar to that reported by Cline and Hu (196Ba, b), which would reduce NAD' and was capable of oxidizing D-fucose to fuconolactone. The latter compound spontaneously hydrolysed to yield D-fuconate as illustrated in Fig. 7. The enzyme had higher activity for D-glucose, D-galactose and D-mannose than for D-fucose and was also capable of oxidizing D-altrose and D-allose. The dehydrogenase was induced by both D-glucose and D-fucose and was believed to function D-Fucose

[Pyranose]

D - Fucono - y

[ Furanose]

u-F'urono-Y-lactone

- lactone

/ m (LactoI1:lsC) u-Fuconate

(Aldolase)

Pyruvate

1)

-Lact nlclchyde

FIG.7. Pathways for D-fucose degradation by a pseudomonad. From Dahms and Anderson (1972d).

CATABOLISM OF UNNATURAL. CARBOHYDRATES BY MICRO-ORGANISMS

47

in catabolism of both sugars by the organisms (Dahrns and Anderson, 1972a). Dahms and Anderson (1 97213) also purified a L-arabino-aldose dehydrogenase which was active with either NAD+ and NADP+ as electron acceptors and also catalysed oxidation of D-fucose to D-fucono~actone. The lactone was hydrolysed to D-fuconate by a lactonase. This particular dehydrogenase was induced by either D-fucose, D-galactose or L-arabinose, and possessed activity for oxidation of a variety of sugars in addition to those mentioned. The D-fuconate which was formed by the activity of either of these dehydrogenases was converted to 2-keto-3-deoxy-~-fuconateby an aldonic acid dehydrase. The dehydrase was partially purified by Dahms and Anderson (19720) and found to utilize either D-fuconate or Larabinate as substrates. The requirement for this enzyme in D-fucose catabolism was confirmed by isolation of a mutant deficient in dehydrase activity which then failed to grow with either D-fucose or L-arabinose as growth substrates (Dahms and Anderson, 1972~). An aldolase was then identified and partially purified which could cleave either 2-keto-3deoxy-D-fuconate or 2-keto-3-deoxy-~-arabinate (Dahms and Anderson, 197211)- The products of cleavage of 2-keto-3-deoxy-D-fuconate were identified as pyruvate and the D-lactaldehyde. These pathways for D-fucose degradation are illustrated in Fig. 7. The similaritiesin structure between L-arabinose and D-fucose apparently resulted in evolution of a catabolic pathway for L-arabinosewhich could be utilized for degradation of D-fucose when that uncommon substrate was present in the medium.

XI. Conclusions I n order to establish a reasonable growth rate on a novel or uncommon substrate, a n organism must possess a means of transporting the new substrate into the cell and must be capable of converting the substrate into a normal cellular metabolite. The natural existing metabolic pathways of the organism can then catalyse further degradation of this intermediate. If a micro-organism is capable of synthesizing enzymes which have evolved for catabolism of naturally occurring carbohydrates but are still capable of catalysing the essential reactions for the new substrate, then the initiation of growth on the new carbohydrate need not require mutation for alteration in the enzyme structures. It is understandable that the most versatile organisms, with respect t o their ability t o utilize uncommon carbohydrates, should be those which possess the ability t o synthesize enzymes having broad substrate specificities. If the required enzymic activities happen t o be constitutive in the organism, or if the novel substrate or a metabolite derived from the

48

R . P. MORTLOCK

substrate is recognized as an inducer of the required enzymic activities, then growth and metabolism may be possible without the requirement for a mutation. If, however, the levels of the required enzyme or enzymes in the organism are not sufficient to permit growth on the new substrate, then regulatory mutations, which either permit constitutive synthesis of the required enzymes or permit the new substrate to act as inducer for the required enzyme, can result in growth of the organism. An additional requirement for growth is that the catabolism of the new carbohydrate should not result in formation of compounds which are toxic to cellular growth or metabolism. The mutation which permits the growth of Aerobacter or Ii'lebsiella strains on xylitol occurs in a regulatory gene for the ribitol pathway and results in constitutive synthesis of the enzyme of ribitol catabolism. Studies with A . aerogenes strain PRL-R3 have shown that the mutation permitting growth on D-arabinose is a regulatory mutation which results in constitutive synthesis of the enzymes of the L-fucose catabolic pathway. On the other hand, with Escherichia coli strain K-12, metabolism of D-arabinose results from a regulatory mutation which permits D-arabinose or some metabolite derived from D-arabinose to act as the inducer of enzymes of the L-fucose catabolic pathway. This latter mutation permits the co-ordinately controlled enzymes of the L-fucose pathway to be induced when cells are incubated in the presence of D-arabinose. Once growth has been established on an unnatural carbohydrate by the means of such regulatory mutations, additional mutations can be selected to permit increased growth rates and rapid degradation of the substrate. The genetic mechanism where growth on a novel carbohydrate initially results from a mutation which has altered an enzyme structure is more difficult to document but still may occur less commonly. It should be noted that, for enzymes which are under negative control, mutations which result in loss of function of the regulator gene can also result in constitutive synthesis of the enzymes controlled by that gene. Since this type of mutational event only requires the loss of function of a gene, it can occur a t relatively high frequency. Interesting problems in regulation can arise if an organism utilizes an enzyme pathway for catabolism of a substrate other than the natural substrate for which the pathway had evolved. A product of the degradation of the new substrate could be an inhibitor of cellular growth or perhaps a repressor of the enzymes required for degradation of the new substrate. Degradation of L-mannose by A . aerogenes, apparently results in the formation of an unnatural intermediate which is toxic to the growth of the organism. Experiments with A . aerogenes strain PRLR3 have shown that catabolism of D-glucose or L-arabinose results in


49

rather severe repression of the enzymes of the ribitol catabolic pathway. Even constitutively synthesized ribitol dehydrogenase is repressed approximately 85% when L-arabinose is utilized a8 a growth substrate. The catabolism of L-arabinose to give L-ribulose 5-phosphate is required for this repression to occur. In spite of the facts that L-arabinose and L-arabitol are degraded through this common intermediate and that ribitol dehydrogenase is required to catalyse the first step in degradation of L-arabitol, cells constitutive for ribitol dehydrogenase maintain high levels of the enzyme during growth on L-arabitol. The relatively poor activity of the enzyme for oxidation of L-arabitol as a substrate results in slow metabolism of L-arabitol and a decreased accumulation of the metabolic intermediates and high-energy compounds which could lead to repression of the ribitol pathway enzymes. It might be postulated that, if mutations were eventually selected altering the structure of ribitol dehydrogenase and permitting more rapid catabolism of L-arabitol, specific regulatory problems might arise and additional mutations would be necessary to modify regulation of the ribitol pathway in order to permit synthesis of ribitol dehydrogenase during catabolism of L-arabitol. Although most of the work done in this area has been descriptive, it might be possible to programme cells for ability to utilize a particular carbohydrate that they had not yet encountered as a growth substrate. For some particular and novel carbohydrate structure, the pathways of catabolism of naturally-occurring substrates of related structure might be examined to identify enzymes whose substrate specificities would permit catalysis of a similar reaction for the novel carbohydrate. Even if activity on the new substrate was slight, mutants might be selected that were constitutive or hyper-constitutive for the desired enzymic activity. Genetic transfer techniques might permit accumulation of several such mutations within a single strain which could then be exposed to the new carbohydrate as a substrate. Even if such genetic programming was not sufficient to permit growth on the novel substrate, it might still decrease the number of mutational events required to permit growth when the organism was exposed to the novel compound as a selective substrate.

XII. Acknowledgements I would like to express my appreciation to P. J. Simpson, Brian S. Hartley, Peter Rigby, Patricia Clarke, Kei Yamanaka and Ken Izumori for making unpublished data and manuscripts from their laboratories available to me. I would also like to express my thanks to R. L. Anderson and T. G. Lessie for reviewing this article before it was submitted for publication.

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The author’s research has been supported by Public Health Service Grant AI-06848 from the National Institute of Allergy and Infectious Diseases. REFERENUES Allison, D. and Anderson, R.L. (1964) Bacteriological Proceedings, 94. Altermatt, H. A., Simpson, F. J. and Neish, A. 0. (1955). Canadian Journal of Microbiology 1, 473. Altermatt, H. A., Simpson, F. J. and Neish, A. 0. (1955). Canadian Journal of Biochemistry and Phy8iology 33, 6 15. Anderson, R. L. and Allison, D. P. (1965). Journal of Biochemistry, Tokyo 240, 2367. Anderson, R.L. and Wood, W. A. (1960). Biochimica et Biophysica Acta 42, 374. Anderson, R.L. and Wood, W. A. (1962a). Journal of Biological Chemistry 237, 1029. Anderson, R. L. and Wood, W. A. (196213).Journal of Biological Chemistry 237,296. Arcus, A. C. and Edson, N. L. (1956).Journal of Biochemistry, Tokyo 64, 385. Barnett, J. A. (1968). Journal of General Microbiology 52, 131. Bissett, D. L. and Anderson, R.L. (1973). Biochemical and Biophysical Research Communications 52, 641. Bissett, D. L. and Anderson, R.L. (1974).Journal of Bacteriology 117, 318. Bisson, T. B., Oliver, E. J. and Mortlock, R. P. (1968).Journal of Bacteriology 95, 932. Bisson, T. M. and Mortlock, R.P. (1968).Journal of Bacteriology 95, 925. Boulter, J. B. and Gielow, W. 0. (1973).Joz~rnalof Bacteriology 113, 687. Camyre, K. P. and Mortlock, R. P. (1965).Journal of Bacteriology 90, 1157. Chakravorty, M., Veigh, L. A., Racila, M. and Norecker, B. L. (1962). Journal of Biological Chemistry 287, 1014. Charnetzky, W. T. and Mortlock, R.P. (1970). BacteriologicaZ Proceedings, 137. Charnetzky, W. T. and Mortlock, R. P. (1972). Abstracts of the Annual Meeting of the American Society for Microbiology, 60 Charnetzky, W. T. and Mortlock, R.P. (1974a). Journal of Bacteriology 119, 162. Charnetzky, W. T. and Mortlock, R.P. (1974b).Journal of Bacteriology 119, 170. Charnetzky, W. T. and Mortlock, R.P. ( 1 9 7 4 ~ )Journal . of Bacteriology 119, 176. Chiang, C. and Knight, S. G. (1961). Biochimica et Biophysica Acta 46, 271. Chiang, C. and Knight, S. G. (1966).I n “Methods in Enzymology”, (S.P. Colowick and N. 0. Kaplan, eds.), Vol. 9, p. 188. Academic Press, Now York. Clarke, P. (1974). Symposium of the Society for General Microbiology 24, 183. Cline, A. L. and Hu, A. S. L. (19654. Journal of Biological Chemistry 240, 4488. Cline, A. L. and H L ~A., S. L. (1965b). Journal of Biological Chemistry 240, 4493. Cohen, S. S. (1953).Journal of Biological Chemistry 201, 71. Cohen, S. S., McNair Scott, D. B. and Lanning, M. (1951). Federation Proceedings. Federation of American Societies for Experimental Biology 10, 173. Dahms, A. S. and Anderson, R. L. (1972a). Journal of Biological Chemistry 247, 2228. Dahms, A. S. and Anderson, R. L. (1972b). Journal of Biological Chemistry 247, 2228.


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Dahms, A. 8. and Anderson, R. L. ( 1 9 7 2 ~ )Journal . of Biological Chemistry 247, 2233. Dahms, A. 5. and Anderson, R. L. (1972d). Joumal of Biological Chemistry 247, 2238. Deupree, J. D. and Wood, W. A. (1970).Journal of Biological Chemistry 254,3988. Deupree, J. D. and Wood, W. A. (1972).Journal of Biological Chemistry 247,3093. Di Girolamo, M., Magliocco, I?., Schiesser, A. and Teece, G. (1958). Giornale d i Microbiologia 5, 11 1. Doudoroff, M., Dehey, J., Palleroni, N. J. and Weimbey, R. (1956). Federation Proceedings. Federation of American Societies for Experimental Biology IS, 244. Eagon, R. G. (1961). Joumal of BacterioZogy 82, 548. Englesberg, E . (1971).I n “Metabolic Pathways”, (H. J. Vogel, ed.), Vol. V, p. 257. Academic Press, New York and London. Englesberg, E., Irr, J., Power, J . andLee, N. (1965).Journalof Bacteriology90,946. Fossitt, D., Mortlock, R. O., Anderson, R. L. and Wood, W. A. (1964).Journal of Biological Chemistry 239, 21 10. Fromm, H. J. (1958).Journal of Biological Chemistry 233, 1049. Fromm, H. J. and Nelson, D. R. (1962).Journal of Biological Chemistry 237, 215. Fromm, H. J. and Bietz, J. A. (1966).Archivas of Biochemistry and Biophy8im 115, 510. Ghalambor, M. A. and Heath, E. C. (1962). Journal of Biological Chemistry 237, 2427. Gibbins, L. N. and Simpson, F. J. (1963).CanadianJournal of Microbiology 9, 760. Gibbins, L. N. and Simpson, F. J. (1964a). Biochemical Journal 91, 9P. Gibbins, L. N. and Simpson, F. J. (1964b).Canadian Journal of Microbiology 10, 829. Green, M. and Cohen, S. S. (1956).Journal of Biological Chemistry 219, 557. Hartley, B. S., Barleigh, B. D., Midwinter, G. G., Moore, C. M., Morris, H. R., Rigby, P. W. J., Smith, M. J. and Taylor, S. S. (1972). Symposium of the Federation of European Biochemical Societies, 29. Heath, E. C. and Ghalambar, M. A. (1962). Journal of Biological Chemistry 237, 2423. Hogeman, G. D. and Rosenberg, S. L. (1970). Annual Review of Microbiology 24, 429. Hollman, S. (1967).I n “International Symposium on Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols”, (Horeclter, B. L., Lory, K. and Takayi, Y., eds.), p. 97. Springer-Verlag, Berlin, West Germany. Horecker, B. L. (1967).I n “International Symposium on Metabolism Physiology, and Clinical Use of Pentoses and Pentitols”, (Horecker, B. L., Lory, K. and Takayi, Y., eds.), p. 50. Springer-Verlag, Berlin, West Germany. Horwitz, S. B. and Kaplan, N. 0. (1964).Journal of Biological Chemistry 239, 830. Horwitz, S. B. (1966). I n “Methods in Enzymology”, (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 9, p. 155. Academic Prcss, New York. Hotta, K. and Kurokawa, M. (1973).Journal of Biological Chemistry 248, 629. Hu, A. S. L. and Cline, A. L. (1964). Biochimica et Biophysica Acta 93, 237. Hully, S. B., Jorgensen, S. B. and Lin, C. C. (1962). Biochimica et Biophysica Acta 67, 21. Ingram, J. M. and Wood, W. A. (1965).Journal of Bacteriology 89, 1186. Ingram, J. M. and Wood, W. A. (1966). I n “Methods in Enzymology”, (5. P. Colowick and N. 0. Kaplan, eds.), Vol. 9, p. 186. Academic Press, New York. Izumori, K. and Yamanaka, K. (1974). Agricultural Biological Chemietry 38, 267.

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Jakoby, W. B. and Fredericks, V. (1961). Biochimica et Biophysica Acta 48, 26. Jakoby, W. B. (1966). I n “Methods in Enzymology”, ( S . P. Colowick and N. 0. Kaplan, eds.), Vol. 9, p. 193. Academic Press, New York. Kersters, K., Wood, W. A. and Dehey, J. (1965). Journal of Biological Chemistry 40, 965. Leblanc, D. J. and Mortlock, R. P. (1971a).Journal of Bacteriology 106, 82. Leblanc, D. J. and Mortlock, R. P. (1971b).Journal of Bacteriology 106, 90. Leblanc, D. J. and Mortlock, R. P. (1972).Archives of Biochemistry and Biophysics 150, 114. Leblanc, D. J. and Mortlock, R. P. (1973).Archives of Biochemistry and Biophysics 156, 390. Lee, N. and Bendet, I. (1967).Journal of Biological Chemistry 242, 2043. Lerner, S. A., Wu, T. T. and Lin, E. C. C. (1964).Science, New York 146, 1313. Lewis, D. H. and Smith, D. C. (1967). New Phytology 60, 143. Lin, E. C. C. (1961).Journal of Biological Chemistry 236, 31. Lin, R. and Cohen, S. S. (1966).Journal of Biological Chemistry 241, 4304. McCorkindale, J. and Edson, N. L. (1954). Biochemical JournuZ57, 518. MacPhee, D. G., Sutherland, I. W. and Wilkinson, J. F. (1969).Nature, London 22, 475. Marcus, L. and Marr, A. G. (1961).Journal of Bacteriology 82, 224. Martinez de Drets, G . and Arius, A. (1970).Journal of Bacteriology 103, 97. Mayo, J. W. and Anderson, R. L. (1968).Journal of Biological Chemistry 243, 6330. Mayo, J. W. and Anderson, R. L. (1969).Journal of Bacteriology 100, 948. Messer, M. and Kerry, K. R. (1973).Science, New Yorlc 180, 201. Mobley, P. W., Metzyers, R. P. and Wick, A. N. (1970). Archives of Biochemistry and Biophysics 139, 83. Moret, V. and Sperti, 5. (1962). Archives of Biochemistry and Biophysics 98, 124. Mortlock, R. P., Fossitt, D. D. Petering, D. H. and Wood, W. A. (19654. Journal of Bacteriology 89. Mortlock, R. P., Fossitt, D. D. and Wood, W. A. (1964).Bacteriological Proceedinge, 95. Mortlock, R. P., Fossitt, D. D. and Wood, W. A. (1965b).Proceedings of the National Academy of Sciences of the United States of America 51. Mortlock, R. P. and Wood, W. A. (19G4a).Journal of BacterioZogy 88, 838. Mortlock, R. P. and Wood, W. A. (1964b).Journal of Bacteriology 88, 845. Mortlock, R. P. and Wood, W. A. (1971). I n “Biochemical Responses to Environmental Stress”, (I.A. Bernstein, ed.), p. 1. Plenum Press, London. Neish, A. C. and Simpson, F. J. (1954). Canadian Journal of Biochemistry and Physiology 32, 147. Nordie, R. C. and Fromm, N. J. (1959).Journal of Biological Chemistry 234, 2523. Nsihizuka, Y. and Hayaishi, 0. (1962).Journal of Biological Chemistry 237, 2721. Oliver, E. J. (1969). Ph.D. Thesis: University of Massachusetts. Amherst, Massachusetts. Oliver, E. J. and Mortlock, R. P. (1969). Biochemical and Biophysical Research Communication 36, 24. Oliver, E. J. and Mortlock, R. P. (1971n).Journal of Bacteriology 108, 287. Oliver, E. 5. and Mortlock, R. P. (1971b).Journal of Bacteriology 108, 293. Onishi, N. and Suzuki, T. (1969). Applied Microbiology 18, 1031. Palleroni, N. J.and Doudoroff, M. (1956).Journal of Biological Chemistry 218,535. Palleroni, N. J. and Doudoroff, M. (1957).Journal of Bacteriology 74, 180. Patrick, J. W. and Lee, N. (1968). Journal of Biological Chemistry 243, 4312.


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Schacker, H., Sarney, J., MoGuire, E. J. and Roseman, S. (1969). Journal of Biological Chemistry 244, 4785. Schaffer, R. (1972). I n “The Carbohydrates”, (W. Pigman and D. Horton, eds.), Vol. lA, p. 69. Academic Press, New York and London. Scher, B. M. and Horocker, B. L. (1966). I n “Methods in Enzymology”, (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 9, p. 100. Academic Press, New York. Shaw, D. R. D. (1956). Biochemical Journal 64, 394. Simpson, F. J. and Bhuyan, B. K. (1962). Canadian Journal of Microbiology 8, 663. Simpson, F. J., Wolin, M. J. and Wood, W. A. (1958). Journal of Biological Chemistry 230, 457. Simpson, F. J. and Wood, W. A. (1958).Journal of Biological Chemistry 230, 473. Sivak, A. and Hoffman, 0. (1961). Biochimica et Biophysica Acta 53, 426. Stirpe, F. and Comporti, M. (1965). Biochemical Journal 95, 354. Stirpe, F., Comporti, M. and Della Corte, E. (1965). Biochemical Journal 95, 363. Teoce, G., Lazzari, G. and Di Girolamo, M. (1957). Giornale di Microbiologia 3, 154. Toustor, 0. and Harwell, S. 0 . (1958).Journal of Biological Chemistry 230, 103. Volk, W. A. (1959).Journal of Biological Chemistry 234, 1931. Volk, W. A. (1960).Journal of Biological Chemistry 235, 1550. Volk, W. A. (1962). Journal of Biological Chemistry 237, 19. Wickerham, L. T. and Burton, K. A. (1948).Journal of Bacteriology 56, 363. Wilson, B. L. and Mortlock, R. P. (1973). Journal of Bacteriology 113, 1404. Wolin, M. J., Simpson, F. J. and Wood, W. A., (1958). Journal of Biological Chemistry 232, 559. Wood, W. A., McDonough, M. J. and Jacobs, C. B. (1961). Journal of Biological Chemistry 236, 2190. Woods, 11’. F. and Krebs, H. A. (1973). Biochemical Journal 134,437. Wu, T. T., Lin, E. C. C. and Tanaka, S. (1968).Journal of Bacteriology 96, 447. Yamanaka, K. and Sakai, S. (1968). Canadian Journal of Microbiology 14, 391.


Peptid es and Mic ro-0rganisms

J. W. PAYNE Department of Botany, University of Durham, Science Laboratories, South Road, Durham DH 1 3LE, England I. Introduction , . 11. Poptides in the Nutrition of Micro-Organisms. A. Introduction. B. Historical . C. Commercial Peptidc Media . D. Strepogenin . E. Modes of Utilization . . 111. Roles of Peptidases in Peptide Utilization A. Introduction. . B. Peptidase Activity and Growth Response to Peptides . . C. Location of Peptidases and Mode of Peptide Utilization D. Functions of Microbial Peptidases E. Distinction Between Hydrolysis and Transport in Peptide Utilization . IV. Peptide Transport in Micro-Organisms . A. Modes of Transport . B. Genetic and Environmental Considerations . C. Methodology of Peptide Transport Studies . D. Distinction Between Amino-Acid and Peptide Transport . E. Structural Specificities of Peptide Transport Systems . . F. Energetics of Peptide Transport G. “Binding-Proteins” and “Membrane Vesicles” . H. Regulation of Peptide Transport . V. Miscellaneous Relationships Between Peptides and Micro-Organisms. A. Toxin Biosynthesis. . B. Toxic Peptides . C. Concept of “Smugglins” . D. Peptide Antibiotics . E. Peptide Ionophores . F. Conjugated Peptides . . VI. Conclusions References 55

56 56 56 57 57 58 61 63 63 64 64 68 71 72 72 74 74 76 77 94 95 97 99 99 99 101 101 103 104 104 104

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I. Introduction It is almost an axiom of microbiology that peptides are extremely valuable in the nutrition of micro-organisms. Accordingly, microbiologists routinely include peptides (peptones) in culture media. Nevertheless, the basis for this routine act of faith resembles somewhat the British constitution, for nowhere is it written down in a definitive and comprehensive manner. Furthermore, many of the early reasons for including peptides in growth media were founded on false assumptions, as were early explanations for their unique nutrient value (e.g. strepogenin, see Section 11, D, p. 58). It is now clear that prior t o their nutritional utilization peptides must be hydrolysed to yield free amino acids, and that this cleavage may occur before or after absorption. Variations in these two essential features of cleavage and transport produce circumstanccs in which particular peptides may give a growth response that is greater than, equal to, or less than an equivalent mixture of free amino acids. This review attempts to give a current assessment of peptide utilization by micro-organisms (mainly bacteria) from the standpoints of cleavage (Section 111, p. 63) and of transport (Section IV, p. 72). Attention is also given to the effects of peptides on other aspects of microbial physiology (Section V, p. 99). 11. Peptides in the Nutrition of Micro-Organisms A. INTRODUCTION For successful growth, bacteria require appropriate sources of carbon, nitrogen and sulphur, together with certain inorganic ions and, in most cases, a chemical source of energy. I n addition, variation in the enzymic make-up of different species may give rise to specific growth requirements. For example, amongst heterotrophic bacteria nutritional requirements may be simple, as with Escherichia coli, or complex like those of the lactobacilli. It is important therefore to relate the observed nutritional efficacy of peptides t o the nutritional requirements of the particular bacterial strain being studied. Organic substances act as biosynthetic precursors and as energy sources, and the efficiency of utilization and the fate of any particular substance is influenced by other constituents of the culture medium. These features alone (without considering differences in peptide cleavage and transport) lead one to expect that the nutritional response of micro-organisms t o peptides will vary significantly among different species, and that environmental conditions and composition of the medium will also affect the growth response. No extensive systematic study has been made of the growth response of micro-organisms to peptides. However, with a few bacterial species a fairly intensive study has been carried out, and it is the general principles suggested by these studies that are considered here.

PEPTIDES AND MICRO-ORGANISMS

67

B . HISTORICAL I n the early days of microbiology, during the debate over “spontaneous generation”, correlations were observed between microbial growth in organic media and chemical changes in the media. These chemical changes were termed “fermentation” and “putrefaction”, and early reports on putrefaction (the microbial degradation of “meatproteins”) are now seen to be relevant t o the present topic. The classic studics of Koch and Pasteur on anthrax, by which they demonstrated that bacteria can be causative agents of infectious diseases, was largely dependent on the development of simple methods for obtaining pure bacterial cultures. I n part this meant the development of specific culture media. Koch argued that successful cultivation of disease-producing micro-organisms was most likely t o be achieved in a medium closely resembling that occurring within the tissues oi an infected host. Accordingly his selective media were based upon meat infusions and meat extracts. These studies fostered development of “nutrient broth” and “nutrient-agar”, which continue to be widely used in general bacteriology. “Nutrient broth” contains about 0.5% (wlv) peptone (an enzymic meat digest), 0.3% (w/v) meat extract (a concentrate of the water-soluble components of meat), and 0.8% (w/v) sodium chloride to give a salt concentration approximating to that found in tissues. It was Koch therefore who first appreciated the importance of peptides in microbial nutrition. Many of Koch’s contemporaries attempted to employ these media (evolved specifically for cultivating “anthrax-bacteria”) for growth of other bacteria, but their attempts met with varied success. It became apparent that the media were not suitable for all species, and were completely unsuitable for some, such as the nitrifying bacteria. Therefore, two important principles were clearly established before the end of the last century. Koch, Naegeli and others showed that media containing peptides (“meat-infusions”, “peptones”) were utilized nutritionally by many bacterial species. On the other hand, Winogradsky and others demonstrated that certain classes of bacteria failed to grow in media containing peptides. C. COMMERCIAL PEPTIDE MEDIA Peptide-containing media are widely used by bacteriologists, but few prepare their own peptide mixtures. The first commercial peptide medium, “Bacto-peptone”, was introduced by Difco Laboratories in 1914 and a wide variety of peptide-containing bacteriological media are now commercially available. However, all conceal *,heir true identities behind names such as Peptone, Tryptone, Tryptosc, Proteose-Peptone, Protone and Casitone.

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To a large extent these different materials are defined not by their compositions but by their nutritional effects. Manufacturers take considerable care to maintain constancy of their products but their criteria are those of general analysis and growth response of selected strains. For most purposes this is adequate ; however, constancy of growth response with certain strains does not preclude variations in medium composition that may be significant in other test systems. Despite the widespread usefulness of these materials, it is nevertheless arguable that their ill-defined composition has hampered understanding of the precise nutritional role of peptides. Because of the varied nutritional requirements of different species, no one peptone is universally satisfactory. This feature makes it possible to prepare selective peptone-containing media for use in the isolation and cultivation of individual bacterial species (Bridson and Brecker, 1970). Information on the specific characteristics of individual peptones are given in the literature of the commercial manufacturers (e.g. “Oxoid” Manual and Difco Manual, 9th edition) and only a few general points will be made here. Peptones are prepared by the enzymic hydrolysis of proteinaceous materials. The exact nature of the peptone is determined by the raw material used and by the methods employed in production. It is to be expected that they will contain variable proportions of amino acids, di-, oligo-, and polypeptides, and this is probably the main distinguishing feature of all-purpose peptones such as Bacto Peptone (Difco),Bacteriological Peptone (Oxoid) and Neopeptone. However, other peptones may be more of a “complete” medium and contain carbohydrates in addition t o nitrogenous materials. For example, Soya Peptone, prepared by enzymic digestion of soya bean, contains bean carbohydrates; and Peptonized Milk, obtained by pancreatic digestion of skim milk also contains carbohydrates. Proteoses are distinguished from peptones by having a higher proportion of “large” peptides, and for this reason they are often nutritionally inferior to peptones. The size of a peptide has a strong bearing on its‘nutrient value for many species (see Section IV, E9, p. 87). In several recent reports peptones have been characterized (using gel chromatography) in terms of the size distribution of their constituent peptides (Desmazeaud and Hermier, 1973; Payne and Gilvarg, 1968b; Ziska, 1967, 1968). D. STREPOGENIN In the continuing story of peptide utilization by bacteria, the chapter on strepogenin was the first in which defined peptides were used in attempts to relate peptide structure with nutritional activity.

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Strepogenin was the name given by Woolley (Sprince and Woolley,

1944) t o material present in liver extract that appeared to be (‘ . . .

necessary for streptococci of group A to generate”. At this time, several authors reported on the growth stimulation of various bacterial species by substances isolated from different sources. Woolley ( 1941) described a growth factor present in liver extracts that was required by certain haemolytic streptococci. A constituent of “peptone” was required for growth of Lactobacillus casei during the “early stage” of incubation, but was not essential for growth during prolonged incubation (Pollack and Lindner, 1943). Smith (1943) reported that a constituent of yeast extract and of peptone, stimulated growth of Streptococcus lactis. Sprince and Woolley (1944) concluded from the evidence in these reports that a similar substance(s)might be responsible for the growth enhancement in each case ;they corroborated this conclusion by showing that stimulatory activity towards all these organisms was shown by a single liver concentrate. Using L. casei as the test organism, it was soon shown that partial acid hydrolysates, or, better, enzymic digests of casein, possessed strepogenin activity, although unhydrolysed, or fully hydrolysed, casein was inactive. Different proteins showed different strepogenin contents and strepogenin activity was influenced by the type of digestion (e.g. peptic or tryptic digestion ;Sprince and Woolley, 1945). Because of the pure protein nature of these strepogenin sources, the slow enzymic release of strepogenin and its various chemical properties, these authors concluded that the strepogenin molecule ‘( . . . may be a peptide, rather than an a-amino acid”. It is important to consider these studies in the context of their time. In spite of the long history of using peptones in growth media, Sprince and Woolley (1944) could still make the statement that : “The occurrence of the growth factor in partially hydrolysed casein was puzzling in view of the fact that unhydrolysed or fully hydrolysed casein was inactive”. It is clear that Woolley pursued these studies in the expectation that the “strepogenin molecule’’would be a specific growth stimulating substance distinct from, but analogous to, vitamins and hormones. At its inception it was referred to as “ . . . the previously described growth factor now called strepogenin . . . ”. For about 15 years, Woolley and his coworkers, and others, sought unsuccessfully t o isolate from protein digests the “strepogenin molecule”, and to synthesize defined peptides thought likely candidates for “the molecule”. I n reviewing these studies, Woolley and Merrifield (1958, 1963) concluded that no simple correlation was to be found between biological activity and amino-acid composition and sequence, but that nevertheless, “Although this lack of structural specificity required for strepogenin activity :is clear, one must not forget that just any peptide will not do. Many are known which are

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inactive”. During these studies, and following unambiguous evidence for the peptide nature of the active principle, the concept of strepogenin as a peptide-like vitamin was enlarged to embrace possible participation in protein synthesis either by direct incorporation of strepogenin into proteins, or by involvement in trans-peptidation reactions. I n retrospect it can be seen that these studies provided (amongst other things) much useful information bearing upon peptide utilization, but the workers failed to arrive at a satisfactory explanation for the frequent nutritional superiority of peptides over equivalent amino-acid mixtures. Such an explanation was provided by Snell and his co-workers. They came to appreciate the fastidious nature of the micro-organisms used in the strepogenin studies, and the ways in which interactions between constituents of their complex growt,h media could influence growth response. For example, it was found that under certain circumstances partial hydrolysates of protein, or peptides of alanine, permitted growth of L. casei, although free alanine was ineffective (Kihara et al. 1952a; Kihara and Snell, 1952). This appeared to occur because other amino acids in the growth medium antagonized utilization of free alanine but did not interfere with utilization of peptides of alanine, or protein digests. A further example was noted with Streptococcus faecalis (Kihara et al., 1952b). In a medium containing high concentrations of vitamin B, (which induces tyrosine decarboxylase in Strep. faecalis), tyrosine peptides were greatly superior to free tyrosine in promoting growth, but the two were nutritionally equivalent in a medium deficient in vitamin B,. Therefore by changing the composition of the medium an apparent requirement for peptides could result. The difficulties in attempting to ascribe the growth-promoting activity of partial hydrolysates of proteins t o a single peptide or group of peptides (as attempted by Woolley and his colleagues)was clearly shown by Prescott et al. (1953)using Lactobacillus delbrueclcii.In basal media peptides of histidine or of serine caused growth stimulation (strepogenin activity). However, if the medium contained excess free serine, the histidine content of the peptides became the determining factor for strepogenin activity, and if the medium were supplemented with excess free histidine the growth promoting activity of peptides correlated with their serine content. The results clearly showed that the strepogenin activity of a peptide depended on the composition of the growth medium. This conclusion was unambiguously confirmed by Kihara and Snell (1960). They considered that the requirement of organisms such as L. casei for particular peptides was caused by the limited availability of more than one free amino acid, resulting from a multiple imbalance in the growth medium. Under one specific set of conditions, any peptide that contained the growth-limiting amino acid would stimulate growth, but only until availability of a different amino


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acid became growth-limiting. A single peptide would not be expected to be as efficient as a partial protein digest, since the latter would more likely contain peptides of all the growth-limiting amino acids. These ideas were confirmed when the authors prepared a growth medium similar to that used in the strepogenin assay, but with various supplements ; the medium stimulated growth of L.casei more effectively than a tryptic digest of casein used as a source of strepogenin. Thus, the requirement for particular peptides (strepogenin) was considered to be adventitious and to be observed only when peptides could satisfy amino-acid growth limitations more efficiently than mixtures of free amino acids. For other reasons (see Sections on hydrolysis and transport p. 61 and p. 72) all peptides that contain the limiting amino acids might not possess equal nutrient value, while a n active peptide might be replaced by appropriate mixtures of other (simpler) peptides. This explanation, that strepogenin peptides” function merely as preferred sources of growthlimiting amino acids and otherwise play no special metabolic role, is the one most commonly accepted today. Nevertheless, attempts to characterize strepogenin factors continue (Baudet et al., 1968 ; Desmazeaud and Hermier, 1972, 1973). ((

E. MODESOF PEPTIDE UTILIZATION It is clear from what has been said already, and from a myriad other studies on the growth of bacteria, that peptides present in the external environment can be utilized nutritionally by most species. Severaldistinct routes may be envisaged by which exogenous peptides can supply amino acids for growth. These are shown in Fig. 1. I n the first mode (A), the peptide is hydrolysed by extracellular, cellwall, or periplasmic enzymes and the liberated amino acids are absorbed through specific amino-acid permeases. In the second mode (B), the peptide is translocated by a specific peptide-transport system and then hydrolysed inside the cell. And finally (C), it is envisaged that both peptide transport and hydrolysis are functions associated with the cytoplasmic membrane. Concerning the first mode (A), evidence is available that few bacterial species produce extracellular peptidases under conditions of controlled growth (see Section 111, C2, p. 56). Furthermore, there is little evidence that peptidases of broad specificity are components of the bacterial wall or that they occur in the periplasmic region (see Section 111, C3, p. 66). It is probable, therefore, that peptide utilization by this process is not of widespread occurrence. Under particular circumstances, such as autolytic conditions giving release of intracellular peptidases, it may play a nutritional role. It should be noted, however, that

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FIG.1. Modes of peptide utilization. The cytoplasmic membrane is represented by the broken lines with tho cell exterior above and interior below. (A) Hydrolysis of peptide either extracellularly or within the wall or periplasniic space, followed by amino acid uptake. (B) Transport of intact peptide followed by intracellular hydrolysis. (CI) Hydrolysis by membrane-bound peptidases followed by aminoacid uptake. (C2) Hydrolysis by membrane-bound peptidases followed by aminoacid uptake by associatod transport sitos. (C3) Membrane-bound hydrolase system serves to transport and liydrolyse peptides.

extracellular proteases are produced by many species ; although they are generally unable to cleave small peptides, it is likely that their secretion permits hydrolysis of exogenous polypeptides to a form compatible with the requirements of modes (B)and/or (C). A considerable body of evidence, including kinetic and genetic studies, supports operation of the second process (B) in bacteria. I n this model, the sites for peptide uptake are distinct from those for amino acids; thus, peptides do not share amino-acid permeases. This will be considered in detail later (Section IV, p. 72). Several variations of the third process (C) are possible, certain of which have been considered by others in relation to intestinal peptide absorption (Matthews, 1972). The first mode ( C l ) is related to A; hydrolysis by peripheral membrane-bound hydrolyses with release into free solution (or to within the cell envelope) of amino acids that are then concentrated via amino-acid permeases. Failure to demonstrate membrane-bound hydrolases (see Section 111, C4, p. 66), lack of competition between amino acids presented as peptides (see Section IV, D, p. 76), and distinctions between amino acid and peptide transport mutants (see Section IV, D, p. 76) all argue against the operation of this process. If it does occur at all, it is an ancillary process of negligible physiological significance. I n mode (C2), peptides may be hydrolysed by


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peripheral, membrane-bound hydrolases, with uptake of liberated amino acids by an associated translocation process, available only t o amino acids released by the hydrolase and inaccessible to amino acids in free solution. It is experimentally very difficult to distinguish between this process and mode (B) coupled with active intracellular peptidases. However, observations that peptides can be transported without cleavage (see Sections IV, E7, p. 84 and 10, p. 90) cannot be accommodated by model C2 (and only by model B), although current information in gcncral does not preclude limited participation of process C2 in the absorption of other peptides. A further theoretical elaboration (model C3) is one in which a membrane-bound hydrolase acts also as a transport protein by binding peptides from the cell wall side and releasing amino acids directly into the cytoplasm. One might speculate further that the relatively high standard free energy of hydrolysis of many peptides might be used to help promote this translocation process. I n practice, the same reservations are placed on model C3 as were considered for model C2 above. The results that follow (see Sections 111,p. 63 and IV, p. 72) support unambiguously the operation of process B (and, in certain instances, process A) during peptide utilization by bacteria. They do not always exclude involvement of additional uptake modes such as are indicated by models C2 and C3. 111. Role of Peptidases in Peptide Utilization

A. INTRODUCTION The search for strepogenin, and later studies, have failed to provide any evidence that peptides per se can be incorporated directly into protein, act as vitamins, or fulfil any unique role in bacterial growth. Nevertheless, it is possible that under certain circumstances particular peptides may enhance or inhibit bacterial growth. For example, growth may be stimulated if peptides were either to bind toxic substances in the medium and prevent their accumulation, or to act as chelators and enhance the uptake of required metals; conversely, growth could be inhibited if any such binding prevented uptake of essential nutrients. I n spite of theoretical possibilities such as these, the evidence is overwhelming that in the nutrition of micro-organisms peptides act merely as sources of amino acids. Their presence in peptide form may, and frequently does, profoundly influence the nutrient value of the amino acids, but this is explicable by reference to their different modes of utilization and does not require an explanation based on specific “peptide effects’’on growth. Given these circumstances, then, peptidases obviously have an essential role in peptide utilization by catalysing the obligatory

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hydrolysis step. Recent reviews have dealt with additional aspects of microbial peptidases and proteases (Sussman and Gilvarg, 1971; Payne, 1974~). Two recent volumes give broad coverage of this class of enzymes and each gives extensive information on microbial hydrolases (Boyer, 1971; Perlman and Lorand, 1970).

B. PEPTIDASE ACTIVITYAND GROWTH RESPONSE TO PEPTIDES It follows from the above that the nutritional response to any peptide is related to the ability of the organism to hydrolyse the peptide (that is, t o their peptidase activity). This feature may usually be unimportant when the amino-acid residues are not essential for growth, or, even if required, when their rate of release more than satisfies the demands of protein synthesis. However, under certain circumstances hydrolysis may be the step that determines overall growth. I n the extreme case, absence of peptidase activity towards a particular substrate may prevent growth if the particular peptide contains an essential amino-acid residue. More likely, if peptidase activity is low the rate of release of a required amino acid may become the growth-limiting process. In contrast to this situation, it is possible that slow hydrolysis may lead to better growth on peptides than with free amino acids if, for example, free amino acids are rapidly degraded. This has been reported by Kihara et al. (1952b) for enhanced utilization of tyrosine peptides arising from the degradative action of tyrosine decarboxylase on the free amino acid. Gale (1945) noted that a strain of streptococci utilized arginine peptides more effectively than free arginine, and attributed this to failure of peptidebound arginine to be degraded by arginine dihydrolase. The observation that a proline auxotroph of E . coli grew better on proline peptides than free proline (Simmonds and Fruton, 1948)was explained by Stone (1953) in terms of a “sparing effect” of proline in peptide linkage. C. LOCATION OF PEPTIDASES AND MODE OF PEPTIDE UTILIZATION 1. General Considerations As indicated in Section 11, E (p. 6l), the form in which a peptide is absorbed depends upon its ability to “run the gauntlet” of peptidase action. The relation between the location of peptidases and mode of peptide utilization is considered here together with appropriate examples. I n any attempt to classify peptidases with reference to their locations in vivo, account must be taken of the fact that enzymes frequently become “redistributed” as a consequence of cell disruption and fractionation. For example, soluble enzymes may become bound to particulate material following disruption or, conversely, ribosomal or membranebound enzymes may become dissociated and appear in the supernatant

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extract. Although this possibility is widely recognized and, “redistribution” may be easy to spot with certain enzymes whose function limits them to particular cellular locations, it is a more difficult problem with enzymes of broad specificity, such as peptidases, that might reasonably be found in any fraction. Certain isolation procedures may also inactivate some peptidases (e.g. those that are membrane-bound) but not others (e.g. cytoplasmic enzymes), a feature that may lead to misinterpretation of their relative importance in peptide utilization. Finally it should be noted that the location of enzymes may apparently vary with growth conditions. Thus, “extracellular” enzymes may appear under those growth conditions that cause an increase in cell permeability, or, alternatively, a change in cation content may influence the amount of “ribosomal” enzymes; such changes may themselves be deleterious to the organism, or they may be responses that subserve useful functions.

2. Extracellular Hydrolases A wide variety of bacteria, moulds, and micro-organisms in general secrete extracellular proteases ; some species produce several different proteases. In many instances the production and characterization of the enzymes has been studied in detail, and this has recently included study of the large pool of protease-specific messenger ribonucleic acid (Semets et al., 1973). I n contrast, only a handful of reports have appeared on extracellular peptidases. In batch culture, the proteases may be secreted during a particular growth phase (e.g. in exponential phase) ; however, the timing and extent of protease secretion is often markedly dependent on environmental conditions and nutritional status. The functions of these enzymes are unclear. In certain spore-forming micro-organisms a role in the sporulation process has been suggested (Mandelstam and Waites, 1968; Schaeffer, 1969). It has been shown with the fungus Aspergillus nidulans that secretion of extracellular proteases is required for utilization of exogenous protein, but not for growth on low molecularweight substrates (Cohen, 1973), and a similar situation no doubt obtains with other species such as Neurospora crassa (Drucker, 1972). Furthermore, ammonium ion and other low molecular-weight nitrogen sources repress synthesis and secretion of protease (Cohen, 1973 ; Drucker, 1972). I n species such as Neurospora (Drucker, 1973), Pseudomonas (Morihara, 1965), Arthrobacter (Hofsten and Tjeder, 1965), Aeromonas (Litchfield and Prescott, 1970a), synthesis and secretion of protease is stimulated by polypeptide substrates in the medium. Extensive evidence exists therefore for their initial role in protein and polypeptide utilization by hydrolysing large molecular-weight substrates to forms that can be directly absorbed (see Section 11,E, modes B, p. 61 and C, p. 62), or can act as substrates for extracellular peptidases.

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Secretion of extracellular peptidase has been reported for strains of Bacillus licheniformis (Hall et al., 1966), Bacillus subtilis (Ray and Wagner, 1972; Wagner et al., 1972a), and Aeromonas proteolytica (Litchfield and Prescott, 1970b; Prescott et al., 1971; Wagner et al., 1972b; Wilkes et al., 1973).I n several instances secretion is enhanced by peptides in the growth medium (Litchfield and Prescott, 1970b; Wagner et al., 1972a). The latter authors speculated that an endopeptidase released during exponential growth could hydrolyse polypeptides, and when exponential growth ceased aminopeptidase activity could be secreted to cleave further the earlier hydrolysis products to amino-acids. This situation exemplifies transport mode A (see Section 11, E, p. 61). Operation of this process of peptidase secretion seems to preclude the need for peptide transport systems. Specific information is lacking on this point, for the above strains, but it would be pertinent to seek an answer.

3. Periplasmic Hydrolases The periplasm refers to a region of the Gram-negative bacterial envelope between the cytoplasmic membrane and the cell wall (Heppel, 1971). It is considered to be the location of a variety of hydrolytic enzymes that are selectively released by spheroplast formation or by a cold ethylene diamine tetra-acetic acid osmotic-shock treatment (see references in Heppel, 1971). At present, it is not clear whether these enzymes exist in a free state in the periplasm or are loosely attached to the membrane. If peptidases were to exist free in the periplasm, they could function in peptide utilization by mode A (p. 62). On the other hand, if they were to occur loosely bound to the cytoplasmic membrane, their involvement would be by mode C1 (p. 62). This distinction seems t o be academic, however, for peptidases appear to be absent from the complement of degradative periplasmic enzymes (Haley, 1968; Matheson and Murayama, 1966 ;Simmonds and Toye, 1966 ;Van Lenten and Simmonds, 1967).

4. Membrane- Bound Hydrolases The extent t o which peptidases exist bound t o the cytoplasmic membrane (extrinsic peptidases), or as integral membrane components (intrinsic peptidases), will determine the contribution of mode C (p. 62) to peptide utilization. To date, and despite fairly extensive investigations, a mere handful of reports testify t o the occurrence of extrinsic and/or intrinsic peptidases in micro-organisms, and in no case is the evidence unambiguous for their membrane location in vivo (see Section 111,Cl, p. 62). It may well be that this accurately reflects the situation in Nature and that peptide utilization by mode C is of no practical

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significance. Nevertheless, it seems prudent to reserve judgement on this point for the present, for the maxim of enzymology, “extract and purify”, is unlikely to apply in “membranology”. More experience with membrane enzymes is required before rejecting the possible existence of membrane hydrolases. For example, extrinsic peptidases may become detached and lose activity during conventional procedures of cell disruption and membrane preparation ; membrane enzymes may be particularly sensitive to conformational changes consequent upon membrane isolation ; intrinsic peptidases may require extrinsic factors for activity that are removed during isolation. Membrane-bound proteases have been reported in E . coli (Regnier and Thang, 1973). Peptidases have been found bound to membranes of Mycoplasma laidlawii (Choules and Gray, 1971; Pecht et al., 1972). The latter authors claimed that theenzymes werelocated on the outside of the membrane, but their results were not unambiguous. Enzymes such as these are to be distinguished from the peptidases commonly found associated with the cell envelope that possess strict specificities for the nnusual types of peptide bonds found in peptidoglycans (Reaveley and Burge, 1972; Schleifer and Kandler, 1972).

5 . Intracellular Peptidases The paucity of reports on extracellular peptidases is more than compensated for by the plethora on intracellular peptidases. The isolation and characterization of these enzymes is considered more fully elsewhere (Boyer, 1971; Payne, 1974c; Perlman and Lorand, 1970; Sussman and Gilvarg, 197 1). Intracellular peptidases may occur as soluble enzymes existing free in the cytoplasm, or bound to ribosomes, or “compartmentalized”. To whichever category they belong they function in peptide utilization by mode B (p. 62). Most micro-organisms produce only intracellular peptidases. It is this class of organism almost exclusively that will be considered in Section 1V (p. 72) on peptide transport, for only with organisms that fail to produce extracellular peptidases can one conveniently and unambiguously demonstrate transport of peptides per se. If E . coli is typical, these micro-organisms possess a range of soluble intracellular peptidases, many with over-lapping specificities (Boyer, 1971; Johnson and Berger, 1942; Matheson et al. 1971; Payne, 1972a, 1 9 7 4 ~Perlman ; and Lorand, 1970; Simmonds, 1970,1972; Sussman and Gilvarg, 1970, 1971; Vogt, 1970; Yaron and Mlynar, 1968; Yaron et al., 1972). Peptidases are found associated with ribosomes after cell disruption but some of this binding is artifactual (Vogt, 1970 ; Matheson et al., 1971), although it may play a role in protein synthesis (see Section 111, D6, p. 70). Studies of peptide utilization by yeast led to the

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suggestion that intracellular peptidases may be compartmentalized and exist within lysosome-like vacuoles and that, prior to their utilization, peptides might need to cross this membrane barrier in addition to the cytoplasmic membrane (Becker et al., 1973; Naider et al., 1974). D. FUNCTIONS OF MICROBIALPEPTIDASES 1. General Considerations Consideration of microbial peptidases has so far been confined to their role in nutrition. However, when attempting to interpret observations on the specificity and location of these enzymes it is important to bear in mind that they probably perform additional functions. Some of these established and suspected functions are considered here.

