Advances in
MICROBIAL PHYSIOLOGY
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Advances in
MICROBIAL PHYSIOLOGY Edited by A. H. ROSE School of Biological Sciences Bath University England
and
J. F. WILKINSON Department of General Microbiology University of Edinburgh Scotland
VOLUME 6 1971
ACADEMIC PRESS - LONDON and NEW YORK
ACADEMIC PRESS INC. (LONDON) LTD. BERKELEY SQUARE HOUSE BERKELEY SQUARE LONDON, W l X 6BA
U.S. Edition published by ACADEMIC PRESS INC. 111 FIFTR AVENUE NEW YORK, NEW YORK 10003
Copyright 0 1971 By ACADEMIC PRESS INC. (LONDON)LTD.
All Rights Reserved No part of this book may be reproducedin any form by photostat, microfilm, or any other means, without written permission from the publishers Library of Congress Catalog Card Number: 67-19860 SBN: 12-027706-0
PRINTED IN QREAT BRITAIN B Y WILLIAM CLOWES A N D SONS LIMITED LONDON, COLCHESTER A N D BECCLEB
Contributors to Volume 6 B. L. A. CARTER,Laboratoryof Molecular Biologyand Department of Bacteriology, University of Wiscon&n, Madison, Wisconsin, U.S.A. S. DAQLEY, Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 55101, U.S.A.
H. 0. HALVORSON, Laboratory of Molecular Biology and Departmenl Bacteriology, Univeraity of Wisconsin, Madison, Wisconsin, U . S . A.
of
ARTHUR Id. KOCH, Department of Microbiology, Indiana University, Bloomington, Indiana 47401, U . S . A .
HENRYKOFFLER,Department of Biological Sciences, Purdue University, hfayette, Indiana, U . S . A . R . W. SMITH, Department of Biological Sciences, Purdue University, hfayette, Indiana, U.S.A.
P. TAURO,Department of Microbiology, Haryana Agricultural University, Hissar, India. R. S . WOLFE,Department of Microbiology, University of Illinois, Urbaruz, Illinois, 61801, U . S . A .
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Contents Contributors t o Volume 6 Catabolism of Aromatic S. DAGLEY I. Introduction .
.
.
v
Compounds by Micro-Organisms.
. A. Inert Compounds in the Economy of Nature B. Aromatic Compounds Made by Man . C. Studies of Enzyme Regulation . 11. The Metabolism of Benzenoid Compounds by Rhodopseudomoms plustris . 111. Enzymic Degradations of Di- and Trihydroxyphenols A. Ortho-Fission Pathways of Catechol and Protocatechuate . B. Metu-Fission Pathways of Catechols . . C. Bacterial Metabolism of Gentisates . D. Degradation of Trihydric Phenols IV. Reactions Converting Aromatic Compounds into Ring-Fission Substrates. A. Hydroxylations . B. Oxidation of Aromatic Hydrocarbons to Catechols C. Modification of Substituent Groups Before Ring Cleavage V. Regulation of Catabolic Sequences A. Physiological Functions and Distribution of the Various Pathways B. Regulation of Ortho-Fission Pathways : Catechol and Protocatechuate . C. Some Methods Used to Investigate Regulation . D. Regulation of the Metu-Fission Pathway for Catechol . E. Evolutionary Significance of Regulatory Mechanisms . VI. Acknowledgements References
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1 2 3 4
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5 7 7 10 14 17
.
20 20 25 27 32
.
.
.
.
Synthesis of Enzymes During the Cell Cycle. B. L. A. CARTER and P. TAURO I. Introduction . 11. Methods for Establishing Synchronous Cultures A. PhasingMethods . B. Selection Methods
32 35 39 41 41 42 42
H. 0. HALVORSON,
.
. . .
.
47 49 50 51
viii
CONTENTS
. 111. Synthesis of Protein and RNA During the Cell Cycle A. Protein Synthesis B. RNASynthesis . . IV. Enzyme Synthesis During the Cell Cycle A. Introduction . B. Synthesis of Enzymes in Prokaryotic Organisms Growing in a Constant Environment . C. Synthesis of Enzymes in Eukaryotic Organisms Growing in a Constant Environment . . D. Induction Capacity in the Cell Cycle E. Speculations on the Molecular Basis of Regulation During the Cell Cycle . . V. Why Does a Cell Divide? VI. The Importance of Temporal Order in Cells . VII. Concluding Remarks . VIII. Acknowledgements . References
.
Microbial Formation of Methane.
.
.
.
57 63 71 75 95 98 99 99 99
R. S. WOLFE
I. An Introduction to the Ecology of Methane Bacteria 11. Isolation of Methane Bacteria A. Enrichments. B. The Hungate Technique . 111. Characteristics of Methane Bacteria . A. Morphological Types B. Species and Their Properties . C . Resolution of Metlmnobacterium omelianskii . IV. Mass Culture Techniques . A. Growth on Hydrogen and Carbon Dioxide . . B. Growth on Formate . C. Growth on Methyl Alcohol . V. Biochemistry of Methane Formation A. Assay System . B. Substrates . C. Methylcobalaniin as Substrate . D. Role of ATP . E . Cobaloximes a s Substrates . P. Role of Coenzyme-M . G. Inhibitors of Methane Formation . H. Reduction of Arsenate I. Mini-Methane Systems . VI. Acknowledgements References
.
53 53 53 55 55
.
107 109 109 110 114 114 118 118 124 124 126 126 127 127 128 130 134 136 138 139 143 144 144 145
ix
(IONTENTS
The Adaptive Responses of Escherichia coli t o a Feast and Famine Existence. ARTHUR L. KOCH I. Introduction . 11. The Speed of Macromolecular Sythnesis . 111. “Extra” RNA in Slowly Growing Bacteria . IV. Description and Operation of Chemostats . A. DesignFeatures . B. Evidence that the “Extra” RNA is not an Artifact Due to . Inadequate Mixing of the Chemostat V. RNA Synthesis in Slowly Growing Bacteria . VI. Tracer Kinetics Interlude . VII. The Growth Cycle Revisited . VIII. Active Transport From Very Low External Concentrations . . A. Uptake by a Motionless Spherical Cell B. Uptake by Spherical Moving Cells . C. Uptake by Rod-Shaped Particles . D. Movement and Mixing Efficiency . E. The Intermediate Region Between Diffusion and Transport Limitation . F. Experimental Determination of Uptake Parameters by Growth Studies IX. General Conclusions . X. Acknowledgements . References
.
Bacterial Flagella.
161 164 169 181 192 196 203 205 207 208 210 214 214 215
R. W. SMITH and HENRY KOFFLER
.
I. Introduction 11. Basal Material and Site of Attachment 111. The Hook . IV. Sheath-Like Structures V. Isolation and Purification of Flagellar Filaments VI. The Protein Nature of the Filament , VII. Immunology VIII. Stability IX. Arrangement of Protein Subunits X. Re-assembly . XI. Synthesis of the Filament
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.
.
147 149 152 159 159
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.
219 223 230 238 239 240 251 260 276 284 295
OONTENTS
X
XII. Mechanisms for the Function of Flagella XIII. Acknowledgements . References
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. .
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314 327 327
Author Index
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341
Subject Index
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.
366
Catabolism of Aromatic Compounds by Micro-Organisms S. DAGLEY Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul. Minnesota 55101 U.S.A. I. Introduction . . A. I n e r t Compounds in t,he Economy of Nature . B. Aromatic Compounds Made by Man U . Studies of Enzyme Regulation . I I . The Metltbolisni of' Berizenoid Compounds by Khodopaeudomonas paluatris . . 111. Enzymic Degradations of Di- a n d Trihydroxyphenols A. Ort?Lo-FissionPathways of Catechol and Protocatechuate . . B. Meta-Fission Pathways of Catechols C. Bactmial Metabolism of Gentisates . D. Degradation of Trihydric Phenols . IV. Reactions Convorting Aromatic Compounds into Ring-Fission Substrates A. Hydroxyllttions . B. Oxidation of Aromatic Hydrocarbons t o Catechols . C. Modification of Substituent Groups Before Ring Cleavage . V. Regulation of Catabolic Sequences A. Phyfiiological Functions and Distribution of t h e Various Pathways B. Rogulation of Ortho-Fission Pathways: Catechol a n d Protocate. chuate C. Some Mothods Used t o Investigate Regulation D. Regulation of t,he Metn-Fission Pathway for Catechol . . E. Evolutionary Significance of Regulatory Mechanisms VI. Acknowledgements . References .
5 7 1
10 14
17 20 20 25
27 32
32
35 39 41 41 42 42
I. Introduction The classification of detailed events in known metabolic pathways, together with discoveries of new reactions, will always invite investigation regardless of the area of metabolism in which they are found. But there are three additional reasons why attention will continue to be given to these reactions which microbes employ for the enzymic degradation of the benzene nucleus. 1
A. INERT COMPOUNDS IN
THE
ECONOMY OF NATURE
First, the Plant Kingdom synthesizes great quantities of natural products that are biochemically inert and are degraded by microbial enzymes. The benzene nucleus furnishes an example of such chemical stability and inertness. It is continually being synthesized by plants ; and if it were not re-opened by the oxygenases of soil microbes, and then degraded, vast quantities of carbon, locked up in stable rings of six atoms, would be taken out of circulation when plants died. It is true that large amounts of rather inert non-aromatic biochemicals are also biosynthesized by plants, and that these also re-enter the carbon cycle through the action of microbes which can insert oxygen into such molecules and thereby initiate their metabolic degradation. However, studies of crystalline oxygenases, admirably reviewed by Hayaishi (1966) and by Hayaishi and Nozaki ( 1969),have made most progress for enzymos obtained from bacteria grown with aromatic compounds, and wcre therefore induced to synthesize abundant quantities of the protein of interest. Thus, the following four dioxygenases that cleave the benzene nucleus have been crystallized : metapyrocatechase (Nozaki et al., 1963), homoprotocatechuate 2,3-oxygenase (Kita et al., 1965), homogentisate oxygenase (Adachi et al., 1966) and protocatechuate 3,4-dioxygenase (Fujisawa and Hayaishi, 1968). Crystalline bacterial mono-oxygenases which attack the non-aromatic substrates lactate (Sutton, 1957), lysine (Takeda and Hayaishi, 1966) and imidazole acetate (Maki et al., 1966)have also been obtained ;bub the mono-oxygenase which hydroxylates p-hydroxybenzoate to give protocatechuate has received particular attention, and crystalline enzymes have been purified from two different strains of Pseudomonas putida (Hosokawa and Stanier, 1966; Hesp et al., 1969) and from Pseudomonas desmolyticu (Yano et al., 1969). From the last-named organism, two crystalline forms of the enzyme were obtained: one was the holo-enzyme and the other, the enzymesubstrate complex with p-hydroxybenzoate. The separate crystallization of such a complex is a notable achievement in enzymology. Slight differences in sedimentation co-efficient8and in the optical rotatory dispersions of solutions examined in the ultraviolet provided evidence for small conformational changes in the enzyme when the substrate was bound. The p-hydroxybenzoate hydroxylase preparations of each of these three groups of workers contained bound FAD and required NADPH, as electron donor. The relevance of these studies to other areas of microbial metabolism is evident from the findings of Trudgill et al. (1966a,b) concerning the degradation of the non-aromatic terpene, camphor, by Pseudomonas putida. A complex containing two enzymes, which
CATABOLISM OF AROMATIC COMPOUNDS BY MICRO-ORGANISMS
3
catalysed the insertion of an oxygen atom between C-1 and C-2 of I)(+)-camphor,was purified ten-fold from this pseudomonad. The electrons required by this mono-oxygenase, which produces lactones from (+)-camphor or 2,5-diketocamphane, were furnished by NADH, ; in this respect the system resembled salicylate hydroxylase (Yamamoto et al., 1965) and differed from p-hydroxybenzoate hydroxylase, which requires NADPH,. Further, in the camphor-lactonizing system, electrons are transferred through FMN, rather than FAD, to enzyme-bound iron. The role of mono-oxygenases as initiators of microbial aromatic degradations is not restricted t o catalysing hydroxylations of the benzene nucleus. I n many naturally occurring compounds the nucleus is substituted by methyl, methoxyl and similar groups. Pseudomonas testosteroni first attacks the methyl group of p-cresol, which is converted to p-hydroxybenzoate (Dagley and Patel, 1957). Another species of Pseudomonas attacks the two methyl groups of 2,4-xylenol, oxidizing each to carboxyl; and the observation that cell-free extracts must be supplemented with NADH,, or NADPH,, before the xylenol is degraded would strongly suggest that the initial attack is catalysed by a monooxygenase (Chapman and Hopper, 1968). Detailed studies have already been made of the enzymology of hydroxylation of fatty acids and of octane in Pseudomonas olevorans (Peterson et al., 1966), and also of the methylene hydroxylation of camphor (Hedegaard and Gunsalus, 1965 ; Katagiri et al., 1968) which is catalysed by an enzyme complex in Pseudomonas putida. It is evident that similar investigations of the enzymes involved in oxidizing methyl and methoxyl groups attached to the benzene nucleus would be of general interest in widening our understanding of the reactions that serve to initiate pathways of degradation in microbes.
B. A~~OMATIC COMPOUNDS MADE BY MAN We may suggest a second line of thought which justifies a continued interest in aromatic degradations. I n addition to what we may learn about the part played by microbes in the general economy of Nature, the investigation of aromatic catabolism is also relevant to problems that arise from the disturbance of natural cycles by the activities of Man. These “problems of molecular recalcitrance and microbial fallibility”, as they are described in the title of a most interesting review of Alexander (1965), are by no means confined to detergents, pesticides and other synthetic compounds that often contain benzene nuclei and whose resistance t o microbial action can constitute a nuisance to Man and a health hazard to other forms of life. Indeed, as Alexander (1965) points out, many organic compounds found in Nature are recalcitrant, possibly
4
9. DAQLEY
on account of features of their chemical structure or combination, or because the conditions that prevail in their environment prevent microbial action. However, if our present knowledge of aromatic catabolism had been available when the compound was first used, we could have predicted without reservations that DDT would be resistant to microbial attack and that its unrestricted use would not have been desirable. Knowledge of the enzymic breakdown of the benzene nucleus, particularly when bearing halogen substituents, will contribute towards an understanding of the stubbornness of particular molecules t o succumb t o microbial action.
c. STUDIES O F ENZYME REGULATION A third general reason for continued interest in aromatic degradations lies in the fact that they provide particularly convenient systems for studying the conditions that determine the derepression of functionally related enzymes. Thus, three separate and distinct types of metabolic pathway are available for converting dihydroxyphenols into metabolites related to the tricarboxylic-acid cycle ; namely, the two pathwaysortho and meta fission-for catechols, and also the reactions that degrade gentisic acid and its derivatives. Each route involves several enzymes that function together as a group; and for any particular compound there are usually several other enzymes that operate for the catabolism of side chains. The existence of these two phases of metabolism, one concerned with the preparation of the nucleus for fission and the other wibh its degradation, can confer a flexibility which is further increased by the possibility of varying the nature of substituents as well as their points of attachment t o the benzene nucleus. Such studies of enzyme induction or derepression have demonstrated a variety of mechanisms by which catabolic enzymes may be derepressed, singly or in batches, by both the substrates and the products of metabolic sequences. This review will deal mainly with advances that have been made, and problems which have arisen, since the review of Ribbons (1965). For an account of the aromatic di-oxygenases, the reader is referred to Hayaishi (1966) and Hayaishi and Nozaki (1969); however, attention is also drawn to recent work which proves that quercetinase is a dioxygenase (Krishnamurty and Simpson, 1970). This remarkable enzyme is synthesized by Aspergillusjavue and other fungi when they are grown with rutin as a source of carbon; and it catalyses an oxidative cleavage of the heterocyclic ring of quercetin to give carbon monoxide and a depside, protocatechuoyl phloroglucinolcarboxylic acid. Quercetinase, therefore, functions early in the degradative sequences of chromones by fungi (Simpson et al., 1963) but it does not cleave the benzene nuclei
CATABOLISM O F AROMATIC COMPOUNDS BY MICRO-ORGANISMS
5
present in these molecules. When ortho and meta fissions of catechols occur, two atoms from one molecule of oxygen invariably combine with adjacent carbons; but Krishnamurty and Simpson (1970) used '*02 to prove that quercetinase incorporated one atom of oxygen a t C-2 and the other a t C-4, C-3 being eliminated simultaneously as carbon monoxide containing no isotope. The reaction mechanism of quercetinase is particularly interesting since it appears to require the formation of an unstable cyclic peroxide as an intermediate. The subjects of this review will be grouped into three main headings : ( 1 ) enzymic degradations of di- and trihydroxy phenols ; (2) reactions converting aromatic compounds into ring-fission substrates ; and (3) regulation of catabolic reaction sequences. First, however, an important advance in microbial aromatic metabolism will be reported which is not accommodated by existing categories ; namely, an entirely new pathway which involves reduction of the benzene nucleus prior to ring fissioning.
11. The Metabolism of Benzenoid Compounds by Rhodopseudomonas palustris
It; has been known for a long time that certain bacteria are able to dissimilate aromatic compounds under anaerobic conditions ; thus, Tarvin and Buswell (1934) showed that methanogenic bacteria decomposed tyrosine and also the following compounds completely : benzoic, phenylacetic, hydrocinnamic and cinnamic acids. Phthalic and salicylic acids and phenol were decomposed to some extent, but benzaldehyde, benzene, toluene and aniline were not attacked by these cultures. Clark and Fina (1952) made the significant observation that methanogenic bacteria grown with benzoate did not metabolize catechol or protocatechuate ; and since these are intermediates commonly formed when the benzene nucleus is degraded by aerobic bacteria, it might be inferred that aerobic and anaerobic sequences bake very different metabolic routes. However, when Proctor and Scher (1960) investigated the anaerobic photometabolism of benzoate by a species of Rhodopseudomonas they reported that, when these organisms were grown in the light, they were also capable of oxidizing benzoate in the dark by a pathway that apparently involved both protocatechuate and catechol as reaction intermediates. The question has now been re-investigated using Rhodopseudomonas palustris, an organism which can be grown photosynthetically with p-hydroxybenzoate, for example, and is then able to photo-assimilate benzoate and all three monohydroxybenzoates a t similar rates by means of enzymes that are inducible but apparently lack substrate specificity
6
8. DAQLEY
(Dutton and Evans, 1969). Para-Hydroxybenzoate but not benzoate can serve as carbon source for aerobic growth in the dark. Hegeman (1967d) showed that the aerobic metabolism of p-hydroxybenzoate by Rh. palustris was initiated by hydroxylation to give protocatechuate, which was then attacked by a 4,5-oxygenase followed by an NADPdependent oxidation of the ring-fission product, u-hydroxy-y-carboxymuconic semi-aldehyde ; pyruvate was the end-product of degradation by cell extracts. The first two enzymes of this pathway were virbually absent from extracts of cells grown photosynthetically a t the expense of p-hydroxybenzoate, and it is therefore evident that neither of these
*.*
I
I1
6 V
IV
111
FIG.1 Roductivo metabolism of bonzoate by RhodopseudortLonas palustris.
enzymes, nor protocatechuate, participates in the photometabolism of p-hydroxybenzoato by this organism. Further, as Dutton and Evans ( 1969) showed, Rh. palustris growing photosynthetically with benzoate does not utilize catechol ;moreover, the metabolism of benzoate is totally inhibited by oxygen. These authors have proposed a new method of aromatic ring metabolism shown in Fig. 1, where benzoate is reduced to cyclohex-1-ene-1-carboxylate(I) which is then metabolized by reactions similar to those employed for the /%oxidation of fatty acids, namely, addition of water 60 give 2-hydroxycyclohexanecarboxylate (11),dehydrogenation to 2-oxocyclohexanecarboxylate (111) and ringcleavage of this compound to yield pimelate (IV). Recent studies show that the ring-fission step is coenzyme A-dependent (W. C. Evans, private communication). The proposed sequence was supported by experiments in which Rh. palwtris metabolized high concentrations of [14C]benzoate in the presence of suspected intermediates added us
CATABOLISM OF AROMATIC COMPOUNDS BY MICRO-ORGANISMS
7
“carriers” of isotope. Convincing evidence for the participation of compounds I, 11, I11 and IV was obtained when they were re-isolated from supernatant culture fluids and crystallized to constant specific activities. Evidence was also obtained for the formation of cyclohexanecarboxylate (V) : this compound might have arisen directly from benzoate by complete hydrogenation of the benzene nucleus, prior to a dehydrogenation to give I. Alternatively a direcb route from benzoate to I is feasible as shown in Fig. 1, although the precise steps by which four hydrogen atoms are taken up remain to be elucidated. The same pathway for the photometabolism of benzoate has been proposed independently by Guyer and Hegeman (1969) who adopted a totally different experimental approach. The parent strain of Rh. palustris is able t o utilize cyclohexanecarboxylate (V) as a source of carbon for aerobic, non-photosynthetic growth, during which compounds I, 11, I11 and IV are apparently formed when the growth substrate is catabolized. By treatment with nitrosoguanidine, mutants were isolated that were no longer able t o grow aerobically with cyclohexanecarboxylate. Some of these mutant strains suffered a simultaneous loss of ability to grow on benzoate anaerobically in the light, but they were able to respond to additions of compounds I, I1 and IV in a manner consistent with the operation of the sequence of Fig. 1 for the photometabolism of benzoate. One strain was shown t o accumulate radioactive cyclohex-lene-1-carboxylate from [14C]benzoate when it grew a t the expense of acetate under anaerobic conditions. These demonstrations of a novel reductive pathway of aromatic ring dissimilation, employed by Rh. palustris growing anaerobically in the light, encourage the prediction that benzenoid compounds may be metabolized in a similar fashion by non-photosynthetic anaerobic micro-organisms such as the methanogenic bacteria.
111. Enzymic Degradations of Di- and Trihydroxyphenols A. Ortho-FIssloN PATHWAYS O F CATECHOL AND PROTOCATECHUATE A preliminary report of Ornston and Stanier (1964), which clarified several obscurities associated with the degradation of catechol and protocatechuate by Pseudornonm putida, was reviewed by Ribbons (1965).A full account of these valuable studies has now been published (Ornston and Stanier, 1966; Ornston, 1966a,b). The two pathways (Fig. 2) show a striking chemical parallelism, but different compounds are involved until the routes converge upon a common metabolite, 8-ketoadipate enol-lactone (y-carboxymethyl-A p-butenolide ; IV, Fig.
I
II
III
V
FIG.2 Bacterial degradation of catechol and protocatechuate by ortho-lkion. In the text, intermediates are designated by the Roman numerals shown beneath the chemical structures, and enzymes are designated by Arabic numerals.
CATABOLISM OF AROMATIC COMPOUNDS B Y MICRO-ORGANISMS
9
2). The enzymes concerned are also different and highly specific for their substrates, so that a muconate or its lactone on the first pathway is not metabolized by enzymes of the second. Two new intermediates were identified by Ornston and Stanier (1966). One of these, P-ketoadipate enol-lactone (IV), was isolated as colourless needles by the action of purified enzymes on j3-carboxy-cis,cis-muconate(11): its melting point differed radically from an isomer which had been synthesized chemically by Eisner et al. ( 1950), namely y-carboxymethylenebutanolide in which the double bond is exocyclic. I n the assigned structure of IV, the bond is endocyclic and this position is in accordance with all the features of the ultraviolet and nuclear magnetic resonance spectra of the compound. fl-Carboxy-&,cis-muconic acid forms two y-lactones ; one of them, which carries a carboxyl group in the /?-position, has been chemically synthesized (MacDonald et al., 1954) and is formed as an intermediate in the degradation of protocatechuate by Neurospora crassa (Gross et al., 1956).Neither of the optical isomers of this lactone was attacked by extracts of P . putida grown with p-hydroxybenzoate. The second of the new intermediates formed in the metabolism of protocatechuate by this organism (Ornston and Stanier, 1966)is the lactone that bears a carboxyl group in the y-position, namely compound I11 of Fig. 2. Unlike j3carboxymuconolactone, which is stable in neutral solutions a t 30°, y-carboxy-y-carboxymethyl-A=-butenolide (111, Fig. 2) loses carbon dioxide rapidly, whether enzymes are present or not, to give /?-ketoadipate enol-lactone (IV). I n the course of these investigations it was necessary to purify several of the enzymes that catalyse reactions shown in Fig. 2. Of the various properties reported (Ornston, 1966a) it is of interest that enzyme 2, which lactonizes 8-carboxy-cis,&-muconate, differed from enzyme 2’ (which serves a similar function specifically for cis-cis-muconate) insofar as it was not stimulated by magnesium or manganese chlorides, neither was it inhibited by 10 mM-EDTA. Enzyme 4, which catalyses the hydrolysis of j3-ketoadipate enol-lactone, was extremely heat-labile. Ornston ( 1966b) crystallized both the cis-cis-muconate lactonizing enzyme (2’)and muconolactone isomerase (3’)and showed that they were highly specific for their substrates. This demonstration was an essential prelude to studies of the regulation of syntheses of enzymes involved in the two pathways. Thus, cells that used the catechol pathway for the metabolism of benzoate gave extracts that were able to lactonize j3-carboxymuconate ( 11). Rigorous purification of the relevant enzymes enabled the conclusion to be drawn that these activities could not be attributed t o non-specific catalysis by enzymes 2‘ and 3’ but were due to the fact that enzymes 2 and 3 were derepressed by /?-ketoadipate
10
9. DAQLEY
(or /3-ketoadipyl coenzyme A) formed during benzoate degradation. Benzoate-grown cells therefore contained all four enzymes : 2,2’,3 and 3’ (Ornston, 1 9 6 6 ~ ) .
B. Me2a-FISSIoN PATHWAYS O F CATECHOLS The pathways for degradation of catechol and 4-methylcatechol by meta-fission, shown in Figs. 3a and b, are known in less complete detail than those for ortho-fissions shown in Fig. 2 (p. 8). Important information relating to meta fission has emerged from studies of the microbial degradation of steroids, and an outline of some of these reserves as actions is also given (Fig. 3c). When androst-4-ene-3,lir-dione growth substrate for Pseudomonas testosteroni or Nocardia restrictus, ring B of the steroid is rupturcd and ring A becomes aromatized with the formation of a seco-phenol (VII) (Dodson and Muir, 1958, 1961). From this point onwards, the elucidation of the fate of ring A becomes a problem in microbial aromatic metabolism. Thus, Sih et al. (1966) prepared the catechol (VIII) and demonstrated that it was rapidly degraded by cell-free extracts of Nocardia restrictw to give ring-fission product (IX); this compound gave ultraviolet spectra reminiscenb of those of 2-hydroxymuconic semialdehyde a t p H 13 and 1.0. The remaining steps in metabolism (Gibson et al., 1966) also involved reactions of the same type as those encountered in the meta-fission pathways of other catechols. First, the rest of the steroid skeleton containing rings C and D was released from the ring-fission product I X (as the acid R-COOH; Fig. 3) by hydrolytic cleavage; similar reactions occur in the degradation of catechol, 3-methylcatechol and 2,3-dihydroxyphenylpropionate when R represents hydrogen, methyl and carboxyethyl respectively (Dagley et al., 1964; Bayly et al., 1966). Hydration of 2oxohex-4-enoic acid (V) then gave 4-hydroxy-2-oxohexanoic acid (VI) which underwent an aldolase cleavage to yield pyruvate and propionaldehyde. Recent investigations of the stereochemistry of intermediates (V) and (VI)in steroid metabolism are pertinent to studies of the degradation of catechol and 4-methylcatechol. Extracts of Pseudomonas sp. grown with phenol or cresols metabolized only half of synthetic 4-hydroxy-2oxovalerate (111)(Dagley and Gibson, 1965) or 4-hydroxy-2-oxohexanoate (VI) (Bayly et al., 1966) and it was concluded that only one of the enantiomers of each compound was biologically active. This was established directly for the second of these two hydroxyoxo acids by Coulter and Talalay ( 1968) who synthesized 2-oxo-ci~-hex-4-enoicacid (V) and showed that it was hydrated stereospecifically by extracts of steroidinduced Pseudomonm tecltosteroni to give 4-hydroxy-2-oxohexanoate.
T
0
? f
w
0
CATABOLISM O F AROMATIC COMPOUNDS B Y MICRO-ORQANISMS
a
x
4 x
0
6 2
11
12
9. DAQLEY
When the latter compound was treated with acid, it lactonized to give one, and one only, of the optical isomers of 2-oxo-4-ethylbutyrolactone, but it was not possible to assign the absolute configuration of the enantiomer in question. The problem has been re-investigated by Collinsworth and Dagley ( 1971) who degraded synthetic 4-hydroxy-2-oxovalerate with extracts of Pseudomonas and then submitted the remainder of the sample, which had resisted enzymic attack, to oxidative decarboxylation with hydrogen peroxide. The product of this treatment, 3-hydroxybutyric acid, was found to be oxidized quantitatively to acetoacetate hy a dehydrogenase ( E C 1.1.1.30)specific for the D-isomer. It therefore follows that the enzymically active form of 4-hydroxy-2-oxovalerate is the L,(S) enantiomer (I11 of Fig. 3). This was confirmed by submitting 4-hydroxy-2-oxovalerate,which had accumulated from catechol enzymically, to the same procedure ; a sample of 3-hydroxybutyratewas given which did not serve as a substrate for this dehydrogenase. Provisionally, the product of hydration of 2-0x0-cis-hex-4-enoate(V) is also shown in Fig. 3 as L,( S)-4-hydroxy-2-oxohexanoate (VI). A second feature of stereochemical interest in Fig. 3 is depicted in t h r conversion of compound IV into compound V. The stereochemistry of this reaction has not yet been established for intermediates in the degradation of 4-methylcatechol, but a shift in the position of the methyl group from one side of the double bond to the other may be inferred from the studies of Shaw et al. (1965)relating to the steroid pathway. They found that, in the presence of EDTA, extracts of steroid-induced P. testosteroni accumulated L-2-amino-cis-hex-4-enoicacid which could be converted, either chemically or enzymically by transamination, into the keto acid ( V ) (Coulter and Talalay, 1968).This keto acid is therefore the cis stereoisomer, whereas in the ring-fission products IV and I X the methyl group would be expected to be trans, as shown. Shaw et al. (1965) discuss a mechanism by which this transformation might take place (Fig. 4). The dihydric phenol formed from androst-4-ene-3,17-dione gives the ring-fission product I (Fig. 4) by meta cleavage. If this product undcrgoes ketonization to give compound 11,there will be a shift of the double bond from C-10(1) to C-1(2),and C-10will now become an asymmetric centre. When C-5 is attacked by water and R - C O O His split off, the double bond again takes up a position at C-lO(1) and the cis-isomer (111)is formed. The amino acid IV isolated by Shaw et al. (1966)would be obtained from compound I11 by transamination and would also possess a cis-configuration. Bayly and Dagley (1969)showcd that partially purified extracts of a fluorescent Pseudomonas sp., grown with phenol, accumulated 0x0enoic acids from catechols. A compound with properties consistent with those expected for compound I1 (Fig. 3) was formed from both catechol
13 and 3-methylcatechol, whereas 4-methylcatechol gave rise to 2-oxohex-4enoic acid (V). The stereochemistry of the latter compound was not investigated by these authors; however, compounds I1 and V were enzymically hydrated to give compounds I11 and V I respectively. All three catechols were readily oxidized, but Cain and Farr (1968) have obtained evidence that 3-methylcatechol was attacked by a separate enzyme which differed slightly in its properties from catechol 2,3oxygenase. When the last-named enzyme was crystallized by Nozaki et al. (1063), catechol and 4-methylcatechol both served as substrates; no studies wihh 3-methylcatechol were reported. CATABOLISM O F AROMATIC COMPOUNDS B Y MICRO-ORGANISMS
I
I1
R’C02H
Iv
UI
FIG.4 is possible mechanism for the reactions that, convert a substituted catecho1 into an oxo-enoic acid. The catechol is intermediate V I I I of Fig. 3, and the numbers around the nucleus show how the carbon atoms were located in the original steroid striicture. The 0x0-onoic acid is 2-0x0-cis-hex-4-enoate(111) which gives rise, by transamination, t o tho amino acid IV. Adapted from Shaw et al. (1965).
Pathways (a) and (b) of Fig. 3 (p. 11)are therefore both initiated by the same enzyme, and this tolerance of the presence of a methyl group in their substrates appears also to be shown by enzymes later in the sequences, since they catalyse the reactions of both pathways with equal facility. However, a precise knowledge of substrate specificities and other properties must await purification of these enzymes. Hitherto, this has been hindered by the fact that most of the reaction intermediates are chemically labile and difficult to synthesize, SO that enzyme assays are not readily devised. Two recent investigations of reaction intermediates are pertinent t o the schemes of Fig. 3. The first of these was concerned with the identity of the meta. cleavage product from 3-methylcatechol. Catelani et al. (1968) incubated this substrate with intacb cells of P. desmolyticum and werc able to isolate yellow crystals which were firmly
14
8 . DAGLEY
identified as 2-hydroxy-6-oxo-2,trans-4,trans-heptadienoic acid. As the authors point out, the trans configuration of the 4,6 double bond probably arose from acidic treatment during extraction of the enzymically formed cis-compound which, according to the evidence of Bayly et al. (1966),ismetabolized toacetic acid and 2-hydroxymuconic semialdehyde. The second investigation (Ribbons and Senior, 1970)relates to the oxidation of 2,3-dihydroxybenzoate by P. Jluorescens t o give 2-hydroxymuconic semialdehyde with simultaneous loss of carbon dioxide. They investigated the action of the enzyme upon 2,3-dihydroxy-p-toluate, namely 2,3-dihydroxybenzoate bearing a methyl substituent a t C-4, and they showed that the benzene nucleus was opened, again with loss of carbon dioxide, to give the ring-fission product of 3-methylcatechol studied by Catelani et al. (1968). Since 3-methylcatechol itself is not oxidized by extracts of P. jluorescens grown with 2,3-dihydroxybenzoate, and since 2,3-dihydroxy-p-toluate is not an inducer of the synthesis of this oxygenase, it is evident that both 2,3-dihydroxybenzoate and 2,3-dihydroxy-p-toluate were cleaved in the 3,4 position by the enzyme. Additional interest in 2,3-dihydroxybenzoate metabolism has been stimulated by the discovery of a new anthranilate hydroxylase which requires NADPH, and forms 2,3-dihydroxybenzoate with the release of ammonia. The enzyme was purified from Aspergillus niger grown in the presence of anthranilic acid (Sreeleelaet al., 1969).
C. BACTERIAL METABOLISM OF GENTISATEB The sequence of reactions by which gentisate is metabolized to fumarate and pyruvate (Fig. 6s) was elucidated by Lack (1959, 1961) using cell-free extracts of a species of Pseudomonas grown with m-hydroxybenzoate (Tanaka et al., 1967; Walker and Evans, 1952). However, Wheelis et al. (1967)found that, whereas P. acidovorans takesm-hydroxybenzoate bhrough the gentisate sequence, P. testosteroni oxidizes the same substrate to protocatechuate which is dissimilated through the meta cleavage pathway. The difference between the two species with respect to m-hydroxybenzoate metabolism reflected a difference in the specificities of their m-hydroxybenzoate hydroxylases : the enzyme of P. tmtoeteroni hydroxylated in the 4-position to give protocatechuate, and that of P. acidovoransin the 6-position to give gentisate. The reactions of Fig. 6a are analogous to those for the mammalian metabolism of homogentisic acid which has been extensively studied, although different enzymes are involved in the two sequences. Thus, gentisate is oxidized to maleylpyruvate ( l ) ,a compound that resembles the product of ring-fission of homogentisate, i.e. maleylacetoacetate, insofar as it gives a single peak in alkaline solution a t about 330 nm.,
W
U H
CATABOLISM OF AROMATIC COMPOUNDS B Y MICRO-ORGANISMS
p y \
-... CI
0
H
Y
U 4.r
16
16
8 . DAQLEY
which is abolished on acidification. The chemical structure of maleylpyruvate was established by its alkaline degradation to maleic and pyruvic acids (Lack, 195.9).Maleylacetoacetate is enzymically isomerized t o fumarylacetoacetate, maleylpyruvate to fumarylpyruvate (11); and both enzymes require reduced glutathione (Ravdin and Crandall, 1951; Lack, 1961). More recently Hopper et al. ( 1968) have shown that a different species of Pseudomonas from that used by Lack (1959) does not isomcrize maleylpyruvate : this intermediate is hydrolysed to pyriivate plus maleate which is then enzymically hydrated t o give n-malate. Hydration of furnarate, produced when isomerization does occur, gives rise to L-malate which is a n intermediate of the tricarboxylic-acid cycle, unlike D-malate. It is interesting that, when the Pseudomonas sp. of Hopper et al. (1968) is grown with L-tyrosine as carbon source, an active glutathione-dependent maleylacetoacetate isomerase is presenb in cell exbracts ; but the ability to isomerize maleylpyruvate is still lacking. The gentisate-degrading enzymes of this Pseudomonas, like those of the meta pathway for catechols described in the previous section (p. l o ) , are active towards substrates even when substituent groups have been introduced. Thus, as shown in Fig. Fib, the same cell-free extracts that degraded gentisate to pyruvate and u-malate also metabolized 3methylgentisate (or 4-methylgentisate) to pyruvate and D-citramalute (IV) which arises by hydration of citraconate (111).Pyruvate and acetylCoA were formed from D-citramalate (and also from L-citramalate) only when extracts were supplied with succinyl-CoA, and it is assumed that citramalyl-CoA (V) is the substrate for the aldolase which gives rise to these products (Hopper et al., 1971). A similar activation system for oitramalate was present in another Pseudomonas sp. grown with itaconate (Cooper and Kornberg, 1964). Hopper et al. (1971) also showed that 3,4dimethylgentisate and 3-ethylgentisate are degraded by these extracts, with the corresponding substituted maleic and malic acids formed as intermediates ; the latter then undergo coenzyme A-dependent aldolase fissions. However, this enzyme system does not degrade unsubstituted D-malate, and the reactions by which this compound is utilized are not clear at present. Ib is evident that the relatively low substrate specificities of the enzymes of the gentisate pathway endow this Pseudomonas sp. with metabolic versatility. The organism oxidizes the methyl group of wL-cresol t o a carboxyl group, for example; and provided C-6 of the m-hydroxybenzoate so formed is available for hydroxylation to give gentisate, other carbon atoms of the nucleus can carry various substituents without impairing ability to metabolize. Accordingly, this organism can utilize a range of xylenols and cresols for growth, in addition to m-hydroxybenzoate or gentisate.
17
CATABOLISM OF AROMATIC COMPOUNDS BY MICRO-ORUANISMS
OF TRIHYDRIC PHENOLS D. DEGRADATION
1. Metabolism of Thymol
The ring-fission substrates in all of the foregoing systems were diliydric phenols. However, two metabolic pathways have been described in which the introduction of a third hydroxyl group prior t o ring-fission is a necessary prerequisite for complete metabolism. Extracts of a soil
I
1
rn
IV
INAD& /20
COzH Q
O
Thymol
H
0QoH I1
CH3.CH.CH3 I Isobutyrate
COzH
+ I
CH:,
Acetate
+ I
CH3 CHz*CO*COzH 2.Oxobutyrate
FIG.6 Bact,erid degradation of thymol.
pseudomonad grown with resorcinol ( 1,3-dihydroxybenzene) did not attack this compound until furnished with NADH, ; hydroxylation then gave 1,2,4-trihydroxybenzenewhich was metabolized to /3-0x0-adipate (Larway and Evans, 1965). The second example is that of thymol degradation (Chamberlain et al., 1967 ; Chamberlain and Dagley, 1968) which appears to be initiated by two successive hydroxylations to give 3-hydroxythymo-1,4-quinol (compound 1 of Fig. 6). The main evidence for this pathway was provided by isolating 3-hydroxythymo- 1,4-quinone (11) which was excreted into the medium when Pseudomonas putida utilized thymol as the carbon source for growth. The quinone, which imparted a deep purple colour t o cultures, was obtained as yellow crystals after ether extraction of the acidified growth medium ; the compound is purple at pH 7.5. Cell-free extracts did not attack compound I1 until NADH, was added ; a ferrous ion-dependent dioxygenase then catalysed ring-fission, with acetate, isobutyrate and 2-oxobutyrate resulting as the end products (Fig. 6).The requirement for NADH, suggested that the quinone (11) was reduced to a quinol (I) which served as the actual
18
9. DAOLEY
substrate for the oxygenase. It was found that non-enzymic reduction of a solution of compound I1 with sodium dithionite gave a compound showing ultraviolet absorption consistent with structure I, but this quinol could not be isolated because it was oxidized very rapidly to the quinone by air. This was probably the reason why compound I1 accumulated during aerobic growth and disappeared late in the exponential phase; non-enzymic oxidation of compound I to compound I1 by air would initially compete with ring-fission, whereas compound I would be reformed later by the NADH,-dependent reductase present in these cells. Of various catechols investigated, 3-isopropylcatechol and 3isopropyl-6-methylcatechol were rapidly oxidized by extracts ; 3methylcatechol and 4-methylcatechol were attacked less readily. However, none of these compounds was metabolized beyond ringfission, an observation which supports the scheme of Fig. 6, where compound I11 differs from the ring-fission products of the above mentioned catechols insofar as it is substituted by hydroxyl a t C-4. Compound I11 would be expected to tautomerize to compound I V , a 2,4,6-triketone which would undoubtedly hydrolyse very readily to give the three carboxylic acids that were isolated. It is of interest that, in every pathway shown in Figs. 3, 5 and 6, two molecules of water are incorporated in reactions that follow ring-fission. In meta-fissions (Fig. 3 ; p. 1 l ) , the first of these reactions is a hydrolysis, the second a hydration ; in the gentisate pathways (Fig. 5; p. 15), these two types are encountered in the same order ; and in Fig. 6 there is no hydration but instead there are two hydrolytic fissions. 2. Metabolism of Gallic Acid Recent work in my laboratory, not yet published in detail, has shown that extracts of a Pseudomonas sp. grown with syringic acid (3,5dimethoxy-4-hydroxybenzoic acid) appear to metabolize gallic acid by the pathway shown in Fig. 7a. Gallate (one mole) is converted into two moles of pyruvate with the consumption of one mole of oxygen and the evolution of one mole of carbon dioxide. Extracts contain a powerful oxaloacetate decarboxylase, but one mole of oxaloacetate is trapped (as malate) when malate dehydrogenase and NADH, are added: one mole of pyruvate is then formed. The ring-fission product (I) was too labile to isolate but its hydration product, 4-carboxy-4-hydroxy-2oxoadipate (11), was synthesized chemically and found to be rapidly degraded t o oxaloacetate and pyruvate by a magnesium-dependent aldolase present in cell extracts. Compound I1 had previously been synthesized by Martius (1943) and was investigated as a possible reaction intermediate of the tricarboxylic-acid cycle.
CO2H
CO2H
Oxaloacetate
HO
HOZC
OH
OH (a) Gallic acid
COzH
COZH
I -?
I1
COZ
i NADP,
(b) Protocatechuic acid
I11
Pyruvate
IV
FIG.7 Bacterial degradation of gallic and protocatechuic acids.
20
9. DAGLEY
Support for bhe pathway of degradation of protocatechuate to formate and pyruvate (Pig. 7b), as proposed by Dagley et al. ( 1964),was furnished by the experiments of Dagley et al. (1968). The conversion of 4-carboxy2-hydroxymuconic semialdehyde (111) into 4-carboxy-4-hydroxy-3oxovalerate (IV) is shown as one step in Fig. 7b, bub by analogy with similar reactions in Fig. 3 (p. 11) this would probably involve the intermediate formation of an 0x0-enoic acid; however, this has not been proved. Hegeman (1967d) has shown that extracts of Rhodopseudomonas palustris, grown aerobically with p-hydroxybenzoate, metabolize protocatechuate by meta-fission and contain an NADPdependent dehydrogenase that oxidizes 4-carboxy-2-hydroxymuconic semialdehyde (111),presumably to give compound I as shown by the dotted arrow of Fig. 7.The suggestion may be made that this alternative pathway could be used by other organisms besides R.palustris, including P. testosteroni which Dagley et al. (1968) investigated. Although P. testosteroni, when grown with p-hydroxybenzoate, contains an enzyme that cleaves formate from compound 111,the cells also contain an NADPdependent dehydrogenase for compound 111. Nishizuka et al. (1962) reporbed a meta-fission pathway for catechol in which 2-hydroxymuconic semialdehyde was similarly oxidized to oxalocrotonate ; however, their sequence involved a second NAD-dependent reaction in which 4hydroxy-2-oxovalerate waa oxidized to acetopyruvate and then cleaved hydrolytically to acetate and pyruvate. If the suggestion is correct that the metabolism of protocatechuate can proceed as indicated by the dotted arrow, and can then follow the sequence of Fig. 7a (p. 19), there would be a single oxidative step followed by a hydration (of compound I)and an aldolase cleavage (of compound 11).It is suggestive that the purified aldolase which cleaves compound I1 will accept compound IV as a substrate ; accordingly, either pathway for protocatechuate might be used by P. testosteroni according to metabolic conditions prevailing during growth.
IV. Reactions Converting Aromatic Compounds into Ring-Fission Substrates A. HYDROXYLATIONS 1. Para-Hydroxybenzoate H ydrox ylase
Mention has already been made of the fact that microbes initiate attack upon chemically inert structures such as camphor, aliphatic hydrocarbons, steroids or benzenoid compounds by introducing oxygen, usually as a hydroxyl group, I n most of the systems studied, one atom of
CATABOLISM OF AROMATIC COMPOUNDS BY MIORO-ORQANISMS
21
an oxygen molecule is reduced to water by an electron donor (reduced nicohinamide or flavin nucleotides, or pteridines) whilst the other abom is incorporated into the molecule to be degraded. Such enzyme systems have been named mixed-function oxidases (Mason et al., 1955) ; and they may also be classified as mono-oxygenases (Hayaishi, 1964) since only one atom of oxygen is inserted. A mechanism for an enzyme in this category, p-hydroxybenzoate hydroxylase, is shown in Fig. 8 and was proposed by Hesp et al. (1969) to account for their observations made with the crystalline enzyme which was completely free from traces of
Enzyme-FADH2-Substrate+Enzyme-FADH2+ Substrate
'f
!
NADP 2k o 2
NADPH2
I
.i.