2. Role i n Peptide Utilization This aspect has been considered in previous sections of this review. I n theory a t least, extracellular, membrane-bound, and intracellular peptidases could all be involved in peptide nutrition in the ways discussed in Section 11, E (p. 61). For extracellular enzymes this may well be their main function. Membrane-bound peptidases (if they occur) may, in addition to a nutritional role, function in cell-envelope biosynthesis, cell division and sporulation. Intracellular peptidases undoubtedly play a role in nutrition by converting absorbed peptides to utilizable amino acids. Kessel and Lubin (1963)demonstrated this function directly when they observed that a glycine-requiring auxotroph of E . coli failed togrow on diglycine following mutational loss of an intracellular diglycine peptidase activity. Although clearly implicating the enzyme as playing a role in nutrition, the result is a little surprising in view of the several different intracellular enzymes that can probably split diglycine. This feature of different enzymes with overlapping specificities makes it difficult to provide direct genetic evidence that mutational loss of a specific peptidase activity leads to a failure to utilize particular peptides. Sussman and Gilvarg (1970) reported that loss of a particular trilysinesplitting activity caused trilysine to become toxic to E . coli rather than of nutrient value. Although it is clear that these enzymes function in peptide nutrition, their presence in micro-organisms growing in minimal media devoid of peptides is consistent with their performing additional roles. 3. Hydrolysis of Inhibitory Peptides The potential biological activities of peptides are enormous (Matthews and Payne, 1974a).Depending on their particular amino acid composition and sequence, peptides derived from protein hydrolysates could have specific affinities for lipids, nucleic acids or proteins (Matthews and

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Payne, 1974a). It is not unlikely that, during the normal course of intracellular protein hydrolysis in micro-organisms, biologically active peptides may be generated. Some may be toxic. This could be a direct toxic effect (e.g. binding t o ribosomes) or indirect, if, for example, they could disrupt the action of (theoretical) peptide regulators and messengers, or interfere with cnzymes that have peptide-like substrates or allosteric sites for peptide-like effectors. Following from these speculations, it is an interesting thought that loss of a peptidase activity, by mutation, might be lethal to a micro-organism not solely because the enzyme performs a useful nutritional function but also because uncleaved intracellular peptides may be inhibitory. A number of examples are to be found of peptides that are toxic because they are accumulated and not cleaved (see Section V, B, p. 99). It appears, therefore, that a n important function of intracellular peptidases may be to hydrolyse peptides that could be deleterious to growth.

4 . Turnover of Intracellular Proteins Protein turnover involves intracellular hydrolysis of proteins to free amino acids and their re-incorporation into newly synthesized proteins. Intracellular proteolysis occurs through the combined action of proteases and peptidases (Pine, 1972).The process has been extensively studied in E . coli and early reports indicated that proteolysis in growing bacteria was negligible, but increased under starvation conditions (Mandelstam, 1960, 1963; Willetts, 1967a). Further studies have shown that during active growth of E . coli a continuous process of proteolysis does occur (to a minimum of 2.5% per hr), that appears to involve repeated cycling of a particular type of protein which comprises 1-7% of the total cellular protein (Goldberg, 1972b; Nathand Koch, 1970; Pine, 1966, 1970,1972; Willetts, 196713). Proteolysis occurs by several distinct processes that vary with nutritional status, type of organism, and growth phase (Pine, 1972; 1973a, b ; Goldberg, 1971a; Prouty and Goldberg, 1972a; Sussman and Gilvarg, 1969; Nath and Koch, 1970, 1971). When cells enter the stationary phase of growth (i.e. for an undefined reason) or growth is ultimately limited by the availability of the source of carbon or nitrogen, or an essential amino acid (with an auxotroph), proteolysis may be affected in different ways. It may remain unchanged (Nath and Koch, 1971; Pine, 1970), it may become more general although not increase in overall rate (Pine, 1967),or the rate may be increased (Goldberg, 1971a; Mandelstam, 1960; 1963; Nath and Koch, 1971; Pine, 1973a, b). Goldberg (1972b) suggested that the proteins that turn over in growing E . coli may be inherently more sensitive to proteases in general. Proteolysis induced by starvation or amino-acid deficiency seems to be carried out by newly synthesized enzymes (it being largely abolished

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by chloramphenicol) ; these enzymes have properties in common with the class of “serine proteases” (Brunschede and Bremer, 1971; Goldberg, 1971a, b ; Prouty and Goldberg, 1972a; Pine, 1973a, b ; Sussman and Gilvarg, 1969). Partial purification of a “serine-protease” from E. co2i was reported recently (Pacaud and Uriel, 1971). It seems likely that during normal growth of E . coli some of the proteolysis is of abnormal proteins; this could serve a protective role t o remove possible deleterious polypeptides or simply represent a salvage operation. Circumstances that lead to greater production of aberrant proteins also lead to enhanced proteolysis. Examples of such abnormal protein are : (i) unfinished polypeptide chains prematurely terminated (by starvation or amino-acid deficiency) ;(ii)proteins containing frequent errors in translation (products of genes carrying nonsense mutations) ; and (iii) proteins containing amino-acid analogues (Goldschmidt, 1970; Goldberg, 1971b, 1972a, b ; Lin and Zabin, 1972; Platt et al., 1970; Pine, l972,1973a, b ; Prouty and Goldberg, 1972a, b). It is to meet therequirements for greater proteolysis posed by these abnormal proteins that synthesis of the special class of protease may be induced. Neverthelcss, inadequate proteolytic activity may lead t o abnormal proteins aggregating to form an intracellular granule (Prouty and Goldberg, 1972b). Study of mutants that fail to degrade rapidly aberrant proteins should help clarify the function of the responsible proteases and peptidases (Bukhari and Zipser, 1973; Shineberg and Zipser, 1973). 5. Additional Functions of Peptidases

Protein synthesis in bacteria is initiated with a n N-formylmethionine residue. However, in E. coli only 45% of completed proteins were found to have N-terminal methionine (Waller, 1963), and only 10% in Bacillus subtilis (Horikoshi and Doi, 1968). It follows that conversion of newly synthesized polypeptide into final protein involves deformylation and generally aminopeptidase action. Deformylation precedes peptidase action and may occur during elongation of peptide chains, or after their release from ribosomes (Pine, 1969). A specific deformylase has been characterized (Adams, 1968; Livingston and Leder, 1969; Takeda and Webster, 1968),and various enzymes have been suggested as the specific methionyl peptidase although none meets all of the criteria expected for this enzyme (Brown, 1973a, b ; Matheson, and Tsai, 1965; Matheson and Dick, 1970; Matheson et al., 1970,1971; Tsai and Matheson, 1965; Vogt, 1970). It seems likely that peptidases and proteases may function in enzyme activation by a process of limited proteolysis. This might be necessary a t certain stages in the cell cycle, or triggered by environmental circum-


71

stances. An example of the first case is observed with the RNA polymerase of Bacillus subtilis during sporulation. Initial loss of vegetative sigma factor changes the template specificity, while subsequent proteasecatalysed modification of a /3 subunit occurs and its place is taken by a smaller polypeptide (Losick, 1972).

E. DISTINCTION BETWEEN HYDROLYSIS AND TRANSPORT IN PEPTIDE UTILIZATION In the overall process of peptide utilization, peptidases perform an essential function. However, it is desirable to consider peptide hydrolysis and peptide transport as quite separate steps in the overall process. As discussed in Section 11,E (p. 61), theoretically they may be coupled in an obligatory way (mode C) but they may also be quite distinct (mode B). Much of the information on peptide transport considered in Section IV (p. 72) provides circumstantial evidence for this distinction, and many of the results are most easily interpreted on this assumption. I n addition, direct evidence for their separate nature is available from studies with E . coli. Kessel and Lubin (1963) provided genetic evidence for the distinction between peptide transport and peptidase activity. They isolated from a glycine-requiring auxotroph two different mutants that had lost their ability to grow on diglycine. One was a transport mutant in which uptake of radioactively-labelled diglycine was severely decreased. This strain characteristically grew when supplied with high concentrations of diglycine but failed to grow on low concentrations. Even this limited growth requires diglycine peptidase activity, and the presence of this enzymic activity in the transport-deficient strain was indicated by the fact that only traces of thc accumulated radioactivity occurred as diglycine. The other mutant was peptidase deficient. Direct assays showed loss of diglycine peptidase activity, and isotopically-labelled diglycine was accumulated one hundred-fold without cleavage. Consequently, this strain was characterized by failure to grow on diglycine at both high and low concentrations. Other observations with E . coli accord with these genetic studies. For example, dipeptides may be accumulated intact under environmental conditions that depress intracellular peptidase activity (Meisler and Simmonds, 1963; Simmonds, 1966, 1970, 1972). I n certain cases the accumulation of intact dipeptides inhibits growth although the same peptides are not toxic when peptidases are present to degrade them rapidly (Simmonds, 1970, 1972; Vonder Haar and Umbarger, 1972; Wasmuth and Umbarger, 1974) ; similar observations have been made with oligopeptides (Payne, 1968 ; Gilvarg and Levin, 1972).

72

J. W.PAYNE

IV. Peptide Transport in Micro-Organisms A. MODES OF TRANSPORT In theory, various mechanisms are available by which peptides may traverse the cytoplasmic membrane of micro-organisms. These are illustrated in Fig. 2. In practice they are of widely different importance.

1. Passive Diffusion In passive diffusion (Fig. 2A), molecules are considered to cross the cell membrane by random molecular motion alone and not to bind to any membrane component. This process does not permit concentration against a gradient although net movement down a gradient is possible. Although this process is quantitatively insignificant for peptide uptake in general, it is possible that certain small neutral peptides may passively diffuse across the cell membrane rather easily. 0

Outside

Membrane

11;0

0

.

0

0

Inside

0

(A)

(D)

(C)

(D)

FIG.2. Modes of peptide transport. (A) Passive diffusion. (B)Facilitated diffusion: catalyses transport of peptide down a Membrane system, indicated thus 0, concentration or electrochemical gradient. (C) Group translocation : peptide is structurally modified during transport. (D) Active transport: similar to (B) but energy, E, is required to transport peptide against a gradient. See text for discussion.

2. Facilitated Diffusion By this process (Fig. 2B) it is envisaged that molecules bind to specific “carriers” during passage across the membrane. Many theoretical models have been suggested for the mobile “carriers”. Pores, lined with ligands in such a way as to achieve substrate specificity, have also been suggested. No direct metabolic energy is required for this process which does not lead to concentration against a gradient. It is possible that, under conditions that prevent energy-coupled (active) transport of peptides, facilitated diffusion may occur.

3. Group Translocation I n this process, (Fig. 2C) the covalent modification of substrate catalyses its transfer across the membrane. Concentration against a gradient

73


can be achieved, although the substrate is accumulated in a chemically modified form; for this reason the mechanism is not a true active transport process. The classic example of a group translocation process is the accumulation of phosphorylated sugars by certain bacteria by means of a phosphotransferase system that catalyses the transfer of phosphate from phosphoenolpyruvate to particular sugars (Roseman, 1972). This particular process is energy dependent, but in theory, group translocation could actually supply energy to the cell if the membrane-bound enzyme modified the substrate by an exergonic rather than endergonic reaction (Roseman, 1972). There are no reports that peptides are transported by this mechanism in micro-organisms. although experiments designed to check the possible contribution of this mode to overall peptide transport have probably not been carried oiit. 4. Active Transport This mechanism (Fig. 2D) resembles facilitated diffusion except that it is coupled to metabolic energy thereby permitting the concentration of substrate against an electrochemical gradient. The process may be “simple” (primary)active transport, in which the substrate alone crosses the membrane (e.g. lactose transpcrt in E.coli), or it may be co-transport (exchange transport) in which substrate movement is coupled to the flux of a separate substance such as H+,Kf or Na+. Evidence to be presented indicates that peptides are taken up by bacteria by an active transport process (See Section IV, F, p. 94). It aids understanding to divide this overall transport process into several separate steps as illustrated in a typical model (Fig. 3). The first steps may be highly specific, and involve recognition and binding of the substrate. During translocation the substrate is in some way, as yet unknown, transferred across the membrane ; in active transport this is coupled to a supply of energy. Subsequently, the substrate is released into Recognition ond binding Outside

0

0 0

Mernbrone Inside

Translocolion

Release and

reody

0 -

0

7

-

7

-e>0 0 0 0

FIG.3. Theoretical steps in peptide transport. Initial step involves binding of exogenous peptide a t a specific site on the membrane. Subsequent steps (some or all of which must be energy linked for active transport) involve transfer across the membrane (translocation), intracellular release, and conversion of system to a form ready for further transport. See text for discussion.

74

J. W. PAYNE

the cell interior. Finally, the system must be made ready for further transport ; this may conceivably require a further step of the cycle or, with a multivalent system, the release step may leave it already primed with a different externally orientated binding site.

B. GENETICAND ENVIRONMENTAL CONSIDERbTIONS When attempts are made to comparc the results of different workers, duc attention should be given to the effects of variations in growth conditions and of strain differences. This can be particularly important with transport systems subject to various regulatory controls. Different bacterial strains often show variations in their transport ability towards amino acids, sugars, and other compounds (Oxender, 1972). I n addition, changes in growth media can alter the transport ability of a particular organism towards various substrates (Kepes and Cohen, 1962), including peptides (Levine and Simmonds, 19G2b). Changes in bacterial physiology caused by environmental alterations and by variations in growth rate during the growth cycle in batch culture (Herbert, 1961; Tempest, 1970) may also affect transport properties. The likely heterogeneous nature of a bacterial population grown in batch-culture should not be forgotten when assessing kinetic evidence for “high-” and “low”-affinity transport systems, and corroborative genetic evidence should bc obtained when possible. C. METHODOLOGY OF PEPTIDE TRANSPORT STUDIES Peptide transport is routinely assessed by measuring the nutritional response of a micro-organism to peptides in the growth medium. This technique obviously requires that the micro-organism possesses no extracellular or periplasmic peptidases so that peptides per se are taken up (see Section 11, E, p. 61); E . coli is an example of this class. Rather than measuring the extent to which peptides may stimulate growth of a particular wild-type strain, it is more informative to use an amino-acid auxotroph, growth of which is absolutely dependent upon an exogenous supply of the required amino acid (which may be free or in peptide form). Thus, growth of such a mutant upon a particular peptide indicates clearly that the micro-organism is able to take up and then to hydrolyse the peptide (Fig. 4).Information obtained by this technique has provided the basis of our present understanding of peptide transport in microorganisms. Detailed information about the transport processes has been obtained from studies of competitive interactions between defined peptides that lead to growth stasis or to a decrease in growth rate of particular auxotrophs. This technique is an indirect way of investigating transport phenomena

PEPTIDES AND MICRO-ORQANISMS

75

80,

Time ( h )

FIG.4. Growth response of Escherichia coli lysine auxotroph (M-26-26) to lysine and peptides of lysine. Growth tubes contained 0,075 pmole per ml of lysine, or lysine residues in the case of tho peptides. Reproduced from Gilvarg and Katchalski ( 1965).

and, although it is undoubtedly better to measure directly uptake of radioactively-labelled peptides by conventional techniques, defined radioactive peptides are generally not readily available and are costly, and time consuming, to prepare. The conclusionswhich can be drawn from the simple growth studies do allow us to specify the particular defined peptides that are best suited €or critical transport experiments using radioactively-labelled substrates. I n the few instances where radioactively-labelled peptides have been used, results have corroborated conclusions drawn from growth tests. When using auxotrophs in these studies one should be aware that starvation for an amino acid can alter membrane permeability. It is well documented that arrest of protein synthesis by amino-acid starvation can disrupt the close coupling between the synthesis of intracellular constituents and the bacterial cell envelope (cell wall plus membrane) that can lead to a general increase in membrane permeability (Shockman, 1963, 1965; Higgins and Shockman, 1970; Knox et al., 1967; Rothfield and Kothencz, 1969; Voll and Leive, 1970; Matzura and Broda, 1968). Inhibition of protein synthesis may also directly change particular transport processes through specific changes in membrane lipids (Higgins and Shockman, 1970; Wilson and Fox, 1971; Holden et al., 1973) or by disrupting the regulation of transport processes that are controlled by the levels of intracellular pool constituents (Holden and Utech,

76

J. W. PAYNE

1967; Elsas and Rosenberg, 1967; Wiley and Matchett, 1968; Grenson et al., 1968; Crabeel and Grenson, 1970; Gross and Ring, 1969). These aspects have been considered in greater detail elsewhere (Payne, 1974b).

D. DISTINCTION BETWEEN AMINOACIDAND PEPTIDE TRANSPORT Numerous reports in the early literature indicated that amino acid and peptide uptake are distinguishable processes, although different interpretations were generally put on the results a t the time. The two uptake processes show distinctive characteristics, the substrates do not compete with one another for uptake, and mutants can be isolated that are deficient in one or other process. Thus, Hirsch and Cohen (1953) reported that growth on leucine of a leucine auxotroph of E . coli was competitively inhibited by related amino acids in the medium but not by peptides of these amino acids, and analogous reports have been made with many bacterial species (Shelton and Nutter, 1964; Kihara and Snell, 1952, 1955). Uptake of labelled amino acids is competitively inhibited by related amino acids but not by peptides containing these residues (Cohen and Rickenberg, 1956). Leach and Snell (1959, 1960) first used labelled peptides in transport studies and showed that accumulation of glycine by Lactobacillus casei was faster and more extensive from peptides than from the free amino acid; in addition, peptide transport was not inhibited by free amino acids. Analogous results have been obtained with a variety of bacterial species (Brock and Wooley, 1964; Kessel and Lubin, 1963; Levine and Simmonds, 1960,1962a; Mayshaket al., 1966; Pittman et al., 1967; Smith et al., 1970; Yoder et al., 1965a). Mutants of E . coli (Kessel and Lubin, 1962,1963 ;Levine and Simmonds, 1960; Guardiola and Iaccarino, 1971), of lactobacilli (Peters et al., 1953), and of Fusiformis necrophorus (Wahren and Holme, 1973), have been isolated that fail to transport a free amino acid but readily take up the amino acid when in peptide form. Bacteroides ruminicola is a “natural mutant” that absorbs amino acids only in peptide form (Pittman and Bryant, 1964; Pittman et al., 1967). Interestingly, this same characteristic is observed in cases of Hartnup’a disease (a mammalian intestinal defect) (Asatoor et al., 1970; Milne and Asatoor, 1974). The alternative class of mutant that is unable to transport peptides but can accumulate free amino acids has been described in E . coli (Barak and Gilvarg, 1974; Felice et al., 1973; Payne, 1968; Payne and Gilvarg, 1971) and in Salmonella typhimurium (Ames et al., 1973). These studies clearly show that amino-acid and peptide transport systems each possess unique components, such as recognition sites, but a t present it is not known whether they are completely distinct or may have certain features in common (e.g. energy coupling). This possibility


77

is best considered with reference to Figs. 2 and 3 (pages 72 and 73). If these transport systems do have common features it is likely that under particular conditions (e.g. energy limitation) the two uptake systems may not function independently. I n addition, as both free amino acids and peptides supply the intracellular pool it is possible that indirect interactions between the two may arise from regulatory control of transport governed by amino-acid levels in the pool (see Section IV, H, p. 77).

E. STRUCTURAL SPECIFICITIES OF PEPTIDE TRANSPORT SYSTEMS

1. Introduction Many bacterial species possess individual transport systems for most protein amino acids. I n addition, they often have high-affinity transport systems for groups of amino acids that are closely related in structure (Oxender, 1972 ;Payne, 1974b). It is energetically better for the organism to absorb amino acids than to synthesize them, consequently amino-acid transport systems are generally constitutive (Kepes, 1964). The selective advantage provided by possession of these transport systems, coupled with the relatively small number of protein amino acids, makes it feasible for E. coli, for example, to possess about a score of amino-acid transport systems. The nutritional advantages to be derived from peptide transport systems may be argued analogously. However, consideration of the possible structural specificities for peptide transport is a different matter. The structural diversity of the protein amino acids, sufficient to allow specific transport systems, is compounded when they are in peptide linkage. In addition, the vast number of different peptides ( 4 x lo2 dipeptides; 6 x lo7 hexapeptides etc.) precludes a comparable number of peptide-transport systems. On the assumption that peptides of all possible amino-acid sequences may occur in the microbial environment, it seems likely that a peptide transport system(s) might possess specificity only for the structural features shared by all peptides and yet sufficient t o distinguish uniquely this type of substrate; most of the studies to be described that have been aimed a t determining these structural specificities have been motivated by this premise. As peptides will be derived mainly from protein hydrolysates, and no specificity in protein sequence is apparent, it is likely that peptides with all possible amino-acid sequences will generally be found in microbial environments. However, if a particular species were to exist in an ecological niche with limited proteolytic activity, only a limited class of peptides might be produced. For example, if only trypsin-like activity occurred, peptide transport systems might evolve with specificity for a C-terminal lysine or arginine residue. Evidence in support of this hypothetical case is, as yet, lacking.

78

J. W. PAYNE

Structural requirements for peptide transport in micro-organisms have been discussed elsewhere (Payne and Gilvarg, 1971; Sussman and Gilvarg, 1971; Payne, 1974b) and will not be considered a t length here.

2. N-Terminal a-Amino Group Several reports in the early literature showed that substitution of the N-terminal u-amino group of a peptide abolished its nutritional activity for bacteria (Table 1,l-6) (Simmonds and Fruton, 1948;Simmonds et al., 1947; Merrifield and Woolley, 1956; Woolley, 1947, 1948; Woolley et al., 1955; Dunn, 1959; Dunn and Dittmer, 1951). However, in no case was it established whether the substituted peptides failed to enter or were resistant to hydrolysis. Gilvarg and Katchalski (1965) provided the first definite evidence on this point. They observed that a-N-acetylated homopeptides of lysine and of arginine (Table 1, 7,s)were nutritionally inactive for appropriate auxotrophs of E . coli even though the substituted peptides were readily cleaved by intracellular peptidases; they concluded that N-acetylation prevented transport. Later results were compatible with this conclusion (Losick and Gilvarg, 1966). (Fig. 5). Although subsequent studies (Payne and Gilvarg, 1971; Payne, 1972b) suggested that the demonstration of peptide cleavage in vitro does not always ensure that peptidase activity in vivo is sufficient to permit auxotrophic growth on the peptide, the conclusion concerning the acetylated peptides was correct.

.re Lysine

%' 4 0 Y

20 -

O

a -Acetyllrilystne

d

TABLE 1. The effect of various a-N-terminal substituents on the utilization, transport and hydrolysis of peptides by Escherichia wli. Standard three letter abbreviations me used for amino acids; sar = sarcosyl (N-methylglycyl); lysn, lys3, lys4 are lys-lys, lys-lys-lys,lys-lys-lys-lys,and similar notation is used for the other homopeptides. n.d. = Not determined ; (+) indicates extensive hydrolysis and transport, and growth equal to (or approaching that) on free amino acid; (-) indicates no hydrolysis, transport or growth; (+) indicates slight hydrolysis, transport or growth. N-Terminal substituent Peptide

Structure

1. Phe-Pro

m 2 -

2. Phe-Pro

3. Gly-Phe; 4. Gly-Phe; 5. Pr-Pro

CE34O-NEGly-Tyr Gly-Tyr

6. Pro-Pro

NH2Ph-CE2-040-NHNH2Ph4E-O-CO-NE-

-

Name

unsubstituted

CC -Gly

28.

27. 28. 29.

30.

Fate of Peptide N-Terminal A K x o t r o p L Charge used Utilized Transported Eydrolysed

Reference

~

+ve

+d

+d

acetyl.

neutral

n.d.

unsubstituted benzyloxycarbonyl unsubstituted

+ve neutral +ve

+n.d.

+n.d.

+d

+d

benz yloxycarbonyl

neutral

n.d.

n.d.

+

+ +

++ +. +++

++ ++ +++ +

unsubstituted

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

,

+ve

acetyl.

neutral

unsubstituted methyl (sar) methyl (sar) unsubstituted methyl (sar) unaubstituted acetyl., succinyla glutarylc unsubstituted dimethyl dimethyl ethyl ethyl propyl propyl butyl butyl isopropyl isopropyl isobutyl isobutyl

+ve +ve +ve +ve +ve +ve neutral -ve -ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve

-

--I

-f

+--I

--I

--I

+.

+.

+.

+.

+.

n.d.

: f f f f f t f f f k

f

Simmonds and Fruton (1948) Simmonds and Fruton (1948) Simmonds etal. (1947) Simmonda e l nl. (1947) Simmonds and Fruton (1948) Simmonds and Fruton (1948) Gilvarg and Katchalski (1965) Gilvarg and Katchalski (!965) Losick and Gilvarg (1966) Payne (1968,1971a) Payne (1971a, 1974a) Payne (1971a, 1974a) Payne (1971a) Payne (1971a) Payne (1971a) Payne (19713) Payne (1971a) Payne (1971a) Payne (1971a) Payne (1974a) Payne (1974a) Payne (1974a) Payne (1974a) Payne (1974s) Payne (1974a) Payne (1974a) Payne (1974a) Payne (1974a) Payne (1974a) Payne (1974a) Payne (1974a)

a N-acetyl-, b N-succinyl-, and c N-glutaryl-substituted peptides may be regarded 85 N-glycyl-, N-aspartyl-, and N-glutamyl-glycine peptides respectively, devoid of a-amino ernunsi d utilized ..-=., .. when nentide _ . _ .is ~ _ ~ (+I. transwrt f+) and hvdrolvsis f+) are assumed: e neutide is eood comnetitive inhibitor of transnort fits own UDtake mav or may not be extensive): f failed to competiti;rklyinhibit uptake of ndrmal-peptides. I

~

~~

~~~~~~~~~

80

J. W. PAYNE

N-Acetylation might prevent transport in various ways : for example by: (i) steric hindrance, (ii) substituting the primary amino group, or (iii)neutralization of the positive charge on the amino group. To try and decide between the last two possibilities, Payne (1971a) prepared a series of peptides in which the charge on the amino terminus was varied ; these substituted peptides (Table 1, 9-18) were tested with E . coli auxotrophs. Certain ofthese substituted peptides can in effect be regarded as normal peptides devoid of a-amino groups (for example, N-glycyl(15), N-aspartyl (16) and N-glutamyl glycine peptides (17). These were all nutritionally inactive, resulting from a failure in transport and not in hydrolysis. Considered from a different stand-point, substituted peptides in which the positive charge on the amino group was neutralized (15) or made negative (16, 17) were not transported. However, the nutritional activity of N-methyl-substituted peptides (10, 11, 13) showed that presence of a primary a-amino group is not essential for transport (eliminating possibility (ii), above). Unfortunately, transport of the a-N-methyl, but not a-N-acyl, peptides did not conclusively demonstrate the requirement for a positively charged amino group because the large acyl groups could be sterically hindered. To try and clarify this point, Payne (1974a)prepared a series of a-N-alkyl peptides (Table 1,19-30) in which the positive charge was retained but some of the alkyl substituents were larger than the inactive acyl substituents. All of the mono-alkyl peptides (Table 1, 21-30) were good competitive inhibitors of peptide transport, and the substituted tripeptides were all nutritionally active (the nutritional failure of the mono-alkyl dipeptides was probably caused by their slow hydrolysis to give free glycine).These results indicated that steric hindrance was an unlikely explanation for the inactivity of the N-acyl peptides, and suggested that the N-terminal group may in fact be substituted, with retention of transport activity, providing the positive charge is retained. However, the additional observation that the positively charged N-dimethyl peptides (Table 1, 19, 20) were nutritionally inactive and devoid of competitive ability suggests a structural requirement for a hydrogen atom on the amino group. Thus, it appears at present that a positively-charged secondary amino group is the minimum structural requirement of the E . coli peptide-transport systems towards the N-terminus. This conclusion is compatible with the observed ability of N-terminal prolyl peptides to use these systems (Payne 1971b). Mechanistic implications of this feature of permease specificity have been discussed (Payne, 1972b). The hope has often been expressed (Payne, 1974b) that results obtained from studies of peptide uptake in bacteria would be representative of most biological systems and would facilitate similar studies in mammalian systems (e.g. intestinal absorption) and several reports have

PEPTIDES AND WURO-ORGANISMS

81

recently appeared encouraging this hope. Acetylation of the N-terminal amino group of glycylglycine resulted in failure to compete for intestinal dipeptide-transport systems (Rubino et al., 1971; Matthews, 1974). N-Methylglycylglycine (sarcosylglycine) was absorbed by mammalian intestine, although uptake and hydrolysis were both decreased to some extent (relative to glycylglycine; Burston et al., 1972). Early studies (Dunn and Dittmer, 1951)with the yeast Saccharomyces cerevisiae (which occupies a place somewhere between the prokaryotic bacteria and complex mammalian tissue) further support the generality of these results. Ability to utilize peptides was destroyed by u-N-acylation, as was competitive ability. However, certain recent studies (Becker et al., 1973; Naider et al., 1974) with Sacch. cerevisiae are a t variance with these results. Thus, several N-acetyl and N-t-butoxycarbonyl methionyl peptides were nutritionally active, although growth was poorer than with the free peptides. Further studies are needed to assess the generality of this result. The competitive ability of the substituted peptides was not checked, so it is possible that these particular substrates could be taken up by a system different from that used for normal peptides.

3. StereospeciJicity D-Amino acids are nutritionally inactive for many bacterial species. Furthermore, bacterial peptidases are generally unable to cleave peptide bonds formed from D-amino-acid residues. For these reasons such peptides are not utilized, and it becomes impossible to employ growth response as a measure of transport ability (see Section IV, C, p. 74). To judge this feature of structural specificity it is necessary therefore t o use labelled D-peptides and/or to measure their ability to compete for transport. Levine and Simmonds (1962a) showed that D-leucylglycine did not inhibit growthof an E . coli leucine auxotrophon L-leucylglycine,and neither did it inhibit uptake of [14C]-~-leucylglycine. A number of dipeptides containing D-residues did not inhibit uptake of [14C]-glycylglycineby E . coli (Kessel and Lubin, 1963). I n lactobacilli, uptake of [14C]glycyl-L-alanine was not inhibited by glycyl-D-alanine (Leach and Snell, 1960) and peptides containing D-residues were not absorbed (Kihara et al., 1961; Shelton and Nutter, 1964; Yoder et al., 1965a, b). Dipeptide transport therefore appears to have a stereospecific requirement for L-residues. Competititon studies (Payne, 1972b) suggest a similar stereochemical requirement with oligopeptides although this may apply only to the first two residues, from the h’-terminus (Shankman et al., 1962; Payne and Gilvarg, 1971; Payne, 1974b). This is not unreasonable, for tripeptides with L,L,L and L,L,D configurations can be superimposed

82

J. W. PAYNE

except for the C-terminal carboxyl group for which the oligopeptide transport system has no requirement (see Section IV, E8, p. 85).

4. a-Hydrogen Atom Several reports suggest that the hydrogen on an u-carbon group of an amino acid is not required for peptide transport. Thus, gly-aib-ala (where aib is a a-aminoisobutyric acid residue, in which the u-hydrogen is replaced by a methyl group) was readily taken up by Lactobacillus casei (Young et al., 1964), and its uptake was competitively inhibited by triglycine (Smith et al., 1970). Similarly, tripeptides with cycloleucine (l-amino-cyclopentane-l-carboxylic acid, which is again devoid of a-hydrogen) as the central residue were rapidly accumulated by L. casei (Young et al., 1964).

5 . Amino Acid Side Chains In theory it is possible that a limited number of different peptidetransport systems could exist with, for example, requirements for predominantly acidic, neutral, basic or aromatic side chains. However, among the vast number of possible peptides most would be of ‘(mixed character” and would lack any overall acidic, basic or aromatic property, making discrimination on these bases difficult. At the other extreme one could conceive of a single system with no specificity towards side chains. This circumstance would satisfy the properties of the vast bulk of peptides of “mixed character”. Although the latter system seems more reasonable, in practice it is difficult to establish conclusively the true situation because of the virtual impossibility of testing all conceivable peptides. At present, genetic studies, and results of competition studies, indicate that the peptide-transport systems of E . coli lack specificity for amino-acid side chains. A fairly wide range of dipeptides have been shown mutually to inhibit each other and to interfere with nutritional utilization (Payne, 1968; 1974b;Payne and Gilvarg, 1971).Many others have been found to inhibit uptake of particular labelled dipeptides (Levine and Simmonds, 1962a; Kessel and Lubin, 1963). Studies with dipeptide-transport mutants (see Section IV, E10, p. 90) support these observations, althoughrather few dipeptides have been used. The mutant characterized by Kessel and Lubin (1963) as unable to transport glycylglycine also failed to accumulate several other dipeptides, and a similar result was reported by Felice et al. (1973) for a mutant unable to accumulate glycylvaline. Similar conclusions have been reached for oligopeptide transport. Evidence that competition between oligopeptides can affect their utilization is found in early reports (Merrifield, 1958; Dunn et al., 1957;


83

Young et al., 1964),and supported by recent systematic studies (Payne, 1968, 1972b, 1974b; Payne and Gilvarg, 1971; Smith et al., 1970). Characterization of oligopeptide-permeasemutants (see Section IV, E 10, p. 90) accord with these observations. These mutants, isolated by virtue of their resistance to certain toxic oligopeptides (Payne, 1968, 1972b; Gilvarg and Levin, 1972; Barak and Gilvarg, 1974; Payne and Gilvarg, 1971; Felice et al., 1973), are unable to utilize any (tested) oligopeptide nutritionally. They fail to take up labelled oligopeptides (Felice et al., 1973) and all oligopeptides so far tested. Although all oligopeptides may share a single transport system in E . coli, they may differ in their affinity for it (Payne, 1968). In lactobacilli, certain dipeptides did not compete with one another for transport (Leach and Snell, 1959, 1960). Whether this reflects the presence of several dipeptide-transport systems, or simply widely differing affinities for a single system, is not clear. Similar observations apply to Saccharomyces cerevisiae which shows varied ability to take up oligopeptides of different structures (Becker et al., 1973; Naider et al., 1974).In the mammalian intestine it has been suggested that more than one dipeptide-uptake system may exist, and that the nature of the charge on the side chain may influence uptake (Matthews, 1974).

6 . a-Peptide Bond Most peptides that occur in the microbial environment are a-linked degradation products of proteins. It seems not unlikely, therefore, that peptide-transport systems will have evolved with a requirement for this linkage. The few studies that have been carried out with peptides linked other than through a-bonds support this expectation. Transport cannot be assessed from growth response because peptides containing 8, y or E linkages, for example, are resistant to peptidase activity (which similarly has a requirement for a-linked substrates), and are nutritionally inactive for this reason alone (Payne, 1972b). Thus, 8-aspartylglycine and /3aspartylalanine, y-glutamylglycine, alanyl-E-lysine, y-glutamyl-elysine were all nutritionally inactive for appropriate E. coli auxotrophs, although the corresponding a-linked peptides were utilized. Utilization of these a-linked peptides was not inhibited by addition of the “unusual” peptides to the growth medium suggesting that they lack affinity for the transport system (Payne, 1972b). Rowlands et al. (1957) reported that y-glutamyl peptides were not taken up by Staphylococcus aureus. 8-Alanyl peptides are, as expected, not utilized by E . coli, although an interesting exception is 8-alanylhistidine (carnosine)which is as effective as free histidine in supporting growth of a histidine auxotroph (Payne, 1973). Lactobacilli (Peters et al., 1953) and corynebacteria (Mueller, 1938)also utilize carnosine. It is possible that a specific transport system

84

J. W. PAYNE

for carnosine may have evolved in enteric micro-organisms ; circumstances favouring this possibility are the nutrient value of B-alanine, the frequent occurrence of carnosine in the gut after meat or fish meals, and the low level of intestinal carnosinase activity.

7. Peptide Bond Nitrogen Atom Preliminary studies indicate that peptides in which the peptide-bond nitrogen is substituted (methylated) are transported by E . coli although the substituted bonds are not hydrolysed by intracellular peptidases (Payne, 1972~). These substrates therefore provide good evidence for the separate nature of the uptake and hydrolysis systems (see Section 111, E, p. 71). Lack of hydrolysis prevents utilization of these peptides but they are effective inhibitors of peptide uptake. For example, glycylsarcosine and diglycylsarcosine inhibit utilization of unsubstituted peptides (Fig. 6). The N-terminal residue in diglycylsarcosine is in fact utilized by a glycine-requiring auxotroph of E . coli (Fig. 7) confirming the transport of this class of peptide. Furthermore, lack of growth with the transport mutant (Fig. 7) (see Section IV, E10, p. 90) confirms that they enter by the oligopeptide permease. Peptides of this type are of considerable interest and of some immediate use. Their resistance to hydrolysis should facilitate kinetic studies of peptide uptake that are normally complicated by rapid hydrolysis of accumulated peptides. Their persistence within the cell may also allow

2

4

6

8

Time ( h )

10

12

14

FIQ.6.The inhibitory effect of glycylsarcosine on the utilization of glycylglycine by the Escherichia coli glycine auxotroph, M-123.( o), Medium + 1 mM glycylglycine ; ( 0)+ 1 m' glycylglycine + 1-3 mM glycylsarcosine; (m)+ 1 mM glycylglycine + 8 mM glycylsarcosine; (0) unsupplemented media, or glycylsarcosine alone (0-6-10 mM).Reproduced from Payne (19720).


86

Time( h )

FIG. 7. Utilization of triglycine and diglycylsarcosine by the Escherichia coli glycine auxotrophs, M-123 and M-123 TOR (an oligopeptide transport mutant). ( 0 ) Medium + 0.625 mM triglycine; (m)+ 3 mM diglycylearcosine; ( A ) unsupplemented media; unbroken lines are for strain M-123 and broken lines for strain M-123 TOR. Reproduced from Payne (1972~).

expression of specific biological activities not normally observed (see Sections 111, D3, p. 68, and V, B, p. 99, and C, p. 101). They have already been used in studies of absorption from the mammalian intestine to show that peptides can be accumulated intact (Addison et al., 1974).

8. C-terminal u-Carboxyl Group In an attempt to judge whether the C-terminal carboxyl group was a structural requirement of the E. coli peptide-transport systems, Payne and Gilvarg (1968a)prepared a series of peptides that lacked this group ; these were lysylcadaverine, dilysylcadaverine, trilysylcadaverine, etc. (cadaverine being the diamine resulting from decarboxylation of lysine). The peptides supported growth of a lysine auxotroph (Fig. 8) showing that they can be absorbed and hydrolysed. The nutritional failure of the higher homologues was caused by their large size; see Section IV, E9, p. 87. The failure of the utilizable peptides t o support growth of a transport mutant (Fig. 9) confirmed that the normal lysine peptides, and these decarboxylated peptides, used the same peptide-transport system. This direct study shows that the system that transports oligopeptidesin E . coli has no requirement for the C-terminal carboxyl group. Other observations support this conclusion. Thus, oligopeptides with C-terminal /I-alanyl residues use the E. coli oligopeptide transport system ;/I-alanine

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J. W. PAYNE 100 -

Dilysyl codaverine

,

:,d;l.-.-.-. I

0

2

4

I

8

6

10

12

14

Time ( h )

FIQ.8. Growth response of Escherichia coli lysine auxotroph, M-26-26,to lysine and to lysylcadaverine homopeptides. Growth tubes contained 0.1 pmole per ml of lysine, or lysine residues in the case of the peptides. Reproduced from Payne and Gilvarg (1968a).

4 ,

Dilysyl cadaverine , Trilysyl cadaverine

/@

4%-, A-

p -A ?A-

A

being in effect aspartic acid without its cr-carboxyl group (Payne, 1973). Oligopeptide amides use the transport system (Payne and Gilvarg, 1968a); thus, oligopeptide amides and esters are utilized by lactobacilli

PEPTIDES AND MIURO-ORGANISM3

87

(Woolley et al., 1955;Merrifield and Woolley, 1956),and trivalyl-methyl ester inhibits uptake of unsubstituted peptides by lactobacilli (Shankman et al., 1962).Yeasts also utilize oligopeptide methyl esters (Naider et al., 1974). The lack of specificity for the carboxyl group was not unexpected. The oligopeptide-transport system ha$ a, requirement (binding site) for the terminal amino group (see Section IV, E2, p. 78) and is therefore unlikely to also have a binding site for the terminal carboxyl group, which would be at a variable distance depending on the chain length of the peptide. However, similar reasoning indicates that dipeptide transport may have requirements (separate binding sites) for both the N- and Cterminal residues which are always at a fixed distance from one another. There is evidence to support this conclusion. Many dipeptide amides are utilized poorly by E . coli even though they are readily hydrolysed (Simmondsand Griffith, 1962;Payne and Gilvarg, 1968a). Glycylglycine-methyl ester was a poor inhibitor of [‘4C]glycylglycine uptake by E . coli (Kessel and Lubin, 1983). Furthermore, lysylcadaverine was utilized by a normal lysine auxotroph (Fig. 8) but not by a mutant that takes in dipeptides normally but specifically fails to absorb oligopeptides (Fig. 9). This suggeststhat lysylcadaverine cannot use the dipeptide permease and enters the normal strain by the oligopeptide permease which is non-functional in the mutant. The different requirement for the terminal carboxyl group appears to be one criterion distinguishingthe di- and oligopeptidetransport systems. The non-involvement of the oligopeptide C-terminus in the transport process has been exploited by attaching impermeant molecules to the carboxyl group and taking them into E . coli by the oligopeptidepermease (Ames et al., 1973; Fickel and Gilvarg, 1973). This concept of peptide “smuggling” is discussed elsewhere (Matthews and Payne, 1974a; and Section V, C, p. 101).

9. Peptide Size The general structure of the microbial cell envelope is likely to be a barrier to the passage of large molecules, including peptides. For microorganisms that secrete extracellular proteolytic ezymes of broad specificiOy (see Section 111,C2, p. 65), this may not be a problem for they possess the means to hydrolyse large exogenous peptides to a size that can traverse the cell envelope. For those micro-organisms that possess only intracellular peptides (see Section 111, CS, p. 67), for example E . coli, large molecules (peptides) are unlikely to be taken up, and there is direct evidence to support this. Gilvarg and Katchalski (1965) observed that the higher homologues of several peptide series (e.g. pentalysine, penta-arginine) were not

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utilized by E . coli (see Pig. 4, p. 75). It was established that discrimination against these homologues did not arise from lack of hydrolysis, inhibitory activity or net charge, and it was suggested that they were too large to be tra.nsported. Discrimination did not appear to be caused by chain length (i.e. five residues) for, with other homologous series, penta- and hexapeptides were readily taken up (Gilvarg and Katchalski, 1965; Payne, 1968). Subsequent studies (Payne a.nd Gilvarg, 1968b) indicated that the hydrodynamic volume was the feature governing uptake. Fractionation of the peptides on Sephadex G-15 gave a measure of their relative hydrodynamic volumes, and showed that, within each -0.21

‘ 0

I

2

3

I

I

4 5 Amino-acid residues

I 6

FIG.10. Correlation between log K , and number of amino-acid residues, for severd peptide series. K , values are a measure of the fraction of the total Sephadex column volume accessible to peptide, and inversely related to peptide size. See text for discussion. Reproduced from Payne andsGilvarg (1968b).

homologous series, addition of an amino-acid residue produced a regular increment in overall size (Fig. 10). The slopes of the plots measure these increments, with glycine < alanine < ornithine w lysine c cadaverine. Correlation of peptide utilization with size (log K d )indicated the particular size at which peptides failed to enter E . coli. This size, predicted from the defined synthetic peptides, was exactly confirmed for the peptides present in a commercial enzymic protein digest (Neopeptone) (Fig. 11). The large peptides (fractions 1-14) were not utilized, whereas the smaller ones (fractions above 15) were utilized ; following acid hydrolysis the amino-acid components 1-14 did support growth (Pig. 11).It is of some practical and economic interest that simply because of peptide size a significant proportion of the nitrogen content of Neopeptone is unavailable to organisms such as E . coli. Measurements of the size distribution

89


of peptides in other commercial peptones suggests that this is a common feature (Ziska, 1967, 1968). A plot of the number of amino-acid residues in peptide linkage versus fraction number (for Neopeptone) indicates that steric exclusion occurs between six and seven residues (Fig. 12),this value being an average of all of the different amino-acid side chains. Other studies have confirmed the failure of large peptides to enter E . coli (Smith et al., 1970). At present it is not known which region of the cell envelope exerts the size restriction. It may be the transport system itself or the peripheral h

e I 2 n. -

In % v)

-

5 L

e 2 0

1200 -

-120

?!

FIG. 11. Growth response of Escherichia coli lysine auxotroph (M-26-20) to “Neopoptone” fractions (separated on Sephadex G-15,as indicated in Fig. 10) before and after acid hydrolysis. Reproduced from Payne and Gilvarg (1968b).

cell wall. The failure of the large peptides t o inhibit uptake of small peptides provides tentative evidence that the cell wall may act as a sieve, preventing large peptides from reaching the cytoplasmic membrane. Unrelated studies have also led to the conclusion that the bacterial wall can act as a permeability barrier to large molecules (Freer and Salton, 1972; Gerhardt and Judge, 1964; Scherrer and Gerhardt, 1964, 1968; Wolf-Watz et al., 1973). The structural differences between the walls of different micro-organisms make one anticipate that their sieving effects would be varied ; relevant studies have been performed with yeasts (Naider et al., 1974; Trevithick and Metzenberg, 1966a, b) and fungi (Gershon et al., 1969). micro-organisma Studies on peptide utilization by certain fastidious micro-organisms sometimes indicate that smaller peptides (2-3 residues) are inferior to

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larger peptides ( 2 5 residues) in stimulating growth (Woolley and Merrifield, 1958,1963; Jones and Woolley, 1962; Hearfield and Phillips, 1961 ; Phillips and Gibbs, 1961 ; Pittman and Bryant, 1964; Pittman et al., 1967; Desmazeaud and Hermier, 1973), or protein synthesis (Wright, 1967; Pittman et al., 1967; Fox, 1961; Hauschild, l965a, b). However, the explanation for this probably resides in the multiple nutrient requirements of these strains that are better met by the larger peptides (see Section 11,D, p. 58). It has been shown that these species also fail to utilize peptides above a certain size (Phillips and Gibbs, 1961 ;

I

10

I

I

I

20

30

40

Fraction number

FIU.12. Distribution of peptide size in Neopeptone fract,ionatedon Sephadex G-15. Number of amino-acid residues in peptide linkage was calculated from ratio of aamino content before and after complete acid hydrolysis. Arrow indicates the region in which peptides are sterically excluded from Eacherichia coli W. Reproduced from Payne and Gilvarg (1968b).

Desmazeuad and Hermier, 1973). These particular aspects, and the general effects of size on peptide utilization by micro-organisms, are considered in detail elsewhere (Payne and Matthews, 1974).

10. Dipeptide and Oligopeptide Permeases The extensive studies of peptide transport in E . coli have shown that all peptides must satisfy certain structural requirements in order to be taken up by peptide permeases. However, a number of results indicate quite clearly that separate systems operate for transport of dipeptides and oligopeptides. The characteristics shared by these systems, as well as their distinguishingfeatures are given in Table 2. The first six common characteristics have been considered already in the cited references. Studies of the energetics of peptide transport indicate that it is an active

91

PEPTIDES A?XD MIOBO-ORGANISMS

TABLE 2. Similarities and differences in the transport of di- and oligopeptides by Escherhhia coli Characteristics shared by both transport systems are listed 1-7, and features that distinguish the two systems are listed 8-13. Transport properties 1. Requirement towards terminal a-amino group ;positively 2.

3. 4. 5. 6.

7.

charged primary or secondary amino group specified Stereospecific for L-amino acid residues No requirements with respect to nature of amino acid side chains Requirement for a-peptide linkages Peptide bond nitrogen may be substituted (methylated) Peptide transport and hydrolysis are distinct cellular functions Energy requiring active transport

8. C-terminal carboxyl group required for dipeptide transport only 9. Size restriction on oligopeptide transport only 10. Dipeptides can share oligopeptide permease but not vice

verso; 11. Existence of oligopeptide permease mutants that still transport dipeptides 12. Existence of dipeptide permease mutants 13. Distinct genetic loci for dipeptide and oligopeptide permeases

Reference Section IV, E2 Section IV, E3 Section IV, E6 Section IV, E6 Section IV, E7 Section 111, E Sections IV, A4 and IV, F Section IV, E8 Section IV,E9 Section IV, E l 0 Section IV, E l 0 Section IV, E l 0 El0 Section IT,

transport process and this feature (Table 2, 7) is considered in a later Section (IV, F, p. 97). The distinction with respect to the C-terminal carboxyl group (Table 2, 8) was discussed previously (p. 85), and the carboxyl-group requirement of dipeptides and lack of specificity with oligopeptides was rationalized on steric grounds. The discrimination against large peptides (Table 2, 9) is obviously specific to oligopeptides because the observed “exclusion limit” is greater than that obtainable by combination of any two protein amino acids (see Section IV, E9, p. 87). I n theory, however, a substituted dipepticle could also be excluded if the substituent(s) were sufficiently large. An indication that dipeptides can use the oligopeptide permease (Table 2,lO) was noted when lysylcadaverine failed to support growth of an oligopeptide permease (opp)mutant (see Section IV, E8, p. 85, and Fig. 9). Loss of the carboxyl group prevented the dipeptide using the dipeptide transport system, and studies with the opp mutant indicated

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this peptide was taken up through the oligopeptide system. Other C-substituted dipeptides are also likely to use the oligopeptide system (Payne and Gilvarg, 1968a, 1971). Direct evidence is available that dipeptides (at high molar ratios) can inhibit the uptake of oligopeptides to a limited extent (Payne, 1968, 1972b; Payne and Gilvarg, 1971). However, normally one fails to observe the reciprocal effect in which oligopeptides inhibit dipeptide uptake. This result is consistent with two features of transport. First, the failure of oligopeptides t o use the dipeptide system ; oligopeptides may be regarded as C-terminal substituted dipeptides, and therefore lack the structural requirements of the dipeptide permease. Second, the fact that the uptake of dipeptides via the oligopeptide permease is proportionally very low compared with uptake by the dipeptide system, and inhibition of this fraction is therefore qualitatively unimportant. However, it would be expected that oligopeptide inhibition of dipeptide uptake might more readily be seen with certain dipeptides (e.g. those with modified C-termini) that apparently can use oiily the oligopeptide system. Unambiguous evidence for the two systems is provided by the existence of mutants that fail to transport oligopeptides but retain normal ability t o take up dipeptides. Payne (1968) first reported the isolation of this class of mutant; they were selected by their resistance to the toxic oligopeptide, triornithine (see Section V, B, p. 99) (Gilvarg and Levin, 1972; Barak and Gilvarg, 1974; Barak et al., 1970, 1973a, b). Only the tripeptide was toxic, and free ornithine, and the di-, tetra- and pentapeptides were not inhibitory. Several other tripeptides of related stucture were also toxic and their degree of inhibition was directly related to their resistance to intracellular hydrolysis (Payne, 1968;Sussman and Gilvarg, 1970, 1971). This finding suggested that the sensitive target exists inside the organism and that the peptides must be taken up to exert their bactericidal action. Subsequent studies have confirmed this conclusion (see Section V, C, p. 101).Although several types of triornithine resistant (TOR)mutants are possible, only the transport-deficient type has been characterized. Extensive studies show that these permease mutants are no longer able to utilize a wide range of defined oligopeptides (Figs. 7 and 8) (Payne, 1968, 1972b, 1974b; Payne and Gilvarg, 1971; Barak and Gilvarg, 1974; Felice et al., 1973; Ames et al., 1973) or to take up labelled oligopeptides (Felice et al., 1973; Payne, 1972b).I n contrast they possess a normal ability to utilize dipeptides (Fig. 8, p. 86) (Ames et al., 1973; Barak and Gilvarg, 1974; Felice et al., 1973, Payne, l968,1972b, 1974b; Payne and Gilvarg, 1971). The same mutant types have been isolated from several strains of E . coli and from Salmonella typhimurium (Ames et al., 1973), sometimes using as selection procedure their resistance to different toxic oligopeptides (See SectionAV,C, p. 101).


93

It is difficult to isolate dipeptide permease (dpp)mutants (Table 1, l a ) , based on resistance to toxic dipeptides, because they also get taken up by the oligopeptide permease. Felice et al. (1973) overcame this difficulty by starting with an oligopeptide transport-deficient (opp) mutant and selecting from it a dpp substrain resistant to toxic dipeptides. With those dipeptides for which extensive uptake is required for growth (Kessel and Lubin, 1963; and Section 111,E, p. 71), or for inhibition (Vonder Haar and Umbarger, 1972; Wasmuth and Umbarger, 1974), uptake through the oligopeptide permease is probably inadequate, and it has therefore been possible to directly isolate dpp mutants. Genetic mapping of the peptide transport systems (Table 2, 13) shows the dpp locus to occur between the pro C marker (at 10 min) and the distinctopplocus (at 27 min) (Feliceetal., 1973; BarakandGilvarg, 1974). These results clearly indicate that dipeptide and oligopeptidetransport systems possess unique components. However, similar reservations apply as to the distinction between amino-acid and peptide transport (see Section IV, D, p. 76) that although distinguishable, the two systems may nevertheless share certain components. At present no single-step mutants have been reported that fail to transport dipeptides and oligopeptides. It is relatively easy to select for such mutants (e.g. by simultaneous resistance t o toxic dipeptides and oligopeptides with different modes of inhibition, or simultaneous resistance to a toxic dipeptide and failure to utilize an oligopeptide). Such mutants could be of particular interest, and some might be deficient in the elusive energy coupling process. 11. Summary The structural characteristics of peptide transport in E . coli are summarized diagrammatically in Fig. 13. The figure is used to illustrate a dipeptide and the N-terminal part of an oligopeptide;numbered features are as follows: (1) the a-amino group may be monosubstituted but a positive charge must be retained; thus, monoalkyl, acyl or dialkyl

FIG.13. Summary of the main structural requiroments for tho transport of poptides into the Escherichia coEi cell. See text for discussion of the numbered features.

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J. W. PAYNE

substituents are not tolerated ; (2) there is a stereochemical preference for L-amino acid residues ; (3) in oligopeptidesthis residue (and probably subsequent ones) may be a D-isomer; (4)it is essential that linkage is by an a-peptide bond; ( 5 ) the peptide nitrogen bond may be substituted (methylated); (6) presence of the a-hydrogen atom is not essential; (7) a free C-terminal a-oarboxyl group is essential for dipeptide transport ; (8) the C-terminal a-carboxyl group is unimportant with oligopeptides, in which it may be substituted or absent; (9) there is no requirement ooncerning side chains, and these may be natural residues, substituted ones or synthetic analogues; and (10)the size of oligopeptidemay become a feature that limits uptake.

3’. ENERGETICS OF PEPTIDE TRANSPORT The various transport modes by which small molecules enter microorganisms were discussed earlier (p. 72). It is apparent from the oharacteristics of peptide transport so far presented that passive diffusion plays a negligible role in peptide upta8ke.The observed dependence of uptake on temperature also argues against passive diffusion (Leach and Snell, 1960; Pittman et al., 1967). However, with certain small peptides at high concentrations it is theoretically possible that significant uptake by passive diffusion alone could occur, and some observations on the uptake of diglycine by transport mutants are consistent with this possibility (Kessel and Lubin, 1963; Levine and Simmonds, 1960). However,the actual transport defects in these mutants were not identified so it is possible that uptake occurred by facilitated diffusion (or even by active transport with a high K,) and it would need competition studies a t high concentrations of peptide to resolve this point. It is theoretically possible that a net accumulation of peptides by facilitated diffusion could be driven by the continuous intracellular hydrolysis of absorbed peptides. Under physiological conditions where energy coupling does not occur, or when it has been eliminated by experimental or genetic manipulation, peptide uptake by facilitated diffusion could still occur. This may be the explanation for the uptake of labelled dipeptides observed with energy starved E . coli (Simmonds, 1966). Indirect evidence for active transport is provided by the observation that peptide accumulation has a requirement for energy. This has been observed, for dipeptides, with many species of bacteria (Leach and Snell, 1959, 1960; Kessel and Lubin, 1963; Meisler and Simmonds, 1963; Yoder et al., 1965s; Mayshak et al., 1966; Simmonds, 1966) and also for oligopeptides (Young et al., 1964; Shankman et al., 1962; Pittman et al.,

PEPTIDES AND IIIIURO-ORGANISMS

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1967; Smith et al., 1970; Payne, 1972b; Felice et al., 1973). Further indirect evidence is the observation that inhibitors of known energycoupling processes inhibit peptide uptake (Leach and Snell, 1960; Shankman et al., 1962; Young, et al., 1964;Mayshak et al., 1966; Pittman et aE., 1967; Payne, 1972b; Felice et al., 1973). Direct evidence for active transport requires demonstration of the accumulation against a concentration gradient of intact, unmodified peptide. The intense activity of intracellular peptidases generally makes it difficultto satisfy this criterion. Nevertheless, it can be alleviated in several ways as, for example by: (i) manipulating growth conditions to minimize peptidase activity; (ii)the use of peptidase mutants; or (iii)use of peptides resistant to peptidase action. Applicable to (i)is the fact that, in E . coli, peptidase activity varies with the phase of growth and culture conditions (Simmonds, 1970, 1972; Payne, 1972a, 1972d). Under conditions of low activity, the accumulation of intact dipeptide has been observed (Levine and Simmonds, 1962a, Simmonds, 1966); in this instance the extent of uptake was similar to that occurring under conditions of extensive peptide cleavage. I n an E . coli mutant lacking glycylglycine peptidase, accumulation of intact glycylglycine was observed (Kessel and Lubin, 1963). I n the third category, examples of peptides that are accumulated but are resistant to hydrolysis are triornithine, and its analogues, and peptides with N-substituted peptide bonds. It is clear, therefore, that the predominant mode of peptide uptake in bacteria is active transport. In common with other active-transport processes the nature of the energy coupling process is not known. The current status of this topic with respect to amino-acid uptake is considered elsewhere (Payne, 1974b). The relative ease with which energydeficient peptide-transport mutants might be isolated (see Section IV, E10, p. 90) makes this system a good tool with which to study the problem.

G . “BINDINUPROTEINS”

AND

“MEMBRANE-VESICLES”.

Investigations into the molecular mechanisms of amino-acid uptake by bacteria have proceeded further than comparable studies of peptide uptake. Amino-acid uptake systems may be categorized empirically into those that do, and those that do not possess binding proteins. Studies reviewed by Heppel (197 1) indicated that when certain bacterial species (e.g. E . coli) were converted to spheroplasts, or were subjected to a specific type of cold osmotic shock, they selectively released a group of proteins (comprising 3-5% of the total cellular protein) that were mostly degradative enzymes and were located at or near the cell surface (periplasm) (see Section 111, C3, p. 66). The location of these

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proteins prompted investigations of their possible role in transport processes. Roseman and his colleagues (Kundig et al., 1966) first reported that following osmotic shock the ability of E. coli to absorb sugars by a specific phosphotransferase system was markedly decreased. However, this arose mainly from release into the shock-fluid of a soluble cytoplasmic protein (H-Pr) of the phosphotransferase system, rather than loss of a periplasmic “transport-protein” (reviewcd by Roseman, 1969, 1972). Pardee (1966) was the f i s t to describe a true periplasmic “transportprotein” in shock fluids; it was involved in sulphate transport in Salmonella typhimurium (Pardee, 1966, 1968). Similar treatments also release proteins involved in the transport of phosphate (Medveczky and Roscnberg, 1969)) galactose (Anraku, 1967, 1968a, b, c) and various amino acids (Piperno and Oxender, 1966; Penrose et al., 1968; Wilson and Holden, 1969a, b ; Anraku, 1968a, b, c, 1971 ; Furlong and Weiner, 1970).

Many of these proteins (excluding that of the phosphotransferase system) were isolated and characterized by their affinity for particular transport substrates ; hence the name “binding-proteins”. Much of the evidence for their involvement in transport is indirect (Pardee, 1968; Kaback, 1970; Oxender, 1972)) and only recently has direct genetic and biochemical evidence been provided (Boos, 1972; Ames and Lever, 1970, 1972). The question arises as to the role of these “binding-proteins” in the transport process. Little firm evidence is available but it has been supposed that they may not merely be involved in the recognition process (see Section IV, A, p. 72, and Fig. 3), but may form part of a solute-carrier complex that carries substrate across the membrane by virtue of a conformational change consequent upon substrate binding (Penrose et al., 1970; Weiner and Heppel, 1971; Boos et al., 1972; Vorisek, 1973). Finally, do “binding-proteins” form part of peptidetransport systems? There is presently no evidence that “peptide bindingproteins” exist. However, difficulties in obtaining appropriate radioactively-labelledpeptides have limited those rather obvious experiments. It is also possible that appropriate studies have been performed but negative results have not been published. B. P. Rosen (personal communication) observed that uptake of [‘4C]-glycylglycinewas decreased following osmotic shock treatment of E. coli, but he failed to detect “binding-proteins” for the dipeptide in the shock-fluid. Understanding the molecular mechanisms of peptide transport requires that the possible involvement of “binding-proteins)’ be clarified. Identification of the products of the d p p and opp genes (p. 93) may be relevant. If “peptidebinding-proteins” do occur, it will be of some interest to compare their binding specificity with the established structural requirements of peptide uptake.

PEPTIDES AND MICRO-ORQANISMS

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An alternative uptake process for amino acids has been revealed by studies with membrane-vesicles. These materials, obtained from many different bacterial species, are devoid of peripheral cell-wall components and of cytoplasmic constituents, and yet retain many of the transport capabilities of the intact organisms (Kaback, 1970. 1972). They lack “binding-proteins” and yet catalyse the uptake of many amino acids. It is clear that these membrane-bound transport systems are distinct from those involving binding-proteins, although this could reflect merely a difference in the relative affinities of different binding-proteins ; other evidence suggests that the process of energy coupling is also distinct in the “membrane-vesicle systems” and “periplasmic binding-protein systems’’ (Berger, 1973).No reports have been published on the ability of membrane-vesicles to take up peptides although this represents an obvious area for study. Payne (1974b) discussed the function of membrane-vesicle systems in the transport of amino acids and peptides.

H. REGULATION OF PEPTIDE TRANSPORT I n contrast to the situation with microbial amino-acid transport, virtually nothing is known concerning the regulation of peptide uptake. However, as both processes supply the intracellular amino-acid pool, it is likely that similar regulatory mechanisms may exist for each, and Payne (1974b) has recently discussed this feature. Kepes (1964) pointed out the energetic rationale for having inducible sugar permeases and constitutive amino-acid permeases. I n general, it appears to be true that specific amino-acid transport systems are permanently present (constitutive) a t maximum concentration so as to absorb rapidly amino acids which, in turn, can regulate the activity of their own (energetically more expensive) biosynthetic enzymes (Oxender, 1972). Nevertheless, regulation of the concentration of amino-acid permeases by changes in their de novo synthesis and turnover rate has been reported. Systems controlled in this way are often of broad specificity, able to handle a variety of amino acids that may be used more to satisfy the general carbon and nitrogen requirements of the cell than to provide substrates for protein synthesis. The inducers of these transport systems are not always free amino acids (Ring, 1960), but may be their degradation products, illustrating the co-ordination between uptake and metabolism (Rosenfeld and Feigelson, 1969; Kay and Gronlund, 1969a, b ; Miller and Rodwell, 1971;Fan et al., 1972).Nitrogen starvation ca.n cause induction of general amino-acid permeases in bacteria and yeast (Pall, 1971;Kuznaretal., 1973).It has been suggested (Ames, 1972)that cyclicAMP may also be involved in the regulation of these “general” amino-acid

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transport systems, and that they may be susceptible to catabolite repression, like certain intracellular pathways. I n addition to the genetic regulation of permeases, evidence exists for the regulation of their activity that may be interpreted as a form of feedback control (Ring, 1970; Grenson, 1973; Grenson et al., 1968; Crabeel and Grenson, 1970; Pall, 1971; Pall and Kelly, 1971. It remains to be seen whether the mechanisms regulating peptide uptake are similar t o those reported for amino-acid transport. Whatever the situation proves to be, one can speculate that the uptake of peptides will influence the uptake of amino acids, and vice wema. Considering the first case, accumulated peptides might indirectly inhibit or stimulate amino-acidtransport in several ways. Feedback inhibition of amino-acid uptake might be exercised by peptide cleavage products (either free amino acids or their metabolites) present in the cytoplasmic pool. An effective decrease in free amino-acid uptake might result from an accelerated efflux of particular amino acids (accumulated in free form) brought about by an intracellular accumulation of amino acids absorbed in peptide form. If pool amino acids can induce the synthesis of amino-acid permeases, then amino acids taken up in peptide form might in turn lead to enhanced amino-acid uptake (Yoder et al., 1965a; Levine and Simmonds, 1962b). Amino acids accumulated as peptides might also stimulate free amino-acid uptake by a process of “counterflow” (Brock and Woolley, 1964). Similar speculations could be made of the possible ways in which amino acids might affect peptide uptake. In view of the demonstrated co-ordination between amino-acid metabolism and transport, it seems pertinent when studying regulation of peptide transport to consider the possibility that it also may be linked t o this co-ordinate process. The rapid intracellular hydrolysis of peptides would appear to preclude a peptide-feedback mechanism, that in any case would lack specificity and might lead to exclusion of required aminoacid residues. Selective amino-acid efflux to remove particular peptidecleavage products that are in excess of cellular requirements seems more feasible, and there is some evidence for this (Levine and Simmonds, 1962a, b ; Shelton and Nutter, 1964).Feedback inhibition of amino-acid uptake would be of no value here (with peptides but not amino acids in the medium), but the regulation of the two processes could be controlled by the relative internal and external concentrations of free amino acid. These possibilities are considered elsewhere (Payne, 1974b). It appears to be essential to maintain a “balanced” amino-acid pool, for it has repeatedly been shown that an unbalanced supply of amino acids disrupts microbial growth. Umbarger (1989)suggeststhis occurs because feedback-inhibition of amino-acid biosynthesis leads to unbalanced synthesis of amino-acyl-tRNA and thus inhibition of protein synthesis.


99

V. Miscellaneous Relationships Between Peptides and Micro-organisms A. TOXINBIOSYNTHESIS Many microbial species produce protein toxins and it is a common observation that peptides stimulate toxin biosynthesis (Kadis et al., 1971). Many commercial peptones were devised specifically for use in toxin production. The mechanisms by which peptides stimulate toxin biosynthesis are generally not understood, although it seems clear that the peptide structures per se are not essential and that they function merely as preferred sources of amino acids (Mueller and Miller, 1956; Miller et al., 1960).In many instances, toxin stimulation is rather specific and occurs in the absence of any demonstrable effect upon growth; this situation is to be distinguished therefore from the nutritional utilization of peptides. The peptides are utilized by the toxin-producing strain but they are channelled into the synthesis of toxins rather than for general cell growth; these circumstances probably occur when overall growth is limited by a specific nutrient depletion. On the other hand, synthesis of tetanus toxin by Clostridium tetani is dependent on a source of histidine peptides, and their omission from the growth medium prevents toxin production although, in this case, growth may occur normally. Many peptides of histidine stimulate toxin biosynthesis. In contrast, free histidine has no stimulatory effect, although it is utilized for growth (Mueller and Miller, 1956). Miller et al. (1960) reported that under toxinogenic conditions the cells produced a peptidase that hydrolysed histidine peptides, and that the most stimulatory peptides were also the most easily hydrolysed. It appears, therefore, that the stimulatory peptides simply act as sources of histidine. Possible reasons for the inaotivity of free histidine were discussed previously (p. 60) in connection with strepogenin peptides. Dalen (1973a, b, c) has suggested that histidine may act as an inducer of staphylococcal a-toxin. Toxin production by Clostridium perfringens is also stimulated by peptides but not by their constituent free amino acids (Adams et al., 1947; Boyd et al., 1948; Murata et al., 1958; Jayko and Lichstein, 1959). Several reports indicate that toxin stimulation is only produced by peptides within a particular size range (Hauschild, 1965a, b; Nekvasilova et al., 1970).

B. TOXICPEPTIDES This review has emphasised the nutritional qualities of peptides but it should be noted that certain a-linked peptides actually inhibit microbial growth. Oligopeptides with large net positive charges bind to the surface of

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certain bacteria causing aggregation and inhibition of growth. Competition between peptides for uptake may limit the transport of perticular peptides that carry amino-acid residues essential for the growth of fastidious or auxotrophic strains ; under these circumstances the competitive peptides would apparently be inhibitory. More specific instances occur in which normal a-linked peptides inhibit microbial growth. Inhibition may occur when a peptide is transported but not hydrolysed (see Section 111, D3, p. 66). Simmonds et al. (1947) noted that glycyl leucine inhibited growth of E . coli; other peptides of glycine and leucine were not inhibitory nor were the free amino acids. The dipeptide inhibited initiation of growth (Simmondsand Pruton, 1949; Simmonds et al., 1951) and organisms with high levels of dipeptidase activity were less inhibited (Simmonds and Pruton, 1949; Meisler and Simmonds, 1963). Umbarger and his co-workers (Vonder Haar and Umbarger, 1972; Wasmuth and Umbarger, 1974) recently showed that glycyl leucine inhibition was reversed by isoleucine and by those precursors of isoleucine beyond threonine in the biosynthetic pathway. Glycyl leucine inhibits threonine deaminase in vitro, probably by binding to the isoleucine site on this regulatory enzyme; in vivo this could lead to an isoleucine limitation sufficient to inhibit growth. A particular mutant resistant to glycyl leucine had an altered threonine deaminase which was resistant to isoleucine feedback and to glycyl leucine inhibition. It is likely that many other peptides might reveal biological (inhibitory) properties if they were not prevented from reaching active concentrations by peptidase action. Inhibitory oligopeptides have been of great use in the isolation of peptide permease mutants. Tri-ornithine is readily taken up, but is resistant to hydrolysis and so can reach an inhibitory intracellular concentration (Payne, 1968). Its mode of inhibition is not clear although it has been established that it affects ribosomal function (in vivo but not in vitro) and specifically prevents protein synthesis (Barak et al., 1970, 1973a, b ; Gilvarg and Levin, 1972). Other oligopeptides can be made to show inhibitory properties by manipulations that decrease peptidase activity and allow intracellular accumulation of intact peptide (Sussman and Gilvarg, 1970, 1971 ; Shankman et al., 1962). Based on these observations, it becomes attractive to design particular peptide structures that are compatible with the general requirements for peptide transport (see Section IV, E, p. 77) but resistant to peptidase action. Peptides with N-methylated peptide bonds represent one such class. Many other cases may be cited in which peptides are inhibitory because they are highly efficient a t bringing toxic substances into the cell. Escherichia coli K12 is specificallysensitive to valine, and particularly to valine peptides (Payne and Gilvarg, 1971 ; Felice et al., 1973). Peptides


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containing amino-acid analogues are often more inhibitory than the free analogues (Dunn, 1959; Smith and Dunn, 1970; Payne, 1972h).

C. THECONCEPTOF “SMUWLINS” The lack of any structural requirements for peptide transport with respect to amino-acid side chains and (for oligopeptides) the C-terminal carboxyl group raises the possibility that other molecules might be attached to these residues and be ferried into bacteria through peptide permeases. If the substituent were itself impermeable, this mechanism offers a means to deceive the bacterial permeability barrier and to smuggle novel substances into the cell. The name “smugglins” has been proposed for peptide complexes of this type (Matthews and Payne, 1974a). Two reports have appeared illustrating the “smugglin” principle. Homoserine phosphate fails to cross the cytoplasmic membrane of E . coli but dilysyl-homoserine phosphate is taken up readily through the oligopeptide permease and is used nutritionally (Fickel and Gilvarg, 1973). Histidinol phosphate is nutritionally inactive for Salmonellatyphimurium because it fails to enter the organism. However, diglycyl-histidinolphosphate is taken up and is degraded to provide histidine to support, growth of a histidine auxotroph (Ames et al., 1973).In each case, it was confirmed that the peptide complex was a “smugglin” (and used the oligopeptide permease) by showing that it was not accumulated by an oligopeptide permease mutant. Similar in principle, but lacking the elegance of these studies, are reports that peptides with modified sidechains are taken up by E . coli although the substituted free amino acids are impermeable (Gilvarg and Katchalski, 1965; Losick and Gilvarg, 1966; Payne, 197213). The concept of “smugglins” is of considerable biological importance. Antibiotic possibilities are obvious, as is the general possibility of bringing into an organism metabolic regulators and effectors. The principle offers an alternative approach to that by which bacteria are made permeable by treatment with EDTA, or toluene, or cold osmotic shock. When designing “smugglins”, the possibility of steric hindrance to uptake should be considered (see Section IV, E9, p. 87). The concept may also have potential application with mammalian cells (Matthews and Payne, 1974b).

D. PEPTIDE ASTIBIOTICS Perlman and Bodanszky (1971) stated that: “ . . . nearly 300 peptide antibiotics have been described in the literature of the past 25 years”. Although clinically important, these represent less than 10% of all reported antibiotics (Berdy and Magyar, 1968). This author shares the

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view of Bodanszky and Perlman (1909) that; “Peptide antibiotics may represent only a few of many yet undiscovered microbial peptides”. These authors emphasize that antibiotic activity is an “artificial”, and probably minor, characteristic of microbial peptides in general, and “antibiotics” could be isolated from any collection of chemicals by applying similar criteria for selection. The structural and chemical characteristics of peptide antibiotics have been detailed by many authors (Schroder and Lubke, 1906; Gottlieb and Shaw, 1907; Perlman and Bodanszky, 1971; Katz, 1971). It is only necessary here to emphasize that, in general, they are different from the usual a-linked peptides considered in this review. They frequently contain D-amino acids, “unusual” residues (e.g. N-methylated amino acids, hydroxy acids, amino sugars), and consequently nonpeptide linkages, and they am frequently cyclic. It is clear that they lack the structural requirements for peptide transport and, analogously, they are generally resistant t o peptidaae action. They are synthesized not by a ribosomal mechanism but by multi-enzyme complexes (Katz, 1971 ; Perlman and Bodanszky, 1971 ; Lippmann et al., 1971). Much has been written on their mechanisms of action (Gottlieb and Shaw, 1967; Gale et al., 1972). The vast biological potential inherent in peptides is well illustrated by the diverse ways in which peptide antibiotics inhibit micro-organisms (e.g. by damage to pre-existing enzymes or structural components, by preventing new enzyme synthesis, or by disruption of the membrane-cell wall structure). Many potential “tmgets” exist although only a single site or chemoreoeptor is generally susceptible. The main concern here is with the possible roles of these microbial peptides. Speculation ranges from their being merely waste products, to performing essential functions (e.g. increasing membrane permeability during sporulation). Indirect evidence for a particular function comes from the fact that they are not produced uniformly throughout the growth cycle, synthesis being increased at particular stages of growth or under particular growth-limiting conditions. It is not clear to what extent they are produced in vivo (in the soil, for example). Many of these peptide secondary metabolites are devoid of antibiotic properties, and it seems unlikely that they are produced primarily to kill alien microorganisms, although this has been suggested by Brock (1966). Bu’Lock (1961) has provided a more general explanation for the widespread occurrence of these secondary metabolites. He envisaged secondary metabolism as a means to keep essential cell machinery ticking over when a particular growth limitation prevented cell multiplication. If the secondary metabolites could in addition have particular properties (e.g. antibiotic character) rather than being merely waste products, so much the better, This point of view was supported by Woodruff (1966), who

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emphasized that antibiotic activity is only one of a number of biological properties shown by secondary metabolites. He suggested that under particular growth-limiting conditions, synthesis of these peptides was triggered by accumulation of “initiation substances”. Dhar and Khan (1971) considered that these “initiators” might be toxic metabolites that could be detoxified by conjugation to form peptide antibiotics.