H202
Enzyme-FAD-Substrate+Enzyme-FAD + Substrate Enzyme-FAD Substrate-OH (protocatechuate)
Substrate (p-hydroxybonzoate)
FIG.8 Hydroxylation of p-hydroxybonzoate. From Hosp et al. (1969).
protocatechuate dioxygenase. I n anaerobic conditions, produced by bubbling helium gas, bound FAD was reduced stoichiometrically by NADPH, in the presence of p-hydroxybenzoate (reaction 1, Fig. 8). The reduced enzyme was quickly re-oxidized when air was introduced ; and during the re-oxidation, p-hydroxybenzoate was converted into protocatechuate (substrate-OH of Fig. 8). This conversion was nearly quantitative when catalytic amounts of enzyme were utilized ; but the yield of protocatechuate in relation to the amount of NADPH, oxidized decreased when substrate quantities of enzyme were present, probably due to competition for reduced enzyme by side-reaction 2. The circular dichroism spectrum of the holo-enzyme differed markedly from that of free PAD, an effect not paralleled in the visible absorption spectra; and i t therefore appears that FAD undergoes either conformational or chemical changes when it is bound to the enzyme. Measurements of changes in circular dichroism spectra, due to additions of various compounds, indicated that p-hydroxybenzoate was bound to the FADenzyme by its carboxyl, but not by its hydroxyl, group. However,
22
8. DAGLEY
substrate specificity was very strict, and only p-hydroxybenzoate was hydroxylated by the enzyme. 2. Hydroxylation of Phenylalanine
The enzymic hydroxylation of phenylalanine has been extensively investigated (Kaufman, 1962, 1966). The system resembles p-hydroxybenzoate hydroxylase except that the natural cofactor is dihydrobiopterin and not FAD ;pteridines resemble flavins in chemical structure and in their enzymic reactions, but they serve only as electron carriers for hydroxylations and not in the normal electron-transport systems. Dihydrobiopterin is enzymically reduced to the tetrahydropteridine which serves as electron donor in the hydroxylation of phenylalanine, being itself oxidized to a “quinonoid dihydropteridine”. I n mammalian systems this compound is reduced back to the tetrahydropteridine by another enzyme, different from the hydroxylase (for a summarizing diagram, see Hayaishi, 1969). On the other hand, these separate enzymes have not been reported for the phenylalanine hydroxylase from Pseudomonaa spp. (Guroff and Rhoads, 1967) which in this respect resembles p-hydroxybenzoate hydroxylase more closely than does phenylalanine hydroxylase from mammals. The pseudomonad system, like the mammalian, requires a tetrahydropteridine and reduced NAD ; but in addition it also has a requirement for metal ions. 3. The “NIH Shift”
I n both the mammalian and pseudomonad systems, hydroxylation of phenylalanine proceeds by a mechanism which has been termed the “NIH shift” (Guroff et al., 1967). This was discovered by substituting phenylalanine with deuterium (or tritium) in the para-position. Reaction 1 (Fig. 9) shows the replacement of deuterium (or tritium) that was to be expected ; reaction 2 summarizes what was actually found, namely migration of D and its retention at C-3 of the tyrosine produced. A sequence proposed by Daly et al. (1968) shows an attack by a hydroxyl radical a t C-4 to give a cationic intermediate; this then undergoes a bond distribution with migration of D so that C-3 now bears both H and D (Fig. 9s). On aromatization, the weaker C-H bond breaks and D is retained. However, whether the “NIH shift” will occur, or whether D will be eliminatied during hydroxylation, depends upon the nature of the ring substituent. Formerly it was thought that the electron-donating or electron-withdrawing capacities of the substituent groups were the deciding factors, insofar as they affected the stabilities of the cationic
CATABOLISM OF AROMATIC COMPOUNDS B Y MICRO-ORGANISMS
23
intermediates postulated in sequence a of Fig. 9. It now appears that the crucial property of the substituent is its ability to ionize (Daly et al., 1968). I n sequence b of Fig. 9, when the substituent X H ionizes, a neutral 2,5-cyclohexadienoid intermediate is formed from which D+ is expelled on aromatization. There is no direct experimental evidence for
0 0 R
R
0 , + 2 H -b
+
4
D
OH
D
OH
DHO
(a) Deuterium migrates and is retained on the nucleus XH
XH
X+H+
I*” Y
XH
OH
(b) Deuterium is released during hydroxylation
FIG.9 The “NIH shift”. Reaction ( 1 ) is a direct substitutionof D by OH. Reaction (2) shows the shift which occurs during an enzymic hydroxylationof phenylalanine (R = CH, -CH(NH,)COOH).Sequence (a): possible mechanism for “NIH shift” when R = OCH,,CI,CH,,CH, *CH(NH:)COOH.Sequence (b): elimination of D when the substituent group can ionize as shown. From Daly et al. (1 968).
cationic intermediates as they are formulated in Fig. 9 and it is possible that arene oxides, such as benzene epoxide discussed below, may be involved; these compounds undergo “NIH shifts” to an extent comparable with enzymic hydroxylations (Jerina et al., 1968a). Moreover, the “NIH shift” is not restricted to para-hydroxylation, and shifts a t other ring positions have been investigated (Daly and Jerina, 1969). 2
24
9. DAQLEY
Much information about the “NIH shift” has been obtained with mammalian hydroxylases, but one observation with Pseudomonas sp. may be singled out as being of particular importance in the general area
-
a-o
01
NADPHz
I
(a) Naphthalsne
\
/
I1
(b) Benzene
0’. (c) Benzene
(d) Toluene
V
VI
VII
VIII
3-Mothylcatecho~
FIG.10 Metabolism of‘~itlphthalerie,bonzene and tolucwc. Pnthways ( c ) and fJ) are confined to bitctoritt.
of bacterial aromatic metabolism. The pseudomonad system converts 4-deutcrophcnylalanine and 4-tritiophenylalarline into 3-deuterotyrosine and 3-tritiotyrosine respectively, and it also gives 3-chlorotyrosine
25 and 3-bromotyrosine with 4-chlorophenylalanine and 4-bromophenylalanine (Guroff et al.,1967). Since halogenated benzenoid compounds are used as pesticides, and studies of their degradation by soil microbes are being actively pursued, the possibility of halogen migration during metabolism should be borne in mind. It may also be mentioned that the well known enzymic conversion of 4-hydroxyphenylpyruvate into homogentisate, which occurs in microbes as well as mammals, is an example of an “NIH shift” of a side-chain substituent. CATABOLISM OF AROMATIC COMPOUNDS BY MICRO-ORQANISMS
B. OXIDATIONOF AROMATIC HYDROCARBONS TO CATECIIOLS 1. Epoxides as Intermediates
Epoxides, or arene oxides, have been suggested as intermediates in the oxidation by rabbits of naphthalene and related hydrocarbons (Booth et al., 1960). Direct proof of the formation of an arene oxide as an intermediate in the biological dihydroxylation of an aromatic compound has now been provided. Jerina et al. (1968b) oxidized naphthalene with rat-liver microsomes and NADPH, and used counter-current distribution to separate and identify the 1,2-naphthalene oxide (reaction 1, Fig. 10) which formed aboub 5% of the oxidized metabolites. Their preparations also contained an enzyme that hydrolysed the epoxide (I) to give trans-l,2-dihydro-l,2-dihydroxynaphthalene (11).I n accordance with similar findings by Holtzrnan et al. (1967), experiments using ‘*02 showed that, when the diol was formed directly from naphthalene, it contained oxygen from the air only a t C-1 whereas the oxygen at C-2 originated exclusively in water. A non-enzymic re-arrangement of compound 1gave 1-naphthol (111). Microbes also metabolize naphthalene by pathway a, Fig. 10. Thus Walker and Wiltshire ( 1953)isolated D-trans-l,2-dihydro-1,2-dihydroxynaphthalene from cultures of a bacillus growing a t the expense of naphthalene. Griffiths and Evans (1965)showed that the same compound was accumulated from naphthalene by cell-free extracts of a soil pseudomonad when NADH, was supplied, and also that it was degraded in the presence of NAD. The reaction sequence of the degradative pathway was elucidated by Davies and Evans (1964);these, and also the reactions by which phenanthrene and anthracene are metabolized by pseudomonads (Evans et al., 1966), were reviewed by Ribbons (1965) and Dagley (1967). It may also be mentioned that Taniuchi and Hayaishi (1963) showed that extracts of P. Jluorescens hydroxylated the benzene nucleus of a quinoline compound, kynurenic acid, bo give 7,B-dihydro7,8-dihydroxykynurenic acid, and they proposed kynurenic acid 7,8oxide as the initial producb of the enzymic attack. It is therefore probable
26
8. DAOLEY
that kynurenate is degraded by microbes (see also Dagley and Johnson, 1963) by reactions that are analogous to those of pathways shown in Fig. 10.
2. Peroxides as Possible Intermediates Investigations of the metabolism of benzene, however, have revealed other alternatives. Pathway b, Fig. 10, is similar to pathway a, giving rise to catechol with benzene oxide (111) and trans-benzene glycol (trans-1,2-dihydro-l,2-dihydroxybenzene, I V ) as intermediates. Jcrina et al. (1968~)have shown that rabbit-liver microsomes catalyse these reactions. There is, however, no evidence that microbes degrade benzene by pathway b, Fig. 10. On the contrary, a notable series of papers by Gibson and Kallio and their colleagues has established sequence c as the metabolic pathway taken by bacteria. Thus partially purified extracts of toluene-grown Pseudomonas putida oxidized [ 4C]benzene when supplemented with NAD and ferrous sulphate ; and when catechol was added during the course of the reaction, and then re-isolated, it was found to carry label. I n a similar experiment, carrier cis-benzene glycol became labelled, whereas trans-benzene glycol did not. Extracts converted both catechol and cis-benzene glycol into 2-hydroxymuconic semialdehyde by meta fission, and they contained an NAD-dependent dehydrogenase for cis-benzene glycol that did not attack the trans isomer (Gibson et al., 1968).Gibson et al. (1970a) also isolated 113 mutant strains of Y.putida that grew with succinate but had lost their ability to grow with toluene. Four of these mutants accumulated a compound having the chromatographic properties of compound VIII, Fig. 10. One strain, growing with glucoso as carbon source, converted toluene vapour into sufficient of the compound to permit the isolation of about two grams of crystals. These were acetylated and then condensed with maleic anhydride to give a bicyclic compound, the nuclear magnetic resonance spectrum of which established unequivocally that the material isolated from the culture was (+)-cis-2,3-dihydroxy-l-methylcyclohexa4,6-diene (VIII). I n accordance with pathway d, Fig. 10, compound VIII was converted anaerobically and stoichiometrically into 3-methylcatechol by extracts of the parent strain of P.putida in the presence of NAD. Finally, Gibson et al. (1970b) grew the same mutant on glucose in the presence of benzene and accumulated cis-benzene glycol ( V I ) which was shown t o be identical to a synthetic sample. I n experiments with leO,, two atoms of atmospheric oxygen were incorporated into compound VI; this is in accordance with pathway c, Fig. 10, and is contrary to the reported sequence (b) for the microsomal oxidation of benzene. These experiments with isotopic oxygen are more conclusive than those concerned with cis-dihydrodiol formation ; for although
CATABOLISM O F AROMATIU COMPOUNDS BY MICRO-ORGANISMS
27
hydrolysis of an epoxide has given the trans isomer in all cases studied in the past, an enzymic and stereospecific opening to give acis-dihydrodiol is a t least conceivable. Arene oxides, such as I and I11 of Fig. 10, can now be synthesized chemically (Vogel and Kliirner, 1968); but the peroxides V and V I I remain as hypothetical intermediates which are only justified by '80-incorporation experiments. The diversity of mechanisms used in microbial hydroxylations is not confined to aromatic hydrocarbon metabolism. Thus, the careful work of Katagiri et al. (1966) on the NADH,-dependent salicylate hydroxylase supports a mechanism similar to that of Fig. 8 (p. 21); in this case, however, carbon dioxide is released ab the same time as an hydroxyl group is introduced into the nucleus when oxygen reacts with the enzyme-FADH,-salicylate complex. Hydroxylation of o-hydroxybenzoate t o give catechol therefore fits into the familiar category of mixed-function or mono-oxygenases. B u t , in contrast, when o-aminobenzoate (anthranilate) is oxidized to catechol, two atoms of oxygen are simultaneously incorporated by a reaction that is probably similar to the first step in pathways c and d of Fig. 10 (Kobayashi et al., 1964). When non-benzenoid ring systems are hydroxylated, the reaction may take yet another course. Thus, the oxygen atom incorporated into nicotinic acid was derived from water, and not from molecular oxygen, when P. jluorescens hydroxylated nicotinate to give 6-hydroxynicotinate (Hunt et al., 1958). Similar hydroxylases appeared to catalyse the conversion of picolinic acid to 6-hydroxypicolinic acid (Dagley and Johnson, 1963) and also the coenzyme Adependent hydroxylation of 2-furoic acid to 5-hydroxy-2-furoate (Trudgill, 1969).
C. MODIFICATIONOF STJBSTITUENT GROUPS BEFORE RINGCLEAVAGE 1. General Observations
Hydroxylation of the benzene nucleus was sufficient to prepare the foregoing substrates for ring fission. Thus, toluene was oxidized to 3-methylcatechol by P. pu.tida and the nucleus was then cleaved (Gibson et al., 1970a). The species of Pseudomonas and Achromobacter isolated by Claus and Walker (1964) probably metabolized toluene by the same reactions. By contrast, a strain of Pseudomonas aeruginosa investigated by Kitagawa ( 1956) appeared to oxidize the methyl group of toluene before hydroxylation occurred, giving successively benzyl alcohol, benzaldehyde and benzoic acid. Some pseudomonads hydroxylate the nucleus of a cresol, leaving the methyl group intact, whereas others oxidize the methyl group to a carboxyl group (Bayly et al., 1966).
28
S. DAOLEY
Cain and Parr ( 1968) found that benzenesult~lionitewas oxidized by pseudomonads that were able t o degrade detergents of the alkylbenzcncsulphonate type. It appeared t h a t a mixed-function oxygeriase formed catechol, and simultaneously released the sulphonic acid substituerit as sulphite. A similar reaction occurred with toluene-p-sulphonate ; sulphite was released, but the methyl group substituent was not attacked prior t o metn fission of the nucleus. However, results with another Pseudonionas sp. sbrongly siiggcsted that the sulphonic acid group of p-toluencsulphonate w7as removed, not as snlphite but as sulphate (Focht and Williams, 1970). I n contrast to the elimination of sulphite or sulphate, ‘ricdje et al. (1969) fontitl that the chlorine substituents of 4-chlorocatechol and 4,~i-tlichlorocnt~ecliol werc rctained during ring opening by Arthrobtrcter sp. grown with 2,4-dichlorophenoxyaceticacid (2,4-D). Ccll extracts formed t h r correqmnding cis,cis-chloroniucoiiic acids ; then chlorinr u’as elimiiiated from position-4 when a maleylacctic acid was formed in each case ; and the chlorine originally in 1)osition6 of 4,6-dichlorocatechol was released when chloromaleylacetic ticid was finally metabolized to succinate. The carbon side-chain of 2,4-D, which is joined by an ether linkage to the benzene nucleus, was removed before the latter was cleaved. Apparently a n oxygenase introduces a hydroxyl group at C-2 of the side-chain, a n d an aldolase-catalysed fission rclenses glyoxylatc (Tiedje and Alexander, 1969). The other product of this cnz.vmic fimion is 2,4-dichlorophc1iol (Loos et nl., 1087) which is liydt.oxyIat,ed to gibe 4,~~-dichlorocat~erIiol (3,rj-dichlorocatechol). 2. Oxidation of Phenylpropa,noid Stmctiires Arining from Lignin.s A high proportion of carbon is returned t o the soil as lignins. But, despite the quantitative importance of this material, detailed information about its biochemistry remains scanty by comparison with the vast amount of knowledge now available concerning other biopolymers. Lignin appears to consist of polymers derived from the phenylpropanoid compound, coniferyl alcohol ; and when these are degraded by soil microhes, the alcohol is released together with its oxidation products such as trans-ferulic acid and vanillin (Freudenberg and Neish, 1968). Accordingly, studies of the microbial metabolism of simple phenylproparioid structures, such as cinnamic, hydrocinnamic, caffeic and ferulic acids, have some relevance t o the problem of lignin degradation. However, little progress has been made with more complex constituents until the recent work of Toms and Wood (1970h) revealed the reactions used by bacteria to initiate tlic degradation of a-conidendrin. Some micro-organisms modify siibstituent groups of phenylpropanoid
C’ATAROLTSM OF AROMATIC COMPOUNDS BY MICRO-ORGANISMS
29
compounds before ring fission, but others do not. Webley et al. (1955) showed that the side chain of hydrocinnamic acid was oxidized by Nocardia opaca, giving benzoic acid, whereas Achromobacter sp. hydroxylated and cleaved the benzene nucleus of hydrocinnamic acid before the intact side chain, as part of the structure of succinate, was released from the ring-fission compound by hydrolysis (Dagley et al., 1965). Similarly Seidmaii et ($1. (1969) found that P. Jluorescens hydroxylated the nucleus of 21-hydroxy-trans-cinnamicacid to give caffeic acid (3,4-dihydroxycinnamic acid) which then underwciit ortho cleavage to give a cis,cismuconate bearing the intact side chain of caffeic acid a t C-3. By contrast, Toms and Wood (1970a) found that part of the side chain of ferulic acid was removed by P . acidovorans before the nucleus was opened (Fig. 11). The reaction sequence they proposed was supported by the observation that vanillin ( I ) and vanillic acid (11) were present in filtrates from cutures grown with trans-ferulatc. Further, cell extracts formed [ I4C!acetatefrom fernlate luGelled in the side chain, and also accumulated compoun(l I1 wlicw furnished with NAD required for the oxidation of compound I. Extracts oxidized vanillate (11)when they were supplemented with Pe’ ant1 GRH, plus formaldehyde which was used to generate reduced NAD by the action of a dehydrogenase that yielded formate (Fig. 11). A prerequisite for the metabolism of vanillate was therefore its denietliylation, involving consumption of one molecule of oxygen and catalysed by a mixed-function oxygenase. This was confirmed by the fact that extracts contained a powerful protocatechuate 4,5-oxygenase which catalysed the uptake of second molecule of oxygen, so t h a t the total was two moles of oxygen per mole of vanillate oxidized to pyruvate. Formate was not oxidized by extracts. The first stcp in the reaction sequence of Pig. 11 was not proved by direct experiment since the proposed liydratioii product of ferulic acid could not by synthesized chemically. For this reason, also, it was not possible to decide whether or not the ready metabolism of cis-ferulate could be explained by the inability of the aldolase to distinguish between the optical isomers that would be expected t o arise from the hydration of cis-or trans-ferulate. Denicthylation of vanillate has bcen investigated by Cartwright and Smith (1067) in the course of studies of the bacterial degradation of compounds related to lignin. The organism used was P. Jluorescens which, like the strain of 1’. acidovorans of Toms and Wood (1970a) formtd formaldehyde and formate from the methyl group of vanillate when adapted to ferulate, but differed in cleaving protocatechuate by ortho fission. Protocatechuate 3,4-oxygenase and vanillate 0demethylase were both obtained in the soluble part of a cell-free extract, but Cartwright and Buswell (1967) were able to separate these enzymes in the preparative ultracentrifuge. A fraction of the extract oxidized +
30
8. DAGLEY
vanillate to protocatechuate when supplemented with NADH,, and on addition of scmicarbazide to trap formaldehyde, 0.6 mole of oxygen was taken up per mole of substrate. This same consumption of oxygen was found when 3-methoxybenzoate and 3,4-dimethoxybenzoatc were each oxidized to give one mole of formaldehyde per mole of substrate. COzH
I
FH II
AH. OH
H*C
CHO
OH
OH tram-Ferulic acid
I
H*COOH
OCH, OH Protocatechuic acid
OH
I1
FIG.1 1 DegrtidtLtiori of l'crulic acid by Peeudomonas ctcidoworans to give protocatcchnntc, acetntc. m t l format,n.
Such preparations differed from those of Toms and Wood (1970a) which catalysed an uptake of one mole of oxygen per mole of formaldehyde formed. This is the uptake to be expected if one atom of oxygen is used to oxidize NADH, and a second is attached t o the carbon of the methyl group. Dcmcthylation in the organisms studied by Cartwright and Smith ( 1967) and Cartwright and Buswell ( 1967) evidently proceeds by adifferent route. 3. Degradation of a-Conidendrin
Two O-demethylations are required during the metabolism of aconidendrin. This lignin model compound, which contains two phenylpropane units, has been extracted by acetone from spruce wood (Erdtmaii, 1944). It is also readily obtained from sulphite-waste liquors in wood pulp manufacture. A number of micro-organisms have been
CATABOLISM OF AROMATIC COMPOUNDS B Y MICRO-ORGANISMS
31
32
S . DACLEY
isolated that arc able to utilize a-conidendrin as sole source of carbon (for references, see Ribbons, 19Gb), and not surprisingly it was found that cells so grown could metabolize several simpler aromatic compounds and could also, on the evidence of chromatography, produce them in traces from conidendrin. However, the reactions that must be elucidated before a feasible degradative sequence can be suggested for a rather complicated molecule of this type are the early steps in the pathway. This elucidation has been accomplished by Toms and Wood (1970b) who investigated a non-fluorescent pseudomonad which grew with a-conidendrin and accumulated enough of compounds I and I1 (Fig. 12) to permit their identification. These are not only new metabolites: they arc new organic compounds; and from samples of 0.1 and 0.06 g. which wcre, respectively, the amounts they isolated, the authors were able to determine chemical structures by the application of the modern physical techniques of mass spectroscopy, nuclear magnetic resonanc(a spectroscopy and infrared spectroscopy. This information made it possible for them to suggest the reaction sequence of Fig. 12 in which a-conidendrin is first oxidized t o a quinone; a double bond is then hydrated and an aldolase-cntalyscd cleavage next gives rise to the ltcto form of compound I, plus goaiacol. Some evidence for the presence of guaiacol in culturc filtrates was obtained, but firm identificiltion was hinderod by its rapid assimilation early in growth. The conversion of the cnol form of compound I into compound I1 is an oxidative step, giving rise t o a naphthalene nucleus. As the authors suggest, opening of the lactone ring of compound I1 would provide a substituted naphthalene which may be metabolized by reactions similar to those elucidated by Davies and Evans (1964). It also seems likely that the ability of soil microbes to metabolize naphthalene, which is a, characteristic not infrequently encountered, may be related to the fact that substituted naphthalenes are formed when these microbes degrade natural products of the type of a-conidendriti.
V. Regulation of Catabolic Sequences A.
DISTRIB~JTION O F PATHWAYS
~ ' I ~ ~ S t ~ ) l ~ ~ ~F(r J: NI C( T ~ IAU IN~S A N D
THE VAltIOllS
When the mefa-clcavage pathway was discovered (Dagley and Stopher, 1959), the reactions for ortho-cleavage were already familiar, and interest
in the new route stemmed mairily from the fact that it provided yet another demonstration of the biochemical versatility of microbes. Although it was not evident what advantages were gained by the microbes themuelvcs in being ablc to brcak open the bcnzene nucleus
CATABOLISM OF AROMATIC C O M P O U N D S BY MICRO-ORUANISMS
33
in different ways, taxonomists hoped to p u t these features to good use. It seemed t ha t i t would be possible to form two categories, ortho cleavers and meta cleavers, from those bacteria such as pseudomonads t h a t are difficult to classify when other criteria are used. This is indeed feasible, bu t only under strictly specified conditions, namely when the pseudomonads are grown with p-hydroxybenzoate and the ring-fission of protocatechuate is then examined (Stanier et al., 1966). With these coritlitions of testing, it was found t h a t the rnetu-cleavage mechanism was confined exclusively to t n o species of non-fluorescent pseiidomonads, namely, P. trcidovorans and 1’. testosteroni, whereas ortho-cleavage of protocatechuate was characteristic of th e entire fluorescent group of pseeudomonacls. No such division can be made when catechol, for example, is used as substrate in the fission test. T ~ L I S Moraxella , culcoacetica (formerly thought to be “vibrio 01’’ ofHapp01d and Key, 1932; but see Fewsori, 1967) was much iiivestigutc~clin early work on ortho-fission. However, the organism employs the ~neta-fissioiienzyme, catechol 2,3-oxygcnase, when i t degrades naphthalene (Grifitlis et ul., 1964). A species of Pseudornonas, when grown with hydrocinnamate and phenylacetate respecand tively, cleaved the benzene rings of 2,3-dihydroxyphenylpropionate 3,4-dihydroxyphenylacetate (Blakley et nl., 1967) b y metu-fission oxygenases; but th e organism was found by Blakley (1967) to cleave protocatechiiate b y ortho-fission when it grew with p-hydroxybenzoate. Likewise Seidman et al. (1969)showcd t h a t protocatechuate and caffeate were cleaved by ortho fission, whereas catcchol and liomoprotocatechnate (3,4-dihydroxyphenylacetate)were attacked by meta-fission oxygenases when synthesis of these enzymes had been induced by growth with the appropriate carbon sources. Feist and Hegeman (1969) found th a t, of 41 strains of P. puiida examined, only eight were capable of performing a rnetn cleavage of catechol. Of these eight strains, six could grow with benzoate, which was m etald ized by an ortho cleavage of catechol in four instances. In two strains, benzoate elicited synthesis of catechol 2,3-oxygenase: one of these was th e organism previously dcsigiiated P. urvilla which had been used as material for the piirification of metapyrocatechase by Nozalti et nl. (1963). Four of th e eight strains could utilize salicylate, and this substrate elicited synthesis of catechol 2,3-oxygeilase in each case. I n fluorescent pseudomonads th a t decompose arylsulphonates, synthesis of the enzymes of the metu pathway for catechol was induced by l)enzenesulphonate, b u t those of th e ortho pathway were induced hy benzoate (Cain and Farr, 1968). B y transferring P. aeruginosa from benzenesulphonate to benzoate as growth substrates, F a r r and Cain ( I 968) obtained cells t h a t contained enzymes of both the ortho an d meta pathways for catechol degradation. These
34
8. DAGlLEY
authors also made the imexpected observation that, whereas catechol itself always elicited a 2,3-oxygenase in uninduced cells, the product of this reaction (2-hyclroxymuconic semialdehyde) induced catechol 1,2oxygenase. If there is one firm conclusion to be drawn from the complexity of findings I havc summarized, it is that ortho- and meta-fissions do not exist merely as alternatives to be chosen a t the caprice of versatile bacteria which arc able to metabolize benzenoid compounds. When these bacteria are presented with an aromatic substrate, the pathway that satisfies growth requirements will be “chosen” by a combination of two factors, namely the mechanisms available for derepression of the enzymes that arc needed, and the substrate specificities of the enzymes themselves. The apparent “choice” will be narrowed both by tight substrate specificities and also by those mechanisms of induction which are very selective because only one or two compounds can act as effective clerepressors. As we have won, t h e enzymes for ineta cleavage, and tliosc for tho gcntisatc pathway, arc relatively tolerant of substituents in the benzene nucleus. Those of the ortho pathways for catechol and protocatcchuate are not : they are highly specific for their substrates, and in some cases are clereprcssed only by particular products of metabolism. Thus, to my knowledge, there are only two reported instances of purified catecholl,2-oxygenases that could tolerate the introduction of an organic substituent into the nucleus. The first concerned the enzyme from Brevibacteriuin fuscum which oxidized both 3-methylcatechol and 4methylcatcchol to give the corresponding methylmuconic acids (Nakagawa et al., 1963). The second example is that of a pyrocatechase from a species of I’seudomonas ; the enzyme oxidized 4-methylcatechol a t about the same rate as catechol, 3-methylcatechol being oxidized much more slowly (Kojima et al., 1967). Chlorine may also be inserted into the nucleus without blocking the action of catechol 1,2-0xygenases from certain species (Evans and Moss, 1957; Tiedje et al., 1969). As mentioned earlier, chloromuconic acids may be further metabolized by certain organisms; but although the ring may be opened, when the substituent is a methyl group it appears that the ring-fission products cannot be degraded. Accordingly, such methyl-substituted catechols and their metabolic precursors do not serve as the sourcesof carbon for the growth of bacteria committed to degrading catechol or protocatechuate by ortho fission. Since the substituted muconic acid arising from the ortho fission of caffeic acid can be metabolized, it may be assumed that the relevant enzymes of the strain of P.jluorescens studied by Seidman et al. (1969) differ markedly in their specificities from those of ortho pathways studied previously. In summary, it appears that bacteria taking the ortho-fission route for catechol and protocatechuate probably
CATABOLISM OF AROMATIC COMPOUNDS BY MICRO-ORGANISMS
35
exercise their biochemical versatility in modifying side chains by the action of non-specific enzymes before they open the nucleus (Kennedy and Fewson, 1968). Later enzymes used in these sequences are then extremely specific. In an extensive survey of metabolism of aromatic acids by fungi, Cain et al. ( 1968) encountered only one organism, a species of Penicillium, which appeared to degrade protocatechuate by meta fission. Most of the fungi examined were able to convert protocatechuate to /3-ketoadipate, with p-carboxymuconolactone as a reaction intermediate. Since they did not degrade /3-ketoadipate enol-lactone, the ortho-fission route for these fungi is quite different from the bacterial pathway of Fig. 2 (p. 8) in which /3-ketoadipate enol-lactone and y-carboxymuconolactone (not /3-carboxymuconolactone) are established as intermediates. Cain et al. (1968) observed that a few of their fungi, after growth with p-hydroxybenzoate, hadno protocatechuate 3,4-oxygenase,but possessed all of the enzymes of the catechol pathway. Patel and Grant (1969), and Grant and Patel (1969), also found that Klebsiella aerogenes decarboxylated p-hydroxybenzoate giving catechol.
B. REGTJLATION O F Ortho-FISSION PATHWAYS : CATECHOL AND P R O T O CATECHU ATE
Twenty-three years ago, R. Y. Stanier published an article entitled “Simultaneous adaptation : a new technique for the study of metabolic pathways” (Stanier, 1947). As a means of obtaining a rapid, preliminary outline of the main features of a new pathway, this approach has been, and still remains, of great value, particularly when due heed is given to its limitations, which were thoroughly discussed a t the time. Briefly, it is generally observed that, when cells are induced to oxidize acompound, they are also capable of oxidizing a t about the same rate those metabolites which lie upon its pathway of degradation. If, in turn, the cells are induced to oxidize one of these metabolic intermediates, provided as a separate substrate, this does not confer the ability to oxidize earlier compounds in the reaction sequence. An explanation for this pattern of behaviour was put forward independently by Stanier (1947), Suda et al. (1949) and Karlsson and Barker (1948).It was proposed that the substrate, and each intermediate in turn, triggers the specific synthesis of the enzyme responsible for its conversion to the next intermediate of the metabolic pathway; induction thus occurs in a stepwise fashion : it is sequential. The earlier observations of Stanier were made for aromatic substrates such as niandelic acid, and subsequent modifications of the theory of sequential induction were also largely due to Stanier
36
9. DAQLEY
and his students and colleagues, Ornston and Hegeman, again investigating the bacterial catabolism of various aromatic compounds. Two modifications of the original theory were found to be necessary. First, enzymes of a section of the pathway may be derepressed co-ordinately : that is, instead of being induced sequentially as individuals, a whole functional group of enzymes may be derepressed together. Second, an enzyme or a group may be derepressed, not by substrates but by products. Hegeman (1967a, b, c) has made a thorough study of these concepts as applied to the degradation of mandelic acid by P. putida. I shall summarize recent investigations of the modes of regulation of synthesis of enzymes that catalyse the bacterial degradation of catechol and protocatechuic acid (Fig. 13). The trivial, but still cumbersome, names of the enzymes are designated as follows : HBH, B H : p-hydroxybenzoate and benzoate hydroxylases ; PO, CO : protocatechuate 3,4and catechol 1,2-0xygenases; CMLE, MLE : carboxymuconate- and muconate-lactonizing enzymes ; CMD, MLI : carboxymuconolactone decarboxylase and muconolactone isomerase ; ELH : /3-ketoadipatc enol-lactone hydrolase ;and T R :6-ketoadipate succinyl-CoA transferasr. Sound studies in molecular biology are usually firmly based upon chemistry and biochemistry, and this principle was certainly recognized in the design of the experiments that established the modes of regulation summarized in Fig. 13. Thcy could not have been performed without the preliminary extensive purification of the enzyme involved : this established the fact that the pathways were specific, each involving reaction intermediates that were not metabolized by the other route. It was also essential t o devise a valid assay for each enzyme, making use of characteristic properties of each compound when, in some instances, they had been obtained for the first time by the action of the very enzymes under investigation. Some of these compounds remain difficult, if not impossible, to synthesize and purify by conventional chemical methods. Figure 13 is designed to contrast modes of regulation in Moraxella calcoacetica and Pseudomonas putida; it does not purport to show all of the information available about these mechanisms. When M. calcoacetica is grown with p-hydroxybenzoate, this substrate derepresscs synthesis of enzyme HBH, and protocatechuate is formed. Then, as shown in Fig. 13, protocatechuate derepresses co-ordinately all of the enzymes (PO, CMLE, CMD, ELH and TR) required to catalyse its conversion into 13-ketoadipyl-CoA. When P. putida is grown with p hydroxybenzoate, Rynthesis of HBH is again derepressed and the protocatechuate formed appears to induce the formation of its oxygenase, PO. This induction was not established unequivocally by Ornston ( 1 9 6 6 ~ ) and is not shown in Pig. 13. However, a t this point, the resemblance with
I
COzH
IBH PH
QH
llf oraxella cnlcoacelica protocatechuate
I
C02H
muconate Pseudomonns putidn
muconate
ELH
muconate Mwnxella calcoacetica muconate
f-
/-ketoadipato Pssudmonas
8-ketoadipyl-CoA
FIG.13 Regulation of tho synthesis in Moraxella calcoacetica a n d Pseudomonas pzitidn of enzymes t h a t degrade benzoate and p-hydroxybenzoate b y ortho-fission. An arrow ( -+) directed from pro1 ocatechuate, muconate or 13-lretoadipato towards an eiizyine tlniiotes thnt synthesis of this onzyme is dereproswd by t)hecompound designntrd. l h z y m o s am abbruvint,od as in t h e text.
38
8. DAQLEY
M . calcoacetica ceases: the remainder of the enzymes are not blockinduced along with P O ;instead, 8-ketoadipate (or its coenzyme-A ester) serves as the co-ordinate derepressor of synthesis of CMLE, CMD and ELH. It is probable that TR is also depressed a t the same time, but this was not investigated by Ornston (1966~). When benzoate serves as growth substrate for either organism, synthesis of B H is induced. By a separate event, in both cases, cis,cismuconate next induces synthesis of catechol 1,2-oxygenase: that is, synthesis of BH and CO is sequentially derepressed in each organism. I n M . calcoacetica, cis,cis-muconate now co-ordinately derepressccl synthesis of the block of enzymes (MLE, MLI, ELH and TR) required for its conversion into @-ketoadipyl-CoA.At this point, the events in P.putida are different, as they were in the protocatechuate pathway; cis,&-muconate co-ordinately derepresses synthesis of MLE and MLI whereas synthesis of EHL, and presumably TR, is derepressecl by 8-ketoadipate or its coenzyme-A ester. There is one interesting comequence of this last event. When synthesis of ELH is derepressed, so is that of CMD and CMLE since they belong to the same co-ordinate block ; accordingly benzoate-grown P.putida contains high levels of two enzymes, CMD and CMLE, which are not used in the metabolism of benzoate. One further problem arises from the schemes of Fig. 13. I n M . culcoacetica, synthesis of enzymes ELH and TR, which are needed for benzoate metabolism, is co-ordinately derepressed, along with two others, by the specific metabolite &,cis-muconate. But these two enzymic activities are also needed for the degradation of protocatechuate, and in Fig. 13 this substrate is shown as effecting their derepression co-ordinately with three other enzymes of the p-hydroxybenzoatc sequence. This apparent contradiction was resolved by the discovery (Chovas and Stanier, 1967) of the existence of two isofunctional enzymes that catalyse the hydrolysis of /I-ketoadipate cnol-lactone, whilst another pair were found to catalyse the activation of 8-ketoadipate. Synthesis of ELH I and TR I is derepressed during the metabolism of p-hydroxybenzoate by ill. calcoacetica, whereas the other members of each pair, namely E L H I1 arid TR 11,are synthesized by this organism when benzoate is metabolized. The two enzymes denoted by ELH differ in certain physical properties; likewise TR I and TR I1 are different proteins, although they catalyse the same reaction. There is one feature of these schemes that would have precluded their acceptance some years ago. The metabolites cis,cis-muconate and j?-ketoadipate are shown as the derepressors of synthesis of enzymes that must operate for their own formation. However, it is now realized that these enzymes are never entirely absent from the bacteria before
CATABOLISM O F AROMATIC COMPOUNDS BY MICRO-ORGANISMS
39
they become adapted; and when they are exposed to benzoate, for example, there is a slow but significant formation of &,cis-muconate and 8-ketoadipate which is sufficient to trigger the derepression of synthesis of the enzymes. Clearly, the small endogenous concentration of a derepressor which may be required for it to be effective will call for caution in the design of experiments. A non-metabolizable inducer may contain an amount of a contaminating metabolite too small to be revealed by respirometry for example, but sufficient to act as an effective derepressor, or else to provide one when it undergoes metabolism. Further, when cells are tested in a respirometer for their ability to oxidize a substrate, it is prudent to set up a control reaction with chloramphenicol present. This antibiotic prevents the very rapid synthesis of new proteins which can occur on exposure to the test substrate, and which may give the erroneous impression that these enzymes were present before the substrate was added. I n any event, i t is vastly preferable t o use sensitive assays for the individual enzymes of interest rather than to rely upon overall measurements of oxygen uptake. C. SOME METHODSUSEDTO INVIETIGATE REGULATION
I shall now summarize briefly the methods used in the extensive studies of regulation in P. putida (Ornston, 1966c) and in M . calcoacetica (CBnovas and Stanier, 1967; CBnovas et al., 1968a,b; CBnovas and Johnson, 1968).Two types of mutants were isolated, those with a metabolic block and also the so-called “permeability mutants”. Although cis,&-muconic acid, for example, is a metabolite, it cannot serve as a growth substrate for wild-type organisms because it cannot enter the cells. In permeability mutants, this barrier to entry is abolished. I can illustrate the use of mutants by considering those which lacked enzyme CMLE in P. putida and therefore could not produce y-carboxymuconolactone. These organisms when exposed to protocatechuate could synthesize protocatechuate 8,4-oxygenase (PO) but not CMD or ELH. However, they were able to grow with ,!I-ketoadipate and they then contained both of the enzymes CMD and ELH. As regards the catechol (benzoate) pathway, exposure of permeability mutants of P.putida to cis,&-muconate elicited the co-ordinate synthesis of MLE and MLI, but not of ELH. On exposure to cis,cis-muconate, P. putida and M . calcoacetica also synthesized catechol 1,2-oxygenase (CO). This observation does not eliminate thc possibility that catechol can also serve as inducer of CO, but Bird and Cain (1968) showed that, although P. aeruginosa synthesized this enzyme when grown aerobically and exposed to catechol, the organism did not have this capacity when grown anaerobically with nitrate a8 the terminal clectron-acceptor. Under
40
9. DAOLEY
anaerobic conditions, therefore, no cis,cis-muconate could bc formed. However, the strain was permeable to cis pis-muconate, and when this compound was added to the culture, it derepressed synthesis of CO, and other enzymes of the catechol pathway, under anaerobic as well as aerobic conditions. Accordingly, muconate but not catechol is the inducer of this enzyme in P.aeruginosa. A fruitful method of investigating co-ordinate induction may be illustrated from experiments concerned with the enzymes of the benzoate pathway in M . calcoacetica (CBnovas and Stanier, 1967). Wild-type cells were grown in media containing benzoate plus various concentrations of succinate, lactate or acetate that exert catabolite repression upon the Synthesis of enzymes in this pathway. I n this way extracts could be prepared from cells that contained a wide range of levels of enzyme activities. Specific activities of MLE, MLI and TR in the various extracts were then plotted against the corresponding values obtained for ELH. I n each case the plot was strictly linear, showing that the four enzymes constituted a “muconate block” of co-ordinately-induced enzymes. No such relationship was obtained for CO, showing that the induced synthesis of this enzyme occurred separately and independently. A similar experiment was performed for M . calcoacetica growing with p-hydroxybenzoate and subject to various degrees of catabolite repression. Specific activities of PO, CMLE, CMD and TR gave linear plots against activities of ELH, but p-hydroxybenzoate hydroxylasc (HBH)activities were not related to those of ELH. Two further experimental findings from these investigations may be mentioned. First CBnovas et al. (1968b) isolated mutants of M . cnlcoacetica which lacked protocatechuate 3,4-oxygenase (PO) but synthesized the remaining four enzymes of the protocatechuate co-ordinate block (CMLE, CMD, ELH and TR) a t high differential rates in the absence of any exogenous inducer. The reason for this behaviour appeared to be as follows. Since the cnzyme P O was missing, protocatechuate accumulated within the cells and derepressed synthesis of the four enzymes (Fig. 13). The source of the protocatechuate was shikimate, an intermediate in the biosynthesis of aromatic compounds required for growth of the cells. It so happens that protocatechuate acts not only as the derepressor for synthesis of the four enzymes mentioned, but also for a nicotinamide nucleotide-independent shikimate dehydrogenase of which it is the metabolic product. It therefore appears that protocatechuate plays a very important role in M . calcoacetica since it controls synthesis of all of the enzymes from shikimate, a compound on a biosynthetic route, down to /?-ketoadipate-CoA, a port of entry int:, the tricarboxylic-acid cycle. The second feature of interest concerns the isofunctiorial cnzymes TR I and TR I1 which, as we have seen,
CATABOLISM OF AROMATIC COMPOUNDS B Y MICRO-ORQANISMS
41
catalyse one and the same reaction (Fig. 13). CBnovas and Johnson ( 1968) discovered a third /3-ketoadipate succinyl-CoA transferase (TR 111)the physiological function of which is to activate adipic acid.
D. REGULATION OF
THE
Meta-FISSION
P A T H W A Y FOR C A T E C H O L
A strain of P. putida isolated by cresol enrichment (Dagley and Gibson, 1965) decomposes phenol and cresols through metu-fission pathways. Peist and Hegeman ( 1969) have used non-metabolizable inducers and suitable mutants of this organism to demonstrate that phenol itself (or a substituted phenol) serves as the co-ordinate derepressor of the whole battery of enzymes that operate for the meta route. By contrast, this strain degrades both benzoate and catechol by ortho fission. Although catechol is an intermediate in the meta route by which phenol is degraded, it is probable that, in this organism as in other pseudomonads, some catechol must be converted into cis,cismuconate before the induced synthesis of early enzymes of the ortho pathway is initiated. No significant concentration of muconate will accumulate in phenol-grown cells because their metapyrocatecliase will already have been fully induced. This organism differed from the strain of P. aeruginosa used by Farr and Cain ( 1968) insofar as 2-hydroxymuconic semialdehyde did not derepress catechol 1 ,%oxygenase. Feist and Hegeman (19G9) also extended the range of alkyl-substituted catechols which the enzymes of meta fission are known to tolerate.
E. EVOLUTIONARY SIGNIFICANCE OF REGULATORY MECHANISMS It may well transpire that studies of enzyme derepression will contribute to our understanding of evolutionary processes. We have seen that different organisms may degrade the same aromatic substrates by alternative routes and it is tempting to speculate that one particular pathway has had a single evolutionary origin and could therefore serve as a marker of evolutionary affinities. Vogel (1965) has reasoned along these lines in reviewing the biological distribution of the diaminopimelic and a-aminoadipic pathways for lysine biosynthesis. However, CBnovas et al. ( 1 967) have made an alternative and stimulating suggestion that “the evolutionary significance of a given biochemical pathway in representatives of several different biological groups can be assessed by means of a somewhat different kind of analysis-comparison of control mechanisms”. As shown in Fig. 13 (p. 37), the control mechanisms of P . putida are entirely different from those of M . calcoacetica. Two other species, namely P . aeru,ginosa and P. multivorans, have been examined and shown to exercise control in the same way as P.putida
42
9. DAQLEY
which, therefore, may by typical of the whole genus Pseudomonas in this respect (C&novaset al., 1967). Now although M . calcoacetica is similar to pseudomonads in many nutritional and physiological features, it differs not only in structure but also most profoundly in the base content of its DNA. This suggests that, despite their many similarities, there is a wide evolutionary separation between Pseudomonas and Moraxella; and the divergence may be reflected in the striking contrasts that are evident when their mechanisms of control of enzyme synthesis are compared.
VI. Acknowledgements The work from the author’s laboratory reported here was supported by U S . Public Health Service grant A107666. REFERENCIEY
Adachi, K., Iwayainn, Y., ‘hnioka, H. and Takeda, 1 ‘ . (1966). Bioclrirn. bioph?ys. A c h 118,88. Alexandor, M. (1965).Adv. appl. Microbiol. 7,35. Bayly ,R. C . and Dagloy, S. ( 1960). Biochem. J. 111,303. Bayly, R. C., Dagloy, 8. and Gibson, D. T. (1966). Biochem. J.101,293. Bird, ,J. A. and Cain, R. B. (1968). Biochem. J. 106,879. Blakley, E. R. (1967).Can.J.Microbio2.13,761. Blaklcy, E. R., Kurz, W., Hdvorson, H. and Simpson, F. J. (1967). Can. J. Microbiol. 13,147. Booth, J., Boyland, E., Sato,T.and Sims, P. (1960). Biochern. J. 77, 175. Cain, R. B. and Parr, D. R. (1968). Biochem. J. 106,859. Cain, R. B., Bilton, R. F. and Darrah, J . A. (1968). Biochem. J. 108,797. CBnovas, J. L. arid JohnRon, B. F. (1968). Eur. J. Biochenb. 3,312. Cdnovas, J. L. and Stanier, R. Y. (1967). Eur. J. Biocheni. 1,289. CBnovas, J. L., Ornston, 1,. N. and Stanier, R. Y. (1967). Science, N.Y. 156, 1695. Cdnovas, J. L., Johnson, B. F. arid Wheelie, M. L. (19688). Eur. J. Biochem. 3,305. CBnovas, J. L., Whoolis, M. L. and Stanier, R. Y. (196%). Eur. J. Biochsm. 3, 293. Cartwright, N. J. and Buswell, J. A. (1967). Biochem. J. 105,767. Cartwright, N. J. and Smith, A. R. W. (1967). Biochem. J. 102,826. Catolani, D., Ficcchi, A. and Galli, E. (1968). E q e r i e n t i a 24,113. Chamberlain, E. M. andDagley, S. (1968).Biochem. J. 110,755. Chamberlain, E. M., Chapman, P. J. and Dagley, S. (1967). Biochem. J. 103, 1 8 ~ . Chapman, P. J.andHopper, D. J. (1968). Biochem. J. 110,491. Clark, F. M. and Fina, L. R.(1952).Archa Biochem. Biophys. 36,26. Clnus, D. and Walker, N. (1964).J.gen. MicrobioZ. 36,107. Collinsworth, W. L.andDagley, S. (1971).Inthepress. Cooper, R. A. and Kornberg, H. JA.(1964). Biochem. J.91.82. Coulter, A. W. and Talalay, P. (1968).J.biol.Chern. 243,3238. Dagloy, S. (1967) In “Soil Biochemistry”, (A. D. McLaron and G. H. Peterson, eds.),p. 287. Edward Arnold, London.
CATABOLISM OF AROMATIO COMPOUNDS BY MICRO-ORGANISMS
43
Dagley, 6. and Gibson, D. T. (1965).Biochem. J . 95,466. Dagley, S. and Johnson, P. A. (1963).Biochim. biophys. Acta78,577. Dagley, S . and Patel, M. D. (1957).Bi0chem.J. 66,227. Dagley, S.and Stopher, D. A.(1959).Bi0chem.J. 73,16~. Dagley, S . , Chapman, P. J., Gibson, D. T. and Wood, J. M. (1964).Nature, Lond. 202,775. Dagley, S., Chapman, P. J. and Gibson, D. T. (1965).Bi0chem.J. 97,643. Dagley, S.,Ueary, P. J. and Wood, J. M. (1968).Biochem. J . 109,559. Daly, J. W. and Jerina, D. M. (1969)Archs Biochem. Biophys. 134,266. Daly, J. W., Jerina, D. M. and Witkop, B. (1968).Archs Biochem. Biophys. 128, 517. Davies, J. I. andEvans, W. C. (1964).Biochem. J . 91,251. Dodson, R.M. andMuir, R.D. (1958).J.A m . chem.Soc. 80,6148. Dodson, R.M.and Muir, R. D. (196l).J.A m . chem.Soc. 83,4631. Dutton, P.L. and Evans, W. C. (1969).Biochem. J . 113,525. Eisner, U.,Elvidge, J. A. and Lindstead, R.P. (1950)J.chem.Soc. 2223. Erdtmm, H. (1944).SvenskPapperstid. 47,155. Evans, W.C. and Moss, P. (1957).Biochern.J . 65,8 ~ . Evans, W. C., Fernley. H. N. and Griffiths, E. (1965).Biochevn.J . 95,819. Farr, I). R. and Cain, 1%.B. (1968).Biochem. J . 106,879. Feist, C.F.and Hegeman, G. D. (1969).J.Bact. 100,869. Freudenberg, IZ. arid Neish, A. C. (1968).“Constitution and Biosynthesis of Lignin”. Springcr-Verlag, New York. Fewson, C. A.(1967).J.gen.Microbiol. 48,107. Focht, D.D. arid Williams, F. D. (1970). Can.J. Microbiol. 16,309. Fujisawa, H.and Hayaishi, 0. (1968).J.biol. Chem. 243,2673. Gibson, D.T., Wang, K. C., Sih, C. J. and Whitlock, H., Jr. (1966).J . biol. Chem. 241,551. Gibson, D. T., IZoch, J. R. and Kallio, R. E. (1968).Biochemistry, N . Y . 7,2653. Gibson, D. T.,Hensley, M., Yoshioka, H . and Mabry, T. J. (1970a).Biochemistry, N.Y.9,1626. Gibson, D. T., Cardini, G. E., Maseles, F. C. and Kallio, R. E. (1970b).Biochemistry, N . Y .9,1631. Grant, D. J. W. and Patel, J. C. (1969).Antonievan Leeuwenhoek 35,325. Griffiths, E. andEvans, W.C. (1965).Bi0chem.J. 95,51~. Griffiths, E., Rodrigues, D., Davies, J. I. and Evans, W. C. (1964).Biochem. J . 91.16~. Gross, S. R . , Gafford, R. S. and Tatum, E. L. (1956).J.biol.Chem. 219,781. Guroff, G . and Rhoads, C. A.(1967).J.biol. Chem. 242,3641. Guroff, G., Daly, J. W., Jerina, I). M., Renson, J., Witkop, B. and Udenfriend, S. (1967).Science,N . Y . 157,1524. Guyer, M. and Hegeman, G. D. (1 969).J. Bact. 99,906. Happold, F.C. and Key, A.(1932)J. Hyg., Camb. 32,573. Hayaishi, 0.(1964).Proc. 6thInternat.Cong. Biochem., Plenary Lectures, p. 31. Hayaishi, 0. (1966). Bact. Rev. 30,720. Hayaishi, 0.(1968).A . Rev. B ~ o c ? L38,21. ~v~. Hayaishi, 0.andNozaki, M. (1969).Science,N . Y . 164,389. Hedegaard, J.and Gunsalus, I. C. ( 1965).J.biol. Chem. 240,4038. Hegeman, G . D. (1967a).J.Buct. 91,1140. Hegeman,G.D.(1967b).J.Bact.91,1155. Hegeman,G.D. (1967c).J. Bmt.91,1161.