E. PEPTIDE IONOPHORES The name “ionophore” was proposed by Pressman et al. (1967) for compounds that can transfer ions across lipid barriers. Many microbial peptides are ionophores. They are frequently antibiotics, a property that can be correlated with their ability greatly to increase the permeability of bacterial membranes to certain ions (e.g. K+, Na+; see Harold, 1970; Bhattacharyya et al., 1971). These cation-specific peptides may be divided on the basis of their structures and properties into two groups, of which valinomycin and nigericin are the best known representatives. Valinomycin has a cyclic structure, and adopts a folded symmetric conformation in which a potassium ion is sequestered at the centre (Pinkerton et al., 1969; Ohnishi and Urry,1970; Haynes et al., 1969; Ivanov et al., 1969; Harold, 1970). It is believed that the various ionophores may function in different ways, acting either as simple carriers that confer lipid solubility on the ions transported (Tosteson, 1968; Pressman et al., 1967; Pinkerton et al., 1969; Ivanov et al., 1969; Shemyakin et al., 1969; Haynes, 1972; Wipf et al., 1969) or by stacking together to form a transmembrane channel with a hydrophilic core (Ovchinnikov et al., 1970; Hladky and Haydon, 1970; Urry, 1971; Krasne et al., 1971). Although these ionophores are of considerable interest as model compounds that mediate ion-specific translocation, there is no evidence that they perform this role in viuo. However, little research has been carried out to establish their physiological function, if any (see Section V, D, p. 101). The facilitated uptake of cations has been reported with several bacterial species (Nelson and Kennedy, 1971 ;Silver and Clark 1971) but ionophores have not been implicated. Iron is accumulated by several species in the form of hydroxamate chelates for which specific transport systems exist (Neilands, 1967; Snow, 1970; Arceneaux et al., 1973; Haydon et al., 1973). In view of this dichotomy between the remarkable oapabilities of microbial ionopheres in vitro and their lack of obvious function in vivo it is of some interest that functional ionophores have apparently been isolated from several mammalian sources (Blondinet al., 1971 ; Binet et al., 1971 ; Hyman, 1970, 1971 ; Goodall and Sachs, 1972; Shamoo and Albers, 1973).

104 J. W. PAPNE Microbial peptide ionophores that can impart electrical “excitability” t o biomembranes are of particular interest, for they currently provide the best operative model of the excitable membranes (e.g. nerve, muscle) in terms of ion translocation and general electrical properties (Mueller and Rudin, 1968, 1969). Alamethicin, a polypeptide produced by Trichoderma viride and of known primary structure, is the best characterized material (Meyer and Reusser, 1967; Payne et al., 1970).Evidence exists that the voltage-dependent conductance induced by alamethicin in lipid membranes requires aggregation of the peptide to form pores (Gordon and Haydon, 1972; Eisenberg et al., 1973). It is to be expected that many more studies will be carried out using these various classes of microbial peptides. 3’. CONJUGATEDPEPTIDES

A wide variety of conjugated peptides (lipo-, glyco-, phospho-, and nucleopeptides)has been isolated from microbial sources. Some are structural components, others may play a role in metabolic regulation, but the functions of most are unknown. The structures and properties of these substances have been considered elsewhere (Matthews and Payne, 1974a).

VI. Conclusions In this review I have attempted to give a description of peptide utilization by bacteria and of the roles played by peptidases and transport systems (Sections11-IV, pp. 56-98). I n the final section (p. 99) I have extended the scope with a view to high-lighting the biological potential of peptides. Simple peptides offer a vast reservoir of biologically active compounds, and techniques already exist for the synthesis of any desired sequence. It is hoped that studies on the structural requirements of microbial peptide permeases and peptidases, and of the effects of peptides on microbial growth, may be useful not only in their immediate context, but that they may also act as a model system to provide information relevant to peptide interactions in more complex biological systems. Studies with mammals already provide some support for this hope (Matthews and Payne, 1974b). REFERENOES Adarns, J. M.(1968).Journal of Molecular Biology 33, 571. Adams, M.H., Hendee, E. D. and Pappenheher, A. M. (1947).Journal of Experimental Medicine 85,701. Addison, J. M., Burston, D., Matthews, D. M., Payne, J. W. and Wilkinson, S. (1974).Clinical Science and Molecular Medicine 46, 30 P.


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The Physiology of the Mycobacteria COLINRATLEDGE Department of Biochemistry, University of Hull, Kingston upon Hull, HU6 TRX, North Humberside, England

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I. Introduction 11. Structure A. Cell Wall : Chemical Composition . B. Cell Wall: Electron Microscopy C. Cell Wall : General Conclusions D. Membrane Structures E. Nuclear Material and Ribosomes F. Polyphosphate Granules . G. Lipidvacuoles III. Lipids : their Structure and Biosynthesis , A. Influence of the Environment on Lipid Formation B. Straight-Chain Saturated Fatty Acids C. Straight-Chain Unsaturated Fatty Acids D. Branched-Chain Acids E. Mycolic Acids F. Effect of Isoniazid on Mycolic Acid Biosynthesis G. Neutral Lipids and Phospholipids H. Glycolipids J. Carotenoids IV. Growth in vivo :Interactions between Invading Mycobacteria and Host Tissues. A. krophage-bacteria Interactions at the Onset of Infection . B. Growth of Mycobacteria in vivo . C. Toxicity of Invading Mycobacteria D. Cellular Immunity and Delayed Hypersensitivity . , E. Other Cell Responses to Mycobacteria and Isolated Fractions F. General Conclusions . . V. Growth in v i t ~ o A. General Observations B. Nutrition, Nutrients and their Assimilation . , C. Growth Factors and the “Non-Cultivatable” Mycobacteria . VI. Aspects of Metabolism A. Central Pathways of Metabolism B. L-Lactate Oxidase C. Energy Metabolism D. Nucleic Acids and Protein Biosynthesis References. Acknowledgements

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116

116 117 118 122 126 127 130 131 133 134 134 136 143 145 147 161 162 164 167 161 161 163 167 170 178 181 183 183 184 197 203 204 206 207

212 216 244

116

C. EATLEDOE

I. Introduction It is now 100 yeam since the first mycobacterium was isolated by Hansen (1874). Somewhat ironically, this was the leprosy bacilli, Mycobacterium leprae, which even today is still resisting all attempts to cultivate it in the laboratory. The tubercle bacillus, M . tuberculosis, was not discovered until eight years later (Koch, 1882)and this has remained object of intensive investigation ever since. The widespread interest in the mycobacteria of course stems from the diseases they cause and, lest it be imagined that tuberculosis is a disease which has now been largely conquered and that leprosy is of relatively rare occurrence, current estimates for the number of cases of tuberculosis and leprosy in the world today are 20,000,000 and 11,000,000,respectively (Bechelli and Dominguez, 1972). The annual estimated mortality rate is equally dramatic, namely 3,000,000 (World Health Organization, 1974). Although respiratory tuberculosis is clearly on the decline in western countries, of increasing occurrence are non-respiratory forms of tuberculosis (e.g. tuberculosis of the bone and joints) and diseases caused by the “atypical” mycobacteria such as M . kansasii which simulates a,mild form of pulmonary tuberculosis. Another mycobacterial infection to be recognized recently is buruli disease which is prevalent throughout eastern Africa and the causative organism has been named ill.ulcerans from the massive ulcerations of the skin which it can cause. Treatment of the diseases caused by these bacteria, like the treatment of all the mycobacterioses, is not easy. Also causing unease is the continuing isolation from tubercular patients of strains already resistant to one or more chemotherapeutic agent. Prognosis for such people can never be too good. The battle against the mycobacteria has a long way to go before it is finally won. This review, however, is not concerned with the diseases caused by the mycobacteria and the way in which these diseases are manifested to a clinician but with the underlying physiology of these bacteria. The term “physiology” has, of course, many interpretations. As my own particular view, I have taken physiology to cover not only the mycobacterial cell, its structure and organization, but also the properties which the organisms must have, first to enable them to survive once having gained entry to a host, and secondly to bring about the various changes which are subsequently manifested in the host. In other words, this review is an attempt to look at mycobacteria from their own point of view rather than from that of the clinician who is at the other end of the microscope. I have deliberately refrained, therefore, from giving any separate treatment of the taxonomy of this group of organisms as taxonomy is an artefact contrived out of a highly selective way of looking at

THE PHYSIOLOC?Y OIP THE MYCOBACTERIA

117

living things. The classificationof mycobacteria is extremely important, from the clinician’s point of view, but unfortunately it tells us very little about the organisms themselves. However, like all “mycobacteriologists” I am indebted to the taxonomists for their continuing efforts to create some order out of the chaos which has hitherto existed with this group of bacteria. I have, therefore, attempted to follow their current recommendations regarding nomenclature (for example, M . bovis is used rather than M . tuberculosis var bovis, and M . marinum for M . balnei) and also what constitutes the limits of the genus. Accordingly, I have excluded from consideration the group of organisms currently known as “ M ycobacterium” rhodochrous. For information regarding matters taxonomic, the reader is referred to the reviews of Cross and Goodfellow (1973) and Stanford and Grange (1974) and to the definitive work of the International Working Group on Mycobacterial Taxonomy whose members have, to date, published four papers with further papers stillinpreparation (Wayneetal., 1971; Kubicaetal., 1972; Meissneretal., 1974; Goodfellow et al., 1974). Also omitted from the review, for more or less the same reason as taxonomy is left out, me epidemiological aspects and a systematic appraisal of the mode of action of the various antimycobacterial agents. As regards the latter I have, in fact, gone some way to discussing these agents whenever a study of them has proved to be helpful in a broader understanding of the physiology of the mycobacteria. Thus streptomycin, rifampin, isoniazid, ethambutol and paminosalicylic acid all get, at least, some mention.

II. Structure The structure of the mycobacterial cell has been the subject of a great deal of investigation. The prime emphasis has been upon the nature of the cell wall which not only is one of the thickest to be found in microorganisms but has a pronounced hydrophobicity due to an abundance of highly complex lipophilic macromolecules within it. Many of these molecules exert profound biological effects when injected into animals (Lederer, 1973) and this has acted as a considerable spur to further study seeking to elucidate the chemical structure of the causative agents. These aspects are described in Section I V (p. 167). The remainder of the mycobacterial cell does not appear to be markedly different from other Gram-positive bacteria, and the presence of most of the usual intracytoplasmic structures, including the various membrane systems, has been recognized.

118

0. RA’l’LEDGE

A. CELLWALL: CHEMICALCOMPOSITION Chemical analysis of the cell wall has proceeded more or less independently of electron microsope studies and, therefore, the contributions of these two approaches can be considered separately. The wall has a very complex structure which can best be described as a macromolecular lipoglycomucopeptide, forming the ground substance, with which a variety of other molecules are associated. Kotani et al. (1959) were the first to examine the entire composition of isolated cell walls although some work had already been carried out on the composition of the basal “wall skeleton” (see p. 130) by Cummins and Harris (1968). Many other groups of workers had worked, however, often for many years, on the structure of components isolated from whole cells and, in so doing, gained valuable information about such materials as the lipids (Anderson, 1943; Asselineau, 1952, 1962) and polysaccharides (Stacey and Kent, 1949) which are now known to be located within the wall. Kotani et al. (1959) showed that the walls from M . bovis BCG contained over 60% of their dry weight as lipid. Some of this lipid could be easily extracted into ethanol-ether (17% of cell wall), and some only into chloroform (21%) whereas some components (22% of the cell wall) were not released until after acid hydrolysis, i.e. were chemically attached to the “wall skeleton”. These results have recently been conf2med by Azuma et aE. (1974) who found that 34% of isolated walls of strain BCG were free (unbound) lipids. Similar results have been obtained for M . rame (Belknap et al., 1961), M. fortuitum, M. kansasii (DeWijs and Jollbs, 1964) and M. lepraemurium (Draper, 1971) but the content of the bound lipids in M. phlei appears lower (DeWijs and Jollds, 1964). The “wall skeleton’’ which remains after removal of the lipids is the murein which has a similar composition to that of the peptidogylycan (or mucopeptide) from other bacteria (see Salton, 1964). Hoare and Work (1967) demonstrated the occurrence of meso-diaminopimelic acid (DAP) in cell hydrolysates from several mycobacteria. Cummins and Harris (1958), using isolated wall fractions, identified arabinose, galactose, DAP, alanine, glutamate, glucosamine and muramic acid as the principal sugars, amino acids and hexosamines in M. phlei, M.megmatis and M . tuberculosis. These results were confirmed by Kotani et al. (1969, 1960) and also by Misaki et al. (1966) who isolated and purified the peptidoglycan from cell walls of strain BCG by periodate oxidation and showed it to consist mainly of alanine, glutamate, DAP, glucosamine and muramic acid. The last two compounds were thought to be acylated. Subsequent work mainly by the groups associated with the laboratories of Lederer, Kato and Kotmi (Kotani et al., 1968, 1970; Adam et al., 1969; Amar-Nacasch and Vilkas, 1969; Petit et al., 1969; Azuma et d.,

119

THE PHYSIOLOGY OF THE MYCOBACTERIA

fi 1+4

+-

N-acetyl- - N-glycolylglucosamine muramic mid

fi 1-+4 -

fi 1+4

N-acety1. - N-glycolylglucosarnine muramic acid

L-AIu

\

@ D-AIu

meso-u,cdiarninopimelic acid'

-

L - A ~

\

meso-u,cdiaminopirnelic acid*

fl D - A ~

\

a,y indicate the carbonyl group used in peptide linkage *Free carbonyl groups are aminated

FIG. 1. Structure of the basal peptidoglycan of the cell wall of mycobacteria.

1970; Wietzerbin-Falszpan et al., 1970) has enabled the structure given in Fig. 1 to be proposed for the basal peptidoglycan of mycobacteria. This structure is, however, a simplification and the peptidoglycan backbone is probably much more complicated. For example, a new tetrapeptide : Ala-Glu-(a-G1y)yDAP and a peptide containing glycine, aspartate, alanine, glutamate and DAP have been isolated from the mucopeptide of M . tuberculosis H37Rv (Kotani et al., 1970). Some galactosamine may also be present in the repeating disaccharide chain (Acharya and Goldman, 1970). The main distinguishing feature of the mycobacterial peptidoglycan is the substitution of a glycolyl group on the N atom of the muramic-acid residue instead of the more usual acetyl group (Salton, 1964). This substitution has been found in all mycobacteria so far examined (Azuma et al., 1970, 1973; Lederer, 1971) and also in Nocardia Eirowani (Guinand et al., 1970) but not in species of corynebacteria or streptomyces (Azuma et al., 1970). Biosynthesis of N-glycolylmuramic acid is probably via: UDP-N-acetylglucosamine + UDP-N-acetylmuramic acid --f UDP-Nglycolylmuramic acid, with the final step being catalysed by a hydroxylase type of enzyme (Petit et al., 1970; Takayama et al., 1970). The peptidoglycan of the mycobacterial cell wall is but a small part of a much more complex molecule. Linked to it is a highly elaborated glycolipid (or lipopolysaccharide) which consists of an arabinogalactan

120

C. RATLEDGE

attached to a mycolic acid (seeSectionIIIE, p. 147). The arabinogalactan is a branched structure (see Misaki et al., 1974) containing mainly 1 + 5 linked D-arabinofuranose units and, what was thought to bc 1 -+ 4 D-galactopyranose units, in the ratio of 2.5: 1.0 (Misaki and Yukawa, 1966). Although subsequent work (Vilkas and Markovits, 1968; Amar-Nacash and Vilkas, 1969, 1970) supported a structure containing 1 -+ 4 linked galactopyranose units, more recent studies, involving PMR spectroscopy, exclude this and proposes the digalactoside as 6-O-cr-D-galaCtOfUranOSyl-D-galaCtOSe(Vilkas et al., 1973). The mycolic acid part of the glycolipid is linked through its carbonyl group to arabinobiose (Amar-Nacashand Vilkas, 1970; Kanetsuna et al., 1969)which the leads into the arabinogalactan chain (see Fig. 2). The linkage of the glycolipid to the peptidoglycan is probably through a phosphodiester bridge to the N-glycolylmuramate (Kanetsuma, 1968) which is supported by the earlier isolation of muramic acid 6-phosphate from M . butyricum (Liu and Gotschlich, 1967) and by the more recent isolation of arabinose 1-phosphate (Amar and Vilkas, 1973). A tentative structure can, therefore, be advanced for the basal macromolecule of the mycobacterial cell wall (Fig. 2). Elucidation of this structure has been assisted by the ability to isolate an “oligomer” of the wall. This oligomer, when originally isolated (Asselinoau, 1951),was thought to be a distinct entity and was called Mycolate of arabinose

t-

SAra(l

-

3)Ara

“1 3

Arabinogalactan (Am, Gal, ; all in furanose form)

+----

---- +

Ara( 1 +5)Ara(1 -+5)Ara(1 + 6)Gal(1-

I

I

0

I I 0 I

Phosphodiester link (on one out of 8 t o 10 moleculcs of NGMA)

HO-P=O

- - - - NAG-NGMA-NAG-NGMA-NAG-NGMA-

I I

I I

L-Ala

L-Ala

D-G~u(NH,)

D-G~u(NH,)

D-G.~u(NH,)

YI

I YI

’/ ’D-L//

YI

mesoDAP(NH,) mesoDAP(NH,) meaoDAP(NH,) / I D.kla/ D-Ah I D.kl&/

m , es;D;:( \

I

L - A ~

FIU. 2. Tentative stmcturc of the cell-mall macromolecule in rnycobcxtcriP,. NGMA indicates N-glycolylmuramic acid ; NAG, N-acetylglucosemine. Adapted from Lederer (1971, 1973) and Vilkas et nl. (1973).

THE PHYSIOLOGY O F THE MYCOBACTERIA

121

Wax D (Aebi et al., 1953). Studies on its structure (Asselineau et al., 1958; Azuma et al., 1969; Jollks et al., 1962; Migliore and Jollks, 1968, 1969;KanetsunaandSanBlas, 1970; Markovitsetal., 1971; Vilkasetal., 1973) have been of considerable value in unravelling the structure of the wall although it is now considered that Wax D arises by autolysis of the wall (Brennan et al., 1 9 7 0 ~ )Wax . D occurs only in old cultures, and is probably liberated from the wall by action of various peptidases, lipases and esterases. It is not surprising therefore that homogeneous preparations of this material have not been isolated (Jollks ?t al., 1962; see also Lederer, 1971 ). The mycolate-arabinogalactan-peptidoglycan complex (Fig. 2) appears to serve as a ground substance with which a large number of other materials can be associated or even covalently attached. Crude cell walls contain large amounts of most protein amino acids (Wietzerbin-Falszpan et al., 1973) but, although these cannot be removed by repeated washing or 8-M urea, some can be removed using hot dodecylsulphate and these probably belong to lipoproteins or glycolipoproteins which are not covalently attached to the wall matrix (WietzerbinFalszpan et al., 1973). Other amino acids can be released by proteolytic enzymes (Kanetsuma, 1968; Misaki and Yukawa, 1966; DeWijs and Jollks, 1964; Kotani et al., 1970; Adam et al., 1972) and the few nonpeptidoglycan amino acids which remain (aspartate, glycine, serine, leucine and threonine have been recognized ; Acharya and Goldman, 1970; Azuma et al., 1974; Draper, 1971 ) are probably the point of attachment of sundry peptides or proteins. One of the mo3t abundant polypeptides in the wall of strain BCG and M . tuberculosis is polyglutamic acid which represents up t o 8% of the total weight of the wall (Vilkas and Markovits, 1972; WietzerbinFalszpan et al., 1973). This molecule has not been found in the saprophyte Jl.smegmatis and its contribution to virulence or inimunogenicity may be important (Wietzerbin et al., 1975). As mentioned earlier, associated with cell walls are many lipids, glycolipids, glycosulpholipids and peptidoglycolipids. The structures of their materials are given in Section I11 (p. 155) but suffice to say that, although these particular lipid materials are not covalently attached to the wall, they make an enormous contribution to the characteristics of the cell surface. Some lipids such as the mycosides (see p. 157) appear to be located on the very surface of the cell wall. Mycoside C has been shown to behave as a receptor site substance for invading mycobacteriophages (Goren et al., 1972; Furuchi and Tokunaga, 1972) and this material has been identified as the capsule around M . lepraemurium (Draper and Rees, 1973) and also M . awium (Draper, 1074) (see Section II.B, p. 123). Esters of trehalose with phleic acids (p. 145) also occur on

122

0.RATLEDGE

the surface of the wall and are produced only when clumping or aggregation of the cells takes place (Asselineau et al., 1972) thus perhaps indicating their involvement in cell-to-cell adhesion. Other lipid materials within the wall include mycobactin (p. 194) which, under certain growth conditions, can constitute up to 10% of the total weight of the cell (Ratledge and Marshall, 1972) and which therefore is probably between 15 to 20% of the weight of the wall itself. Mycobactin functions as an iron transporter (or ionophore), and doubtless other carriers for the uptake of nutrients into the cell must also exist within the wall. A number of polysaccharides also occur within the wall, most abundant of which are glycogen-type cc-glucans (Chargaff and Moore, 1944 ; German et al., 1961; Misaki and Yukawa, 1966; Amar-Nacasch and Vilkas, 1970; Antoine and Topper, 1969a, b, c ; Draper, 1971). The glycogens from M . phlei, M . smegmatis and M . tuberculosis have been studied in detail by Antoine and Tepper ( 1 969a, b, c) and can reach very high molecular weights (up to 2 x lo8 daltons). Mannan and an arabinomannan, and possibly an arabinogalactomannan, have also been reported (Ohashi, 1970; Kotani et al., 1971). Some lipopolysaccharides are also within the wall but are sufficiently water soluble to diffuse out into the culture mcdium (Saier and Ballo~i,196%). Although a great deal of work has been accomplished on the chemical constitution of the mycobacterial cell wall, it is not yet possible to extrapolate these findings with any degree of confidence to give an accurate interpretation of what is seen of the wall by electron microscopy.

B. CELL WALL: ELECTRON M~CROSCOPY Probably the single, most valuable study of the ultrastructure of the mycobacterial cell wall is that of Imaeda et al. (1968). These authors consider the wall to consist of three unit layers (see Fig. 3) and attempted to identify the various components of those layers by differential extraction techniques. Their results, i t must be said, are somewhat at variance with the findings from the above-mentioned direct chemical approaches (see Lederer, 1971). The out-rmost layer of the cell wall is composed o f a network of fibrile or filaments which have been recognized in all mycobacterial species so far examined (White, 1965; Imaeda et al., 1968; Rieber et al., 1969; Gordon and White, 1971). These fibrils are most clearly seen in cells from young cultures, and an example from M . phlei is shown in Fig. 4. In old cultures the fibres tend to be less irregular in distribution and to be fewer in numbers (Imaeda et al., 1968, 1969). Imaeda et al. (1968) consider these filaments to consist of a glucose-rich (i.e. glucan) lipo-


123

FIG.3. Proposed structure of the mycobacterial ccll wall. Layer I, indicates the lipopolysaccharide layer composed of fibrils and homogeneous substances : layer 11, lipopolysaccharide-lipid-proteincomplex conskting of fibrils and a membranous matrix ; layer 111, lipopolysaccliaride-peptidoglycancomplexes consisting of fibrils within D thick layer. PS indicates tho periplasniic space, of doubtful existence but it may be up to 10 nm across according to some workers; CM, tho cytoplasmic membrane. Adapted mainly from Tmltcda et al. (1968).

polysaccharide around which a secuiid and independent lipopolysacchsride accumulates which is rich in arabinogalactan. White (1965))however, has shown these filaments to be a peptidoglycolipid, the identity of which, a t least in M . avium and M . lepraemurium, appears to be mycoside C (Fig. 18b, p. 157; Draper and Rees, 1973; Draper, 1971). The middle layer of the mycobacterial wall consists, according to Imaeda et al. (1968)) of a further fibrillar network and a membranous matrix. This is a lipopolysaccharide-lipid-proteincomplex. The innermost layer is a lipopolysaccharide-mucopeptide complex consisting again of fibrils which appear to contain mycolic acid embedded in a thick layer which would correspond t o the macromolecule of the basal wall structure. Takeya and his coworkers have also proposed that thc basal peptidoglycan-lipid molecule forms a thin and innermost layer of the cell wall giving shape and rigidity to the cell itself (Takeya and Hisatsune, 1963; Takeya et al., 1963). Winder and Rooney (1970a) have shown that polysaccharides extracted with dilute alkali from Jf. bovis BCG can be resolved into three distinct fractions of markedly different molecular size, and have suggested that these fractions could arise from the three layers of the cell wall. Although there may be disagreements with the findings of Imaeda et al. (1968),their proposed structure (Fig. 3)nevertheless gives a valuable guide as to how the cell wall may be organized. The wall is, without doubt, extremely thick (estimates of about 20 nni have been given; Keike and Takeya, 1961) and most electron micrographs do show it as a layered structure (Keike and Takeya, 1961; Imaeda and Ogura, 1963; Imaeda et al., 1969; Petitprez et al., 1967). A typical electron micrograph of M . smegmatis (Fig. 5 ) shows that outside the cytoplasmic membrane

124

C. RATLEDQE

FIG.4. Electron micrograph of Mycobacterium phlei showing a surface network of interlacing filaments. The preparation was negatively stained with potassium phosphotungstate. Magnification x 200,000. From Gordon and White (1971). Reproduced with kind permission of Professor R. G. White.


125

there is possibly a discrete periplasmic space separating it from the wall. The wall of M . smegmatis appears to have a thin inner electron-dense layer, a less electron-dense middle zone which is much thicker, and a final electron-dense peripheral layer.

FIG.5. Electron micrograph of a thin section of Mycobacteriurra srnegmatis showing the trilayered structure of the cytoplasmic mcmbrane (CM),the wall (W) and the possible occurrence of a poriplasniic space (PS).The cells were embedded in araldite, sectioned and then staincd with uranyl acetate and lead citrate. Magnification, ~ 1 6 5 , 0 0 0Unpublished . data of P. V. Patcl, Jnnicc Miindy and C. Ratledge.

A somewhat similar la,yerccl structnre of the wall of M . leprae has been shown (Edwards, 1970) although this organism, and the related 1cl.lepraemurium, have a further electron-transparent zone outside the wall proper (Yamamoto et al., 1958; Hanks, 1961). Thiszone or capsule can be extremely wide (Fig. 6 ; see also Brown and Draper, 1970; Thanh et al., 1971 ) and has been considered to act as aprotectionagainst destruction by phagocytic cells (Draper and Rees, 1970). The material of the capsule, which can be stained with Sudan Black B indicating it to be a lipid, can be easily removed merely by a brief wash with xylene (Fisher and Rarksdale, 1973). Draper and Rees (1973) found the capsule to contain, or consist, of mycoside C in the form of fibres which could perhaps represent an extension of the surface fibres of the cell wall as seen by Gordon and White (1971). Hasegawa (1971), however, has suggested that the capsule may contain protein as well as lipid.

126

C. RATLEDGE

FIG.G(a). Electron micrograph showing tho morphology of Mycobacteriurn leprae in a mousc lymph node. Magnification, ~ 4 0 , 0 0 0(b) . Interpretation of the electron micrograph through acrosssoction oftlie bactorium and lymph node : 1 indicates the bacterial cytoplasm; 2, plasma mombrane consisting of two dense layers on rither sido of a less dense one; 3, elcctron-dense layer of cell wall ;4, electron-transparont layer of coll wall; 5 , tenuous electron-dense zone outside cell wall; 6, zone of low electron density; 7, lysosomal membrane: 8, host-cell cytoplasm. From Edwards (1970) with kind permission of the author. The original doubt about the identifiration of structure 7, the lysosomal mombrane, has now been resolvcd and this structure is now known t o oxtend right round M . leprae (Rosa Edwards, personal communication).

C. CELLWALL: GENERALCONCLUSIONS Thc wall of the mycobacterial cell may be considered a series of complex lipid-containing substrates giving a structure which is probably not


127

less than 20 nm thick in any mycobacterium. The wall may be even thicker if the capsule, which is known to be present with some mycobacteria, is considered as an extension of the lipid-rich surface filaments. The high content of lipid in the wall is of obvious importance to enable the cells to resist the digestive enzymes of host tissues when mycobacteria grow parasitically. Furthermore, when cells are faced with periods of starvation or aridity, the wall prevents dehydration and thus contributes to their survival. The nature of the wall, however, must be an obstacle to the transport of nutrients into the cell, and this is probably exacerbated by the extensive clumping of the cells due to their hydrophobicity. Such events may be responsible for the slow rate of growth of most mycobacteria. The resistance of mycobacteria to chemical agents, such as dilute acids and alkalis, as well as to some chemotherapeutic agents, is again the consequence of an ultra-thick water-repellent outer casing. The wall of the mycobacteria is also responsible for the characteristic acid-fastness of these bacteria and differences in the degree of acidfastness between species (Murohashi and Y oshida, 1968) are probably due to different cell-wall properties. The component responsible for acid-fastness has not yet been identified. Qoren (1972) has suggested that the lipids within the well absorb the stain but then impede penetration of the subsequent alcoholic acid wash so that the dye remains unbleached. The wall contains many compounds, either free or attached to the peptidoglycan backbone, which can be antigenic, immunogenic or toxic when injected into host animals. Other compounds may serve to enhance the activity of these materials, i.e. act as adjuvants. An understanding of the nature of these materials is of obvious importance as they are often implicated in the pathogenicity of mycobacteria. This aspect is covered in detail in Section I V (p. 167).

D. MEMBRANESTRUCTURES Two principal membrane structures have been observed in all mycobacteria so far examined ; they are the cytoplasmic membrane and the mesosome.

1. Cytoplasmic Membrane and the Periplasmic Space The cytoplasmic membrane appears to be the conventional threelayered structure (protein-lipid-protein) about 8 nm thick, which occurs in most other cells. The cytoplasmic membrane is probably best seen in sphaeroplasts (e.g. Thacore and Willett, 1966; Willett and Thacore, 1967; Adamek et al., 1969) or in cell wall-deficient forms (Mattman, 1970; Bykov et al., 1972) where the wall is no longer present,

128

C. RATLEDGE

Excellent electron micrographs of sphaeroplasts of M . smegmatis and burst sphaeroplasts (“ghosts”) of M . phlei have been published (Adamek et al., 1969; Asano et al., 1973) which show the triple-layered structure of the outer cell membrane. Koike and Takeya (1961) describcd the cytoplasmic membrane of Mgcobacterium strain Jucho as being separated from the cell wall by a low-density (periplasmic) space, whereas Imaeda and Convit ( 1962) and Imaeda and Ogura (1963), examining M . leprae, M . lepraemurium and a Mycobacterium sp., were unable to recognize such a zone. Edwards (1970), also using M . leprae, and Kats et al. (1972) using M . tuberculosis, would concur with this view although workers examining M . smegrnatis (see Fig. 5 and also Ariji et al., 1972) and M . phlei (Petitprez et al., 1967) would perhaps disagree. With 31. phlei this periplasmic space was assessed a t about 10 nm wide (Petitprez et al., 1967). Some of the more recent electron micrographs of Imaeda (Imaeda et al., 1969) have also shown evidence for a periplasmic space in M . smegmatis and M . tuberculosis but as this was not the subject of the investigation no comments were made. However, as Edwards (1970) has remarked, it only needs a small amount of plasmolysis to occur before, or during, the preparation of sections to give the appearance of a finite periplasmic space. Perfect sections of bacteria illustrating continuous contact between wall and membrane are rare (Edwards, 1970) and advocates of the existence of a periplasmic space must therefore be cautious that they are not dealing with an artefact. We can, however, be certain that the wall and the membrane in the mycobacteria are distinct entities as they can be easily separated either by plasmolysis (deliberate or accidental ; see Imaeda and Ogura, 1963; Imaeda et al., 1969) or by the formation of stabilized sphaeroplasts (see above).

2. Mesosomes The presence of intracytoplasmic coiled and multi-layered lamella structures has been recognized in many mycobacteria, although a t first these were thought to be equivalent to the mitochondria of eukaryotic cells (Shinohara et al., 1957; Zapf, 1957). Mesosomes can often be seen in electron micrographs (see Fig. 7) to be formcd by invagination of the cytoplasmic membrane. Thus, the dimensions of the unit membrane of the mesosome are the same as those of the cytoplasmic membrane. Although a wide variety of shapes and distributions of the mesosome can be recognized, some without apparent connection with the outer cell membrane, this is probably due to the angle of sectioning taken through the cells. Several authors, however, have commented upon the frailty of the mesosome. Mesosomes may alter their configuration with different fixation techniques or can be disrupted and rcdistributed


129

FIG. 7. Electron micrograph showing mesosomes in Mycobacteriurn srnegrnatis. The cells were negatively stained. Magnification, ~ 6 0 , 0 0 0Note . the connection of the unit membrane system of the mesosomc to the cytoplasmic membrane. Unpublishod work of P. V. Patel, Janice Mundy and C. Ratledge.

within the bacterium by plasmolysis created by careless handling of the bacteria prior to fixation (Silva, 1971; Burdett and Rogers, 1970; Highton et al., 1973). Caution must therefore be exercised in reaching any definitive conclusion about the structure and function of mesosomes. Mesosomes have been recognized in M . avium (Shinohara et al., 1957), M . kansasii (Schaefer and Lewis, 1965), M . leprae (Brieger, etal., 1959; ImaedaandConvit, 1962; ImaedaandOgura, 1963;Edwards, 1970; Thomas, 1973), M. lepraemurium (Chapman et al., 1959; Imaeda and Ogura, 1963; Whitehouse et al., 1971; Thomas, 1973), 41. phlei (Asano et al., 1973; Drews, 1960; Petitprez, et al., 1967; Silva, 1971), M . smegmatis (Rieber et al., 1969; Imaeda et al., 1969; Ariji et al., 1972; Mison et al., 1969; and see also Fig. 7), M . tuberculosis (Imaeda et al., 2969; Ariji et al., 1968, 1972; Kats et al., 1972), $1. paratuberculosis (Smith, 1969) and Mycobacterium strain Jucho (Koike and Takeya, 1961). Methods for the isolation and partial purification of membrane units have been developed independently in the laboratories of Trnka and Brodie using ill. smegmatis and M . phlei, respectively (Mison et al., 1969; Asano et al., 1973). A prerogative of both methods is the ability to produce satisfactory protoplasts of the selected organism (Adamek et al., 1969; Mizuguchi and Tokunaga, 1968, 1970) but this procedure is not always as straightforward as it may be presented. Many functions for the bacterial mesosome have been proposed (see Reusch and Burger, 1973; Salton, 1971) and a detailed discussion of these is beyond the scope of the review. However, one such proposed function is that mesosomes are connected with septum formation in a dividing cell, and several examples of this in different mycobacteria are available (Imaeda and Ogura, 1963; Ariji et al., 1972; Petitprez

130

C. RATLEDGE

et al., 1967; Edwards, 1970). Figure 7 (p. 129) illustrates the beginning of cell division in M . smegmatis. Ariji et al. (1968) have shown that several

respiratory enzymes, including succinate dehydrogenase and cytochrome oxidase, as well as ATPase and acid phosphatase, were localized mainly on the mesosomes and therefore the mesosome may be an important organelle in energy-production processes.

E. NUCLEAR MATERIALAND RIBOSOMES Imacds et al. (1969) described the nuclear network of M . smpgmatis as consisting of filaments approximately 3 nm in diameter which were uniformly distributed in the central portion of the cytoplasm. Similar filaments are shown in Fig. 5 (p. 125) and have also been recognized in M . lcprae (Edwards, 1970). There is, however, some confusion as to what is “nuclcar material” (i.e. DNA) and what are ribosomes or collections of ribosomes (i.e. polysomes or polyribosomes). Thus, Gale and McLain (1963) and Whitehouse et al. (1971) described the “nuclear material” of M . smegmatis and M . lepraemurium, respectively. as consisting of coarse granules. This material is probably not nuclear material as such but ribosomal material. Distinct filamentous material could be seen in the electron micrographs of Gale and McLain (1963) but was termed “fibrillar cytoplasm”. The general consensus of opinion would, however, interpret this material as the bacterial nucleoid or nucleus (see Fuhs, 1965, 1969; Bisset, 1970). Ribosomes have been recognized in the cytoplasm of mycobacteria as small, dense spherical particles measuring approximately 16 nm in diameter according to Imaeda et al. ( 1 969) who examined sections of whole cells of M . smegmatis, and 20-25 nm in diameter according to Worcel et al. (1968) who worked with purified ribosomal preparations from M . tuberculosis. Both groups of workers interpreted these bodies as being the intact 705 ribosome. Smaller particles 6 x 12 nm and 8 x 12 nm were also seen in the cell by Imaeda et al. (1969), and these were thought to be the 305 and 505 ribosomal subunits which Trnka et al. (1968) had previously shown to exist from sedimentation patterns of ribosomes from strain BCG on linear sucrose gradients. Worcel et al. (1968), however, could not recognize the ribosomal subunits until their preparations had been depleted of Mg2+.This action caused dissociation of the 705 ribosome and swelling of the individual subunits; the 305 subunit was measured as 15-20 nm in diameter and the 505 subunit as 20-30 nm in diameter which was, in fact, larger than the original ribosome when in the presence of Mg2+. Trnka et al. (1968) and Kiss and Tomcsanyi (1973) also found that a t least 10 mM Mg’+ was required to maintain the integrity of the ribosome. Aggregation of two or three 7 0 s ribosomes into a polysorne was also observed by Imaeda

T H E PHYSIOLOGY O F T H E MYCOBACTERIA

131

et al. (1969) but these could not be demonstrated by the analytical techniques of Trnka et al. (1968). The structure of mycobacterial ribosome was pursued by Worcel et al. (1968) who, by a combination of electron microscopy and a determination of sensitivity of isolated subunits to ribonuclease activity, have proposed the ribosomal model shown in Pig. 8. The proteins of Mg2+

IOmM fl

Protein

FIG.8. Proposed model of the ribosomr of Mycohacterium tuberculosis. The model exphins the ribonuclease reslaterice of thc 705 ribosome, made by “moutli-tomonth” attachrnorit of the 50s and 30s subunits, w well us the ribonuclcr,sc sensitivity of the isolitted s11l~uriit;s arid of the c x p a n d d Mg2+depleted nbosomcq. Tho proposed model for tlir 70s ribosome isolated in 10 inM Mg2+may not reprcscrit the biodynaniic structurc of ribosorne during protein synthesis. From Worrcl et az. ( 1 9 6 8 ) .

the ribosomes of M . phlei have been found to be similar to those from several other prokaryotes (Sun et al., 1972) although the 305 subunit did not contain a high molecular-weight protein, detected in other bacteria except Bacillus stearothermophilvs, which had previously been considered to be involved in the binding of messenger-RNA and transfer of RNA t o the ribosome (Van Duin and Kurland, 1970).The involvement of the ribosome in protein biosynthesis is described in Section V I (p. 214).

I?. POLYPHOSPHATE GRANULES Intracellular “bodies” or granules of varying sizes and opacity to Xrays in the electron microscope have been reported for almost every niycobacterium studied. Many interpretations of thcir nature have been given, ranging from spores to mitochondria. It now seems apparent, however, that the main elechon-dense bodies which can be seen in mycobacteria are polyphosphate granules. Polyphosphate granules are sometimes referred to as containing metaphosphate but this is highly unlikely and any metaphosphate which can be recovered is probably produced as an artefact by hydrolysis of polyphosphate (Harold, 1966; Dawes and Senior, 1973).

132

C. RATLEDOE

Glauert and Brieger (1955) were the first t o give a substantiated identification of the electron-dense granules by using light and electron microscopy on the same samples of M . phlei. Winder and Denneny (1954), however, had already suggested that this may be the case as they had been able to show a direct correlation between the amount of polyphosphate present and the number and size of the granules in M . sinegrnatis and M . tuberculosis. Polyphosphate granules have also been identified (sometimes retrospectively) in electron micrographs of M . avium (Brieger and Glauert, 1956a; Knaysi et al., 1950; Shinohara et al., 1957), M . bovis BCG (Mudd et al., 1951, 1956; Merckx et al., 1964; Shinohara, 1955a, b ; Takeya et al., 1954), M . kansasii (Schaefer and Lewis, 1965), M . leprae (Imaeda and Convit, 1962; Imaeda et al., 1962, 1963; Edwards, 1970), M . lepraemurium (Whitehouse et al., 1971), M . paratuberculosis (Smith, 1969), M . smegmatis (Gale and McLain, 1963), M . thamnopheos (Mudd et al., 1951, 1956) and 31. tuberculosis (Brieger and Glauert, 1956b; Kolbel, 1958). Under the electron microscope, polyphosphate granules appear as opaque bodies, dnvoid of any internal structuring or limiting membrane and, if subjected t o high-electron bombardment, will characteristically bubble and then volatilize or may even explode. They exhibit a wide range of sizes, from 7 0 to 500 nm (Mudd et al., 1951 ; Shinohara et al., 1957) though Smith (1969) noted with ill. paratuberculosis that, apart from the usual polyphosphate granules (70-1 80 nm diameter), smaller bodies (10 to 20 nm in diameter) could also be seen which he suggested were small accumulations of polyphosphate. Similar reports of small electron-dense granules in other mycobacteria (Kolbel, 1958 ; Shinohara, 1955a; Takeya et al., 1954; Whitehouse et al., 1971) have led to the suggestion that the variation in size of polyphosphate granules could be due to variations in growth conditions or caused by differences in the growth substrates used. Mudd et al. (1956) showed that, when both $1. thmmnopheos and BCG were grown with an oxidizable substrate, such as glycerol or malate, the larger granules were formed, but if BCG was depleted of the granules by manipulation of the cultural conditions, the granules re-appeared in the minute form. Shinohara (1955a, b) likewise found that unfavourable conditions led to a marked increase in the proportion of the minute granules in M . bovis strain BCG, and Whitehouse et al. (1971) concluded that in 41.lepraemurium the small polyphosphate granules were a consequence of the low growth rate and diminished metabolic activity of this species. The polyphosphate within the granules is probably associated with organic material, as the quantity of polyphosphate in the cells is insufficient to account for the total volume of the granules (Glauert and Brieger, 1955). It is not clear, however, what this organic material may be,


133

and a potpourri of RNA, lipid, protein and Mg2+could even exist (Widra, 1959). VC’inder and Denneny (1954) suggested that polyphosphate in &I. smegmatis and ill.tuberculosis could be associated with RNA as these two materials could not be easily extracted separately. Drews (1962) proposed an association with a protein matrix, and Schaefer and Lewis (1965) showed that, when mycobacteria grew on medium containing oleic acid, the polyphosphate granules appeared to be enclosed in lipid deposits. These views may not, however, be mutually exclusive. The possible role of polyphosphate in energy metabolism is discussed in V1.C (p. 211).

G. LIPIDVACUOLH~W Burdon (1946) was probably the first to demonstrate that AW.tubercuZ~siscould take up a fat-soluble dye, Sudan Black B, to stain intracellular material. Such material appears to be absent from M . leprae (Bermann, 1953; Chaussinand and Viette, 1955; Fisher and Barksdale, 1973). These fat bodies, or lipid vacuoles, probably correlate with the moderateand low-electron-dense bodies which have been described in many mycobacteria. These are often ofthe same size as the large polyphosphate granules although minute electron-transparent entities have also been seen (Whitehouse et al., 1971). I n most cases, the nature of these inclusioiis has not been elucidated, and it is therefore a matter of speculation that they are all of a similar compoAition. Several authors have described these bodies as being limited by a singlelayered membrane (Kolbel, 1958; Gale and McLain, 1963; Brieger and Glauert, 1956) which would suggest a close analogy with the lipid vacuoles studied in other organisms (see Matile et al., 1969). Indeed, Gale and McLain (1963) found that these inclusions were stained intensely with Oil Red 0,a fat-soluble dye, indicating a lipid composition. Smith (1969) and Edwards (1970) made similar conclusions about the large vacuoles they observed in M . paratuberculosis and M . leprae, respectively. The “spherules” described by Merckx et al. (1964) and the “cell-sap vacuoles” described by Knaysi et al. (1950), both in M . tuberculosis, map also be lipid vacuoles. Other authors have been more cautious in their interpretations of these bodies. Whitehouse et al. (1971) could not observe a limiting membrane to the electron-transparent vacuoles seen in M . lepraemurium and, because these bodies did not collapse during the drying of thc specimens for electron microscopy, concluded that they were not filled with liquid. This latter observation, however, ignores the fact that the lipid (if indeed it is lipid) necd only he fluid a t the growth temperature (e.g. 37°C) and would solidify, perhaps into a microcrystalline form, a t

134

C. RATLEDGE

room temperature. Solidification of the lipid within these electrontransparent bodies may therefore contribute to the variety of appearances which are observed for these bodies by electron microscopy. Another important factor which could dictate the appearance, size and distribution of lipid vacuoles is the presence of fatty acids in the growth environment. Schaefer and Lewis (1965) have shown that massive lipid globules quickly form within M . kansasii, M . marinum, M . avium and related species when grown in a medium containing either Tween 80 (polyoxyethylene sorbitan mono-oleate) or oleic acid. As Tween 80 is often used in growth media for cultivation of mycobacteria, its contribution to the morphology and contents of the cell cannot be ignored.

111. Lipids: their Structure and Biosynthesis The importance of lipids to the physiology of the mycobacterial cell is probably three-fold. Firstly, lipids, especially the mycolic acids, form an integral part of the structure of the cell wall; secondly, the high content of lipid associated with the cell wall gives the cell many of its outward characteristics (e.g. hydrophobicity, impenetrability to many materials) ;and thirdly by their more specific immunogenic and antigenic properties, many of the interactions between host-tissue and invading mycobacteria are seen to have their origins. The first two aspects have already been discussed in Section I1 (p. 118) and the last aspects are discussed in detail in Section I V (p. 161). Lipids, of course, fulfil many other functions such as acting as storage compounds and being involved in membrane structure and function but, as tthese functions are shared with most other organisms, these aspects will only be briefly mentioned here. This section then is devoted mainly to an account of the structure and biosynthesis of the major lipids within the cells. The subject has recently been reviewed by Goren (1972). A. INFLUENCE OF THE ENVIRONMENT ON LIPID FORMATION A number of reports have shown that the amount of lipid synthesized by mycobacteria can vary according to the growth conditions. Addition of Tween 80 to the medium can double the quantity of lipid (Stinson and Solotorovsky, 1971) which is an important observation as Tween 80 is often added routinely to culture media, both liquid and solid. Tween 80 can be hydrolysed and the oleic acid incorporated into triglycerides (Weir et al., 1972). Tepper (1965) showed that when glycerol, but not) glucose, was used as carbon source non-essential lipid (perhaps triglycerides) as well as glycogen accumulated in M . phlei. Earlier reports


135

also showed that glucose-grown M . tuberculosis had 60 to 80% less lipid than glycerol-grown cells (Frouin and Guillaunie, 1923; Terroine and Lobstein, 1923)and this was confirmed by Antoine and Tepper (1969~); glycerol-grown cells contained up to 23% lipid (and up to 14% glycogen) whereas glucose-grown cells contained maximally 15% lipid (and only about 7% glycogen). There was little significant change in lipid content between nitrogen-limited cells and cells grown under nitrogen-sufficient conditions although this often occurs with yeasts and fungi (see Hunter and Rose, 1971). Glycogen, however, was virtually absent from the latter cells. With M . phlei, the lipid content did fall, however, from 24% to 12% when the medium was changed from a nitrogen-limited one to one in which nitrogen was present in excess, although this difference was less when ammonia was substituted for asparagine in the medium (Antoine and Tepper, 1969a). The content of lipid in mycobacteria may also vary with the age of cell, being usually highest in the later stages of growth (Asselineau, 1951;Tepper, 1965; Antoine and Tepper, 1969 a, c ; Winder andRooney, 1970a). The nature of the lipids, not surprisingly, also changes during the course of growth; Bennet and Asselineau (1970) found that, in M . tuberculosis, although mycolic acids were synthesized in parallel with growth rate, synthesis of other fatty acids declined. The concentration of oleic acid in cells fell markedly during growth, and this has been confirmed by Chandramouli and Venkitasubramanian ( 1973)who examined M . smegmatis and Mycobacterium 607 as well as M . tuberculosis. Production of branched acids (either mycocerosic acid or tuberculostearic acid) increased in all organisms examined by the two research groups although contents of cardiolipin and phosphatidylethanolamine decreased in old cultures of Mycobacterium 607, M . bovis BCG (Khuller et al., 1973) and M . smegmatis (Chandramouli and Venkitasubramanian, 1973). The content of the mannoside-phospholipids, and in particular the dimannoside, however, increases with age of culture (Khuller et al., 1973). However, the appearance of some of the macromolecular lipids in old cultures must be treaced with caution. For example, the appearance of Wax D (see p. 121) in cultures is now thought to be due to autolysis of the cell wall (Brennan et al., 1970c)and this would certainly explain the variability of different Wax-D preparations. Also the use of static cultures of mycobacteria as a source of material must be viewed with some reservations as such cultures produce, even in the early stages of growth, a pellicle wherein the cells must, of necessity, be heterogeneous. Analysis of these cultures for any component may well give erroneous results as cells at the top of the pellicle, although in contact with air, are probably deficient in nutrients whereas those a t the bottom of the pellicle, though having an unrestricted supply of nutrients from the medium

136

0.RATLEDQE

with which they are in direct contact, are nevertheless probably starved of oxygen. Premature autolysis of cells in both locations is likely, and only those cells in the centre of the pellicle may achieve any sort of balanced growth. These objections, which apply equally to analysis of enzyme activities as well as assays of structural components, are of course circumvented by the use of shaken cultures provided this is coupled with the harvesting of cells from cultures before they enter the stationary phase of growth. But alas this is rarely done. Allied to the desirability of using correctly grown cultures is the problem of lipid extraction from the cells. Because of the high content of bound lipids (i.e. lipids not released by direct solvent extraction) in most mycobacterial cells, some hydrolytic procedure must inevitably be incorporated into any scheme devised for the complete defattening of the organism. Early schemes of Anderson (1939, 1941, 1943) and Aebi et al. (1953) provided protocols whereby an array of different fractions could be isolated from the bacteria (soluble fat, waxes, A, B, C and D, phospholipids and bound lipids). Unfortunately these fractions are heterogeneous and, moreover, simple lipids such as triglycerides are often found distributed between various fractions (Brennan et al., 1970~).Schemes based on more conventional chloroform-methanol extractions, which simultaneously remove neutral and phospholipids which can later be separated by differences in solubility in cold acetone, followed by a mild acid hydrolysis of the cell debris to remove bound lipids, are to be preferred (Brennan and Ballou, 1967; Brennan et al., 1970~).

B. STRAIGHT-CHAIN SATURATED FATTY ACIDS Fatty acids including mycolic acids probably do not exist in a free form within the cell. Such traces as can be found during analysis are probably attributable to the action of esterases, lipases or phospholipases (Sartory and Meyer, 1947; Cattaneo, 1955)during the extraction procedure. As lipases are often more active when in an organic solvent such as ether and are temperature-stable (Andrejewand Tacquet, 1965), their quick de-activation often presents a problem. Fatty acids therefore usually occur as esters, and some of these structures which sometimes can be very complex are described in Section 1II.G (p. 152).

1. Distribution Mycobacteria contain straight-chain saturated fatty acids ranging from C, to C,, although C24is the longest acid usually found in saprophytes (Anderson, 1939; Asselineau, 1953, 1962; Barbier and Lederer, 1954).The most abundant of these acids is palmitic acid. ReGned techniques of gas-liquid chromatography have enabled many

TEE PHYSIOLOGY OF THE MYUOBAUTERIA

137

minor fatty acids to be recognized in mycobacteria. All of the oddnumbered saturated fatty acids from C,, t o C,, have been identified in various mycobacteria (Campbell and Naworal, 1969; Walker et al., 1970; Thoen et al., 1971a, b). The report of tridecanoic acid (C13)in 11.1. kansasii (Cattaneo et al., 1965; Lucchesi et al., 1967)is thought to be mis-identificationof a branched-chain acid (Thoen et al., 1971a)although this particular acid has been recognized in M . phlei by Campbell and Naworal(l969).

2. Biosynthesis The biosynthesis of saturated fatty acids in mycobacteria is at present being studied in detail by two groups. That led by Dr A. H. Etemadi uses M . smegmatis as its principal organism, and the other, led by Professor K. Bloch, uses an organism labelled as M . phlei, ATCC 365, but which has been identified as a strain of M . smegmatis (Hendren, 1974; see p. 140). To avoid confusion, however, this latter organism will be referred to in this review as M . phlei 356. The results of the two groups of workers are not entirely complementary with regard to the subcellular organization of the enzymes involved in the process and, for the time being, these differences must be attributed to intra-species variation. Research with other mycobacteria in this area would therefore be welcome. Incorporation of [',C]-acetate, with extracts from several mycobacteria, into a wide range of acids from C, to C,, gave preliminary evidence for a pathway of biosynthesis based on acetate (Kusunose et al., 1960; Pierard and Goldman, 1963; Winder et al., 1964; Khan and Venkitasubramanian, 1964).However, the first indication that biosynthesis in myobacteria may not be like that in other bacteria came when Brindley et al. (1969) isolated a multi-enzyme fatty-acid synthetase complex of high molecular weight from M . phlei 356. This differs from other probaryotic organisms which synthesize fatty acids by individual nonaggregating enzymes involving an acyl-carrier protein as the attachment molecule for the growing fatty-acyl chain (Vagelos, 1971; Packter, 1973). The enzyme system of M . phlei 356 also differs from the fattyacid synthetase complex of animals or yeasts (Lynen, 1967) in that its reaction products were bimodal, i.e. C,,and C14acids were produced as acids being the predominant end-products with CIJ, c18, C,, and found in lesser quantities. A feature like this could be expected to be widespread amongst the mycobacteria as the longer-chain acids are the probable precursors of the mycolic acids (see Section III.E, p. 149) and the short-chain acids are probably associated with neutral lipids, phospholipids and some glycolipids. Purification of the fatty-acid enzyme complex (Vance et al., 1973a)

138

U. RATLEDGE

showed that this bimodal pattern was due to the activity of a single homogeneous multi-enzyme complex. The enzyme, when purified to homogeneity, had a molecular weight of 1.39 x lo6 daltons, and readily dissociated in buffers of low ionic strength into inactive species which only partially re-aggregated into an active form when returned to a solution of a high ionic-strength. The enzyme, when in 0.01 M-phosphate buffer, had a half-life of one minute a t 4OC. The complex showed a simultaneous requrement for both NADH and NADPH (H. B. White et al., 1971). Optimal activity of the enzyme was attained by adding a mycobacterial polysaccharide (see p. 141). Either a polysaccharide containing 3-0-methylmannose (MMP) or one containing 6-0-methylglucose (MGLP) was active, the former being the more effective (Ilton et al., 1971).The polysaccharides lowered the K , value of the synthetase for acetyl-CoA nine-fold, and that for malonyl-CoA four-fold, and it was proposed that the polysaccharide functioned by binding the long-chain acyl-CoA produced by the synthetase and thereby relieved product inhibition of its activity (Vmce et al., 1973a). Formation of a complex between palmityl-CoA and MMP or MGLP was subsequently established (Machida and Bloch, 1973). Figure 9 shows the proposed hydrophobic interaction between the two compounds. These stimulatory effects were also found to be shared by cyclo dextrins containing 6, 7 or 8 D-glucose residues and which are not derived from mycobacteria (Machida et al., 1973). A polysaccharide with a similar function has been isolated from N . tuberculosis (Mehdi et al., 1974). The polysaccharides (MMP or MGLP) also influence the bimodality of the products of the synthetase ; in their presence a shift from a low (25%) t o a high (85%) proportion of shorter-chain acids (centred on C,,J occurred simultaneously with a stimulation of the overall rate of synthesis (Flick and Bloch, 1974). However, in the absence of any polysaccharide, similar shifts in the proportion of the short-chain acyl end

mo ABOUT 0.52nrn

n

:

0

I

fSCO*

I

0 I

0 I

0 I

...~..o..~...o...~...o..~..,o.*. O H ~ H OH&

OHOH

OHW

FIG. 9. Proposed hydrophobic interaction botwaen the paraffln chain of fatty acyl-CoA and the 6-0-methyl groups of 6-0-methylglucose. For a complete struoture, see Fig. l l a , p. 142). From Machida and Bloch (1973) with kind permission of Professor K. Bloch.

139

THE PHYSIOLOGY OF THE MYGOBACTERIA

AcetylCoA n-Malonyl.CoA

CI6-ENZYME -Elongation

___f

Cz4-ENZYME

nCoA

Transacylase 1

Transacylase I1

11

11

MMGt-MGLP

MMG .C~~-COA MGLP C16-CoA

.

FIG.10. Proposed scheme of fatty-acid synthesis in Mycobacteri~irnphlei ATCC 365. TE indicates a thioesterase ;MGLP, 6-0-methylglucose-containing lipopolysaccharide; MMG, 3-0-methylmannose.Based on the work of Vance et al. (1973a) and Flick and Bloch (1974).

products could be achieved by increasing the acetyl-CoA :malonyl-CoA ratio in the assay system from 0.41 : 1 to 150: 1. A separate feature of the synthetase from M . phlei 356 is its ability to elongate palmityl-CoA or stearyl-CoA to a C24 acid. This process requires, in addition to MMP or MGLP, free coenzyme-A and an elongation factor (El?) which was found to be heat-stable protein of molecular weight 10,500 daltons, and which functioned as thioesterase. By virtue of this activity, the elongation factor can adjust the intracellular concentrations of palmityl-CoA and free coenzyme-A to values which are favourable to chain elongation (Vance et al., 1973b). A proposed scheme for fatty-acid biosynthesis based on these results is shown in Fig. 10. To explain the bimodal fatty-acid pattern, two long-chain transacylases with different substrate specificities are proposed, Addition of MMG or MGLP will shift the equilibrium of the transacylase I in favour of free palmityl-CoA. Some inhibition of this enzyme may however be produced by the polysaccharides. The concentration of free coenzyme-A is critical for chain elongation, and i t can be readily furnished by the thioesterase whose specificity is highest for C,, and C,, acids. Complex regulatory mechanisms clearly operate with the synthetase, but as yet it is not known to what extent the various factors can regulate the activity and specificity of the synthetase under physiological conditions. Palmityl-&A, however, has been implicated as a feedback inhibitor of the first reaction (carboxylation of acetyl-CoA) which is also sensitive to changes in the ADP: ATP ratio (Erfle, 1973).

140

0.RATLEDOE

Etemadi and his coworkers, in an examination of fatty-acid biosynthesis in M . smegmatis, were unable to find any aggregation of enzymes into a multi-enzyme complex. Instead, individual enzymes such as malonylCoA :ACP transacylase could be recognizod (Etemadi and Josse, 1972; Josse et al., 1973).Both enzymes could be separated (Berset and Etemadi, 1973a) and could also be distinguished from acetyl-CoA:ACP transacylase and caprylyl-CoA:ACP transacylase (Berset and Etemadi, 1973b). It is difficult to reconcile these findings with those of Bloch, but earlier work from the latter’s laboratory did report the isolation of an acyl-carrier protein from M . phlei 356 which was chemically related to the acyl-carrier proteins found in other bacteria, but this material did not function in de novo fatty-acid synthesis from acetyl-CoA but only in the elongation of palmityl-CoA to C,, and C,, products (Brindley et al., 1969;Matsumura, 1970;Matsumura et al., 1970).This elongating system is, however, quite independent of, and separate from, the elongating ability of the fatty-acid synthetase complex already discussed (Vance et al., 1973a).Clearly the results of Etemadi are concerned with de novo fatty-acid synthesis, and it may be possible that the instability of the synthetase complex in M . phlei 356 becomes exaggerated in another mycobacterium to the point where quaternary structure is not retained after disruption of the cells. If this is so, examination of the disaggregated fragments from the fatty-acid synthetase complex should reveal the presence of acyl carrier protein. Allied to fatty-acid biosynthesis is the biosynthesis of substituted aromatic acids such as 6-methylsalicylic acid. This acid occurs in M . phlei both in the free form (Ratledge and Winder, 1966) and as part of mycobactin P (Snow, 1970),and also in the mycobactins from M . aurum and M . thermoresistible (Snow, 1970) but not in other mycobacteria where salicyclic acid is found instead (Ratledge and Winder, 1962, 1966; Snow, 1970). 6-Methylsalicylic acid is synthesized from acetate and malonate in plants and fungi by an enzyme complex which is similar in organization to, but which can be distinguished from, the enzyme complex involved in fatty-acid biosynthesis (Dimroth et al., 1970; Kannangara et al., 1971). In M . phlei a similar route based on acetate also exists (Hudson et al., 1970; Dain and Bentley, 1971). K. Bloch (personal communication) has confirmed that crude extracts of M . phlei ATCC 354 and M . phlei ATCC 10142 (both bonaJiderepresentatives of M . phlei) incorporate acetyl-CoA and malonyl-CoA into 6-methylsalicylate, but those from M . phlei ATCC 356 cannot, thus indicating that this organism is not M . phlei. As this particular organism produces mycobactin S, it is evidently a strain of M . smegmatis (Hendren, 1974). Mycobaccterium phlei has no capacity to synthesize salicylic acid even when presented with the immediate precursors which, as has been shown

THE PHYSIOLOGY OF THE MYOOBAOTERIA

141

with extracts of M . smegmatis, are chorismic acid and isochorismic acid (Marshall and Ratledge, 1971, 1972). Further details concerning these aromatic acids are given in Section V (p. 195).

3. Nature of the Polysaccharides Involved in Fatty-Acid Biosynthesis Ballou and his coworkers have isolated and characterized a 6-0methylglucose-containing lipopolysaccharide (MGLP) from M . phlei and also M . tuberculosis and M . smegmatis (Saier and Ballou, 1968a,b, c; Keller and Ballou, 1968). The molecule from M . phlei (sic.)ATCC 356 contains 18 hexose units with various acyl groups (acetyl, propionyl, isobutyryl, octanoyl and succinyl) attached. The number of succinyl groups ranges from zero t o three moles/mole MGLP and thus can give rise to four forms of the material (Gray and Ballou, 1972; Smith and Ballou, 1973). The structure of the molecule, shown in Fig. l l a , is thought to assume an unusual helical conformation with the succinyl groups esterified to one end of the polysaccharide and is, perhaps, chelated to a metal ion which is tucked inside the helix (Smith and Ballou, 1973).Although the order of the acylgroups on the polysaccharide is as yet unknown, biosynthetic studies have shown that they are not randomly distributed (Narumi et al., 1973; Grellert and Ballou, 1972; Tung and Ballou, 1973). The second polysaccharidefrom M . phlei (sic.)ATCC 356 which stimulates fatty acid synthesis was isolated simultaneously by Gray and Ballou (1971)and Ilton et al. (1971).The molecule (MMP)is characterized by its content of 3-O-methylmannosewhich occurs along with mannose in a 5 : 1 molar ratio. Acyl groups do not appear to be attached to the molecule. The structure has been partially elucidated by Gray and Ballou (1971), and the most reasonable structure is that of a nona- or deca-cyclic polysaccharide (see Fig. 1 lb). The manner in which the polysaccharides influence fatty acid biosynthesis has already been briefly discussed. The properties of both polysaccharides are such that they could readily associate at membrane interfaces or align themselves between the hydrophobic and hydrophilic surfaces of proteins. The recent report of Silcock and Tove (1973) of a polysaccharide from adipose tissue which stimulates phospholipid synthesis in microsomes may indicate that regulation of lipid synthesis by polysaccharides is not restricted to the mycobacteria. 4. Other Systems of Biosynthesis Two other fatty-acid synthesizing systems have been described in M . tuberculosis. The finst system (Kanesmasa and Goldman, 1965)

142

0.RATLEDOE

x T

II

FIQ.11. Structure of polysaccharides involved in fatty-acid biosynthesis in mycobacteria. (a). Structure of the lipopolysaccharide-containing6-0-methylglucose, showing a helical structure involving chelation to a central metal ion (M),perhaps CaZ+. From Smith and Ballou (1973) with kind permission of C. E. Ballou. (b). Two possible structural forms of the polysaccheride containing 3-0-methylmannose. D - M ~ ~residues o s ~ are indicated by 0; 8-O-methyl-~-mannose residues by 0 . The mannose residues aro linked a (1 --f 2) and the 3-0-methylmannose residues are linked a (1 + 4).From Gray and Ballou (1971).

condenses two molecules of a fatty acid such as octanoate in a head-totail manner to form a fatty acid such as palmitate, while the second system (Wang et al., 1970) elongates long-chain fatty-acyl CoA esters from C, t o C,, using acetyl-CoA in a step-wise mechanism. The first system is apparently unique and its importance in cell metabolism

THE PHYSIOLOGY OF THE MYCOBAUTERIA

143

is obscure as octanoic acid or its coenzyme-A or acyl-carrier protein esters would not be expected to exist within the cell to any extent. The true substrate for this enzyme may therefore be completely different. The second system is very likely allied to the system already described for the elongation of palmityl-CoA, but curiously appears to function only with shorter chain acyl-CoA esters. C. STRAIGHT-CHAIN UNSATURATED FATTY ACIDS Mono-unsaturated acids in mycobacterial lipids have been recognized for some time (Anderson, 1939). Hung and Walker (1970) examined the structure of the mono-unsaturated acids which occur in M . bovis BCG and ill. smegmatis. The acids found (with the positions of their double bond) are given in Table 1. Biosynthesis of unsaturated fatty acids in bacteria usually proceeds by C,-elongation of D(-)/3-hydroxydecanol-acyl-carrierprotein which leads to the formation of cis-vaccenic acid (C,,d I ) and not oleic acid (C1,d9).However, M . phlei and probably other mycobacteria contain a d9 desaturasc which converts stearyl-CoA and, to a lesser extent, palmityl-CoA to oleyl-CoA and palmitoleyl-CoA (Fulco and Bloch, 1964). Some other bacteria, however, also possess desaturase systems of a similar type for production of mono-unsaturated acids (Fulco et al., 1964; Quint and Fulco, 1973). Kashiwabara and Sat0 (1973) have studied the system in Jf. smegmatis in more detail, and have suggested that the desaturase, which is sensitive to cyanide inhibition, is linked t o NADPH via a n FAD-requiring NADPH-cytochrome c reductase. A further but unknown carrier, which is not a cytochrome or a non-haem iron protein, may interpose between the two enzymes. The desaturase may be linked directly to molecular oxygen to complete the reaction : stearyl-CoA + NADPH + H+ 0, -+ oleyl-CoA NADP+ 2H20. As the system is very sensitive to reagents which react with sulphydryl groups (Fulco and Bloch, 1964), this could be an indication that the substrate fatty acid is attached to the desaturase via a covalent bond between the carboxyl group of the substrate and the thiol group of the enzyme. Such a linkage, it has been argued, would explain why the desaturase always functions a t the distance along the fatty acid chain, i.e. a t carbon atoms 9 or 10 (see Quint and Fulco, 1973). This would certainly explain the occurrence of some of the unsaturated acids in mycobacteria (see Table 1 ) but, to explain the occurrence of the CL0d1',C2,dL3,C2,d'5 and C,,dl' acids, C, elongation of oleic acid is the obvious pathway. It would be interesting to know if this elongation could occur on the same fattyacid synthetase complex used for elongation of saturated acids. The occurrence of several isomers of tetra- and pentadecenoic acids in M .

+

+

+

TDLE 1. Mono-unsaturatedfatty acids identiiied in Mywba&&cm megmatis and M y w bacterium bo&. From Hung and Walker ( 1970) Mywbacterium megmatia n-tetradec-A9-enoicacid n-hexadec-Alo-enoicacid n-heptadec-A9-enoicacid n-octadec-Ag-enoicacid n-nonadec-Ag-enoic8.cid n-eicos-A1l-enoicacid n-docos-Al3-enoicacid n-tetracos-A15-enoicacid

Mycobacterium bovis n-tetradec-As-enoicacid n-tetradec-A7-enoic acid n-tetradec-As-enoicacid n-tetrad ec-A lo-enoicacid n-pentadec-As-enoicacid n-pentadec-A7-enoicacid n-pentadec-As-enoicacid n-pentadec-Ag-enoicacid n-pentadec-A lo-enoicacid n-hexadec-Ag-enoicacid

n-hexadec-Alo-enoicacid n-heptadec-Ag-enoicacid n-octadec-Ag-enoic acid n-nonadec-A9-enoicacid n-eicos-Ag-enoicacid n-eicos-AlO-enoic acid n-eicos-Al1 -enoic acid n-docos-A13-enoicacid n-tetracos-A's-enoic acid n-hexacos-A"-enoic acid

THE PHYSIOLOQY OW THE MYCOBACTERU

145

bovis is a possible indication of other, as yet unknown, pathways for producing unsaturated acids. Polyunsaturated acids of a unique type have been found in M . phlei. Asselineau et al. (1969) found a homologous series with the general formula : CH,-( CH2),--(CH=CH--CH2--CH2),--COOH where n E 12 or 14 and m = 5 . 6 or 4

The main component of these acids was h e ~ a t r i a c o n t a - A ~ *16s20 *.~~* penta-enoic acid. The acids, given the name phleic acids, have also been found in M , smegmatis but not in two strains of M . tuberculosis or in one strain of M . bovis BCG (Asselineau et al., 1969, 1972).

D. BRANCHED-CHAIN ACIDS Mycobacteria contain a large number of methyl-branched fatty acids (Cason and Miller, 1962; Campbell and Nowaral, 1969; see also Polgar, 1971 for a recent review of this particular subject). They appear to fall into two main categories: iso- and anteiso-acids (i.e. o-and w-1 methyl acids) and acids containing one or more methyl groups located further within the chain. Both types have quite different biosynthetic origins. Iso- and anteiso-acids probably arise fkom leucine and isoleucine which are elongated by successive additions of malonyl-CoA or malonylacyl carrier protein, depending upon the type of synthetase thought to operate (Kaneda, 1963; Lennarz, 1961):

0

II

R-C-S-COA

6 malonyl CoA

R-(CH2-CH2)6-COOH

I

R = CH3-CH-CH2leucine gives D-16.methylpalmitic acid; or

CH3

I

CH3-CHZ-CHisoleucine gives ~-14-methylpalmiticacid.

The other branched-chain acids are mainly tuberculostearic acid (D-10-methylstearic acid), phthienoic acid (2,4,6-trimethyltetraco-A2enoic acid) and mycocerosic acid (2,4,6,8-tetramethyloctacosanoic acid). The last two acids appear to be confined to M . tuberculosis and H.bouis,

146

C. RATLEDGE

and phthienoic acid moreover is only found in virulent strains (Polgar and Robinson, 1951 ;Polgar, 1954a, b ; Asselineau et al., 1959; Asselineau and Bennet, 1964). Tuberculostearic acid, however, is much more widespread having been found in M . tuberculosis, ill.phlei and M . smegmatis (Anderson, 1939, 1941; Lennarz et al., 1962; Jaureguiberry et al., 1964). The acid has also been found in two species of streptomycetes (Hofheinz and Grisebach, 1965). Its synthesis has been studied mainly in M . phlei. It is formed from oleic acid using S-adenosylmethionine as the active donor of the C, unit (Lennarz et al., 1962; Lederer, 1964) but the oleic acid must be esterified to a phospholipid (Jaureguiberry et al., 1966; Akamatsu and Law, 1968, 1970). 0

II

CHzCHzCH(NH2)COOH

0 CH20C-(CHz)7C=C-(CHz)s-CH3

II I R-COCH

0

II

I

I I

+

H H

I

CH,-S+

CH2OPOCH2CHZNH+

I

0Oleyl phosphatidylet~ianolainiiie

S-Adenosylmethionine

00

II

R-C-0-CH

II

CHzCHzCH(NH2)COOH

CHz-C-(CH2)7-C-CH,-(CH,),-CH3

I I

0

II I

I

I1

CH2

+&I

CHzOPOCHzCHzNH~

adenosyl

0-

K

0

0

II

R-C-0-CH

I1

CHZO-C-

I I

0

NADPH

NADP+ H

I I

(CHZ)7-C-CHz-(CHZ)s-CH3 (333

II CHzOPOCHzCH,NH: I 0-

Tuberoulostearyl phosphatidylethanolamine

Because of the variety of homologues based on tuberculostearic acid which occur in mycobacteria, Campbell and Naworal (1969) have suggested that the S-adenosylmethionine methyltransferase may also

147


methylate other unsaturated acids besides oleate, and that chain elongation of tuberculostearate itself may also occur. Phthienoic and mycocerosic acids are probably formed by elongation of a long-chain fatty acid with propionic acid (Gastambide-Odier et al., 1963, 1966; Lederer, 1964).A tentative biosynthetic route is: CH,--(CHZ) .-COOH

-

+ 4CHz-COOH I

CH3 0

0

0

0

CH,-( CHI),-C-CH-C-CH-C-CH-C-C-COOH

I

CH3

II

II

II

II

I

CH3

I

7-T

I

CH,

CH3

NADH NAD-'

CHI--( CH~),-CH~-CH-CH~-CH-CH,-CH-CH-CHz-CH-COOH

I

I

CH3

CH3

I

CH3

I

CH3

Mycocerosic acid

E. MYCOLICACIDS

1. Structures All strains of mycobncteria elaborate a family of high molecularweight a-alkyl-/3-hydroxylfatty acids terms mycolic acids, which were originally discovered by Anderson (1929).These acids are usually found esterified, often to arabinose, as part of the cell wall. Azuma et al. (1974) found that 97% of the lipid extracted from the walls of strain BCG was, in fact, mycolic acid. Other mycolate-containing substances are cord-factor and various glycolipids (Section III.H, p. 154). This subject has been reviewed by Etemadi (1967a, b) and by Polgar (1971). The general formula of a mycolic acid is : OH

I

R,-CH-CH-

I

COOH

RZ

R, is essentially invariant in any group of mycolic acids, and is usually n-C,,H,, for avian and saprophytic strains and n-C,,H,9 for human and bovine strains (Asselineau and Bennet, 1964; Asselineau, 1962; Kusamran et al., 1972). Pyrolysis (Asselineau and Lederer, 1950) of the acid produces a mero-(meros=part)-mycolic aldehyde, i.e. R , CHO, which can be examined more easily (Etemadi, 1964, 1967~).There are many variations within the mero-aldehyde chain and the principal types which have been found are given below.

148

U. RATLEDGE

(a) Unsaturated mycolic acids. Mono-unsaturated C,, acids have been found so far only in M . smegnzatis (Krembel and Etemadi, 1966a, b): CH3-(CHz),74H=CH-(CHz)

17-R

I

OH

I

R = -CH-CH-COOH

I

(CHa)"-CHswith n = 21 or 23

Longer di-unsaturated acids also occur in this species (Etemadi et al., 1964, 1967): Rl

I

CH~-(CH~)I~-CH=CH-(CH~)~,-CH=CH-C(CH,)~~-R

I1

R' = CH3- or H-; R as in I

(b) Cyclopropane mycolic acids. Acids with one or two cyclopropane rings are produced by several species ( M . avium, M . kansasii. M . paratuberculosis, M . phlei, M . smegmatis and M . tuberculosis; Minnikin and Polgar, 1966a, 1967a; Laneelle and Laneelle, 1970; Lamonica and Etemadi, 1967a, b ; Etemadi et al., 1967). The general formula of the dicyclopropane mycolic acid is : CH3-( CH~),-CH-CH-(CH~),-CH-CH-(CH~),-R \ / \ / CHZ CHz

I11

x = 17or19,y=12or14andz=13or 17:RasinI

I n M . smegmatis, one of the cyclopropane rings (that nearest to the carboxylic end) can be replaced by an unsaturated bond (Etemadi et al., 1967). Cyclopropane rings have a specific configuration but this may be either cis- or trans- according to the origin of the mycolic acid (Minnikin, 1966b; Minnikin and Polgar, 1967b).

(c) Other variations. Methoxy- and keto- groups have been located in certain mycolic acids usually as 8 replacement for a cyclopropane ring as in structure I11 (Minnikin and Polgar, 1967b; Laneelle and Laneelle, 1970; Kusamaran et al., 1972). They are usually accompanied by an adjacent methyl group : CH30 either

I 11

-CH-C-

CH30CH3 or

I

I

Iv

-CH-CH-

Mycobacterium phlei has also been shown to contain a mixture of mycolic acids in which the terminal methyl group of the mero-aldehyde chain is replaced by a carboxylic acid group (Markovits et al., 1966). Bowever, such acids are shorter than the usual mycolic acids, and are


149

probably derived by oxidation of a mycolic acid containing an ester link such as that isolated from M . paratuberculosis (Laneelle and Laneelle, 1970): CH,

I

OH

0

C€€S-(CH,) 1,--CH-O-C-(

II

I

CH,),-CH-CH-COOH

I

V

C22H+5

As mycolic acids with shorter chains in the mero-aldehyde part of the molecule and with alkyl groups no longer than C,, in the a-side chain (see structure I , p. 148 and Fig. 12, p. 150) also occur in corynebacteria and nocardia (Etemadi, 1967b), various workers have successfully used these differences as chemotaxonomic characters (Lechevalier et al., 1971a; Kanetsuna and Bartoli, 1972; Mordarska et al., 1972). Of particular value has been the ability to use the nature of the mycolic acid to distinguish between true mycobacteria, nocardia and organisms belonging to the “rhodochrous” complex (Lcchevalier et al., 1971b; Goodfellow et al., 1973; Azuma et al., 1974b; Minnikin et al., 1974).

2. Biosyntheeis The a-side chain together with the a-carbon atom and terminal carboxy group of a mycolic acid are derived from C,, or C,, straightchain fatty acids. The origin of the meromycolic acid on to which the fatty acid is condensed is enigmatic. By a study of the incorporation of [1-’4C] palmitate or [1-l4C] stearate into the simpler corynomycolic acids (foundin corynebacteria), some indication of the likely pathways of biosynthesis has been given (Gastambide-Odier and Lederer, 1959, 1960; Walker et al., 1973). Cell-free extracts of Corynebacterium diphtheriae activated and condensed two moles of [1-l4C]-palmitate by a Claisen-likeprocess to form an ester of trehalose with 2-tetradecyl-3keto-octadecanoic acid (VI) (Walker et al., 1973; Prom6 et al., 1974). CH3-(CH,) ,,-1’CO-CH-’4CO-Trehelose

I (CH2)13 I

VI

II

CH3

Etemadi (1967a, b) has suggested the following scheme to embrace the biosynthesis of these acids in corynebacteria, nocardia and mycobacteria (see Fig. 12). In each case the final condensation will probably proceed as described above by Walker et al. (1973). Elongation of the mero acid by successive additions of C, units does not satisfactorily explain the occurrence of groups such as a cyclopropane ring or an unsaturated bond a t specific points in the mero acid chain and, furthermore, would suppose formation of a fatty-acid

150 nl CH3COOH

-

0.RATLEDQE c16 or

CI8 acid

C16,Clsfatty acids

n2CH,COOH

corynomycolic acids

C,,, C14,C16or CI8 acid

C,,-meronocardic acids

nocardic acids

Cbo-mycolicacids

.1

c24, c26 acid

C,,-meromycolic acid

mycolic acids

FIG.12. Proposed biosynthesis of mycolic acids by a C2 elongation pathway.

chain to a length which is biosynthetically highly improbable. As an alternative, Asselineau et al. (1970) and Bordet and Michel (1969) have suggested that preformed long-chain fatty acids (probably unsaturated) condense together in a head-to-tail manner probably by the system described by Kanemasa and Goldman (1965). Variations of the groups within the mero-acid chain can be subsequently brought about, for example, by methylation using S-adenosylmethionine (Etemadi, 1967a, c) thus suggesting that the various mero acids which can be isolated from a single species represent a series of related biochemical intermediates (see Fig. 13). Three CI8fatty acids

1 1 1

(i) elongation?/desaturation

(ii) activation

(iii) condensation

CH,) 1,-CH=CH-(

CH3-( CH,) i,-CH=CH-( C26HdZO2 C H d C H 2 ) 17--CH=cH-(CH2)

-1

14--CHEICH-(CHz)

2 S-adenosylmethionine

CH,--(CHz)1 ,--CH--CH-(CH2) \/

CHI

CH2)17-CO OH

-1

14-CH-CH-(CH2) \ / CHI

17-CHOH-CH-

17-CHOH-

I

COOH

CR-COOH

I

C24H49

FIG.13. Proposed biosyuthesis of mycolic acids by a head-to-tail condensation pathway,

151


F. EFFECTOF ISONIAZID ON MYCOLICACID BIOSYNTHESIS Although a variety of proposals have been advanced (see reviews of Winder, 1964; Youatt, 1969) to account for the mode of action of the highly potent antitubercular drug, isoniazid (isonicotinic acid hydrazide ; Pig. 14) the most recent and most convincing evidence suggests that its tuberculocidal action is due to an inhibition of mycolic acid biosynthesis (Winder and Collins, 1970; Takayama et al., 1972 ;Wang and Takayama, 1972). Winder and Collins (1970) showed that, within one hour exposure of M . bovis BCG or M . tuberculosis H37Ra t o 0.5 pg and 1.0 pg isoniazidl ml, respectively (concentrations about equal to their minimum inhibitory concentration), complete inhibition of mycolic acid biosynthesis occurred as judged from incorporation of 14C from [‘4C]glycerol into mycolic acids. Incorporation of carbon into other cell fractions was unaffected. This effect was not observed with an isoniazid resistant strain. Takayama et al. (1972) confirmed these findings, and subsequently showed that uptake of isoniazid into sensitive cells was cxtremely rapid and, with 0.3 pg isoniazid/ml, mycolic acid biosynthesis was inhibited by 50% in 40 minutes’ exposure (Wang and Takayama, 1072). With 0.9 pg isoniazid/ml, 50% inhibition was achieved in 10 minutes exposure. Preliminary data with cell-free extracts of M . smegmatis suggest that isoniazid may be acting in the earlier parts of the pathway of mycolic acid biosynthesis (Winder and Tighe, 1973) but other preliminary work suggests that it may be the synthesis of the high molecular-weight lipids which may be affected (Takayama and Schnoes, 1974). Related to the structure of isoniazid are two further antitubercular drugs, ethionamide and isoxyl (Pig. 14), both of which affect M . tzcbercuZosis similarly to isoniazid (Rist, 1960; Schnefer, 1960) and have also been found to act as inhibitors of mycolic acid synthesis (Winder et al., 1971). As there is no cross-resistance produced between ethionamide and isoniazid, their sites of inhibition are not identical. It is of interest that other antimycobacterial agents, streptomycin, p-aminosalicylic acid and CONHNH

NH-C-HN Isoniazid (isonicotinic acid hydrazide)

II

S Ethionamide Isoxyl [Thiocarlidc] (2-ethylthioisoni~otinamide) (4,4’-bis(isopentyloxy) thio carbamilide)

FIU.14. Structuralformulae of three antitubercularagents.

152

(1.

RATLEDOE

or ethambutol, had no effect on mycolic acid synthesis (Winder et al., 1971). I n attributing mycolic acid biosynthesis as the target for the mode of action of isoniazid, it is relevant to point out that the observed effects: (i) are extremely rapid; (ii) can only be reversed with difficulty (Takayama et al., 1974); (iii) are brought about by low concentrations of the drug; and (iv) can account for the specificity of isoniazid towards mycobacteria. These criteria are not fulfilled by alternative proposals, the main one being that isoniazid interferes with nicotinic acid function, to which it is structurally related, by inhibiting NAD+ metabolism (Zatman et al., 1954a, b) through an activation of NADase (Bekierkunst and Bricker, 1967; Sriprakash and Ramakrishnan, 1970). Although NADase in M . tuberculosis does increase in activity, this is only when relatively high concentrations of isoniazid ( 5 to 10 pg/ml) are used. Furthermore, this enzyme is absent from extracts of M . bods BCG which is equally sensitive to the effects of isoniazid, and hence it cannot be a universal target site for isoniazid (Winder and Collins, 1969). Inhibition of mycolic acid biosynthesis will obviously cause damage to the cell wall and ultimately degeneration of cell structure, and such morphological changes have been observed by scanning electron microscopy of cells treated with isoniazid (Takayama et al., 1973b). The effects brought about by isoniazid help to explain many of the earlier observations which were made concerning the action of isoniazid. Thus the increases in the contents of soluble carbohydrate in treated cells (Winder et al., 1967; Winder and Rooney, 1970a),the loss of acidfastness (Middlebrook, 1952), decrease in hydrophobicity (Youatt, 1965),extractability of triglycerides (Winder and Rooney, 1968)and the increased ease of preparing cell walls from isoniazid-treated cells (Winder and Collins, 1970)can all be attributed to a collapse of the wall structure.

G. NEUTRAL LIPIDSAND PHOSPHOLIPIDS In many mycobacteria, triglycerides constitute the major neutral lipid of the unbound lipids (Vilkasand Rojas, 1964;Winder and Rooney, 1968). Other neutral lipids may include additional fatty-acid esters and various mono- and diglycerides (Smith et al., 1960), but these may be artefacts due to the action of unchecked lipases during extraction procedures (see p. 136). Waxes also occur in M . tuberculosis (Asselineau, 1962;Wang et al., 1972)and in most human and virulent bovine strains, a wax of phthiocerol dimycocerostate also occurs (Smith et al., 1960). The principal mycobacterial phospholipids are cardiolipin, phosphatidylethanolamine, phosphatidylinositol and a series of mannophosphoinositides(Fig. 16;Asselineau and Lederer, 1960; Pangborn and

THE PHYSIOLOQY OP THE MYCOBACTERIA

153

Mannose

I

0

II

OH

I

CH3(CH,)n-C-O-CHz

II

0

O

H

I [Mannose],

FIQ.15. Structure of mannophosphoinositidesfound in mycobscteria. n = 14 or 16 (usually);z = 0 to 4 giving mono- to pentamannosides. Linkages are (1 + 6) up to z = 3 and the h a 1 mannose is linked ( 1 + 2).

McKinney, 1966; Pangborn, 1968; Saier and Ballou, 1968a, b, c; Bahrs, 1972; Sasaki and Takahashi, 1972). Some phosphatidylglycerol, phosphatidylserine and lysophosphatidylethanolamine occur in small amounts (Labelle and Walker, 1973). Phosphonolipids (i.e. with a C-P bond replacing the C-0-P of a phospholipid) have been isolated from 31.tuberculosis (Raghupati Sarma et al., 1970) and the presence of a glyceryl ether in the phospholipid fractions has been suggested (Chandramouli et al., 1971). Biosynthesis of triglycerides and phospholipids (save for the mannoinositides) has not been studied in much detail in mycobacteria. The positional distribution of the fatty acids on the triglyceride and phospholipid are similar in the 1-and 2-positions (Walker et al., 1970).Position-2 is usually occupied by residues of palmitic acid, and long-chain acids (Cz0to CZ6)only occur on the 3-position of glycerol and hence are found in triglycerides only. Tuberculostearic acid only occurs a t position 1 of phospholipids which is in keeping with earlier observations that this acid is almost exclusively confined to the phospholipids (Brennan and Ballou, 1967; Akamatsu and Law, 1968; Walker and Howard, 1968). Mannophosphoinositides (Fig. 15) have been found in mycobacteria (Lee and Ballou, 1964) and related genera such as corynebacteria (Brennan and Lehane, 1971; Khuller and Brennan 1972a), nocardia (Pommier and Michel, 1973; Khuller and Brennan, 197213) and propionibacteria (Brennan and Ballou, 1968b). The structure, as given in Fig. 15, may be further complicated by two additional acyl groups being attached (Pangborn, 1968) of which one is probably on the 6-position of a mannose residue (Brennan and Ballou, 1968a).Sasaki and Takahashi (1972) separated two dimannophosphoinositides from M . tuberculosis containing different fatty-acid substituents, and showed that each compound gave an individual serological response when tested with sera from patients with tuberculosis.

154

C. RATLEDQE

Biosynthesis of these materials has been elucidated by Brennan and Ballou (1967, 1968a),Hill and Ballou (1966), Takayama and Goldman (1969, 1970), Takayama and Armstrong (1971) and Takayama et al. (1973a), and the pathway as far as is currently known is summarized inFig. 16. 2 GDP-Mannose 2 phosphoryldecaprenol

-I

2 Mannosyl- 1.phosphoryldecaprenol

Phosphatidylmyoinositol

d

Phosphatidylmyoinositol dimannoside (dimannophosphoinositide)

--I

AcYI-COA

Acylated dimannophosphoinositide Acyl-CoA

3 Mannosyl-1-phosphoryldecaprenol

Diacylated dimannophosphoinositides

Acylated pentamannophosphoinositide

FIQ.16. Pathway of biosynthesis of mannophosphoinositides in mycobacteria.

H. GLYCOLIPIDS Mycobacteria contain a number of quite different glycolipids. The three main types are cord-factor (6,6’-dimycolyl-cr-~-trehalose ; Fig. 17a),acyl glucoses such as 6-corynomyco~y~-a-~-g~ucose (Fig. 17b) and a series of mycosides (A, B, C) having no common structure (Fig. 18). Related to cord-factor is an unusual sulpholipid (Fig. 17c). Cord-factor, although originally isolated from virulent mycobacteria (No11 et al., 1956), has since been isolated from most mycobacteria (Azuma and Yamamura, 1962, Azuma et al., 1962) and esters with nocardomycolic acid and corynomycolic acid have also been found in nocardia (Ioneda et al., 1970; Yano et al., 1971) and corynebacteria (Lederer and Pudles, 1951; Senn et al., 1967). The properties of cordfactor are discussed in detail in Section 1V.C (p. 167).

THE PHYSIOLOGY OF THE MYUOBACTERIA

CH,O-mycolate

H

155

OH

(a)Cord-factor (6,6’-dimyco~yl-~-~-trehalose) CHI

I

0 (CH,), II I1

I I

CH,0C-CH-CHOH-(CH2)n-CH3

HO H

I

H

H OH for Corynebacterium diphtheriw n = 14; m = 13 (80%) and 11 (20%); for Mycobacterium megrruztk n = 14 (70%) and 12 (30%);m = 3

(b)Acylglucose ( 6 corynomycolyl-a-D-glucose) From Brennan et al. (1970b). 0

II

HO

H

R-C-O-CH2

II

0 1 C15H3,-

(a) Sulpholipid (NH,SL-I) from Mycobaoterium tubercuzosb H37Rv. From Goren

et aZ. (1971). FIG.17. Structure of glycolipids found in mycobacteria.

156 (1. RATLEDQE Other esters of trehalose include those with phleic acid residues (see p. 145). These esters differ in the number of estersed hydroxyl groups which may be up to eight in number (Asselineau et al., 1972).The complete “lipid P” has a molecular weight of 4400 daltom and has a high degree of unsaturation (40 double bonds). The routes of synthesis of the various acylated trehaloses are unknown. Winder et al. (1972) concluded from turnover studies that free trehalose could not be a precursor unless there was more than one intracellular pool of this disaccharide. Alternatively, acyltrehaloses may be synthesized from trehalose phosphate, which is a precursor of free trehalose (Matula et al., 1971), and which is synthesized from glucose 6phosphate plus UDP- (or GDP-) glucose (Lapp et al., 1971; Elbein and Mitchell, 1975). Trehalose 6-monomycolate, recently isolated from M . tuberculosis (Kato and Maeda, 1974),may be a possible intermediate in this pathway. Acylglucoses occur only when glucose is used as the sole carbon source (Brennan et al., 1970b) and are also found in several Gram-negative bacteria (Smith and Mayberry, 1968; Brennan et al., 1970a, b). Brennan et al. (1970b) isolated 6-corynomycolyl glucose from both C . diphtheriae and M . smegmatis (Fig. 17b) and an unidentified 6-acylglucosefrom M . bovis BCG. Traces of palmitic acid and stearic acid were found in hydrolysates of all of the acylglucoses. It was thought that these compounds were unlikely to be precursors or degradation products of cord-factor as they could not be found in glycerol-grown cells. As they occurred in place of triglycerides, it was proposed that they may function as storage compounds. Although glycolipids containing sucrose have recently been found in corynebacteria and nocardia, spp., grown on sucrose, similar compounds were not found in M . avium (Suzuki et al., 1974). Glycosyldiglycerides (mainly mono- and di-glucosyldiglycerides) occur in M . smegmatia (Schultz and Elbein, 1974)and may be involved in cell-wall polysaccharide formation. Palmitate and oleate were identified as the two main substituents on the glycosyl moieties. Very little is known of the biosynthesis of these materials (see Shaw, 1970). In a preliminary communication, Winder and Tighe (1973a) found that coenzyme-A was not needed for acylation of glucose although ATP was beneficial. Schultz and Elbein (1974) showed that UDPglucose was the preferred source of glucose for synthesis of the glycosyl &glycerides. Mycosides are specific to the mycobacteria and occur in a variety of structures (Fig. 18a). Although they are mainly found in photochromogenic mycobacteria ( M . lcansasii: mycoside A; M . marinum (syn.) batmi : mycoside G ;and M . scrofulaceum (syn.) marianum : mycosides C ) and bovine strains (mycoside B ; see Goren, 1972), a lipid closely related to myoosides A and B has recently been found in several attenuated

167


(a). Mycosides A and B. From Oastambide-Odierand Sarda (1970) and Gastambide-Odieret al. (1966)

OCH, CH2)+-CH-CH-CH,-CH~ I

&O~(CH~),,-CH-CHz-CH-(

I

I

,&,&,

I

CH3

Acyl = palmityl and mycocerosyl

rnycrn.de A : n = I6 to 20; sugar is a trisaccharide of 2-O-methyIfucose, 2-0-methylrhamnose and 2,4-di-0-methylrhamnose m y c o d e B : n = 14 to 18; the sugar is 2-0.methylrhamnose

(a). Myooside C, from Mycobacteriurn scrofulaceum. CH,-(

From Vilkas el al. (1968).

CH,)zz-CH=CH-CH-CONH-CH-CONH-CH-CONH-CH-

I

OCH,

I

YHz

I

CH3-CH I

I

CONH--CH-CHZ

I

CH3

m 3

yI I

3,4-di-O-methylrhamnose

FIU.18. Structures of mycosides found in mycobecteria.

strains of M. tuberculosis (Goren et al., 1974b).The structure of this lipid is the same as mycoside A (or B) but without the terminal sugar. The acyl substituents are still residues of mycocerosic acid and palmitic (or stearic) acid. Mycosides of the C-type present a greater number of individual variations than mycosides of the A or B type, and are better described as peptidoglycolipids. As an example, the structure of mycoside C, from M . scrofulaceum is given in Fig. 18b. For the mycoside C,, from M. butyricum, the terminal sugar is replaced by 2,3,4,-tri-O-methylrhamnose (Vilkas and Lederer, 1908). For other variations see Goren (1972). Although little is known of the origins of these materials, all of the amino acids so far isolated are of the D-configuration.

J. CAROTENOIDS Several recent and excellent reviews have appeared on microbial carotenoids (Batra, 1971;Goodwin, 1972;Liaaen-Jensen and hdrewes, 1972). Most pigments detected in mycobacteria have been shown to be carotenoids, or related compounds, and the expression of pigmentation has formed a useful taxonomic oharacter (Runyon, 1969) although,

158

U. RATLEDOE

because of the variability of the pigments in some strains, this may not be an entirely reliable feature (Gordon and Rynearson, 1963). Carotenoids have been found to be widely distributed among most of the saprophytic mycobacteria, but have also been isolated from M . tuberculosis (Takeda and Ohta, 1944) and M . Zeprae (Takeda and Ohta, 1940). The major carotenoids are listed in Table 2 ; several other carotenoids have been found but usually only in trace quantities (see Tanaka et aZ.,1968b; Tarnok and Tarnok, 1972; Goodwin, 1972; David, 1974a). There does not appear to be any single carotene which is universally abundant in mycobacteria. /?-Caroteneis the main component found in M . kansasii and M . marinum (David, 1974a),lycopene the major component of the red Ill. Zycopenogenes (Tarnok and Tarnok, 1971) and 4keto-y-carotene the major pigment of M . smegmatis (Tanaka et al., 1968b). Carotenoid synthesis can be stimulated in several mycobacteria, in particular M . kansasii and M . marinum, by a short exposure to light (Rilling, 1964; Batra and Rilling, 1964; Wayne and Doubek, 1968), that is the organisms are known as photochromogens. I n other mycobacteria, carotenoids are produced irrespective of exposure to light (e.g. M . phlei, M . smegmatis). Several mycobacteria, however, are classed as non-chromogenic, e.g. M . xenopi, M . avium, A!. gastri and M . terrae (see Runyon, 1959; Cross and Goodfellow, 1973), but it is probable that they too, contain a t least some traces of carotenoids. Minute amounts of coloured polyenes have even been recovered from mutants which have been isolated as apparently devoid of pigmentation, e.g. M . lcansasii var. album (see Tarnok and Tarnok, 1972). Just as “non-chromogenic” mutants can be isolated, there have been several reports on increased carotenoid production following treatment of M . phlei with ultraviolet radiation or N-nitrosomethyl urea as mutagens (Konicek and Malek, 1967; Voznyakovskaya and Daraseliya, 1972). Production of different geometric isomers of lycopene in M . phlei after mutation has also been reported (Hochaannova et al., 1971). Improved production of carotenoids has been reported by growing M . megmatisonn-alkanes (C,,-C,,;TanakaetaZ., 1968a);themaximum concentration of carotenoids attained in this work was about 0.3 mg/g cell dry weight. The events leading to photo-induction of carotenogenesis have been discussed by Batra (1971).More recently Johnson et al. (1974) using an untyped species of mycobacterium have shown that the key enzyme, which is wholly dependent upon an exposure to light before it shows activity, is prephytoene pyrophosphate synthetase (see Fig. 19). Synthesis of geranylgeranyl pyrophosphate synthetase ,the immediately preceding enzyme, is increased several-fold by photo-induction, but

TABLE2. Distribution of principal carotenoids in mycobacteria B-Carotene

M . smegmatis (syn. lacticola) M . phlei

y -Carotene

M . kansasii M . marinum (syn. balmi) M . aquae M . scrofulaceum M . aurum M . intracellulare M . phlei

[-Carotene 4-Keto-y-Carotene Leprotene (isorenieratene)

M . phlei M . smegmatis M . phlei

Lycopene

M . leprae M . tuberculosis M . phlei M . smqmatis (syn. lacticola) M. lycopenogenes

Neurosporene Phytofluene Xanthophylls

M . marinum M . smegmatis (syn. lacticola) M . phlei M . phlei

M. smegmatis (syn. lacticola)

Goodwin (1972) Chargaff and Lederer (1935) Ingraham and Baumann (1934) Tarnok and Tarnok (1971) Tarnok and Tarnok (1971) Tarnok and Tarnok (1971) Tarnok and Tarnok (1971) Tarnok and Tarnok (1971) Tarnok and Tarnok (1971) Ingraham and Baumann (1934) Chargaff and Lederer (1935) Goodwin (1972) Tanaka et al. (196813) Grundmann and Takeda (1937) Goodwin and Jamikorn (1956a, b) Takeda and Ohta (1940) Takeda and Ohta (1944) Goodwin and Jamikorn (1956a, b) Grechushkina et al. (1968) Tarnok and Tarnok (1971) Tarnok (1972) Tarnok and Tarnok (1971) Goodwin (1972) Goodwin and Jmnikorn (1956a, b) Schlegel (1958, 1959) Goodwin and Jamikorn (1956a, b) Grechushkina et al. (1968)

c-l

cn W

160

(1.

RATLEDQE

mevalonic acid

isopentenyl pyrophosphate (C,)

dimethylallyl pyrophosphate (C,)

geranyl pyrophosphate (Clo)

1 1 1

farnesyl pyrophosphate (CIS) geranylgeranyl pyrophoaphnte synthetase

geranylgeranylpyrophosphate ( C l 0 ) prephytoene pyrophosphate synthetase

prephytoene pyrophosphete (C4o)

1 phytoene

phytofluene

(-carotene

neurosporene

lycopene

xanthins

1

a-carotene

y-carotene

1%

4-keto-y-carotene

pcarotene

leprotene

FIG.18. Pnthway for bioaynthesis of carotenoida in mycobaateria. Key enzymes for photo-induotionare indioated.

THE PHYSIOLOGY OB THE MYUOBAUTERIA

161

synthesis of the enzymes necessary for synthesis of the C,, polyprenylpyrophosphates and isopentenyl pyrophosphate is unaffected. Somewhat similar, though less-definite, conclusions have been reached by David (1974b) working with M . kansasii. Antimycin A rather than light can stimulate carotenoid synthesis in M . marinurn (Batra et al., 1971). Although antimycin A is also an inhibitor of the electron-transport sys . tem at the cytochrome level (seeSection VI.C, p. 208), there appears to be no connection between these two events. The function of the carotenoids is not entirely certain (see LiaaenJemen and Andrewes, 1972) particularly as “non-chromogenic” mutants can easily be isolated which show no loss of growth capacity. However the most firmly substantiated role is that of photoprotection (Liaaen-Jensen, 1965) and the ability to produce carotenoids would therefore be only of advantage when the mycobacterium grows in an open environment. Indeed, Tsukamura (1963a, b) found that strongly pigmented strains M . kansasii (scotochromogens)were better able to withstand the damaging effects of ultraviolet radiation than Iess-pigmented strains. Konicek and Malek (1967),however, found no significant differences among scotochromogenic and achromogenic strains of M . phlei in their sensitivity to ultraviolet radiation.

IV. Growth in

Pivo: Interactions Between Invading Mycobacferia and

Host Tissues

I n this section, consideration will be given to the changes brought about in the host tissue as a direct result of the invading mycobacteria and to the processes which must take place within the mycobacteria to enable them to survive within the host. A detailed analysis of the defensive mechanisms of infected animals against mycobacterial infections is, however, strictly outside the scope of this review, but a discussion of the factors involved in the development of acquired immunity and allergic responses in animals is given as a great deal of knowledge of the causative agents from mycobacteria is now available.

A. MACROPHAQE-BAUTERIA INTERACTIONS AT THE ONSETOF INFEUTION Several recent reviews and monographs have appeared concerning the general physiology of the macrophage (Bowden, 1971;Vernon-Roberts, 1972;Carr, 1973).The role of the macrophage, specificallyin tuberculosis, has been reviewed by Berthrong (1970) and Joseph and Voisin (1972). Mycobacteria enter the host tissue usually by inhalation into the lungs where they tend to remain because of their preference for an environment where the oxygen tension is high. The invading bacteria are rapidly

162

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phagocytosed by the macrophages. Early work sometimes showed that migration of macrophages could occur in vitro, but this phenomenon could not be readily or reproducibly demonstrated (Harris, 1953). However, the more recent studies of Symon et al. (1972) have shown that migration of the macrophage to the sites of infectionis caused by generation of a chemotactic factor@)which, with M . tuberculosis at least, is dependent upon the presence of serum. Presumably there are materials within the serum which become activated by a bacterial factor (termed a cytotaxigen by Keller and Sorkin, 1967), which then stimulates migration of the macrophages towards the bacterium. Although several mycobacterial fractions (proteins derived from culture filtrates of M . tuberculosis, lipid-extracted cells and a cytoplasmic fraction from M . tuberculosis) were all active in causing movement of macrophages, it could not be resolved whether this was because all of the fractions contained a single cytotaxigenic substrate or whether each contained a separate active material. Preparations of Wax D were inactive when tried in such tests. Production of the chemotactic factor from guinea-pig plasma by M . tuberculosis was prevented by pretreatment of the plasma with several complement inhibitors or by heating the plasma (Symon etal., 1972). DonaldandPound (1971,1973) also foundthat injectionsofa hard wax fraction from tubercle bacilli into rabbits cause a proliferation and migration of reticulo-endothelial cells. These latter results, however, were achieved only by using extremely high amounts (10 mg) of material which are probably far in excess of those likely to be produced during an infection and, therefore, may have only limited significance. I n comparison, the results of Symon et al. (1972) were achieved using only 10 pg material/ml of test solution but, as this was against isolated macrophages and not the whole organism, one must again ask if such concentrations are likely to be attained during the early stages of an infection. Aggregation of macrophages around the site of infection often continues until a giant cell is formed. This is the classical “tubercle” or granuloma which will also contain epitheloid and lymphoid cells. Formation of the tubercle can, perhaps, be seen as the culmination of the chemotaxis of many macrophages, and a great deal of work has been done to attempt to clarify which mycobacterial material is the principal cause of it. As granuloma formation is a defence mechanism of the body and results in an increased resistance to re-infection, this aspect of the tissues’ response to an invasion of mycobacteria is discussed more fully in relation to cellular immunity and hypersensitivity (see Section IV.D, p. 170). Suffice to say at this point that granuloma formation can be induced not only by heat-killed cells of M . tuberculosis (Youmans and Youmans, 1964) but by specific fractions from mycobacteria. Mycobacteria, within the macrophage, are enclosed by the endolytic

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vacuoles or phagosomes. If the bacteria are damaged or non-viable, secondary lysosomes then fuse with the phagosomes to produce digestive vacuoles or phagolysosomes within which degradation of the ingested material takes place (Cohn and Pedorko, 1969) probably by means of proteases and other enzymes (Kondo and Kanai, 1973 ;Kanai and Kondo, 1972b). However, the pathogenic tubercle mycobacteria are well-adapted intracellular parasites, and their ability to survive digestion has been attributed by Armstrong and Hart (1971) to the bacteria somehow preventing fusion of phagosome with lysosome rather than to their ability to resist the attack of the hydrolytic enzymes of the phagolysosome, as was suggested by Leake and Myrvik (1970) who employed M . megrnatis as a test organism. Dead, damaged or non-virulent bacteria cannot prevent formation of the phagolysosome, and they consequently undergo digestive degradation (Hart and Armstrong, 1974). This view must be amended slightly when M . lepraemurium (and presumably M . leprae) is the invading species. I n this case, fusion of the secondary lysosomes and phagosomes is not prevented (Brown and Draper, 1970; Hart et al., 1972) and yet the bacteria are still able to survive and multiply (Chang et al., 1067). Protection of M . lepraemurium and M . leprae against lysosomal enzymes is presumed by Brown et al. (1969) and Brown and Draper (1970) to be by the large lipoidal zone or capsule which surrounds the bacteria (see Section II.B, p. 126). A similar but not as extensive a zone occurs with M . tuberculosis (Dumont and Sheldon, 1965; Merckx et al., 1964; Armstrong and Hart, 1971) and affords some resistance against enzyme attack, but, in the main, the leprosy bacilli and virulent tubercle bacilli have developed different methods of achieving intracellular growth. The ability of tubercle mycobacteria to multiply within the macrophage depends upon the virulence of the strain used, but this can be modified experimentally by using certain surfactants which act upon the host cell rather than the bacteria (Hart, 1968). The bacteria, a t best, grow relatively slowly but, if left unchecked, will eventually destroy the host cell. The liberated bacteria will be rephagocytozed by other macrophages and the process can continue until the bacteria become widely distributed a t many foci and ultimately will cause death of the host animal (see Dannenberg et al., 1972).

B. GROWTH OF MYCOBACTERIA in vivo

1. General Observations The supply of nutrients, which includes oxygen, to any bacterium within a phagosome is of obvious importance if multiplication of that bacterium is t o proceed. Conceivably, transport of nutrients from the tissue

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to within the bacterium could be the rate-limiting step in its development, and the more a bacterium insulates itself against the degradative action of the lysosomal enzymes, e.g. by synthesis of thick lipoidal walls, the slower becomes its rate of growth. The entire subject of in vivo nutrition of bacteria when within the phagocytic cells of the host is probably the largest single area of ignorance in the whole of our knowledge concerning the physiology of the mycobacteria. Clearly this is a crucial area where knowledge should be sought as it ia only by understanding the true behaviour and requirements of the bacteria when growing in vivo that shall we learn how to prevent their multiplication and, hopefully, how to cause their death. The nature of the compounds used as intracellular nutrients for tubercle bacilli is unknown. Lecithin has been suggested (Kanai and Kondo, 1972a) as one such compound, it being a common component of both the host cell membrane and egg yolk which is often used as a potentiating nutrient (i.e. improves the growth but is not strictly “essential”) for direct isolation of tubercle bacilli from pathologic specimens. As mycobacteria grown in vivo are coated with host-derived acid phosphatase (Kanai, 1967; Kanai and Kondo, 1970), phosphorylated compounds (e.g. carbohydrates and nucleotides) possibly could be hydrolysed and the products adsorbed by the cells as sources of carbon and phosphate and sometimes nitrogen. The true purpose of this phosphatase is, however, unclear. Cholesterol, which has been found in M . tuberculosis (Younger and Noll, 1958) and M . lepraemurium (Sakurai and Skinsnes, 1971) grown in vivo, probably originates from the host (Asselineau, 1962), but it can be esterified with fatty acids (Razin and Shafer, 1969; Schubert et al., 1969; Tume and Day, 1970). The degree of esterifhation progresses with the development of the tubercular infection in mouse lungs (Kondo et al., 1971; Kondo and Kanai, 1974). Whether cholesterol can also be degraded in either M . tuberculosis or N.1epraemuriurrL to supply small carbon fragments for cell synthesis is not known. The cholesterol acyl esters, which are formed, will almost certainly alter the conformation of the mycobacterial cytoplasmic membrane (Nes, 1974) and the changes in its permeability which may follow could be to the advantage of the bacteria, Other host lipids, such as triglycerides or small quantities of free fatty acids, may also be used for growth by some pathogenic mycobacteria (McCarthy, 1974). Several studies have sought to compare the metabolism of mycobacteria grown in vivo with those grown in vitro (see reviews by Hanks, 1968; Bekierkunst, 1968a; Kanai and Kondo, 1974). One of the more interesting observations appeared t o be a complete lack of cytochromes (measured spectrophotometrically) within M . bovis var. BCG and M . lepraemurium grown in vivo (Kusaka et al., 1964). This supposed de-

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ficiency was used to explain why these bacilli, when isolated, had no respiratory response to glucose or its intermediates (Segal and Bloch, 1956; Segal, 1964; Brezina et al., 1967), and Hanks (1968) constructed an elaborate hypothesis suggesting that the in vivo mycobacteria had a “leaky” membrane structure which allowed metabolites to enter and leave the cell by simple diffusion. Subsequent and more careful work has, however, shown that, with at least M . lepraemuriunz, a complete sequence of cytochromes does exist (Mori et al., 1971 ;Ishaque and Kato, 1974; Kato et al., 1974). The earlier failure t o detect cytochromes in this organism, and presumably M . bovis also, is attributed to the cytochromes being in a totally reduced state. Bubbling with oxygen for several minutes was needed to restore them to their oxidized form in whole cells (Ishaque and Kato, 1974). The concentration of the cytochromes in the in wivo-grown bacilli is lower than in bacilli grown in witro probably because of a decrease in the amount of iron available to the cells (see p. 191). This certainly occurs when M . megmatis is grown in vitro with low concentrations of iron (C. Ratledge, unpublished work) and also in various other organisms such as Corynebacterium diphtheriae (Righelato, 1969), Hydrogenomonas eutropha (Drozd and Jones, 1974) and Candida utilis (Light, 1972; see also Coughlan, 1971, for review). Changes in the availability of iron to the bacteria will, of course, lead to changes in their metabolism such as an increased dependence upon glycolysis as noted by Schade et at. (1968) with Staphylococcus aureus, and similar changes for in vivogrowing M . tuberculosis have been described by Segal and Bloch (1956) and Brezina et al. (1967). Ramifications from changes in energy metabolism could obviously be widespread. There are, however, almost certainly further adaptations of tubercle bacilli to an in vivo environment (Segal, 1964) to account for all of the changes which are observed, but these changes are only phenotypic as they can be easily reversed by a single passage through culture medium (Segal, 1967). 2. Acquisition of Iron

The supply of iron, and indeed other trace metals, to mycobacteria when in the host tissue is as much of a problem to the organism as the acquisition of other nutrients. There is, however, reason to suppose that it is the supply of iron to mycobacteria which limits their growth rate, and which could even be an essential feature of their pathogenicity. Several reports have detailed that an injection of iron into animals infected with a micro-organism can increase the growth rate of the microorganism and hence increase its virulence (Bullen et al., 1968 ;Bullen and Rogers, 1969; Rogers et al., 1970; Elin and Wolff, 1974; see also reviews

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0.RATLEDOE