44
S. DAQLEY
Hogciiiari, U. D. (1967~1).Arch. MikrobioZ. 59, 143. Hcsp, B., C I L ~ V IM. I I ,and Hosokawa, K.(1969).J.b i d . Clrena. 244, 5644. Holtzmnii, J.,C~illotto,J.I t . a ~ d M i l l l eCi. , W. (1987).J.A?rt.c/~etri.*S’oc.89, 6341. Hopper, D. J., Chapman, P. J. and Ihgloy, S. (1908). B ~ o c h e mJ. . 110,798. Hopper, D. J., Chapman, P. J. and Dnglcy, S. (1971). I n tho press. Hosokawn, K. arid Staiiier, R. Y. ( 1 9 6 6 ) J . bioZ.C’heni.241,2453. Hunt, A. L., Hiighoa, I).14:. tmd Lowcnstcin, J. M. (1968). Biochern. J.69, 170. Jorina, D. M., Dttly, J. W. m d Wltkop, 13, (1968a).J. Am. ~ h r s n . ~ S o9O,(j523. c. Jerina, D. M., Dtily, J. W., Witliop, U., Znltzrnitii-Ni1.t.iihc.rg,P. t t i i d l J d ( ~ i i f i * i t ~ r i t l , S. (1968b).,J.Am, clrrmSoc. 90, 6526. Jerina, I).M., Uitly, J . W . , Witkop, I%., ~,ltzmuri-Nirt.iihcrfi, P. nnd L J d t ~ i t i * i c ~ ~ S. itl. (1968c).Archs RiorireiiL. Biopliys. 128, 176. I<arlason, J. L. niid Ihrkor, H. A . (1‘348). J. biol.L‘/wrib. 175, 913. Katngiri, M., Ttikiirnori, S., Suzitki, Iliemethanogenic organism and tho S organisin grown in ethanol-yeast extract-tryptone agar; C, tho S organism grown in an ethanol-ycast oxtract-tryptone agar slant for four days. Reproduced froin Archi?). fiir Mikrobiologie wit.h perriiiasion.
initiated by Bryant t o establish the characteristics of the isolate (Bryant et al., 1967). The organism was unable to oxidize ethyl alcohol and as a result to reduce carbon dioxide to methane ; however, hydrogen was oxidized readily with reduction of carbon dioxide t o methane. To solve the problem as to how alcohol was used by the original culture, dilutions of this culture were carried out by Bryant in roll tubes of the rich medium with ethanol as the substrate, but without hydrogen in the gas phase. Under these conditions an organism was isolated which would oxidize ethyl alcohol but not hydrogen. The resolution of the culture is shown in Fig. 17. The long slightly curved rods shown in (A) are the cells of the methanogenic organism, whereas the short rods shown in (C) represent the ethyl alcohol oxidizer. Cells from a typical culture of M . omelianskii from the ethyl alcohol-
MICROBIAL FORMATION O F METHANE
123
carbonate mineral medium are shown in (B). Thus, the organism known as i~l~thanobarillus omdianskii does not exist ; instead the culture represents a symbiotic association of two organisms neither of which is
able to grow significantly in the ethyl alcohol-carbonate mineral medium of Barker. The methanogenic organism i s designated Methanobacterium strain 3I.o.H. and appears t o be closely related t o Methanobacterium formicicum, one difference being that the former organism does not utilize formate. The ethyl alcohol-oxidizing organism has been designated “S” organism, and represents a previously undescribed species. The “S” organism is a Gram-negative, motile, anaerobic, rod which oxidizes ethyl alcohol to acetate and hydrogen ; it is inhibited by the hydrogen which it makes, and in the ethyl alcohol-carbonate mineral medium the “S” organism requires JlPthanobacteriurn M.0.H. to use the hydrogen which accumulates. Thus two organisms affect tlie scheme presented in Fig. 1 6 with molecular hydrogen bciiig tlie intermediate between the two organisms. Resolution of this culture has opened new vistas. The r81e of molecular hydrogen as a major substrate for methane formation in nature is apparent. The possible direct oxidation of higher alcohols or fatty acids other than methyl alcohol or acetate by methane bacteria now must receive careful study t o establish that sueh oxidation is carried out by a single species. On the other hand, resolution of the culture provides evidence that substrate coupling between different species in anaerobic ecological niches may not be fully appreciated, especially as to the r8le of molecular hydrogen as an intermediate. For instance the “S” organism readily couples with Methanobacterium ruminantium for the overall oxidation of ethyl alcohol to acetate and the reduction of carbon dioxide to methane (Bryant et al., 1967). Furthermore, Methanobacterium species which oxidize hydrogen may be coupled with Desulfovibrio species in the absence of sulphate when pyruvate is used as substrate (M. P. Bryant, personal communication). The formation of methane, especially in ruminants, has been considered to be a wasteful process, and considerable effort has been devoted in many laboratories t o find ways to prevent this 8-107; “energy loss”. However, the alternative to methane formation is hydrogen accumulation. We have discussed above the precarious nature of the equilibrium for certain oxidations from which molecular hydrogen is produced. Wolin (1969) has considered the thermodynamics of substrate coupling between two species which together effect an unfavourable oxidation at a significant rate. Under natural anaerobic conditions the methane bacteria which oxidize molecular hydrogen may be considered actually to “pull” the degradations in microbial food chains by displacing unfavourable equilibria.
124
R . 9. WOLBE
IV. Mass Culture Techniques A.
(:ROWTH ON
HYDROGEN AN11 C A R B O N I ~ O X I D E
A system has been devclopcd for the mass cultivation of hydrogenoxidizing methane bacteria on mixtures of hydrogen and carbon dioxide under strict mnerobic conditions (Bryant, el nl., 1968). Although initial
FIG.18. Appnrntrin for providing n gn#mixtrirr of H2 and C ' 0 2 to a 12-1. forincmtor (Bryarit et al., 1968). A, gas proportioiier; H, electric f i ~ r ~ i n c(', e ; roduced copper; n, rribbrr stopper wired in plncc; E,stninlass-steel holder for sterile filter; F, port for reiriovirig rarnplas ; G , port for receiving iriociilation from another fcrnicntor ; H, effluont w r i t to hood; I, inorrilation port; (1, 2, nnd 3) screw-clamp vnlves. Reproducal froiii the Joiirtrul uf Bacteriology w i t h perrniamori.
attempts to grow methane bwteria under these conditions yielded erratic results, the important parameters eventually were defined. The technique includes a very simple system of mixing gases in a gas, ballfloat flowmeter-proportioner followed by passage of the gas mixture through a column of heated copper filings to remove oxygen. 1'0 avoid generation of carbon niorioxide the temperature of the copper was maintained below 350". A diagram of the apparatus attached to :L 14-1. ferrnentor is shown in Fig. 18. After inoculation of the fermeritor with 200-400 ml. of culture the gas mixture is passed through the fermentor at a rate of 200 ccl. per minute with a stirring rate of 400 rev./min. As the
MICROBIAL FORMATION O F METHANE
125
culture grows the rate of gas input is increased so that a t the time of maximal cell-crop about 500 t o 600 cc. of gas per minute is being added. A yield of 50 t o 60 g. wet weight of cells is obtained per 12 1. of medium. Growth is slow compared to that of Escherichia coli in nutrient broth,
40
80
HOURS FIG. 19. lielation of cell mass, liptake of hydrogen aud carbon dioxide, and formation of methane during a growth period of 24 to 96 hr. (Itoberton and Wolfe, 1970). Symbols: . , hydrogen uptake; 0 , carbon dioxide uptake; A, methano formation ; A , cell mass. Liquid volume 11 1. Initial cell mass and insoluble components of the medium amountcd to 4.7 g. per 11 1. of medium. Reproduced from the Journal of Bacteriology wit'h pcrmission.
requiring about three days to reach maximal growth. The growth rate of Methanobacterium on hydrogen-carbon dioxide gas mixtures is faster than that of Methanosarcina on methanol, but the final cell yield is not as great. The stoichiometry of hydrogen oxidation and carbon dioxide reduction by Methanobacterium in a 12-1. fernientor is shown in Fig. 19. These
126
R. S. WOLFE
data were obtained in R study by Roberton and Wolfe (l!170). The amount of carbon dioxide used exactly equals the amount of methane formed, whereas the ratio of hydrogen oxidized t o methane formed is about 3.7: 1. The theoretical ratio of 4:1 has not been obtained experimentally, nnd these results arc believed t o indicate that a s m a l l portion of reducing power is obtained from com1)onents of tlic growth In(’( 1’111111. It should be emphasized that, although the growth rate and cell yicld of Methmobactwium are wcll below that of m m y bacteria, the yields which are obtained are good for methtme bacteria and are sigiiificantJy Iwttcr than thosc of rriaiiy strivt anaerobes. The miss culture has bccn sculcd up successfully t o the 250-1. stage. From such a fermentor a kilogr;im of wet cells may be obtained from a single batch ferment at‘ion.
B. GROWTHON VORMATE Growth of mc4littnc ha ria on formate ns the substrutc ~)rcw~iits difficulties. One problem conceriis pH value ; initially t h e mediiim is bufiercd and formate is added as tlie sodium or pot,assiuni d t . As rnctubolism of formate occurs, carbon dioxide arid mcthane are formed. The medium becomes all~alirie,and addition of appropriate amounts of formic acid tidjust~sthe p H value and provides new substrate; thus, a pH-stat may bc used for the addition of substrate in the mass culture of formate-oxidizing inethnrie bacteria. A second problem which may he iicute at tlie tthst)tuhe or flask stage, when forriiatc is used, conccriis tlie high gas I)rctJsurc. wliich may be produced within the growth vessel. Pour moles of formate are decomposed forming one molc of rncthime mid three rnoles of uirbon dioxide. While c+nrbondioxide is significantly soluble especially as the pH valiic rises, methane is I’oorly soluble, and the pressure within tlic vcssel increases. LVhcn the solid stopper is removed t o add fresh substrate, the pressure release of the gases within the growth vcsscl must hc mnde with caution ; the stopper may be blown outJof the tube or flask, and the carbon dioxide which is dissolved under prcssure is relciiscd, c+ausingviolciiitnfrothing of the rnediun~.Likewise, addition of sterile formic acid m u s t be made slowly so that dissolved carbonatch is converted t o carbon dioxide at i~ rate whicah avoids excessive frothing of’ the culture medium.
c. G R O W T H O N METHYLA l , ( w H o r , Methanomrcinn h a d w i has been cultivated successfully in a methanol broth medium t o the 200-1. stage by Blaylock and Stadt,rnun (I!IB(j). Produc%ionof met hnne and ear1)on dioxide was followcd during growth of the c d t u r r s , atid mctli;tiiol W M S & l e d to niaint;~iniL c~oiic.C.iitriLtioiiof 0.5”/oi n the growth medium ; yields of’ 1400 g. wet weight of cells wcrc1
MICROBIAL FORMATION O F M E T H A N E
127
obtained. Although growth was slow ( 7 to 14 days) tlie cell yields which were obtained represent the highest level for inass culture of n methane bacterium which so far hi~sbecn obtainrtl. (:rowth on acetate is very slow. Thus, tlie technology now is available for the mass culture of these strict anacrobes, so that it study of tlie enzymology of these organisms need not, be limited 11y a dearth of cell tissue.
V. Biochemistry of Methane Formation A. ASSAY SYSTEM Metlia,ne may be assayed quickly and accurtitely in minute amounts by gas c.liroiriatograpli?.. W'itli this technique we routinely have used a silicn-gel column attached to a hydrogen flame detector ; hydrogen, carbon dioxide, and methane are easily separated on this column. We have found that a common 1 -cc. tuberculin syringe, which is fitted with a %&gauge needle and in which the piston is very lightly coated with silicone high-vacuum grease, makes a cwnvenient and inexpensive gas syringe. JVe have used for a reaction vessel a modified Warburg flask (Wolin P t nl., 1 W3a) in which a serum cap is fitted tightly in the neck of the flask (Fig. 20). A gas atmosphere is passed into the flask through a syringe needle which is attached to a manifold from which an oxygen-free gas mixture of hydrogeii and carbon dioxide is provided. When the atmosphere in the vessel is completely anaerobic, additions are made to the side arm as gassing is continued. A cell suspension or cell extract is added to the side arm and tipped into the main eoinpartnient. The side arm is rinsed and tipped into the main compartment ; then appropriate additions of substrate or (+ofactorsmay be added t o the side arm. The flask is isolated by removal of'the gassing needle and by closure of the side-arm vent. The re:tction is started by tipping the contents of the side arm into the rnain compartment as tlie flask is placed in a water bath shaker a t 40". At appropriate times a 0.4-cc. sample of gas atmosphere is renioved through the serum vap into the gas syringe arid is injected into the gas chromatograph. To study the niicrobial formation of methane by whole cells a culture is harvestcd, and cells are concentrated by cmtrifugation. Cells are resuspended in carbonate or phosphate buffer of ncutral p H value. To avoid a prolonged lag in tlie reaction rate once the substrate is provided to the cells, exposure to air must be kept to it niinimurn as the cells are harvested and suspended. Buffers should be freed of oxygen by boiling or by sparging oxygen-free gas through the buffer. Oxygen poisoning of the cells may result in a lag of several hours before the cells recover arid are able to produce methane.
128
R . 5. WOLFE
Active cell-e~t~racts may bc prepared from freshly harvested cells or froin frozen cells which have been stored a t -20". Although the Hughes press has been used successfully to prepare extracts, we have found that a sonic probc is more convenient. For breakage of Methanobacterium M.o.H., oells are suspended in 0.05 M-phosphate in a ratio of about, 1 g. of cell per ml. of buffer and are sonicated for about 4 min. No precautions to exclude air are necessary during sonication, providing that the extracts are placed under hydrogen during subsequent centrifugation and storage. Cells of Methanobacterium ruminantium are very dificult t o break whereas cells of Methanobacterium M.o.H., Methanobackrium
Syringe and needle
.
/)L/
1111
ions
Extract-.. FIG.20. Tho reaction vessel for following methane formation (Wolin et al., 1903a). The vessel is a standard Warburg flask of 20-ml. capacity fitted with a rubber serum cap. Reprodiicod from the Journal of Biological Chemktry with permission.
formicicum, wid Methanosrcrcina barkpri ure relatively easy t o disrupt by sonication. B. SUBSTRATES The first substrate which was found to be active in the formation of methane by cell-free extracts was pyruvate (Wolin et al., 1963a). With this substrate it was possible to work out the optimal parameters for the preparation of cell extracts. For early studies with the culture of M . omelianskii this included the use of high molarity phosphate buffers in the range of 0.5 to 1.0 M t o produce active extracts. Subsequently, by use of 114C] pyruvate, it was found that only the carboxyl group of'
MICROBIAL FORMATION OF METHANE
129
pyruvate was a precursor of methane in cell extracts. Recently McBride (1970) has shown by use of an alkali trap that the carboxyl group of pyruvate is converted to methane via free carbon dioxide as an intermediate. Additional substrates which yield methane are listed in Table 2. Carbon dioxide is readily converted to methane in the presence of hydrogen. However, essentially nothing is known about the activation and reduction of carbon dioxide in methane bacteria. Since no I4CI intermediates have been detected prior to the methyl level of reduction, when [“Cj carbon dioxide is used in a Calvin-type experiment, it would appear that the activated C, unit is firmly bound during reduction and does not readily dissociate. Formate is a good substrate for methane formation by most species of Methanobacterium ; present evidence suggests that the formation of carbon dioxide and hydrogen from TABLE2. Substrates W hicli Form Methano In Cell Extracts
*coz CH,CO*COOH *CH,OH CHNHZ COOH 5*CH3-H.+--Folate 5,10, *CH2-H,-Folate *CHS-B,2 H*COOH *CH30H H*CHO
*
C atom converted to CH4.
formate is a primary step in its conversion to methane. Formate is inert when exposed to whole cells or extracts of Methanobacterium M.0.H. This organism apparently lacks formate dehydrogenase (M. P. Bryant, personal communication). Methanol is an excellent substrate for the growth of Methanosarcina barkeri and is converted t o methane by extracts of this organism (Blaylock and Stadtman, 1966), but it is not a substrate for cells or extracts of a variety of other methane bacteria. Formaldehyde also is converted t o methane in extracts of this organism but this reaction has not been reported for extracts of other methane bacteria. When L-[I4C] serine was added t o cell extracts of Methanobacterium M.0.H. (Fig. 21) only carbon-3 was found to be a precursor of methane (Wood ct al., 1905). 0-Phosphoserine also served as a substrate, whereas D-Serine, homoserine, a-methylserine, and 0-methylserine were inactive. Results of these experiments suggested that carbon4
130
H . 8. WOLFE
of serinc might bc transft.rret1 ant1 reduced via totraliydrofolate deriva-
tives. When this pssiI)ility was examined i n extrarts of V .omdirinskii, carbon-3 of serine was fouiid to be transferred t o tctrahydrofoliite to form N-5,hr-10-methylene tetraliydrofolizte by tlie enzyme, serinc transl~ydroxymetliylase.Serine hydroxymethylase is not believed to be involved here, sinre free formaldehyde was inert when added to tlicse extracts. 1V-6,N-lO-MethyIene tetrahydrofoltite was reduced to N - 5 methyl tctriihydrofolate by t i reductasc i n tlie extracts which wiis '2,
specific for NADH, (Wood et aZ., 1965). As shown i n Fig. 21, extracts readily r o n v e r t d N-51 ''CH,ltetrahydrofolatc t o I4CH4.T n extracts of lllpthrznosarcinabarkwi the methyl group of N - 5 -methyl tetrahydrofolnte also was converted to methane but was not, iis active a, ~weriirsorof methane as fonnaldehydc (Stadtman, 1967).
C?. METHYI~COBALAMIN AS SUBSTRATE Rlaylock ant1 Stadtmaii ( l!)63) first, employed niet~liylrobalaminas a substrate for rncthnnc formation i n extracts of Aleti~anosarcina.Subsequently thiN coinpound was fouiid to bc an excellent source of methyl
MICROBlAL FORMATION OF METHANE
131
groups for the formation of methane by extracts of Mpthanohacterium (Wolin et al., 1963b) where t h e reaction was found to be dependent upon the addition of ATP. Fig. 22 prcsents the formula of methyl/cobalamin; this cobamide possesses a dimrthylbenzimidazole moiety as the lower axial ligand. The methyl group which constitutes the upper axial ligand of the cobalt atom is the precursor of methane. This group is easily rendered radioactive by employing [ 14C]methyliodide in the synthesis of methylcobalamin from vitamin B , *. Methyl Factor-I11 also is an active substrate; this compound has an hydroxyl group on the 5-position of the
benzimidazole moiety and is the natural cobamide isolated from cultures of M . omelianskii (Lezius and Barker, 1965). To investigate the importance of the lower ligand in methyl donation t o the methane-forming system in Mpthanohacterium, methyl Factor-B was tested as a substrate. The formula for this cobinamide is shown in Fig. 23; the benzimidazole moiety of the lower ligaiid has been hydrolysed away. However, the methyl group which constitutes the upper ligand was found to serve as a precursor of methane as shown in Fig. 24. It is evident here that the formation of methane from methyl Factor-III (methyl-Co-5-hydroxybenzimidazolylcobarnide), the naturally orcurring cobamide in 41. omdianskii, or from methyl Factor-B is dependent upon ATP. The constituents of the lower ligand are of little importance in determining
132
R. S . WOLFE
FIG.23. Structural formula of arl~toii'Ot}iylCo~iiiaiiiidO.This coinpowid is coinrnonly known as methyl-Factor-B.
~~
~
- Methyl - Factor ID
0
20
40
MINUTES
5.0- Methyl- Factor B
0
40 MINUTES
20
60
FIG.24. Vorniation of [14C]methanefrom [I4C]methylFactor I11 (a)and formation of methane from inethyl Factor B (b)(Wood et al., 1966). (a)The reaction mixtures contained crude extract ; [14C]~nethyl Factor 111 : ATP (whoreindicated). Reaction at 40" undtw H1. (b) The rotiction mixtures coritaincd critdo cxtract, inethyl Fttctor B, potassium phosphato bitffcv, pH 7.0, ATP (whore indicated).Iteaction at 40" under HS. lieproducod from Biocliemiatry, N . I'. with permission.
MICROBIAL FORMATION O F METHANE
133
the abilit'y of a Inetliylcobaniide or incthyl/cobinaiiiitlc t o servc iLs a methyl donor (N'ood et uZ., 1966). When metl~ylcobalaniinis used as a siibstrate for methane formation by cell extracts it is possible t o follow thc1 progress of tlic reaction by the appearmce of the brown colour of B , Zr, i~ product of the derncthylation of met,hyIcobalamiii. The reaction as carried out by extracts of illethnnobnctpri?im is represented in Fig. 25 wlitw tlic cobaniidc winpound is
ATP Cell-extract
(BIZ,)
(CH3-B12)
FIG.25. C'olivorsiori of the xnctliyl groiip of ~iietliylcnbiilarrririto ~netlittneby extracts of nilethur2obnc.teriiLtn M.o.H .
Methanosarcina barker1
(Blaylock and Stadtman) Ferredoxin Corrinoid protein Protein X Heat-stable cofactor
METHANOL I
8125
ATP Mg1+
+C H 3 - 6 1 2
H2
Frc:. 26. ('oii~ponontsof' the iiieLh>I-transfrr reaction in extracts of Methamsarcina hcirkeri as dt.tc.riniwd by ISliiylnrk iind Statltrnan (191iG).
re~~resented in a sirnplified form as used by Ljungdahl et al. (1960). A hydrogen atmosphere and ATP are esseritial for the formation of mcthanc. This rcac*tionhas bcen difficult to resolve into its components (Wood and IVolfe, l ! M h ) . In extracts of the culture of Ill. omdianskii a cobamide protein was implicated i n the methyl-transfer reaction (Wood and Wolfe, 1!)6Gb). So far no evidence for this protein has been obtaiiied in extracts of hydrogen-grown cells of Methanobacterium M.o.H . Considerable progress toward resolution of a methyl-transfer reaction in 1Clethu~~osarcit~a has been oFtiLined by Hlaylork (1968). Here methanol
134
I t . 8. WOLFE
was used as the rnctlryl donor and R , zs was tlic. inetlryl :tccq)tor. 'l'hc enzymic w i n Iwncnts which were identified inclutled ferredoxin, n corrinoid rotein in, tind an unidcnt ified protein ; other components included an unknown heat-stable cofactor, A'I'P, Mg", and a hydrogen atmosphere. This reaction is represented in Fig. 26 and is obviously a complex system. At present it is not known how many of these c ~ m ponents may be involved in tlic formation of metliane. 1). ROLEOF ATP All systems wlrirh so far have been examined in MPthanobuctrriunt or Melhanosarcina require the addition of ATP for t h e formation of mcltliane from a variety of methyl donors. 'I'he role of ATl'in the terminal mc~thyltrtinsfer reactions which lead to the formation of rnctliane is not known. In early experiments it t i p peared that substrate levels of A'I'P were required to activate the methyl group and reduce it t o methane (U'ood and Wolfe. I HOOa). High levels of ATPase in the cell extracts of Jldhariobacleriurn r i d e these experiments difficult. A recent study by Roberton nnd Wolfe (1'309) presents results which indicate that c*atalytic ribther than substrate amounts of ATP are required for the conversion of tlic rrietliyl group of rncthylcobnlamiri t o methane. When a hexokiuase trap was added to the reaction mixture, results indicated that, onc.eA'I'P had reacted, free ATP wtis no longer required. These conclusions were drawn from the experiment shown in Pig. 27. In this experiment methane formation from carbon dioxide and hydrogen by cell extracts of Methanobacterium M.0.H. was followed. When A'I'P and the hexokinase trap were added sirnultanconsly, essentiully no methane was formed. A small nniount, of methane was formed when thc trtip was added two minutes after the addition of A'I'P. When the trap was added a t 5 min. and at 20 rnin. after the addition of A7'P the amounts of nietliane formed were 35 and 650/,, respectively of the control, indicating that free AL 'P ' is not required once it has initially rearted. The enzymic mechanism for the generation of ATP in methaiic bacteria is unknown ; a reaction which yields ATP has not been defined. Hydrogenoxidizing organisrris surh as species of Methanobacterium appear to br good candidtttes for oxidative phosphorylation, since hydrogen is oxidized anaerobically, and it is by no means obvious how ATP is generated. Yet a thorough attempt by Roberton t o demonstrate ATP generation in extracts of this organism produced negative results. By following ATP pools in whole cells, Roberton and Wolfe (1970) showed that a linear relationship existed between ATP and methane produced. These data are shown in Fig. 28 and indicate that there is a direct relationship
135
MICROBIAL FORMATION OF METHANE
6(
5
-4 In 0) d
-wE 3
=%
z a
I
t-
w
I 2
1I
(
0
10
20
30
40
50
60,
70
TIME (min.)
FIQ.27. Time dependence of the inhibition of methane formation by a hexokinase trap (Roborton and Wolfe, 1969). The gas phase was 80% H2and 20% COz. The main compartment of each flask contained cell cxtract suspended in potassium TES buffer, pH 7.1. Flasks A, B, C, and D contained in side arm I, potassium TES buffer (pH 7.6), sodium ATP, glucose, and MgSO, ; and in side arm I1 potassium TES buffer (pH 7.6), glucose, MgSO, and hexokinase. Flasks E and F contained in a single side arm potassium TES buffer, glucose, and MgSO,; and in addition flask E contained ATP, and flask E' contained sodium glucose 6-phosphate and sodium ADP. At zero time, flasks were placed in a water bath a t 38'. At 5 min., the contents of side arm I (flasks A-F) were tipped into the main compartment. The contents of side arm I1 were tipped into the main compartment a t : flask A, 5 min. ; flask B, 7 min. ; flask C, 10 min. ; flask D, 25. Reproduced from Biochimica et Biophysica Acta with permission. 6
136
R . 9. WOLFE
between energy charge as defined by Atkinson and Walton (1967) and the methane formed.
4-
32-
IOL L1-LLLLW-J
0
5
10
15
20
METHANE FORMED ( p moles/min./g. dry wt. cells)
FIQ.28. Relationship of ADP and ATP pools in whole colls to rnethano formud, showing a direct correlation betweon “energy chargo” and mothano formation.
E. COBALOXIMES AS SUBSTRATES For a number of years Schrauzer has been studying the biological implications of cobnloxime derivatives (Schrauzer and Windgusscn, 1967). The first example of a biological system which could ut’l‘ J ize one of tliesc derivatives proved to be the methane-forming system of Methanobacterium M.0.H. (McBride et al., 1968). Methyl-Co-(aquo)bis(dimethylglyoxime) was found t o scrvc as a methyl donor in cell extracts. No methane was formed, however, unless catalytic amounts of B I 2r were added in addition t o ATP; the dependence of the reaction on these compounds is shown in Fig. 29, and the reaction is presented in Fig. 30. When a vnriety of cobaloxime derivatives were tested in the presence of ATP and BI2,,the rates of methane evolution were found t o be dependent on the in-plane ligands. For instance, methyl-Co(benzimidazole)bis(dimethylglyoxime)was only about one third as
MICROBIAL FORMATION OF METHANE
---)
-+
137
f A T P +B,,.,
-ATP
min.
FIG. 29. Dependonce of I4CH4 formation from 14CH3-Co-dimethylglyoximato rnonoanion on vitamin BIzrand ATP (McBride et al., 1968). Reactions contained extracts, ATP, 13, 2r, (whereindicated), methyl- 14C-(aquo)bis(dimethylcobaloxime) and TES buffer, pH 7.0. Reactions were run under H, at 40". Total reaction volume 1.25 ml. Reproduced from the Journal of the American Chemical Society with permission.
ATP, Ha
CH,f
FIQ. 30. Convorsion of the methyl group of methyl-14C-(aquo)bis(dimethylcobaloxime) to methane by extracts of Methanobacterium M.0.H. (McBrido et al., 1968). lteproduced from the Journal of the American Chemical Society with permission.
138
R. S. WOLFE
active as methyl-Co-(aquo)bis(dimethylglyoxime). In evaluation of the implications of this "biological activity", a cautious view should be taken until the requirement for B, Zr has been elucidated since methylcobalamin can be formed chemically in this system; it has not been unequivocally established that this reaction is unimportant in the formation of methane from methyl-Co-(aquo)bis(dimethylglyoxime).
F. ROLEOF COENZYME M Recently, evidence for a new coenzyme of methyl transfer in methane bacteria has been obtained by McBride (1970) and McBride and Wolfe (1970, 1971). I n certain experiments where [ ''C]methylcobalamin was used as a substrate for the formation of methane not all counts were recovered as methane. The missing counts were found t o be trapped in the reaction mixture. Experiments designed t o elucidate the nature of this phenomenon yielded evidence that the counts were trapped primarily in one compound. A method of isolation and purification of this factor has been worked out. Cell extracts which have been resolved for the factor by anaerobic dialysis do not form methane from methylcobalamin ; addition of the factor back t o the resolved extract allows the formation of methane t o proceed. The name, coenzyme M, is proposed for this coenzyme which is involved in methyl transfer (McBride, 1970). So far the following properties have been determined for the new coenzyme : Co-M is acidic; contains phosphate; is adsorbed t o Dowex-1 but not to Dowex-50; is insoluble in acetone, ether, or chloroform; has a A,,, at 260 nm ; is not fluorescent ;is ninhydrin negative ;is enzymically methylated and demethylated. The compound can be isolated in t h o nonmethylated state and can be enzymically methylated, then purified. Thus, a compiirison of the methylated compound wit,h the unmethylatcd state should provide a handle as to information on the structure and on the site of methylation. The enzymic methylation of Co-M (Fig. 31) is followed by use of ['4C]methylcobalamin as the methyl donor, dialysed extract, subRtrate amounts of Co-M, ATP, and a hydrogen atmosphere. Addition of tripolyphosphate inhibits the demethylation of Co-M and so allows the stoichiometric amounts of [ 14CH,]Co-Mto accumulate. The requirements of ATP and a hydrogen atmosphere are not understood, but it is conceivable that the reaction mechanism may involve a phosphorylation as well as tt reductivemethylationof Co-M.NADPH,hasbccnfoundtosubstitute for a hydrogen atmosphere in the reaction mixture. I n following the demethylation reaction ['4CH,1Co-Mis used as asubstrate and the formation of [14C]methane is determined (Fig. 32). Here resolved extracts require ATP, Mg, ['4CH,]Co-M and a hydrogen atmosphere t o form
139
MICROBIAL FORMATION O F METHANE
[I4C]methane.Thedemethylation of CH,-Co-M is a reductive demethylation, and again the rSle of ATP in this reaction is not understood. So far Co-M has been detected only in methane bacteria. Coenzyme-M exhibits a vitamin-coenzyme relationship and serves as a growth factor. M. P. Bryant (Bryant and Nalbandov, 1966)has studied a growth factor for Methanobacterium ruminantium which was purified from rumen fluid. This work was abandoned when amounts insufficient for further study were obtained. However, Co-M has been found t o substitute for this growth factor. Blaylock ( 1968) has described a heat-stable, dialysable
ATP
Dhlysed Cell extract Tripolyphosplub
(CH3-B
(BIZ.)
12)
FIG.31. Diagram of the methylation of coenzyme M (Co-M)by extracts of Methanobacterium M.0.H. Tripolyphosphate inhibits the demethylation of Co-M.
AT P
'CHS-CO-M
Hz
Cell extract
CO-M + 'CH4
FIG.32. Diagram of the demethylation of methylcoenzyme M (CH,-Co-M) methane by extracts of Methanobacterium M.0.H.
to
cofactor which is required in the enzymic transfer of the methyl group of methanol to B , 2s. This cofactor may be identical to or be a form of Co-M. Since we could not find Co-M in extracts of Glostridium sticklandii, an organism in which the Blaylock cofactor was reported, we assumed that the factor was not the same as Co-M. However, a subsequent personal communication by T. C. Stadtman states that there may be inhibitors in the clostridial extract which inhibit action of the cofactor.
G. INHIBITORS OF METHANEFORMATION The relation of various inhibitors of methane formation t o ATP pools in cells of Methanobacterium M.0.H. has been studied by Roberton and
140
R. 9. WOLFE
Wolfe (1970). Among the most sensitive organisms to oxygen are the methane bacteria; the mechanism of inhibition of these bacteria or of anaerobes in general by oxygen is not well understood. The results of an experiment in which cells of Methanobacterium M.0.H. were exposed to
CH4 I
0
I
I
I
I
I
2
3
4
5
6
HOURS FIG.33. Effect of air on adenine nuclootide pools and methane production in whole cells (Roberton and Wolfe, 1970). Initially a mixture of H, and CO, (80:20) was bubbled through the cell suspension. At the arrow, air was bled in with tho H2:CO, mixture. Temperature 24'. A , Methane formation ; A , ATP ; 0, ADP ; 0,AMP; V , A T P A D P + AMP. Reproduced from the Journal of Bacteriology with permission.
+
141
MICROBIAL FORMATION OF METRANE
small quantities of air after the cells were forming methane actively is shown in Fig. 33. The cell suspension was stirred under an atmosphere of hydrogen and carbon dioxide ( 4 : 1). Due to exposure t o air during harvesting the cells were in oxygen shock for about 2.5 hr. ; after this time, methane formation started. As methane formation increased the
40
-
20
-
'01
do
I
I
150
100
I
200
MI NUTES FIG.34. Effect of 2,4-dinitrophenol (DNP), carbonyl cyanide-m-chlorophenylhydrazone (CCP) and pentachlorophenol (PCP) on methane production in whole cells (Roberton and Wolfe, 1970). Cells suspended in potassium TES buffer solution (pH 7.4) were placed in the main compartment of a Warburg flask, and uncoupler in 0.6 ml. buffer was placed in the side arm. The flasks were gassed with a mixture of H, and CO, (80: 20) a t room temperature, tipped and shaken in a 37" water bath. Methane formation was measured. v , Control; 0 , 2 x df DNP; 0 , 1 0 - 4 ~ D N P ; A , 4x 1 0 - S ~ C C P ; 1 0 - 4 ~ P C P0;, 5 x MPCP.Reproduced from the Journal of Bacteriology with permission.
.,
AMP pool decreased, and the ATP pool increased. When the ATP pool and methane formation reached a maximal level, air was added (as indicated by the arrow) a t a rate of 1-5 cc. per minute into the influent hydrogen :carbon dioxide gas mixture which was passed over the cells a t a rate of 30 cc. per minute. Methane formation was inhibited gradually, and as this occurred the ATP pool decreased and the AMP pool increased.
142
R. 9. WOLFE
The total adenine nucleotide pool essentially was constant throughout the experiment. Uncouplers of oxidative phosphorylation produced a very similar picture. A decrease of ATP pool levels was associated closely with a decrease in methane formation. The effect of various levels of carbonylcyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol, or pentachlorophenol on methane formation by whole cells is shown in Pig. 34. Reversal of the inhibition caused by 2,4-dinitrophenol was found to occur in titne
t I
/e
t
///
v
I2'O
I
0
CHCI3
20
I
40 [I/S]x 10-'Af
cc14
CH2C'2
I
60
FIQ.35. Competitive inhibition of methane formation from methylcobalamin by chlorinated hydrocarbons (Wood et al., 19BBb). Reaction mixtures contained extract, methylono chloride, chloroform or carbon tetrachloride, ATP, potassium phosphate buffer (pH 7.0) and variable levels of methylcobalamin. Gas phase H,; incubated a t 40"for 16min. Reproduced from Biochemistry, N . Y . with permission.
and was due to reduction of the cotnpound as was indicated by a change in its absorption spectrum. A similar decrease in the ATP pool also was observed when thc inhibitor of methanogencsis, chloroform, was added t o cell suspensions. Bauchop ( 1967) discovercd that certain chlorinated hydrocarbons (chlorinated methanes) were potent inhibitors of methane formation in suspensions of rumeii fluid. By use of cellextracts, Wood et al. (196813)found that methylene chloride, chloroform, and carbon tetrachloride were competitive inhibitors of methane formation a5 presented in Pig. 35. These compounds also were shown to react chemically with B, 28 to form a series of chloroinethylcobalamina. A cobaniide protein from extracts of the culture of M . omelianskii was propylated by the method of Brot and Weissbach
143 (1965), and after purification was shown to stimulate the formation of methane from methylcobalamin by dilute cell extracts. The chloromethanes also were found t o inhibit competitively another cobamide protein, 5-methyl-tetrahydrofolate homocysteine transmethylase from Escherichia coli (Wood et al., 1968b). Penley et al. (1970) have continued studies on alkylated cobamides and have synthesized various fluoromethyl cobalamins (CFC1,-B, ; CF,Cl-B, ; CF,-B I ,) which were shown t o be competitive inhibitors of CH,-B,, in the formation of methane by extracts of Methanobacterium M.0.H. Although a protein was readily propylated and isolated from ext,racts of the culture of M . omelianskii (Wood and Wolfe, 1966b), it has not been detected in extracts of Methanobacterium M.0.H. (McBride, 1970); this fact raises questions about the r61e of the cobamide enzyme in methyl transfer or in methane formation. This finding also appears t o open again the question as to the mechanism of inhibition of methanogenesis by the chloromethanes. MICROBIAL FORMATION OF METHANE
,
,
H. REDUCTION OF ARSENATE Extracts of Jlethanobacteriurn have been shown t o catalyse reactions in which an active methyl group is transferred t o acceptors such as arsenate (McBride and Wolfe, 1969). When extracts are incubated in a hydrogen atmosphere with methylcobalamin, arsenate, and ATP, a volatile arsine derivative is formed. Arsines are difficult and dangerous to work with; they are extremely poisonous, and are rapidly oxidized in air. The pertinent methylated arsenic derivatives are presented in Fig. 36. Fortunately, they have an intense garlic odor, so the investigator is warned of their presence. In reaction mixtures which contained arsenate the reaction mixture turned brown, indicating the transfer of the methyl Arsines: CH3
I
H-AS-H Methyl arsine
CH3
I
H-As--CH, Dimethyl arsine
CH3
I
H~CAS--CH~ Trimethyl arsine
Oxidized derivatives: CH3
I
HO-AS-OH
II
0 Methyl arsonic acid
CH3
I
HO-As-CH~
II
0 Cacodylic acid
CH3
I
H,C-As--CH
I1
3
0 Trimethyl arsinic acid
FIG.36. Structural formulae of tho methylaminos and their oxidized derivatives.
144
R. 9. WOLFE
group from methylcobalamin ; methane formation was inhibited. Of the various methyl donors tested as substrates only methylcobalamin was capable of forming an alkyl arsine ; of the arsenic derivatives tested as substrates only cacodylic acid was reduced directly to an alkyl arsine without the addition of methylcobalamin. However, ATP and a hydrogen atmosphere were required, and the final alkyl arsine derivative was identified as dimethyl arsine. The reductive pathway is shown in Fig. 37. Wood et al. (1968a) have presented evidcnce that small quantities of activated methyl groups may be transferred from methylcobalamin to mercury by extracts of Methanobacterium M.0.H. t o form toxic methylmercury compounds.
-
OH HO--Ae-OH +5 I
2.3
II
CH3-B12
A +3 d H
B12r
A
II
0
0
CH1-B12 HO-As-OH
II 0
B12r
A 2e
CH3 HOZL!E-CH,
I1
0
CH3 & _3
-31
As--CH]
I
H
h a . 37. Pathway for tho formation of dimethylnrsine by extracts of Methanobacterium M.0.H.
I. MINI-METHANESYSTEMS Postgate (1969) has reported the synthesis of minute amounts of methane by extracts of Desulfovibrio, Desulfotomaculum, and Clostridium pasteurianum in the presence of sodium pyruvate. I n contrast t o extracts of Methanobacterium in which the carboxyl group of pyruvate is the precursor of methane, the methyl group of pyruvate is the precursor of methane in extracts of the above-mentioned organisms. McBride (1970) suggests that the detectable amounts of methane might be low due t o the reaction of nctivated methyl groups with SH-groups in extracts of the sulphate-reducing organisms. Formation of methane by these organisms appcars t o be by way of a unique mechanism. Certain mammalian tissues also produce methane when provided with approprinte wbstrates. Dost and Reed (1967) found that the N-methyl group of N-isopropyl-a(2-methylhydrazino)-p-toluamide was converted t o respired methane when the labelled compound was given intraperitoneally to rats.
VI. Acknowledgements
It is a pleasure to acknowledge the rBle of my colleagues, S. R. Elsden, M. J. Knight, E. A. Wolin, M. J. Wolin, W. J. Brill, A.M. Allam, J. M.
MICROBIAL FORMATION OF METHANE
145
Wood, M. P. Bryant, K. F. Langenberg, P. Cheeseman, A. M. Roberton, and B. C. McBride in the development of this problem. I n addition I thank P. H. Smith for kindly providing phase-contrast photomicrographs of methane bacteria from sludge, and M. P. Bryant for helpful discussions on nutrition. REFERENCES Atkinson, D. E. and Walton, G. M. (1967).J. biol. Chem. 242,3239. Barker, H. A. (1936).Arch. Mikrobiol. 7, 420. Barker, H. A. (1940).Antonie van Leeuwenhoek 6,201. Barker, H. A. (1956).“Bacterial Fermentations”, p. 1, John Wiley and Sons, Inc., New York. Bauchop, T. (1967).J.Bact. 94, 171. Blaylock B. A. (1968).Arche Biochem. Biophye. 124, 314. Blaylock, B. A. and Stadtman, T. C. (1963).Biochem. biophys. Res. Commun. 11, 34. Blaylock, B. A. and Stadtman, T. C. (1966).A r c h Biochem. Biophys. 116,138. Brot, N. and Weissbach, H. (1965).J. biol. Chem. 240, 3064. Bryant, M. P., McBride, B. C. and Wolfe, R. S. (1988).J. Bact. 95, 1118. Bryant, M. P. and Nalbandov, 0. (1966).Bact. Proc., 90. Bryant, M. P. and Robinson, I. M. (1961).J . Dairy Sci. 44, 1446. Bryant, M. P., Wolin, E. A., Wolin, M. J . and Wolfe, R. S. (1967).Arch. Mikrobiol. 59, 20. Dost, I?. N. and Reed, D. J . (1967).Biochem. Pharmac. 16,1741. Hungate, R. E.(1960).Bact. Rev. 14, 1. Johns, A. T. and Barker, H. A. (1960).J. Bact. 80,837. Knight, M., Wolfe, R. S. and Elsden, S. R. (1966).Biochem. J . 99,76. Langenberg, K.F.,Bryant, M. P. and Wolfe, R. S. (1968).J. Bact. 95,1124. Lezius, A. and Barker, H. A. (1965).Biochemktry, N.Y. 4, 610. Ljungdahl, L., Irion, E. and Wood, H. G. (1966).Fedn Proc. Fedn. Am.Soca ezp. Biol. 25, 1642. McBride, B. C. (1970).Dissertation: University of Illinois, Urbana, Illinois. McBride, B. C. and Wolfe, R. S. (1969).Bact. Proc., 130. McBride, B. C. and Wolfe, R. S. (1970).Fedn Proc. Fedn. A m . Soca exp. Biol. 29, 344 Abs. McBride, B. C. and Wolfe, R. S. (1971).Biochemistry, N . Y . In press. McBride, B. C., Wood, J. M., Sibert, J. W. and Schrauzer, G. N. (1968).J. A m . chem. SOC.90,5276. Omelianski, W. L. (1916).Annls Inst. Pasteur, Paris 30,56. Paynter, M. J. B. and Hungate, R. E. (1968). J . Bact. 95, 1943. Penley, M. W., Brown, D. G. and Wood, J. M. (1970).Fedn Proc. Fedn. A m . Soca exp. Biol. 29,344 Abs. Postgate, J. R. (1969).J. gen. Microbiol. 57,293. Roberton, A. M.and Wolfe, R. S. (1969).Biochim. biophys. Acta 192,420. Roberton, A. M.and Wolfe, R. S. (1970).J. Bact. 102,43. Schnellen, C.G.T. P. (1947).Dissertation: Tech. University, Delft. De Maasstad, Rotterdam, Publisher. Schrauzer, G. N. and Windgassen, R. T. (1967).J . A m . chem.Soc. 89, 1999. Smith, P.H. (1966).Developments in Industrial Microbiology 7 , 166. Stadtman, T. C.(1967).A . Rev. Microbiol. 21, 121. Stadtman, T. C.and Barker, H. A. (1949).Arch8 Biochem. 21,266.
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Stadtman, T. C. and Barker, H. A. (1951). J. Bact. 62,269. Steggerda, F. R. and Dimmick, J. F. (1966). Am. J. din. Nutr. 19, 120. Tseng, S. F. (1970). Masters Thesis: University of Illinois, Urbana, Illinois. Wolin, M. J. (1969). Abstracts of 158th American Chemical Society National Mooting, New York, Section MIRC No. 19. Wolin, E. A., Wolfe, It. S. and Wolin M. J. (1964). J. Bact. 87, 993. Wolin, E. A., Wolin, M. J. and Wolfe, R. S. (1963a).J. biol. Chem. 238, 2882. Wolin, M. J., Wolin, E. A. and Wolfe, R. S. (1963b). Biochem. biophys. Res. Comrnun. 12,465. Wood, J. M., Allarn, A. M., Brill, W. J. and Wolfe, R. S. (1965). J . biol. Chem. 240.4664. Wood, J. M., ICennedy, F. S. and Rosen, C. G. (1968a). Nature, Lond. 220, No. 5163, 173. Wood, J. M., Kennedy, F. S. and Wolfe, R. S. (1968b). Biochemiatry, N.Y. 7 , 1707. Wood, J. M. and Wolfe, R. S. (1966a).J. Bwt. 92, 696. Wood, J. M. and Wolfe, €3. S. (1966b). Biochemktry, N . Y . 5 , 3698. Wood, J. M., Wolin, M. J. and Wolfe, R. S. (1966). Biochemistry, N.Y. 5 , 2381.
The Adaptive Responses of Escherichia coli to a Feast and Famine Existence ARTHUR L. KOCH Department of Microbiology, Indiana University, Bloomington, Indiana 47401, U.S.A. I. Introduction . 11. The Speed of MacroniolecularSynthesis. . 111. “Extra” RNA in Slowly Growing Bacteria . IV. Ilescription and Operation of ( ’hemostats A. Design Features . B. Evidence that the “Extra” RNA is not an Artifact Due to Inadequate Mixing of tho Chemostat V. RNA Synthesis in Slowly Growing Bacteria . VI. Tracer Kinetics Interlude . VII. The Growth Cycle Revisited . VIII. Active Transport From Very Low External Concentrations A. Uptake by a Motionless Spherical Cell . . B. Uptake by Spherical Moving Cells. C. Uptake by Rod-Shaped Particles . D. Movement and Mixing Efficiency . E. The Intermediate Region Between Diffusion and Transport Limitation . F. Experimental Determination of Uptake Parameters by Growth Studies . IX. General Conclusions . X. Acknowledgements . References .
. .
.
147 149 162 169 169 101 164 109 181 192 190 203 206 207 208 210 214 214 216
I. Introduction The ancestors of modern Escherichia coli probably have been occupying mammalian intestines ever since the beginning of the Jurassic, 2 x lo8 years ago. The botal bacberial generations involved are of the order of los0.Geneticists interested in evolution of higher species of organisms such as any of E . coli’s hosts have 1020 to IO3O fewer organism generations of organisms to explain the evolution of that mammal than does the geneticist interested in the evolution of the common colon bacillus. I n fact, the evolutionary possibilities are so great that it is reasonable to 147
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expect that toduy’s E . coli is maximally adapted (in the sense of being finely tuned) to its habitat. Biochemists and molecular biologists have studied E . coli so devotedly that there can be no doubt that it is the best understood of any living creature. However, our knowledge is almost entirely confined to the properties the organism exhibits when it grows unrestrictedly with doubling times of one hour or faster. I n its natural ecosystem, an average doubling time of once or twice a day is all that is permitted by the volume of, and flow through, the intestine. Because of the power of Malthusian growth, much of its life is spent under chronic starvation. From this fact and the consideration that selection must act almost purely in favour of rapid growth for an organism in such an ecological niche, E . coli must be highly efficient in utilizing nutrients and effective a t growing under what in human terms would bc called diseased states of under-nutrition, malnutrition, or Kwashiorkor. The purpose of this review is to examine several facets of the efficiency of the organism in growing in a harsh competitive environment. We would like to see how closc to the theoretical chemical, physical, and biochemical limits the organism operates. Effective use of resources to produce functioning organisms implies that macromolecular synthesis is properly divided between structural and enzymic units on one hand and the means of production that include ribosomes and t-RNA on the other. The problem is formally equivalent to that faced by a human society which must decide how much of the gross national product to plow or t o invest back into the land improvements, schools, factories, and other capital equipment t o keep the economy expanding and how much to devote to just as essential but less immediately catalytic elements such as automobiles and medical care. I n an expanding economy all elements must increase, but the manufacture of means of production themselves must be more sensitively geared to the rate of expansion than to the actual quantity of goods produced. The analysis of this problem presented below shows that our laboratory strains of E . coli are not constructed to be ultimately cfficicrit a t allocating resources for nucleic acid and protein synthesis from the point of view of what is best in an ideal, non-fluctuating continuous culture. I hope to make a plausible case that this seeming imperfection is simply the difference between strategy and tactics. The micro-organisms have not only been selected for ability to grow under chronic starvation, but also for ability to respond quickly to unannounced and irregular windfalls of food. Selection is still directed almost solely toward growth. However, a t one time, the emphasis is on ability t o accelerate growth, and a t a later time the emphasis is on coping with a deceleration of
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growth. The capability to respond to fluctuations in the environment and the cost to the organism of such adaptability will be examined. We will skip the normal processes of feedback inhibition and induced or repressed enzyme formation. The other major problem that enteric bacteria have had to solve is the effective abstraction of essential small molecular-weight nutrients from an environment which contains compounds at very low concentrations. This leads to an analysis of the efficiency of the transport machinery that allows E . coli to compete with its neighbours for carbon and nitrogen sources, growth factors, or trace elements. It is concluded that the transport mechanisms for several compounds have evolved t o the degree where diffusion through the viscous natural environment is limiting. These various subjects bring us to grips with chemical kinetics, growth kinetics, tracer kinetics, and diffusion kinetics. Fortunately, these subjects can be presented without a great deal of mathematics. The basic derivations have been given in the literature. I have appropriated formulae to be used for the present purpose, and I present computations to give a feel for the theoretical considerations. I wish to thank the National Science Foundation, which allowed us to buy a Wang computer to do the arithmetic. It must be admitted that t,his paper is not a literature review, but a frankly chauvinistic assemblage of a variety of experimental observations, thoughts, derivations, and philosophies produced in this laboratory over a number of years with the help of people who start as students but end as colleagues. Some of the data presented will never be published separately but are included here because they bear on the present discussion. The variety of topics presented here, I hope, all relate t o the biology of enteric bacteria forced by their environment to grow slowly much of the time. 11. The Speed of Macromolecular Synthesis
I n economic contexts, a society achieves highest efficiency when it extracts the maximum amount of product from a workman or a machine a t the lowest total cost. If the workman or the machine is expensive compared with factors such as raw material and power, then higher efficiency is achieved by increasing the product per unit time per worker or machine. I n an expanding economy, the means of production are expensive because they are large and complex and have to be continually made. Applying the same considerations of efficiency to the enteric bacteria, we feel that they should have evolved very rapid rates of translation, even if it is a t the expense of a high cost of maintaining elevated levels of
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m-RNA, t-RNA, amino acids, and energy supplies. We would also expect the bacteria to have an abundance of RNA polymerase. All this is so that the ribosome, which on proration is the heaviest and therefore the most expensive part of the protein-synthesizing machinery, will be used a t highest efficiency and therefore lowest total cost t o the cell (see Table 1). Evolution would also select for very efficient activating enzymes for they too represent a costly item in the cell's economy. TABLE1. Overhead costs for protein synthesis
Investment in capital equipment DNA D N A polymerasob Mossenger-RNA R N A polymeraseC Transfer-RNA Activating enzymesd Ribosomal-RNA(6S Ribosomal protein Maintenance costs m-RNAm
Cost to m a k e one now cell i n units of lo8 daltons per parontal cell' 60 1.5 7.5
+ 1 6 s + 23s)
N
4.1 25 160
200 110
N
30
Depreciation costas The calculatione are bawd on the following wumptions: thecell isgrowing withadoubling time of 60 min. and has a dry weight of 2.6 x g. Most of the cell composition data have been taken from McQuillen (1965). * CalculatedfromLehmanetaZ.(1958). Calculated from MaitraandHurwitz (1967). * There are 40 different activating enzymes. Each has a molecular weight of about lo5 daltone and there are 4000 molecules of each species per cell (Fangman et al., 1965; Calendar and Berg, 1966). The m-RNA hae a half life of one min. A single high-energy bond is expended per molo of phosphate which could have been used t o make approximately 20 daltons of stable cell material. a
I n the course of evolution, the translation mechanism itself m u d have become faster and faster; in micro-organisms, the step time of translation is 16 amino acids per second (Lacroute and Stent, 1968). For a review of earlier work, see Kelley and Schaechter (1968). For mammals, the step time is two per second (Dintzis, 1961), and we might speculate that the mammal has not been selected t o be ultimately fast at protein synthesis bub selection has been on other qualities, such as mobility and wisdom. Transcription is also very fast in micro-organisms. The average step-time in vivo is about 48 per second for the addition of a
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nucleotide. This is three times faster than the step time for adding an amino acid (Bremer and Yuan, 1968; Manor et al., 1969; Winslow and Lazzarini, 1969a; see Geiduschek and Haselkorn, 1969, for a review). There is a real need t o make the stable kinds of RNA as fast as possible because they are parts of the means of production. There are two ways to speed up the rate of synthesis: one is to have multiple copies of the genes for ribosomal-RNA. I n a bacterium such as E . coli, there are 5-6 (Spiegelman and Yankofsky, 1965). There are many more (300-500) in a larger amphibian or mammalian cell (Wallace and Birnstiel, 1966; Steele, 1968) and there are still many more than that in the yet larger oocyte as the result of gene amplification (see Gall, 1969). The other method of speeding up synthesis is to increase the rate at which the DNA-dependent RNA polymerase transcribes r-RNA. It is argued, however, that, as far as message synthesis and its economics go, there is no basic reason for messenger synthesis to be as fast as it is. Most of the evidence suggests that the average step-time for the synthesis of RNA for ribosomes is just 8s fast as the step time for messenger synthesis. If the cell has gone to the trouble to evolve a faster polymerase for stable synthesis, it would be cheaper to make a copy from the same genes and use it also for messenger synthesis. There is good evidence that there is only one kind of RNA polymerase in the cell, although it may be modified by different initiation factors based on the properties of rifamycin- and temperature-sensitive mutants. However, there is a secondary, but much more compelling, reason for messenger-RNA synthesis to be very fast. I n micro-organisms, transcription and translation are tightly coupled. This means that translation of a message commences while transcription is in progress. Therefore, the ribosome can add no more than one amino acid to the nascent peptide chain while the polymerase is adding three nucleotides to the growing messenger molecule. This would suggest that RNA synthesis proceeds as fast as is physically possible in terms of diffusion forces or in terms of kinetic steps of enzyme reactions. Protein translation mechanisms as presently evolved are either really faster than, or just as fast as, one-third the messengersynthesis step time. This consideration focusses on the DNA-dependent RNA polymerase as the rate-limiting step for growth. I t s inherent speed may determine both how fast ribosomes can be made and how fast they can function. What limits the enzyme’s speed is surely not the polymerization process itself, because DNA-dependent DNA polymerase works in vivo much faster (more than 1000 nucleotides per second). Therefore, based on this biological argument, I predict that the unwinding and concomitant rewinding of DNA during RNA synthesis will turn out t o be a rate-limiting step to the overall process of protein synthesis under in vivo conditions in enteric bacteria.