by Weinberg, 1970, 1971, 1974; Bullen et al., 1972, 1974; and Glynn, 1972). Szabo (1971) demonstrated similar results for infections of M . tuberculosis in mice, and also showed that administration of 8-hydroxyquinoline to the animals could afford some protection a2ainst the bacteria. This agent, presumably, chelates with iron and renders it unavailable to the bacteria. Similarly Fletcher and Goldstein (1970) found increases in the numbers of M . fortuitum infecting mice after intravenous injections of iron. Iron, however, when given as a complex with a dextrin of high molecular weight, could not exacerbate the infection probably indicating a failure of the large molecule to reach the site of bacteria. Thus, the supply and acquisition of iron appear to be of paramount importance to mycobacteria growing in vivo. It is perhaps of some relevance in this matter to note that, in cases of untreated pulmonary tuberculosis, patients become anaemic and the iron content and total iron-binding capacity of the serum diminish (Tani, 1965, 1970; Kokkola and Tani, 1972) thus again suggesting an association between the state of the disease and the iron concentration in the body. However, the relationship between anaemia and tuberculosis cannot be explained solely by the tubercle bacilli merely depriving the body of iron, as the amount of iron involved must be only a small percentage of the total in the body. The role of iron in bacterial infections, with particular emphasis on the mycobacteria, has been reviewed by Kochan (1973). Kochan et al. (1969) have shown that growth of M . tuberculosis, in an experimental medium containing a high concentration of blood serum, was limited solely by the availability of iron. The main tuberculostatic agent within the serum was transferrin, which is the iron-binding protein usedin the release of iron from ferritin (the store of iron with animal tissues). Transferrin appeared to chelate all of the iron which otherwise would be available to the mycobacteria, and this could also be imagined to occur in the in vivo situation. Specific compounds therefore have to be synthesized by mycobacteria to gain release of iron if they are to succeed as intracellular parasites. Kochan et al. (1971) attempted to correlate this anti-transferrin compound with mycobactin which has been well established as a very powerful iron-binding compound produced by almost all mycobacteria when grown (in vitro) under iron-deficient conditions (Snow, 1970). However, for Kochan’s view to be correct, mycobactin synthesized by the mycobacteria must diffuse from out of the cell into the surrounding environment in order to chelate the iron held by transferrin (or even ferritin). This, in fact, can only occur when a surfactant is included in laboratory medium, otherwise the lipophilicity of the mycobactin molecule is sufficiently high to keep it entirely as an intracellular molecule (Ratledge and Marshall, 1972). I n the absence of such


167

factants in the milieu of the macrophage, mycobactin cannot function in the manner proposed and the acquisition of iron must be by other means. Other known extracellular iron-binding compounds produced by mycobacteria are*salicylic acid, and sometimes 6-methylsalicylic acid (Ratledge and Winder, 1962,1966)and a recently discovered oligopeptide containing e-N-hydroxylysine, termed ochelin (Machan and Ratledge, 1975). These compounds, like mycobactin, are produced in highest concentrations during iron-deficient growth, and seem more fitted to serve as the in vivo iron-chelating compounds because of their much higher solubilities in water. The iron bound to these compounds would be passed to the mycobactin remaining within the boundary layers of the mycobacteria, and thus acquired for growth and development. Further details concerning iron metabolism are given in Section VB4(b), p. 192.

C. TOXICITY OF INVADING MYCOBACTERIA The pathological effects resulting from mycobacterial infections are well known (see, for example, Skinsnes, 1968).Attempts have been made to equate these effects with the production of a definite toxin in the same way as has been established for infections of, for example, clostridia, staphylococci and streptococci. Middlebrook et al. (1947) and Dubos and Middlebrook (1948) drew attention t o the fact that production of intertwining “serpentine” cords of mycobacteria, growing in vitro, was confined to virulent strains, and that the material responsible for this was located on the periphery of the cell. A subsequent search for this material which could be associated with toxicity led to the finding of two materials, one termed “cord-&tor” (Bloch, 1950; Bloch and Noll, 1953)which was identified as trehalose 6,6’-dimycolate (Noll et al., 1956) (Fig. 17a, p. 155),and the other as a family of sulpholipids (Middlebrook et al., 1959; Fig. 17c, p. 155). Of these two materials, only cord-factor has received any subsequent justification for its anticipated toxicity but even this is still not established without equivocation. The sulpholipids, although initially implicated as being toxic (Middlebrook et al., 1959; Ito et al., 1961; Gangadharam et al., 1963)when sufficiently purified, have no toxicity even when injected a t high doses of 50 to 100 pg/injection (Goren, 1970). Kato and Goren (1974a, b) found that the sulpholipids may act synergistically with cord-factor to enhance its toxicity. Goren et al. (1974a) have also shown that, in an examination of 40 strains of M . tuberculosis taken from patients with tuberculosis, elaboration of the sulpholipid by a particular strain correlates very closely with its virulence as measured towards the guinea-p g. Phthiocerol dimycoserosate, although

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thought to be associated with virulence, was also found to be widespread among attenuated strains (Goren et al., 197413).

1. Toxicity of Cord-Factor Bekierkunst (1968b, 1971) showed that extremely small quantities (1 to 5 pg) of purified cord-factor elicit an aggregation of macrophages in the lungs of mice, following either an intravenous injection or injection into footpads (Bekierkunst et al., l969,1971a),which was identical to that caused by whole bacteria (Bekierkunst, 1968b). Predating this work was the finding that mice could, in fact, be killed by several intraperitoneal injections, each of 5 pg, of this material (Bloch, 1950). Cord-factor, however, is not, as was first thought, confined to only virulent bacteria, and the cord-factors from M . phlei, M . smegmatis, M . fortuitzcm and other atypical strains were equally as toxic as those from virulent mycobacteria when administered to mice (Azuma and Yamamura, 1962 ; Azuma et al., 1962). Although cord-factor, therefore, fulfils the most important criterion of a toxin, namely to be lethal a t very small concentrations and, thus, it may well be reasonable to think of it having this role during the progression of the disease, one must be cautious in attributing the pathogenesis of tuberculosis solely to this material. Studies on the biochemical basis of this toxicity have shown that cordfactor, either in vivo (Kato, 1968; Kato and Fukushi, 1969) or in witro (Kato, 1969, 1970), can modify the membrane system of mitochondria and impair the associated process of oxidative phosphorylation. The vulnerable target would appear to be site I1 in the chain of coupling of electron transport to phosphorylation (see p. 211; Kato, 1970) but very high concentrations of cord-factor (50 to 100 pglmg mitochondrial protein) had t o be used to demonstrate these effects. For the time being at least, one must, therefore, be cautious in equating these effects with the prime cause of cord-factor toxicity although the recent report by Goren et al. (1974a) that sulpholipids can potentiate the toxicity of cord-factor towards the membranes of the phagosome and lysosome may be of considerable importance. If the uncoupling of oxidative phosphorylation a t site I1 is realistic, it could explain the decrease in activity of some NAD(P)+-linked dehydrogenases in the liver of cord-factor-treated mice (Murthy et al., 1967) and perhaps even be the primary cause of the lower concentrations of NAD+ in tissues (Artman et al., 1964; Bekierkunst and Artman, 1962 ; Shankaran and Venkitasubramanian, 1970). Other effects following injections of cord-factor are clearly secondary consequences of its action but may be attributable to a primary inhibition of energy production. For example, the decrease in glycogen and the increase in lipid


169

contents of liver, and the decrease in activity of gluconeogenic enzymes (Murthy and Venkitasubramanian, 1967; Shankaran and Venkitasubramanian, 1970), are clearly interrelated and known to be controlled by the prevailing energy state of the tissue (Atkinson, 1969). Some of these effects brought about by cord-factor are also manifested by injections of whole mycobacteria, again supporting the contention that cord-factor fulfils a strategic role during the course of a tubercular infection. Furthermore, Toida (1973a, b ; 1974)has reported that the decrease in activity of pyrazinamide deamidase in mouse liver is suppressed by a tuberculous infection, and that this can be reproduced using purified cord-factor. Pyrazinamide (pyrazinoic acid amide) is a second-line antituberculosis agent (Crofton, 1969). It is rapidly transformed by a deaminase found in animal tissues into ammonia and pyrazinoic acid, which is then excreted in the urine. Chemical modifications to the structure of cord-factor reveal that the fatty-acid moiety may be varied between wide limits without seriously impairing its toxicity (Noll, 1956; Lederer, 1957),but changing the sugar moiety from trehalose to glucose (i.e. using glucose 1- (or 2-) mycolate) leads to a complete loss of activity. However, sucrose 6,6’-dimycolate and a-methyl-n-glucose 6-mycolate had a toxicity only about three times less than authentic cord-factor and had a limited ability to affect oxidative phosphorylation in mitochondria (Kato and Asselineau, 1971). Interestingly, the naturally occurring trehalose G-monomycolate (Kato and Maeda, 1974) was much less toxic than the dimycolate ester, having an L.D.,, value in mice of 452 pg but was still able to uncouple respiration and phosphorylation a t site 11. Further work with semisynthetic cord-factor analogues has indicated which are the important functional groups involved in toxicity. Hydroxyl groups at carbon atoms 2 , 3 and 4 (using stereo-isomers of methyl glucose 6-mycolate) play a definite role, whereas the glycosidic linkage a t carbon 1 has no specificity (Asselineau and Kato, 1973). Evidence against cord-factor being an important agent in the pathogenesis of tuberculosis, notwithstanding its occurrence in non-pathogenic species, is that, although a precipitating and neutralizing antibody to cord-factor can be produced in mice and rabbits by vaccination with a methylated bovine-serum albumin complex with cord-factor (Kato, 1972), a similar precipitin antibody in man or animals infected with virulent tubercle bacilli cannot be detected (Kato, 1973a). This would indicate that the antibody against cord-factor may not be associated with an infcction with virulent bacteria and thus cord-factor may have no connection with the pathogenesis of tuberculosis. Further work is still obviously needed t o verify this conclusion although without doubt cord-factor is a highly toxic material.

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D. CELLULARIMMUNITY AND DELAYED HYPERSENSITMTY These two processes may be described briefly as follows. Immunity is the acquired resistance to the disease (tuberculosis, leprosy and other mycobacterioses) in which the macrophages have been activated, have proliferated and possess an enhanced capacity to destroy further invasions of bacilli. Activation of the macrophages results in a n increase in their content of lysosomal (i.e. digestive) enzymes and perhaps other bacteriocidal properties (see Dannenberg, 1968; Dannenberg et al., 1968; Shima et al., 1972). In tuberculosis, hypersensitivity takes the form of a delayed allergic response which is defined as an immunological state in which lymphocytes and macrophages show a sensitivity to tuberculin or materials containing tuberculin-like substances. Hypersensitivity to components other than tuberculin, however, can be generated and distinguished from the tuberculin-type; this is discussed on p. 176. There is still a good deal of controversy as to whether acquired immunity is just another manifestation of delayed hypersensitivity. The two processes certainly develop simultaneously following an initial infection with an attenuated strain of a mycobacterium. Mackaness (1964, 1967, 1968, 1971) and Dannenberg (1968) would argue that cellular immunity is the same as delayed-type hypersensitivity on a small but multifocal scale. I n other words, when the tuberculin skin test is done, the reaction which follows is a manifestation of the host’s cell-mediated reaction to a massive dose of the antigen. And when there is an infective focus within the tissues, the cell-mediated reaction which follows is a reaction directed primarily a t the antigen but ultimately a t the organism which is carrying the antigen. This view is, however, challenged by the demonstration that each phenomenon can be elicited by different mycobacterial substances, and thus are independent responses of the host tissue (see Youmans and Youmans, 1969b; h a c k e r et al., 1969c; Takahashi, 1969; Berthrong, 1970; Kanai and Kondo, 1972a). It is not, however, the intention here to attempt either a reconciliation of these different points of view or to take up one side or the other in this issue and, although in the discussion which follows the two phenomena are kept separate, this is for convenience and simplicity.

1. Immunity Immune responses of thc cell-mediated type, rather than the humoral type, are widely accepted to play a major part in determining the eventual outcome of tuberculosis in human subjects. The outcome of leprosy would also appear to be so governed (Rees, 1970a; Turk and Bryceson.

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1971; Choyce, 1972). Aspects of cell-mediated immunity have been recently reviewed by several authors and these should be consulted for detailed discussions of the cellular mechanisms which are involved (Laskin and Lechevalier, 1972; North, 1973; World Health Organization, 1973). It has been recognized for many years that, once animals including man are infected with tubercle bacilli, they become resistant to reinfection and that macrophages from immunized animals have a higher ability t o inhibit the intracellular growth of mycobacteria than those from unprotected animals (see, for example, Lewis and Sanderson, 1927; Lurie, 1939; Mackaness, 1954).Furthermore, protection can be conferred by using, as a deliberate primary infection, viable or even non-viable cells of attenuated mycobacterial strains such as M . bovis BCG and M . microti (Rich, 1951; Rosenthal, 1957; Weiss, 1959a, b, 0). When various isolated fractions of mycobacteria were first shown to be immunogenic (see Crowle, 1958) the search began to elucidate which compound, or compounds, were responsible for this phenomenon. No clear answer has yet been obtained in spite of the considerable efforts of several groups of workers. One of the main difficultiesis the diversity of methods used in the testing and evaluation of the various compounds (see Smith et al., 1971). Confusion may also arise if there is more than one type of cell-mediated immunity as has been indicated by the work of Coppel and Youmans (1969)and which is discussed on p. 180. Even the strain of mouse or other animal used in experimental tuberculosis can be an important factor as some strains are clearly more susceptible to infection than others (Youmans and Youmans, 1972b);rats, in particular, are highly resistant t o tuberculosis (see Lefford et al., 1973). The principal compounds which so far have been implicated as immunogens will now be discussed. (a) Cell-wall fractions. Ribi and his coworkers (Ribi et al., 1967, 1971; h a c k e r et al., 1967, 1969a, b ; Brehmer et al., 1968) have shown that oil-treated cell walls from strain BCG given intravenously afforded a, high level of resistance against airborne infection in mice and monkeys (Fig. 20). Although the animals vaccinated with cell walls failed to gain weight during the period immediately after vaccination, after challenge with airborne virulent M . tuberculosis, the cell-wall vaccinated monkeys gained more weight than the BCG-vaccinated animals and the nonvaccinated controls. The wall preparations could be inactivated by extraction with organic solvents or treatment with alkali, but activity could be restored by recombination with Wax D (p. 120; h a c k e r et al., 1969b). From these results Ribi (1971) suggested that the mycobacterial cell walls, though inactive themselves, contain important components

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U. RATLEDGE 3.5

-

I

-1

PCO\,

n

0

2

4

4

Challenge

Vaccination 6

8 10 12 Time (weeks)

Autopsy

14

16

I8

FIG.20. Protection of rhesus monkeys against tuberculosis using isolated mycobacterial cell walls. Rhesus monkeys vaccinated intravenously with cell walls of Mycobacterium .bovis or with viable Mycobacteriunz bovis BCG and challenged with 21 viable units of Mycobacteriuna tuberculosis H37Rv by an airborne route. Non-vaccinated control, ; vaccinated with whole viable cells of strain BCG, o ; with cell walls of Mycobacteriuna bovis, 0 ; with cell walls of strain BCG, a. From Ribi et al. (1971).

of the full immunogen, and that Wax D (and the oil with which all these preparations must be mixed with to show protection) was functioning mainly as an adjuvant. These roles may, of course, be reversed. Wax D could be the main immunogen, as has been proposed by White (1967), and a component of the walls the adjuvant as indicated by Adam et al. (1974).Further work (Anacker et al., 1973)has indicated that several lipid fractions, including Wax D and cord-factor preparations, all have a common component which upon further fractionation yields four fragments, two of which, when again mixed with inactivated cell walls, could protect mice against infection by airborne virulent tubercle bacilli. The chemical structures of these two purified components have been determined as cord-factor and a related glycolipid (Azuma et al., 1974; Ribi et al., 1975). Thus the active principle may be cord-factor itself. (b) Cord-factor. The toxicity of cord-factor and its ability to induce granuloma (i.e. tubercle) formation in mice have already been discussed (Section IV.C, p. 168). Cord-factor when given to mice intravenously can protect mice against a subsequent challenge of virulent M . tubercu-

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Zosis also given intravenously (Bekierkunst et aZ., 1969). If cord-factor is given intraperitoneally or via the footpad, no protection results probably because diffusion of it to the various organs of the body, which may be vulnerable to a subsequent infection, is necessary for immunity to be established. Indeed, acquired resistance to tuberculosis seems to be of a local character (Dannenberg et al., 1968; Bekierkunst et al., 1971b). One must, however, be cautious about extrapolating results obtained with experimental animals to humans as the spread of bacilli (and probably individual mycobacterial fractions also) is known to be by different routes (Pierce et al., 1953; Ratcliffe and Palladino, 1953). Kato (1973b)showed that, when rabbit serum containing an antibody to cord-factor (induced by vaccination with a methylated bovine-serum albumin complex with cord-factor; Kato, 1972) was passively transferred into mice, it conferred resistance against infection with virulent M . tuberculosis. However, this resistance was quite different from that caused by live BCG vaccine in that the latter material does not produce antibodies to cord-factor (Kato, 1972). This evidence has already been used to argue (p. 169) against cord-factor being involved as a toxin during a tuberculous infection and can, of course, also be used against cord-factor’s involvement in immunogenicity. Supporting this contention is the failure of cord-factor to induce granuloma formation in rabbits (Moore et al., 1972)or guinea pigs (see Bekierkunst and Yarkoni, 1973)although living strain BCG can do so. I t is thought by Bekierkunst and Yarkoni (1973)that, in these latter animals, the substrate to which cord-factor and other granulomagenic substances (like Wax D) are bound is important to the induction of granuloma. Thus, cord-factoritself cannot be the immunogenic substance though in combination with other substances, as suggested by the work of Meyer et aZ. (1975a, b) and Ribi et al. (1975),it may be an answer to the search. (c) Ribonucleic acid. An extensive examination of the immunogenesis of tuberculosis has been conducted by G. P. Youmans and A. S. Youmans and their colleagues. Part of their early work (see Fig. 21) established that living avirulent M . tuberculosis were several hundred times more effective as immunizing agents than comparable doses of heat-killed cells, implying that the immunogenic agent is heat-labile. During the ensuing investigations they have presented extremely strong evidence supporting mycobacterial RNA as the specific immunogen (see Youmans and Youmans, 1969b).When RNA is carefully prepared from ribosomal fractions to avoid degradation, its immunogenic activity is similar to that obtained with viable cells of M . tuberculosis. As little as 0-5 pg protects up to 60% of vaccinated mice (Youmans and Youmans, 1966a, b, 1969a). Although these preparations contain

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C. RATLEDOE

1001

1

80

Amount of H37Ro cells in mg. moist weight

FIQ.21. Immunizing capacity of living and heat-killed two week-old cells of Mycobacterium tuberculosis H37Ra (avirulent) for mice against infection with Mycobacterium tuberculosis H37Rv (virulent). Intraperitoneal vaccination with livingcells, 0 ;with heat-killed cells, A. Mice were infected via the intravenous route with 1.0 mgvirulent cells (4 x lo7viable units) four weeks after vaccination. From Youmans and Youmans (196913).

approximately 65% RNA and 35% protein, the protein when separated from the RNA was not itself immunogenic (Youmans and Youmans, 1971) whereas the RNA, freed of protein by enzymic digestion, can be fully immunogenic (Youmans and Youmans, 1966b; also unpublished results quoted in Youmans and Youmans, 1974). This concept is supported by the results of workers who have shown that other homologous preparations of RNA from Salmonella typhimurium (Venneman, 1972), Staphylococcus aureus and Pseudomonas aeruginosa (Winston and Berry, 1970), Diplococcus pneumoniae (Thompson and Snyder, 1971) and Neisseria meningitidis (Thomas and Weiss, 1972) are highly immunogenic. The mechanism by which mycobacterial RNA may induce immunity has begun to be investigated. Active sites on the RNA can be masked by polyamines (Youmans and Youmans, 1972a). Ribonucleic acid must exist in a double-stranded form to be active (Youmans and Youmans, 1970); after treatment with pancreatic ribonuclease, which hydrolyses single- but not double-stranded RNA, the main ribosomal component essential for immunogenesis sediments a t 23 S (i.e. has a molecular weight of approx. 450,000 daltons; Youmans and Youmans, 1973). Other evidence (Youmans and Youmans, 1974) suggests that the mechanism whereby mycobacterial RNA exerts its effect could be similar to that used by RNA tumour viruses (see Green, 1970). Work is in progress t o elucidate if mycobacterial RNA can transform the macrophages and

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other cells to be resistant to further infection and, in this connection, the results of Fong et al. (1963) and Oprescu et al. (1972) are of obvious interest. These workers have each shown that ribosomal RNA taken from the macrophages of immunized animals can passively transfer resistance to other macrophages both in vitro and in uiuo. Sato and Mitsuhashi (1965) have demonstrated that the same processes occur to produce resistance against salmonellosis. (d) Other substances. Takhashi (1969) has suggested that mycobacterial polysaccharides can act as immunogens in rabbits and guinea pigs. Tuberculin and other proteins, phosphatides and Wax D were ineffective agents in his test system. Although the polysaccharides which were examined were not fully purified they contained little nitrogen or phosphorus. Some support for Takahashi’s conclusions comes from the earlier work of Crowle (1968, 1964) and Hedgecock (1965). However, none of the preparations, including those of Takahashi, is any more effective, and in many cases somewhat poorer, than cells of BCG used as a comparative immunogenic control. Tsumita et al. (1970) have described a partially-purified, watersoluble protein fraction which is immunogenic for mice and guinea pigs. As diaminopimelic acid was not detected, the protein does not originate from the peptidoglycan backbone of the wall. However, this material contained about 3% polymannophosphoinositide (p. 153) and 5% of a polysaccharide containing arabinose, mannose and galactose and, therefore, contributions to the observed activities of the material may not be due entirely to the protein component.

2. Hypersensitivity The usual type of hypersensitivity which is discussed in connection with mycobacterial infections is the allergic reaction of sensitized hosts to tuberculin. This usually takes about two days after an injection of tuberculin for the reaction to be fully expressed, and hence is usually termed delayed-type hypersensitivity. This subject, which has applications well removed from the field of tuberculosis, has been reviewed in depth in a monograph by Turk (1967). The questions to ask in this connection would therefore seem to be: what agent or agents are involved in the initial sensitization? ; what is the nature of the antigen tuberculin? ; and, can hypersensitivity be induced by any materials other than tuberculin? (a) The agents involved in initial sensitization. The initial sensitization of the host, mediated through the macrophages, can be by whole cells,

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either virulent or attenuated, viable or killed, or can be by using tuberculin itself. If tuberculin alone is used (Raffel, 1948; Raffel et al., 1955) it must be given along with Wax D which acts as an adjuvant for the antigen (seeSection IV.E, p. 178). Neither tuberculinnor Wax D preparations are active when given alone. Although various workers have since examined Wax D, and related structural components, for their ability to induce delayed hypersensitivity, any positive results so obtained (Choucroun et al., 1960; Kourilsky et al., 1965) seem abtributable to contamination of the lipid with small amounts of protein (Tanaka et al., 1967; Koga and Pearson, 1973) and extensive examinations show that Wax D contains little or no detectable tuberculin-sensitizing material (Tanaka et al., 1971; Ishibashi et al., 1971; Koga and Pearson, 1973). Thus, tuberculin appears to be the unique antigen which can sensitize a host for subsequent demonstration of classical hypersensitivity. This sensitization is extremely specific for, although a type of hypersensitivity can be generated by mucopolysaccharides sharing some common component with tuberculin itself, this latter hypersensitivity is manifested only to a subsequent exposure of the same material and not to tuberculin itself (Koga and Pearson, 1973). Polysaccharides, lipopolysaccharides and mannophosphoinositides are, however, most certainly antigenic (see, for examples, Schaefer, 1940; Cummins, 1962; Kotani et al., 1971; Pangborn, 1968; Khuller and Subrahmanyam, 1971, 1972), but recent studies have shown that these materials are probably not involved in the production of delayed hypersensitivity (Birnbaum and Affronti, 1969). The occurrence of antibodies to these compounds, including tuberculin, in patients sufferingfrom tuberculosis is also welldocumented but, because the immunogens can also be derived from saprophytic or avirulent mycobacteria, there appears to be a widespread occurrence of these humoral antibodies in people who have never given any manifestation of the disease (Bardana et al., 1973). Thus, as yet no single antibody test has been found which is positive in tuberculous patients and is consistently negative in healthy subjects.

(b) T h e nature qf tuberculin. Although tuberculin has long been recognized asaheterogeneousmixtureofproteins (Seibert, 1949; SeealsoLong, 1968) its active principle is still not yet certain. However, Kuwabara and Tsumita (1974) have elucidated the primary structure of a tuberculinactive protein from M . tuberculosis. The protein was a single polypeptide of molecular weight 9700 daltons and contained 89 amino-acid residues with one disulphide bridge (Kuwabara, 1975a, b). It therefore should not be long before this work is extended, and we learn if a particular peptide sequence must occur for a protein to be active like tuberculin. The value of tuberculin as a skin-test antigen for testing patients for


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previous exposure to tuberculosis has been known since the work of Koch (1890). Initially the preparations of tuberculin were extremely variable, but improvements in protein chemistry led to the establishment of a standard tuberculin preparation which is now in widespread use. This preparation is known as purified protein derivative (PPD) which is sometimes qualified as PPD-S to denote it as the recognized standard product. Tuberculin has usually been prepared from culture filtrates of mycobacteria (Augier et al., 1974), but cytoplasmic extracts and cell walls have activity (as a skin test antigen) equal or greater than PPD (Larson et al., 1961, 1966,1968; Counts and Kubica, 1968; Glenchur et al., 1973) and the most recent results would equate the antigenic material of the cytoplasm with a protein, or proteins, of the ribosome (Baker et al., 1972, 1973; Ortiz-Ortiz et al., 1971 ; Loge et al., 1974). Baker et al. (1973) have shown by immuno-electrophoresisthat the culture filtrate from M . tuberculosis H37Rv, the cytoplasm, crude ribosomes and the 605 and 30 S ribosomal subunits of M . bovis BCG all contain many common antigens. Furthermore, Loge et al. (1974), working with M . smegmatis, have shown that the most specific agent for provoking tuberculin-type delayed hypersensitivity is a 305 particle which can be stripped of much of its protein to leave a still active 1 6 s particle with only 11% protein; this is the core protein of the original ribosome. Work is still needed, however, to establish that similar antigenic proteins can be isolated from M . tuberculosis or M . bovis BCG. The biological activity that is attributed to the cytoplasm and culture filtrates can, therefore, be traced to a ribosomal origin. It would be interesting to know if the ribosomal core protein could reproduce the interesting effect which Sultzer and Nilsson (1972) and Nilsson et al. (1973) have recently demonstrated for PPD. These workers showed that, in witro, DNA synthesis in spleen cells taken from nonimmunized mice and guinea pigs could be triggered by PPD. The cells which were stimulated into mitosis were the bone marrow-derived (B) lymphocytes. The proliferation of B cells then led to an increased production of non-specific immunoglobulins, thus showing that PPD (and tuberculin) contributes t o the non-specific enhancement of antibody production during natural and experimental tuberculosis infections. The mycobacterial ribosome, therefore, is a most remarkable entity. Its ribonucleic acid is a key factor in the induction of immunity to tuberculosis and its core protein is implicated as the prime source of the antigen eliciting the classical delayed hypersensitivity response. These two important physiological properties are quite distinct, and neither material appears to be involved in the production of the other phenomenon (Neiburger et al., 1973).

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E. OTHERCELLRESPONSES TO MYCOBACTERIA AND ISOLATED FRACTIONS 1. Mycobacterial Adjuvants Many of the immunogenic and antigenic compounds which have been isolated from mycobacteria elicit their effects only when administered in conjunction with an adjuvant. Again, many substances behaving as adjuvants have been isolated from mycobacteria and as some, such as Wax D or cord-factor, have also been implicated as immunogen or antigens, the complexity of the various interactions when crude fractions or whole cells are administered to animals is enormous. Some illumination into this matter, however, is being achieved by the greatly improved ability to isolate homogeneous compounds which exhibit a high specificity of adjuvant activity. A n elucidation of their structure and their relationship to the larger heterogeneous macromolecules will be a major contribution in this area. As the chemical and biological basis of adjuvants has been the subject of an extensive monograph by Jollhs and Paraf (1973), and has also been reviewed in detail by White (1972), only a brief description of their nature and properties need be given here. The importance of mycobacterial adjuvants extends beyond their ability to promote subsequent responses within a host tissue to tubercle bacillus or its various fractions. Promotion of antibody production to an antigen totally unrelated to mycobacteria can occur by injecting into an animal killed mycobacteria suspended in mineral oil and emulsified with water containing an antigen (Preund et al., 1937). A delayed hypersensitivity to the antigen also develops. If the antigen is omitted from the emulsion and “Freund’s adjuvant” (i.e. BCG in mineral oil emulsified in water) only is injected into rats, adjuvant or allergic polyarthritis will develop (Pearson and Wood, 1959). Whole mycobacteria as the adjuvant can be replaced by fractions of the bacteria and, once again, it is clear that there are anumber of materials which can probably act in this way. Kotani et al. (1960) showed that mycobacterial walls could replace whole cells as an adjuvant, and White et al. (1958) found that lipid-extracted mycobacteria could function similarly. Further work highlighted the heterogeneous Wax D (p. 120) as the likely culprit (White et al., 1964). Hiu et al. (1969) found that the content of mycolic acids was important for adjuvant activity of Wax D, and that hydrogenation of the fatty acids improved this activity still further (Hiu and Amiel, 1971). More recently, however, Adametal. (1972, 1973) and Migliore-Samour and Jollhs (1972, 1974) have isolated from mycobacterial walls, lipid-extracted cells and from whole cells several water-soluble fractions which still retain adjuvant activity even though they do not contain mycolic acids. As all fractions show some chemical

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similarity with the basal cell-wall (i.e. Wax D) structure, they can all be considered as being derived from the cell wall. Kamazawa et al. (1972) have also isolated an adjuvant material, described as chromatographically homogeneous, from Wax D. Similar adjuvant materials probably reside in the chemically similar walls of the nocardia and corynebacteria (Azuma et al., 1972).Adam et al. (1974)in further studies have found that the adjuvant function is attributable to the monomeric peptidoglycan of the cell wall (Fig. 22) and have shown that the properties of this material are also given by the corresponding compound from the cell walls of E . coli. The material from E . coli contains N-acetylmuramic acid residues instead of those of N-glycolylmuramic acid, and further work has elucidated that the minimum structure needed for adjuvancy is probably N-acetylmuramyl dipeptide (Ellouz et al., 1974). fi 1+4 N-acetylglucosamine

-

N-glycolylmuramic acid

I I D-glutamateENH2 I L-alanino

meao-diaminopimelicacidaNH,

I

D-ahnine

FIG.22. Structure of adjuvant fraction, the monomeric peptidoglycan, from the cell walls of Mycobacterium smegmatis.

An examination of the properties of the water-soluble adjuvant from mycobacteria walls shows that, although it can also induce hypersensitivity to some antigens such as ovalbumin (Adam et al., 1973),it does not induce hypersensitivity to tuberculin (Chedid et al., 1972) and, as one would anticipate, is completely unrelated to the antigenic ribosomal proteins (Section IV.D.2, p. 177). Other adjuvant materials from mycobacteria which have been recently implicated include cord-factor, which Bekierkunst et al. (1971b) found to stimulate the response in mice to injection of sheep red blood cells, and ribonucleic acid. With the latter material, antibody production to bovine y-globulin could be stimulated (Youmans and Youmans, 19720)as well as the induction of experimental allergic encephalomyelitis (Gumbiner et al., 1973) and delayed hypersensitivity to PPD (Casavant and Youmans, 1975). Synthetic polynucleotides (poly A:U or poly I :C) produced identical responses, and these results are similar to those obtained by other workers using preparations of RNA from a variety of sources (see Beets and Braun, 1971).

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2. Stimulation of Immunity to Other Diseases and the Speci$city of Mycobacterial Immunizing Fractions Animals infected with M . tuberculosis or immunized with attenuated strains of mycobacteria not only show an increased resistance to further infection by M . tuberculosis but are more resistant to infection with unrelated bacteria such as brucella, klebsiella, staphylococcus, pasteurella, listeria, salmonella and the anthrax bacillus (see Youmans and Youmans, 1969a). Resistance can also be produced using strain BCG after methanol extraction (Weiss et al., 1964) or crude cell-wall fractions (Fox et al., 1966; Lederer, 1971, 1973). However, under certain conditions, a difference in protective abilities of certain mycobacterial fractions can be seen. Thus Coppel and Youmans (1969) found that rnycobacterial ribosomes and cell walls were equally effective in generating resistance to infection with K . pneumoniae, but only cell walls produced any protection against L. monocytogenes indicating that immunity can be both of a general type as well as of a specific form. Non-specific immunity stimulated by mycobacteria applies not only to infections with bacteria but also to the development of malignant tumours. Old et al. (1959) were the first to discover this effect; when sarcoma 80 was implanted into animals previously infected with strain BCG, the subsequent death of the animals was delayed much beyond that in animals which had not been inoculated with strain BCG, Weiss et al. (1961) later reported similarly for T-32 mammary carcinoma, and Rees and Potter (1972) have also found that heat-killed M . smegmatis (syn. butyricum) can produce a specific enhancement of transplantation immunity if added along with the tumour transplant. These experimental findings have been successfully extended into clinical trials for treatment of acute lymphoblastic leukaemia and other malignant melanomas (Math6 et al., 1964, 1969; Morton et al., 1970; Davignon et al., 1970; Nathanson, 1972; Sokal et al., 1972; Bast et al., 1974). Several studies have been carried out to elucidate which are the prime mycobacterial compounds responsible (Weiss et al., 1966; Esber et al., 1971 ; Kuperman et al., 1972). Not surprisingly, as a non-specific type of immunity is indicated, the mycobacterial cell wall or associated materials are once more strongly implicated. Cord-factor appears to be the most promising single entity although Wax D has also been reported as being able to stimulate resistance against tumours (Amiel and Hiu, 1971). With cord-factor, Bekierkunst et al. ( 1 9 7 1 ~ 1974) ; showed that it could suppress the development of tumours induced by urethane in the lungs of mice to a similar degree as living strain BCG. Several synthetic analogues of cord-factor (trehalose 6,6’-dipalmitate, sucrose 6,6’-dimycolate, a-methylglucose 6-mycolate, fl-methylglucose 6-mycolate and trehalose

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6-monopalmitate) were all as equally effective as cord-factor itself indicating that neither trehalose nor mycolic acid is essential for the activities observed (Yarkoni et al., 1973). Presumably though a glycolipid structure is needed. Yarkoni et al. (1974) have since demonstrated the ability of cord-factor to repress growth of Ehrlich ascites tumour cells implanted in mice. It seems possible that preparations of cord-factor, which are not totally homogeneous even after purification, and Wax D, however, share some common components which are biologically active. Meyer et al. (1974) isolated a toxic lipid, termed P,, from both sources and found that if this was administered to guinea pigs with a cell-wall skeleton preparation, regression of hepatoma was brought about. If cell-wall skeleton alone was given, the tumours were only suppressed and did not regress. The cell-wall skeleton material still retained the mycolic acid-arabinogalactan peptidoglycan characteristic of the wall structure and Wax D (Azuma et al., 1974a). The most reasonable explanation for repression of tumour growth is that the antigens which are associated with the tumour itself fail to provoke antibody formation within the host tissue. The tumour therefore is “accepted” by the body. However, an injection of strain BCG provokes a general non-specific type of immunity and, as a result, the macrophages are stimulated into a “rejection” of the tumour. Granulomatous reactions (Rios and Simmons, 1972; Mitchell et al., 1973) and antibody production (see Alexander, 1970) eventually lead to the suppression of the tumour’s growth. As these events are the manifestations of nonspecific immunity, agents other than strain BCG can also elicit these responses, although among bacteria strain BCG appears to be the most suitable for immunotherapy (Math6 et al., 1969; Mackaness et al., 1973).

P. GENERALCONCLUSIONS A great deal of confusion still exists over what components of the mycobacterial cell can elicit a particular reaction when injected into host tissue. The seeming ability of some components of the mycobacterial cell to be able to act simultaneously as immunogens, antigens and adjuvants undoubtedly stems from the use of heterogeneous fractions which, though often indicated to be pure) seldom are. At best, one may be dealing with a mixture of like materials. Separation of the individual components of such mixtures is obviously difficult and is probably the stumbling block which limits real progress in understanding the chemical nature of the active compounds. Of course, one must also recognize that true synergistic effects can occur between individual components and several examples of activity being restored to a particular fraction

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(3.

RATLEDGE

when its separated parts are recombined have been quoted. The ability of some components of the mycobacterial cell to act as adjuvants (that is enhancing the activity of other compounds but without themselves being active) alongside other active compounds from the same source complicates an already difficult picture. In spite of all of these difficulties, real progress towards correlating chemical structure and biological activity does appear to be being made currently in several laboratories throughout the World. To attempt a concise summary of the preceding data is impossible. There is strong evidence that mycobacteria can generate a broad nonspecific resistance to many infections and can also produce a much more specific immunity (see Youmans and Youmans, 1969b). These two types of immunity are probably generated by different components of the cell, i.e. a cell-wall component and a ribosomal component, respectively. These two materials must, therefore, be able to elicit different responses in the host tissues. The current view would appear to indicate that the target cell is the lymphocyte which can produce a variety of products each with a different function according to the stimulus it receives (David, 1971). Each lymphocyte product then produces a further reaction in the host cell. Klun and Youmans (1973) have hypothesized that one of these lymphocyte products might be able to stimulate fusion of lysosome and phagosome organelles in the macrophage which Armstrong and Hart (1971) have shown to be a prerequisite for destruction of the tubercle bacilli (see Section IV.A, p. 163). Other lymphocyte products may stimulate delayed hypersensitivity as well as chemotaxis of the macrophages (David, 1971; Nathan et al., 1971, 1973). This latter function obviously accords with the work of Symon et al. (1972) which showed that chemotaxis of macrophages was produced by mycobacteria only in the presence of serum (Section IV.A, p. 162). If it is accepted that there can be more than one type of immunity, then this could help to explain why there has been so much disagreement as to the nature of the immunogenic materials. Different workers obviously adopt quite different criteria for the demonstration of various phenomena and disagreements will undoubtedly persist until some sort of standardization is imposed or accepted. Thus, from several different lines of evidence, the mycobacterial ribosome and the cell wall would appear to be the principal structural components within which are materials which can elicit most if not all of the reactions discussed in this section. If one had to select for further study only three compounds out of all those mentioned here, one would perhaps suggest ribosomal RNA, the core protein/s of the ribosome and cord-factor as being the likely progenitors of specific immunity, delayed hypersensitivity and non-specificimmunity, respectively. This, however,


183

must be stressed as a purely personal view. I n the field of mycobacterial adjuvants, the picture seems to be a t the point of resolution through the current researches of the teams of Lederer, Jollds and Ribi.

V. Growth in nitro A. GENERAL OBSERVATIONS Apart from the mycobacteria belonging to the “non-cultivatable” taxa (see Section V.C, p. 197), most mycobacteria can be grown satisfactorily in simple chemically-defined medium. The rates of growhh of mycobacteria, however, vary enormously from the slow growing M . tuberculosis and related species to the rapidly-growing saprophytes such as M . phlei and M . smegmatis. If grown without any agitation, all mycobacteria grow as crusty pellicles on liquid medium and may take up to 8-10 weeks to reach full growth for M . tuberculosis or as little as 4 or 5 days for M . smegmatis. Given suitable sources of carbon and nitrogen in a medium, improved growth rates can usually be attained by agitation of the cultures particularly with slower growing cultures. Bowles and Segal (1956) found that rotary shaking (200 rev/min; 2-5 cm diam. circle of shaking) shortened the lag phase of M . tuberculosis from 40 days to about 13, and Lenert et al. (1958) and Weiss (1959d) have reported similar improvements, although Ratledge and Winder (1962) found that use of similar conditions made no difference to the growth of M . srnegmatis (see Fig. 23, p. 185). Addition of surfactants such as Tween 80 to medium tends to improve the growth rate of mycobacteria by helping to prevent aggregation of cells. Once clumping begins, free diffusion of nutrients to many cells is prevented, and the growth rate usually approximates to an arithmetical progression. Winder and Rooney (1970b) found that this condition, described as “cellular crowding”, was characterized in M . bovis BCG by a constant carbohydrate content and an increased DNA-to-protein ratio in the cells. However, over a range of growth rates, the RNA to protein ratio remained relatively constant indicating that the growth rate of this species is presumably controlled by factors other than the rate a t which proteins can be synthesized. This does not apply to the faster growing M . smegmatis where the concentration of ribosomes is probably the limiting factor (Winder and O’Hara, 1962; Winder and Coughlan, 1971). The improvement in growth rate by adding Tween 80 is, however, less than that attained by shaking the culture vessels; Weiss (1959d) reported a maximum increase in cell yield after 15 days of growth of M . tuberculosis of only about 30% under either shaken or static conditions. The amount of Tween 80 which is needed is usually about 1%

184

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(w/v), although some workers (e.g. Lyon et al., 1970) found 0.02% was sufficient to maintain dispersion in shake cultures of M . smegmatis whereas others (R. J. White et al., 1971) have found that 4% was needed with the same organism. Tween 80 however is not a passive surfactant and it can affect metabolism and cell composition. Stinson and Solotorovsky (1971) found that Tween 80 could be readily hydrolysed by M . avium and the oleic acid then incorporated into lipids. Tween 80, at 1% in the medium, led to a 45% increase in cell volume and a doubling of the total lipid content. Weir et al. (1972) have shown that the oleic acid may be incorporated into the triglycerides or even exist unchanged as globules with the cytoplasm. Growth of mycobacteria in mechanically-stirred fermenters is somewhat difficult (Weiss 1959d). The main problem to be overcome is that the hydrophobic nature of the cells’ surface prevents true dispersion. Cells flung out of the fermenter tend to adhere to the upper surfaces and thereby provide an even better surface on to which other cells can stick. Thus, medium can be gradually depleted of cells without much growth being achieved. Mycobacterium phlei seems an exception, and reports of it being grown, without Tween 80, in 200-litre fermenters have been given (Brennan and Ballou, 1967). On a smaller scale, M . smegmatis, M . phlei and M . bovis BCG have been grown in one-litre vortex-aerated fermenters of the type described by Marshall et al. (1973). Growth of M . smegmatis under different conditions of agitation is shown in Fig. 23. It will be seen that, although the lag period is not shortened, the rate of growth is considerably faster and the cells attain a much higher final density. The reason for the increase in growth is clearly due to improved dispersion of the cells, and the increase in cell yield is perhaps because of the increased fixation of carbon dioxide due to the continuous aeration of these stirred cultures. Fixation of carbon dioxide is important in the metabolism of mycobacteria (Wherry and Ervin, 1918) and can contribute appreciably to the total cell carbon (Long et al., 1955).

B, NUTRITION, NUTRIENTS AND THEIRASSIMILATION 1. Carbon Sources Long (1958) has reviewed in some detail the carbon and nitrogen sources which mycobacteria can utilize. Without doubt the most important source of carbon for successful growth in vitro is glycerol which can be utilized without exception by all mycobacteria. Other carbon sources such as glucose, fructose, acetate, pyruvate, citrate or propanol can be used by many, but not all, mycobacteria, whereas utilization of sucrose, succinate, malate, malonate, fumarate and butanol appears to be more narrowly distributed (Tsukamura et al., 1960; Tsukamura and

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Time ( h )

FIG.23. Growth of Mycobacterium smegmatis under various conditions of agitation. Without shaking, A ; from Ratledge and Winder (1962): with rotary shaking, o ; from Ratledge et al. (1974) :grown in a vortex-aerated,stirred 1-1itra fermenter, 0 ; K. A. Brown and C. Ratledge, unpublished data. The same medium was used on each occasion, i.e. glycerol + basal salts without addition of Tween 80.

Tsukamura, 1966; Kubica et al., 1972; Meissner et al., 1974). Ability to use these compounds and others is, of course, often important taxonomically and diagnostically. Tepper (1965) has shown that, with M . phlei, growth on glucose or glycerol is at the same rate with respect to DNA and protein synthesis. However, due to uptake of glycerol not reaching saturation at any concentration tested and glucose uptake being saturated a t low external substrate concentrations, the excess intracellular carbon arising when glycerol is the carbon source leads to increased formation of lipids and glycogen storage materials and thus t o higher cell dry weights (Tepper, 1968).

186

0.RATLEDQE

Utilization of hydrocarbons by authentic species of mycobacteria (M. smegmatis, M. phlei, M . fortuitum, M. marinum and M. tuberculosis) was examined by Lukins and Foster (1963). Representatives of all five species could utilize n-alkanes from C,, to CI6,and all except M. tuberculosis could utilize alkanes down to C,. Only M . smegmatis could utilize the gaseous hydrocarbons, n-propane, n-butane and n-pentane, but could not grow on hexane, heptane or octane. Mycobacterium marinum could also grow on isopropylbenzene, n-butylbenzene and tert-butylbenzene. Grange (1974) also examined a number of authentic mycobacteria and found some, including M . smegmatis, could be grown on alkanes as short as hexane. Beam and Perry (1974) found that M . vaccae could utilize not only n-tetradecane, but also n-dodecylcyclohexaneand n-heptadecylcyclohexane,but could not grow on cyclohexane or other cycloparaffinic hydrocarbons. Other hydrocarbon-utilizing mycobacteria have been described, such as M . paraficum (Davis et al., 1956) and M . ceroformans (Krassilnikov, 1971), as well as many untyped mycobacteria (e.g. Popova et al., 1973). These can utilize long-chain alkanes (C1, to C1,) as well as ethane at low concentrations such as may be encountered in a natural environment in certain soils (Brisbane and Ladd, 1972). Whether these are “genuine” mycobacteria is uncertain. Wayne et al. (1971) thought M . paraficum could possibly fit into the M . scrofulaceum taxon, but Krassilnikov et al. (1973) described the mycolic acids of M . lacticolum var. aliphaticum as containing only 30 to 34 carbon atoms. These acids would therefore resemble those which are isolated from corynebacteria (p. 150) and indicate that the isolate is not a mycobacterium. Utilization of carbon sources by non-pathogenic mycobacteria existing in a natural environment outside a host organism appears to have been little studied. Lukins and Foster (1963) found some strains of M . smegmatis,M . marinum and M .fortuitum which could exist as chemolithotrophs using carbon dioxide and molecular hydrogen as carbon and energy sources provided other nutrients including ammonium ions were present. Kazda (1973a, b) found that the potential pathogens M . intracellulare and M . avium could survive and grow slowly on the nutrients in moorland waterpools, and no doubt other mycobacteria could do so as well (Beerwerth, 1973).

2. Nitrogen Sources Various nitrogen sources can be used for growth; some species can Use nitrate or nitrite as sole sources (De Turk and Bernheim, 1968) and this is often used as an identification test for mycobacteria (Virtanen, 1960; Bonicke et al., 1970). Most species can use ammonium salts (Long, 1958) although improved rates of growth are produced using amino

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acids or amides as nitrogen sources. In particular asparagine, glutamate and aspartate are particularly good stimulators of growth (Youmans and Youmans, 1954). Asparagine seems widely preferred as a nitrogen source with both the amino- and amide-nitrogen atoms as well as the carbon being incorporated into cell constituents (Long, 1958, Lyon et al., 1969).However, Lyon et al. (1970, 1974) could show little difference between growth of M . tuberculosis on asparagine or ammonium chloride each at 5 mM; the best source of nitrogen out of several tested appeared to be alanine. As asparagine was utilized relatively inefficiently, disappearing from the culture medium extremely rapidly and, with such low concentrations being employed (the usual concentration of asparagine is about 40 mM), cultures containing only asparagine must have quickly become nitrogen-deficient. Indeed, these cultures probably had insufficient organic nitrogen available even at the onset of growth, as Winder and Rooney (1970b) found that the critical organic nitrogen concentration in the medium was between 15 and 35 mM, below which the growth rate of M . bovis BCG was curtailed. These experiments of Lyon et al. (1970) may therefore not have been adequate tests of the ability of asparagine to act as a nitrogen source, but only served to indicate its effect on the early phases of growth. Further experiments using higher and non-limiting concentrations of various amino acids would obviously be welcome, Tarnok (1970) made the interesting observation that the activity of asparaginase, which has been studied in detail by Andrejew et al. (1974, 1975), was inhibited by glycerol but that utilization of glutamate was not. This result may indicate that the inefficient and rapid utilization of low concentrations of asparagine observed by Lyon et al. (1970) was because metabolism was not proceeding by degradation with asparaginase. Tarnok’s result does, however, clearly explain why the lag period of growth of M . tuberculosis was shortened from 14 days to 10 days when glutamate was substituted for asparagine in glycerolcontaining medium as observed by Bowles and Segal(l965); under such circumstances glutamate is used during the initiation of growth even though glycerol is not assimilated until the growth rate becomes well established. Bowles and Segal(l965) also showed that glucose, if present with other carbon sources, was inhibitory t o growth and that citrate, when added, exerted no effect whatsoever on the growth rate or utilization of the other carbon compounds. Tepper (1965) has also concluded that glutamate is better than asparagine as a source of nitrogen for M . phlei. Interestingly, Lyon et al. (1967)and Sehrt and Iwainsky (19688, 1969)found that, although synthesis of the glutamate-transport systems of M . tuberculosis and M . smegmatis had to be induced, these took only a few hours to be fully expressed. Iwainsky and Sehrt (1968) have indi-

188

C. RATLEDGE

cated that, in M . smegmatis, /3-alanine and y-aminobutyrate may be even better than glutamate or asparagine as nitrogen sources. Amides including urea can be used by some species as a source of nitrogen and in some cases, such as acetamide, as a source of carbon also (Draper, 1967). The ability to hydrolyse certain amides, especially urea, nicotinamide and pyrazinamide, is often used taxonomically (Bonicke, 1959; see also Wayne et al., 1972; Meissner et al., 1974) and extensive studies have been carried out on the factors influencing hydrolysis of aliphatic amides and urea by Sehrt and Iwainsky (196813, 1970, 1972; Iwainsky and Sehrt, 1969; 1971).

3. Inorganic Elements The requirements for the major inorganic elements potassium, phosor magnesium and sulphur (as for maxiphorus (as mum development of mycobacteria has been known for some time (see Long, 1958). Of these four, only potassium does not appear to have been investigated in much detail. The uptake of 32P(as PO,3-) has been examined, and its distribution in some cell constituents (pentose- and hexose phosphates, nucleotides, NAD+ and NADP) has been quantitated (Reutgen and Iwainsky, 1967). Although uptake can be severely inhibited by arsenate and azide, the recognized antitubercular agents (isonicotinic acid hydrazide, paminosalicylic acid, streptomycin, isoxyl and ethambutol) in general had little effect except at very high concentrations (Iwainsky et al., 1968). Uptake of 32Pinto phospholipids, particularly cardiolipin and phosphatidylethanolamine, has also been followed (Akamatsu et al., 1967; Chandramouli and Venkitasubramanian, 1973b). Spitznagel (1961) and Spitznagel and Sharp (1959) found that magnesium depletion caused branching of the cells, and therefore could be implicated in cell-wall synthesis (Webb, 1970). Polyphosphate granules were not formed in magnesium-deficient cultures, although lipid vacuoles could be seen. Deficienciesof sulphate caused a cessation of DNA and protein synthesis, but surprisingly not of RNA synthesis; cells as a consequence failed to divide and became greatly elongated. Similar long forms caused by a deficiency of iron in M . smegmatis have also been observed (Winder and O’Hara, 1962) as well as in E . coli (Ratledge and Winder, 1964). The requirements for trace, or minor, metals by mycobacteria are probably qualitatively no different from those of other bacteria (see Rouf, 1964) and, although not all bacteria require the same amounts of a particular metal (Waring and Werkman, 1943; Weinberg, 1970; Lankford, 1973), presumably all mycobacteria can be expected to have quantitatively similar requirements. The amounts of a trace metal which


189

must be added to a medium to give full growth is obviously dependent upon the amount of that metal already present in the medium as a chance contaminant. Thus, although early workers recommended addition of iron to medium a t 10 pg/ml (Sauton, 1912), this was obviously an empirical amount. A requirement for iron by mycobacteria has, however, been confirmed by several workers (Edson and Hunter, 1943; Goth, 1945, Turian, 1951 ; see also Long, 1958). For M . phlei the amount required for full growth has been quoted as 1.0 pg/ml (Turian, 1951) and 3.75 pg/ml (Edson and Hunter, 1943), and for M . smegmatis and M . bowis BCG 2-0 pg/ml was found adequate (Winder and Denneny, 1959; Ratledge and Winder, 1962) although 1.0 pg/ml may be close to the minimum requirement for full growth of M . smegmatis (Ratledge and Hall, 1971). Zinc is also required by mycobacteria (Drea, 1956; Dekker and Huitema, 1958; Williston et al., 1958; Winder and Denneny, 1959; Sternberg et al., 1964) and, under carefully controlled conditions (i.e. by removing traces of zinc from the prepared medium), the minimum amount required for full growth is between 0.2 and 0.4 pg Znz+/ml (Winder and O’Hara, 1962; Ratledge and Hall, 1971). Manganese is needed by mycobacteria but only in minute quantities, and early recommendations for its inclusion in medium (Dekker and Huitema, 1958; Williston et al., 1958) were based on a “better-be-safethan-sorry” attitude. However, Gonocharevskaya et al. (1 961) found 0.12 pg Mn2+/mlstimulated growth of M . bowis BCG, while Ratledge and Hall (1971) found with M . smegmatis that only 5.5 ng Mn2+/mlwas needed. Without addition of this metal the cell yield decreased about 20 to 50% (Ratledge and Hall, 1971; C. Ratledge and M. J. Hall, unpublished work).Although a requirement for Ca2+was indicated by Edson and Hunter (1943), this has not been substantiated by other workers (Winder and Denneny, 1959; Ratledge and Hall, 1971). Other trace metals (A13+,Ag2+,B033-,Ba2+,Co2+,Cr3+,Cu2+,Moo4’--,Ni2+,Pb2+and V0,-) have been tested for growth-promoting properties (Turian, 1951 ; Winder and Denneny, 1959; Ratledge and Hall, 1971). Although some are undoubtedly required for certain enzymes, the quantities are too small to be quantitatively determined and so their effects on growth are not significant. Ghys (1967) made some preliminary observationfl on uptake of various radioactive isotopes of Fe, Zn, Mn, Co, Sr, Cs, Pm, Eu, Hg and I. All isotopes were taken up at least to some extent by strain BCG and, of particular interest, was the observation that iron uptake was less when high concentrations of iron were used whereas the cells appeared to have an uncontrolled avidity for Zn to the extent that, if sufficient of the metal ions were provided, adsorption would continue until a toxic intracellular concentration was produced.

19G

a. RATLEDOE

4. Trace Metal Metabolism A brief summary of the consequences of trace element deficiency in mycobacteria is given in Table 3. Of the various metals, metabolism of iron and zinc has been studied in the most detail principally for two reasons. First, such studies can give a unique insight into the organization of the mycobacterial cell ; secondly, because these elements are essential for growth and development of the organism (see Section IV.B.2, p. 165), elucidation of how they are acquired may reveal possible target sites for future chemotherapeutic agents, particularly if the means of acquisition are dissimilar from those of the host tissues. These two aspects are quite distinct and can therefore be discussed separately. (a) Work of Winder and his associates. The analytical work of Winder and O’Hara (1966) showed that, with M . smegmatis when growth was limited by a deficiency of iron, the cells contained 64 pg Fe/g dry weight suggesting that this was the lowest concentration of iron with which cells could still function. If iron was not limiting, then the cell content was 224 pg Fe/g dry weight. With zinc, the corresponding contents were 11 pg and 43 pg/g dry weight, respectively. The cells are therefore able to adapt, at least to a certain extent, under conditions of trace-metal depletion but, as subsequent work has shown, this is only at the expense of modifications in cell metabolism which can, in the first analysis, be recognized by changes in cell composition. Winder and O’Hara (1962) reported an increased amount of polyphosphate and ATP in zinc-deficient cells of M . smegmatis suggesting that energy- and phosphate-using reactions were inhibited rather than respiration. In iron-deficient cells, the coproporphyrin content increased probably as a direct lack of iron for its conversion into haem. This is supported by the finding of a low concentration of cytochromes in these cells (C. Ratledge, unpublished work) though Brown (1975) has found a virtual absence of porphyrin from these cells. The main effect, however, of iron deficiencyis a decrease in the DNA to protein ratio (Winder and O’Hara, 1962) which was subsequently found to be associated with marked increases of an ATP-dependent deoxyribonuclease (Winder and Coughlan, 1969, 1971) and a DNA polymerase (Winder and McNulty, 1970). The former enzyme of M . smegmatis was found to be similar to the ATP-dependent deoxyriboCCUS (Winder and Lavin, nucleases in E . coli and D ~ ~ ~ O C Opneurnoniae 1971) and hence was thought to be involved in recombination repair and perhaps also in excision repair. Further work on characterization of the enzyme (Winder and Sastry, 1971; Winder et al., 1973) has provided additional evidence in keeping with this view. The DNA polymerase

TABLE 3. Metabolic consequences of trace-element deficiencies

Element Sulphur

Amount required for full growth (/ml)

Consequencesof deficiency

0-4 mg (as sulphate) Long forms of cell produced; DNA and protein

Magnesium

1-0 mg

Iron

1.0 11g

synthesis decrease Cell branching; polyphosphate granules absent Long forms of cells produced ; DNA synthesis declines Increased activities of DNAse and DNA polymerase Iron-containing enzymes decrease in activity

Reference Spitznagel(l961) Spitznagel and Sharp (1959) Winder and O’Hara (1962)

E!

2

0

zinc

0.4 11g

Increased polyphosphate and ATP levels Zinc-containingenzpnes decrease in activity

Winder and Coughlan (1969,1971) Winder and McNulty (1970) Winder and O’Hara (1964) Winder et ol. (1961) Hudson and Bentley (1969) C. Ratledge (unpublishedwork) Brown (1973) Winder and O’Hara (1962) Winder and O’Hara (1964)

Manganese

0.12 pg 5.5 116

Cell yields lowered Mycobactin synthesis decreased

Gonocharevskaya et aZ. (1961) Ratledge and Hall (1971)

Isofavonoids increase Low content of cytochromes with low content of coproporphyrin

P

w

lMl z 20

F

d +I

m

192

0.RATLEDOE

of M . smegmatis has also properties similar to those of DNA polymerase I from E. coli in having 5‘ + 3’ exonuclease activity suitable for an excision repair function (McNulty and Winder, 1971). The relationship between the increased activities of both enzymes and the decreased DNA content in iron-deficient M . smegmatis was resolved by finding that hydroxyurea could induce the same effects as iron deficiency including that of cell elongation (Winder and Barber, 1973). However, as treatment with nalidixic acid could also lead to a decrease in the DNA to protein ratio without causing an increase in the specific activities of the two enzymes, the two enzymes themselves cannot be the cause of DNA degradation. Instead, iron limitation and hydroxyurea treatment might cause an inhibition of synthesis of ribonucleotide reductase which, in E. coli, is known to contain iron as an essential component (Ehrenberg and Reichard, 1972; Atkin et al., 1974), and thus leads to an alteration in the pool of deoxyribonucleotides.The abnormal DNA which results from this change in precursors induces an increased production of repair enzymes. Thus, the decrease in DNA is a secondary effect of iron deficiency. As a direct effect of metal deficiencies it is not surprising to see many metallo-enzymes drop in activity. Winder and O’Hara (1964) showed that activities of both haem-iron and non-haem iron-containing enzymes were diminished under iron-deficient growth conditions. Under zincdeficient growth conditions, glycerol dehydrogenase and lactate oxidase (lactate dehydrogenase) both declined in activity. Tryptophan synthetase also behaved similarly (Ratledge and Winder, 1961). Although these results suggest the participation of zinc as a cofactor in these enzymes, definite proof of this is still needed.

(b) Iron assimilation and transport. The problem facing all organisms in dealing with iron is the insoluble nature of this metal at physiological pH values, and a variety of iron-chelators have evolved to overcome this problem in micro-organisms (see Lankford, 1973; Neilands, 1973, 1974). The chelators are usually water-soluble and are found extracellularly, being produced in greatly increased quantities if iron is limiting growth of the organism, i.e. they function as scavengers of iron. The ironchelate complex which is formed in the medium is thought to be the form in which the metal is transported into the cell. Mycobacteria however have evolved a different procedure probably because of the very thick lipid-rich layers of the cell envelope. The principal iron-chelating agent in mycobacteria is mycobactin which is a lipid-soluble material and was originally isolated from M . pklei as a growth factor for M . paratuberculosis (Francis et al., 1953) (see Section V.C.3, p. 200). All other mycobacteria which have so far

THE PHYSIOLOGY OJ? THE MYCOBACTERIA

193

been examinedproduce a mycobactin, and the structures of many of them have been elucidated by Snow and his colleagues (Snow, 1970).Structures of the mycobactins are shown in Fig. 24. X-Ray crystallography of ferrimycobactin P has recently been carried out (Hough and Rogers, 1974) which shows that the iron atom is exposed in a splayed V-shape cleft in the mycobactin. An examination of a constructed model of the molecule offers an insight into the exceptionally high stability of the complex and also the ease of release of the iron atom (see below). Mossbauer spectroscopy of mycobactin has also been determined (Spartalian and Oosterhuis, 1974). A n attempt at a chemical synthesis of an analogue of mycobactin has been made though without achieving the correct iron-chelating centres (Carpenter and Moore, 1969). Some synthetic studies on formation of the salicyloyloxazoline moiety of the molecule (Black and Wade, 1972) and on the formation of the hydroxamic acid units (Black et al., 1972) have been made. Studies on the biosynthesis of mycobactin itself have, as yet, proceeded no further than following incorporations of recognizable components of mycobactin, such as lysine (Allen et al., 1970; Tateson, 1970) and salicylic acid (Hudson and Bentley, 1970; Ratledge and Hall, 1970), into mycobactin. Distribution of mycobactins appears to be confined to the mycobacteria although similar materials occur in the closely related nocardia (Patel and Ratledge, 1973; Ratledge and Snow, 1974). Mycobactins do not however occur in the organisms known (erroneously) as "Mycobacterium" rhodochorus (Ratledge and Patel, 1974) or in other bacteria within the Actinomycetales family (Ratledge and Chaudhry, 1971). Mycobactin acts as the prime receptor site for soluble iron coming into contact with the mycobacterial cell (Ratledge, 1972; Ratledge and Marshall, 1972). Insoluble or colloidal iron cannot be accepted by mycobactin (Ratledge et al., 1974). The ferrimycobactin complex which is formed moves through the cell envelope by creation of a concentration gradient and, after the iron has been released, the mycobactin returns across the envelope by the creation of an equal and opposite concentration gradient (Ratledge and Marshall, 1972). The affinity of mycobactin for Fe"' is extremely high (a stability constant in excess of 1030has been calculated; Snow, 1970), and iron has to be released therefore by a specific mechanism which has been found to be a NAD+-linked reductase converting Fell1 mycobactin to Fe" mycobactin (Ratledge and Marshall, 1972 ; Brown and Ratledge, 1975b). The Pel1atom has little or no affinity for the chelate and is able to leave the molecule and be inserted into porphyrins as the final step in haem biosynthesis (Brown, 1975 ; Brown and Ratledge, 1975b). Mycobactin is extremely lipophilic with a very low solubility in water (Snow, 1970) and it resides almost entirely within the cell envelope with

T

r

-0

Organism Mywbacterium aureum Mywbacteriurn fortuitum Mywbacterium thermoresistible Mycobacterium marinum Mywbacterium marinicm Mywbacterium phlei Mywbacterium terrae Mywbacterium smegmatis Mycobacterium tuberculosis

Mycobactin isolated

A F* H M N P R

S T

Rl

R2

13 A 17,11 A 19,17 A 1 2 17 cis A 19 A 17, 15 c k A 19 A

CH3 H CH3 H H CH, H H H

P

Substituents R3 H OH3 CH3 CH3

CH3 H H H H

? R4

R5

CH3 CH3 CH3 C17H35 C17H35 C2H5 CH3 CH3 CH3

H H H CH3 (333

CH3 CH, H H

FIG.24. Structure of the mycobactins. (a) General st,ructure of the ferric mycobactins. (b) Substituents of the various mycobactins. Side chains R, are alkyl groups having the number of carbon atoms shown (double bonds are indicated where known); alkyl groups with different numbers of carbon atoms have been identified in trace quantities. From Snow (1970)with kind permission of the author. *Occurs as mixture with mycobactin H.

P w U

0

m


195

none being detectable in the culture medium (Ratledge and Marshall, 1972). As mycobactin cannot react with insoluble or colloidal iron (Ratledge et al., 1974), the problem is how does the organism solubilize the iron into a form which is subsequently acceptable to the mycobactin? A likely compound for this role is salicylic acid which is produced in increased concentration during iron-deficient growth of M . smegmatis and M . tuberculosis (Ratledge and Winder, 1962 ; Hudson and Bentley, 1970; Antoine and Morrison, 1968). I n M . phlei, however, 6-methylsalicylic acid occurs instead (Ratledge and Winder, 1962, 1966; Hudson et al., 1970) paralleling the aromatic moieties which are found in the respective mycobactins. Salicylate is synthesized via the shikimic acid pathway (Ratledge, 1964, 1969; Hudson and Bentley, 1970) and the two enzymes for synthesizing salicylate from chorismic acid via isochorismic acid are only active when iron is limiting growth, thus confirming its involvement in some capacity in iron metabolism (Marshall and Ratledge, 1971, 1972). That the organism has a requirement for salicylic acid was shown by the isolation of appropriate auxotrophic mutants of M . smegmatis (Holland and Ratledge, 1971 ; Ratledge and Hall, 1972). It was conjectured, therefore, that the function of salicylate was that of an extracellular iron chelator, and experiments using washed suspensions of cells containing mycobactin or model systems using mycobactin dissolved in octanol showed that, of several iron-chelating compounds tested, salicylate was one of the best (Ratledge et al., 1974). However, when similar experiments were carried out in the presence of phosphate ions, salicylate failed to function in this way, instead the iron came out of solution as an insoluble precipitate. Salicylate therefore is only able to function as an extracellular iron chelator in circumstances where competing ions are absent. Such an environment possibly exists within the macrophages of host tissues when mycobacteria are growing in vivo but not, of course, in conventional laboratory media. This unexpected result led to a search for further iron-binding compounds which could solubilize iron in the presence of phosphate. Such a compound has been found which is highly water-soluble and is an oligopeptide containing e-N-hydroxylysine, allo-threonine and fl-alanine (Macham and Ratledge, 1975). This compound, given the trivial name hydrochelin, appears to be the correct extracellular iron chelator. But having found this compound, one then must ask what is the function of salicylic acid? As salicylic acid is not metabolized t o any compound other than mycobactin (Ratledge and Hall, 1970, 1972) it must function catalytically. During an investigation of the mechanism of action of the anti-tubercular drug p-aminosalicylic acid (Ratledge and Brown, 1972)) the drug was found to be most effective whenever the

196

0.RATLEDQE

intracellular concentration of salicylic acid was low. The observed effects of p-aminosalicylic acid suggested that it interfered with the acquisition of iron, and it was therefore proposed that, as salicylate might function as a means of transferring Fe" from Fe"-mycobactin to an acceptor enzyme (see Fig. 25), the drug could act as a competitive inhibitor of this process (Brown and Ratledge, 1975a).The antagonistic effect of p-aminobenzoic acid on the action of p-aminosalicylic acid which has been the main evidence in favour of the drug being an antimetabolite of p-aminobenzoate and hence of folic acid biosynthesis (see Winder, 1964), is explained by the finding that p-aminosalicylic acid is transported into the mycobacterial cell on the p-aminobenzoatetransport system (Brown and Ratledge, 1975a).

OUTSIDE

CELL BOUNDARY LAYERS

INSIDE porphyrin

I

LFe'Ihaem

Fel"hac

mycobactin c--mycobactin Fe"'hydroche1in Fe "mycobactin hydrochelin Fe"'mycobactin

__t

Fe'"mycobactin

Fe"'

Fra.25. Hypothesis for iron transport and assimilation. Adapted from Ratledge a n d Marshall (1072), Macham et al. (1976) and Brown and Ratledge (1976a, b). The role of salicylate in this scheme has still t o be substantiated.

The mechanism of iron transport is given in Fig. 25. It has been almost entirely worked out using iron-deficient cells in which the concentration of all of the participating components is high. However, the same mechanism also exists in iron-sufficient cells indicating that there is probably only one route for iron assimilation in mycobacteria (Brown, 1975).


C. GROWTHFACTORS AND

THE

197

“NoN-CULTIVATABLE” MYCOBACTERIA

Many compounds have been cited as exerting a favourable effect on the growth of mycobacteria (Long, 1958). These include egg yolk (possibly due to its lecithin or cholesterol content although other factors within it have been described; Yamane, 1957), lipids such as sphingomyelin, serum proteins and even polysaccharides derived from coconut (Ramakrishnan et al., 1957). The effect of Tween 80 as a surfactant on the growth of mycobacteria is also well described and has already been discussed (Section V.A, p. 183. All of these compounds only potentiate growth and therefore are not strictly growth factors. The requirement for growth factors is confined to those organisms which are host-dependent and cannot be grown in the laboratory without the addition of a particular compound. The requirements of these “non-cultivatable” species, M . leprae, M . lepraemurium and M . paratuberculosis, are discussed in this section of the review.

1. Mycobacterium leprae The leprosy bacillus has proved to be the most intractable of all mycobacteria to cultivate in the laboratory. A major development was considered the ability to transmit experimental leprosy to animals by inoculation of infected tissue into either the footpads of mice (Shepard, 1960a, b), the mouse ear or the footpads of the hamster or rat (Waters and Niven, 1965; Hilson, 1965). Only a limited amount of multiplication occurs in these situations which is often difficult to quantitate, although Talwar et al. (1974) suggested that incorporation of [3H]thymidine into the bacterial RNA can be used for this purpose. A subsequent improvement in growing this organism has been the ability to develop the systematic or lepromatous form of the disease in armadillos (Kirchheimer and Storrs, 1971 ; Convit and Pinardi, 1974). Ng et al. (1973) suggested that M . marinum, which can be cultivated in the laboratory with ease, could be used as a useful model of mouse footpad infection with M . leprae. The patterns of disease produced by the two bacteria appeared similar. An invaluable review of the advances which have been made with experimental leprosy has been written by Rees (1970b), and Klingmuller (1974) has recently described the history of this organism since its discovery by Hansen in 1874. Mycobacterium leprae is thought to derive its energy and nutrients from the phagocytic cells in which it multiplies more or less on a “lendlease” basis created by alterations in the permeability of the cell wall, thereby allowing flow of nutrients both into and out of the bacteria (Hanks, 1966, 1968). However, Kato et al. (1973) suggested that this organism may be a chemo-autotroph on the basis of the absence of a-

198

C. RATLEDOE

ketoglutarate dehydrogenase and the presence of ribulose diphosphate carboxylase which catalyses the reaction:

+

+

D-ribulose 1,5-diphosphate CO, + H20+ 2 ~-3-phosphoglycer&te 2H+

This reaction has only previously been found in autotrophic bacteria. The absence of a-ketoglutarate dehydrogenase from M . leprae, which also is missing from M . lepraemurium (Tepper and Varma, 1972), indicates that the tricarboxylic acid cycle is functioning only biosynthetically and not for respiratory purposes as well. Obligate autotrophs have a similar deletion (Smith et al., 1967). There is, therefore, strong evidence that M . leprae may behave in an in vitro situation quite differently from previous indications, and this may explain the failure to cultivate this organism successfully in vitro. It should be noted, however, that Lukins and Foster (1963) were able t o grow strains of M . smegmatis, M . marinum and M . fortuitum as chemolithotrophs (see p. 186) and thus the behaviour of M . leprae may not be as exceptional as is thought. Adding to this confusion is the recent suggestion of Fisher and Barksdale (1973) that, on the basis of cytochemical evidence, M . leprae may not be a true mycobacterium at all. This view has also been taken by Uyeda (1064a, b) and could be supported by the occurrence of a unique enzyme in M . leprae. This enzyme is o-diphenoloxidase (Prabhakaran, 1973) which is thought to be associated with the predilection of this organism for growth in the skin and in peripheral nerves (Prabhakaran et al., 1968, 197 1, 1972) by enabling dihydroxyphenylalanine of the host to be utilized (Ambrose et al., 1974). Further work is obviously needed to clarify this issue, but whether or not M . leprae is a mycobacterium still does not explain the failure to achieve reasonable growth in laboratory media. The attempts to cultivate M . leprae in vitro have centred around providing a complex array of nutrients and growth factors. Successful growth has been achieved in tissue-cultured macrophages (Chang and Neikirk, 1965; Garbutt, 1965), but attempts merely to sustain cells in a viable state in laboratory media have taken considerable effort (Pattyn, 1971). However, Olitzki et al. (1971) have reported that mycobactin S, the growth factor for M . paratuberculosis (Fig. 24, p. 194), together with sonicated M . smegmatis and with a phospholipid from strain BCG, promoted significant increases in the cell numbers of M . leprae in different liquid medium. Mycobactin S only partially contributed to the growth effect as, when it alone was present, continued growth was not sustained (Olitzki et al., 1972). Of several surfactants tested for growth-promoting properties, dimethyl sulphoxide apparently enhanced multiplication in liquid and in semi-solid agar medium (Olitzki et al., 1973). This group of workers conclude (Olitzki et aZ., 1972) that there are two equal possibilities which must be examined simul-


199

taneously to account for the difficulty in growing M . leprae : ( 1 ) growth inhibitors may be present in laboratory medium and a t least two components, tyrosine and glutamate, of Eagle’s medium were found to be detrimental to growth (Olitzki and Levy, 1972); (2) a search for possible growth factors from other more competent mycobacteria, in which capacity mycobactin, phospholipids and other materials are only partially satisfactory. I n all cases where in vitro growth of M . leprae or M . lepraemurium is claimed, it is imperative that the pathogenicity of the organism a t the end of its growth should be retested by passage through a susceptible animal in order to be certain that avariant hasnot emerged.

2. Mycobacterium lepraemurium The difficiilties surrounding the growth of M . leprae have resulted in much attention being given to the rat leprosy bacillus as an alternative organism for studying comparative pathology, immunology and chemotherapy. No doubts about the authenticity of this species as a mycobacterium have, however, been raised (Fisher and Barksdale, 1973). Even its cell-wall structure is typically that of a mycobacterium (Draper, 1971; Azuma et al., 1973; Draper and Rees, 1973). However, many attempts to cultivate this bacillus in vitro have failed; for example, over 250 different media were tried by Tepper (1971) and a system for periodically changing the medium without removing the bacteria (Sula and Dubina, 1971) failed to establish growth. A major development came when Hart and Valentine ( 1 963) reported elongation of M . lepraemurium in laboratory medium containing glycerol, sucrose, asparagine, basal mineral salts, casamino acids supplemented with albumin. Elongation was very dependent upon the pH value with the maximum effect being from about pH 7 to pH 6.3, which may co-incide with the intraphagosomal pH value encountered during growth in oivo. Addition of mycobactin did not produce any significant change in the observed elongation patterns. More recently, however, two independent advances of considerable importance have been made. Rightsel and Wiygul (1971) developed small diffusion chambers which allowed two-way permeation of low molecular-weight materials. When M . lepraemurium was placed inside such a chamber along with animal tissue cells (human embryo, mouse fibroplasts or mouse macrophages) and then implanted in mice or guinea pig, slow multiplication of the bacteria occurred. Further work established that, although the presence of animal cells within the chamber was beneficial for growth, they were not essential (Wiygul and Rightsel, 1971). An interesting observation was that guinea pigs were less effective hosts than mice, perhaps being a reflection of the non-susceptibility of guinea pigs to natural infections of this bacterium.

200

C . RATLEDGE

The other development concerning growth of M . lepraemurium has been the finding by Nakamura (1972)that addition of a-ketoglutarate and cytochrome c to a basal salts medium containing sucrose, glycerol, citrate, pyruvate and glutamate led to successful growth. Addition of goat serum, haemin, cytochrome c and cysteine produced further improvements in the rate of multiplication (Nakamura, 1974).Scanning elcctron micrographs of the bacteria taken during growth (Yoshii and Nakamura, 1974) are shown in Fig. 26. Even under the best conditions found by Nakamura (1974),the doubling time of the bacteria was between 8 and 14 days which, although close t o the value of l l days found by Rightsel and Wiygul(1971))is still extremely slow. We are, therefore, still some way from being able to cultivate this organism to give sufficiently large populations to enable detailed investigations of its metabolism t o be made, although some attempts a t this have been carried out by Tepper and Varma (1972)using cells derived from culture in vivo. These studies have provided unequivocal evidence for the ability of' 21.1. lepraemurium to be able to utilize exogenous substrates, and thus support the contention that this organism, given the appropriate conditions, could be cultivated in vitro.

3. Mycobacterium paratuberculosis The fastidious M . paratuberculosis ( M .johnei) is the cause of chronic enteritis in cattle and certain other ruminants. A growth factor from growth-competent mycobacteria was shown to be required for its isolation and cultivation in vitro by Twort and Ingram (1913).This growth factor was subsequently isolated and purified by Francis et al. (1953) and named mycobactin (see Fig. 24,p. 194).The requirement for mycobactin appears quite specific and cannot be replaced by other naturally occurring iron-binding compounds (Morrison, 1965 ; Snow, 1970). However, Woolley and McCarter (1940)found that phthiocol and other naphthoquinones (related to vitamin K ) could stimulate growth, and more recently Coletsos (1971)found that vitamin K could act as a growth factor when a t 1.7 ng/ml. However, Francis et al. (1949)failed to observe any significant effect when vitamin K-like materials, extracted from M . tuberculosis, were tested with M . paratuberculosis. Other mycobactin-requiring mycobacteria have since been isolated from wood pigeon (H. E. Ottosen, and A. MacDiamid, personal communications t o Wheeler and Hanks, 1965). The basic mycobactin molecule varies slightly from species to specics (Fig. 24,p. 194)and the response of M . paratuberculosis to each one has been tested (Snow and White, 1969;Wheater and Snow, 1966; Snow, 1970).Mycobactin S appears to be the outstanding one and shows significant growth stimulation when added to medium a t 0.3 ng/ml whereas


201

FIG.26. Scanning electron micrographs of Mycobacleriuna lepraemuriuni grown in a cell-free modium. Bacteria were grown as a smear o n a glass slide immersed in aliquid medium : (a)Showselongatcd bactcrie after growth in synthetic medium for two weeks; D = division of bacteria, LB = lateral budding: (b) Shows appearance of the mass of bacteria in the peripheral area of the smear after eight weeks’ growth. From Yoshii end Nakamura (1974), with permission of the authors.

202

0.RATLEDGE

others, such as mycobactin R, are required a t 3 ng/ml. In all cases, however, about 30 ng of mycobactin/ml are needed for maximum growth of M . paratuberculosis (see Fig. 27). The requirement for mycobactin presumes an incompetence on behalf of M . paratuberculosis to assimilate iron, since the function of the molecule is to solubilize iron into a form which can pass through the lipophilic layers of the cell envelope (Section V.B.4b, p. 192). However, the solubility of iron in an aqueous environment is dependent upon the pH value; e.g. at pH 7.0, the solubility of free Fell' is M whereas,

70

2

e'-

20

I

I

10

15

0

Mycobact in concentration ( n g /ml)

FIG.27. Growth of Mycobacteriurn paratuberculosis (syn. johnei) in the presence

.;

of various concentrations of different mycobactins. Points show the increase in

nephelometer reading after 14 days' growth, each point being the mean of triplicate determinations. Growth response given by mycobactin S, ; mixture of mycomycobactin M, A ; mycobactins F and H from Mycobacteriurn fortuitum, bactin H, v ; mycobactiii R, 0.From Snow (1970), with kind permission of the author.

at pH 5.0, it is M, that is a million-fold increase. Not surprisingly, Morrison (1965) found that the mycobactin requirement could be circumvented by employing growth medium a t pH 5.5. Presumably the concentration of free iron at this pH value is sufficient to satisfy the requirements of the bacteria, and iron may be able to enter the cell by simple diffusion a t a sufficient rate not to limit growth. I n addition, Morrison (1965) reported the production of growth-promoting compounds during autoclaving of the medium ; these might conceivably have been ferric complexes of glucose or glycerol which may be able to function as iron carriers. Wheeler and Hanks (1965) confirmed Morrison's results and also showed stimulation of growth in vivo of M . paratuberculo& and the wood-pigeon mycobacteria by adding Fe(NO,),. Similar results have also been reported by Merkal and Curran (1974). Like all

THE PHYSIOLOQY OF THE MYCOBACTERIA

203

organisms, however, a variety of other factors could also be shown to improve the in vivo growth rate although none of these could be claimed to be essential. The argument can be advanced (Hanks, 1966) that, as M . para,tuberculosis (and other mycobacteria) flourish in the phagosomes of the macrophages wherein the pH value is likely to be between 5.0 and 5.5, and as the requirement for mycobactin is circumvented by such a low pH value, the organism when in vivo may not be dependent upon mycobactin for growth. This would certainly answer the awkward question : whence does M . paratuberculosis obtain its mycobactin? Snow (1970) answered this by supposing that mycobactin could arise from non-pathogenic mycobacteria residing in the alimentary tract or elsewhere in the ruminant. There assuredly does not appear to be any shortage of saprophytic mycobacteria in the soil or on the grass which, being continually ingested by animals, would provide a constant source of mycobactin. There is, however, no answer as yet to this problem, but clearly, if mycobactindependent mycobacteria can grow in vivo without mycobactin, then mycobactin-independent mycobacteria would have no need t o synthesize mycobactin to assist them in their acquisition of iron. It will, therefore, be of considerable interest to determine if mycobactin is, in fact, produced during in vivo growth of such organisms as M . tuberculosis.

VI. Aspects of Metabolism Ramakrishnan et al. (1972) have recently reviewed the intermediary metabolism of mycobacteria and, although their treatment of certain areas may have been a little perfunctory, nevertheless the review encompassed almost every single facet of this subject and should therefore be referred to for information which is not covered in this section or elsewhere in this review. This section, therefore, is not a n attempt to review the entire intermediary metabolism of all mycobacteria but concentrates mainly on those aspects which have not yet been discussed and are thought to be central, or somewhat particular, to the physiology of the mycobacteria. Apart from pathways of biosynthesis leading to compounds which are unique unto mycobacteria, mycobacteria possess very few novel enzymes. One such enzyme, however, is L-lactate oxidase which is discussed in the second part of this section. Although certain pathways of energy-yielding metabolism in mycobacteria are also found in other organisms, an understanding of how mycobacteria derive their energy is of considerable importance and this is discussed in the third part of this section. Finally, a brief account of the organization of genetic

204

d. RATLEDOE

information and protein biosynthesis is given even though our knowledge of these processes has come from research with other organisms.

A. CENTRAL PATHWAYS OF METABOLISM Enzymes for metabolism of glucose via the Embden-Myerhof-Parnas pathway have been found in M . tuberculosis and M . phlei when grown on glucose or glycerol, thus indicating their ability to function also in gluconeogenesis (Bastarrachea et al., 1961; Le Cam et al., 1969; Le CamSagniez and Sagniez, 1973). The fructose 1,6-diphosphate aldolase of 41, tuberculosis has been shown by Jayanthi Bai et al. (1974, 1975) to change from Type I to Type I1 in going from conditions of high oxygen tension (e.g. shake-cultures or in a fermenter) to low oxygen tension (i.e. in stationary cultures). The two types of aldolase have different kinetic parameters, pH profiles and requirements for a metal-ion cofactor. Type I1 aldolase is the usual bacterial aldolase and is activated by K+. As glycerol was being used as substrate, the change in the type of aldolase synthesized was probably to ensure an increased rate of gluconeogenesis under the highly aerobic conditions, for there is then a more rapid production of energy than under conditions of low oxygen tension. The entry of glucose 6-phosphate into the pentose cycle (i.e. via glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) occursin M.phlei (Sutton, 1963; Le Cam et al., 1969)but not apparently in M . tuberculosis (Le Cam et al., 1969).However, this latter observation was determined using glycerol-grown cells so that one must be cautious in making an interpretation of its significance. Gluconic acid can also be metabolized to 6-phosphogluconate in M . phlei (Szmona and Kowalska, 1970). Other results confirm that more than 75% of glucose dissimilation in M . tuberculosis is by way of direct glycolysis (Ramakrishnan et a&., 1962; O’Barr and Rothlauf, 1970). Glycerol, when used as a carbon source, is metabolized to glycerol 3-phosphate via glycerol kinase (Pande et al., 1967) and then to dihydroxyacetone phosphate via an NAD(P)-dependent glycerol 3-phosphate oxidoreductase (Winder and Brennan, 1966). The failure of Goldman (1963)to detect this enzyme in extracts of M . tuberculosis may have been due to the use of old cultures. Winder and Brennan (1966)indicated that the oxygen tension of the cultures, particularly in relation to cells within a large clump or pellicle, is likely to be very low a t the end of growth, and they could find no activity of glycerol dehydrogenase, which Goldman could easily detect, except in old cultures. Dihydroxyacetone phosphate is converted into pyruvate by the usual sequence of enzymes. Of these, pyruvate kinase has been isolated and purified from M . phlei and its kinetic properties studied (Dudouet et al.,

205


1973). Just as other workers have found with the enzyme from other sources (see Atkinson, 1969))the enzyme from M . phlei is subject to allosteric regulation. Adenosine triphosphate was found to be an inhibitor, and AMP an activator. Somewhat surprisingly, however, fructose 1,B-diphosphate had no effect on its activity (Dudouet et al., 1973). Mycobacteria possess a tricarboxylic acid cycle which functions both for biosynthetic and energy-production purposes (see Ramakrishnan et al., 1972).The enzymes of the glyoxylate by-pass (isocitrate lyase and inalate synthase) also function under appropriate conditions. Interestingly, Murthy et al. (1973)found that the activity of isocitrate lyase increased in the later stages of growth of M . tuberculosis, thus indicating that a high degree of lipolysis was probably occurring and generating the necessary acetyl units to derepress synthesis of the enzyme. As mentioned previously (Section V.C, p. 198), the absence of a-ketoglutarate dehydrogenase from M . leprae and M . lepraemurium, but the presence of other enzymes of the tricarboxylic acid cycle, have been taken to infer a lack of respiratory function of the cycle in these two organisms (Tepper and Varma, 1972;Kato et al., 1973).

B. L-LACTATE OXIDASE Mycobacteria appear t,o be unique in possessing a specific decarboxylase which converts 1,-lactate into acetate. The enzyme, L-lactate oxidase (decarboxylating E.C.1.1.3.2), was first isolated by Edson (1947) who observed that oxygen had to be present a t the onset of the reaction for acetate to be produced; if not, pyruvate was formed. However, pyruvate is not a substrate for the enzyme as was confirmed by both Cousins (1956)and Sutton (1954,1957)who purified and characterized the enzyme from M . phlei. Sutton (1957)suggested that the enzyme could produce an intermediate with pyruvate which could be decarboxylated only if oxygen was present, otherwise it would dissociate. This view has only recently been substantiated by Lockridge et al. (1972). The enzyme utilizes molecular oxygen, which Hayaishi and Sutton (1957)showed by use of *O, is incorporated into acetate. The reaction catalysed is therefore : CH,CHOHCOOH

+ I8O2

-

+

CH,C0180H C 0 2

+ H2"0

Thus the enzyme is a mono-oxygenase with the substrate serving as an internal electron donor. The enzyme contains FMN as its prosthetic group (Sutton, 1955). Preliminary studies on its molecular weight (Sullivan, 1968) indicated it to be within 300,000to 400,000daltons, and more recent information

20G

C . RATLEDQE

(P. A. Sullivan, personal communication) has confirmed this. The enzyme from M . srnegmatis consists of eight identical subunits each containing one FMN molecule with a molecular weight of about 43,000 daltons. Takemori et al. (1974)) however, find that the enzyme from M . phlei has only six subunits each of similar molecular weight (54,000 t o 57,000 daltons) and each containing an FMN molecule. As yet it is not clear why there should be these differences between these two species of mycobacteria. Further properties of the enzyme have been investigated independently by Takemori et al. (1969, 1973), using crystalline preparations from

1

Enz-FMN

-4

CH3CHOHCOOH

J.

Enz-FMN-CH&HOHCOOH

I

4

[Enz-FMN-CH3COCOOH] Enz-FMN

anaerobic

. Enz -FMN.CH3COOH

+ COz + HzO

CH,COOH

Enz-FMNH,

CH,COCOOH

FIG.28. Proposed mechanism for action of the L-lactate oxidase from mycobttcteria. From Lockridge et al. (1972) and Takemori et al. (1973).

M . phlei, and by Sullivan and his coworkers (Sullivan, 1968; Lockridge et al., 1972) using similar preparations from M . srnegrnatis. Their work has enabled the scheme given in Fig. 28 to be proposed as the minimal mechanism. Takemori et al. (1969, 1973), in fact, proposed a second unstable intermediate immediately before the anaerobic formation of pyruvate, but Lockridge et al. (1972) believe that this is due to the use of high concentrations of phosphate which is a competitive inhibitor of the enzyme. Their own investigations, using a stopped flow apparatus, could discern only one such intermediate (i.e. enzyme-bound pyruvate) in this step.


207

The main problem concerning this enzyme is not explaining its mode of action but in providing a satisfactory explanation for its occurrence. The enzyme occurs in all recognized mycobacteria (Murthy et al., 1973; Clarke, 1971; Szymona et al., 1967a, b ; Szymona and Szumilo, 1968), but not in M . rhodochrous (Clarke, 1971) which is not now considered to be a true mycobacterium. Induction of synthesis of the enzyme varies enormously according t o the species of mycobacterium used; in Mycobacterium 279, activity was increased only three-fold by growing cells on lactate instead of on glycerol or glucose (Szymona and Szumilo, 1968) whereas Clarke (1971) found that, in M . smegmatis NCTC 523, there was a 150-fold increase in activity after changing the substrate from glycerol to lactate; but in M . smegmatis NCTC 8157, there was only a 50% increase in activity. Acetate, pyruvate, citrate and glutamine can also act as inducers for synthesis of this enzyme (Szymona and Szumilo, 1968). The little evidence that there is suggests that the enzyme should not be a favoured means of converting lactate into acetate as this is an energetically wasteful reaction, nor can it be a means of producing pyruvate under anaerobic conditions as the relevant rate constant is too low (Lockridge et al., 1972). However, it is the only enzyme of sufficient activity which can metabolize lactate ; the presence of lactate dehydrogenase has been firmly established in these mycobacteria but has about a t the most 1% of the activity of the oxidase (Szymona and Szumilo, 1968; Clarke 1971). The only other enzyme which can metabolize lactate is a D-lactate dehydrogenase whose properties have been described (Szumilo and Szymona, 1972), but this has no reactivity towards the L-isomer and, in any case, still has only about 10% of the activity of the oxidase (Clarke, 1971). Although the enzyme can be seen to be required when lactate is used as sole source of carbon, the high activity of the enzyme when other substrates, such as acetate, are used remains inexplicable unless the enzyme functions in the cell in a manner different from that supposed a t present. The high activity of the enzyme in some mycobacteria growing on glycerol or glucose may be because of an inability to regulate the enzyme for, under these conditions, the enzyme would appear to be completely redundant.

C. ENERGY METABOLISM 1. Oxidative Phosphorylation and Electron Transport Elucidation of the electron-transport chain and the sites of coupling of phosphorylation of ADP in mycobacteria has been established by the extensive work of Brodie and his coworkers using M . phlei. Subcellular particles which can carry out this complex process have been isolated

208

C. RATLEDCE

from M . phlei. (Brodie and Gray, 1957); these are usually refcrred to as coupled ele ctron-transport particles. Non-phosphorylating particulate preparations are sometimes produced, and are terincd electron-transport particles (see Brodie and Gutnick, 1972). Ariji et al. (1968) have demonstrated the occurrence of succinate dehydrogenase and cytochrome c as well as ATPase activity on the mesosomes and sometimes on thc cytoplasmic membrane of M . tuberculosis. Unlike most other bacteria, M . phlei has three pathways of electron-transport leading to cxygen, compared with the usual two. These are shown in Fig. 29. All pathways converge a t the level of cytochrome b. Brief details of the three pathways are as follows. (a) Succinoxidase pathway. Succinate is oxidized by a flavoprotein dehydrogenase which was first isolated from ill.phlei by Brodie and Gray (1957). The enzyme system is localized in the coupled electron-tmnsport

Succinate

I

flavoprotein (fps)

.1

light-sensitive component

I

J.

Subsfrate

Malnte

I I

I I

Malate-vitamin K rcductaso

NADP+

FAD+-phospholipid

NAD+

non-haem iron protein? flavoprotein

non-haem

cytochrome b

I

cytochromo ( c ,

cytochrome ( a

+ c)

+a3)(o)

I 0 2

FIG.29. Electron-transport chain of Mycobncteriun&phlei.


209

particles. Succinate dehydrogenase can be released from the electrontransport particles by treatment with silicotungstic acid (Kalra et al., 1971). Irradiation a t 360 nm inhibited a component of the pathway after the flavoprotein, but before the non-haem iron region of the chain (Kurup and Brodie, 1966, 1967). Activity could be restored by addition of an unknown soluble protein (Asano and Brodie, 1964; Kurup and Brodie, 1966). The participation of non-haem iron on the pathway was found using o-phenanthrolinate to trap Fe" formed with succinate as the electron donor (Kurup and Brodie, 1967). (b) NAD+- and NADP+-linked oxidation pathway. There appear to be a large number of enzymes which are capable of oxidizing NADH in

mycobacteria as well as in most other micro-organisms. Concentrations of NAD(P)+ and NAD(P)H have recently been determined in M . smegmatis, M . tuberculosis and M . phlei (Seshadri et al., 1973; Le Cam et al., 1969; Le Cam-Sagniez and Sagniez, 1973). When NADP+ is linked to oxidation of a substrate, the ensuing NADPH is re-oxidized via a transhydrogenase using NAD (Murthy and Brodie, 1964). The existence of a flavoprotein in the NADH oxidase which required an artificial electron acceptor, such as menaquinone, was demonstrated by Brodie and Gots (1951, 1952). However, the only quinone which can restore NADH oxidation to quinone-depleted particles of M . phlei was found to be trans-vitamin K (Gutnick et al., 1967), and Azerad and Cyrot-Pelletier (1973) have shown that this has the same absolute configuration as the saturated isoprenoid unit of the menaquinone from M . phlei [7'S(-)MK-9(II-H2)]. The sequence of the respiratory carriers on the pathway leading into cytochrome b was determined mainly by using respiratory inhibitors (Asano and Brodie, 1964). (c) Malate-vitamin K reductase pathway. Oxidation of malate to oxaloacetate can be effected by different bacteria in a t least two different ways. The commonest method is the reaction catalysed by a soluble malate dehydrogenase linked to NAD+. In other cases, a particulate malate oxidation system may exist either in place of the dehydrogenase system or in addition to it. Organisms having this second mechanism include Axotobacter agilis, Micrococcus lysodeikticus, Serratia marcescens, PseudomonasJluorescens, Ps. ovalis (see Francis et al., 1963) and M . phlei (Asano et al., 1965; Asano and Brodie, 1963). I n M . phlei, the enzyme catalysing this NAD+-independent oxidation of malate has been solubilized and shown to have a requirement for vitamin K, a phospholipid and FAD (Asano and Brodie, 1963, 1965; Brodie and Adelson, 1965; Imai and Brodie, 1973) and possibly to contain non-haem iron as well (Kurup and Rrodie, 1967). The enzyme is termed malate-vitamin K

210

C. RATLEDQE

reductase. It has a molecular weight of 164,000 daltons (Imai and Brodie, 1973) and appears to be part of the membrane structure of the electrontransport particle (Imai and Brodie, 1974). Andrejew et al. (1972) have also found this enzyme in M . fortuitum but failed to detect any significant activity in M . bovis BCG or M . avium. Mycobacterium phlei also contains a L-malate-NAD+ oxidoreductase (Asano and Brodie, 1964; Andrejew et al., 1973) but curiously M . fortuitum possesses an NADP+-linked enzyme (Andrejew et al., 1973). Why the rapidly growing mycobacteria require two pathways for malate oxidation, and the slower growing bacteria only one, is as yet unknown and further studies on this area of metabolism are obviously needed (see Prasada Reddy et al., 1975). (i)Common Terminal Pathway The electron-transport sequence of the terminal respiratory carriers, involving cytochromes b, c and a, has been elucidated from studies of their reduced steady states as well as from the studies with respiratory inhibitors. Complete reduction of cytochrome b can only be caused by adding substrates to operate the three individual pathways simultaneously. There appear to be at least two types of cytochrome b ; one type may be associated with the succinate pathway and the other on the NAD+ pathway. Electron transfer can occur from the cytochrome b(NAD)to the cytochrome b~succinoxfdase), and preliminary evidence suggests that they may be located on opposite sides of the membrane of the electron-transport particle (Cohen et al., 1973a, b). The terminal cytochrome oxidase has been prepared from M . phlei and contains cytochrome 0, in addition to cytochromes a a , (Revsin et al., 1970). Cytochromes a3 and o are both sensitive to carbon monoxide. Two cytochromes sensitive to carbon monoxide have also been found in M . lepraemurium, besides cytochromes a + a3,b, and c (Ishaque and Kato, 1974; Kato et al., 1974; Mori et al., 1971).

+

(ii) Sites of Phosphorylation The low P :0 ratios of isolated electron-transport particles of M . phlei, which has remained unexplained for some time, may be because the membrane of the particle is turned inside out during the sonication of the cells (Hirata and Brodie, 1972; Brodie et al., 1972; Asano et al., 1973). When protoplast ghosts are prepared, however, these show the same orientation of the membrane as in the original cell, but unfortunately do not., show increased P :0 ratio probably because the membrane still presents a permeability barrier to several substances, e.g. ADP (Asan0 et al., 1973). Thus, the best bacterial preparations still compare poorly with the tightly-coupled systems of mitochondria. However, even in bacterial systems which show a P :0 ratio of less than unity, more than


21 1

one site of phosphorylation may still be functioning (Asano and Brodie, 1965). The sites of phosphorylation are considered (Brodie and Gutnick, 1972) as being between: (i) NAD and flavoprotein fp(FA,,); (ii)MK-9 and cytochrome b ; (iii) cytochrome c and 0,. Efficiencies of phosphorylation are therefore less when succinate and malate (via the malate-vitamin K reductase pathway) are the electron donors (Kurup and Brodie, 1966; Asano and Brodie, 1965). All sites of phosphorylation are probably on the inside of the membrane of the coupled electron-transport particles (Hirata and Brodie, 1972).

2. Polyphosphate-Linked Enzymes Independent of the oxidative phosphorylation processes, ATP or various phosphorylated compounds can be produced by reactions linked directly to inorganic polyphosphate. Inorganic polyphosphate is probably synthesized when the demand for high-energy phosphate drops, i.e. during the late stages of growth and while there is still a surfeit of carbon available for energy production and, of course, inorganic phosphate itself. The resulting polyphosphate is stored as large granules which are easily recognizable in whole cells (Section II.F, p. 131). Suzuki et al. (1972) partially purified an ATP:polyphosphate phosphotransferase from M . smegmatis which catalysed the reaction : ADP

+ (POA

ATP + ( P O & . I

The enzyme was inhibited not only by ATP, which may have been expected, but also by ADP and AMP. The exact role of the enzyme therefore is uncertain, and further studies on its function are clearly needed. Winder and Denneny (1957) reported a related enzyme, also in M . smegrnatis, which could catalyse transfer of a phosphate unit to AMP: AMP

+ (PO,),,

-

ADI’

+ (P0T)n-i

The enzyme showed a strict specificity for AMP. An earlier report of Winder and Denneny (1955)) which suggested that the enzyme could synthesize ATP from ADP was, however, found to be erroneous (Winder and Denneny, 1957). Szymona (1962) has confirmed the presence of AMP : polyphosphate phosphotransferase in extracts of M . phlei. Other enzymes, however, have been recognized which can also make use of inorganic polyphosphate without the formation of ATP. For example, a specific glucokinase from M . phlei and other mycobacteria (Szymona, 1962; Szymona and Ostrowski, 1964; Szymona et al., 1967b; Szymona and Widomski, 1974) catalyses the reaction : glucose + (PO,-),

+glucose 6-phosphate + (P03-)n-l

This enzyme may account for the preferential utilization of polyphosphate for RNA synthesis (Szymona, 1974). A separate fructokinase catalysing a similar reaction has also been

212

C. RATLEDGE

reported (Szymona and Szumilo, 1966).In both cases, formation of ATP and activity of a conventional hexokinase were not involved. The occurrence of polyphosphate hexokinases in a wide variety of bacteria, other than mycobacteria and members of the Actinomycetales, has been found (see review by Dawes and Senior, 1973). Glycerol may also be phosphorylated using polyphosphate and AMP as a primer (Winder and Denneny, 1957) but this may be with ADP being formed as an intermediate. There seems no reason, however, why other compounds could not be similarly phosphorylated by direct linkage to inorganic polyphosphate, e.g. ribose to ribose 5-phosphate, or even t o 5-phosphoribosyll-pyrophosphate, or even acetate to acetyl phosphate as an intermediate in the formation of acetyl-CoA. Such reactions would certainly be energetically more sensible than a simple degradaticn of polyphosphate t o inorganic phosphate using polyphosphatases. D. NUCLEIC ACIDAND PROTEIN BIOSYNTHESIS 1. D N A and Transfer of Genetic Information

The sizes of the chromosomes from a variety of mycobacteria have been determined from renaturation kinetics as ranging from 2.5 x lo9 daltons to 4.5 x l o 9 daltons (Bradley, 1972, 1973). Improved methods for the efficient release of DNA (Mizuguchi and Tokunaga, 1970) and its purification (Hill et al., 1972) have been reported. Although no direct investigations on DNA replication in mycobacteria have been carried out, it is reasonable to suppose that the mechanism is similar to that in E. coli (Klein and Bonhoeffer, 1972). Repair to damaged DNA in mycobacteria is again inferred to be similar t o that in E . coli (see Berndt, 1973, for review) and this is supported by the isolation of a DNA polymerase from M . smegmatis with properties similar to those of DNA polymerase I of E . coli which is known to function in excision repair. The properties of this enzyme have already been discussed (p. 190). Transfer of genetic information can occur in mycobacteria either by lysogeny, transduction, transformation or by conjugation. No direct studies on the mechanism involved in the recombination of genetic material have been made, although an ADP-dependent deoxyribonuclease has been isolated from M . smegmatis and shown to be similar to the ATP-dependent exonuclease from E. coli which is involved in genetic recombinations (Winder and Coughlan, 1969). Further information on the control of this enzyme is given on p. 190; it should be noted that ATPdependent deoxyribonuclease from M . smegmatis has a substantially higher specific activity than that from E . coli (Winder et al., 1973a). The process of transferring genetic information by lysogeny has been described by several workers (Juhasz, 1967, 1968, 1970; Juhasz and


213

Bonicke, 1966, 1970; Gelbart and Juhasz, 1969; Mankiewicz, 1961 ; Mankiewicz et al., 1969; Jones and White, 1968; Grange and Bird, 1975). The establishment of transduction, however, has been more difficult due to the lack of suitable markers on the genome. This deficit has, unfortunately, now been overcome and the process has been demonstrated simultaneously by Sundar Raj and Ramakrishnan (1970, 1971) and by Gelbart and Juhasz (1970,1973) using two suitable auxotrophic mutants and an appropriate transducing phage, and has subsequently been confirmed by Saroja and Gopinathan (1973). Transformation with isolated free DNA was established in mycobacteria by Katunuma and Nakasato ( 1 954) who showed that streptomycin resistance could be transferred to streptomycin-sensitive strains using DNA extracted from resistant cells. Juhasz et al. (1971) confirmed this process in M . phlei, and reported that about 20% of the total transfer of genetic material in their transduction experiments was occurring by transformation. Conjugation between mycobacteria with subsequent recombination of the genomes was demonstrated by Mizuguchi and Tokunaga (1971) using Jucho and Lacticola strains of M .smegmatis, and this was later confirmed between the Rabinowitchii and Jucho strains which had different phage susceptibilities and colony appearances (Tokunaga et al., 1973). Many of the difficulties associated with following the transfer of genetic material stem from the paucity of stable auxotrophic mutants which bear easily recognizable but disparate markers. Several techniques, however, have been devised for the isolation of such mutants (Radochova et al., 19G6 ; Konickova-Radochova and Konicek, 1974 ; Holland and Ratledge, 1971) and the first steps towards constructing a linkage map of the mycobacterial chromosome have been taken (Suga and Mizuguchi, 1974).