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This line of inquiry has been taken about as far as possible. I end this section by noting that efficient use of the protein-synthesizing machinery would require, in very slowly growing bacteria as in rapidly growing bacteria, a rapid rate of messenger-RNA transcription to permit protein synthesis coupled to transcription to take place rapidly.
In. “Extra” RNA in Slowly Growing Bacteria It was thought until five years ago (see Maalne and Kjeldgaard, 1966, for a review) that the number of ribosomes per genome was directly proportional to the growth rate constant, and, therefore, that the cell was as economical as it could be. This was consistent with the idea that ribosomes worked with constant efficiency, independent of the nutritional environment of the cell. One cell growing twice as fast as another was to have twice as many ribosomes, so that it could synthesize the same amount of protein in half the time. Improved analytical measurements showed that this is just not so (see Koch, 1970, for a review). But the coup de grdce of the constantefficiency hypothesis is measurements of the in vivo rate of protein synthesis when carbon-limited chemostat cultures are suddenly enriched (Koch and Deppe, 1971 ; Fig. 1). I n much less time than the stable RNA content can change appreciably, the rate of protein synthesis of chemostat-grown cells previously growing with a 10-hr. doubling time increases seven-fold. After that, the capability for protein synthesis increases in parallel with new net RNA synthesis. Although the efficiency with which RNA is used for protein synthesis is much smaller in carbon-limited chemostat growth than it is a few seconds after enrichment, the speed a t which a ribosome works as measured by the step time for the addition of an amino acid to a growing peptide chain appears to be just the same. This conclusion is drawn from experiments (Coffman et al., 1971) using an ultra-sensitive fluorometric assay in which inducers for /3-galactosidase synthesis are added t o unlimited batch cultures (Fig. 2) and to carbon-limited chemostats (Fig. 3). Thc delay time from the instant inducer is added until the completion of the first polypcptido chain in either batch cultures with succinate or glurose as carbon source or in chemostats is invariant a t 95 sec. in cells with doubling times ranging from 50 min. t o 13 hr. Strong evidence that transcription and translation are coupled in all of these cases comes from the experiment,al observations that it takes ricarly that long (80-90 scc.) for either transcription (see Geiduschek and Haselltorn, 1969) or translation (Lacroute and Stent, 1968) of the m-RNA for p-galactosidusc in bacteria growing at the fastest doubling time. The same statistical analysis applied to the data in Fig. 3 shows that neither the step time for
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transcription nor translation is slowed by even a factor of two as the growth rate is decreased nearly 30-fold. The conclusion from Fig. 1 is that “extra” RNA is preseiit in a rapidly utilizable, though unutilized, form in these slowly growing cells. Another possibility is that some cells might be inactive in protein synthesis; this
Time (rnin )
FIG.1 . Rate of protein synthesis in Escherichia coli after “shift-up” of a glucoselimited chemostat culture. Measurc:ments of rate of uptako of radioactive tryptophan in 2 min. pulses were madc bofore and after an enrichment that shifted a culture with a 11 hr. doubling time to onc with a 40-min. doubling time. Tho theoretical curve was calculated as described in Koch and Deppe (1971) on tho assumption that, after the shift, ribosomal officioncy rises quickly and then remains constant.
can be eliminated by the findings of Koch and Coffman (1970) that all cells in chemostats make /3-galactosidase within a very few minutes after induction. Even when the doubling time is extended to 24 hr., two-thirds of the cells immediately make enzyme on induction while the remaining one-third synthesizes protein within the next 3 hr. Yet another possible alternative t o postulating unutilized protein-synthetic
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machinery would be that the ribosomes make protein, which is then broken down. While there is intracellular turnover even in growing bacteria, we have found (Nath and Koch, 1970) that only a limited class of proteins are actually degraded, and this synthesis could at most use a small fraction of the ribosomes engaged in the synthesis of stable proteins in the cell.
FIU.2. Kinotics of induction of ,Ll-galactosidase synthosis in batch-grown Escherichia coli strain ML3 (Coffmanet al., 1971). Both curves show a detectable increase above the basal level a t 96 rt 6 sec. Noto the highly expanded scales in both dimensions made possible by using the sensitivo fluorometric assay. Open circles indicate data from succinate-grown batch cultures ; closed circles, glucosecontaining batch cultures.
Thus, cells in the carbon-limited state of slow but balanced growth make r-RNA, t-RNA, ribosomal protein, and possibly other proteins needed for protein synthesis that they do not use to full efficiency. I n bhe contrasting situation of sulphur-limited chemostat growth, although the ratio of total RNA to dry weight is just a little lower than that observed under carbon limitation, the “extra” RNA is not rapidly
THE ADAPTIVE RESPONSES OF ESCBERZCEIA COLZ
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converted into materials functioning in protein synthesis (Koch and Deppe, 1971; Fig. 4). Moreover, the capacity of the cells for protein synthesis is the same in these cells before and after a nutritional enrichment. This means that the few ribosomes present in a functioning state in these cells were being used with the same efficiency under the chemostat conditions as in the rich medium. Still in this case, as in the previous one, the cells had synthesized RNA that they could not use.
Time after addition of isopropylthiogalactoside (sec.)
FIG. 3. Kinetics of induction of 8-galactosidase synthesis in succinate-limited chemostat cultures of Eecherichia coli ML3. Essentially the same delay time is observed after inducer has been added and before detectable enzyme polypeptides are found as with the batch cultures described in Fig. 2. 0-0 indicates data from aculturewitha 7.0-hr.doubling time; 0-0, a 12.6-hr.doubling time; A-A, a 24-hr. doubling time.
Is this trivial or profound? Maybe there are inadequate control mechanisms present in the cells to shut off sufficiently r-RNA and t-RNA synthesis so that, when the growth rate is decreased, there is a build up of RNA. A major point of my theoretical paper (Koch, 1970) was that, in order that there be no extra RNA, the control would have to act by restricting bacteria growing with a 30-hr. doubling time to make RNA slower than bacteria growing with a 30-min doubling time by the Eiquare of the ratio of the growth rate constants, or 602 = 3600 times slower. Perhaps the control just cannot turn that far down because it is simply not that effective. It is well known that, in many cases of induced or
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repressed enzyme synthesis, significant amounts of enzyme arc produced in the repressed state. The basal level may be 3000 times lower than the fully derepressed level, as in the case of /3-galactosidase in E . coli strain ML. But this is a particularly favourable case and most other systems do not show this wide st dynamic response. Maalrae (1970) has argued that the ribosomal-RNA content of the cell may reflect concomitant
Tlme (min 1
FIG.4. Rates of iiucleic acid and protein synthesis after a “shift-up” of a 20-hr. sulphate-limitod chcrnostat culture of Eecherichia coli strain B U-. The enrichment medium contained glucose, sulphate, vitamins, and a mixture of amino acids, and yielded a doubling time of 30 min. The RNA pulses of 2 min. duration are shown with thin horizontal linos, and the protein-pulse incorporation data of 2 min. duration by thick horizontal lines. The thin curve was fitted to tho RNA data aa descrihod in Koch and Deppe (197 1 ) . The thick continuous h e is the theoretical curve for the specific rate of protein synthesis calculuted from tho cquatioii fitted to data for the specific rate of RNAsynthosis. It can bc concluded that new protoin synthosis capacity parallels now not RNA synthesis.
synthcsis of RNA to go with and be limited by ribosomal protein more than it reflects nucleic acid synthesis alone. At the present time, either hypothesis is tenable. At the teleological level, the “extra” RNA may represent an anticipatory response of the organism in the perennial hope that circumstances will improve. Below it is shown that this “extra” RNA has a great selective advantage in a fluctuating environment. Of course, this explanation would not work for the sulphur-limited cells, essentially sulphur-containing amino acid-limited cells, which make RNA that not only does not function under the growth condition but also does not
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appear to be able to function when the cells are put in a rich environment. On the presumption that the cell could have evolved control mechanisms so that exactly the minimum number of ribosomes would be made and that they could function with maximal efficiency to support any growth rate, what is the cost to an organism to produce the “extra” RNA? This cost should logically be expressed as the fractional decrease in the growth-rate constant or in the increase in the length of time to double. The cost of the “extra” RNA would be qualitatively different both for different growth-limiting conditions and €or different growth rates. It is usually found (see Koch, 1970) that the ratio of total RNA ( T )
Growth-rate constant ( A )
Frc:. 5. RNA contents of cells as a function of the growth-rate constant. See text for oxplnnet>ion.
to protein or to total cell dry weight (w) is a linear function of the growthrate constant, A. This is shown diagrammatically in Fig. 5 and mathematically as :
Equations have been developed (Koch, 1970), and are developed in another way in a later section of this review, to show that the efficiency with which the RNA of the cell is mobilized to produce cell constituents, designated by k,is equal to A(w)/(r). Therefore : k:=-
A a + bA
The constant-efficiency hypothesis of Maalee and Kjeldgaard ( 1966) corresponds t o (I = 0 and hence k = l / b and is shown as a Iine passing
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ARTHUR L. KOUH
through ( r ) / ( w= ) 0 when A = 0. On the other hand, if b is zero, ( r ) / ( w is ) independent of the efficiency, and k is directly proportional to A. This case corresponds to the horizontal straight line in Fig. 6. Actual values lie in between, and in many cases have the same value of ( r ) / ( wfor ) the same value of h no matter how achieved. This generalization, first proposed by Schaechter et al. ( 1958) is usually, but not universally, true. Let us assume in a particular case that the cell has to synthesize a fraction z of useless material for every unit amount of cellular dry weight. If it costs as much t o make any one type of cell constituent as another, this useless material decreases the growth rate from h to ( 1 - x)A; therefore, the slowing in growth rate is : 100
__
(1 - x
- loo)%
Consider very slowly growing cultures where ( r ) / w is 0.1 instead of essentially zero us expected for the constant-efficiency hypothesis. If unused RNA is all that is made in excess as might be the case for the
;;(
sulphate-limited chemostat cells, the slowing is
-
~
loo)% = 11%.
If unused ribosomal protein is also made, as in the carbon-limited chemoloo stat cells, the slowing is - 100 yo = 18*3y0. If the cell also makes
( ~
0.845
)
an equivalent amount of activating enzymes in the proportions listed in Table 1 (p. l50), then the slowing is
(i;i5lOO)y0 -___
-
=
30.7%.
Under phosphate limitation, the “extra” RNA synthesis presents a scvere cost to the organism. About 100 micromoles PO:-/g. dry weight of the cell is in DNA. Essential roles are also served for a like amount (150 micromoles/g. dry weight) of phospholipid, and 200 micromoles/g. dry weight are used for soluble cofactors and intermediates in the cell. Even a t zero growth rate, cells in a balanced carbon-limited culture would use 300 micromoles/g. dry weight for RNA. I n such a case, 40% of the ccll’s phosphate can be in the “extra” RNA. I n our hands, phosphatelimited E’. coli with a 22-hr. doubling time has essentially the same total RNA to dry weight ratio as glucose-limited cells (K. Bernstein arid A. L. Koch, unpublished observations). This means that “extra” RNA accounts for nearly half of the phosphorus-containing compoiinds in these growing cells. A cell growing but not producing “extra” RNA could therefore grow about twice as fast as our laboratory strain of Escherichia coli B U-in a phosphate-limited chemostat. Actually, from these compositional facts, mutants obeying the constant-efficiency hypothesis would grow times faster than the actual organisms. Consequently, in the phosphate-limited chemostat
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with a 22-hr. doubling time, such mutants should increase by a factor of 6.1 x lo6 relative t o the standard strain in 31 days. If there were initially one in lo6, by the end of this period, there should have been a significant drop in the RNA content. Such an experiment was performed (K. Bernstein and A. L. Koch, unpublished data) with strain B U-, and the composition of the cells either in the chemostat or when subcultured into medium supporting a 60-min. doubling time was the same as the initial strain. The conclusion t o be drawn is that such mutations, if they exist a t all, are rarer in our laboratory strains than about one per million. Before we discuss this further, it is necessary to show that the ((extra” RNA is not an artifact of the design and operation of our chemostats. Details of the chemostat which we employ are presented in the next section. This section is included not only for that reason, and because we feel that we have designed LL very easy-to-build useful apparatus of high versatility and flexibility which should make it much easier for chemostat cultures to be routine in microbial physiology, but also because it will make i t easier to consider some of the additional problems that the study of slowly growing cultures entails.
IV. Description and Operation of Chemostats A. DESIQN FEATURES Since the chemostat was invented (Novick and Szilard, 1950; Monod, 1950), almost as many types of apparatus have been constructed and made to function successfully as there have been workers in the field. For a number of years, I looked a t a possible number of designs and even contributed an idea for the design of one (Kubitschek, 1954). Most of them were messy, costly, difficult to sterilize, easily contaminated, and occupied bench space. However, over the last five years we have developed what we think is an extremely convenient and flexible unit which is shown in Fig. 6. It embodies modifications and suggestions of Richard Ecker, Penelope Gumapas Clark, Robert Coffman, and Thomas Norris. The most important feature of the apparatus is the mechanism to control flow rate. This is a pressure differential operating through a fixed resistance tubing. This approach has been chosen, instead of mechanical pumps and measuring devices, because the law of gravity is repealed less often than the law stating that electricity will continually come out of the plug in the laboratory wall. For the necessary flow rates, the resistance to flow need be fairly high, and short pieces of fine capillary sooner or later clog. Therefore we chose long lengths (10-20 ft.) of relatively large diameter Teflon tubing as the resistance to flow. Much of
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ARTHUR L. KOCH
the difficulty due to clogging is obviated since particulate material passes through and does not collect on the Teflon eventually to cause occlusion of the tubing. With true American ingenuity, the apparatns is designed to be assembly-line produced with interchangeable parts, so that growth chambers of different size, different flow rate tubings, and different reservoir bottles can be put together from previously sterilized parts kept available mi the shelf. Figure 0 is largely self explanatory. Further
Ion resistance tubing Air filter
Stainless steel weight attached to shuker clamp
Fra. 0. Uoeigii of chomostnts usc:d in my Inbortttory.
details will be made availablc to tliosc requesting them and are inclutled in Norris (1970). There are, however, two features requiring further comment here. One is the levelling or overflow device. First, it is bent away from the point of entry of new media in order to minimize the possibility of removing the freshly added medium before it becomes well-mixed with the entire contents of the growth chamber. Secondly, because mixing is effected by air bubbles, there is an accumulation of foam a t the surface. Bacteriacongregateat air-waterinterfaces, and therefore these organisms are concentrated in the froth. This means that, for very slow growth in a chemostat where a very large volume of air is
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passed through the culture per generation, the effluent has the expected concentration of organisms, but, the growth chamber itself has a lower cell density. Under these circumstances, the doubling time is much shorter than that calculated from the flow rate and the growth chamber volume. We eliminated the problem by blowing a 2 mm. diameter hole 1.5 cm. above the end of the levelling device. I n operation, when the fluid level rises so that a film of a surface bubble occludes this hole, the vacuum system sucks bubble-free fluid from the bottom of the tube. The second special feature of the apparatus is the length of Teflon tubing between the growth chamber and the collection vessel. The large overflow vessels are not sterilized because we thought that no micro-organism would be able to make its way against the flow of air and contaminate the culture. However, motile contaminants do swim or crawl upstream and eventually bake over the chemostat (K. Bernstein, unpublished observations). Insertion of a length of Teflon tubing has completely eliminated this problem, presumably because the organisms cannot hold on to this plastic. Before speculating on the advantage that motility gives an organism under certain growth conditions, it is to be noted that inspection of living cultures in the phase-contrast microscope for very rare motile contaminants of our laboratory strains (generally non-motile) is an easy routine way to check for contamination. Although the Teflon resistance tubing works very well and is highly reliable, sometimes it is desirable for short-term experiments to be able to vary the flow rate over a range not accessible by varying the hydrostatic head. For this reason, we also use a peristaltic pump of variable and well controlled speed. This too can be used interchangeably with the other items.
B. EVIDENCE THAT THE “EXTRA” RNA IS NOT A N ARTIFACTDUE TO INADEQUATE MIXINGOF THE CHEMOSTAT Every time a drop falls into the one-litre chemostat operated as indicated above, the limiting nutrient must be shared among the 2-3 x 10’ inhabitants. To assure that each individual receives his fair share is a Herculean task and requires a Christ-like distribution system. The chemostats as we operate them take a second or two for a drop of ink to become apparently uniformly dispersed throughout, but the eye is a crude instrument for making this assessment because it tells about largescale mixing and not about the mixing at the size-scale of an individual bacterium. The aerator has been designed to form large bubbles that give mass fluid movements so that there will be no backwaters where the organisms would be starved for a substantial period of time, and prob-
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ARTHUR L. KOOH
ably the mixing time on the microscopic level cannot be much longer than 10sec. If mixing were instantaneous, every time a drop of about 0.023 ml. entered thechemostat, the concentration ofthe limiting nubrient would be increased by 2.3 x times the concentration of the reservoir solutions. When glucose (0.02%) is the limiting nutrient as in most of our x 1110 p M = 0.0255 experiments, the increment would be 2.3 x p M . The average concentration in the chemostat culture can be calculated from the formula presented first by Novick and Szilard (1950). In the steady state, the growth-rate constant, A, must equal the washout rate = w / V ,where w is the flow rate and V is the volume of the chemostat, and the actual substrate concentration in the chemostat must satisfy :
At slow dilution rates, the substrate concentrations will be well below the K value for the uptake process (20 p M for glucose), so that the growth rate constant is given by :
For a typical example of our operation, Amax. is equal to 0.693/50 min. to a, doubling time of 60 min. in ordinary batch cultures. When 20-hr. chemostats are considered, A = 0.693/20 hr. = 0.00068/min., and the average substrate concentration turns out to be 0-83p M . We can now turn the calculations around and ask how long it would take the cclls to consume 0.83 p M glucose, if no more were added. The rate a t which nutrient is consumed is AS. Therefore : = 04139/min. corresponding
hs=
0,693 x 1110 p M = 0.641 PM 20 x 60 min. min .
Comparing this with the steady-state concentration of 0.83 p M it follows that the half time for the consumption of residual glucose, if flow were stopped, is : 0-693 0.83 p M 0 6 4 1 micromoles/min. ~
= 0.90
min. = 54.1 sec.
Even though drops fall every 2.4 sec., there will be a significant change during this time. Assuming first order uptake, we can calculate that the concentration range is 2*9y0,with a standard deviation of about 1%. Even if mixing is not instantaneous, the given curves are still correct if the glucose is delivered quickly to a fraction of the total bacteria. This is because first-order utilization of glucose will occur at
THE ADAPTIVE RESPONSES OF ESCEERZCEIA COLZ
163
even a 20 times higher rate than this average concentration because
R = 20pM. Even a 3% fluctuation in external glucose concentration greatly exaggerates the instantaneous variability experienced by the organisms,
Time ( m i n )
FIG. 7. 8-Galactosidase synthesis in a glucose-limit,ed chemostat culturo of E8cherichin coli I3 U- starved of a carbon source. The horizontal lines show the amount of enzyme producod in 30-min. induction periods at 37"starting at various times aftm cells were removed from a glucose-limited chemostat culture. Basal levels measured at times indicated are shown by circles. Induction rates corrected for basal level are shown by the vertical bars which span one standard deviation above and ono below the mean. The solid line was computed by the least squares procedures, and its slope corresponds to a half life of 15.2 min. This lino has been offset in such a way that it would havo an intorcopt equal to the amount of enzyme that would actually be produced if the induction had taken place in the chemostat in 30 min.
because the cell is further buffered by the pools of intermediates formed from the exogenously supplied limiting nutrient. But the half time calculated above, 54.1 sec., is not sufficiently long compared with the mixing speed to remove the faint suspicion that, even in our chemostats, cells may be subject t o alternate feast or famine. There are three kinds of evidence which we can muster against this allegation. First, we added
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ARTHUR L. KOUH
a magnetic stirrcr inside the chemostat which greatly increased the effectiveness of the mixing (note that this stirring action produces a mixing at right angles to that produced by the bubbles) and found that values for ( r ) / ( w )and the glucose yield remained the same with and without the extra mixing (Norris, 1970). The second test is to measure uptake of isotope into nucleic acids and into protein in samples taken from chemostats which have been given the isotopic compound, but none of the growth-limiting substance. I n these experiments, uptake is linear for several minutes at the same rate as whcn the isotope is added to the chemostat. Thus the organisms maintain enough reserves to buffer themselves over a much larger period than the mixing time in the chemostat. The third test is to take cells from the carbon-limited chemostat and add isopropylthiogalactoside to induce the cells to form pgalactosidase in the absence of any additional carbon source. Figure 7 shows the amount of enzyme found after 30-min. induction periods spanning different time intervals after the cells are no longer continuously supplied with glucose. Enzyme production decreases quickly by a factor two in less than three minutes from the rate occurring in the 10-hr. chemostut and then declines with a half life of 15.2 min. (T. Alton and A. L. Korh, unpublished observations). This second phase represents utilization of a limited class of the continuously turning over “rapidly degrading proteins” (Nath and Koch, 1970) but also guarantees tho cells continuoiw metabolism over times very long compared with the mixing time.
V. RNA Synthesis in Slowly Growing Bacteria I n the previous sections of this review, we havc been forced to accept the idea that slowly growing bacteria make “extra” RNA which, under some circumstances at least, is present in cells in a functional or nearly functional state. It is clear that this “extra” RNA is not accounted for as material in dead cells or even in temporarily inactive cells in the population. It is not functioning to make proteins that are broken down, nor is it functioning under conditions where it takes a longer step time to add an amino acid to a growing peptide chain. Below it is shown that there is a high selective advantage to the cell in having unused proteinsynthesizing machinery ready to function. This is because it decreases the lag phase when the opportunity to grow rapidly occurs. I n this section, I consider what is known about the control of the rate and nature of the synthesis in E . coli under starvation and during slow continuous culture conditions. I n part, this is an oft reviewed topic, but now there is very clear agreement on certain issues. For cxample, there is general agreement that m-RNA synthesis continues during amino-acid starvation of stringent
THE ADAPTIVE RESPONSES OF ESCHERICAIA COLI
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E. coli (see Edlin and Broda, 1968, and Geiduschek and Haselkorn, 1969, for recent reviews). Stringent organisms under amino-acid starvation accumulate little RNA, although they are capable of synthesis of RNA when challenged with a protein inhibitor such as chloramphenicol. This fact has been known and exploited for a decade (Kurland and Maalere, 1962). Recently, Nierlich (1968) and Winslow and Lazzarini (1969b) have shown that the rate of total RNA synthesis on amino-acid starvation is not decreased nearly as much as the apparent net rate. From the discussion of tracer kinetics below, this is perfectly understandable in terms of the net synthesis theorem. I n both laboratories, the total rate of RNA synthesis was computed by isolating and measuring the specific activity of the triphosphate precursors of nucleic acids. When the work of Nierlich is corrected for the temperature difference of his control and experimental cultures (according t o the temperature dependence that he measured (D. P. Nierlich, personal communication)), a two-fold inhibition is observed. Winslow and Lazzarini found that, after an initially larger inhibition, the total rate of RNA synthesis is decreased three- to four-fold on amino-acid starvation. We have recently performed a different kind of experiment to get a t the same type of information (B. Dancis, unpublished observations). Cells of E . coli were deprived of required amino acids and were allowed to take up high specific activity 32PO:- for 1 min. Further uptake of label was then prevented by a greater than lo4-fold dilution with 3'PO:-. The culture was divided into two portions, and the amino acids were restored to one. It was found that the isotope present in the metabolic pool a t the end of the 1-min. uptake proceeded into nucleic acid a t the same rate for the next 4 min., identically in both cultures, Since the specific activity of the precursor pool was initially identical it follows that the total rate of RNA synthesis of all species is the same (*10%) during amino-acid starvation as during growth. The discrepancy between our finding no decrease in total synthesis and Nierlich's two-fold or Winslow and Lazzarini's three-fold decrease has elicited criticism and self-criticism on the part of the various workers. Certainly our basic experiment is readily repeatable, but it leads to an unambiguous interpretation only for times short compared with the life of messenger-RNA. For purposes of completing this section, we will assume that the total rate of RNA synthesis is not decreased on amino-acid starvation, although this is certainly not fully proven a t this time. With this hope, we shall explore the consequence of the hypothesis that amino-acid starvation does not affect RNA synthesis at all, i.e. r-RNA and nascent t-RNA are made normally and then broken down. It would then be necessary to assume further that, in relaxed organisms or in the presence of agents like chloramphenicol, the degradation is less
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ARTHUR L. KOOH
severe and particles with a larger proportion of r-RNA accumulate. They might be stabilized by direct binding with other cell proteins, with ribosomal protein removed from pre-existing ribosomes, or with the drugs themselves. There is another reason to believe that degradation of r-RNA is an important process under many circumstances. This is because Friesen (1966) and Stubbs and Hall (1968) found that essentially the same proportion of pulse-labelled radioactivity hybridized as messenger during growth as during starvation of amino acids of stringent organisms. Friesen’s conclusion that there was co-ordinate control of several types of RNA synthesis was disputed by other workers. Here we suggest that synthesis is uncontrolled (and therefore incidentally co-ordinate), but that further control is extended through degradation. The idea that ribosome synthesis might be controlled in such a way that r-RNA would be synthesized in excess and then some degraded was first proposed by Rosset et al. (1966) and documented by them in the case of phosphate starvation (Julien el al.) in 1968. Control a t the level of degradation was also proposed by Ehrenfeld and Koch (1968), who suggested that it might not be trivial but rather a normal biological control mechanism. It was found that penicillin-induced sphaeroplast preparations of E . coli in a rich adequate medium can synthesize macromolecules and eventually achieve a state where they synthesize ribosomal subunits of almost exactly the size of functional subunits and then degrade a t least the RNA down to the acid-soluble level. If this hypothesis is correct, studies on relaxed mutants can be used to investigate more directly control of synthesis because, in these cases, RNA is made and degraded more slowly. Neidhardt (1963) observed that, although these organisms produce an excess of RNA during aminoacid limitation, they have the same amount of RNA per unit amount of protein when grown in the chemostat whenever cell growth is limited by some factor other than availability of an amino acid. We not only confirmed his findings (Clark, 1967) but extended this observation to very slowly growing cultures. It was found that relaxed cells in slowly growing chemostat culture limited by an amino acid produce an extremely large amount of RNA, up to 16 times the amount associated with either stringent organisms limited on the same medium or the relaxed organisms limited by a carbon source. Most of the excess RNA was released from viable cells and found in apartially degraded state in the growth medium, while the cells themselves had almost a normal amount of RNA and a normal sucrose density-gradient profile. It must be pointed out, however, that these observations were made after only two doublings in the chemostat. The experiments could not be prolonged because of reversion of the amino-acid mutants.
THE ADAPTIVE RESPONSES OF E S C H E R I C H I A COLI
167
Together, these findings could easily be consistent with the hypothesis that the wild-type cell a t slow growth rates makes r-RNA, but immediately degrades and recycles the nucleotides. On the other hand, the bulk of this RNA is not recycled as rapidly during amino-acid starvation or in chemostat amino acid-limited cultures of relaxed organisms. It is also not recycled as rapidly in any of the cases of abnormal particle accumulation. I n the rest of this section, I wish to re-examine the rate of m-RNA, r-RNA, and t-RNA synthesis as a function of growth rate in stringent organisms in balanced growth. It is felt that these rates too are substantially independent of the growth-rate constant. For m-RNA synthesis this conclusion follows when we combine the data which show that the content of m-RNA is nearly the same in bacteria growing in balanced batch or chemostat cultures a t any growth rate with the observation that the half-life of messenger is the same a t all growth rates. Forchhammer and Kjeldgaard (1968) showed that the ratio m-RNA to r-RNA in batch-grown cells of E . coli was constant. They measured the m-RNA by its ability to stimulate amino-acid incorporation in a messenger-depleted Nierenberg system. I n extension, Norris and Koch ( 1972) have found that 3% of the RNA of bacteria growing with doubling times ranging from 60 min. to 10 hr. hybridizes as m-RNA. Until recently, there had been some speculation but very little data in the literature about the half life of m-RNA in slowly growing bacteria. Norris (1970) measured the life of the lactose-operon message as a function of growth rate with a modification of method developed by Kepes (1963). Cells in balanced growth were induced with 5 x lop4 M-isopropylthiogalactoside for 20 sec. The culture was rapidly filtered and resuspended in medium lacking inducer ; this prevents further initiation events. Samples were diluted into chloramphenicol-containing medium a t the indicated times (Fig. 8). The relevant time t o be extracted from such data is the average life span; this is the time when the rate of polypeptide completion is half maximal, i.e. when the tangent to the curve is 0.5 of the tangent a t the steepest point. This time is about 200 sec. and has a scatter of 20 sec. regardless of doubling time of the culture. In a growing culture, the total rate of synthesis of m-RNA is only slightly greater than the rate of degradation arid we can treat them as essentially equal. The rate of degradation is the product of the concentration of m-RNA and the degradation rate-constant (the latter is equal t o 0.693 divided by the apparent half-life of the message). Even if the apparent half-lives of all messenger molecules are not the same, if each class is like the lactose messenger in that its degradation is independent of the growth rate-constant of the culture, we can infer that the rate of synthesis of all m-RNA is proportional to the content of m-RNA or 7
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ARTHUR L. KOUH
r-RNA. This in turn varies at most several fold per unit of dry weight as shown in the middle line in Fig. 5 (p. 157). It follows, then, that the total rate of messenger synthesis per genome varies no more than this same several-fold over conditions where the value of the growth rate-constant varies from zero to the maximal value in glucose-containing media.
0
I
0
120
I
I
240
I
I
I
360
I
480
I
600
''d00
Time (sec.)
FIG.8. The life span of the /I-galactosidase message is independent of growth rate. The ordinate is p-galactosidaae activity expressed as percent of the maximum value resulting from a 20-sec. pulse induction of Eecherichia co2i strain B U-. The abscissa is time after addition of inducer. The average life span can be derived from these data (see text). o indicates data for glucose-containing batch cultures (1-hr. doubling time), for glycerol-containing batch cultures (70-min. doubling time), A for cells grown in a glucose-containing chemostat which were resuspended in 0.029% glucose plus 10 pg. uracil/ml. (10-hr. doubling time), A for cells grown in a glucose-containing chemostat and resuspended in M-B after filtration (10-hr. doubling time), and x for DL-alanine-containing batch cultures (6-hr. doubling time). The maximum /I-galtlctosidase activities in absolute units were 17.67, 166.17, 48.43, 48.43, and 224-66 micromoles o-nitrophenyl galactoside/g. min., respectively.
Reasons for suggesting that r-RNA is synthesized and then degraded in slowly growing carbon-limited chemostat cultures at some stage in ribosome maturation can be drawn from the data in Table 2 (Norris and Koch, 1972). Cultures of E . cola strain B U- in balanced growth with various doubling times were pulse labelled for 30 sec. and the RNA from these cultures was then hybridized to DNA embedded in membrane filters. Three methods were used to determine the fraction of the label hybridizing as message. I n the first, increasing proportions of pulselabelled RNA was used to titrate the E . coli genome according to the method of Kennel1 (1968). In this method, plateaux appear when suffi-
169
THE ADAPTIVE RESPONSES OF ESCHERICHIA COLI
cient copies of the redundant stable species of RNA are present in the sample t o saturate corresponding DNA sites. In the other two methods, the RNA was hybridized with a very large excess of DNA, and the rRNA and t-RNA prevented from hybridizing by competing nonradioactive r-RNA and t-RNA. No matter whether the competing RNA is mixed with the radioactive RNA or pre-hybridized, the same decrease in radioactivity hybridizing is observed. TABLE2. Relative R a t e s of Synthesis of Messenger-RNA aa Compared with Total R N A as a Function of Growth R a t e i n Escherichia coli Amount of messenger-RNA
Doubling time
Ti trstion
(hr.)
%
1 5
10
Batch Chemostat Chemostat
55
62 70
Simultaneous competition % f S.D. 53.8 f 1.6 -
-
Pre-competition yo f S.D. 57.1 f 1-6 65.0 f 1.6 73.2 f 1-7
[3H]- or ["C-Guanine waa used to label RNA in cells from a culture growing at the indicated doubling times. The RNA was isolated and hybridized t o DNA-bearing filters. Since the rate of the hybridization reaction is dependent on the concentration of complementary sequences of RNA and DNA, all experiments were performed at sufFiciently high RNA concentrations, a t a given RNA to DNA ratio, t o yield maximal hybridization of the radioactive RNA. The competition experiments used 0.25 pg labelled RNA and 500 pg DNA. Control experiments indicated that, if the competing t-RNA and r-RNA were present at 10 and 20 pg or higher respectively in 1.2 ml. of reaction mixture, the amount of labelled RNA hybridizing did not change. Hybridizations were carried out in 6X SSC buffer for 24 hr. at 70" and were treated with RNAse before counting aa described by Kennel1 (1968). Data taken from Norris and Koch (1972).
It can be seen that, by these methods, there is only a slight increase in the proportion of m-RNA to the total pulse-labelled RNA as the growth rate is decreased 10-fold. Since, as indicated above, the rate of synthesis of m-RNA is similar a t various growth rates, the rate of synthesis of the so-called stable forms of RNA a t various growth rates must be similar too. Therefore, in very slowly growing cultures where only a slow net RNA synthesis can take place, this must be much smaller than the total rate of synthesis of what are usually termed stable species of RNA.
VI. Tracer Kinetics Interlude Although isotope methods have been fundamental to the development of microbial physiology, there have been enough misconceptions and
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inappropriate interpretations of uptake and turnover data that I include this section in the hope that it will help the reader to make better use of his own data and also to understand the experimental results given below. When an isotopic compound is given to a metabolizing system, such as a growing bacterial cell, it will be taken up and distributed in a way that depends on the amounts of the various components in the system and on the velocities of a number of physiological processes. Usually, experiments are carried out to follow the appearance of radioactivity with time in a single chemical component or a single combination of chemical components such as the total trichloroacetic acid-insoluble material. The mathematical formula for the radioactivity in both cases is made up of a sum of exponential terms. From the exponents, estimates of the apparent half lives of the total system can be obtained. I n fact, this collection of apparent half lives is the only kind of information that is obtained. No additional information can be gained by varying the experiment with a different isotope administration schedule ; no matter how the experimental procedure is varied, whether the experiment is done by supplying the isotope continuously or as a brief pulse, the same apparent half lives are involved. If the system is complicated and is composed of many reactions, there are a number of apparent half lives. But I stress that the half lives belong to the system, not necessarily t o the components measured. It is true that the accuracy with which one can measure different apparent half lives depends on which chemical component is measured and how the tracer is administered. Sometimes one method of labelling is preferable to another but, in principle, they all give the same kinds of information. I n order t o obtain the true half life of a component from tracer data alone, even when timed samples of it have been isolated and counted, the specific activities of the component’s immediate precursor as well as those of the component must be known as a function of time. Usually, the experimenter is interested in the true half life of the component actually measured and not in the more general properties of the system, because the true half life tells him how fast the components of interest are actually made and/or broken down. The problem of tracer kinetics is to relate the true half lives to the apparent values. These general comments can be made clearer by a few examples of metabolic systems. The simplest metabolic system can be represented : Vb
AAB+C
vc
Scheme I
All we mean by this scheme is that B is made from A at a velocity Vb, and that B undergoes further changes t o yield products that never
THE ADAPTIVE RESPONSES OF ESCEERICHIA COLI
171
return to either A or B. When the biological system is such that the velocity of synthesis of B, v b , is constant and also equal to the rate of further metabolism, V,, the system is said to be in a “Dynamic State” (Schoenheimer, 1942). In this case, the amount of component B, designated by a lower case b, must be constant. If initially B is quickly labelled by supplying a brief pulse of radioactive A , and after the incorporation phase is completed the loss of radioactivity in B followed, the specific activity will decrease according to :
B = B,e-vb‘/b
(5)
where B, is the specific radioactivity a t a time we designate as t = 0, which we could choose as any time after the radioactivity in A has again become zero. The other labelling schedule that is used to do such experiments is to raise the specific radioactivity of A suddenly from a value of zero to a value of A , and then keep it constant at that value. Then the specific activity of B at any time, t , after the discontinuous step increase in precursor specific activity is given by :
B = A,( l-eYb‘lb)
(6)
I n both of these cases, if we measure B a t various values oft, the kinetic analysis yields information about V b / b .In this simple system of Scheme I, the true half life of component B is obtained in either of these ways of labelling and is 0.693/ V J b . I n fact, the term half life got its introduction into biochemistry from this kind of pulse labelling procedure and is t*he time for B t o become 0.5 B,. Substituting B = 0.5 B, into Equation 5 and taking natural logarithms of both sides leads to In (0.5 B,/B,) = - Vbto.,b. This in turn leads to lo.5 = 0 * 6 9 3 / V b / b . Many metabolic systems of interest are such that the scheme must be made more complicated by the addition of an intermediate pool : vb
A + X a B
1
Scheme I1
v-b
Frequently this intermediate pool contains only a small amount of material compared to the rest of the system, and for that reason the pool turns over rapidly. For the limiting case of an infinitesimal pool, the mathematical problem posed by Scheme I1 was solved (Koch, 1962) with just a little more algebra than in the previous case. Only one apparent halflife is involved in the solutions. This is surprising until the details of the differential equations that have been set up are examined, but the shape of the curves for the specific activity in B versus time for either
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ARTHUR L. KOUH
isotope administration schedule is precisely like those predicted by Equations (1)and (2). The curves or appropriate plots on semilogarithmic paper give a straight line, from which a single half-time can be extracted. But for this metabolic scheme it is not a true half life and does not correspond to 0.693/Vb/b. Rather it turns out to be the true half life divided by vx
(V, + V-b) Scheme I1:
where the new velocities are designated in a modification of
Although two new parameters are involved in the metabolic scheme,
V-b and V-,, they can be dissected only with the aid of additional kinds of experimental data-not, of course, by doing more experiments in which the isotope content of component B alone is measured. I have designated the ratio of the true half life to the apparent as the “recycling factor”. If the recycling factor is small, because breakdown and resynthesis of B are fast, B does not appear to turn over as rapidly as it is actually formed and broken down. I n this situation a molecule of B breaks down to X and then the resultant pieces are likely to be reassembled into new molecules of B. The recycling factor is the ratio of the velocity of formation of the pool from the exogenous source t o the velocity of formation from all sources. If V , is zero, this recycling process can take place indefinitely and the specific activity of B would not change, no matter how fast B is synthesized and degraded. Metabolic schemes can be amplified and made more complicated with more intermediate and metabolic processes to correspond more closely to the real biological cases. The systems so far discussed were in a “Dynamic State”, but we can also handle exponential growth or “unbalanced” states of growth. As the schemes and growth pattern become more complicated, the uptake-time courses also become more complicated, and it becomes more difficult to fit apparent half-life parameters to the curve. But, after fitting, all the information that can be extracted is a series of apparent half lives which are functions of a number of reaction velocities of the metabolic system and the amounts of certain of the cell components. I n fact, the metabolic properties of a component chemically isolated and measured may be masked by a component in rapid isotopic equilibrium with it. As a case in point, consider RNA metabolism in E . coli, idealized as follows :
”HE ADAPTIVE RESPONSES OF ESCHERICHIA COW
173
111
T
In this scheme, P stands for “pool” (the equivalent of X in Scheme 11). While there may be many components in this pool, if they are all rapidly interconvertible, we can treat them as a single larger pool. We have also used E (for “eosome”) to be an intermediary stage in ribosome production (McCarthy el al., 1962).There appear to be several intermediate states in ribosomeproduction (seeSchlessinger and Apirion, 1969),but aseachturns over rapidly under ordinary growth conditions, they will all have nearly the same specific activities and also may be treated together. Whether components can be treated together or not depends on the time scale of the experiment. For short-term experiments where the specific activities of individual components are measured separately, the half times of all of the isolated stages of ribosome maturation could be worked out. But, as pointed out above, it would require the specific activity of precursor and product as a function of time to compute the true turnover characteristics of each component. On the other hand, for most of the applications to be made here, where the data are measurements of total radioactivity in trichloroacetic acid-insoluble material with no distinction between m-RNA, t-RNA, and r-RNA, considering even one intermediate is superfluous, as is shown below. For Scheme 111, differential equations have been written and solved for the case of agrowing bacterial culture (Koch, 1968). Into the resultant equations, assumed values for the composition of the cells and velocity of synthesis of messenger and the radioactivity-time curves for the different cell components were computed on a desk calculator. It should be noted that information about the apparent half life of messenger can be gleaned from the specific activity measurements of the messenger fraction as shown in the specific activity curves of Fig. 9. Similarly, a term involving the apparent half life of messenger, M , dominates the specific activity of the pool, P.Since this pool is the source for eosomal material, a term in the equation involving the apparent turnover rateconstant of messenger appears in the equation for eosomal material. It turns out that this term is more important than the term having to do with eosomal turnover per se. Therefore, the identical apparent rate
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ARTHUR L. KOOR
constant can be inferred from the data for either messenger pool or the eosome material. For the chosen parameters in Figs. 9 and 10, the apparent half life of messenger is 8.4 times longer than the true half life because of recycling factors. The recycling factor for messenger turnover is identical to the recycling factor for the previous case. Let me make this clear. By looking at the radioactivity in eosomal material, one learns almost nothing about eosome biosynthesis but a great deal about the combined process of the turnover and recycling of messenger. On the
Time (in units of a doubling time)
FIU.0. Plots of specific activity v e r m time for the pool and different classes of RNA in bacteria. These curves are calculated for the theoretical case of an infinitesimal pool, P. The amounts of RNA assumed to be in the various oompartments are: pool = 0.01, m-RNA = 0.08, eosomal-RNA = 0.02, t-RNA = 0.16, andr-RNA = 0.76. It is also assumed that the rate of synthesis of m-RNA is 10 times the rate of ribosome formation. For comparison, the simple turnover curve, which every component would follow if there were no pools and no breakdown, is shown by the dotted line. Calculation taken from Koch (1068).
other hand, the equations show that, when the specific activities of t-RNA or of r-RNA, which are stable end products of biosynthesis, become one half the final value, the time is nearly equal to the doubling time of bacteria. Therefore, little additional information can be gathered from measuring the specific radioactivity of these latter components. In the plot shown in Fig. 10 (total radioactivity ver8u8 bacterial growth) different information is apparent. Although the curves for each component are quite complicated, the striking point is that the sum for all species of nucleic acids is mathematically quite simple and this sum, increases linearly with bacterial growth. Apparently much information has been lost by this grouping together of all of the nucleic acids. The
x,
175
THE ADAPTIVE RESPONSHS OF ESCHERICEIA COLI
slope of the line of the sum of total radioactivity in all species is a measure of net rate of RNA synthesis (anabolic minus catabolic) and is completely independent of the total or anabolic rate of RNA synthesis. Of course, this must be true after extensive growth when all cell components have the same specific activity and new radioactivity in the cells corresponds to a net increase in the amount of radioactivity. It can also be true a t shorter times when messenger is still increasing in specific activity. This can be seen from the following argument. The exogenous isotopic
Messenger -RNA
0
10 Bacterial growth
2.0
3
(%= e“’-1)
FIG.10. Plots of total activity w e r w bacterial growth. The same hypothetical example at3 in Fig. 9 is here shown on a different basis. The ordinate is the product of specific radioactivity shown in Fig. 9 and the amount of the component at each particular time. However, instead of the results being plotted against time, they are plotted against the fractional new growth since the start of the experiment. This plot against growth as abscissa is called 8 “Monod” plot and corrects for the exponential character of growth. Calculation taken from Koch (1968).
compound enters the cell, mixes with the pool and proceeds into nucleic acids. If the pool has a constant size, then the rate of entry from the outside must equal the net rate of removal. The rate of entry is a measure of net synthesis because new building blocks are not needed for the recycling synthesis. If the pool is very small, even at very early times after the isotope compound is supplied to the culture medium, a negligible fraction of the isotope taken up by the cell will be free in the pool and therefore unincorporated into macromolecular trichloroacetic acidinsoluble material. Consequently, the linear uptake of isotope into the
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ARTHUR L. KOOH
cell with growth will lead to an almost linear increase in the measured quantity of the total isotope incorporation into macromolecular linkage. Comparing Figs. 9 and 10 we find a paradoxical situation. The former shows that the rapidly turning-over components quickly acquire high specific activities. For the example chosen, even after a time period as long as one-tenth of a generation, only 76% of the total radioactivity in the trichloroacetic acid-insoluble form in the cell is in stable RNA. But Fig. 10 shows that no information about the rate of synthesis or the half life of these rapidly labelled components is obtained from total activity data. Rather, total radioactivity measures largely the less rapidly labelled stable component, since synthesis of r- and t-RNA accounts for the majority of the net increase; message level hardly contributes a t all. This conclusion that net synthesis alone may be measured by total activity measurements even at early times after isotope administration has been called the “net synthesis theorem’’ (Koch, 1971). It was first pointed out by Nierlich (1967) and analysed kinetically by Koch (1968). The conditions that must be met for an incorporation experiment t o measure purely net synthesis, even a t very short times, are : (1) the amount of material in the intermediate pool must be very small compared with cell macromolecules ; (2) the size of this pool must not change during the measurement ;
( 3 ) the pool must be formed only from the external source and from breakdown of macromolecular components ; (4) the velocities of all processes must not be changing during the
experiment ;and (6) the pool must lead to the measured macromolecules and not to other metabolic products. An indication that measurements at early times yield correct assessments of net rate is the identity of the early time rate with the differential
rate obtained after extensive growth when the theorem must hold of necessity, whether or not conditions 1-6 are met. Such identity is not absolute proof because, if the pool turn-over is responsive to factors other than those assumed above, such as exchange with the medium or exchange with other components not measured together with the macromolecular components, the recycling of messenger through the pool would be largely uncoupled. This uncoupling would lead to an early increase in radioactivity in the messenger fraction (Koch, 1968) that could under certain circumstances compensate quite precisely for the lag due to the pool itself. Recently it has been necessary to extend the kinetic analysis because our experimental studies have shown that the initial rates of incorpora-
outside
inside M
J
R
Scheme IV
A set of differential equations was set up for this scheme for the case of balanced growth, and these equations were solved exactly with no approximations. Calculations are shown in Fig. 11 for a culture of E . coli growing with a 60-minute doubling time, based on a set of parameters consistent with the known amounts of m-RNA, t-RNA, r-RNA, and precursor pool material as well as an average of the literature estimates of the velocity of messenger synthesis. It can be seen that the radioactivity incorporated into total nucleic acids as a function of time is dependent upon the extent of exchange with the outside medium. If there is no exchange but only entry, the incorporation curve extrapolates to the ordinate at -0.03, corresponding t o the negative of the amount of soluble pool. Another way to express this
’
is to say there will be a lag of - = (0’03)(60) = 2.6 min. which is the V 0*693(1-03] length of time it takes for the synthesis of total cell material from the material in the pool. At the other extreme, if there is an infinitely fast exchange process, the incorporation curve extrapolates to +0.03, corresponding to the size of the messenger pool. This is equivalent to a “negative” lag of 2.6 min. In this fast exchange case, the messenger turns over quickly and exhibits its true turnover rate, i.e. there is no need for a recycling factor correction. Of course, intermediate rates of exchange give intermediate lags. To account for the very short lags, such as those observed with [14C]guanine (e.g. Koch, 1965), the velocity of
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exchange must be about equal to the velocity of net synthesis. A shorter lag than expected from the amount of material in the pool was first noted by Britten and McCarthy (1962), who postulated a smaller bypass pool leading directly from exogenous uptake into nucleic acid. Now we can see that the fact that there exists both a messenger fraction
0
0.05
o,io
o 1'5
Relative growth ( NO
0.20
0.25
1
FIG.11. "Monod" plot of total RNA for baateria growing at moderate rates. The plot is similar to that of Fig. 10. However, in this plot, turnover of the pool is no longer neglected. Furthermore, a set of parameters corresponding to those measured for Escheriohia ooli growing with a 60-min. doubling time waa chosen. In particular, it is assumed that the rate of messenger synthesis is 60% of the total rate of nucleic acid synthesis. The lag before a steady differential rate is established varies from -2.6 to +2.6 min. depending on the magnitude of the exchange process. It was assumod that the amount of RNA in the pool is 0.03 and the amount of m-RNA is 0.03.
and an exchange process accounts for the observation that the lag is shorter than predicted from the size of the pool. We have seen in the previous section that it is quite likely that the rate of messenger synthesis is not greatly decreased in slowly growing bacteria. It also appears that the amount and average life of messenger do not ohange very muoh with ohanges in the growth rate constant. On this basis, the calculations were repeated for a hypothetioal culture
179
THE ADAPTNE RESPONSES OF ESCEERICEIA COW
growing ten times more slowly but with the same rate of messenger synthesis, and the same size pool of soluble material and messenger nucleic acid. These computed results are shown in Fig. 12. The important conclusions that can be drawn by comparing Fig. 11 and Fig. 12 are
5 Time ( m i d
FIG.12. “Monod” plot for a slowly growing culture. The data in this plot indicate a doubling time 10 times longer than that in Fig. 11. The velocity of messenger synthesis is 10 times the net rate of synthesis so that the former retains the absolute value shown in Fig. 11. The abscissa is time, not the relative growth of a true “Monod” plot. However, over the range of time displayed, the amount of new growth is roughly proportional to time. The numbers on the curves indicate values for V,/Vm.
that a small exchange velocity causes much more deviation from the predictions of the net synthesis theorem in a slowly growing culture than in a rapidly growing culture, and initial isotope incorporation when net synthesis is slow may be very much larger than the net rate. This is exactly what is seen in slowly growing chemostat cultures, as shown in
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ARTHUR L. ROOH
Fig. 13. In this experiment [8-14C]guaninewas added to a glucoselimited chemostat with a doubling time of 12 hr. at a final concentration of 6 p M . The incorporated radioactivity at various times up to 5 hr. was expressed in units of micromoles x lo4 per g. dry weight. To correct for the exponential growth character, a modified Monod plot was constructed by multiplying these values by eAcand then plotting these products against the fractional increase in growth that would have been observed in a batch oulture of cells growing at these rates. It can be seen that there is an initial overshoot in isotope incorporation, suggesting
FIG.13. Inoorporation of guanine by chemostat cultures. See text for explanation.
some exchange. The resultant slope is the differential rate, which for the dashed line is 20.2 x micromoles/g. These data are taken from Norris (1970), where it is calculated that there are 10.6 x lo4micromoles of guanine in the RNA of one gram dry weight of E. coli growing at this rate. The discrepancy is fully accounted for by two factors. First, guanine at this concentration is converted to adenine. The specific activity of adenine is 0.6 of that of the guanine in the nucleic acid after 2 min. incorporation and also 0.6 after 6 hr. Secondly, there is a small contribution due to radioactivity in the DNA. The ratio of slope to intercept is 0.042 although the extrapolation only gives a very approximate estimate of the intercept. If exchange were very efficient, this would
THE ADAPTIVE RESPONSES OB ESCEERICEIA C O W
181
imply that 4.2% of the nucleic acid of the cell is in the turning-over pool. The averageof estimates for the m-RNAcontent in cultures with a 60-min. doubling time is 3%. Since exchange exists but is not highly efficient, we might conclude that there must be considerably more than 4.2% in the turning-over pool. Above, we cited experiments for believing that the messenger pool is not greater in slowly growing chemostat cells. Tentatively we take this as supplementary evidence that a pool of intermediates in ribosome biosynthesis is continually being formed and then degraded in slowly growing bacteria. This brief introduction into the problems of isohope turnover of bacterial nucleic acids can be summarized as follows. Isotope incorporation may exhibit little or no lag. I n such cases, even short incorporation experiments give information about net rate of RNA synthesis and are independent of messenger and pool turnover and do not give any information about the velocity of messenger synthesis. This is so in cultures of E . coli growing at moderate rates because the two processes of exchange with the outside medium and messenger turnover give an increased initial rate of incorporation that just compensates for the decreased initial rate of incorporation arriving from the lag due to the pool of nucleic acid intermediates. This can be a useful situation since it allows the study of stable RNA synthesis more rapidly and accurately than can be done by chemical determinations. However, in any given situation, it is essential to prove that short-pulse incorporation data reflect net RNA synthesis. This is more likely to be the case in relatively rapidly growing cultures than in slowly growing or non-growing conditions where exchange and messenger turnover give a falsely high initial incorporation.