2. Ribonucleic acid Bicsynthesis and S'ite of Action of Rifampin Ribonucleic acid biosynthesis in mycobacteria, like that in other organisms, is by a DNA-dependent RNA polymerase. The occurrence of this enzyme in M . smegmatis, M . bovis and M . tuberculosis has been shown mainly in conjunction with studies on the mechanism of action of rifampin (rifampicin; R. J. White et al., 1971; Konno et al., 1973a; Trnka and Smith, 1970; Mison and Trnka, 1972; Schainen and Antoine, 1974). As with the system from E . coli, which has been extensively investigated (see Burgess, 1971 ; Wehrli and Staehelin, 1971 ; Riva and Silvestri, 1972), rifampin in M . tuberculosis has been found to affect only the initiation of polypeptide chain formation and not chain elongation (Konno et al., 1973a). With M . smegmatis, although incorporation of phenylalanine was completely inhibited within six minutes of adding the antibiotic to cells, and uracil incorporation was inhibited within two minutes, rifampin

214

0.RATLEDGE

kills this species very slowly (R. J. White et al., 1971). The minimum inhibitory concentration of rifampin for M . bovis BCG and most other mycobacteria, including M . leprae, is, however, about 500 times less than that for M . smegmatis (Rynearson et al., 1971; Woodley et al., 1972; Shepard et al., 1972). The difference in sensitivity to rifampin may be due to an altered permeability of the cells to the antibiotic (Mison and Trnka, 1972), and this explanation has also been advanced to account for the emergence of resistant strains of M . tuberculosis (Le Cam et al., 1971). However, in E. coli, it is the affinity of rifampin for the B-subunit of RNA polymerase which becomes diminished in a resistant mutant due to a change in the amino-acid sequence of the protein (Rabussay and Zillig, 1969; Iwakura et al., 1973). Recent results (Dworsky and Schaechter, 1973) suggest that RNA polymerase in E. coli is also involved in maintaining the structure of the bacterial DNA and attachment of DNA to the cell membrane. Rifampin decreases the number of attachment points of the DNA to the membrane from about 20 to 5, but only in sensitive strains and not in resistant ones. Furthermore, as RNA polymerase also participates in the initiation of DNA synthesis (Brutlag et al., 1971), rifampin may ultimately interrupt the processing of genetic information a t various levels. There has also been some suggestion that, in mycobacteria, rifampin may inhibit protein biosynthesis on the ribosome (Trnka and Smith, 1970; Shaila et al., 1973)) and Konno et al. ( 1 9 7 3 ~have ) shown, in an electron-microscope study of the effects of rifampin on M . tuberculosis, that the typical structures of the mesosomes and cytoplasm are lost and the ribosomes become coarse and irregular. Nuclear material, the plasma membrane and cell wall all appear unchanged, however. It is suggested that both RNA polymerase and protein synthesis are being inhibited to cause these changes. There is no reason to suppose, however, that rifampin, or indeed any other antimicrobial agent, may just have only one site of action. Clearly, though, some sites of action will be more sensitive to the agent than others.

3. Protein Biosynthesis and Mode of Action of Streptomycin and Other Antibiotics The structure of the ribosome in mycobacteria has already been described (Section III.E, p. 131). The ribosomal-directed synthesis of proteins has been studied in mycobacteria a t a fairly superficial level mainly following polyU-directed synthesis of polyphenylalanine with extracts of M . smegmatis, M . tuberculosis or M . bovis BCG (Worcel et al., 1968; Trnka et al., 1968; Rieber and Imaeda, 1969; Trnka and Smith, 1968, 1970; Trkna and Mison, 1971; Yamada et al., 1972a). Trnka and Mison ( 1 971) observed little difference between the charac-


216

teristics of ribosomes isolated from M . megmatis and those from strain BCG, although polyphenylalanine synthesis was the more rapid in the former species as may have been expected from its faster growth rate. The concentration of Mg2+appears to be critical at 18 mM for optimum protein synthesis in strain BCG and M . tuberculosis (Shaila et al., 1973; Trnka and Mison, 1971 ;Worcel et al., 1968) and to be at 10 to 15 mM for optimum synthesis in M . smegmatis (Reiber and Imaeda, 1969; Trnka and Mison, 1971). Shaila et al. (1973) have examined protein synthesis in a little more detail using M . tuberculosis H37Rv. Incorporation of amino acids into proteins was stimulated by tRNA isolated from phage-infected M . smegmatis, although by comparison tRNA from E . coli was over five times more effective. The amount of tRNA required appears very high (c. 50 to 100 pg/mg ribosomal protein) in all of these systems but this is thought to be because of a low mRNA content in the preparations. Using a poly U-directed system, streptomycin was found to be as potent as an inhibitor of protein synthesis as others have found it to be in E . coli (Flaks et al., 1962). This is in keeping with the mode of action of streptomycin which has been worked out mainly in E . coli (see Pestka, 1971); streptomycin irreversibly binds to the ribosome and blocks protein elongation (Biswas and Gorini, 1972; Wallace et al., 1972; Wallace and Davis, 1973). It also produces an ambiguity in translating RNA into protein (Davies et al., 1966) which is unconnected with the first effect. Although information is not available as to whether streptomycin exerts the same effects in mycobacteria, Konno et al. (1973b) showed that, in M . bovis BCG, the related aminoglycoside antibiotic, kanamycin, inhibits polypeptide synthesis at the extension of the protein chain, and this is followed by a breakdown of the polysomes and detachment of mRNA. Resistance to streptomycin in E . coli is caused by an alteration in the P-10 core protein of the ribosome which governs binding of the antibiotic to the ribosome (Ozaki et al., 1969). Thus, isolated ribosomes from resistant bacteria show a decreased sensitivity t o the effects of streptomycin but without a significant change in protein-synthesizing ability. A similar situation has been found with ribosomes from M . tuberculosis (Shaila et al., 1973) and M . smegmatis (Masuda et al., 1974) and thus gives circumstantial evidence in favour of identical modes of action of streptomycin in mycobacteria and E . coli. Many other antibiotics are known to inhibit ribosome function (see Weisblum and Davies, 1968; Pestka, 1971, 1974) and although some, such as tetracycline and chloramphenicol, are ineffectual as antimycobacterial agents, this appears to be due to their lack of uptake by cells rather than a failure to act intracellularly (Shaila et al., 1973). Viomycin

216

a. RATLEDGE

and capreomycin, which are sometimes used in the chemotherapy of tuberculosis, have been shown to inhibit protein synthesis at the ribo,some level in M , smegmatis and M . tuberculosis, respectively (Yamada et al., 1972b; Trnka and Smith, 1970).Resistance to viomycin, which is rare in E . coli, has been found in M . smegmatis to be associated with changes in the structure of either the 505 or 30s ribosomal subunit (Yamada et al., 197213). CzHS.CH .NH .CHI .CHz.NH .CII .CzHS

I

CHzOH

I

CHZOH

FIG.30. Formula of ethambutol (2,2-ethylenedi-imino-dibutan-l-01).

Ethambutol (Fig. 30), which is a potent and specific antitubercular agent (see British M.R.C. Co-operative Study, 1973), is structurally related to the polyamines, and Bacalao and Rieber (1972)have reported that, in M . smegmatis exposed to ethambutol at 10 pg/ml, some RNA components were selectively altered. In particular, a component migrating on polyacrylamide gel electrophoresis slower than the 23s component and another migrating between the 235 and 16s components could not be recognized in ethambutol-treated cells. There was no evidence that this change was by an increase in nucleolytic activity or a change in specificity, but may be due to a type of non-co-ordinated control of RNA metabolism. Recent studies on uptake of ethambutol have shown it capable of binding to a heterogeneous group of sites, but only one (unknown) of which is directly related to its biological activity (Beggs and Andrews, 1974).

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Note added in proof The extensive account by Iwainsky and Kappler (1974) of the biochemistry of mycobacteria should be consulted for greater information regarding intermediary metabolism than is given in this review. This monograph also contains a wealth of practical detail regarding the differentiation of the various species. Lederer has up-dated his earlier review on the structure of the mycobacterial cell wall and has discussed its various immunostimulating properties (Lederer et al., 1975). The discovery of a novel linkage between two DAP residues on different peptide groups of the wall peptidoglycan (p. 119) begins t o illustrate the likely complexity of this structure (Wietzerbin et al., 1974).Bergerson et al. (1975) and Flick and Bloch (1975)have elaborated upon the importance of the mycobacterial polysaccharides in the regulation of longchain fatty acid synthesis. The complex which is formed between palmitoylCoA and MMP (p. 138) may prevent and even reverse the disaggregation of the fattyacid synthetase complex induced by palmitoyl-CoA. Of importance in understanding how mycobacteria survive and multiply within macrophages (p. 161) is the observation by Lowrie et al. (1975) that cyclic AMP may be secreted by the pathogen to inhibit fusion between phagosome and lysosome. This would normally result in the discharge of the bactericidal lysosomal contents into the proximity of the bacteria. Further evidence for the dependence of macrophages on T lymphocytes for their activation and mobilization during infection with M. tuberculosis continues to be presented (North, 1974; Collins et al., 1975). The areas of disagreement in the controversy concerning the relationship between delayed hypersensitivity and immunity in tuberculosis have been discussed in a series of articles (Youmans, 1975; Lefford, 1975; Salvin and Neta, 1975). If these two phenomena are linked, and if ribosomal proteins are the instigators of delayed hypersensitivity (p. 177), then this may explain why the immunogenicity of a streptomycinresistant strain of BCG is very low in mice (Collins and Montalbine, 1975) as streptomycin resistance is due to changes in the P-10 core protein of the ribosome (p. 215). Work on the bioenergetics with M . phlei has implicated a soluble protein fraction in translocation of energy-rich phosphate bonds across protoplast ghost membranes to exogenous ADP (Lee et al., 1974). The process of energy production seems distinct from those involved in the transport of amino acids (Kosmakos and Brodie, 1974; Hirata et al., 1974; Prasad et al., 1975) although this is a view not shared by all workers in this field (see Hamilton, 1975). Activity of NADH oxidase in M . phlei is modulated by the prevailing NAD+/ NADH ratio which may thus regulate electron flux through the entire respiratory chain (Davis et al., 1975).

REFERENCES Bergerson, R., Machida, Y . and Bloch, K. (1975). Journal of Biological Chemistry 250, 1223. Collins, F. M. and Montalbine, V. (1975). American Review of Respiratory Diseases 111, 43.

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Collins, F. M., Congdon, C. C. and Morrison, N. E. (1976).Infection and Immunity 11, 67. Davis, W. B., Zlotnick, B. J. and Weber, M. M. (1976).Journal of Biological Chemistry 250, 1648. Flick, P. K. and Bloch, K. (1976).J o u m l of Biological Chemistry 250,3348. Hamilton, W. A. (1976).Advances in Microbial Phyaiology 12, 1. Hirata, H., Kosmakos, F. C. and Brodie, A. F. (1974).Journal of Biological Chemistry 249,6966. Iwainsky, H. and Kiippler, W. (1974). Mykobakterien: Biochemk und biochemische Differenzierung J. A. Barth, Leipzig. Koemakos, F. C. and Brodie, A. F. (1974).Journal of Biological Chemistry 249, 6966. Lederer, E.,Adam, A,, Ciorbaru, R., Petit, J. F. and Wietzerbin, J. (1976). Molecular and Cellular Biochemistry 7, 87. Lee, S. H., Kalra, V. K. and Brodie, A. F. (1974).Journal of Biological Chemistry 249,8048. Lefford, M. J. (1976).American Review of Respiratory Diaeaeea 111, 243. Lowrie, D. B., Jackett, P. 5. and Ratcliffe, N. A. (1976).Nature, Lo* 254,600. North, R.J. (1974).Infection and Immunity 10,66. Prasad, R.,Kalra, V. K. and Brodie, A. F. (1976).Journal of Biological Chemistry 250, 3690. Salvin, S. B. and Neta, R. (1976).American Review of Respiratory D ~ X M 111, M 373. Wietzerbin, J., Das, B. C., Petit, J. F., Lederer, E., Bouille, M. L. and Ghuysen, J. M. (1974).Biochemistry, New York la, 3471. Youmans, G. P. (1976).American Review of Respiratory Disewea 111, 109.

ACKNOWLEDGEMENTS

I would like to thank Drs. C. E. Ballou, K. Bloch, Rosa Edwards, R. W. Hendren, M. Nakamura, G. A. Snow, P. A. Sullivan, R. G. White, F. G. Winder and G. P. Youmans for their kindness in providing material for use in illustrations and for giving information on their work in progress. I would also like to thank Dr. L. P. Macham for his help and comments with the preparation of this article.

Effect of Endogenous and Exogenous Factors on the Primary Structures of Bacterial Peptidoglycan K. H. SCHLEIFER, W. P. HAMMES and 0. KAKDLER Lehrstuhl far Mikrobiologie and Botanisches Institut der 1Jniversittit Manchen, Manchen, West Germany I. Introduction

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.

246 246

11. Effects of Endogenous Factors A. Growthphase . B. Peptidoglycan Structures of Vegetative Cell Walls Compared with the Spore Cortex C. Changes in the Peptidoglycan Structure during a Morphological Life Cycle . D. Genetic Variations of the Peptidoglycsn Structure .

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111. Effects of Exogenous Factors . A. Aerobic and Anaerobic Growth . B. Amino-AcidComposition of the Growth Medium C. Ionic Environment . D. Osmotic Value of the Growth Medium . E. Antibiotics . F. Growth-Inhibiting Concentrations of Glycine and amino Acids

268 268 260 276 277 279 281

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IV. Concluding Remarks V. Acknowledgement References

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287 288

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I. Introduction The composition and primary structure of peptidoglycans have been intensively studied over the past fifteen years. This work provides a detailed picture of the chemical structure of this polymer. There are general features common to all peptidoglycans, such as the basic composition of the glycan chains and their substitution with short peptides containing alternating D-amino acids. On the other hand, a remarkable variation has been found in the amino-acid composition and sequence of peptidoglycans of Gram-positive bacteria (Schleifer and Kandler, 1972). These differences in the primary structures, i.e. the different peptidoglycan types, appear to be of great value for classification of Cram-positive bacteria (Schleifer and Kandler, 1972). It is therefore important for taxonomists to know whether and to what extent the primary structure is subject to genetic variations and phenotypic modifications. Another special field that requires more knowledge about the peptidoglycan structure and its variability is growth physiology. The question of how the morphology of a cell is determined and maintained cannot be solved without a detailed understanding of the chemical and structural basis of the cell-wall components and their possibilities for variation. Therefore, i t is not surprising that aberrant morphological forms like the L-forms, or morphological changes in the course of cell development such as the endospore and cyst formation or the rod-sphere transition, have stimulated discussions about specific alterations of the rigid layer of the cell wall. Finally, the various “cell wall-active” agents (antibiotics, glycine, D-nmino acids) must be mmtioned. It has been supposed that these agents affect not only the biosynthesis but also the chemical structure of the cell-wall polymers. It is the purpose of this review to assemble data showing to what extent the primary structure of the peptidoglycan may change due to the effects of endogenous and exogenous factors. 11. Effects of Endogenous Factors

A. GROWTH PHASE The structure and composition of peptidoglycans are quite stable throughout the growth cycle of bacteria. Only small quantitative alterations of the amino-acid and amino-sugar compositions of $he cell wall of Bacillus subtilis 168 were found during growth (Young, 1965). The contents of alanine and galactosamine in the cell wall reached their maximum in mid-to-late log-phase cultures. The simultaneous increase in the contents of these two compounds may indicate that primarily the teichoic-acid fraction of the cellwall is altered, since

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galactosamine is found solely within the teichoic acid and alanine is also a component of this polymer. Thus, the changes observed in B. subtilit? are probably caused by changes in the cell-wall tejchoic acid and not by alteration of the peptidoglycan. Schwarz and Leutgeb (1971) studied the peptidoglycan of Escherichia coli from both log- and stationary-phase cells. They found that the peptidoglycan of stationary-phase cells is significantly more cross linked than that of log-phase cells. Moreover, cells from stationary-phase cultures contain in their peptidoglycan more tripeptide subunits lacking the C-terminal D-alanine than do log-phase cells. We determined the quantitative amino-acid composition and the extent of cross,linkage of the peptidoglycan of Bijidobacterium infantis (L-LYS-G~Y type) and Lactobaci1lu.y plantarum (m-Dpm-direct type ; m-Dpm indicates a meso-diaminopimelic acid residue) from log- and stationary-phase cells and did not observe any differences (unpublished results). In the case of aerobically grown cells of Microbacterium lacticurn, the ratio of glutamic acid to threo-3-hydroxyglutamicacid (3-Hyg) in the cell wall was altered during the growth cycle. I n log-phase and early stationaryphase cells, this ratio was about 1:10, whereas in late stationary-phase cells it increased to about 1 :4 (K. H. Schleifer and 0. Kandler, unpublished results).

B. PEPTIDOQLYCAN STRUCTURES OF VEGETATIVE CELLWALLS COMPAREDWITHTHESPORECORTEX Endospore-forming bacteria contain two independent layers of peptidoglycan, namely peptidoglycan of the vegetative cell wall and that of the spore cortex. Although the qualitative chemical composition of these two peptidoglycan structures is identical in the case of Bacillus subtilia and most other bacilli (Warth, 1965; Warth and Strominger, 1969, 1971, 1972; Tipper and Gauthier, 1972), several differences have been found in their primary structures (Warth and Strominger, 1969, 1971,1972). The peptide subunits of the vegetative cell-wall peptidoglycan consist of MurNAc-L-Ala-y-D-Glu-(L)-m-Dpm-(L)-D-Ala residues which are cross linked between the amino group of the masymmetric carbon of meso-diaminopimelic acid and the carboxyl group of the D-alanine of an adjacent peptide subunit. The carboxyl groups of the D-centre of meso-diaminopimelicacid are amidated, and the non crosslinked peptide subunits terminate in C-terminal meso-diaminopimelic acid. The peptidoglycan is cross linked to a degree of about 60% (Hughes, 1970; Warth and Strominger, 1971). The composition of the spore peptidoglycan differs considerably from this (Warth and Strominger, 1972; Fig. 1). Only 35% of the muramic-acid residues are substituted

35% /

1B %

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A

A

\

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0

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H

NHAc

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I H

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1

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I1

O

Muramic 6-lactam

co I

I L-Alo

1y

D-Glu

4 rn-Dpm

1

-

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T FIG.1 . Repeating units of the peptidoglycan from spores of Bacillus subtilk according to Warth and Strominger (1972). The amount of each unit is expressed as a percentage of the total. The cross-linkage structure is put in parentheses since only 19% of the tetrapeptides are cross linked.

ENDO- AND EXOGENOUS 'AAUTORS ON BAUTERIAL PEPTIDOGLYUAN

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by a peptide subunit, and these subunits are not amidated. Moreover, the non cross-linked peptide subunits terminate in C-terminal D-alanine and not in C-terminal meso-diaminopimelic acid. Of the muramicacid residues, 18% are substituted by a single L-alanine residue. The remaining 47% of the muramic-acid residues form muramic &lactam in which the d a c t y l moiety on C-3 is linked to the amino group on C-2 (Fig. 1).Therefore, only 50% of the total muramic-acid residues are N-acetylated. The spore peptidoglycanis slightlycross-linked.Anaverage of only 7% cross-links-based on the total number of disaccharide units-has been found in the spore peptidoglycan of B. subtilis (Warth and Strominger, 1972). A similar cortex peptidoglycan structure has been found in spores of B. meqaterium, B. cereus T, B. stearothermophilus and Clostridium sporogenes (Tipper and Gauthier, 1972). The differences in the peptidoglycan structure of the cell wall and the spore cortex may be related to their different functions. The far greater cross linkage of the cell-wall peptidoglycan makes it less elastic and contributes to its function as a rigid supporting structure. The spore peptidoglycan, on the other hand, should be less resistant to stretching and possess a large net negative charge (Warth, 1965) due to the many free carboxyl groups of the peptide subunits (Fig. 1).Such a structure should possess gel-like properties. At low ionic strength, the cortex exists in an expanded, highly hydrated state, whereas in the presence of divalent cations like Ca? this polyanionic gel should considerably shrink. Thus, its properties would be consistent with the requirements of the theory of a contractible cortex (Lewis et al., 1960). The peptidoglycans of the cell wall and the spore cortex of B. sphaericus differ not only in structure but also in chemical composition (Hungerer and Tipper, 1969). Vegetative cel1,walls of B. sphaericus contain Llysine and D-aspartic acid residues in their peptidoglycan, whereas the spore peptidoglycan contains meso-dianiinopimelic acid instead of L-lysine residues and lacks D-aspartic acid residues. The cell-wall peptidoglycan reveals the typical glycan moiety, namely p-l,4-linked residues of N-acetylmuramic acid and N-acetylglucosamine. All of the muramic acid residues are substitut4edby the tetrapeptide : L-Alay-n-Glu-~-Lys-~-Ala. An a-amidated D-aspartic acid residue is involved in the cross linkage. It is bound through its /3-carboxyl group to the €-aminogroup of L-lysineof one peptide subunit and through its amino group t o the carboxyl group of D-alanine of an adjacent peptide subunit. About 55-65% of the peptide subunits are cross linked (Hungerer and Tipper, 1969). The non cross-linked peptide subunits lack D-alanine residues and terminate in a C-terminal L-lysine residue. On the other hand, the spore cortex peptidoglycan of B. sphaericus is similar to that of B. subtilis (Tipper, 1969a; Tipper and Pratt, 1970). Of the muramic+

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K. R. SCHLEIFER, W. P. HAMMES AND 0 . KANDLER

acid residues, 64% are lactamized, 18% carry a single L-alanine residue and the remaining 28% are substituted by the tetrapeptide: L-Ala-yD-Glu-m-Dpm-D-Ala. A similar difference in the chemical composition and structure may exist in B. pasteurii. The primary structure of the cell-wall peptidoglycan from several strains has been studied (Ranftl and Kandler, 1973). The mll-wall peptidoglycan is devoid of mesodiaminopimelic acid, but contains L-lysine as the diamino acid residue. The peptide subunits are cross' linked through D-aspartyl-L-alanine or p-D-aspartyl-L-serine interpeptide bridges. The spore peptidoglycan of these organisms contains meso-diaminopimelic acid as observed in other bacilli. A complete study of the primary structure was, however, not carried out. If the biosynthesis of the spore-cortex peptidoglycan follows the general pathway of wall peptidoglycan biosynthesis, some modifbations must be included (Tipper and Pratt, 1970; Tipper and Gauthier, 1972). For example, non cross-linked tetrapeptide may be cleaved by endopeptidase action to give muramic-acid residues with single L-alanine residues. The formation of a muramic lactam might result either from the action of N-acetylmuramyl-L-alanine amidase followed by transacylation, or from de-N-acetylation and transamidation. Moreover, cortex synthesis in B. qhaericua requires an additional enzyme for incorporation of meso-diaminopimelic acid to the nucleotide-activated peptidoglycan precursor. This diaminopimelic acid-adding activity has been detected in sporulating cells of Brsphaericus (Tipperand Pratt, 1970).

C. CHANGES IN

THE

PEPTIDOQLYCAN STRUCTURE DURINGA MORPHOLOGICAL LIFE CYCLE

A variety of bacteria are distinguished by a characteristic cycle of development in which they undergo a change in morphology. Prosthecate bacteria, like Caulobacter spp., develop, in the course of their life cycle, cellular appendages which are extensions of the cell walls (Staley, 1968). The wall of the cell and stalk of Caulobacter spp. displays a typically Gram-negative multilayered structure in profile (Poindexter and CohenBazire, 1964). The peptidoglycan type of cell walls of Caulobacter spp. has not been determined. Since meso-diaminopimelic acid is present (Kandler and Zehender, 1966), it can be assumed that the directly cross-linked meso-diaminopimelic acid type is present as in other Gramnegative bacteria. Schmidt and Stanier (1966) studied the mechanism of stalk elongation. Treatment of stalked cells with lysozyme converted the cells to sphaeroplasts and, after a somewhat longer period of lysozyme action, the stalks also lost their rigid outlines. Schmidt and Stanier

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(1966) concluded from these results that the stalks contain a rigid peptidoglycan layer, and they suggested that the stalk wall is a “stronger structural entity” than the cell wall, since lysozyme affects the integrity of the stalk wall only after a longer exposure. The “stronger structural entity” of the stalk wall may depend on alterations in the peptidoglycan structure, e.g. a greater extent of cross linkage, or on alterations in non-peptidoglycan material. It is also interesting to note that the stalks were unaffected by penicillin, whereas the cells were converted to sphaeroplasts. This indicates that the stalk wall itself is no longer the site of peptidoglycan synthesis. This was also confirmed by radioautographic studies which have shown that synthesis of the stalk is restricted to a narrow zone at. its juncture with the cell proper (Schmidt and Stanier, 1966). Some bacteria do not divide by binary fission but by formation of a bud (daughter cell) at the end of stalk (hyphae)that arises from a mother cell. Typical budding bacteria are species of Hyphomicrobium and Rhodomicrobium. The chemical composition of the walls of Hyphomicrobium sp. and H. neptunium was determined by Jones and Hirsch (1968). The walls showed a chemical composition typical for Gramnegative bacteria. The peptidoglycan layer of H. neptunium was isolated, and the molar ratio of the peptidoglycan components determined. The chemical composition of the walls resembles that of other Cramnegative peptidoglycans with the exception of significant amounts of glycine (0.31 moles of glycine/mole glutamjc acid) which were found in Hyphomicrobium peptidoglycan but do not occur in peptidoglycans from other Gram-negative bacteria. Jones and Hirsch (1968) suggested that the basic peptidoglycan structure is similar to that of Gramnegative bacteria, i.e. to the directly cross-linked meso-diaminopimelic acid-containing peptidoglycan type, but one third of the peptide chains should, in addition, contain a glycine residue. The authors did not comment on differences in the peptidoglycan structure of the buds and the hyphae. It would be interesting to study these two morphological forms separately, to see if the occurrence of glycine residues in the peptidoglycan is restricted to just one of them. Two apparently unrelated orders of bacteria, the Azotobacteriales and the Myxobacteriales, are able to form rest,ing bodies. The cell walls of these resting forms (cysts or microcysts) are considerably thicker than those of the vegetative cells. Extensive electron micrographic studies have revealed that the mature cysts of Axotobacter vinelandii are surrounded by thick coats consisting of two layers, namely the exine, an electrondense outer layer, and the intine, an electron-transparent inner layer (Parker and Socolofsky, 1966; Koo et al., 1969; Cagle et al., 1972). The exine and intine layers were purified by differential and iso-

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K . R. SCIILEIFER, W. P. HAMMES AND 0. KANDLER

pycnic centrifuga.tion, and subjected to chemical analysis (Lin and Sadoff, 1969). Both layers are composed of a complex of carbohydrate, protein and lipid. The exine contains, however, three times as much protein as the intine, and the lipid was mostly in a bound form, whereas it was free in the intine. Unfortunately, it is unknown if one of these two layers contains peptidoglycan. The presence of typical constituents of peptidoglycan, such as muramic acid or diaminopimelic acid, was obviously not checked. Lin and Saddoff (1969), however, mentioned that “the exine contained large amounts of glutamic acid, glycine, alanine, and lysine, which are known to be major components of peptidoglycan”. If this conjecture could be verified, a lysine-containg peptidoglycan would be present in the cyst, whereas the vegetative cell wall would have a peptidoglycan containing diaminopimelic acid (Kandler and Zehender, 1956). Thus the peptidoglycan type of the vegetative cell wall and that of the cyst would differ. In this respect it would be a similar situation to that in certain bacilli ( B .sphaericus, B. pasteurii), where the peptidoglycan types in walls of vegetative cells and spores are different. The fruiting myxobacters possess a complex life cycle. The flexible, rod-shaped vegetative cells aggregate on solid surfaces and form a fruiting body. These fruiting bodies are either simple structures consisting of spherical microcysts (myxospores),as in the case of myxococci, or more complex structures consisting of large mu1ticellular macrocysts on specialized stalks, as exemplifiedby species in the genus Chondromyces. The peptidoglycans of the vegetative cell wall and the wall of the microcyst have been studied in Myxococcus xanthus (White et al., 1968; Johnson and White, 1972) and in Sporocytophaga myxococcoides (Verma and Martin, 1967). The peptidoglycan type is the same as in Gramnegative eubacteria, i.e. a directly cross-linked meso-dianiinopimelic acid type. The extent of peptide cross-linkage, however, is significantly higher. More than 50% of the meso-diaminopimelic acid residues are involved in cross linkage of the peptide subunits in vegetative cells of M.xanthuus, or even as many as 70% in vegetative cells of Cytophaga hutchinsonii and S. myxococcoides, whereas in the peptidoglycan of E . coli only about 30% are cross linked. The flexibility of the vegetative cell-wall of S. myxococcoides is in agreement with the occurrence of “naked tubes of murein (peptidoglycan) monolayers”, whereas in the vegetative cells of M. xanthus the arrangement of the peptidoglycan in a discontinuous layer seems to be responsible for the flexibility. I n the latter case, it has been suggested (White et al., 1968) that the peptido glycan layer exists as patches separated by a non-peptidoglycan material composed of lipid and peptide material. During the conversion of the vegetative rod to the spherical microcyst, several structural changes apparently occur. The cross linkage

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of the peptidoglycan is increased in comparison with that of the vegetative cell peptidoglycan. This can be deduced from the decrease in the content of free amino groups in the diaminopimelic acid residues and the decrease in the content of monomer-sized fragments released by lysozyme, from 2OYi of the total amount of peptidoglycan in vegetative cell walls to 7% in microcysts of 31.xanthus. At the same time the amount of larger-sized fragments (tri- and tetramers) increased. During the transition froin the vegetative rod t o the spherical microcyst, the thickness of the peptidoglycan layer increases in S. myxococcoides from 2 to 9 nm. The microcyst wall of this organism consists of almost pure pcptidoglycan, which may be made up of several superimposed and cross-linked monolayers of peptidoglycan (i'erma and Martin, 1967). The vegetative cell walls and microcysts of 31. xanthzes, on the other hand, contain equal amounts of peptidoglycan despite the synthesis of new peptidoglycan during microcyst formation. At the same time increased autolytic activity is found, indicating that specific alterations necessary for changing the shape of the peptidoglycan layer from a rod to a sphere take place. The higher extent of cross linkage of the pepticloglpcan and, in particular, the synthesis of a new outer layer (capsule) containing galactosamine and glycine may be responsible for the heightened rigidity and mechanical strength of the microcyst. Organisms in the genus Arthrobacter are characterized by a simple life cycle. The rod-shaped cells divide by fragmentation into spherically shaped cells upon entering the stationary phase of growth. After inoculation into fresh medium, the spheres revert to rods. The extent of the life cycle of Arthrobacter depends not only upon the growth stage but, under special conditions, also upon the composition of the growth medium. I n complex media, the typical sphere-rod transition is observed during growth. I n a mineral salts-glucose medium, some arthrobacters grow only in spherical form, whereas in media containing sufficient peptone or succinate the same organisms grow only as rods (Ensign and Wolfe, 1964). The chemical composition of cell walls of spheres and rods of A . crystallq.poietesATCC 15481 was studied by Krulwich et al. (1967a, b). I n order to exclude differences in cell-wall composition which were not related t o the morphogenesis but to the growth stage of cells, growth conditions were chosen in which both spheres and rod-shaped cells were obtained in the logarithmic phase of growth. Spheres were cultivated in salts-glucose medium and rods either in peptone- or succinatecontaining medium. The overall chemical composition of the different cell walls was quite similar with the exception that the walls of spherical cells differ from those of rod-shaped cells by the presence of small amounts of residues of glycine, aspartic acid and serine. Further purification indicated that glycine is a constituent of the peptidoglycan

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K. R. SCHLEIFER, W. P. HAMMES AND 0. KANDLER

whereas serine and aspartic acid may be contaminants from residual membrane components or proteins. Enzymic degradation of the peptidoglycan showed that the polysaccharide backbone (glycan) of peptidoglycan from spherical cells was smaller in size than that of rod-shaped cells. The spheres contain, on an average, less than 40 hexosamine residues per chain and the rods 114 to 135 hexosamine residues per chain (Krulwich et al., 1967a).An N-acetylmuramidase was found as an autolytic enzyme in cell walls of A . crystallopoietes (Krulwich and Ensign, 1968). The autolytic activity in sphere walls was much higher than in rod walls. Thus the decrease in the length of the glycan chains correlates well with the increase in N-acetylmuramidase activity during the rodsphere transition. Studies on the peptide moiety of the peptidoglycan revealed that both the sphere- and the rod-peptidoglycans contain identical peptide subunits, namely : L-Ala-y-D-GluNH,-L-Lys-D-Ala, which are cross linked by interpeptide bridges. The interpeptide bridges of rod peptidoglycan are formed exclusively by single L-alanine residues. This indicates that the peptidoglycan of rod-shaped A . crystallopoietes belongs to the L-Lys-L-Ala-type.The sphere peptidoglycan also contains predominantly L-alanine bridges, but a small percentage of the interpeptide bridges may consist of L-alanyl-glycyl-glycine residues. It is not known whether the occurrence of L-Ala-Gly-Gly bridges is a typical feature of the sphere peptidoglycan or if it depends on the composition of the growth medium. Studies in our laboratory on the chemical composition of the peptidoglycan of cell walls of A . crystallopoietes ATCC 15481, grown in yeast extract-glucose broth and harvested during the exponential (rod-shaped) or stationary growth phase (spheres), failed t o reveal differences between sphere and rod peptidoglycans (F.Fiedler, unpublished results). Significant amounts of glycine were not found in total hydrolysates of sphere cell walls. Partial acid hydrolysis of sphere cell walls did not yield any peptides containing glycine. The peptidoglycan was of the L-Lys-L-Ala type for both rod and sphere cell walls. Thus, the only significant difference between rod and sphere peptidoglycan may be the different chain length of theglycan. Itis, however,quite possible that the shorter glycan chains in the spheres are only degradation products which arise during isolation of peptidoglycan as a consequence of the high N-acetylmuramidase activity of the spherical forms. This possibility is all the more to be considered since the peptidoglycan was prepared from cell walls whose autolytic activity was obviously not destroyed. Krulwich et al. (1967a)suggested that the shorter chain length of the sphere glycan is more compatible with the structural flexibility required by a spherical surface, whereas the more rigid structure of rod-shaped cells may require longer glycan chains. The occurrence of long glycan

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chains in curved rods of Spirillum serpens (Kolenbrander and Ensign, 1968) and short glycan chains in the spherical cells of Staphylococcus aureus (Tipper et al., 1967) and S. epidermidis (Tipper, 1969b) seems t o support this hypothesis. I n more recent studies by Hughes (1970) and Warth and Strominger (1971) on the peptidoglycan structure of Bacillus licheniformis and B. subtilis, very short glycan chains were found in these rod-shaped organisms. These findings contradict a general correlation between cell shape and average chain length of the glycan. D. GENETICVARIATIONS O F THE PEPTIDOGLYCAN STRUCTURE The first mutant known to be defective in the biosynthesis of peptidoglycan was strain 173-25 of E. coli described by Bauman and Davis (1957). I n the absence of meso-diaminopimelic acid this strain grows in osmotically balanced medium as an L-form and reverts t o the rod form after addition of meso-diaminopimelic acid. In this organism a very early stage of peptidoglycan synthesis is affected, namely the synthesis of meso-diaminopimelic acid. Recently many other mutants have been found which are defective in different steps of the biosynthesis and accumulate different nucleotide-activated peptidoglycan precursors (Good and Tipper, 1972; Lugtenberg et al., 1971; Miyakawa etal., 1972). Since the normal balance between synthesis and enzymic hydrolysis of peptidoglycan is disturbed, these cells lyse. The peptidoglycan of such mutants has not been investigated up till now. One may suppose that such a peptidoglycan should contain a certain percentage of incomplete peptide subunits, since accumulated incomplete precursors may be incorporated under these abnormal conditions. Indeed, in vitro studies with enzyme preparations from E . coli (Izaki et al., 1968) and Qaffkya homari (Hammes and Neuhaus, 197413) showed that UDPMurNAc-tripeptide and tetrapeptide are incorporated into peptidoglycan. However, the rate of their incorporation is decreased. Lehmann and Martin (1972) investigated the peptidoglycan of a mutant, derived from Proteus mirabilis which had been treated with N methyl-N '-nitro-N-nitrosoguanidine. The rounded irregularly shaped cells of this organism are similar to L-forms. The purified peptidoglycan contained the typical amino acids and amino sugars in the expected molar ratios, but the extent of its cross linkage was only 13%, compared with 29% in that of the wild type. NOsuch differences were found in morphological mutants of E . coli. I n the osmotically-stable spherical forms of temperature-sensitive rod mutants ( E .coli K12) grown a t non-permissive temperature, the overall composition and the extent of cross linkage of the peptidoglycan were found to be identical with those of the rod form grown a t the permissive temperature (Henning et al., 1972).

256

K. H. SCHLETE’ER, W. P. HAMMES AND 0. KANDLER

There is no convincing evidence that a qualitative change in the primary structure of peptidoglycan is caused by induced mutation, in spite of a report of Korman (1966) on pleiotropic mutants. These mutants of Staphy1ococcu.r aurew exhibited a decreased amount of glycine, but an increased content of serine, in their peptidoglycan. We re-investigated three of these mutants, and found their properties to be typical of S. epidermidia (Schleiferand Kocur, 1973). They contain increased amounts not only of serine, but also of glycerol teichoic acid, in their cell walls. In addition, they possess an L-lactate dehydrogenase which is specifically activated by fructose 1,6-diphosphate, whereas S. aureus strains contain ribitol teichoic acid and L-lactate dehydrogenase not activated by fructose 1,6-diphosphate.It is difficult to imagine that these mutants have been derived from S. aureus by single-step mutations. The ba.sicdifficulty in trying to detect experimentally induced qualitative peptidoglycan mutants is the lack of a suitable screening technique for such mutants. One possible approach to this problem is selection of mutants which are resistant to “cell wall-active” agents. We isolated spontaneous mutants from three different strains of M . luteus ( M . lysodeikticus) whose resistance to lysozyme was 20-fold greater than that of the parent strains. Cell wells of these mutants were only slightly more resistant to lysozyme than those of the parent strains. However, differenceswere not found with regard to the amino-acid composition or the extent of cross linkage of the peptidoglycan (H. Stetter and 0. Kandler, unpublished results). The reason for thc lysozyme resistance of these mutants might be the same as reported by Brumfitt et al. (1958) for M . luteus, namely an increased 0-acetyl content in the cell wall. They found that the cell walls of the resistant mutant contained more than 100 times the number of 0-acetyl groups than the cell walls of the sensitive parent strain. Removal of the 0-acetyl groups made the wn,lls sensitive to lysozyme. Although experimentally induced peptidoglycan mutants have not been found, there is no doubt that mutations leading to qualitatively different peptidoglycans do occur. Otherwise it would be difficult to understand why Gram-positive organisms should have developed such a great variety of peptidoglycan types in the course of their evolution. From a study of the peptidoglycans of closely related strains, several variations in the amino-acid composition of the polymer are known which are most likely due to the occurrence of alleles having arisen by mutation. The most common variations are discussed in the following paragraphs.

1. Variation in the Diamino Acid of the Peptide Xubunit In most bacteria, all of the peptide subunits of the peptidoglycan contain the same diamino acid, but in some cases two different diamino

ENDO- AND EXOGENOUS FACTORS ON BAUTERIAL PEPTIDOGLYCAN

257

acids occur alternatively at the same position. I n the genus Ri$dobacterium the peptidoglycans of the species B. adolescentis and B. globosum contain L-lysine and L-ornithine. The ratio of the two amino acids varies among different strains, but it is fairly constant if different batches of cell walls of one and the same strain are examined. When L-lysineor L-ornithineat a concentration of 0.5% is added to the medium, the ratio is only slightly changed (about 10%) in favour of the amino acid added (G. Stern and 0.Kandler, unpublished results). It seems likely that, in the various strains examined, the enzymes responsible for attachment of the diamino acid residues to the UDPMurNAc-dipeptide exist in several genetically determined copies exhibiting different affinities towards lysine and ornithine. It is also of course possible that two different enzymes, specific for ornithine and lysine respectively, are present in genetically determined different amounts. One may assume that the genes coding these enzymes occur in several alleles in the strains of the two species mentioned.

2. Variation in the Amino-Acid Composition of the Interpeptide Bridge In some bacteria the interpeptide bridges of the peptidoglycan are not identical, but vary with respect to one particular amino acid. The most common instance is the replacement of L-alanine or glycine by serine. A well documented example is Leuconostoc oenos. The peptidoglycan in this bacterium is of the L-Lys-L-Ser-L-Alatype, which implies the following molar ratio of amino acids : D-Glu:L-LYS:Ala :L-Ser = 1 : 1:3 : 1. Some of the 24 strains investigated exhibit a close approximation to this ratio, but other strains contain significantly more than one mole of serine per mole of glutamic acid and correspondingly less than three moles of alanine. The following ratios of L-Ser :D - G ~ uwere found : 1.0 and 1.1 (4 strains each), 1-2 (6 strains), 1.3 (5 strains), 1.4 (2 &rains), 1-13 (2 strains) and 1-8 (1 strain). Analysis of the amino-acid sequence of these peptidoglycans (E. Lauer and 0. Kandler, unpublished observations) has shown that the L-alanine residues of the interpeptide bridge can be replaced more or less completely by L-serine residues. Thus the peptide bridges of some strains contain predominantly L-Lys-L-Ala-L-Ser and less L-Lys-L-Ser-L-Ser(L-Ser:D-Glu = 1.0-1.5), whereas in others this ratio is reversed (L-Ser:D-Glu= 1.5-2.0). When several batches of cells of a particular strain grown on the same medium were investigated, the ratios of L-Ser :D - G ~ ufound were similar. Table 1 shows the molar ratios ofthe amino acids of the peptidoglgcan of strain L. oeno8 83 and of three subcultures obtained from single colonies of this strain. The ratio of L-Ser :Ala is practically identical in all cases. This may indicate that a set of alleles code enzymes with different affinities towards L-serine or L-alanine in the various strains of L. oenos. The enzyme which adds the

258

K. H. SCHLEIFER,

W. P.HAMMES AND 0.KANDLER

second amino acid of the interpeptide bridge is probably not subjected to such variation, but rather is quite specific for L-serine. Addition of L-serine or L-alanine to the medium causes only a slight change in the amino-acid composition of the interpeptide bridges, whereby the proportion of the amino acid added rises (Table 1). A similar situation is found in species in the genus Staphylococcus, where serine may replace glycine to a differing extent depending on the genetic make up. I n this instance, however, the modification caused by manipulating the concentration of glycine or serine in the medium is much more pronounced than with L. oenos. The phenotypic modification may even mask the genotypic differences (vide infra). Another example of the occurrence of multiple alleles is probably provided by strains of Micrococcus mucilaginosus (Bergan et al., 1970). As shown from the quantitative amino-acid composition of the cell walls (Table 2) and the peptide pattern of their partial acid hydrolysates, the interpeptide bridges consist in all strains of single amino-acid residues which may be either L-Ala, L-Ser or Gly (Schleifer and Kandler, 1972). Within these strains, one amino acid can be replaced in part or completely by another one. In three strains (CCM 2417, 2486, 2487) only r,-alanine serves as the interpeptide bridge; in strains CCM 2392 and 2393 some L-alanine residues are replaced by L-serine and in strains CCM 2391 and 2485 most of the interpeptide bridges consist of L-serine and, to a minor extent, of glycine. A partial replacement of L-alanine residues in the interpeptide bridges by L-serine is also common among streptococci. I n Strep. agalactiae, which contains peptidoglycan of the Lys-L-Ala, type, and in Strep. bovis and Strep. equinus, which contain the Lys-L-Thr-L-Alatype, some of the L-alanine residues are replaced by L-serine. The degree of L-alanine replacement is again characteristic for the particular strains (Schleifer and Kandler, 1972). Although it seems most likely that the variations mentioned have arisen as a result of mutations, direct evidence for such mutations is not available at present.

III. Effects of Exogenous Factors A. AEROBIC AND ANAEROBIC GROWTH

O’Brien and Kennedy (1971) observed that t h e walls of anaerobically grown cells of Staphylococcus aurew were more sensitive to the action of lysozyme, lysostaphin, and Bacillus subtitis 168 I- autolytic enzyme than walls of aerobically grown cells. Based on this differenco in the rate of lysis, the authors postulated that the cell walls or surfaces of anaerobically and aerobically grown cells are different. The aminoacid and amino-sugar compositions of these cell walls were not deter-

TABLE1. Amino-Acidand Amino-SugarCompositions of the Peptidoglycans of Leuconostoc oenos Strain 83 and Three Subcultures (I-111) Obtained From Single Colonies Molar ratio (Glutamate = 1) Strain 83 83/III 83/11 83/I 83/I

Addition tothe medium

pmoles Glu/mg cellwall

Serine

None None None

0.089 0.075 0.079 0.084 0.080

1.3 1.3 1.2 1.3 1.2

0.8 0-7 0.7

0-074

1.4

None

L-Alaaine

Muramic acid

Alanine

Glucosamine

Lysine

Ammonia

0.7

2-3 2.4 2-4 2-5 2.6

0.9 0.8 0.8 0.8 0.6

1.0 1.0 0.9 1.0 1.1

0.5 0.5 0.4 0.6 0-7

0.7

2.3

0-8

1.0

0.7

0.7

(0.50/,,W/V)

83/I

L-Serine (0*5%,W/V)

260 K. € SCHLEIFER, I. W. P. HAMMES AND 0. KANDLER TABLE 2. Amino-Acid and Amino-Sugar Compositions of the Peptidoglycans of Micrococcw mucilaginoaus

Molar ratio (Glutamate = 1) Strain CCM 2417 CCM 2486 CCM 2487

CCM 2392 CCM 2393 CCM 2391 CCM 2486

Lysine

Alanine

Glycine

Swine

0.96 1.0 0.99 1.02 1.02 0.98 0.97

2.75 2.88 2.86 2.67 2.30 1.85 1.73

-

-

-

-

0.74 0.86

0.35 0.67 0.15 0.14

-

Muramic acid Glucosamine 0.88 0.92 0.98 1.0 0.93 0.90 0.92

1.1 1-35 1.20 1.05 1.04 1*4 0.90

mined by these authors. We analysed walls of Staph. aurew, strain Copenhagen, derived from cells grown in the same medium under aerobic and anaerobic conditions. The amino-sugar and amino-acid compositions of both cell-wall preparations were quite similar (Table 3), and even the peptide patterns of partial acid hydrolysates of these cell walls were identical, indicating that no significant differences exist in the amino-acid sequence of the peptidoglycan. Differences in the sensitivity of these cell walls t o lysostaphin were also not as drastic as described by O’Brien and Kennedy (1971). We could, however, corroborate the findings of O’Brien and Kennedy that the walls of “anaerobic grown” cells are slightly more sensitive to lysostaphin than walls of aerobic grown cells (Fig. 2). A remarkable change in the wall composition of aerobically and microaerophilically grown cells was found with Microbacterium lacticum. Aerobically grown cells contained predominantly threo-3-hydroxyglutamio acid, whereas microaerophilically grown cells contained predominantly glutamic acid in their walls (Schleifer et al., 1968a). This indicates that hydroxylation of glutamic acid depends on the oxygen supply during growth. I n addition, it is also dependent on the growth phase (vide supra). B. AMINO-ACID COMPOSITIONOF

THE

GROWTH MEDIUM

The chemical composition of the peptidoglycan can be modified in certain bacteria under conditions where different amino acids are limiting growth, or using an unbalanced growth medium. The modifications, however, are not as dramatic as those found in cell-wall polysaccharides (Ellwood and Tempest, 1972). A few chemically similar amino acids can be oxchanged when the cell-wall amino acids occur in limiting amounts in the medium and related amino acids are added in

TABLE3. Molar Ratios of Amino acids and Amino Sugars of Trypsin-Treated Cell Walls of Staphylococczcs aureus, Strain Copenhagen, Grown Under Aerobic or Anaerobic Conditions Molar ratio (Glutamate = 1) Cells grown under :

Cell wall treatment

Lysine

Alanine

Glycine

Muramic acid

Glucosamine

Trypsin

1.0

2.08

5.27

1.05

1.7

0.95

2.00

5.07

1.0

1.6

1.0 0.96

2.10

5.27

1.0

1-8

2.03

5.05

0.9

1-6

Aerobic conditions Trypsin

+ dinitrophenylation

Trypsin

+ dinitrophenylation

Anaerobic conditions

Trypsin

262

K. H.SOHLBIFER, W. P. HAMbfES AND 0.KANDLER

01 0

, 10

,

20

,

,

30 LO Time (min)

,

SO

, 60

,

FIG.2. Lysis by lysostaphin of walls of Staphylowccue aurew.9, strain Copenhagen, cells grown under aerobic (x) or anaerobic ( 0 )conditions.

rather high concent.rations. Thus, hydroxylysine can be incorporated instead of lysine into the peptidoglycan of Streptococcus faem1i.s or Leuconostocsp. (SmithandHenderson, 1964;Smithetd., 1965;Shockman et al., 1965)during growth in a lysine-depleted medium which is supplemented with hydroxylysine. In a diaminopimelic acid auxotrophic mutant of E. coli, lanthionine, a monosulphur analogue of diaminopimelic acid, can be incorporated into the cell wall (Knusel et al., 1967). Addition of L-serine to the growth medium influences the content of both serine and glycine in cell walls of staphylococci (Zygmunt et al., 1967; Browder et aZ., 1968). More detailed studies on the influence of the amino-acid composition of the growth medium on the composition and amino-acid sequence of the peptidoglycan have been carried out in our laboratory. The most pronounced modifications were found among staphylococci (L. HUSS, unpublished observations; Schleifer, 1969; Schleifer et al., 1969; G. Rauch, unpublished observations). Staphylococcus aurezls strain Copenhagen, Stqhy~ococcu8sp. strain 24 and Staphylococcus sp. strain 66 were studied in more detail. Fragments of the primary structure of the peptidoglycan of these three strains are depictedin Fig. 3. They are distinguished by the chemical composition of their interpeptide bridges. The interpeptide bridge of dtqh. aweus consists of five glycine residues (Ghuysen et al., 1965;

ENDO- AND EXOGENOUS FAUTORS ON BAUTERML PEPTIDOGLYCAN

263

-G-M-G-

1

L-Ala

1

D-GIu-NH,

P

L - L y s z G l y c GlycGlycGly-Gly+(a) Gly t Gly t L - S e r c G l y c G 1 y - b ) Z L - A l a c GlycGly-GlycGlyc(c)

4 D-Ala

1

(D-Ala)

D - Ala

l I t

L- Lys I

I

FIG. 3. Fragments of the primary structures of peptidoglycans found in Staphylococcus azcreua strain Copenhagen (a),Staphylococcus sp. strain 24 (b) and Staphylococcus sp. strain 66 (c).

Tipper et al., 1967);that of strain 24 also contains pentaglycine interpeptide bridges, but some of the glycine residues are replaced by L-serine (Schleifer et al., 1969). The interpeptide bridge of the peptidoglycan of strain 66 consists of a tetraglycyl-L-alanine peptide (Schleifer et al., 1968b). Staphylococczlsaurezcs, strain Copenhagen, was grown in yeast extractpeptone-glucose broth (0.5% Cenovis yeast extract; 1% peptone from casein, Merck; 0.5% glucose) and walls of bacteria from a stationaryphase culture were prepared. The amino-acid composition of the peptidoglycan was determined, and it was observed that the molar ratio of glutamic acid to glycine was not 1 :5 as found by Tipper et al. (1967)but only 1:2.9 (Table 4). The content of alanine, on the other hand, was increased. Instead of a glutamic acid :alanine mtio of 1 :2.1, we obtained a molar ratio of 1 :2.6. Comparing our culture conditions with that of Tipper (1969~)and Tipper et al. (1967), we found that the composition of the growth medium was quite similar with the exception of the brands of yeast extract and peptone. We used Cenovis yeast extract and Merck peptone from casein (abbreviated as Cenovis-Merck medium) whereas Tipper (Tipper et al., 1967; Tipper, 1969c)used Bacto-yeast extract and Bacto-peptone (abbreviated as Difco medium). The effect of different combinations of peptones and yeast extracts on the amino-acid composition and primary structure of the peptidoglycan of Staph. aureus, strain Copenhagen, is summarized in Table 5. Using the Difco medium, we obtained an amino-acid composition similar to that found by Tipper et al. (1967).Even in the presence of only one of the Difco products, glycine values of five moles per mole of glutamic acid were found. Determination of the N-terminal amino acids of the cell-wall peptidoglycan by dinitrophenylation indicated that, in cells

w m

TABLE 4. Molar Ratio of the Amino Acids and Amino Sugars of Peptidoglycan from Cell Walls of Staphylococcus aurew, Strain Copenhagen, (Ester Alanine Free)

Preparation

Muramic acid Ammonia

Glutamate

Lysine

Glycine

Alanine

Glucosamine

Tipper et al. (1967)

1*o

1.0

5.1

2.1

2-1

1.0

1.1

K. H. Schleifer, W. P. Hammes and 0. Kandler

1.0

1.02

2-9

2.6

2.05

0.98

1.12

@ 9

.v

B

kiU ?

ENDO- AND EXOGENOUS ITACTORSON BACTERUL PEPTIDOOLYUAN

265

TABLE5. Effect of Different Yeast Extract Peptone Media on the Primary Structure of the Peptidoglycan of Staphylococcw aurew, Strain Copenhagen Nature of the yeast extract (Y) andpeptone(P)

Molar ratio (Glutamate = 1)

N-terminal amino acid Occurrence of

Lys

Ala

Gly

Lys“ Ma” Glyb

N6-~-Ala-~-Lys

Y

P

Difco Difco

1.04

2.16

6-2

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