VII. The Growth Cycle Revisited Almost every textbook and treatise in microbiology has a section discussing the growth cycle. The reader is instructed that a culture of organisms goes through a seven-stage life cycle similar to that of an individual higher organism (Shakespeare, 1623). It is certainly true that, upon dilution into fresh medium, an old broth culture of an enteric organism exhibits the proverbial lag phase, logarithmic phase, stationary phase, and death phase. The end of lag phase can be usefully called the accelerating phase, and the beginning of stationary phase can be called the decelerating phase. Similarly, the death phase can be subdivided into a loss of viability phase and an autolysis phase. However, this is not the universal rule. The same organism which has these population cycles in broth exhibits no lag and no decelerating phase when grown in media containing a limiting concentration of glucose (Cohenand Arbogast, 1960). For quite long periods of time (weeks
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ARTHUR L. KOUH
if stored in the refrigerator), glucose-limited cultures of E . co2i enter the logarithmic or exponential phase immediately upon the addition of (or dilution into) glucose. It has been observed (A. L. Koch, unpublished observations) that the rate of RNA synthesis returns to the full maximal rate in 100 sec. even in a culture that has been completely starved of carbon sources for 7 hr. When glucose is limiting, growth ceases as abruptly as it began. As we shall see below, the growth rate changes almost discontinuously as the glucose becomes nearly consumed. Because the apparent Michaelis-Menten constant for the uptake process is so small, there is almost no growth taking place at an intermediate rate between that characteristic of unlimited glucose and that of no glucose a t all. The latter phenomenon accounts for the former one. Because of the abrupt cessation of growth, almost all macromolecular synthesis ceases. In the usual growth cycle, there is reasonably extensive growth at less than the maximal rate. During this time, the rates of protein and nucleic acid syntheses are different from those characteristic of balanced growth, and the proportion of RNA to dry weight in the cell decreases. I n the glucose-limited batch growth case, the cell cannot revise its composition through continuing growth except to a very small degree by breakdown of a limited class of proteins (Nath and Koch, 1970) and a very slow breakdown of some ribosomes. There are three fundamentally different aspects to the usual growth cycle which can be experimentally and conceptually dissociated. These are the problem of macromolecular synthesis, the problem of cell division, and cell viability. I n the present review we shall avoid the issue of the control of cell division and cell-size changes as well as the issue of cell viability, but shall see how far we can go in understanding the manner in which rates of macromolecular syntheses vary throughout the cell cycle. I n a manner similar to our previous treatment (Koch, 1970; Koch and Deppe, 1971),we can write that the rate of dry weight synthesis depends on the amount of ribosomal-RNA.
This is inocuous enough; all that this relation specifies is that, when the amount of ribosomal-RNA doubles, the rate of dry-weight synthesis also doubles. The symbols (w) and ( r )designate the amount of dry weight and ribosomal-RNA per unit volume of culture. Of course, ribosomes actually make protein. Therefore, in this equation, the proportionality constant, k, combines the rate constant for protein synthesis per unit ribosomal-RNA and the ratio of dry weight to protein. The latter is the same under almost all conditions of growth so that k reflects the former
THE ADAmIVE RESPONSES OF ESCEERZCEIA COLI
183
factor, i.e. ribosomal efficiency. Similarly, for ribosomal-RNA, we can write :
doc ( w ) at
=
where c is the rate constant forribosomal-RNA synthesis per unit amount of dry weight of cell substance. Like k, c is a composite term made up of the products of the rate constant for the synthesis of r-RNA per unit amount of DNA and the ratio of DNA to dry weight. This ratio is experimentally found to be quite constant, so that changes in the value of c reflect largely changes in RNA synthesis per unit amount of genetic DNA. I n balanced growth, k and c must be constant by definition. There is a close connection between k and c and the growth-rate constant, A. To observe this, multiply both equations together and divide by ( r )(w). This yields :
(%)&)
= kc
(9)
During balanced growth, not only are k and c constant and independent of time, but d(w)/(w)dt andd(r)/(r)dtare both equal to A. I n fact, according to Campbell’s (1957) definition of balanced growth, any, all, or any combination of extensive properties of the cells, z,would satisfy the equation : d(1n z)/dt = d(z)/zdt = A Consequently both factors on the left side of Equation (9) must equal
A. Therefore : A2 = kc
(10)
With a little more algebra we could show that, during balanced growth:
( r ) / ( w= ) m k
(11)
So far we have paraphrased what has been said before by Hinshelwood (1952) and Koch (1970). I n these papers, equations were developed for
the transient response after the shift of the environment of the culture, where k and c were abruptly altered to new values appropriate to the new medium. After the discontinuous changes to the new values, k and c were assumed to remain constant. We found that this model did adequately handle the case of the enrichment of glucose-limited chemostat cultures because it was experimentally ascertained that k and c do change abruptly on the shift, but shortly thereafter they do remain constant (Koch and Deppe, 1971). However, these equations could not handle the case for the enrichment of sulphate-limited chemostat cells, since in this case, c, the rate constant for RNA synthesis, changes only gradually
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ARTHUR L. KOUH
after enrichment, A special treatment was developed by Koch and Deppe (1971) t o handle this situation, but that treatment could only apply when c changes with time in a very special way. Mathematically, the treatment also required that k be rigorously constant. Because of these limitations, I have set up a completely general treatment using the calculus of finite differences. This approach does not lead to an analytical solution, but rather to a computer programme. From our point of view either is just as good if it can show us what is the important basis of the biological phenomenon and point out what is inconsequential and what is merely the mathematical consequence of the basic phenomena. Equation (7) written in the finite calculus instead of the infinitesimal calculus becomes :
(4" = (w)"-l + hl-l(r)"-l
(12)
where the subscript n refers to the nth. time interval from a reference point. Equation (8)similarly becomes : (TI" = (%-I
+ c"-I(w)n-l
(13)
From these expressions, if (w),-,, ( T ) , , - ~ , k,,-]and c,-~ are known, we can calculate what (w), and ( r ) ,will be after one time interval has elapsed. The desk computer can do this very quickly ;therefore, we can afford to use very small time intervals and to carry out the calculations for many such successive small time intervals. In this way, the computer very closely approaches the infinitesimalcalculussolution of the same problem. For the computer, we imagine growth to be a large number of small discontinuous steps, and in the latter approach we imagine growth aa a continuous process. When these equations, known as recursion relations, are fed into the Wang 370 computer, with and c,-~ chosen as positive and independent of n, (w) and ( r )increase with time, and, after an initial adjustment period, increase exponentially with the doubling time predicted by Equation (10) andavalueof(r)/(w)givenbyEquation(11). It is really not strange that the recursion relationships of Equations (12) and (13) lead to exponential or logarithmic growth of (w)and ( r ) . They do so for the same reason that money invested at a constant interest rate grows exponentially. I n fact, exactly the same argument applies after the proportion of ( r ) to (w) becomes constant, because the k- ( r )
(4 becomes the interest rate per time interval for dry weight and c-(w (r) is the interest rate for RNA. Both, of course, have to equal A. Therefore :
THE ADAPTIVE RESPONSES OF ESCHERICHIA C O W
185
and :
These two equations are an alternative method of writing Equations (10) and (1l),as can be readily checked. We can also arrange the programme so that values for k and c change either discontinuously or progressively. Although much more can be and will be done, so far we have only used linear changes or discontinuous changes of these parameters. The latter is done by simply halting the programme and changing the constants. The former is done by altering the programme to subtract a constant from k,-, and another constant from c,-, to get new values for the next re-iteration step.
Time (hr)
FIQ.14. Theoretical growth curve. This ciirve was produced by a computer, 8s described in the text. The schedules for k and c were inserted into the programme which generated the growth curve of dry weight per ml. culture ( w )and the @)/(to) ratio asa percentage.
The purpose of this kind of approach is to see how and when values of
k and c must change to account for the culture growth-cycle. We want to do this not only to understand the classical observations of Henrici (1923), Hershey and Bronfenbrenner (1938), Morse and Carter (1948), and Cohen and Arbogast (1950),but also to fit the accurate data obtained in our laboratory by Kenneth Bernstein. With his data, we have a better idea of what is occurring, because the rates of protein and RNA synthesis were simultaneously measured. The growth curve is really a cyclic process; where we start depends on where we ended the last cycle. An idealized growth curve constructed to our specifications by the computer is shown in Fig. 14. We imagined
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ARTHUR L. KOOR
that the inoculum was taken from a stationary-phase culture. During late exponential and early stationary phases, some growth takes place, but RNA synthesis lags behind. Teleologically, the cell does not need ribosomes or the rest of the protein-synthesizing machinery if only a small amount of protein is to be made. On this basis, we instructed the computer that initially the ( r ) / ( w )ratio of the inoculum was one-half that of a balanced growing culture. To simulate Bernstein’s system, we told the computer to use values of k and c that we had calculated from Equations (14) and (16), so that when the culture achieved balanced growth the doubling time would be one hour and the ( T ) / ( wratio ) would be 0.2. The computer, minute by minute, constructed the initial part of the growth curve shown in Fig. 14. It exhibits a brief lag during which the ( r ) / ( w )ratio rises rapidly. The lag phase is briefer than that shown in most microbiological texts because we did not choose the parameters characteristic of a stationary-phase culture in rich broth or the parameters of enteric bacteria growing in rich medium. In a very rich medium, the cells would grow faster during the exponential phase, and the ( r ) / ( w )would be larger. Secondly, the usual growth curve is based on viable count ; for viable count, the lag is longer than for dry weight because the small stationary cells grow large and increase their dry weight before they divide (Hershey, 1938). There is a third reason for the lag being short. For the curve shown in Fig. 14, we had instructed the computer to use those values of k and c that would apply for balanced growth starting from the instant the inoculation was made. If there is a recovery period during which values of k and c increase towards their balanced-growth values, the lag would have been longer as discussed in connection with Fig. 17 (see p. 191). As growth continues, the environment of the cells eventually becomes depleted of nutrients or polluted with cell products, and various aspects of growth must decelerate. From the arguments given above, the RNA synthesis rate-constant as measured by c must decrease even further than the dry weight or protein synthesis rate-constant, k. It must do this if the ( r ) / ( w ratio ) is to decrease (see Equation 11). I n the example shown in Fig. 14, we instructed the computer first to decrease progressively the value of c so that it dropped slowly to one-third of its balanced growth rate, while k remained constant. It can be seen that this drastic change in the rate of formation of the protein-synthesizing machinery leads almost imperceptibly and only very slowly and progressively t o a decrease in the apparent growth rate. I n part this is because preformed ribosomes, t-RNA, and activation enzymes continue to function and produce cellularmaterial. The ratio of ( r ) / ( wstarts ) to drop, but the change is not as severe as the change in c. Eventually, we assumed that the
THE ADAPTIVE RESPONSES OF ESCHERICHIA COLI
187
conditions become so poor that the ribosomes and the rest of the proteinsynthesizing machinery cannot function as efficiently as before and the value of k must fall. Our conceptual argument is that c must fall further and faster than k ; otherwise the ( r ) / ( w ratio ) will not continue to fall. So, in the example shown in Fig. 14, we set c at 0.025 the balanced rate and k at 0.10 the balanced rate. Finally, we progressively decreased both values toward zero, keeping the proportions constant. We believe that this model shows all the essential features of the culture growth cycle with respect to levels of dry weight, protein, DNA, RNA, or any other cell constituent not specifically involved in fixing cell size. The conditions chosen for the calculation approximate some, but by no means all, biological situations. Special attention is called to the possibility that the value of c might decrease a long time before the value of k. So far we do not have evidence of a situation where this decrease in the value of c can precede the decrease in k by the time interval assumed in the calculation, but the fact that such a severe change causes such a little change in the apparent growth should cause concern in microbial physiologists whose sole test that a culture is in balanced growth is that the growth of cell material is apparently exponential. A great deal of the experimental literature in this area may be found by consulting Dean and Hinshelwood (1966) and Powell et al. (1967). The calculation is only important in that it stresses the importance of the biological mechanisms responsible for controlling the values of k and c. We could alter the parameters and the schedule of when and how the parameters change, thereby simulating any particular growth curve. We could also let k and c become negative to simulate endogenous metabolism, autolysis, and turnover. Under our conditions, these are very minor processes. Actual experimental results are shown in Figs. 15 and 16 (Bernstein, 1970). Both show growth curves of a culture of a stringent strain of E . coli K12. I n the first, the culture is in balanced growth in a glucosecontaining medium with an excess of the various amino acids that this strain requires. Eventually growth slows down, not because the culture has used up glucose or because aeration is limiting, but because of the production of valine which is inhibitory to this strain. I n this culture the RNA synthesis rate constant, c , as measured by guanine-pulse incorporation, possibly falls a little before the rate constant, k,but at about the same time as a detectable change in the apparent slope of the logarithmic growth curve. The proportions of RNA to dry weight decrease throughout the late exponential or early stationary phase because the value of c decreases proportionately more than k, although the decreases take place at about the same time. I n Fig. 16 the cells were given the identical growth medium with the one exception of a small and limiting concen-
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ARTHUR L. KOOH
tration of histidine. The hietidine-uptake system in these organisms is not highly avid ( K = 0.844 pg./ml.) so there is a relatively long period of growth during which the cells are decreasing their growth rate because histidine is becoming progressively more limiting. With a small contribution to continued synthesis of some polysaccharide after the histidine is exhausted, the growth curve can be fitted to the Monod formulation of transport-limited growth (see p. 211). Also shown on the same graph are estimates of the rate constants based on the analytically determined amounts of RNA and dry weight. From these data, accurate determinations of the values of k and c using Equations (12) and (13) over time
Time (hr.)
FIG.16. Growth curve of a stringent strain of Eechem'chia co2i K-12 CP78 in a medium containing glucose and an excess of amino acids. Growth ( A )of the strain in the presence of 80 pg. leucine, threonine and arginine per ml., 40 pg. histidine per ml. and 0.2% (w/v) glucose and 1 pg. thiamine per ml. was followed turbidimetrically. Uptake of guanine into trichloroacetic acid-insoluble material over 8-min. periods was also followed throughout the growth ( 0 ) . The results were expressed on the basis of mg. dry weight and are therefore proportional to c. The inoculum was made from an exponentially growing culture in the same medium. Date from Bernstein (1970).
intervals of 1 hr. were calculated and are shown as horizontal lines in the figure. In this case of an amino-acid limitation of a stringent organism, it is clear that the elective shut-off of RNA synthesis precedes and is more drastic than the necessary shut-off of the ability of the ribosomes to function due to lack of histidine. Returning to the lag phase of growth, if we continue to restrict ourselves to the synthesis of cell constituents and exclude consideration of viability changes and control of cell division, the lag depends on the
189
THE ADAPTIVE RESPONSES OF ESCBERICEIA COLI
Histidine concentration (,ug./ml culture) 400
I008 07 0605 04 03
02
’
i
lO0li”l”’
’4 80
Z
I
I
2ot
’
I
----
50
30
i
01 005
0.03
2
i n -/
iL
: -
FIG.16. (irowth CIII’VO of a stringent strain of Escherichia coli K-12 CP78 with histidine liniitat,ioii. Growbh conditions are as for Fig. 15 except that the histidine coriceiitjratioriwas 1.0pg./ml. The culture wm grown 105-foldat low cell densities in this medium hofore the experiment started. The smooth curve was fitted to the Monod formula discussed below with A,,,,,. = 0.683/56 min. and K = 0.844 pg. histidinn/ml. It is prosumed the discrepancy for the last points is due to polysaccharidc deposition. The horizontal lines represent estimates of k (dotted) and c (solid) bascd oil en arielytical rletcnnination of ( r ) and (to) at almost hourly intorvals. from: and
where n rofws to thc sampling point, mid n - 0.5 refers t o theoxtrapolatedmid-point. DaLn froin 13criistoiti(1970).
( r ) / ( w ratio ) of thc i~ioculumand on how fast the cell’s mechanisms can cause k and c t o be changed to become equal to those values associated with balanced growth in new media. Previously I have presented a formula (Koch, 1970) that would apply if any “extra” RNA becomes functional after the shift, and if values of k and c are discontinuously shifted to the new values. For this case, the lag time r is :
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ARTHUR L. KOUH
where g is given by : 9 = (r)/(w)/(r)‘/(w)‘
(17)
The primed values refer to the conditions at the time of the shift, and unprimed values refer to balanced-growth conditions after the shift when anew steady state has been established. If the ribosomal efficiency, k, has the same constant value both before and after the shift, g is the ratio of the growth-rate constant after the shift to that before the shift. Clearly, if g is large because (r)’/(w)’is small in the stationary-phase growth medium, T approaches l / h In 2. By definition, ( l / X ) In 2 is the doubling time under the new growth conditions. This result is a very useful and practical one because, by simply measuring the intial phases of a growth curve, the amount of “extra” RNA that can function in protein synthesis can be estimated. Thus, if the lag is less than one new doubling time, Equation (16) permits the calculation of g and Equation (17) permits the relative RNA constant before and after to be computed. This can be done with only one assumption namely that the RNA produced in the previous growth condition can function in the new environment. We have found (Koch and Deppe, 1971)that these equations described very well the lag phase of carbon-limited chemostat cultures after a shift to rich medium. This expression does not work if the change in the value of c is not instantaneous. A more elaborate expression was derived for this special case, but it cannot be generalized for the reasons given above. Therefore, we present in Fig. 17 computer calculations for those cases where values for k and c are both initially zero and rise towards their balanced growth values slowly. For these calculations we have assumed that both aonstants increase proportionately. Other assumptions might be made; however, here we simply point out that the timecourse can be estimated by the length of the lag phase if all cells are viable. I n fact, if this mathematical analysis had been available before, it would have been clear that the “constant efficiency” hypothesis could not be true for the slowly growing cultures of Salmonellu studied by Kjeldgaard el al. (19SS), since the turbidity curves after a “shift-up’’ had almost no lag. We conclude this section by assessing the cost to the organism of having “extra” RNA and the cost of not being able to adjust abruptly values for k and c to the optimal values. The “extra” RNA in those organisms where it quickly becomes functional costs the cell in the sense that it must grow between 10 and 20% slower during chemostat-type of growth, depending on what growth factor was limiting as we showed above. But the “extra” RNA almost decreases the growth lag on a “shift-up” to zero. This means a factor of two head-start or (t 100%
THE ADAPTIVE RESPONSES OF ESCEERICEId COLI
191
gain when compared against prudent, but unwise, organisms that had only made as many ribosomes as could be gainfully employed under the chronic starvation conditions. On this basis we see that the “extra” RNA, instead of being a liability, gives the organism retaining it a 300% advantage per day (if its host eats three square meals). On the other hand, if there are of the order of two cell generations per day of cells in the
FIG.17. A computer treatment of the lag phase of growth. The initial phases of growth are represented. The dotted lines are calculated for the case where there is an exceea of functional ribosomes in the cells in the previous growth medium. If the excess is large there is no lag. The solid lines have been calculated on the assumption that the inoculation culture had been grown in the previous medium so long that the values for both k and c were zero. On sub-inoculation, these values are assumed to rise progressively and linearly to the balanced-growth values, taking the indicated time to beoomo stable. Values of g are equal to (r)/(w)/(r‘)/(w’) (Equation 17).
intestine of a mammal, the prudent micro-organism would obtain an advantage of 20-40% per day if the host were to browse continuously with its pyloric sphincter continuously patent so that true chemostat conditions would result. On this basis, there is every reason for evolution to select for organisms that can quickly alter values of k and c in a typical intestinal flora.
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ARTHUR L. KOOH
The fact that sulphate-limited chemostat cells cannot alter values for k and c quickly probably means that sulphate limitation and/or sulphurcontaining amino-acid limitation has not been important in the evolution of E . coli.
VIII. Active Transport From Very Low External Concentrations During much of their evolution, enteric organisms have been selected for their ability to extract almost the last metabolizable molecule from their environment. A better variant would outgrow his neighbours very quickly and in turn be replaced by still more effective organisms. Effectiveness would include, among other considerations, the efficiency of conversion of a metabolite into cell material. Let us presume for this discussion that the yield coefficient, &, is as large as evolution could make it and focus our discussion on E , the efficiency with which the cell can clear the surrounding medium of nutrient. We will define E as a rate constant, that is, as the equivalent number of volumes of medium that can be cleared of nutrient by a unit volume of cytoplasm per unit time. Units of E will be reciprocal time, and we will quote E values in units of l./sec. High values for E can be obtained by decreasing the size of the organism, by changing its shape to make it asymmetric, by increasing the number of units of the transport machinery per unit membrane area, and by increasing the intrinsic capability of the unit transport mechanism in the cell membrane. I n addition, values for E can be increased through motility. There is a definite limit to how far each of these factors can be extended either because of some physical restriction or because the cost to the organism becomes too great in terms of material, energy, and competition for available membrane with other transport mechanisms. I n the classical investigation of tryptophan-limited growth in continuous cultures (Novick and Szilard, 1950; Novick, 1958), a series of variants, each having an advantage only a t lou7 concentrations of tryptophan, were observed t o succeed each other. Just why the successful variants were more effective in growing a t low concentrations of tryptophan was never reported. However, in another chemostat system (Novick and Horiuchi, 1961; Horiuchi et al., 1961), which employed lactose as the limiting carbon source, the nature of the evolutionary steps is understood. First, constitutive organisms took over the population; they had the advantage of full permease production while the parental wild type was only partially induced a t the low concentration of lactose existing in the steady-state chemostat. Then, the constitutive mutant was replaced by a, constitutive hyper-producer resulting from duplication of the lactose operon.
THE ADAPTIVE RESPONSES O F ESCEERICHIA COLI
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Both of these responses to low concentrations of lactose are extremely typical of many situations in biology; both are different methods of increasing the redundancy of the biological system. They both yielded more of the same kind of permease on the membrane of the cell. It is also clear that these observed responses are only of adaptive value in the constant environment of the chemostat, not in the vicissitudes of real life. I n a mammalian intestine, there may sometimes be no lactose although another carbon source may be present. Therefore, it is clearly of selective value to shut off electively permease and j3-galactosidase synthesis. Similarly, it is also quite clear that the hyper-producer with the multiple dosage of lactose genes is a t a disadvantage in the presence of high concentrations of lactose; in fact, it is reported that such hyperproducers are killed under these conditions. This, it has been presumed, is because high concentrations of lactose interfere with the synthesis of a cell-wall component. I n part, because experimental attempts to study microbiological evolution in the laboratory are sparse, these are the only kinds of evolutionary progress that have been observed actually taking place. Many other kinds of changes must have taken place in the past. Some of these havenot takenplacein the laboratory because evolution had already pushed that line of development as far as it could go, and others might have happened if the experimenter had been several orders of magnitude more patient. It is worth while enumerating the kinds of changes that must have taken place during evolution. First, growth on a limiting concentration of substrate eventually should result in a very efficient utilization of this resource for the cell’s needs. Of course, this austerity would mean st multitude of changes in the cell. For the tryptophan limitation, it would mean selection for proteins of all kinds with a lower tryptophan content ;for any carbon limitation of a heterotroph it would mean most efficient extraction of energy. It would mean decreased endogenous metabolism. In short, it would lead to many changes, each resulting in a small increase in the value of &. It would also mean that the cell had to have more effective transport mechanisms. There are ways other than the two mentioned above to increase the redundancy of transport machinery. I n addition to constitutivity and duplication of genes, the rate of initiation of transcription events might be increased. The number of translation events per message might be increased either by attaching more ribosomes per message or by endowing the message for the permease in question with a longer half life. The fact that such mutations have not appeared and taken over the population during lactose limitation must mean that initiation in this operon already takes place as often as possible. I n addition, it means that the half life of a particular message cannot be
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ARTHUR L. KOUR
specifically lengthened. These statements are consistent with the known facts that 4% of the cell’s protein is in the form of 8-galactosidase when the single copy of the 8-galactosidase gene is derepressed. This is such a large rate that it probably means that the initiation rate and the number of transcription events are already maximum. It also implies that the average life of the messenger is not easily altered. Another group of changes are those involving cell size and shape. Enteric organisms possess a mechanism to adjust the average cell size to changing conditions of nutrition, with poor growing conditions leading to shorter and thinner cells. It was partly because of the existence of this mechanism that Koch and Schaechter (1962) postulated that cell division is controlled by a mechanism sensitive to the cell size, but set to trigger at different cell sizes under different physiological conditions. However, it is apparent that a further decrease in cell size is not under simple mutation control. There should be a mutant class that remains small even in rich medium, although we are not familiar with any attempts to find such. A final group of evolutionary changes would include the actual improvement of the permease either with respect to its affinity for substrate or its maximal velocity of transport. Improvements in the effectiveness of a permease, or of any enzyme, have not been observed in any experimental attempb to reconstruct evolution. J. Landridge (personal communication) has achieved an alteration in specificity, but not the improvement of a permease for the specificity t o which it is already optimally atuned. Once again I feel that this is because such elaborations have already taken place. I n fact, as far as the 8-galactoside permease system is concerned, I have already argued in the literature (Koch, 1964) that the permease addition to a transporter mechanism was an improvement over the primaeval transporter system ; the system was also improved by the addition of an energy coupling system to speed further entry a t low concentrations of substrate. Increasing the number of permease systems per unit of surface does not change the properties of the individual permease units. Under widely varying circumstances, the transport system will seem to exhibit kinetics reminiscent of the simple enzyme law of Michaelis and Menten (1913), i.e. at very low substrate concentrations, uptake is proportional to concentration, but a t higher concentrations the uptake is saturated. These statements can be given mathematically as :
v=-
v.s K’+S
where v is the velocity of entry via a membrane transport system, V is the maximum velocity, K’ is the concentration giving half maximal
THE ADAPTIVE RESPONSES OF ESCEERICEZA COLI
196
rate, and S is the external substrate concentration. A similar equation has been given on p. 162. In terms of the growth rate constant, A, and the maximum growth rate constant, Amax.. Probably neither of these processes is truly described by a rectangular hyperbola, and certainly K , K’, V , and Amax. are not fundamental quantities. Thus, for any particular detailed mechanism for transport involving many steps, e.g. combination of subunits with permease, transfer to carrier, diffusion of the carrier, and release from the carrier,
External substrate concentration/concentratlon giving half maximal rate (S/K’)
FIG.18. A plot showing transportation limit of growth. The solid lines illustrate the uptake capability of a cell satisfying the Michaelis-Menten hyperbolic relationship. Both curves have the same value of K’. The upper solid line is for a cell with twice the transport capacity, so that V , = V1. The dashed lines show the growthrate constant. In this theoretical example, we have imagined that even the lower level of maximum transport at high concentrations of substrate is sufficient not to limit growth. Therefore, A,,,,,. is the eame in both cases. However, the substrate concentrations giving half maximal rates are different and are smaller than the K‘ value of the uptake system. No diffusion limitation is assumed in this case.
both the apparent V and K’ values are functions of the rate constants of all of the constituent processes and their back reactions. For an explicitly stated mechanism, the relationship between the rate constants for the steps and values of V and K’ can be readily written down (see for example Koch, 1967). Values for K and A,,,. in the growth-rate equation are also complex. In fact, this general saturating kind of dependence has been called the Liebig (1843) or the Blackman (1906) “law of the minimum” longer than enzymologists have talked about Michaelis and Menten (1913).
196
ARTHUR L. KOUH
If transport limits growth at all substrate concentrations, then we oan expect t o see an increased growth rate at all substrate concentrations. If transport limits growth only a t low substrate concentrations, an increase in transport capability is translated into an increased apparent avidity (lower K value) of the total growth process. I n such a case, the value of K for growth depends on both the K’ and V values for bransport and on Amax. and is not simply equal to K’. Although the curve will have a zero order and first order region, just how one region grades into the other is not clear. I n fact, the curve may make a sharper break than predicted by an hyperbola. These concepts are shown graphically in Fig. 18. Also, this graph shows that simply having more numerous pumping sites results in the successful variants being able to grow faster than the previous variant a t the same low concentration and, a t the same time, leads to a lower value for K . Thus, under conditions equivalent to continued chemostat limitation, they would grow faster than their forebears. Eventually such selection would lead to a take-over of the population, as indicated above. If this evolutionary process were to continue to its logical conclusion, the cells would eventually contain on their cell membrane active transport mechanisms of sufficient capability to deplete the immediate environment of the cell of molecules of the limiting nutrient. I n this extreme, the rate of uptake a t low external substrate concentrations would no longer be limited by the transport capacity of the cells, but rather become limited by the diffusion of the substance from the bulk medium up to the cell membrane surface. I shall now examine how closely micro-organisms have adapted towards this limit.
A. UPTAKE BY A MOTIONLESSSPHERICAL CELL Figure 19 depicts a spherical cell well separated from other bacterial cells during steady-state uptake. It shows that there must be the same total quantity of nutrient transported across any closed spherical surface concentric with the centre of this cell as is transported from the medium immediately adjacent to the cell into the cell membrane. This in turn must equal the rate of transport across the cell membrane and the rate of utilization inside the cell. Thus the total flux through a spherical surface at any distance from the surface of the cell has to be the same as that at any other distance. Because the flux in the medium can only be due to diffusion, the concentration gradients that drive diffusion must change to produce this equality of flux. Consider the case in which the evolution of the transport system and the consumption system has become so effective that the cell is limited by the rate of diffusion of nutrient up to the cell surface. This happens
THE ADAPTIVE RESPONSES OF ESCHERICHIA COLI
197
when the pumping system scavenges very effectively and keeps the surface concentration essentially zero. The mathematical treatment of the adsorption of diffusing particles by a sphere that adsorbs or reacts with every molecule that approaches it was given many years ago by von Smoluchowski (1915,1917).After the sphere is introduced into a medium containing a uniform concentration of particles, the concentration of particles decreases in the neighbourhood of the sphere until a constant concentration profile is established, the concentration being zero a t the cell’s surface a t all times after introduction of the sphere.
1
FIG. 19. Steady-state uptake by a spherical cell. A hypothetical motionless metabolizing cell is shown in the centre of the diagram. The arrows indicate flux of a nutrient. The same total flux must pass through a closed spherical surface concentric with the centre of the sphere.
The equation for the concentration profile is quite complicated mathematically; however, as shown in Fig. 20, a time-independent profile is eventually established. For an object the size of a bacterium with a radius (R)chosen to be 0.8 pm., and for the uptake of small organic molecules like glucose, lactose, or tryptophan in dilute aqueous solution a t body temperatures, the Diffusion Constant is about 6 x cm.*/sec.,and the time to approach a steady-state concentration profile is very short. The concentration profile during the steady-state turns out to be very simple mathematically, i.e. it is a rectangular hyperbola :
c = s (1 - ;),
198
ARTIIUR L. ROOH
where C is the concentration at a distance, r, away from the centre of the cell and R is the radius of the spherical cell. We use, as before, S for the bulk concentration. Fick’s first law of diffusion can be written :
where Do is the Diffusion Constant in the medium and q is the quantity taken through the surface of area, A , of the sphere. Therefore, for any of the hypothetical spherical surfaces of Fig. 19, the area as 4nr2 and the t,,=o+
g?:?:?
Ratlo of distance ( r ) from centre of sphere to the radius (R) of the sphericol cell
FIG.20. Concoritrntion profiles around a sphcrical cell. I f a spherical cell of radius R and of infinite uptake capacity is instantaneously placmd into a uniform concentration (8)of particles, then the concentration a t various times and distancrs is indicated in the graph. Each line is for a fixed time (indicated in the figurc as to values) aftcr tho discontinuous introduction of tho sphere. To be generally risoful this time is a normulixed time (to = D t / R 2 )I. n a medium of low viscosity, assuming Do (the Diffusion Coofficient) = 6 x cm.z/soo. and R = 8 x cm., s to of 1 corresponds to 0.00106 scc.
value of dC/dr can be obtained by differentiating the expression for the concentration profile. This is easily done because S and R are constant. These substitutions yield : SR v, = D0(47rr2)- = 4.rrDoRS (21) r2
The subscript on v, designates bhe velocity of uptake by a single sphere. For purposes of considering and comparing uptake and growth data we would like t o reformulate this equation to express the equivalent number of volumes of solution cleared per unit time per volume of the cell. This quantity, which above we designated as the E value, is the ratio of the equivalent volume of solution cleared of substrate per unit
199
THE ADAPTIVE RESPONSES OF ESCHERICHIA COLI
time per spherical particle (v,/S) divided by the volume of the sphere (413)~ R3:
As a minimal value, let us substitute Do with 6 x cm.*/sec. and cm. in Equation (22). The E value is 3 x 6 x R by 0.8 x (0.8 x 10-4)2or 2800/sec. Thereforc, a perfectly efficient organism under these circumstances could physically clear 2800 times its own volume in a second in ordinary growth media at 37'. This maximal value can then be compared with available data in the literature concerning uptake and growth a t low concentrations of nutrilites. For uptake or transport data under conditions of slow rate of substrate addition (S Q K ' ) ,Equation (18)becomes: 2,
-
V (S) K'
=-
This equation can be written to permit calculation of E values by dividing by ( S ) .However, since v and V are usually expressed as micromoles per gram dry weight of cells per minute, and not per volume, of cells, it is necessary to convert the results onto a volume basis by multiplying by the dry weight content per unit volume of cell substance, W . We express this in units of g./ml. Then if K is given in units of M or micromoles/litre : micromoles 1000 ml. g. dry wt. V W g. dry wt. m i n litre ml. cells E = - iK' micromoles 60 sec./min. (24) \ litre
)
=
VW K
16.6 I (set.)-'
For growth rate data, Equation (3) can also be used by converting Amax. to V in the usual units by the relationship : 1
__
min.
Lax.
MQ
g . g. dry wt. lo6 micromoles g. of substrate micromoles
-
~~
where Q is the yield coefficient of the substrate and M is its molecular weight. If the curves for either uptake or growth are not exactly perfect hyperbolas, it is the first order rate constant a t low substrate that should be used. The results calculated from data taken from the literature and from 8
200
ARTHUR L. KOCR
our own unpublished data for the uptake of a number of substrates by E . coli are reported in Table 3. Uridine is taken up and transformed faster than any other compound listed. Also, it is evident that there are very effectivepumps for adenosine and cytidine. These aminated nucleosides are taken up, rapidly deaminated and the resulting inosine or uridine largely released from the cell. This suggests that, in the gut, nucleosides have been important sources of sugar and that these aminated nucleosides are and have been an important and critical nitrogen source for E . coli. The amino acids listed are also effectively used. As expected, glucose is also effectively used. Lactose utilization is ten times slower than that for glucose even in the constitutive organisms studied, when grown in batch culture. Chemostat-limited cells are much more efficient. These data suggest that, while aminated nucleosides, glucose, and amino acids have been consistently present in small amounts in the cell’s environment, lactose, while intermittently present in the mammalian intestines, has not been chronically present a t low persisting levels. This is not too surprising a conclusion, considering the properties of the mammalian hosts. There is some experimental uncertainty in the values of K , R, Q , Amax., Do, and W . Also there is error introduced by assuming that rod-shaped bacteria behave as equivalent spheres, but it is clear from Table 3 that no transport system allows the cell to approach maximum efficiency of E = 2800/sec. I n view of what was said above this is a surprising result. Why should these cells not be diffusion limited, but seemingly be transport limited in their natural habitat? There are a number of possibilities that we must consider. First, we may not have tested the right substrate; some other substrate may be chronically limiting in vivo. The second possibility is that the enteric organism may not be food-limited in its natural habitat, but be limited by antibiotics produced by other organisms, or by predators, both bigger than itself, such as protozoa, or smaller than itself, such as the bacteriophages. The third possibility is that the strains of Escherichia coli employed in the various studies used to produce the values calculated in Table 3 have all spent many years under laboratory conditions, where growth is not chronically limited as it is in the intestine. They might have, therefore, become decadent and slothful. For example, most strains of Escherichia coli isolated from nature are motile ; most of the strains used for the data in Table 3 are not. A variety of loss mutations may have accumulated in laboratory strains. Fourthly, selection may have operated not at the level of a single isolated cell, but largely a t the level of microcolonies of organisms. Inspection of the data arid the formula 3D,/R2 shows that a spherical microcolony containing 60-100 individuals would be diffusion limited for many of the compounds
Velocity of uptake (V)
Solute
Michaelis constant (K‘ or K)
micromoles g. dry wt. min.
Carbohydrates : Glucose (growth studies)
Volume of solution cleared of substrate /unit time/cell (E)
WC.-l
Reference
22 19.4’ 121” 3lC 500
54.8 53.2‘ 4.8” 3lC 1
Monod (1942)
13.4
1
44.8
Valine
24.3
8
10.2
Proline
15.7
1.5
33.2
Piperno and Oxender (1966) Britten and McClure (1962) Britten (1965)
Nucleosides : Adenosine
54.6
2.7
67.6
4.6 3.3
80.8 119.5
Lactose (growth studies) Thiomethylgalactoside Amino acids : Leucine
Cytidine Uridine Efficiency of perfect efficient sphere cm.2/sec. R = 0.8pm., Do = 6 x
367 311” 175” 29lC 148
112 118
R = O . S p n . , Do = 12 x 10-8cm.2/sec.
‘Glucose-limitedbatchculture;growthdetaf!howninFig.23 (p.211). bLactose-unlimitedgrowingculture;growthdatashowninFig. 24 (p, 212). Lactose-chemostat culture; doublingtime 2 hr.; growth datashowninFig. 25 (p. 213).
2800 56
Kepes and Cohen (1962)
Peterson and Koch (1966) Peterson et al. (1967) Peterson et al. (1967) Theory for dilute solution Theory for viscous media with relative viscosity of 50
202
ARTHUR L. KOOH
tested. Finally and most likely, the diffusion constants measured in a medium consisting of colon contents are very much higher than those measured in dilute aqueous solution. The relative viscosity of the colon contents is many times that of water. This is because of the StokesEinstein Relationship which states that Do is inversely proportional to viscosity and directly proportional to the absolute temperature. To gain some appreciation of the kinds of viscosities needed to make cells diffusion-limited for many of the compounds reported in Table 3, we note thab ordinary kerosene or olive oil has a viscosity sufficiently greater than that of water so that, if diffusion had to proceed in a kerosene or olive oil environment and transport remained unaltered, diffusion would be limiting. To appreciate how viscous a medium would have to be, another example within the usual experience of most people is that of winter-weight SAE 10 oil. At body temperature it has a viscosity of 50 centistokes while the viscosity of water is only 0,695 centistokes. Most of the contents of the large intestine have viscosities as high or higher than this. I plan to test explicitly all of these explanations, but for the rest of this essay I shall presume that the cells are well separated from other metabolizing cells but in a highly viscous medium where they are frequently diffusion limited in growth for several kinds of low molecularweight nutrients. However, I must also assume that the organism spends a good portion of its evolutionary history in media of lower viscosity where it is transport limited. I n both of these circumstances, small size is an advantage. Since E = 3Do/R2,halving the size quadruples the efficiency. This equation, of course, applies if the membrane contains adequate transport capability t o maintain the surface concentration a t infinitesimal values. However, even if the membrane cannot maintain the surface concentration at zero, smaller size increases the surface: volume ratio. If the substrate is not significantly depleted a t the surface, then it can be assumed that the amount taken in by the spherical cell is proportional to the surface area :
V , = P4r R2S (26) where P is either the permeability coefficient in some cases or the permease capability per unit membrane area in other cases. The same arithmetic as before yields : E
3P = -V8 /(4/3)rR3 =-
S R If transport capability is almost at the diffusion limit, the efficiency will change slightly more rapidly than the inverse first power of the radius. This is because it is easier to be diffusion-limited when diffusion is essentially in one dimension than when it is in two or three dimensions, I n
THE ADAPTIVE RESPONSES OF ESCHERICEIA COLI
203
a large cell, diffusion is essentially normal to the surface ; in a small cell, tangential diffusion can also aid in bringing nutrient to the cell surface. Clearly, micro-organisms are usually small for just these two reasons : (a) to increase the diffusion-limited value of E ; and (b) to increase the surface-limited value E. Either is of less importance a t high substrate concentration when enteric organisms can afford to be and are bigger. B. UPTAKE BY SPHERICAL MOVINQ CELLS Many enteric bacteria are motile. In fact, almost all isolates of E . coli from the intestine are motile. At first thought, this makes sense because such cells can graze and then move on. On second thought, it does not make sense when the viscosity is low. The calculations in Table 3 indicate that for many compounds the cells are not diffusion-limited; therefore, if the cells do not expend energy on travel, low molecularweight substrates will come to them faster than they can be consumed. Seemingly, under these circumstances, motility would only become an important advantage if the cells consumed compounds of much larger molecular-weight than glucose, which would diffuse so slowly that motility in going to the food could be competitive with Brownian motion bringing the food to a sessile organism. After all, sitting still and clearing 2800 volumes per second is hard to improve on for even the greediest enteric organism. It makes good sense for organisms such as protozoa and jaguars to be motile in order to catch large particles such as coliforms and peccaries instead of waiting for Brownian motion or the prey’s own motility to bring them into contact. The purpose of this section is to give a semi-quantitative treatment of the effects of motility on diffusion-limited uptake. A spherical cell, as pointed out in connection with Fig. 3, when displaced into a region of uniform concentration, depletes the surrounding medium until the concentration profile comes into a time-independent state. Afterwards, the flux due to diffusion inwards a t all distances from the centre of the cell equals transport a t the surface of the cell and consumption internally. Initially, after the cell is introduced into a new environment, the consumption by the cell is much larger. If the capacity of the pump is infinite, the rate of consumption will be infinite but, of course, only for an infinitely short time. I f a spherical cell is moved a t regular intervals of time AT, far and quickly enough such that the cell is now bathed in a uniform environment of the bulk medium where the concentration of nutrients is S, the velocity of uptake would be increased 2R (Koch, 1960) by a factor of 1 -which we will designate by (2.
+ &DA~
Consequently, the efficiency, E , would also be increased by the same
204
ARTHUR L. KO(1H
factor. Although this expression was derived to consider the effects of Brownian motion, sedimentation of the cells in the earth’s gravitational field, and cell motility on adsorption of virus, the same formulation applies to nutrient uptake. However, since the diffusion constant of bacteriophage is smaller by about two orders of magnitude than the diffusion constant of the lower molecular-weight nutrients, the time, AT, required to move the particle several times its own diameter to produce
lo5.0 O
I
2 0c 0
2
B
050 2 .-
Cell
00510-9
I 10-8
moilon
I 10-7
10-6
10-5
10-~
I
10-~
I
I
I O - ~ lo-’
I 10-O
I
Dimensionless velocity (pR2/LDo)
FIG.21. Effect of motility on diffusion-limited uptake by a sphere and a cylinder. The absciasa is a normalized velocity, where p is the true velocity, R is the radius, 1 is the length (= 2R for a sphere) and Do is the Diffusion Constant in the medium. The ordinate is the factor, B , which when multiplied by 3Do/R2gives the efficiency, E . The range of velocities given to the cell by motility and Brownian motion are indicated in ordinary growth medium. I n highly viscous medium, the range for cell motility does not change because values for p and Do increase proportionately. For Brownian motion, the dimensionless velocity is greatly decreased in highly viscous medium. The velocities refer to that of the cell relative to the medium very close to the cell.
the same percentage increase in uptake of the nutrient would be two orders of magnitude more than for the uptake of virus. Thus, motility provides a much greater chance that a bacterium will become caught by a virus than it increases the chance of the cell finding low molecular-weight nutrients. For a rough numerical calculation, assume that a motile spherical cell moves twice its own radius in 0.1 sec., the time we estimate for AT. This means that the velocity, p, is 16 pm./sec. Twice the radius is far enough so that the cell will be in essentially fresh medium with bulk ooncentration, S. For the same values of R and Do used above it follows that : 2x 8x = 1.12 Q=l+ x 0.1 43.4 x 6 x
THE ADAPTIVE RESPONSES OF ESCHERICHIA COLI
205
However, when the relative viscosity of the environment is 100, G will be 2.2. The first value of G is irrelevant because it was calculated for the case where the uptake was not diffusion limited and movement could cause no increase, but the second value should be highly relevant for selection of colon bacteria. Over many bacterial generations in natural habitats, a 120% advantage, even if effective only a portion of the time when the viscosity was high, could cause a virtually complete replacement of the non-motile types with motile types. There is no need to belabour this calculation further. It is only an approximation because motile organisms move continuously not discontinuously. Furthermore, most of the bacteria of interest are not spherical, but are rod shaped. However, in Fig. 21, for comparison with the treatment of rods given below, I present G as a function of the ditLR2 I n the case of the sphere, the mension-less velocity parameter -. DO1 length of the particle, I , is equal to 2R. G is calculated from :
C. UPTAKEBY ROD-SHAPED PARTICLES The general solution of the diffusion equation for a finite rod-shaped particle adsorbing all the molecules striking it has never been obtained. However, the solution to the problem of an infinitely long, stationary cylinder has been given. The solution involves several kinds of Bessel functions. Fortunately, numerical values have been computed and have been published (Carslaw and Jaeger, 1947; Jaeger, 1956). From these I have prepared Fig. 22, a graph for the long rod, similar t o Fig. 20 (p. 198) for the sphere. There is an important difference between the case of the sphere and that of the long rod. In the former, a steadystate profile is quickly established while in the latter a steady state is never achieved. Rather, the profile becomes progressively more gradual. Therefore, the velocity of uptake must decrease indefinitely with time. A filament is doomed to starvation unless it can depend on its own motility or circulation in its environment by convection or currents of other kinds. For the present problem, we need to calculate the velocity of uptake from the concentration profiles and Pick’s diffusion law. This is very difficult applied mathematics, but even if we could compute this it would change from instant to instant as the concentration profile changed. The total flux per unit area from time zero to any arbitrary time, t , has been calculated (Goldenberg, 1966). It is this integrated flux which
206
ARTHUR L. KOUH
is of help in the present connection. If a rod-shaped bacterium moves along its axis with a constant velocity, then the forward margin of the bacteria is exposed to the bulk concentration of the nutrient, whereas the rear margin is exposed to the same concentration gradient that would have been produced by discontinuously moving the bacterium and then allowing uptake to take place for the same length of time it actually takes the organism to move continuously past a fixed point. Thus, by dividing the integrated flux by this time, the average flux into the moving
FIG.22. Concentration profiles around a long oylindrical cell. So0 caption for Fig. 20 (p. 198) for an explanationof the symbols.
bacterium is obtained. When Goldenberg’s ( 1956) notation is altered to apply to our present needs, the amount taken up per unit time and unit volume for a moving cylinder is given by :
E
30 (a) R2
=-
where B is a factor analogous to that used above in the case of a moving sphere t o correct for the increased uptake caused by the movement. Mathematically the factor is complicated, but that need not concern 11s here. Graphically the factor is shown in Fig. 21. It is apparent that a rod-shaped organism must move at a speed such that the abscissa1 value of pR2/D,Z is about unity in order just to equal the uptake of a resting sphere of the same radius. For an organism 2 pm. long and 0.44 pm. in radius, this velocity in dilute aqueous solution is: x 2x = (0.44 x 10-412 = 0.62 pm.lsec.
6x
= 0.62
x
cm./sec.
THE ADAPTIVE RESPONSES OF ESCHERICEIA COLI
207
or about a third of its length in a second. Micro-organisms have been clocked at up t o 50 pm./sec. If high viscosity decreased values of p and Do by the same proportion, then motility is a very important aid in uptake especially since the effects of Brownian motion are much less. If the bacterium is slowed in some way other than by increasing the viscosity of the medium, the number of volumes that can be taken up per unit of time becomes smaller. However, the resting steady-state value of zero is only very slowly and very asymptotically reached. Brownian motion of the bacteria, if they are no longer than several micrometres, produces movement that prevents uptake from going to lower values then those roughly corresponding to abscissa1 values of to at low viscosity. Brownian motion would be of less aid in a high-viscosity medium, and very long non-motile filaments would be extremely inefficient in a still environment of high viscosity. Since the mathematical calculation has assumed the cell t o be a long cylindrical shape, the calculation is in error for short cylinders because uptake from the ends has been neglected. For short cylinders, and especially for short moving cylinders, I have underestimated the uptake. Very short cylinders would approach the behaviour of spheres. For moderately long cylinders, because no micro-organism moves fast enough to produce turbulent motion, laminar flow is involved. Therefore, the end-effect increase can be approximated by assuming a value for the length of the cell which is larger than the true length. This extra length should be calculated to give an extra area corresponding to the area of the leading and trailing faces. Thus, we added to I, the true length, an increment A1 obtained from the relationship : 27rR2 = 27rR. A1
(31) Al= R (32) This relationship assumes the faces to be flat. A different correction is needed for hemispherical ends. The correction should be used in the abscissa of Fig. 21 (p. 204). From the shape of the curve, it can be seen that, in almost any case, it will hardly affect the values of G or E . Thus, end effects are small for moving cylinders unless very short cylinders are considered, where the theory is inapplicable for other reasons.
D. MOVEMENT A N D MIXING EFFICIENCY As already mentioned, flow is laminar and not turbulent around any moving micro-organism. This means that the fluid volume in contact with the cell is not exchanged as fast as is implied by the calculation given above. There, we had imagined that we had simply moved the organism to a new portion of the environment. On this basis, higher
208
ARTHUR L. KOOH
velocities are needed to achieve a given value of (7 than those indicated in Fig. 21. On the other hand, the velocity of significance for the present purposes isnot that which would be measured by a remote fixed observer, but rather velocity relative to some nearby point in the growthmedium. So just as a self-propelled boat moves much faster relative to the water passing through the propeller or being pushed by the oars than relative to the shore, a self-propelled bacterium comes into contact with more of its environment than is apparent from its net velocity viewed through a microscope. Obviously, there are a number of engineering problems that need to be attacked to make an accurate appraisal of the mixing times. At this juncture, it may be wise only to comment that the peritrichous flagella of the eubacteria seem to be much more ideally suited to stirring up the local environment in a very viscous medium than they appear to be designed to function to move a microbe from one place to another.
E. THEINTERMEDIATE REGIONBETWEEN DIFFUSIONAND TRANSPORT LIMITATION I n the steady state, the flux from outside the cell, the flux through the cell wall, the flux through the membrane, and cellular consumption must be equal. The flux depends on the total driving force which is the concentration differential between the bulk medium and that inside the cell. This can be likened to a resistance movement in just the same way that Ohm’s law relates the current to thevoltage and the resistance. Fick’s law, in its finite difference form as in Equation 33 :
AC (33) AR is of the form of Ohm’s law if we call D , A / A R the reciprocal of the resisv=-
tance. This reciprocal is called the conductance in the study of electricity. If there is more than one resistance element either in the electrical or in the diffusion case, and if they are in series as in our case, the total resistance determines the flux and is the sum of the separate resistances. Thus, if there are a number of chemical and diffusion reactions in transport, as for example in the model for the permease transport system analysed by Koch (1967), the flux a t low concentration is proportional to the concentration where the proportionality constant is : 1
where the values of k are first-order rate constants for the successive steps in the process. I n the example cited, k , is the rate constant for
THE ADAPTIVE RESPONSES OF ESCHERICHIA COLI
209
substrate with the permease, k, is for the transfer t o the transporter, k, is for diffusion, and 00 on. The negative subscripts refer t o the back reactions. I n the case where the forward and backward process have the same rate constant, this simplifiesto : 1
which is the same form as the rule for adding the electrical conductances of a series of resistors. This means that the step with the smallest conductance is the most important in determining the total flux in a steady-state system. For von Smoluchowski-limited external diffusion up to a sphere, the conductance term (see Equation 21, p. 198) is 4vD0R.For simple membrane permeability it is 4vPRo2(see Equation 26, p. 202), or, as indicated above, we can subdivide the conductance amongst many steps in the permeation process. Internal consumption, if it is first order throughout the inside of the sphere, is given by : q cosh q - sinh q 4rDiR( sinhq where q is :
(Koch and Coffman, 1970); V , is the uptake by a single sphere and D, is the diffusion inside the cell. As long as the steps are first order and do not saturate, we can combine conductances by summing the reciprocals and then taking the reciprocal of the sum. We can then use this in calculating the flux in the cases where many steps are each partial bottlenecks. In those cases considered above, one step alone was the bottleneck. This rule of combining conductances turns out to be applicable t o uptake by a sphere even during motion (Koch, 1960). The proof of this is quite difficult and will not be given. For our purposes, this means that, even if the concentration at the surface of the sphere during the steady state does not become zero because the cell has more than one bottleneck and therefore that Equation 21 (p. 198) does not apply, an equation with the combined conductance factor but the same value of G applies as would apply if the cell was still. This means that the arguments proposed above for the case when external diffusion was completely limiting also have validity even when uptake a t the cell surface is only adequate to deplete partially the substrate a t the cell surface.
210
ARTHUR L. KOUH
F. EXPERIMENTAL DETERMINATION OF UPTAKE PARAMETERS BY GROWTH STUDIES For uptake processes with avid affinity constants (where the value of
K' is small) or where the maximal capacity is very high (the value of V is large), the apparent Michaelis-Menten constant for growth is very low. I n these cases, it is very difficult to follow growth at concentrations giving intermediate growth rates, because the organisms consume and alter the nutrient concentration during a period when the number of organisms hardly changes. Therefore, it is necessary to resort to low concentrations of cells and sensitive methods for their measurement. I n this section, I describe our experimental approach t o this problem and some of our results relevant t o the previous section. Growth is followed in a Cary model 16 double-beam spectrophotometer ab 420 nm. This instrument is extremely stable, permitting accurate absorbance measurements even days after the cuvette was blanked. For growth studies, the cuvette holder is thermostatically controlled as is also the cuvette compartment. The cuvette holder has fittings for circulating water, and an electric motor coupled to a magnet for rotating a Teflon-coated magnet in a 2 cm. x 2 cm. cuvette. The output from the spectrophotometer is accurately converted into a voltage proportional to absorbance, which is then recorded; usually one inch corresponds to 0.01 di. Noise level in its final configuration of the experimental set-up is about one-fiftieth of an inch. Zero suppression can be accurately subtracted in increments of 0.1. For the present application, we never go above A = 0.3. Even so, for most accurate results, we use a correction for the fact that the apparent absorbance due to turbidity is not linear with concentration. We find that (w) = 0.1361 A + 0.03719 A2 in a l-cm. cuvette. On dividing by 2, it applies just as well t o the 2-cm. cuvette. This formula applies within 1% up to A = 1.1. It applies to E . coli growing at a variety of growth rates and t o Bacillus megaterium. Theoretically the same relationship should apply to any cell inside this size range unless i t contains a large amount of substance with a high index of refraction (Koch, 1961). The Cary model 16 is a well collimated instrument; therefore, a higher absorbance is measured than would be measured in less well collimated instruments where some scattered light is also intercepted by the phototube. Relative to an ideal turbidimeter, the Cary model 16 or a Zeiss PMQ I1 are both about 93% efficient for E . coli at 420nm (A.L. Koch, unpublished measurements). The problem of batch-culture growth, where the growth rate constant, A, changes because of substrate utilization, was worked out by Monod (1942). If the growth-rate constant is given by Equation 3 (p. 162),
211
THE ADAPTIVE RESPONSE9 OF ESCBERICHIA COLI
Monod's relationship, in terms of the quantities already defined, can be given as : 2=-(
(A,.,
K + C o + & ( w ) (4 K --In-~ m m . co + Q(w)o W O Qo + 1
In co + Q ( 4 0 - Q(w) (34) CO
)
This equation contains a number of parameters, but most of them Co, (w),,, and&)are obtained as an integral part of each experiment.
0-051 0
I
I
30
60
Time (rnin)
FIQ.23. Time-course of growth of Eschevichia coli ML-308 in media containing limiting and non-limiting concentrations of glucose. The curves are tracings of the chart recording. The geometrical construction described in the text is used to calculate the point on the curve where the growth-rate constant (A) is one-half that of unlimited culture. Also shown is the best fit for the data to the Monod equation (closed dots). The growth curve actually bends more sharply than predicted by Equation 34 (p. 211). Over this limited range a larger estimate of growth-rate constant, A,,, than measured for the control and a larger K fit best. The values of E calculated with either set of A,,. and K estimates are nearly the same, 68/sec. v8 53*2/sec.,respectively.
A computer programme was set up to compute the time corresponding to any value of ( w ) ,given an estimated value of K . With this programme it is a simple matter by trial and error to choose the value of K that best fits the data. More sophisticated procedures have so far not been needed. We have found very helpful the following graphical procedure' which gives nearly the same results as the computer. Conditions are adjusted so that a culture is given such a small amount of the essential nutrient that growth ceases within the span of the recording. A second recording
212
ARTRTJR L. KOOH
on the same piece of graph paper is obtained on the same scale for a culture given an excess of the limiting nutrient. A copy of an original recording and the geometrical construction are shown in Fig. 23. We approximate that point on the limited growth curve where the slope is about half that of the corresponding unlimited growth curve a t the same value of (w). Usually, this can be closely estimated by visual inspection.
Time (hours)
FIG.24. Fit of lactose utilization to the Monod equation. A culture of Eacherichia ooli ML-308 growing on lactose was harvosted and the cells washed once and stored on ice. Forty-eight minutes before the time taken as zero on the graph, the culture was allowed t o grow in the presonco of 73 pM-lactose at 36". The dry woightconcentration data were fitted with theh,,,. of the control culture with 0.2% (w/v) laotose corresponding t o a doubling time of 51.5 minutes. The K value for the line shown is 143 pM-lactose. In an independent experiment with the same batch and with the aame number of cells, the initial growth rate at different concentrations of lactose was followed for a time sufficiently short that utilization could be neglected. When these data were treated by the standard Lineweaver-Rurk double reciprocal plot, a K value of 121 p M was calculated. The A,,,. and R values in the data shown in the figure gave a value of E of 3.04 per second.
Then the tangent or the normal to the unlimited growth curve is drawn through that point. Half-silvered mirrors are very helpful in drawing the normal. Then the slope of the tangent is halved or the normal is doubled by geometric construction. The procedure with the normal was used with the data in Fig. 23. Then a line parallel to the tangent or perpendicular to the normal is drawn tangentially to the limited curve. The value of (w) at this point is designated (w) ,/2 and corresponds to the amount of cell material when the amount of nutrient just supports half maximal
213
!THE ADAPTIVE RESPONSES OF ESCEERICEIA COLI
growth. The difference between this value and the final value of (w), divided by Q is an estimate of K. If the original guess of ( w ) , ,was ~ inaccurate, the calculation must be repeated using the new value.
0 '
3 Time ( m i d
FIG. 25. Growth curves of lactose-limited chemostat cells of E.9cheriohicc coli ML-308. Cells from a lactose-limited culture in which the doubling time waa 64 min. at 37' were placed in the growth chamber in the spectrophotometer. In the data on curve A, the cells grew with a 69-min. doubling time in the presence ofhigh concentration of lactose (760 p M ) and thiodigalactoside (250pM). Curve B shows data for cells growing in media containing a low concentration (76 p M ) of lactose. The solid dots are fitted to the Monod equation with A,,,,=. = 0.693/34*4min. and K = 31 p M . These values lead to a value of E of 21*O/sec.at 36'. Note that the maximum doubling time of the control cultures was 56 min. Curve C shows data for growth on 76 pM-lactose in the presence of 25 p.M-thiodigalactoside. This concentration of thiodigalactoside is expected to inhibit uptake of lactose to 0.326 of the maximal growth-rateconstant, baaed on K = 20p.M for thiodigalectoside and K = 70 p M for lactose uptake (Kepes and Cohen, 1962).Curve D shows growth on 76 pitI-lactose and 250 pM-thiodigalectoside. This concentration of thiodigalactoside inhibits the rate of entry of lactose to 0.0744 of the maximal, or a decrease of 13.4-fold. Actually the growth rate was decreased from 0.693/56 min. to 0.6931223 min., e f&ctor of 3.9-fold.
From the value of K and the corresponding A,, . value, the efficiency can be calculated. Several examples are given in Table 3 (p. 201) of data computed from Figs. 23,24, and 25. The values of E for growth in glucosecontaining batch cultures are virtually identical when computed from data 30 years old or from our recent experiments, which embarrassingly
214
ARTHUR L. KOOH
required about a 100 times larger capital investment. The results with lactose-grown cells show an interesting side line. Batch growth of E. coli ML-308 on lactose yields cells with one-tenth the efficiency of cells from rapidly growing lactose-limited chemostat cultures. The organisms are constitutive for p-galactosidase and permease production. However, in the presence of high concentrations of lactose in batch cultures, they are catabolite-repressed to a high enough degree as to have a decremed efficiency.
IX. General Conclusions Escherichia coli is a creature wonderfully adapted to its ecological niche. This review has examined several aspects of its fitness for survival in the lower intestine, and has shown that E. coli is optimally designed for efficient growth in its high viscosity habitat with its highly sporadic nutrient input. It is now clear that control of protein synthesis is both at the level of the control of synthesis of the translation machinery and at the level of the efficiency with which it is used. I n addition to envisaging controls on the rate of ribosome formation, a t the transcription level, we must now consider controls a t the level of the catabolism of r-RNA (and possibly nascent t-RNA). The problem has become more complex regarding the efficiency with which the ribosomes function in protein synthesis. The limitation in carbon-limited chemostat cells, and presumably also under balanced growth conditions with poor carbon sources, is not a t the level of the machinery for protein synthesis: there are adequate ribosomes and t-RNA for much more protein synthesis. Hopefully, there exists a profound and interesting control mechanism(s) so that the cell can have reserves that can be instantly mustered; possibly, there is only a trivial limitation for energy or some amino acid(s). With respect to uptake, it now appears that a number of transport systems have evolved to a pitch of efficiency such that further improvement would be useless in high viscosity medium. However, motility and especially movement causing local stirring probably do aid uptake. It would appear from the analysis presented here that more efficient transport systems might be found in organisms habitually occupying a lower viscosity medium, and that further improvement of permease systems might be achieved under evolutionary chemostat conditions.
X. Acknowledgements Work in this laboratory has been supported by the National Science Foundation and by the United States Public Health Service, currently
THE ADAPTIVE RESPONSES O F E S C B E R I C H I A COLl
215
under NSP GB-7846 and USPHS AI-9337. The major debt of gratitude goes to my sometime students, Elvera Ehrenfeld, Nicholas Peterson, Penelope Gumapas Clark, John Boniface, Thomas Norris, Kamal Nath, Carol Deppe, Robert Coffman, Barry Dancis, Kenneth Bernstein, and Thomas Alton. Science progresses on ideas whether they be right or wrong; sometimes the ideas are truly joint, sometimes they are personal property. Among such property rights are : That organisms might electively control functional ribosome formation a t a very late stage of particle maturation even a t the size and complexity of 30 and 505 ribosome subunits ; Elvera Ehrenfeld. That the speed of transcription and translation a t slow growth rates could be estimated by measurements of the first appearance of the completed polypeptide chains of an induced enzyme ; Thomas Norris. That during amino acid starvation of stringent organisms and possibly in other cases as well, control would be exerted not a t the level of anabolism, but at the catabolism even for so-called stable RNA species such as r-RNA and t-RNA ;Barry Dancis.
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Hershey, A. D. and Bronfenbrenner, J. ( 1 9 3 q . J . gen. Physiol. 21,721. Hinshelwood, C. (1952).J.chem.Soc. 745. Horiuchi, T., Tomizawn, J. and Novick, A. (1961). Biochim. biophys. Acta 55, 152. Jaeger,J.C.(1956).J.math.Phys.34,316. Julien, J., Rosset, R. and Monier, R. (1968).Bull.Soc. Chim. biol. 49,131. Kelley, W. S. and Schaechter, M. (1968).Adv. microbial physiol. 2,89. Kennell, D. ( 1 9 6 8 ) J .molec. Biol. 34,86. Kepes,A. (1963).Biochim. biophys. Acta76,293. Kepes, A. and Cohen, G. N. (1962).I n “The Bacteria”, (I.C. Gunsalus and R. Y. Stanier, eds.), Vol. IV, pp. 179-221. Academic Press, New York. Kjeldgaard, N. O., Maalee, 0. and Schaechter, M. (1968).J.gen. MicrobioZ. 19,607. Koch, A. L. (1960).Biochim. biophys. Acta 39,311. Kooh, A. L. (1961).Biochim. biophys. Acta 51,429. Koch, A. L. (1962).J. theor. Biol. 3,283. Koch, A. L. (1964).Biochim. biophys. Acla 79,177. Koch, A. L. (1965).Nature, Lond. 205,800. Koch, A. L. ( 1 9 6 7 ) J .theor. Biol. 14,103. Koch, A. L. (1968).J.theor. Bio2.18,105. Koch, A. L. (1970).J. theor. Biol. 28, 203. Koch, A. L. ( 1 9 7 1 ) J .theor. Biol. in the press. Koch, A. L. and Coffman, R. (1970).BiotechnoZ. Bioengng. 12, 651. Koch, A. L. and Deppe, C. S. (1971).J. molec. Biol. in the press. Koch, A. L. and Schaechter, M. (1962).J.gen. Microbiol. 29,435. Kubitschek, H. E . ( 1 9 6 4 ) J .Bact. 67,264. Kurland, C. G. and Maalee, 0. ( 1 9 6 2 ) J .molec. Bio2.4.193. Lacroute, F . arid Stent, G. S. ( 1 9 6 8 ) J .molec. Biol. 35,166. Lehman, I. R., Bessman, M. J., Simms, E. S. and Kornberg, A. (1958). J. biol. Chem. 233,163. Liebig, J. (1843). “Chemistry in its Application to Agriculture and Physiology”. Peterson, Philadelphia. Maslee, 0. (197O).Symp.Soc. dev. Biol. 29, in the press. Maalee, 0. and Kjeldgaard, N. 0. (1966). “Control o f Macromolecular Synthesis”. W. A. Benjamin, New York. Meitra, U. and Hurwitz, J. ( 1 9 6 7 ) J .biol. Chem. 242,4897. Manor, H., Goodman, D. and Stent, C:. S. (1969).J. molec. Biol. 3 9 , l . McCarthy, B. J., Britten, R. J. and Roberts, R. B. (1962).Biophys. J . 2,67. McQuillen, K. (1965).Symp.Soc.gen. Microbiol. 15, 134. Michaelis, L. andMenten,M. L. (1913).Biochem. Z.49,333. Monod, J . (1942). “La Croimance des cultures bact6riennes”. Hermann et Cie, Paris. Monod, J. (1960).Annla Inst. Paateur, Paria79.390. Morse, L. and Carter, C. E. ( 1 9 4 8 ) J .Bact. 58,317. Nath, K . and Koch, A. L. ( 1 9 7 0 ) J .bioZ.Chem. 245,2889. Neidhardt, F . C. (1963).Biochim. biophys. Acta 68,365. Nierlich, D. P. (1967).Science,N. Y. 158,1186. Nierlich, D. P. (1968).Proc. natn. Acad.Sci. U.S.A. 60,1345. Norris, T. E . (1970).Ph.D. Thesis :IndianaUniversity, Bloomington, Indiana. Norris, T. E. and Koch, A. L. (1972).J. molec. Biol. in the press. Novick, A. (1958).In “Perspectives in Marine Biology”, (A. A. Buzzati-Travorm, ed.), pp. 833-546. University of California Press, Berkeley. Novick, A. and Horiuchi, T. (1961).CoMSpring Harb. S y m p . quant. Biol. 26, 539.
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Bacterial Flagella R. W. SMITH AND HENRYKOFFLER Departmeijt of Biological Sciences, Purdue University, Lafayette, Indiana, U . S . A . I. Introduction . 11. Basal Material and Site of Attachinelit . 111. TheHook , IV. Sheath-Like Structures . V. Isolation and Purification of Flagellar b’ilaments VI. The Protein Nature of the IWament . VII. Immunology , VIII. Stability . IX. Arrangement of Protein Subunits . X. Re-assembly , XI. Synthesis of t h e Filament . XII. Mechanisms for the Function of Flagella . XIII. Acknowledgements . References ,
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219 223 230 238 239 240 251 260 276 284 295 314 327 327
I. Introduction Bacterial flagella have become of great biological interest because they areuseful modelsinstudiesdealing with theconversionof chemical energy to motion and with molecular aspects of morphogenesis. Although the existence of flagella and their role as organelles of locomotion were first suggested by Ehrenberg (1838) and later described by Cohn (1872), Dallinger and Drysdale [1875, cf. Houwink and van Iterson ( 1950)],Warming [ 1875,cf. Houwink and van Iterson ( 1950)],and Koch (1877), a major impetus to recent studies of their structure and function was provided by Adrianus Pijper. Pijper attempted t o revive the theories of van Tieghem (1879), Kurth (1883), deBary (1887), and Hueppe ( 1896) that bacterial flagella are passive, useless appendages and are the result, not the cause, of cell locomotion (Pijper, 1938,1940, 1946, 1947, 1947a, b, 1948, 1948a, 1949, 1949a, b ; Pijper et al., 1953; Pijper and Abraham, 1954; Pijper et al., 1955, 1956; Pijper, 1957, 1957a). Alternate interpretations of Pijper’s observations and data supporting the opposing viewpoint have been presented from numerous laboratories (JohnsonandBaker, 1947; Brskov, 1947; Boltjes, 1948,1948a; Houwink 219
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R . W. SMITH AND HENRY KOFFLER
and van Iterson, 1950; Rinlter et al., 1950; DeRobertis arid Peluffo, 1951; Mallett el nl., 1951 ; Rinker, 1957; Stocker and Campbell, 1959; Stocker, 1957 ; van Iterson, 1953). Most investigators now accept the view that bacterial flagella are active organelles of locomotion. Before Pijper’s attack on the notion that flagella participate actively in motility, most, reports dealt with their antigenicity (Smith and Tenbroeck, 1904; Gruschka, 1922; Yokota, 1925; KauffmannandMitsui, 1930), the effect on motility of media (Dimitrijevic’-Speth, 1929; Kramer and Koch, 1931 ; Colquhoun and Kirkpatrick, 1932) and temperature (Mironesco, 1899 ; Matzushita, 1901 ; Kossel and Overbech, 1902 ; Nicolle and Trenel, 1902 ; Neustadtl, 1917 ; Brauii and Lowenstein, 1924; Braun and Weil, 1928), their utility in taxonomy (Arkwright, 1927; Moltke, 1927), the swarming phenomenon (Weil and Felix, 1917; Seiffert, 1920; Moltke, 1929; Russ-Munzer, 1936), and cell velocity (Ljuchowotzky, 1911 ; Sanarelli, 1919; Ogiuti, 1936). Hardly any information on the chemistry of flagella was reported prior t o 1950. More recently, work on bacterial flagella has been covered in reviews on rather specialized areas of investigation, for example, isolation (Koffler, 1967), synthesis (Kerridge, 1961), self-assembly (Kushner, 1969), genetics (Iino and Lederberg, 1964; Iino, 1969a), structure as related to function (Weibull, 1960; Rogers and Filshie, 1963; Burge and Holwill, 1965; Jahn and Bovec, 1965; Newton and Kerridge, 1965; Lowy et al., 1966; Oosawa d al., 1966; Klug, 1967; Lowy and Spencer, 1968), and importance in tuxononly (Leifson, 1960, 1966; Ithodes, 1965). Since this summary was begun, well written reviews on the biochemistry of bacterial flagellu have also been presented by Joys (1968) and Doetsch and Hageage (1968). Individual flagelltt are too thin to be visualized by ordinary light microscopy without special staining techniques. On the cell, however, they tend to form bundles or aggregates that may be seen by dark-field microscopy (Fischer, 1895; Migula, 1900; Reichert, 1909; Neuman, 1925; Pijper, 1930; Weibull, 1950a, c). In suspension, flagella occur as individual spirals which upon drying collapse into filaments that describe a sine wave (Fig. 1) with a wavelength of 2 to 3 microns and an amplitude of 0.25 to 0.60 microns (Pietschmann, 1942; Leifson et al., 1955 ; Leifson, 1960). Generally, the wavelength is approximately four times the amplitude (Leifson and Palen, 1955). Earlier pictures of dried bundles of flagella indicated that individual filaments associate with one another in a sidewise fashion (Houwink and van Iterson, 1950; Weibull, 1960). Mitani and Iino ( 1965, 1968) demonstrated that in suspension each filament in a bundle is spirally coiled. The bundles then appear t o be twisted into t t helicttl shape. ,J. R. Mitchen and H. Koffler (unpublished results) have observed that wire models of flagellar filaments consisting of
BACTERIAL FLAGELLA
22 1
FIG,1. Proteus wulgariv negatively stained with phosphotiiiigstic acid x 35,000. D. A. Abrain and H. Koffler, unpriblishctl obsc>rvatioris.
222
R.
W.
SMITH AND HENRY KOFFLER
spinning heliaea may intertwine without bccoming ~nt~angled as long as the sense of the helices is the same and opposite to the direction of rotation. Similar observations have been made and are discussed by Lowy and Spencer (1968). The interactions between spinning helices have been extensively studied and reconstructed in models by Jarosch (1966,1967,1968). Koch [1876, cf. Houwink and van Iterson (1960)l was the first to stain
and photograph bacterial flagella. Subsequent improvements in staining techniques considerably increased the ease with which flagella in dried preparations could be observed (Loeffler, 1889, 1890; Shunk, 1920; Gray, 1926; Conn and Wolfe, 1938; Hofer and Wilson, 1938; Leifson, 1938; Fisher and Conn, 1942; Leifson, 1961; Rhodes, 1968; Leifson, 1960; Blendon and Goldberg, 1966; Caldwell et al., 1966). A s techniques improved, reports appeared describing filaments with morphologies which departed from the “normal”. For instance, while examining cells of Bacillus cereus immobilized in tragacanth, Pietschmann ( 1942) noticed helical filaments whose pitch of 1-2 microns differed from the pitch of 2.4 microns of the normal structure, and also showed a form of Proteus vulgaris flagella with two different pitch lengths in the same filament. Leifson (1961) reported that the length of flagella may vary with the medium and age of the culture; he also found a variant with EL pitch one-half normal denoted by the term “curly”. Leifson and Hugh ( 1963) reported additional variations in morphology such as straight filaments, filaments hooked at the distal end, and filaments coiled into a circle. Flagellar filaments of P.rnirahilis are completely changed from normal to curly by adjustment of the pH value from 8.0 t o 6.0 (Leifson et al., 1966). Mixed types (but not with intermediate pitch lengths) are found a t pH values between 6.7 and 7.2. These same authors described a semi-coiled variety with a pitch characteristic of the curly type but possessing an amplitude twice that of curly filaments. Leifson and Palen ( 1966) also found variants of Listeria some of which were non-flagellated while others were non-motile and had flagella with either an abnormally small amplitude or straight filaments. They also found an abnormal form that has short, coiled filaments, which produce only a slow erratic type of motility. The aberrant forms revert t o a normal morphology a t a rate of lo-* to per division. Later, Leifson (1961) noted that all the aberrant morphologies except curly could be induced in some organisms by the addition of 6-10% formaldehyde. Apparently, the abnormal forms can arise not only by mutation but also by treatment with non-mutagenic compounds (formaldehyde, hydrochloric acid). Electron microscopically, the flagellum can be seen to consist of the following three morphologically distinct parts : a basal structure that is closely associated with the cytoplasmic membrane and cell wall, a hook,
BACTERIAL FLAGELLA
223
and the main spiral filament, apparently a tube, the wall of which is constructed of the protein named flagellin by Astbury et al. (1955). Since the filament is the most prominent feature of the bacterial flagellum, it has been studied most extensively, and a large body of knowledge has been accumulated regarding its nature. The hook and basal structure, on the other hand, constitute only minor portions of the organelle, and relatively little is known about them, although the recent purification of hooks promises more tangible information regarding that region. Studies on the basal structure, moreover, have been complicated by its internal location, its intricate connection with membranous material, and its fragility. Because of the ease with which this structure is converted to artifacts, a description of its true nature is just beginning to emerge. We shall refer to these differentiated substructures specifically as the basal structure, the hook, and the filament, with the term “flagellum” reserved to indicate the entire organelle. Preparations of isolated ‘flagella” are predominantly filament material although, depending on the method of isolation, basal regions and hooks are also present in varying degrees. Initial electron microscopic observations supported observations made by light microscopy. Filaments, 120 to 200 A in diameter and several microns long, described a sine wave, a predictable phenomenon after the collapse of a spiral filament. Since the bulk of recent work involves the use of electron-opaque negative stains in which the preparation is submerged, many estimates regarding the diameter of the filament are probably low. The maintenance of a regular wave length by filaments after separation from the cell body and after drying suggests a t least a certain amount of rigidity in the structure. However, there is still some question as to the degree of rigidity in vivo (Lowy and Spencer, 1968). Significant distortion is encountered when samples are dried for electron microscopy. For example, the wavelength of filaments in dried samples may be from 1.3 to 1-6 times that observed by dark-field microscopy (IinoandMitani, 1966). 11. Basal Material and Site of Attachment Due to the limited resolving power of the light microscope, the manner in which flagella are attached to the cell remained obscure for a long time. Historically, flagella were thought t o arise from superficial layers of the cell (Babes, 1895; Bunge, 1895, Hinterberger and Reitman, 1904; Meyer, 1912), the cell wall (Pijper, 1949b), the cell membrane, the cytoplasm (Ellis, 1903, 1903a, b ; Lasseur and Verneir, 1923), and a blepharoplast inside the protoplast (Fischer, 1897 ; Yamamoto, 1910; Prenant, 1915; Butschli, 1902; Fuhrmann, 1910; Yuasa, 1936). Eventu-
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W. SMITH
AND HENRY KOFPLER
ally electron microscopy revealed that flagella indeed penetrate the cell wall but left unresolved the method of attachment on or inside the cell rnembrane (Mudd and Anderson, 1942; Johnson et al., 1943; Rinker arld Koffler, 1949; Houwink and van Iterson, 1950; Bisset and Hale, 1951 ; Salton iind Horne, 1 % 1 ) . Flagella are tightly attached to the cytoplasmic membrane, as can be observed when the membrane withdraws from the wall during autolysis (Abram et al., 1965, 1966). Furthermore, flagella are still tittarhetl to the protoplast after removal of the cell wall (Mudd and Anderson, 1941 ; Weibull, 1953; Wiame et al., 1955). The proximity and attachment of the flagellum to the cytoplasmic membrane probably represent design features essential to flagellar function. First, regardless of whether motion is brought about mechnnically or by conformational changes in flagellin, some structural changes requiring energy must be involved, and it is most plausible that the initial energy input occurs at the level of the membrane where many energy-transfer reactions take place. Second, if motile bacteria are capable of responding to environmental stimuli resulting in attraction or avoidance, the cytoplasmic membrane may be the medium through which such information is transmitted. Third, for logistic reasons it is unlilrely that flagellin and other flagellar constituents are synthesized throughout thc whole cell. It seems reasonable that they may be synthesized near the site at which they will subsequently be assembled into the organelle, perhaps on polysomes attached to the cytoplasmic or other membranes. At the moment, however, the observations of Murray and Birch-Anderson (1903) cannot be reconciled with such a view. Apparently flagella are attached to the cell by a basal structure either a t the cell mcrnbrane or in the cytoplasm (Mudd et at!., 1942; van Iterson, 1947; Lofgren, 194%;Houwink and van Iterson, 1950; Bisset and Hale, 1951). Van Iterson (1953) suggested that basal structures may not be presciit in all species, while Pijper (194913, 1957) went further and thought all such ityparcnt structures to bc artifacts since they are most easily seen after autolysis and withdrawal of the cytoplasm from the cell mcmbrttne. Observation of basal structures is almost impossible in intact rclls; identification is difficult even in the envelope of lysed cells unless their location is indicated by the presence of the external portion of the flagclluni (see Pig. 2). However, the existence of a structure in the basal region of some, if not all, flagella appears certain in light of the studies by AbrtLm et ul. (1961, 1965, 1966), van Iterson et al. (1966), Hoeniger et al. (1966), Cohen-Bazire and London ( l M 7 ) , and Abram (1968) Riisal regions have heen reported in Proleus spp. (Houwink and , vai: iternon and Lecne, 1904; Hoeiiivan lt(wmi, l!M); l ’ r c ~ i w ~195%; ger, 1965; Ahram c.t d . , l!MX; van Tterson et al., 1966; Hoenigcr c.f a / . ,
BACTERIAL FLAQELLA
225
FIG.2. Protevs wztk~ari.9nutolyscd by growth at 4" for 8 vcelis. Nrgntivcly sttiiiied with phosphotu~igaticacid x 75,000. From rriipublish(d tlntn of E. .I. McGroarty, H. Koffler and R. W. Smith.
226
R. W. SMITH AND HENRY KOPPLER
1966 ; Abram 1968), Spirillum spp. (Pease, 1966 ; Grace, 1954 ; Lofgren, 1948 ; Williams and Chapman, 1961 ; Murray and Birch-Anderson, 1963 ; Abram, 1969), Vibrio spp. (Mudd et al., 1942; Das and Chatterjee, 1966; Ritchie et al., 1966; Tawara, 1964; Glauert et al., 1963; Takagi and Asaki, 1960; van Iterson, 1947 ; Tawara, 1957), Chromobacterium sp. (Sneath, 1956)) Aerobacter sp. (Thornley and Horne, 1962), Leptospira sp. (Nauman, 1967), Rhodospirillum spp. (Cohen-Bazire and London, 1967), Rhodopseudomonas palustris (Tauschel and Drews, 1969), and Elctothiorhodospira mobilis (Remsen et al., 1968). The major questions now
pertain to their detailed structure and function. Descriptions of size and shape vary considerably and with almost no information available as to their function much work remains to be done. The first reports on morphology describe the basal structures as spherical, bulbous, dense granules (Lofgren, 194%; Houwink and van Iterson, 1950 ; Grace, 1954; Tawara, 1957).The flagella of Vibrio riLdchnikowii appear to terminate in a basal disc or cup, 300 t o 350 A in diameter, located just inside a granule-free area of the cell membrane (Gluuert, et al., 1963). A cup-shaped basal structure, 640 A in diameter, has been described for V . cholerae (Das and Chatterjee, 1966). Studying the same organism, Grace ( 1954) previously reported a spherical basal structure about 1,000 A in diameter. The basal structure in 1'. fetus appears as a cone-shaped structure with a maximum diameter of approximately 500 A (Ritchie et al., 1966).The cell membrane also appears differentiated about the area in which flagella are inserted (Abram et al., 1965,1966; Ritchie et al., 1966). Ritchie and Bryner (1969) recently presented a more elaborate description of the basal structure in V .fetus. Two discs, 200 A in diameter, are spaced about 100 A apart with the outer disc attached to a differentiated region of the cell wall and the inner one attached t o the cell membrane. Tawara (1957) noticed that filaments of 1'.comma are attached to dense granules, 1,500 to 2,000 A in diameter, located in the cytoplasm. The size of the granules appears to decrease as the cells get older. This latter observation may prove pertinent when more information is obtained concerning t+ Bynthesis of the basal structures. Grace (1954) reported that all the filaments in bundles of flagella of Spirillum spp. arise from a single basal structure whereas in other organisms each flagellum is attached to a single basal structure. These results were supported by Pdase (1966). Later, Murray and BirchAnderson (1963) in sectioned S. serpens stained with phosphotungstate noticed that flagella arise from knobs inside the cell membrane and pass through the membrane individually. Abram (1969) has now clearly demonstrated in S . serpens that each filament is joined to a single basal structure, 390 to 430 A in diameter. In thin sections a polar membrane appears to be located parallel to and 200 A inside the cell membrane
BACTERIAL FLAGELLA
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proper (Murray and Birch-Anderson, 1963). The polar membrane is linked to the cell membrane by fine bars and appears to be discontinuous immediately beneath the areas at which flagella are inserted. The region adjacent to the sites of insertion appears to be free of ribosomes and membranous organelles. The biological significance of these observations made in sectioned material cannot yet be evaluated. To our knowledge, it has not been possible to observe the polar membrane in intact cells. The basal structures of P. mirabilis are spherical with a diameter of either 20 m p (Hoeniger, 1965))25 t o 46 mp (van Iterson et al., 1966),or 50 mp (Hoeniger et al., 1966). The frequent observation of paired basal structures and of single structures one-half the normal size may suggest that they are self-reproducing structures. The same authors show that there is only one flagelluni per basal structure, that the basal structures are fragile, and that they are located close to but separate from the cell membrane. I n penicillin-treated swarming cells, thin strands are seen connecting the basal structures and cell contents. In autolysed or elongated cclls of P.vulgaris, filaments are attached to spherical structures, 110to 140Aindiameter(Abrametal., 1965).Asin V.fetus,themembrane appears to be differentiated about the area of insertion. The biological significance of this observation still remains to be demonstrated. The heterogeneity in size reported for the basal structure probably is due to the tearing of the cell membrane with a portion of the membrane folding about the basal structure of remnants of the basal structure. Although this factor certainly explains variations in size of basal regions torn from cells during removal of filaments, the actual size of the basal region within the protoplast is larger than indicated in the report of Abram et al. (1965) since the structures observed most likely do not constitute the complete basal region. I n autolysed elongated cells, strands or fibres are seen which appear to connect the basalstructures (8bram etal., 1965). As mentioned previously, these interconnecting strands were also seen by Hoeniger et al. (1966). Possibly, these are analogous t o the special membranes connecting the basal structures of S . serpens (Murray and Birch-Anderson, 1963) and Rhodospirillum spp. (Cohen-Bazire and London, 1967). Certainly the speculation that these form a reticulum on which the flagellar proteins may be synthesized or that they function in the act,ivation or co-ordination of the movement of the filaments warrants careful examination although a t t h e moment there is no convincing evidence to support this hypothesis. Flagellar filaments attached to cells of P. vulgaris damaged by Rdellovibrio bacteriovorus 109 originate within the protoplast from spherical bodies, 390 t o 430 A in diameter (Abrarn, 1968). A structure similar t o a disc or double discs, 160 to 180 a in diameter and 40 to 60 a thick, joins to the proximal end of the hook
228
R . W. SMITH A N D HENRY KOFFLER
by a stalk 30 to 40 A thick. Surrounding this structure is a thin, fragile layer of‘material 26 to 35 A thick. Doughnut-shapcd objects, 40 to 50 A in diameter, can be seen on the inner side of the cell membrane and appear to be specifically associated with the sites of origin of flagella. They are observed in large numbers on fragments of membrane that retain numerous flagella. Apparently, the basal region lies immediately inside the cell membrane with specialized structures attached to the inner side on the membrane. I n Rh. rubrum, Rh. molischianurn, and Rh. fulvum, hooks are connected to paired discs in the basal structure by a short, narrow collar that passes through both the cell wall and the membrane (Cohen-Bazire and London, 1967). The paired disc is then connected to a second paired disc. The structures are joined by a “flagellar membrane” similar t o the polar membrane of Murray and Birch-Anderson ( 1963). The basal structure of Ectolhiorhodospira rnobilis Pelsh. consists of il pair of discs 80 tf thick, 160 to 200 tf apart, and connected by a thin rod (Remsen et al., 1968).The outer disc is 200 to 250 A in diameter and about 200 A thick and may be composed of two smaller structures, 100 A thick, separated by a distance of 60 A. The inner disc is 200 tf in diameter and 100 A thick. Each basal structure appears t o be associated with a larger basal disc, 400 to 500 b in diameter. Eight to ten of the basal discs further appear to be associated into a polar plate, 2,500 A in diameter. The fine structure and origin of the basal region in Rhodopeudomonae palustris has been studied in thin sections by Tauschel and Drews (1969). They describe a spherical basal structure with a core in its centre. Ten spokes, 85 to 90 A in length, attach the inner and outer boundaries. Three types of basal regions are defined which are differentiated by size, location, and membrane involvement. The first type is 460 A in diameter bounded by a layer distinguishable from the cell membrane. The second type is 600 to 650 A in diameter, located between the cell membrane and wall, and is surrounded by a membrane that is continuous with the cell membrane. Two layers of membrane surround basal structures of the third type which have a diameter of 1,000 to 1,100 b and are located in the cytoplasm close to the cell membrane. These appear to be attached to the cell membrane by membranous stalks. Tauschel and Drews propose that these types represent various stages of engulfment of the basal structures by cell membrane material. The basal structure cannot be clearly defined in all organisms studied so far. For example, the basal material in cells of various Bacillus species, when it can be observed a t all, appears to be so heterogeneous both in size and shape as to defy accurate description (Abram et al., 1964, 1966). While it is possible that some organisms do not possess basal structures, from n comparative point of’view this would be unexpected. I n those
BACTERIAL FLAGELLA
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cases where none is observed, it is more likely that most of the structure itself deteriorates under favourable experimental conditions and what remains are its remnants surrounded by torn pieces of cell membrane. In any case, in these organisms too, the firm attachment of the flagellum to the cytoplasmic membrane can be readily demonstrated. A t this point, our knowledge concerning the function of the basal region is regrettably limited. Possible functions include the synthesis and/or polymerization of flagellar constituents, especially the protein subunits of the filament, mediation and control of the movement of the flagellum, and finally anchorage of the filament to the cell body. Roberts and Doetsch (1966) report that monotrichic organisms are able to synthesize flagellar filaments in the presence of chloramphenicol a t concentrations 100-fold greater than required t o inhibit cell division. They postulate that the basal granule is the site of the synthesis of flagellin and is impervious to chloramphenicol. Monotrichic, unlike peritrichic, organisms continue t o synthesize flagellar protein after infection with a lytic phage. The authors suggest the existence in the basal structure of satellite DNA resistant t o attack by the phage deoxyribonuclease. These observations also may indicate a difference in the manner in which monotrichic and peritrichic organisms synthesize or assemble flagellar proteins or regulate the formation of the flagellum. However, it also seems reasonable that a rate of synthesis of flagellar protein drastically lowered by phage infection might be sufficient to provide building blocks for the single flagellum of the monotrichic organism but insufficient for the larger task of the multiflagellated organism. Although significant data to the contrary now exist, there have been claims that cells of Salmonella typhirnurium (McClatchy and Rickenberg, 1967) and B. subtilis (Martinez, 1963, 1966) are capable of forming flagella in the absence of RNA synthesis. The presence of a long-lived messenger RNA (m-RNA) stabilized by association with the proximal end of the flagellum was proposed. However, as will be discussed in detail later, the synthesis of flagellin, hence the subsequent formation of filaments, does require the concomitant synthesis of RNA. Based on indirect evidence a t best, many workers have proposed that the basal structure is the site of polymerization of the flagellar proteins (Stocker and Campbell, 1959; Kerridge, 1960, 1961; Asakura et al., 1964; Hoeniger, 1966; Iino and Mitani, 1966; Oosawa et al., 1966; Joys, 1968). Morrison (1961) noticed that cells of Escherichia coli are nonflagellated when grown a t 37" but develop flagella after transfer of the culture to 20". The cells become motile a t 20" even in the presence of 100 pg. chloramphenicol/ml. (the bacteriostatic concentration is 0.8 pg./ml.). The flagella become non-functional when the culture is placed
930
R . W. SMITH AND HENRY KOFFLER
back at 37”. Morrison proposes that the filaments arc packed into the basal structure analogous to a “jack-in-the-box” during growth a t 37”. When the culture is placed a t 20”, they “spring out” and the cells become motile, Most likely, after transfer of the culture to 20”, chloramphenicol fails t o shut off protein synthesis, and thus the formation of flagella from newly synthesized flagellar proteins takes place. Another possible function is u mechanical one involved in the rotation of the filaments. Jahn and Bovee (1965) consider it possible but unlikely that the basal structure rotates thereby imparting a spinning motion t,o the filament,. Uoetsch (1966) suggests that basal structures may contain rotating helices coupled to the filament. A spinning motion might bc achieved with the involvement of special structures in the basal region. Many other workers have argued that the basal structure may function as a source of energy andlor as a “signal box” for the movement of filaments (Astbury et al., 1955; Rinker, 1957; Beighton ef al., 1!)5X; Enomoto, 1966; Klug, 1967). If energy is supplied from the bmal structure, one might expect the occurrence of oxidation-reduct,ion reactions. Initial experiments suggested that this might be the case since reduced tellurite seemed to be deposited a t the sites of insertion of the filaments (van Iterson and Leene, 1964, 1964a). Further studies, however, failed t o demonstrate a correlation between these sites arid thc regions of tellurite reduction (van Iterson et aE., 1966; Abram et al., 1966). However, it seems plausible that the early transfer of energy relevant t,o motility, regardless of its mechanism, occurs a t the cytoplasmic membrane. The interface between the membrane and the basal structure is a most important region that deserves a great deal of future study. Perhaps the most obvious function thus far attributed t o the basal structure is that of an “intracytoplasmic anchoring element” (ChhenBazire and London, 1967). No doubt this is true; however, much work remains before conclusions can be made concerning the complete role of the basal structure in synthesis and motility. 111. The Hook
The other distinct flagellar structure is the hook, which can be distinguished from the main filament not only by its morphology but also by differences in fine structure (Fig. 3), antigenic nature, and greater stability to a variety of agents. The differences in the relative stability of hooks and filaments have been exploited in the isolation of hooks. In some organisms the surface of the hook, but not the filament, appears to be covered by an additional structure. The presence of a bent region, termed the “hook”, which connects the proximal end of the filament to the basal structure has been documcnterl
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in I’roleus spp. (Houwink and van Iterson, 1950; Rinker, 1957; Rogers and Filshie, 1963; van Iterson et al., 1966; Hoeniger et al., 1966; Abram et al., 1965; Abram, 196X), ~Spirillumspp. (Houwink, 1953), Bacillus spp. (Rinker, 1957; Abram et al., 1964), Clostridium sp. (Betz, 1967), Salmoiiella sp. (Kerridge et al., 1962), l’ibrio spp. (Glauert et al., 1963; Ritchie ef al., 1966), and Rhodospirillurn spp. (Cohen-Bazire and London, 1967). In R.yumilus the hook amounts to about 1% by weight of the flagellum and about 0.02% of the total cell weight (Mitchen, 1969; Mitchen and Koffler, 1969).Apparently, the hooks penetrate the cell wall
FIG.3. Intact. filtmient, and Iiook of flagellum of Bacillus stearothermophilus 194. Ncgativdy stained with ursnyl acetate. x 500,000. Taken froin Abrarn et al. (1966).
(Houwinlt and van Tterson, 1050), and occasionally remain attached to filaments shaken froin the cells. The bonds between subunits at the juncture of the hook and filament must be less strong than those existing either in other regions along the filament or between the hook and basal structure, since breakage at the point a t which the hook and the filament become differentiated is commonly observed (Fig. 4).In our hands, for example, only about 2Oo/O of the isolated filaments of B. pumilus possess hooks. Presumably, the remainder are left attached to the cell body or are broken from the filaments during the isolation procedure. Rogers and Filshie (1963) describe hooks or “rootlets” of Profeus sp., 600 by 150 A, which they presume t o come from inside the cell. The fine structure at the surface of the hooks appears different from that of the filament. The surface of the hook has the appearance of subunits, 30 to 40 a in diameter, arranged in a helical fashion. Similar structures in the surface of the filanierit were not detected even after sonication, treatment with detergelits i ~ n dprotctwcs, p H shifts, and freezing and thawing. A definite zone
232
R. W . SMITH AND HENRY KOPFLER
Fru. 4. J!’lt~gelltir propurt~tiori from llncillue pinilus trcstcd with 06(;, ethyl alcohol. Negatively stJaiiiedwith potassium phospliotuiigstate ; x 330,000. Takon from Abrarn et al. (1970).
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of differentiation is also observed between hooks and filaments of P. vulyaria (Abrani ct al., 1965) and AS.typhi?nuriirrrr (Lowy, l!H%). In certain filaments the region normally hooked may bcl straight arid tapered (Lowy, 1965). Marx and Heumann ( 1962) noticed a “pin-like connecting element” joining the filament to the cell in Pseudornonas echinoides. The significance of these straight “hook” regions is not known. J. R. Mitchen and H. Koffler (unpublished) have observed that certain electronopaque substances used in negative staining for electron microscopy, such as uranyl acetate, uranyl oxalate, and ammonium molybdate, irreversibly straighten isolated hooks in vitro. Other stains such as phosphotungstate and uranyl acetate-cthylenediamirie tetra-acetate (EDTA)do not have such an effect and hooks retain the same curvature both in unstained and in shadow-cast preparations. Most likely, this morphological change is an artifact,. However, i t might have bearing either on the basis of bacterial motility or the directional control of cell movement if similar deformations occur on living cells. Interestingly, axial filaments of Leptospira also contain hook-like regions (Nauman et al., 1969). Non-motile mutants of Lcptospira were found that either lack the hook region or have straight “hooks”. The fine structure of hooks of Proteus vulgaris flagella differs from that of the filament; also the hook is larger in diameter than the filament (Abram et al., 1965). Hooks of P . mirabilis are described as being 300 to 400 k in length with the same diameter as the filament, i.e. 120 k (Hoeniger, 1965). Abram ( 1968) noticed a “collar”, apparently continuous with the cell wall, at the proximal base of hooks of P . vulgaris. The collar appears as a double plate, 150 to 180 a in width. Betz (1967) described a collar close to the hook-filament junction in cells of C. sporogenes. The collar has the appearance of a truncated cone with the wide base facing toward the cell body. It was suggested that the collar serves as rtn “eyelet” or “grommet” where the hook passes through the surface of the cell. The fine structure of hooks of 1‘. metchnikovii differs from that of the filament in that a distinct hexagonal pattern of globular units is seen on the surface of the hook (Glauert et al., 1963). Hooks of B. pumilus are less distinctly differentiated from the filament, having a width of 110 k as compared to about 120 k for the filament. A slight constriction is noticed between the hook and filament, however. The differences in the arrangement of subunits in hooks and filaments suggest that the hooks either are constructed of different building blocks than the filament, or that the building blocks, if identical, exist in different conformations, perhaps stabilized by some non-flagellin component. I n either case, one can expect hooks and filaments to have different properties. Indeed, hooks are more stable to acid, alcohol, and heat than are
“4
It. N’. SMITlI A N D HENRY KOFFLER
filaments (Abram et d.,1966, 1967). This relatively small difference i n stability hits been utilized t o isolate and purify hooks from ~)rcparations containing hooks still attached to filaments (Abram et nl., 1907 ; Mitchen, 1969; Mitchen and Koffler, 1969). Acid, acid-alcohol, and heat cause breakage at the juncture of hooks and filaments; some of the hooks still contain membrane and basal material. Further purification is achieved by treatment with deoxycholate or other membrane-solubilizing agents and by density-gradient centrifugation. Recently, we have
FIG.5 . Hooks isolated from flagrlla of BnciZZu.4 p u ~ n i l u s by tho diffwcntial solubilization procediire described in the text. Nttgutiwly stained M ith potnasiiim phosphotungststc. ; x (30,000. Uripiit)lishod obscnmtioris of . I . It. Mitchcvi, I t . M‘. Sinith and H. Kofflcr.
found that treatment with Triton X-100of pH 3-insoluble material from flagellar preparations that previously had been fragmented by freezing and thuwing solubilizes contaminating materials and leaves the hoolts intact (Mitchen P t n l . , 1970). The hooks then m e further purified by denisty-gradient centrifugation through renografin. The final preparation is shown in Fig. 5. Measuremcnts on purified hooks of H. pumilus show them to have a length along their curved axis of approximately 660 A (J.R. Mitchen and H. Koffler, unpublished). Hexose, pentose, and nucleic acids are not
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detectable in purified hooks, but of course it is possible that these and other materials are removed during the isolation procedure. The relative stabilities of filaments and the hook region in axial filaments of Leptospira are similar to those of B. pumilus (Nauman et al., 1969). Treatment a t pH 2.4, pH 12.0, or with 6 M-guanidine, 67% dimethylsulphoxide, 6 M-urea, or 50% ethanol results in the disintegration of the axial filaments but leaves the hook region intact.
FIG.6. Intact filamont itnd hook of BncilZue p u n d u e treated with antisera prepared by injection of purified flagellin into rabbits. Negatively stained with potassium phosphotungstate ; x 250,000. Unpublished data of L. Oiler, H . W. Smith and H. Koffler.
Further evidence for differentiation between the hook and filament has been presented by Lawn ( 1967). Antiserum prepared against flagellated intact cells from which the antibodies against the 0 antigens had been removed by absorption with deflagellated cell bodies does not react with hooks. E. McGroarty and H. Koffler (unpublished work) have extended this finding by showing that when flagella isolated by mild lysis of cells of P.vulgaris are treated with antiserum against purified flagellin only the filament, but not the hook or remnants of the basal structure, react (Fig. 6). Dimmitt and Simon (1970) have also found that hooks do not
236
R. W. SMITH AND HENRY KOFFLER
react with antisera prepared against the protein in the filament. In addition, they prepared antisera against purified hooks and demonstrated that these antibodies do not react with the filament. Theseresults, how-
R
-
3H FLAGELLIN 14C
2500
HOOKS
-
0-0
- I2500
-
:.
-10000
2000-
c
E
h
k
In
c C
u)
c C
3
\2
500
I
I
I
I
I
10
20
30
40
50
Fraction number FIG.7 . Elution pattern of tryptio peptides of Bacilltre p t w d ~ t filnmrnt, x ntitl hook proteins from Ihwox-50 ion rxchnngo colunui.
ever, do not permit one to decide whether the hooks consist of flagellin in a different state of conformation or of protein(#) different from those found in the filament ; it is also possible, though not likely, that the hook is covered by non-flagellin material. To answer these questions, we have
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isolated both hooks and flagellin from cultures of B. pumilus uniformly labelled by growth in the presence of either I4C- or 3H-glucoseas a sole carbon source (Mitchen et al., 1970). The 14C-hookswere mixed with 'H-flagellin and vice versa. The 'H-hook protein does not migrate with the 14C-flagellinin sodium lauryl sulphate-containing polyacrylamide gels. The hook protein moves more slowly, an observation that may indicate t,hat the hook protein has a higher molecular weight than flagellin or that the sodium lauryl sulphate does not completely disintegrate the hooks. The 14C-ho~k-3H-flagellin mixtures after disintegration a t pH 3 were digested with trypsin, and the peptides separated on an ion-exchange
Fraction number
FIG.8. Elritiori pattorn of fractions 50 to 200 of tryptic peptides of Bacillus pumilus filament arid hook proteins from Dowex-50 ion-exchange column.
column. The elution patterns are shown in Figs. 7 and 8. The resultant I4C- and 'H-peptide profiles demonstrated that the hook protein and flagellin have a surprisingly similar primary structure, although a few differences were found. More work is needed to define these differences with respect to amino-acid composition and sequence of these specific peptides. The function of the hook region is unknown. Several repork suggested the presence of RNA in the hook region although more recent findings indicate that this probably is not the case. Martinez (1963) reported the occurrence of a ribonuclease-resistant RNA in crude preparations of flagellar filaments. The RNA was precipitated by antisera against flagella in the crude preparation but not after chromatography on Sephadex G-50, u step that appeared to destroy the hooks. He proposed that the hooks may contain RNA presumably for the synthesis of flagellin. In another report, the synthesis of flagellin in cells of B. subtilis 168-15 appeared to continue in the absence of RNA synthesis (Martinez, 1966). This suggested an abnormally stable m-RNA for the synthesis of
238
R. W. SMITH AND HENRY KOFFLER
the flagellar protein. Martinez speculated that this stability may be due to the inclusion of the RNA inside the hook. As mentioned previously, McClatchy and Rickenberg (1967) also postulated a stable m-RNA associated with the proximal end of the flagellum. These suggestions were ext,ensions of their conclusion that the synthesis of flagellin does not require the concomitant synthesis of RNA. However, this does not appear to be the case as will be discussed in detail later, As mentioned before, preliminary experiments have failed to reveal any RNA associuted with isolated hooks of B. purnilus (J. R. Mitchen and H. Koffler, unpublished results). In considering any role of the hook in protein synthesis, moreover, one needs to keep in mind that bacterial ribosomes range in size from 140 to more than 200 A. The upper limit of the range of the external diameter of hooks is about 150 A ; therefore, ribosomes, as we know them, probably do not exist within the hook.
IV. Sheath-Like Structures Sheath-like structures with distinct architectural features and other properties sometimes are associated with flagella of several species. It is doubtful whether they have any function. DeRobertis and Franchi (1951, 1952) reported a thin trypsin-digestible sheath on filaments of Bacillus brevis. Sheaths also occur on filaments of Vibrio spp. (van Iterson, 1953). The entire sheathed filament has a diameter of approximately 300 A with the sheath being 57 thick (Glauert et al., 1963; Dus and Chatterjee, 1966). The sheath can be removed from the filament by washing with water (Glauert et al., 1963), acid, or urea (Gordon and Pollett, 1962; Follett and Gordon, 1963). It has a layered structure similar to and continuous with the cell wall (Glauert et al., 1963; Gordon and Follett, 1962; Follett and Gordon, 1963). A similar sheath, 75 A thick, has been reported on filaments of Bdellovibrio bact~riovorus (Seidler and Starr, 1967, 1968; Abram and Shilo, 1967). This sheath also appears t o be continuous with thc cell wall, extending over the entire length and possibly beyond the distal end of the filament. Sheath-like structures havc also been seen to be associated specifically with the hooks of Bacillus hrevis and 11. circulans, and filaments of B. stearotherrnophilus 2184 havc some differentiated material on thcir surface (Abram et al., 1966). The sheath-like structures associated with the hooks appear striated and cwmposed of six bands whereas the surface material on the filaments of strain 2184 appears as mats of fibrous material approximately 100 A thick. Sheaths have also been observed on filatnents of Leptospira sp. (Naumun, 1O W ) , Prot~itsvulgaris (Lowy and Hanson, l964), and Pseudomonws spp. (Lowy, 1965; Lowy and Hanson, 1965). The shcaths
BACTERIAL FLAGELLA
239
of Protous i d g a r i s are less than 50 A thick with periodic indent.I d t',1011s 011 their surface a t intervals of approximately 100 A (Lowy and Hanson, 1964). They appear to be organized in a helical manner. Marx and Heumaun (1963) describe a structure consisting of two left-handed helices with a pitch of about 200 A wrapped about filaments of Pseudomoms rhodos. This structure has been also described as being a band about 25 A thick helically wrapped about the filament (Lowy and Hanson, 1965). The role, if any, of the sheath in the structure and function of flagella is not known. Future observations may prove that the sheath occurs more frequently than thought until now ; because of its apparently delicate nature, the sheath may escape notice. Also, during the process of staining samples negatively for electron microscopy the sheath may accept a positive stain from such chemicals as potassium iodide: ammonium molybdate (Follett and Gordon, 1963), and phosphotungstate (Elek et al., 1964) rendering them invisible.
V. Isolation and Purification of Flagellar Filaments De'Rossi (1905) observed that motile cells of Salmondn t?yp?msa became non-motile after they had been shaken t o remove flagella. Purification of the free flagella can then be obtained by differential centrifugation (Orcutt, 1924,1924a).Generally, Gard (1944) is considered to be the first t o obtain flagellar filaments in high yields and purity using this method. Further purification may be obtained by precipitation with salts (Weibull, 1950, 1950b ; Koffler and Kobayashi, 1956, 1957 ; Kobayashi et nl., 1957; Ririker et al., 1957) or ethanol (Uchida et al., 1952; Koffler and Kobayashi, 1957). Weibull (1948, 1950b) obtained saltprecipitated filaments of Proteus vulyaris that were 16.3 to 16.5% nitrogen, 0-03 to 0.04% phosphorus, less than 0.2% carbohydrate, about 0.7% fat, and 1.0% ash, and concluded that 95% of his preparation consisted of protein. The protein appeared t o have a constant composition based on amino-acid analyses and the determination of N-terminalresidues. (Weibull, 1948, 1950b ; Kobayashi et al., 1957). Other methods used for purification of filaments involve freezing and thawing (Comes, 1957) and chromatography on diethylaminoethylcellulose (DEAE-cellulose) columns after extensive fragmentation (Martinez, 1963, 1963a). An important technique in the purification of filaments to be discussed in detail later is that of re-assembly (Abram and Koffler, 1963,1964; Adaet al., 1964; Asakuraet al., 1964).By adjustment of the pH and ionic environment with or without primer, depending upon conditions, flagellin from solubilized filaments can be made t o re-assemble into flagellar filaments which appear normal. Repeated acid treatment,
240
R.
W. SMITH AND
HENRY KOFFLER
re-assembly, and washing, essentially a recrystallization procedure, results in filaments estimated to be 99y& or more pure. As mentioned previously, both basal structures and hooks are occasionally torn from the cell and remain with the filaments after shaking. I n samples prepared solely by centrifugation and salt precipitation, these proximal structures are probably also present, though in small amounts relative to the yield of filament obtained. Hooks and possibly portions of the basal structure remain in the insoluble fraction a t pH 2.5. After removal of this acidinsoluble material, therefore, re-assembly is an effective and convenient technique for the isolation and purification of flagellar filaments, though strictly speaking they no longer represent “native” material.
VI. The Protein Nature of the Filament Boivin and Mesrobeanu (1938) suggested that bacterial filaments are composed of protein since they are insoluble in trichloroacetic acid. Although Pijper (1957a) considered them to consist mainly of capsular polysaccharides, many reports soon verified that the filament is protein in nature (Weibull, 1948, 1949, 1950b; Rinker and Koffler, 1949, 1951; Uchida et al., 1952; Koffler and Kobayashi, 1956, 1957; Kobayashi el al., 1957 ;Rinker et al., 1957). Purified flagellin can be easily obtained by solubilization of flagellar filaments below a pH value of about four, and subsequent removal of the insoluble material by centrifugation or filtration. Before acid treatment, filaments precipitate over a wide range of ammonium sulphate concentrations (Koffler and Kobayashi, 1956, 1957). This apparent heterogeneity prior to acid treatment probably exists because of differences in the length of filaments in a given preparation and the amount of nonflagellin components present. After disintegration of the filaments in 0.01 N-HCl, however, the proteins precipitate within a very narrow range of salt concentrations a behaviour that suggests a fair degree of homogeneity. Purified preparations contain only small amounts of pH 2-insoluble material, hexose, pentose, nucleic acid, or ash. Weibull(l949)predicted that, as a group of related proteins, flagellins from different species should have similar amino-acid compositions. Although the amino-acid composition of each specific flagellin is unique, Weibull ( 1948, 1949) determined spectrophotometrically that flagella of Protew vulgaris and Bacillus subtilis contain 1-8 to 1.9% tyrosine and no tryptophan. Less than 0.05% cysteine was found in flagellin of either organism. The composition of flagellin from several organisms is shown in Tables 1 t o 3. All flagellins so far examined contain no or only a few residues of cysteine, tryptophan, tyrosine, proline, and histidine (Kobayashi et al., 1957; Rinker et al., 1957; Rinker, 1957; McDonough, 1966;
TABLE1. Amino-acid compositions of flagellins from strains of Bacilli, expressed as residues per lo5 grams Bacillus species
Thermophilic strains of Bacillus' X-l"
ZChiti i formisa pumiluk
subtilis' 168
subtilisb 19
58 121 0 40 86 63 18 14 5
56 122 0 40 87 63 18 14 5
-
-
1
8 46 35 151 133 155 9
50 67 12 45 40 155 127 151 7
51 69 12 44 39 156 127 152 7
57 112 0 37 95 70 26 15 5 0 51 81 11 42 37 139 116 138 8
908
92 1
908
910
902
74 123 0
Glycine Alanine Cysteine Valine Leucine Isoleucine Methionine Phen ylalanine Tyrosine Tryptophan Threonine Serine Histidine Lysine Arginine Aspartic acid Glutamic acid Ammonia, amide Proline
81 111 0 36 89 63 19 17 5
65 111 0 29 82 6i 28 18 0 -
-
59 51 9 51 34 146 135 134 10
63 76 9 35 43 135 138 163
53
Total Residues
916
-
.subtili@ 23
r
44
80 61 28 16 0 5i
D. Abram, M. Farquhar,andH. Koffler, unpublished. Calculated from data of Martinez etol. (1967).
brevis'
cireukm9
194
10
CD
44 106 0
44 137 0 26 93 61 17 12 7
154 116 1.54 4
68 67 10 54 35 133 135 188 5
67 117 0 42 74 52 20 22 4 4 112 60 8 63 27 145 98 151 6
60 116 0 31 83 63 19 19 9 0 93 76 7 37 45 123 121 137 12
53 140 0 52 77 66 23 19 6 0 78 43 7 49 35 140 113 132 13
5i 121 0 21 90 72 33 13 7 0 70 58 13 33 47 130 134 129 4
79 132 0 62 63 69 14 13 9 2 106 73 9 34 33 140 94 146 6
897
904
921
914
916
903
938
45
91 58 15 12 9 3 63 78 6 4i 44
-
F J W 2184
k 2 f F
:
TABLE2. Amino-acid compositions of flagellins from gram-negative bacteria, expressed a s residues per lo5 grams 1Q
SWlOG1
Proteus cuk7ari.P
Glycine Alanine Cysteine Valine Leucine Isoleucine Methionine Phenylalanine Tyrosine Tryptophan Threonine Serine Histidine Lysine Arginine Z-N-Methyllysine Z-N-Dimethyllysine Aspartic acid Glutamic acid Ammonia, amide Proline Total Residues a
69 101 0 52 80 54 3 19 8 -
"I I
Protms vu&nri@
Spirillrrm serpensc
,Ypirillum serpensd
rr
85
-
97
145
o
-
54
34
-
82 56
-
I I
50 32 14 24 9 0 86
I I
-
3
20 8
-c
-
7
14
-
-
-
90 83
-
-
59
84
-
59 33
56 31
1 35 46
0 34 45
-
-
-
4
69
70 120
24
-
64 26
-
67 110 0 73 73
55 7 21 15 0 91 85 0 35 26 30
81 141 0 61 54 45 5 14 23 0 100 58 3 35 25 26
81 lid 0 63
86 118 0 63
I 0
81
49 7 15 22 0 101 56 3 60 27 0
47 5 14 24 0 107 66 3 35 26 26
153 96 139 12
149 98 152 9
962
957
-
-
-
-
-
-
165
120
-
100
-
137 4
-
165 89 216 7
166 92
-
161 105 130 3
10
153 95 145 10
915
931
933
942
958
954
I04 155
-
+P
SWllPO
14
1,2 1,2 i SL64'2 Salmonella Salmonella Salmonelln Salmonella Salmonella Salnioiwlla i adelaidee adelaidef typhimi~rium~typhimunum'typhimurium~typhi~~~uriun~d Arizona'
64 69 55 5 21
-
SLSil
-
-
*-
-
-
15 24
2 40
85 116 0 64 56
47
5
3
14 23
m
O
E
110 69 2 33
26
-
967
D. Abram. 31. Farquhar, and H. Koffler, unpublished.
* Calculated from data of Chang et al. (1969).
Calculated from data of Martinez et nl. (1967). Calculated from data of Glazer el d.(1969).Based on 18 residues of arginine per molecule of 40,000 for Spirillum eerpens and 6 residues of phenylalanine per molecule of 40,000 for Salmonella typhimurium. Ada et al. (1964). Calculated from data of Parish and Ada (1969). Calculated froin data of McDonough (196.5).
'
F
$ d
m
TABLE3. Amino-acid compositions of flagellins from strains of Salmonellae, expressed as residues per lo5 granis Salmonella paratyphi B"
Saln~ortellr abortus-equi" SL169 S L l 6 l S L l i 4 SL168 SL165 SL166 S L l 6 i S L 8 i i SL23 1,2 a h s e,h g,p i i e,n,x
Salmonella senftenberg" SLi36 g,s,t
Salmonella Averages of ell essen' organisms reported Numherof Mole SL588 g,m residues percent ~
Glycine Alanine Cysteine Valine Leucine Isoleucine Methionine Phenylalanine Tyroeine Tryptophan Threonine Serine Histidine Lysine Arginine Z-K-Methyllysine Aspartic acid Glutamic acid Ammonia, aniide Psoline
81 144 0 59 75 4i 6 15 23 0 101 62 3 59 26 0 153 97 142 10
i2 85 120 130 0 0 63 72 71 74 47 40 8 1 0 15 16 21 21 0 0 105 115 69 59 5 4 36 43 2i 29 24 0 147 14i 98 101 149 138 19 14
Total Residues
961
947
960
126 0 57 80 32 3 15 19 0 112
82 134 0 69 i4 4i 8 16 22 0 103
il
il
3 34 26 23 144 96 139 16
4 37 25 23 142 97 149 14
73 115 0 72 68 57 3 23 16 88 80 0 37 24 34 161 90 159 8
959
968
951
80
Calculated from dnta of McDonough (1965).
83 121 0 62 82 48 5 14 22 0 106 67 3 33 25 25 150 98 142 10
79 113 0 63 '79 47 6 14 22 0 110 '70 4 59 26 0 150 94 152 12
954
947
80 130 69 66 46 5 16 2i 0 104 il 3 37 23 23 140 95 142 15
68 112 0 65 66 54 6 21 15 0 90 81 0 34 24 38 169 93 153 9
75 111 0 68 65 55 6 25 16 0 94 81 0 33 21 40 l5B 89 156 i
14 0 87 69 5 46 33 14 149 106 151 9
952
945
944
941
0
il
122 0
--
JO
76 56 12 li
~-
7.5 13.0 0 5.8 8-1 6.0 1.3 1.8 1.6 0 9.2 7.3 0.5 4.9 3.5 1.5 15-8 11.3 16.0 1.0
100
m
g Y
E 3
E M
244
It.
W. SMITH
AND HENRY KOFFLER
adit et al., 1!)64, 1967). Earlier work suggested that the flagellins of P. t~ulgarisi ~ n dseveral members of the genus Bacillus contained one cysteine residue per molecule (Mallett, 1956 ; Koffler and Kobayashi, 1956; Kofflcr el al., 1956; Rinker, 1957). Later work, however, indicates that this is unlikely (Abram and Koffler, 1962; D. Abram, H. Koffler, and M. Farquhar, unpublished results; R. W. Smith and H. Koffler, unpublished results ; Chang et al., 1969). Hydrolysates of flagellin previously oxidized with performic acid (Schram et al., 1954) contain less than 0.5 residues per 100,000 g. of a material that chromotographically behaves like cysteic acid. When oxidized by the procedure of Moore (1963) cyst,eic acid or a cysteic acid-like material can be found in larger but still considerably less than stoichiometric amounts. I n addition, it is not possible to demonstratc free sulphydryl groups in flagellin either by reaction with ~,5’-dithiobis-2-nitrobenzoic acid or by titration with nitroprusside. Furthermore, exhaustive carboxymethylation of flagellin a t pH 7 with 14C-iodoaceticacid in the presence of 6 M-guanidine hydrochloride and dithiothreitol yields no carboxymethylcysteine ; under these conditions this agent should also react with cysteine if this amino acid were present. In any case, it is most unlikely that disulphide bonds are involved in either intramolecular or intermolecular bonding The N-terminal amino acid in the flagellin from P. vulgaris (Koffler et al., 1956; Koffler and Kobayashi, 1956; Rinker, 1957; Chang et al., 1969) and Salmonella spp. (Ada et al., 1964) is alanine. Only one-half of an alanine residue per molecular weight of 40,000 is found to be N-terminal, however (Weibull, 1953a; Chang et al., 1969). Possibly, half of the amino groups on the N-terminal alanine residues are blocked, although these were not detected (Chang et al., 1969). Flagellin from members of tho genus Harillus contain methionine as the N-terminal amino acid (J. Stenesh and H. Koffler, unpublished results; Sala and Koffler, 1967). Many workers appear convincd that the filament is composed of a single protcin (Oosawa et al., 1966; Ada et al., 1967; Martinez et al., 1967; Joys, 1968; Lowy and Spencer, 1968). However, the occurrence of two protein-containing regions following double diffusion (Comes and Valentino, 1956; Comes, 1957; Ada et al., 1964), immunoelectrophoresis ( R . W. Smith and H. Koffler, unpublished results), disc gel electrophoresis in the presence of 6 M-urea (Gaertner, 1966), preparative polyacrylamide electrophoresis (Sullivan, 1968), chromatography on DEAE-cellulose (Sullivan et al., 1969), and electrophoresis on celluloseacetate strips (M. Farquhar and H. Koffler, unpublished results) suggests that filaments of a t least some organisms may be composed of more than onc protein. For example, when examined by analytical ultracentrifugation, flagellin from acid-disintegrated filaments of B. purnilus behaves as if homogeneous. Nevertheless, H. Suzuki and H. Koffler
BACTERIAL FLAGELLA
246
(unpublished results) and Sullivan et al. (1969) found that this flagellin may be separated into two fractions under carefully selected conditions of pH and ionic strength. At pH 1 . 7 , i i protein fraction dcsignated as “B” precipitates. The protein in this fraction migrates as a single bandin disc gel electrophoresis identical to the faster moving of the two bands observed in samples of acid-disintegrated filaments. The supernatant liquid contains a small amount of protein B but consists mainly of a protein designated as “A”. The precipitate formed a t pH 4.7 dissolves if t h e pH is increased to 7 . 0 . Actually, Abram and Koffler (1964) previously had observed the occurrence of this precipitate but did not pursue this observation further. These two protein components are most conveniently separated on a DEAE-cellulose column a t pH 8 with a gradient of sodium chloride (Sullivan el al., 1969). Even early in this work, the possibility that these two fractions represent two different configurational states or degrees of polymerization of the same molecular species seemed unlikely since the two bands exist even after boiling of the protein or after polyacrylamide electrophoresis in the presence of 6 M urea. Furthermore, after separation and isolation, each component remains homogeneous despite treattnent a t various pH values (A. Sullivan and H. Koffler, unpublished result,s). That A and B are indeed different proteins has been firmly established by amino-acid analysis and examination of the tryptic peptides obtained from the separated proteins (Sullivan, 1968; Sullivan el al., 1969; Smith and Koffler, 1969). Their composition and apparently also amino-acid sequences are similar but distinct. After separation on ion-exchange columns, the tryptic peptides from either isolated A or B proteins or from mixtures of )H-labelled A protein and ‘‘C-labelled B protein (and vice versa) are similar yet show some differences; 17 peptides from a total of approximately 26 in A and 31 in B appear similar or identical. Each protein has nine methionine residues. Six of these appear to be common to both proteins with regard to the nature of the methionine-containing tryptic peptides. Each protein, however, contains three uniquely located methionine residues. Furthermore, the A and B proteins may be differentiated with respect to the heat stability of their intramolecular organization as determined by optical rotatory dispersion measurements (D. Klein and H. Koffler, unpublished results). The midpoint of the region in which the B protein undergoes a reversible temperatureinduced transition from a-helix to random coil occurs five to eight degrees lower than that of the A protein. The existence of the two flagellins in a single culture may have some similarity to the phenomenon of phase variation in Salmonella, and provide an advantageous system to study the regulation of flagellin synthesis. In Salmonella two distinct structural genes for flagellin are
246
R . W. SMITH AND HENRY KOFFLER
controlled SO t l i i L t 0 1 1 1 ~0 1 1 ~ ' functions a t it givcii time. (:t'O\+ktl o f t 1 (*tllttlrc from a single cell after 300 to 500 generations results in a11 equilibrium mixture of cells producing either one protein or the other (Stocker, 1949). In B. yumilus the A and B proteins normally occur in a 7 :3 ratio (H. Suzuki and H. Koffler, unpublished results) ; either both proteins itre synthesized by a single cell or each is made by cells capable of synthesizing one or the other protein. While our observations are not conclusive in this respect, they suggest that in B . purrdus individual cells produce the A and B proteins simultaneously. In 13 separate trials, cultures that had been grown for 30 to 60 generations from single-colony isolates (i.e. probably but not certainly derived from single cells) produced thc A and B flagellins i n a 7 : 3 ratio; departures from this ratio might] be expected if each protein were made by specific cells. The question as to whether the two proteins occur in the same or different filaments is still unresolved. An interesting discovery is that 14C-lysineincorporated into flagcllin of S. typhimurium during growth may be recovered as lysiiie and E-Nmethyllysine (NML) (Ambler and Rees, 1959; Ambler, 1960). The unusual amino acid, NML, is not found in any part of the cell except ill flagellin where, in 8. typhimurium, it amounts to about 4% of the flagellin molecule by weight (Ambler, 1960). Lysine and NML are generally present in approximately equal amounts. Flagellin of 19. arlelaitle contains I5 lysine and 11 NML residues (Ada ~t nl., 1967; Parish and Ada, 1969). Half of the molecules of S. typhirnurium flagellin appear to contain one residue of r-N-dirnethyllysine (Glazer P t al., 1969). 'I'hc authors suggested that the small amount of dimethyllysine found rnuy be due to a non-stringent control of the degree of methylation. Martinez (1963) arid Glazer et al. (1969) have also identified NML in flagellin of Spirillum serpens. Methylation probably occurs after incorporation of lysine into the primary structure of the protein. Kerridge (1963) reportcd that radioactive NML can be recovered from t h e flagellin of Salmoiielln typhimurium grown in the presence of either 14C-lysineor methionine. There is no dilution by non-ridioactive NM I, in the growth medium (Kerridge, 1960). 1)-Methionine and DL-cthionine i n hibit the incorporation of the radioactive methyl group from methioiiinc but not the incorporation of Iysine. Stocker et al. (1961) &scribe a gene that controlsthepresenccofNML in flagelliiiofSnlrnoiLellnspp.In phase 1 flagellin, the nml locus is closely linked to, but separate from, H1, the structural gene for phase 1 flagellin. On the other hand, the locus controlling the presence of NML in phase 2 flagellin is not closely linked to H d , the structural gene for phase 2 flagellin. A special triplet of nucleotides that codes for NML probably does not exist in the structural gerles for flagellin. I n all likelihood, the t m l locus codes for an enzyme t h a t mcthyl-
BACTERIAL FLAGELLA
247
ates c*ertain lysine residues in flagellin but apptrently not in other cellular proteins. It is not known whether this is due to the specificity of the enzyme for flagellin or compartmentalization within the cell. N-hlethyllysine cannot be enzymically attached to transfer-RNA isolatcd from calf liver and as expected is not incorporated into protein. In calf thymus nuclei there exists an enzyme that catalyses the methylation oflysine residues in proteins (Kim and Pail\-,1965). Some flagellins appear to be glycoproteins. Abram and Koffler (1963a) report that those isolated from strains 10 and 50 of B. stearotherwaophilus contain a carbohydrate component that persists during purification of the flagellin. Filaments of Spirillurn serpens contain 1 * 5 O / , carbohydrate measured as glucose equivalents that appears tightly bound since it cannot be separated from the protein by disintegration of the filaments and chromatography on Sephadex (:-50 (Martinez, 1963). Weibull ( 1R19, 1950b) noticed that preparations of flagella of B. subtilis contained 1 to 2"/0 carbohydrate. McDonough ( 1965) found that preparations of filaments of Salmonella spp. often include up to 10% carbohydrate; however, he considered this to be due to contamination with the lipopolysaccharide 0 antigen, even though the carbohydrate component persists after acid disintegratior? DEAEcellulose chromatography, and re-assembly. The presence of' a small amount of any component always presents a perplexing problem with respect to its significance. As will be discussed later, nearly all antisera prepared against flagellar proteins contain a small but detectable amount of antibody against the somatic 0 antigen. Therefore, the preparations injcctetl must have included the O antigens as contaminants. Perhaps, these could be related to the sheath structures often found on filaments, although the contamination could easily result from the presence of stnall fragments of the cell membrane or wall. I n most cases non-protein components are insoluble at pH 2 arid may be separated frotn the flagellar proteins by such a treatment (Weibull, 1950; Koffler and Kobayashi, 1956; Kobayashi et al., 1957; Ada P t al., 1964). Before the true glycoprotein nature of sotne of the flagellins can be accepted, the covalent linkage between the presumed carbohydrate component and t he peptide needs to be demonstrated directly. In any case, this observation is niore likely to be interesting in studies dealing with the biosynthesis of glycoproteins than flagellar function, since most of the filaments examined so far do not seem to contain bona$de carbohydrate components. Minerals are probably not involved in the structure of either flagellin or flagellar filaments (Vegotsky et al., 1965a). Only trace amounts of cdcium, copper, iron, magnesium, manganese, potassium, and zinc can be found by atomic absorption spectroscopy in isolated flagellar 10
248
R . W. SMITH AND HENRY KOFFLER
filaments of P. nulgnria, B. puinihs, and the thermophilic strain 21 84 of H. stenrothervr~o~)~~ilus, and in purified P. imlgaris flagellin. I n some experiments, calcium and magnesium occurred in larger amounts ; however, treatment with ethylenediaminetetraacetic acid (EDTA) reduced these inorganic elements t o less than one mole of calcium or magnesium per mole of flagellin without altering the stability of the filaments. The small amounts of mineral elements found most likely represent fortuitous contaminants rather than real constituents of flagellar filaments. As expected, the levels of magnesium and zinc in preparations that after purification contained only minute amounts can be increased substantially by dialysis against solutions containing these mineral elements. J. Stenesh, M. E’arquhar, H. E. Abron, and H. Koffler (unpublished results) have calculated the molecular weights of flagellins from various mesophiles (13. pumilus, B. subtilis, B. brevis, B. licheniformis, and B. sp. X-1) and thermophiles (B. stearothermophilus strains 2184, 10, CD, PJW, and 194) using data obtained by the Archibald method (Archibald, 1947), Sedimentation and diffusion analyses, and fingerprints of peptides produced by tryptic digestion. In all cases, the calculated molecular weight of the tnonomeric subunits fell in the range 30,000 to 50,000. Flagellins of R. subtilis and H. stearothwmophilus strains 10, l!H, and 2184 have molecular weights between 40,000 and 50,000, whereas the others ranged between 30,000 and 40,000. The molecular weight of flagellin of strain 2184 is between 45,000 and 60,000 based on data from equilibrium sedimentation in the presence of 6 M guanidine hydrochloride and disc gel electrophoresis in the presence of sodium lauryl sulphate (L. R. Yarbrough and H. Koffler, unpublished results). Flagellins of P . vulgaris (Weibull, 1948, 1949), Bacillus spp. (Weibull, 1949; Martinez et al., 1967), Salmonella spp. (Ambler, 1960; Lowy and McDonough, 1964; Asakura et al., 1964; McDonough, 1965; Ada et al., 1!)67 ; Parish and Ada, 1!)69), and Spirillurn serpens (Martinez et al., 1967) are reported to have molecular weights between 35,000 and 40,000. Erlander et al. (1‘360) using approach-to-equilibrium techniques reported that flagellin of P. vulgaris exists as a dimer above p H 4.5 or lower if salts are present and that the molecular weight of the monomer is 20,000. The number of peptides produced after tryptic or chymotryptic digestion, however, is inconsistent with a molecular weight of 20,000, and suggests a molecular weight closer to 40,000 (Abron, 1966). Other investigators (A. 7’. Ichiki and R. J. Martinez, personal communication; Chang et al., 190!)) have found a molecular weight of 40,000 for flagellin from P. vulgaris. Although data obtained in our laboratory by equilibrium sedimentation in the presence of 6 M-guanidine hydrochloride indicate a molecular weight of 20,000, gel filtration through
BACTERIAL FLAGELLA
249
agarose also in the presence of guanidine hydrochloride a t either acid or neutral pH values and cleavage with cyanogen bromide yield data that indicate a subunit molecular weight of 40,000 (L. R. Yarbrough and H. Koffler, unpublished results). The reasons for this discrepancy are not fully understood in any case; unlike the flagellins from various strains of Bacillus, the conformation and charge of this particular flagellin must be uniquely responsive to low pH to explain its peculiar behaviour during ultracentrifugation. Peptide maps produced after tryptic digestion of flagellins from P. vulgaris and the five mesophiles and five thermophiles mentioned above are unique except that an L-shaped pattern formed by several peptides was noticed in maps of all species examined (Abron, 1966; M. Farquhar and H. Koffler, unpublished results). These probably carry the greatest positive net charge as judged by their electrophoretic mobility. This does not necessarily mean that the flagellins of the Bacillus species have areas of common amino-acid sequence although peptides are produced that have similar electrophoretic mobility and solubility properties. So far, no data are available that would speak meaningfully either for or against the existence of common aspects in the primary structure of various flagellins. Ada et al. (1967) noted that tryptic digestion of flagellin from cells of Salmonella adelaide released 39 peptides. Only 20 of the trypsin-susceptible bonds were quantitatively broken, however. The fact that all of the theoretically susceptible bonds were not broken probably was responsible for the appearance of a non-dialysable trypsin-resistant material. A similar fraction results during the tryptic digestion of flagellin of B. pumilus (F. H. Gaertner and H. Koffler, unpublishedresults). By weight, the trypsin-resistant material of B. pumilus amounts to 15 t o 20% ofthe undigested molecule. The amino-acid compositions are similar except that the trypsin-resistant material is much more acidic than the whole molecule (R. W. Smith and H. Koffler, unpublished results). The trypsin-resistant fragments aggregate into straight fibres, 50 to 60 a in diameter, which in turn associate in a side-by-side fashion (Fig. 9). These fibres retain their integrity a t p H values from 2 to 13 and in 9 Murea or 6 M-guanidine. They dissolve in concentrated trifluoroacetic acid, but form a precipitate when the acid concentration is lowered by dilution below 50% v/v. Isolated filaments of flagellin obtained by disintegration of filaments under mild conditions are not digestible by trypsin, pepsin, or papain without prior “denaturation” (Koffler and Kobayashi, 1956; Kobayashi et al., 1957) although members of the genus Bacillus and probably other bacteria produce one or more proteinases that are capable of digesting flagellins in only slightly denatured conditions. I n spite of the fact that we routinely attempt t o denature
250
R . W . SMITII A N D HENRY KOFFLElt
PIG.9. Structurrs formed by self association of ‘‘corer'' pcpttdc formed during txyptic digestion of flagollin from &2Cih!lLS pz~milua.Not0 the intact filaincnts for sizo cwnptmsnu. Nvgativcly stlttritvl with phosphotungsttttu; x 83,000. Proin 1111piihlivhrtl dtLta of I t . W. Smith and H. I i o f f l t ~ .
BACTERIAL FLAQELLA
251
flagellin by treatment a t 100” for 15 min. prior to the addition of trypsin, after cooling to 37” certain regions may again become inaccessible to trypsin. The homogeneity of the resistant fragments is supported by the ability to form an ordered fibre structure and the presence of only one N-terminal amino acid, glutamic acid (methionine is the single Nterminal amino acid in the whole molecule). Patterns established by equilibrium centrifugation indicate homogeneity with respect t o size. Because of its apparently ordered structure, this trypsin-resistant fragment may turn out to be useful in the determination of the structure of flagellin.
VII. Immunology Malvoz ( 1 897) noticed that>cultures of flagellated cells typically gave flocculent precipitates when treated with a suitable antiserum. Based on the type of agglutinate obtained in serological reactions, two separate classes of antigens could be distinguished in Bacillus typhosus (Salmonella typhosa) (Joos, 1903), later recognized as somatic and flagellar in origin (Smith and Reagh, 1904; Beyer and Reagh, 1904; Smith and Tenbroeck, 1904). Initially coined to differentiate swarming cells of Proteus from non-swarming, the terms “H” (Hauch) and “0” (ohne Hauch) came to stand for the flagellar and somatic antigens respectively (Weil and Felix, 1917). These antigens can be distinguished by the physical appearance of the antigen-antibody complex and their relative stability to heat. Evidence cited by early investigators to prove the association of the H antigens with the flagellar filaments was derived mainly from experiments that demonstrated the simultaneous loss of H-agglutinability and filaments or motility due to heating. Unheated cells give a characteristic large flocculent agglutinate, which is easily dispersed (Gruschka, 1922; Balteanu, 1926; Nelson, 1928). After being heated a t 60” to 70” for 15 to 20 min., cells agglutinate in a fine granular manner when exposed to antisera ; this precipitate is relatively resistant to disruption by shaking. Heating lowers the titre (Balteanu, 1926) due to the disintegration of t,he filaments (Nelson, 1928). Antiserum prepared against purified filaments does not agglutinate heated cells or nonflagellated mutants (Orcutt, 1924a; Balteanu, 1926). Gard et al. (1955) demonstrated that heating detaches the H antigen from the cell but does not9 destroy its reactivity with antisera. Unless cells are washed after heating to remove the solubilized flagellin, antibodies against flagella may still precipitate with the flagellin released from the disintegrat,ed filaments. Other early workers also considered the H antigens to be associated with the filaments (Braun and Schaffer, 1919; Joetten, 1919; Feiler, 1920; Orcutt, 1924; Yokota, 1925; Henderson, 1932). Much
252
R . W. SMITH AND HENRY KOFFLER
of their work has been reviewed by Craigie (1931). Apparently thc H antigens are completely distinct from other cell antigens (Schutzc, 1932; Belyavin et nl., 1951 ;Vennes and Gerhardt, 1959).Several workers have thought that the H antigens are located in other cell structures ax well as the filaments (Jenkins, 1946 ; Tomcsik and Baumann-Grace, 1956; Martinez, 1963). The explanation probably lies in the observation that preparations of flagella are almost invariably contaminated with small amounts of somatic antigens (Gard et al., 1955; Weinstein, 1959; Makela and Nossal, 1961; Winebright and Fitch, 1962; Fitch and Winebright, 1962;Adaetal., 1963; Nossaletal., 1964). The mechanism of agglutination has received considerable attention. Gard (1937) and Gard and Erikson (1939) demonstrated that antibodies against flagella immobilize the filaments. I n dark-field microscopy, Pijper (1938) noticed that, in the presence of antisera, filaments becotnc thickened due to the deposition of a granular deposit. Mudd and Anderson (1941) demonstrated by electron microscopy that the thickening is due t o the absorption of antibody molecules. They concluded, along with Pijper (1938), that the actual process of agglutination is a non-specific mechanical event brought about by an increased tendency of the cells to stick together. Absorption of antibody molecules onto the surface of flagellar filaments has been described and photographed in greater detail by Elek et nl. (1964), Wilson et al. (1966), Lawn (1967), and Goto et al. (1967). Menolasino et al. (1966) report that binding of antibody causes loss of the helical configuration and shortening of the filament. However, this is not observed in the work of Goto et al. (1967), DiPierro and Doetsch (1968) or in our laboratory (J. R. Mitchen, E. McGroarty, and H. Koffler, unpublished results) and probably is not a specific effect caused by the binding of antibody. Giesbrecht et al. (1964) observed that antibody molecules can bind free filaments together. Divalent, but not monovalent, antibodies immobilize cells by cross-linking flagellar filaments (Greenbury and Moore, 1966; DiPierro and Doetsch, 1967, 1968). The cross-linking is reflected as a large increase in viscosity when filaments are mixed with antiserum (Read et al., 1956; Read, 1957; Koffler, 1957). Immune univalent globulin fragments produced by digestion of the antibody molecule with pepsin do not affect motility ; however, they do protect the filaments from immobilization by divalent antibody (DiPierro and Doetsch, 1967, 1968). Thus, occupation per se of the antigenic sites on the filament does not prevent the function of flagella or cause agglutination. Motion is returned t o filaments that are immobilized by divalent antibody after treatment with proteolytic enzymes. Apparently, this is due to removal of the cross-linking globulin protein from the surface of the filaments. Using 1311-labelledantibody against flagella, Greenbury and Moore (1 966) estimate that between 130
BACTERIAL FLAQELLA
253
and 200 antibody molecules participate in linking with the flagella of an individual cell when half of a culture of S. typhimurium is immobilized. Further, on the assumption that the average filament is 5000 A long and 120 11 wide, the area on the filament surface occupied by one monovalent antibody fragment is approximately 690 A2. Dimmitt et al. (1968) calculate that in B. subtilis an average of one antibody molecule is bound per 45 A of filament length. A filament 9 pm. long would then contain a t least 2,000 antigenic sites. The fact that the subunits in the filaments are also approximately 45 A in diameter does not necessarily mean that there is only one antigenic site per subunit. At least nine antigenic specificity-determining sites have been identified in the flagellin of phase 1 Salmonella that has the g, n antigens (Yamaguchi and Iino, 1969). These were defined by factor analysis using cross-absorption tests following intragenic recombination within the structural gene for flagellin. Four of the antigenic factors are strain specific and five are common to two or three strains. Each determinant maps as a unit and in a linear fashion within H1, the structural gene for phase 1 flagellin. Since these are defined by cross absorption tests, a separate antibody molecule must exist for each determinant. A flagellin molecule, therefore, is capable of reacting with any one of several types of antibody molecule. However, for steric reasons, it is improbable that in the polymerized form a flagellin subunit reacts with more than one antibody molecule a t a given time. On the contrary, since the antibody molecule is large, it probably reacts simultaneously with several flagellin subunits and thereby prevents other antibody molecules from reacting. Elek et al. (1964) observed in electron micrographs that antibody-coated filaments were separated from each other by a distance of 180 A ; this indicates a layer of antibody 90 a thick on each filament. These workers proposed that the reactive sites lie a t the ends of the long axis of the antibody molecule. The molecules of antibody on the filaments would then resemble the bristles of a bottlebrush. Similar observations were made by Goto et al. (1967) in that the surface of the filaments appeared to be coated with a 95 A thick layer of antibody molecules. Assuming this t o be less than half the length of the 75 antibody molecule, they propose that the molecule forms a loop on the surface of the filament by the reaction of combining sites located a t the ends of its long axis. Again, this makes possible the blocking of several subunits in the filament by one antibody molecule. Andrewes ( 1 922) was the first t o describe a phase variation in which a culture with a particular H antigen is reversibly altered so as to produce an entirely different antigen. His observations were verified by Kauffmann and Mitsui (1930) and have been described in detail in a series of articles from Edwards’ laboratory (Edwards and Bruner, 1938, 1942 ; Edwards and West, 1945; Edwards and Moran, 1946; Edwards et al.,
254
R. W. SMITH AND HENRY KOFFLER
1948 ; Edwards, 1950; Bokkenheuser and Edwards, 1956 ; Edwards et al., 1856, 1957, 1960, 1962; Fife et al., 1961 ; Douglas et al., 1!)62). Although most strains exhibit only two distinct phases, cultures with four have been described (Edwards et al., 1962). Variation occurs spontaneously. It has been reported, although without substantial evidence, that variation can be induced at a much higher frequency by incorporation of agglutinating antiserum into the growth medium. Several workers consider this to be an unnatural process and the induced antigens to have no relationship with naturally occurring ones (Kauffmann, 1936; Kauffmann and Tesdal, 1!137; Gnosspelius, 1939). As will be discussed later, the process of phase variation is controlled at the level of gene function. The proteins in filaments of different antigenic phases may be similar but have their own distinctive composition. Sixteen of 30trypticpeptides of flagellin possessing the 1,2 antigens are identical with those of flagellin with the i antigen although they are unrelated immunologically (McDonough, 1962). Antigens that cross-react (e.g. e,h and e,n,x,or g,m) have similar but not identical amino-acid compositions (McDonough, 1!)65). I n the same report it was shown that proteins with the g antigen lack histidine and that the amino-acid composition of a particular antigenic type appears to be constant. Paterson (1939) noticed a crossreaction between filaments of a number of strains of Listerella with antiserum prepared against any one. All strains appear to have a common antigen plus one or more others that permit definition into three groups. Kauffmann (1950) and Nakaya et ul. (1952) suggest that each filament may contain many different antigenic sites. Purified filaments of S . enteritidis with antigens I, IX, XI1 : g,m :- react quantitatively with antisera prepared against filaments of S. oranienburg with antigens VI, VII: m,t:- (Nakaya et al., 1852). No reaction was obtained with filaments possessing the e,n,x antigens. It was concluded that both the g and the m antigenic sites are located on the same filament. This does not necessarily imply the presence of two types of flagellin in the same filament since one flagellin molecule may have several antigenic determinants. Using absorption tests, Davies (1951) defined five groups of H antigens in 22 strains of B. polymyxa. One group is common to nearly all strains while the other groups are shared by two or more strains. Filaments of a given strain may possess a number of different antigenic groups. Some Pseudomonas spp. may produce morphologically different but antigenically similar polar and lateral flagella (Leifson and Hugh, 1953). This observation would seem to indicate that similar or identical protein molecules may form filaments of varying morphology. A similar situation occurs in Chrornobacterium sp. except that the polar flagella have a different antigenic composition than the lateral ones
255
BACTERIAL FLAGELLA
(Sneath, 1956). The cells may be producing two types of flagellar protein each located in a specific portion of the cell or one protein capable of assuming two conformations resulting in the exposure of different antigenic sites.
i.0
3.0
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U
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.-cm !.O .-P 0
0)
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: ..-m
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2
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0
0)
.-c2C
0)
2 .-c C
-gm
.O
1.0
c
z
mg. Antigen nitrogen added
FIG. 10. Precipitin reaction of flagellar filaments with serum (0.5 ml.) against flagellar filaments. The flagellar filaments were highly purified isolates from cells of froteuz vulgaris. Antisera were obtained from rabbits immunized by six successive injections at two or three day intervals of one millilitre of 0.9% sodium chloride suspensions (0.18 mg. nitrogen per ml.) of purified filaments and bled seven days after the last dose. Tho quantitative precipitin reactions were performed according to Kabat and Mayer (1948). One millilitre of antiserum was added to varying amounts of antigen diluted to constant volume with 0.85 % sodiurn chloride. The reaction mixtures were incubated at 37" for one hour, and tho11 a t 4' for 18 hours. Precipitates were removed by centrifugation at 2,000 x g. for 0.5 hours. Nitrogen in the precipitate was determined by a modified Kjeldahl method. Supernatant fluids were tested for the presence of excess antigen or antibody. There is excess antibody in the supernat,ant liquids regardless of antigen concentration. The addition of this antiserum results in the almost instantaneous formation of a gel, and it is likely that in spite of stirring some antibody molecules are kept, from reaching antibody-binding sites. 0 - 0 , Total mg. nitrogen precipitated ; 0- 0,mg. antibody nitrogen precipitated.
256
R . W. SMITII AND HENRY KOFFLEB
That soluble flagellin differs from the polymerized form in immunological properties was indicated by the work of Read and his colleagues using quantitative precipitin reactions as criteria (Read et al., 1956 ; Read, 1957 ; Koffler, 1957). As expected, the antigen-antibody curves are distinct. The reaction of filaments from cells of Proteus vulgaris with antibodies against filaments (Fig. 10) results almost instantaneously in
AG EXCESS (0-1)
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mg. Antigen nitrogen added b'ra. 11. Precipitin reaction of flagellin with serum (1.0 ml.) against flagellin. Flagelliri was prctpared and the procedure used was as in Fig. 10. Similar results were obtained when flagellin prepared by disintegration of filaments by heat ( B O O , 30 minutes) was usod. Tho ratio of antibody nitrogen to antigen nitrogen a t eciuivalurice is about 13. 0 - 0 , Total mg. nitrogen precipitated ; 0- 0, mg. antibody nitrogen precipitated.
gels that are difficult to stir. Probably because of the physical condition of this complex, some antibodies always remain in the supernatant liquid. The antibody nitrogen to antigen nitrogen ratio a t the point of maximum reaction is near one. Soluble flagellin, obtained by the disintegration of filaments a t pH 2.5, removal of the acid-insoluble material, and subsequent neutralization, when reacted with homologous antisera gives precipitin curves characteristic for soluble proteins
257
BACTERIAL FLAQELLA
(Fig. 11). I n the absence of antibody this flagellin does not re-aggregate under the conditions of the experiment, though of course its condition during the immunization of the animal is unknown. There is an equivalence point a t which the antibody nitrogen to antigen nitrogen ratio is 12.7; antigen excess results in a decrease in the amount of the precipitation. Since the quantity of antigen a t the point of maximum reaction 0.09-
0.09 U
AG EXCESS (+3)
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Fro. 12. Precipitin reaction of flagellin with serum (0.5 ml.) against flagellar filaments. Flagellin was prepared by disintegration of flagellar filaments from cells of Proteus vulgaris a t pH 2.5, centrifugation at 25,000 x g . for three hours a t O", and neutralization with sodium hydroxide. Similar results are obtained when flagellin prepared by disintegration of filaments by heat (60",30 minutes) was used. Otherwise the procedure was identical to that given in Fig. 10. The presence of antigen (AG) or antibody (AB) excess was indicated by values ranging from +1 (slight) to $4 (copious). The antigen-antibody complex is granular rather than a gel. Thnre is always either excess antigen or antibody present in supernatant liquids. The ratio of antibody nitrogen to antigen nitrogen a t maximum precipitation of antibody is six. 0-0, Total mg. nitrogen precipitated; 0- 0,mg. antibody nitrogen precipitated.
for the flagellin-antiflagellin system is much less than that needed for the filament-antifilament system and the precipitate dissolves in antigen excess, the reaction can be fairly easily overlooked. The reaction of soluble flagellin with antibodies against polymerized flagellin gives precipitation curves characteristic for soluble proteins but there is always either an antibody or antigen excess (Fig. 12). Maximally, soluble flagellin reacts with less than one-fifth of the antibodies against filaments
258
R.
W. SMITII AND
HENRY KOFFLER
(cf. Fig. 12); a t an antibody nitrogen to antigen nitrogen ratio of about ti to 7 flagellin binds markedly fewer antibodies against filaments (about 15 t o 20°h) than homologous antibodies (cf. Pig. 11). The reaction of flagellar filaments with antibodies against flagellin (Fig. 13) behaves similarly to the homologous reaction (cf. Fig. 10). However, since in both reactions there always exists an antibody excess, one cannot be certain ‘26
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mg. Antigen nitrogen odded FIG. 13. Precipitiii reaction of flagellar filamoiits with serum (0.5 ml.) against flagollin. Flagelliri was prepared by disintegration of filaments by heat ( B O O , 30 minutea). Otherwise the procedure was as in Fig. 10. There is an excess of a11tibody in tho supernatant liquids regardless of antigen concentration. 0-0 Total mg. nitrogen precipitated; 0- 0,mg. antibody nitrogen precipituted.
that polymerized flagellin binds antibodies against soluble flagellin as well as it does homologous antibodies. Using different methods, others confirmed the notion that the filament possesses antigenic determinants not accessible in “soluble” flagellin (Ada et al., 1964, 1967;Martinez et al., 1967; Grant and Simon, 1968a; Ichiki and Martinez, 1968); especially impressive is the work of Grant and Simon (1968a) who used I3’Iiodinated purified immunoglobulins against flagellar filaments as a specific reagent. It seems that new determinants are created by the
UACTERIAL FLAQELLA
259
association of flagellin molecules. Although the specific residues that constitute the antigenic site may be contributed by adjacent flagellin subunits without conformational change, it is also possible that new sites are exposed by each flagellin molecule as it changes shape during assembly. Cleavage of flagellin of S . adelaide with cyanogen bromide results in no detectable loss of antigenic determinants (Parish et al., 1969). Four fragments are produced by this cleavage that have molecular weights of 18,000, 16,000, 5,500, and 4,500 (Parish and Ada, 1969). The largest fragment reacts with antisera against flagellin to the same extent as the intact flagellin molecule. A small amount of reactivity was seen with the 12,000 molecular weight fragment while the two smallest fragments had no activity (Parish et al., 1969). Antisera prepared by injection of the 18,000 molecular weight fragment into rabbits or rats also reacts with polymerized flagellin, an observation that suggests that this portion of the flagellin molecule is exposed in the polymer and is not involved in inter- and intramolecular foldings. Evidence is now evolving to indicate the existence of more than one type of immunoglobulin relevant to the flagellin system. For example, Martinez et al. (1967) noted that the absorption of antisera against filaments eliminates the ability of the sera to react with flagellin but not with intact filaments. Ichiki and Martinez (1969) defined four types of antibody molecules as follows : ( 1 ) antiflagella, immobilizing, neutralized only by intact filaments ; (2) antiflagella, neutralized by intact filaments and by flagellin ; (3) antiflagellin, immobilizing, neutralized by intact filaments and flagellin ; and (4)antiflagellin, neutralized only by flagellin. Probably it will be feasible t o purify antibodies against soluble and polymerized flagellin and to use them as specific reagents in studies on the morphogenesis of the flagellum. The availability of flagellin in soluble and insoluble form also has well served research on the nature of the immunogenic process. For example, it was found that peak titres are obtained more rapidly upon injection of flagellin of S. typhosa into rats, although much higher titres are attained when intact filaments are used (Winebright and Fitch, 1962 ; Fitch and Winebright, 1962). Filaments of S . adelaide also appear to be more immunogenic than the monomeric protein (Ada et nl., 1963; Nossal et al., 1!)04; Lind, 1968). Injection of filaments into rats induces the formation of two types of immune globulin (Ada P t al., l!lA3; Nossal et al., 1964; Nossal and Austin, 1!)66). A mercaptoethanol-sensitive 1 9 s macroglobulin is produced during the first week after injection. After eleven days, most of the antibody consists of a mercaptoethanolresistant 75 globulin. Flagellin is more effective at inducing peak 7s titres (0.01 pg. required of intact filaments), although final titres are
200
R . W. SMITH A N D HENRY KOFFLER
much higher when filaments are used. Flagellin was found to be incapable of inducing t,he 19s macroglobulin. Vlagellins readily induce a state of tolerance in rats (Nossal et al., 1965, 1887; Mitchell and Nossal, 1W6; Austin and Nossal, 1966; Ada and Parish, 1968). Apparently, the solubilized flagellins are more potent inducers of tolerance than are the intact filaments. Complete tolerance can be induced by daily injection of as little as lo-* pg. flagellin per g. body weight. This property may prove troublesome if one wished to produce precipitating antibodies to flagellins. For comparison, Shellam and Nossal (1968) note that flagellin is l o 6 times more effective in inducing the state of tolerance than is bovine serum albumin. Rats tolerant to flagellin have been reported to break tolerance when the protein is injected with an adjuvant (Lind, 1$%8).The size of the antigen dose apparently is important. A low-zone tolerance is induced in rats by daily injection of lo-' pg. of protein per g. body weight (Shellam and Nossal, 1968). A second, high-zone effect is produced upon injection of pg./g. body weight. The immune response results when levels intermediate to these are used. Further complications arise since these doses required to induce low-zone and high-zone effects may change as much as loo-fold over a six-week injection schedule.
VIII. Stability While the stability of the flagellum, of course, is of' functional significance, its main interest currently lies in the insight that this characteristic might provide regarding the structure of the constituent molecules and the nature of their interactions. The effects of various environmental conditions on the structure of flagellar filaments were reviewed by Lacey (1961). Most of the early work concerning stability dealt with the nature of a thermolabile H antigen, which even then was regarded as being associated with the filament (Braun and Schaffer, 1919; Joetten, 1919; Feiler, 1920; Gruschka, 1922; Yokota, 1925; Balteanu, 1926). Nelson (1928) and Craigie ( 1 931) concluded that heating a t 100" destroys the physical integrity of the filament, but does not affect the antigenicity of the constituent protein even after 2 hr. Therefore, thermal disintegration of the filament can be followed by loss of agglutinability of intact cells (McCoy, 1937), and also by a decrease in viscosity (Adye and Koffler, 1953; Adye, 1!)54; Adye et al., 1957; Stenesh and Koffler, 1962), light scattering (Martinez and Rosenberg, 1964))and sedimentability (Stenesh and Koffler, 1962). Filaments of ~Salmcmellatyphimuriuin are destroyed by heating a t 60" (Kerridge et al., 1962) with the formation of monomeric flagellin (Asakura et nl., 1964). There is a large change in optical rotation
BACTERIAL FLAGELLA
261
as filaments from cells of Proteus vulgaris and various strains of Bacillus are exposed t o temperatures sufficiently high to cause a decrease in the viscosity of the suspensions (D. Klein, H. Koffler, and J. F. Foster, unpublished results). Although much of the rotation is probably due to the helical content of the molecule, as will be mentioned later, additional rotation may be generated by the aggregation of the subunits in the filament. At the moment it is not clear whether the transition in spectropolarimetric parameters with an increase in temperature precedes or accompanies the disintegration of the filament. I n other words, it is still unknown whether the subunits undergo a conformational change that then results in the falling apart of the filament, or whether the increase in temperature causes the breakage of bonds that results in the separation of subunits from the filament and subsequent conformational changes in the monomers thus released. The relative stability of flagellar filaments has been a useful property in studies regarding the nature of heat stability in thermophilic organisms. McCoy (1937) noticed that filaments of the thermophile Clostridium thermosaccharolyticum are stable a t 78" for 50 min. whereas those of the mesophile CE. butyricum disintegrate a t 58". Filaments of P . vulgaris and B. subtilis are destroyed in less than 5 min. at 70" (Adye and Koffler, 1953; Adye, 1954; Adye et al., 1957; Koffler et al., 1957), while filaments of thermophilic strains of Bacillus are stable for at least 20 min. under these conditions. Neither labilizing factors in filaments of mesophiles nor stabilizing factors in those of thermophiles can be demonstrated. Mixtures of mesophile and thermophile filaments behave predictably; thermophile filaments, therefore, do not likely contain diffusible materials capable of protecting mesophile flagella. The stability of isolated but otherwise native filaments is not significantly affected by treatment with trypsin, deoxyribonuclease, ribonuclease, thioglycolate, Dowex-50, Dowex-2, glass beads, or EDTA a t pH 4.5, 7 , or 10. Mesophile flagellin (from filaments held a t p H 2 and 26" for 30 min., heated a t 60" for 30 min., or disintegrated by sonication), the ash from mesophile flagella, or crude cytoplasmic isolates from sonicated mesophile cells do not reduce the stability of thermophile flagella. Comparable preparations (except that filaments were heated a t 80" rather than 60" to obtain flagellin) do not provide mesophile filaments with the stability typical of thermophile flagella. The heat stability of filaments isolated from cells of a given species appears t o be inherent in the structure of the filaments themselves, and probably ultimately resides in the primary sequence ; that is in the location, type, and number of bonds that stabilize the intramolecular structure. The initial observation that flagellins from rnesophiles contained a larger number of ionizable groups than therrnophile flagellin (Koffler,
262
R. W . SMITH AND HENRY KOFFLER
~t d ,1!)57) could not be supported by more extensive studies (Mallctt, 1956; D. Abrarri, H. Koffler, and M. Farquhar, unpublished results). A summary of our analyses 011 flagellins from various mesophiles and thermophiles is shown in Table 4.Three out of five rncsophile flagellins ‘rABL€?
4.A C O I I l ~ X W I H O I Of l tllr l~llllJlO-tWld COlKlpll8ltlOll of fliLgC?lllllfl’oltl I I l ( ~ S O p h l ~ 1 C and thorrnophilic strains of Ii’acillux. Roxitlucs per lo5 grarns __
~
-
GI ycino Alaninc Cystoine Vnlirio Loiicinc Imloucino Motliioniric Phonylnlanirio Tyrosirio Tryptophtw Thrconino Sorino HiRtidirio Lysino Arginino Aapartic ac*id-f AHpnrcigino Ciliitaniic acid I (:lritciminc Ammonia, nniiclo Prolino Total cationic Total rositlrwx Tryptic pcpt ides M ~ I\\.t. . x 10-3
~
M
Amino ari d -
~
~
-
~
T ___
44(67)8O 106(111)123 0 30(40)44 80(8li)91 58(63)68 17( 10)28 12( 16) 18 O( 5)Q 0(0)3 5 0 ( 58)65 5 1(67)78 6(9)12 36(46)5 1 34(40)44 135( 150)154 1l6( 133)138 134(153)15!5 4(7)10 88(94)97 S78( 910)921 30(32)39 32(88)45
__
52(60)67 118(121)14X 0 31(42)62 63(75)90 52(66)72 14(20)43 13( 19)22 7(9)11 0(0)4 70(91)11 1 42(60)70 7(8)13 33(37)63 28(36)47 133( 137)144 94( 110) 134 129(137)lSl 4( 6)13 78(92)Q7 9O2(916)941 27(33)45 32(35)49
- I+ic.illuSv 81). S1, B.~ t r ~ h c ~ t i i ~ c ) r 13. m zpxu , r n i l w , If. x i d ) l t l i ~untl , 11’. h r r z w . T - Five theinwphtk RtlltlllHOf ~ h ’ t l l t ~194, s . 10, C‘D, FJW ant1 21 84.
M
The first iiriiiilicr r q ~ r t ~ s t m t hs r lowest vduo obtained for uiiy of thv fiagollltls 1, lthltl narh group, thc riurnher in p i mthwm8 reproclotits tho inoaii value, niltl the thirrl t h r hlghheshape of small waves (Abram et al., 1964a; see Fig. 14). Apparently, the stability of intermolecular bonds between subunits varies depending 011 the molecular sites a t which subunits arranged in a given geometry interact, andspecificdisintegration products reveal the strongest interactions prevailing under given experimental situations. For example, Champness and Lowy (1 968) conclude that bonds in the axial dimension are more stable than those in the lateral direction since, during drying of suspensions of filaments, the change in the equatorial diffraction pattern is more marked than the
BACTERIAL FLAGELLA
269
change in the near-meridian pattern. Figures 15 to 17 illustrate the preferential breaking of lateral bonds resulting in the formation of fine fibres. Occasionally one sees filaments with large pieces missing (Fig. 18). The explanation for this type of disintegration is not known. Maximum stability of flagellar filaments is reported to occur a t pH 8.5 for P. vulgaris (Weibull, 1948) and pH 7.8 for S. serpens (Martinez and Rosenberg, 1964). While Weibull (1951) reported that filaments are stable in dist>illedwater, Martinez (1963a) finds that filaments of P. vulgaris, 8. serpens, and €3. subtilis SB-19 disintegrate in the absence of salts and that they are stabilized by addition of phosphates to 0.01 M at pH 7-0. In our experience, native filaments of B. pumilus may be stored in distilled water a t 0" for a week or more without noticeable disintegration. After acid disintegration and re-assembly, the reconstituted filaments are more stable during prolonged storage, perhaps due t o the removal of a proteolytic enzyme that is frequently found in association with native filaments (Farquhar, 1966; Farquhar and Koffler, 1968). Intact filaments attached to the cell body may be more resistant to acid than isolated ones. Stocker and Campbell (1959) noted a heterogeneity in the acid lability of filaments of individual cells. Filaments on cells of Vihrio methnikovii do not disintegrate a t pH 2 (Follett andGordon, 1963; Gordon and Follett, 1962). Flagella of several species when still attached to cells survive treatment with phosphotungstic acid or uranyl acetate a t pH 2 (Lowy and Hanson, 1965) and pH 3 to 5 (Rinker and Koffler, 1949,1951).In our laboratory, we have noticed an unexpectedly low recovery of flagellin in the supernatant liquid from suspensions of cells of B. pumilus treated a t pH 2 (F. H. Gaertner, J. Bui, and H. Koffler, unpublished results). Leifson ( 1 960) and Hoeniger (1965a) report that the morphology of filaments attached t o cells can be altered by slight increases in the hydrogen ion concentration of the growth medium. Filaments of Proteus sp. exhibit a normal pitch length a t pH 6. A gradient in the number of filaments with one-half the normal pitch (i.e, curly) is noticed, however, as the pH value is lowered until a t pH 5 only the curly morphology is seen. The shortened pitch length is probably due to conformational changes in the flagellin molecules within the filament rather than an effect on the synthetic mechanism. However, since all observations were made on cells that had been incubated for several hours in a growth medium of the desired pH value, it is difficult to know whether the morphological changes observed were due to direct or indirect effects of pH. Most likely, though, the effect occurred after the filaments had been formed. Otherwise, many normal filaments would have been observed a t pH 5 even after 2 hr. incubation since many of the filaments 011 a given cell had already been formed prior to the change in pH.
270
It. W . SMITH A N D HENRY KOFFLER
Pic:. 16. l%~gc4lnr filtnricrits f'roin Racillitu p i ~ ~ n i l utmatcd u with 50% t+lianol -1 N-IICL. Arriis M it h tniridlca of fibrcas and uoii-fibrous clcctron-lucid material nro found togctlicbr tvith intact Aitgollar filaments. Tho prcparation wtia nogntivcly staiiied with phosi~~iotiiiigstatc. ~200,000.D. Abram arid H. Kofflor, uiipublished obsorvations.
BACTERIAL FLAaELtA
27 1
Frct. 16. lplagellar filainnrits from Bacillus punzilus trentcd w i t h 50% cthnnol + N-HC1. Specimens, prcparcd from H dilute suspension of flagellar filainciits, contain marly well soparated groups of fibres. The liiiear orgaiiizatioii of these groups indicates that native flagellar filaments are disintegrated into fine fibres. Often tho filaiiicwts are frayed nnd irregiiltdy clispcwcd. 'rho cliainetcr of the fiiie fibres is 2.5-3.0 tiin. ~200,000. D. Abram a i d H. Kofflcr, uiipiiblished obsrrvations.
272
R . W. SMITII AND HENRY KOFFLBR
FIG.17. Flngellnr filnmonts from Bacillus purnilue treated with 50% cthaiial + N-HCI. Tho preparation wus Hhadow cast with pnllndiuni. ~ 8 3 , 0 0 0TI. . AlmLin uric1 H. Koffler, urputdi~hcdohservntioiis.
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As filaments disintegrate, the pH value of the solution slowly increases (Weibull, 1950d; Koffler, 1067). Based on the data of Vegotsky et al. ( 1965), during complete acid disintegration, eight hydrogen ions are taken up per moleeule of P. vulgaris flagellin of molecular weight 40,000. Hydrogen ion uptake follows pseudo-first order kinetics. No intermediate size particles were observed in the ultracentrifuge, an observation that favours a fully co-operative mechanism for the acidinduced disintegration of filaments. It is not possible a t present to eliminate a rapid zipper mechanism as opposed to an explosive one for the disintegration of the filament. Optical rotatory dispersion measurements (Klein et al., 1968, 1969, 1969a) and difference spectroscopy data (R. W. Smith and H. Koffler, unpublished results) indicate that soluble flagellins have conformations different from subunits in the intact filament,. The conformational change, therefore, may occur prior to actual disintegration of the filament. The major question concerns the stage of disintegration a t which hydrogen ions are bound, i.e. during the conformation change of the subunit or its release from the structure of the filament. To which of these steps do the pseudo-first order kinetics observed by Vegotsky et al. (1965) apply? Unfortunately, we do not know the exact sequence of events that occurs during disintegration of the filament. We feel, however, that the conformational change in the subunits could weaken intermolecular attractions and cause the filament to fall apart. In the course of acid-catalysed disintegration, a protonbound intermediate should exist and this may occur either prior to or during the conformational change. If true, the pseudo-first order kinetics may not have resulted from the actual removal of the subunits from the structure of the filaments but from proton uptake before or during the conformational changes. As mentioned previously, intact flagella remaining on the cell may be more resistant to acid disintegration than broken filaments. If disintegration proceeds from the proximal to the distal end of the filament, attachment to the cell or perhaps the presence of the basal region or the hook may stabilize the filament. This may be tested in several ways. One should be able to isolate entire flagella, i.e. with basal region, hook, and filament intact. The stability of these preparations to disintegration by acid, heat, and other chemical and physical agents could be examined. As will be discussed later, relatively intact flagella are more frequently torn from cells grown in the presence of p-fluorophenylalanine (R. W. Smith and H. Koffler, unpublished ) purifiedflagella from these cells is signifiresults). The heat stability ( T dof cantly greater than that of “normal” filaments. Although this increased stability may be due to incorporation of the analogue into the flagellin subunits, i t is also possible that t h e greater frequency of organelles observed containing hooks and basal materials may be responsible.
274
R . W . SMITlI A N D HENRY KOFPLBIt
Pru. 18.
BACTERIAL FLAGELLA
275
Treatment a t alkaline pH values is also capable of disintegrating flagellar filaments (M'eibull, 1948; Mallett, 1956; Koffler, 1957; Erlander et al., 1960; Lowy et nl., 1966). Martinez and Rosenberg (1964) noticed a decrease in T , values of filaments of h'pirillum s e r p e n s at pH 7-8 and above. Flagellar filaments were completely disintegrated a t room temperature in 0-25 M-sodium hydroxide (Martinez et al., 1967). Surface-active agents inhibit motility by disintegration of filaments (Lominski and Lendrum, 1942). As mentioned previously, filaments are destroyed by sodium dodecylsulphate (Mallett and Koffler, 1955, 1957 ; Mallett, 1956; Koffler, 1957 ; Koffler et al., 1957). Kopp and Muller (1!(65) noticed that motility-inhibiting cmcentrations decrease from a range of 20 to 30 mM for sodium hexylsulphate to 0.1 to 0-5 mM for sodium tetraderyl sulphate. The four-carbon compound has no effect. Inhibited cells are non-flagellated presumably due to disintegration of the filamen ts by these agents. Filaments are also disintegrated by guanidine, urea, and acetamide (Koffler et al., 1057; Koffler, 1957; Stenesh and Koffler, 1962; Roberts and Doetsch, 1 !Mi; Mallett, 1 %6), cetyl pyridinium chloride (Mallett, 1956), alcohols (Mallett, 1956, J. Stenesh and H. Koffler, unpublished results; Abram et nl., 1966, 1!)67), dioxane (Stenesh and Koffler, 1962), acetone (Asakura et al., 1964), and sonication (Kerridge et al., 1062; Koffler et al., 1057). Various proteolytic enzymes do not attack undenatured filaments (Koffler and Kobayashi, 1956; Kobayashi et al., 1957; Stocker, 1957; Stocker and Campbell, 1959). Kerridge et aE. (1962) report that filaments of Salrrionelln typhimuriurn are disintegrated by chymotrypsin, however. Farquhar ( 1 066) and Farquhar and Koffler (1968) demonstrated the presence of one or more proteolytic enzymes associated with flagella of several species that cannot be separated from the flagella by washing and differential centrifugation. When suspensions are heated above 37" there is an increase in ninhydrin-positive substances ; these changes can be shown to be associated with the hydrolysis of flagellin to peptides. From experiments in which boiled flagella were used as substrate and native flagella as source of enzyme, it was concluded that heating allows proteolysis due to denaturation of the flagellin and not activation of the Vra. 18. Fragiiiriits of flngrlliw filaments of Bacillus pumilus frozen III liquid nitrogrti arid tliawcd at room trmprraturr. In addition to the breakage ofthe filaments shnrt gtqxi o r "rrotletl" regions oftrri appear along thr surface of the filaments. The polar tiattire of the filaineirt 14 rwrdotit from observations showing that the distal c i i c l of the frnginmt 19 split while thc proximal end is blunt. The threeiittiioinctcr core visthlc in alrnost rvrry fragtnent only srldom extends ttito thr hook. The, piqxwatioii was ncgatively stained with phosI-'hotmtgstat~..~ 3 3 3 , 0 0 0 . D. Abrarn and H. Koffler, unpublished observations.
276
R . W. SMITH AND HENRY KOFFLER
enzyme ; neither the boiled nor the native flagella above show proteolysis. However, the association of the enzyme(s) with flagellar filaments apparently does not significantly alter the T, values for filaments obtained by viscosimetry. Unlike native organelles, heat-denatured filaments (e.g. 100”for 15 min.) are susceptible to a variety of proteolytic enzymes.
IX. Arrangement of Protein Subunits Recently, a great deal has been learned about the architecture of the filament largely with the aid of electron microscopy and the techniques of X-ray and optical diffraction. However, each approach has its own limitations, and our knowledge regarding the nature of the filament is likely to remain incomplete until the three-dimensional structure of the constituent protein is determined. The use of electron-dense stains has permitted the observation of globular or ovoid subunits, yet the tendency of such heavy-metal compounds to disrupt the normal arrangement of protein subunits raises some question as to the validity of conclusions based on topographical examination alone. In addition, the submersion of particles in the stain tends t o result in underestimates of their dimensions. Since the front and back images often are superimposed, subjective interpretations are not always reliable. I n this respect, optical diffraction methods combined with “filtering” techniques and the use of electron micrographs of the same specimen taken from several positions are likely to lead t o a more definitive analysis of the geometry in which constituent subunits are arranged. The ability of negative stains t o penetrate crevices has been most helpful in demonstrating the existence of a central region in the filament that appears to be different from the remainder of the filament, but one needs to establish carefully that the stain is located inside the filament rather than in surface indentations. Because of the thinness of individual filaments, the preparation of cross sections is difficult, though the chances for success can be increased by sectioning bundles of flagella. A great deal can be learned from the slow disintegration of filaments under mild conditions and examination of the products formed. However, this approach raises the question whether the resulting structures represent the native organization and also whether a model “reconstructed” from its pieces has any semblance t o the real organelle. X-Ray diffraction studies regarding the strucures of flagella are complicated by the difficulty in obtaining crystals. Flagellin normally crystallizes in the form of flagellar filaments, which have specific dimensions. These do not represent sufficiently large crystals to be useful crystallographically. Other crystal forms are possible (Abram and
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Koffler, 1964) but i t is debatable whether knowing the architecture of flagellin in such “abnormal’)forms would result in meaningful interpretations regarding its “normal” structure. However, X-ray diffraction studies are helpful in determining the size of the subunits, even if such studies are handicapped by the difficulty of orienting filaments in the form of macrofibres. As early as 1888, Biitschli and later Reichert (1909) proposed that the filament has the shape of a cylinder with a line of contractile elements running helically about it. Such helical features in the filament received attention much later as techniques for electron microscopy became more powerful (DeRobertis and Franchi, 1951, 1952; Starr and Williams, 1952; Labaw and Mosley, 1954, 1955; Preusser, 1958). The remarkable helical structures shown in these reports must be viewed with some caution, however, since in all cases, only shadow-cast preparations were examined. The resolution obtained with such samples is not sufficient, even today, to permit identification of features in the surface of flagellar filaments. hlost likely, these observations represent sheath structures. Based on features on their surface, DeRobertis and Franchi (1951, 1952) claimed filaments of Bacillus brevis to be a double helix composed of two strands, 50 to 70 A in diameter, coiled into a major helical structure, 100 to 120 A wide, with a pitch of 410 A. I n the case of filaments from an unknown diphtheroid subunits appeared to be packed into a threestranded, left-handed helix with a diameter of 190 A and a pitch of 500 A (Starr and Williams, 1952). Labaw and Mosley (1955) calculated that the filament of Brucella bronchiseptica must be a triple-stranded helix with a diameter of 139 A. Filaments of Pseudomonas echinoides were described as left-handed double helix (Marx and Heumann, 1962). Strands apparently arising from intact filaments were seen by Braun (1956) in flagellar filaments of Escherichia coli. When flagellar filaments are exposed to a variety of conditions, such as acid, alkali, alcohol, acid-alcohol, buffered osmic acid, formaldehyde, glutaraldehyde, uranyl acetate, freezing and thawing, sonication, or heat, various proportions of the preparation, depending upon conditions, form thin fibres (Abram et al., 1964a). These fibres are distinctly wavy, especially when the position of individual filaments on microscope grids during disintegration as indicated by their appearance has been undisturbed; these waves are much smaller in length and amplitude than those formed by collapsed flagellar filaments. As mentioned previously, the formation of fibres is accompanied by an increase in viscosity, and is most readily observable when the agent is applied only briefly, a t low temperatures, and in low concentrations. Fairly often during disintegration the filaments appear as if they were uncoiling. The wavy appearance of the fibres probably reflects a helical arrangement of subunits within
R. W. SMlTli AND HENRY KOFPLER 278 the filament, Of course, the packing of glohular or ovoid subunits to form the walls of a hollow tube o n l y gives the impression on the surface of the filament that the subunits arc arranged in the form of helical strands. Fine fibres revult from careful disintegration of the filament not iiecessarily because the filament is composed of a number of fine fibres hut because of differences in thc stiibilitg of thc axial and lateral bonds c i t h c ~ in type, number, or location. $;ssentially, one necds to keep i n mind that flagellar filaments are probably formed by the sequential polymerization of subunits and not by the wrapping of presynthcsizcd fine fibres. hi thc filaments each monomer is surrounded by iieighbouring subunits in such a manner that arrangements between them can be vicwed as occurring between subunits adjacent along the long or short axis of the filament. It is most unlikely that molecular affinities betwcen monomers in “hcud to tail” and “side by side” arrangements are idcntical. In difierent physical and chemical environments, one or the other type of intcraction might be expected to dominate. The wavy fibres observed by Abram et al. (1964a), therefore, reflect ths interactions among subunits in an arrangement that proved t o be stronger than other possible interactions under the conditions of disintegration used. Two apparently distinct arrangements of flagellin subunits in the surface of filaments of a given species were seen in samples negatively stained with uranyl acetate (Lowy and Hanson, 1964, 1!)65). Filaments of the flagella of Proteus vulgaris appeared to possess two types of arrangements, one consisting of a n alternating sequence of subunits packed to resemble longitudinal lines with short regions where the lines are interrupted, generally at 600 t o 750 A distances, and a second typc cornposed of globular subunits in a helical arrangement (Lowy and Hanson, 1064). Lowy and Hanson (1965) later modified the description of the two types of subunit packing. Type A appeared to consist of globular subunits packed into longitudinal rows but joined together i n a helical fashion. Neither globules nor helices are seen in the B structure, the most prominent feature being thick longitudinal rows. Filaments consisting entirely of the A or the B configuration were observed in cells of Pseudorrionus jluormcwu, Bacillus subtilis, Proteus vulgaris, or flalmonella t!yphimurium, but only in Pseudomonas rhodos was i i single filament observed with both typcs of substructure. The 13 type was seen in parts of the filament surrounded by sheath material and the A type in unsheathed sections. Despite the several types of subunit arrangements reported, Lowy and Spencer (1068) have concluded that differences in surface features merely reflect differences in the conformation of tho subunits. It is proposed by them that the geometrical packing of the subunits into the filament is the same for all types of flagella. h i o and Mitani (1!167,
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1 !)671$) and Martinez rt nl. (1!)68) have reported that rnisseiise mutation on thr. struc%ui.algene for flagellin can cause tlic formation of filaments with a n altered iuor1)Iiology. As sugg:csted before, the 1)riniarysequence of amino acids detcrmiiies the conformation of the subunits which in t u r n tleterniinw the niorpliologg of the filament. Burgue t o the low rwolutioii obtained with sliadow-cast preparations, most observations of fine structure in the surface of the filament have been inadr using electron-dense negative stains such as 1)otassium l)Iiosl)hotungstate, potassiuni borotungstate, aniitioniuni molybdate, uranyl acetate, or a uraiiyl acetate-ED'I'A com])lex. One needs t o keep in miiid tlie possibility that these cheniicals have distinct effects on the morpliolog. and fine structurc of specimens. For example, Abram and Koffler (l!)(i4)noted that ti*entmcntwith uranyl acetate, but not urangl acctatc-E I>TA, results iii partial or c ~ m l ) l e t edegradation of flagellar filanients. Also a s nic~ntioned])reviously, J. 11. Illitchen a n d H. lioffler (unpu).)lislicdresults) observc t h a t uranyl acetate, uraiiyl oxalate, a n d am nioniuni molybdate muse a n irreversible straiglitening of flagellar hooks ; ~ ~ l i o s ~ ~ l i o t m i g ant3 s t a t curanyl aeetate-HI)TA have no noticeablc effect. 'I'lierefore, it is difficult to assess the significance ofresults obtained with rz single staining technique. In particular, in the case of flagcllar filaments, uraiiyl acetate often c*aiisesalterations not seen with other chemicals, a n d the differeiitiatrd regions observed by Lowy a n d Hanson ( 1 W-k, I M S ) probably represciit areas in ~vliiclithe reagent disrupted t h e organization of t h e subunits. In iiegatively stained pre1)arations of flagella a n d thin sectioiis of' cells of 5'. t,yphi/uuri11911, the subunit arrangement a1)peared t o rcflect a five-fold s y m n e t r y (Kcrridge et c d . , 1 ! W ) Spherical . subunits with a diameter of 45 I!. were seen after treatiiieiit of filaments by sonication, heating, or wit Ii sodium dodecylsulp,hate. These observations could be 11
2x0
R. W. SMITH AND HENRY KOFPLEH
iiitcrprcted as describing filaments composed of either three helical or five parallel strands of subunits. I n the helical structure, the axial separation of the globules appeared t o be about 41 A with a 50 A lutcral separation of the longitudinal rows. Elck el nl. (1964) report that the subunits are packcd into the filament, with a centre-to-centre distance of 45 8.A row spacing of 50 to 60 8 appears inore likely, however, from the X-ray diffraction results of Champness (1968) and Champness and Lowy (1!)68 and personal communication). A model consisting of' ovoid subunits arranged in a rhomboidal fashion to form a cylindrical shell was proposcd for the A or bead-type filanierits based on electron micrographs and X-ray diffraction results (Champness and Lowy, 1!)68). This model is similar to that dcscribed by Lowy and Hsnson (1965) except that a wider separation of subunits than originally thought is observed and explained by the packing of ovoid rather than spheriral subunits. In thc B or line-type filamentls, monomers appear to be arranged in rows not parallel to the filament's axis but slanted two to three degrecs. Intciisity differences in diffraction patterns of filaments with the bead-type substructurc and those with linc-type indicate that the subunits themselves are not, similar. The visually apparent differences i n surface structure arc due, then, not to different packing orientations of similar subunits hut to arrangements dictated by the dissimilar nature of the prirnary structure and conformation of the moiiotners I)eing packed. Filaments of Vibriofehs, about 120 in diameter, appear to consist of subunits packed into a maximum of four parallel strands (Ritchie rt nl., 1966). Lateral bonding between subunits is suggested since, in filaments broken by freezing and thawing, unravelling or fraying into fibres is not observed. As previously mentioned, under certain conditions flagellar filaments of B. piriilus and other members of Ikicillus appear to unravel iiito wavy fibres that may represent single strands of subunits which a t least in the case of 13. pumilus tend to be ovoid (Abram d nl., 1W-h). Under alkaline conditions or i n filaments isolated from old cultures, segments can be observcd that when embedded in phosphotungstate appear to consist of six electron-lucid, hexagoiially arranged, ovoid subunits surrounding an electron-opaque centre (Fig. 19). The diameter of these hexagons is approximately that of the flagellar filaments. IV'hilc these structures may relmscnt the arrangement of subunits in cross section, they may also be longitudinal pieces o f the tubular filanicnt without a subunit in the centre of'the hexagoiial arrangement. Moreover, it is impossible to exclude the possibility that they actually are artif'acts due to the aggregation of single subunits or chains of subunits occurring in the micro-environment that exists on the grid during electron microscopic exarriiiiation. In any case, the existeiicc of these structures may
a
B A C T E R I A L BLAQELLA
281
I’’1u. 19. Flagellt~rfilnnic.iits isolntcd from an “old” sporulatiiig culture of Bacilltis pzrinilus 111 a incdiiiin rc~itlerctlalltaliiie (about pH 9.5) by cell metabolism. Hexagons usiially are associated with filaments that oftcti reveal a “herringbone” pattern. The prcparatioil as negatively stained with phosphotungstate. ~ 2 3 0 , 0 0 0 . D. Abram tiiid H. Kofflcr, unpublislied observations.
282
H. W . SMITH AND IIENRY KOFFLER
reveal the most stable interactions between subunits under the conditions of isolation and examination used. Prom electron micrographs, Polevitsky (1944) suggested that flagellar filaments of many species are “hollow tubes”. The presence of such a hollow centre is suggested by X-ray data (Swanbeck and Porslind, 1964) and by penetration of the filament by electron-opaque staining materials (Kerridge et al., 1962; Abram and Koffler, 1963; Glauert et al., 1!)63; Abram et al., 1964; Claus and Roth, 1964; Hoenigcr, 1065; Ritchie et al., 1966). An electron-opaque line, a t times discontinuous, can be observed in the ccntrc of flagellar filaments, especially in short fragments (U. Abram and H. Koffler, unpublished). This indicates that the centre is either empty or consists of different material than flagellin. Since filaments reconstituted from purified monomers (see below) consist essentially entirely of flagellin yet show the electron-opaque centre, the first explanation is more plausible and we conclude that the flagellar filament is a tube, the walls of which consist of flagellin. By tthe same token, the occurrence of ((doughnuts” with electron-opaque centres serves as evidence for the tubular nature of the filament, since they probably are short fragments standing on the grid so that their longitudinal axis is perpendicular to it. Apparently, electron-dense stains penetrate shorter pieces of flagella more easily than long filaments; this may explain why a hollow centre has not been observable by some workers (Lowy and McDonough, 1964; Lowy and Hanson, 1964, 1965; Klug, 1967; Raimondo et al., 1968). It is likely that the conditions to which the specimens are exposed has some effect on the penetrability of heavy metal stains. Clearly, it stands to reason that shorter pieces are penetrated more readily than native filaments. The possibility that the centre consists of non-flagellin material capable of interacting with the heavy metal stain exists (Lowy and Hanson, 1965; Lowy et al., 1966), but the fact that fragments of purified filaments prepared by re-assembly of flagellin subunits show an electron-dense centre makes this possibility unlikely. While the functions of a hollow centre are not yet clear, two appear feasible. First, if elongation of the filaments occurs by addition of subunits at the distal end, as will be discussed later, translocation of the subunits may be brought about by passage through the tube. Secondly, the ability of chemical substances to reach individual flagellin subunits by diffusion through the centre may be relevant to mobility. Unless the subunits in a filament are arranged in a random fashion, which seems improbable, the differences in molecular topography between opposite “ends” of each flagellin molecule are likely to impose a morphological polarity on the filament. The appearance of fragments obtained by freezing and thawing, desiccation and hydration, or exposure to GO”, sonication, or hydrochloric acid treatment strongly
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FIG.20. A region of a grid onto which intact cells of Bacillus stearothermophilus 194 were placed. This fortuitous observation apparently resulted from uneven drying of the sample on the grid. Filaments that originate in one cell showpolarizedorientation of almost all the parallel pieces. The preparation was negatively stained with phosphotungstate. x 167,000. D. Abram and H. Koffler, unpublished observations.
284
H. W. SNTT11 AND HENRY KOFFLFLElt
indicates such polarity since in ncarly all caws one end of each fragment is split or fruycd (Abram et nl., IQBla,196ti~).When the frapmcnts still retain the relative position that they might have l i d in thc unbn~kc~11 filarncnt the other end usually appears complcmentury to thc split elid (Fig. 20). Thc split cnd is distal, as can be obscrvcd when fi'agmwits of isolated filunieiits still attached to hook8 arc cxamiiicd at various stage8 of disintegration in eitu 011 electron microscope grids in such cascs in whioh the relative position of each resultiiig fragment within tho filnment is still retained. Sincc tlic hook clcarly indicates the ~~x~oximsl cwl of tho flagellum, it is ImMible to identify thc split and of the hook RR well as those of filamentous fiqqncnts as bcing thc diRta.1 termini. Using this criterion, Avakura et al. (lfM8) dernoiir~tratedthat the olongation of' the filament is unidirectional and occurs at the distal end, since soluble fl. typhimuriwn flagellin molecules rn added to tho split ends of picm of filamenh that wcrc uscd as primttr. Howerer, i t in still puzzling why the split (i.e. distal) end of a tubular frugmcnt would not give the complementnry apparancc (i.c. look like the proxirnal and) whnn thc tubular structure is turned 00". Subunits located at the diRtal end must have different tnolecular sites exposed than those located at the proximal end, and it is conceivable t h a t the exposcd sitcs interact differently at each cndmsulting in a distinct apIntarccna..
X. Reassembly The most characteristic property of flagellin is itR ability to assemble into flagellu-like filaments. The re-aggregation of flagellin obtained by disintegration of flagellar filninonts at pH 2 into ordered tubular struutures was first reportcd in an oral prcsemat.ioa at thc Intc?rnatiaiinl Conppss of Jlicrobiology by Abram and Ihffler (I!M2) ill coiincuth with a paper on the amino-acid composition of flagellar filRment from several strains of UacZZZua (sec also Abram and Koffler, IO(j3, 1I)B:Ia). Unlike native flagellar filainents these wcre Btraight. It soon becainc appamnt (Abram and Kofflcr, 1083, 1083a, 1M4)that the nature of the ~~~
Frci. 21. (a). Straight sitriicturr*sYormtd at pH 4-4during tlialpiN of flngvlliii iti 0.06 N-HCI againfitdietilled water at 26'. Aggwgiitrn iip to 20pm. long have hwn obaorvod; thc width vwiw from 150 to 1,000 A. Segatively vtuirictl with iimnyl aoetate-EDT.-\. x20,OOO. Taken from Abratn and KlJftk!r (1964). (b). Stniotiirra formed in 0.05 .M -potaeeiiim phnsphato buffer at pH 4.9. Both flagella-lib nritl ntraight, Rtructtirce a m awn. Sliiulon-cnst with pn.llnditiin. x30.000:hkim frnm A tmtm axid ICofflw ( 1 904). (a). Homo aa Plate (b) except iic'gativnly ntriwd with 11r~tiy1 alc!c*tntct-ICl)'I'A. x 120,000.Ttikonfrom Abram nnd Koffler (1964). (d). Flqelln-li ko filament forined ia 0.06 M-potriwiuin phosphate Ijuffer ut pH 1.4. ~20,000.'raken from Abram and Koffler (1004).
BACTERIAL FLAGELLA
FIG.21.
285
286
R . W . SMITII AND HENRY KOFFLER
final product is determined by circumstances prevailing during assembly and that, under appropriate conditions, essentially instantaneous and complete polymerization of flagellin into normal-looking, but longer than normal, filaments can be accomplished (Fig. 21). Pavourable conditions for the re-assembly of flagellin from B. purnilus into flagellar-like filaments are a protein concentration of 2 mg. or morr per ml. (at a concentration of 5 mg./ml. the reaction is virtually installtaneous), 0.0275 M-phosphate buffer a t pH 5.4 to 5.6, and a temperature of 26" (Abram and Koffler, 1964). Within a pH range of about 4 to 1.9, ribbon-like straight structures of varying thickness are formed that not only differ from reconstituted flagella-like filaments in morphology and probably fine structure, but also in stability. Ribhon-like structures are also formed at low temperatures, below pH 5.1 exclusively, and at pH 5.4 to 5.8 in conjunction with normal-looking filaments. When ribbonlike structures are incubated under conditions desirable for the assembly of "normal" filaments, they becornc converted to flagellar-like filaments ; electron microscopically they seem t o arise directly from the ribbons, a still puzzling transformation. On the other hand, normal looking filaments when exposed to conditions suitable for ribbon formation will not convert to ribbons unless the polymer is first converted to nionomeric form. I t seems that, probably depending upon the conforms'lt'1011 and/or electrostatic charge of flagellin, this protein is capable of assembling into several organized structures of which the helical filament is the natural and probably most stable form. Flagellin of H. stearothwmophilus which is capable of assembling under much more varied conditions than is that of B. pumilus (Abram and Kofller, 1964) forms normalappearing filaments optimally between 2G' arid around 70', but straight tubular structures having the same thickness as normal filaments a t 2'. Polymerization of flagellin can also be achieved by salting out procedures, such as cycles of freezing and thawing or by the addition of' strong salt solutions, for example, in the case of flagellin from Snlmo)idlo typhirnuriurn ammonium siilphate t o about 33 saturation (Ada clt al., 1964). The polymers obtairicd in this manner, after dialysis against water, tend to be only slightly curved and preparations include many short pieces. Flagella-like filaments can be formed in witro, as indicated, from rnonoIners. I n fact, as mentioiicd previously, flagellin from cells of B. pumilus consists of two molecular species, A and R, occurring in tlic ratio of 7 to 3. Flagellins A and B, purified by column chromatography using DEAE-cellulose (Sullivan ~t nl., 1W O ) , separately can be reconstituted into flagella-like filaments under conditions at which clearly no flagellar fragments can be present t o serve as seed. However, the conditions for re-assembly seem to bc fairly specific for the various flagellins.
BACTERIAL FLAGELLA
287
For example, flagellin from Proteus vulgaris does not assemble at all under optimum conditions for B. pumilus flagellin. However, at p H 7 to 8.5 a t a potassium phosphate buffer concentration of n.4 to 0.6 M , 1OOq/, reconstitution of monomers derived from filaments by acid disintegration into long filaments can be accomplished without seed, probably by a process similar to saltingout (Martinez et al., 1967). Dependent upon conditions under which flagellin is prepared and reconstituted, fragments of flagellar filaments may be necessary as seed. Monomers from R. t?yphimuriuna, released b y heating flagellar filaments in 0.15 ill-sodium chloride and 0-01 dl-phosphate a t p H 7 at 60°, are incubated together with 0.2 t o 0.3Ilm.-loiigfraginents (“seed”), obtained from filaments by sonication, upon which the monomers can polymerize (Asakura et al., 1964, 1966; Asakura, I9fi8). While there is little doubt that all the flagellins so far studied are capable of polymerizing into filamentous structures without seed when conditions are appropriate, the use of seed has been an elegant way of studying the logistics and kinetics of assembly (Asakura et al., 1‘364, 1960; Oosawa et al., 1D6fi; Asakura, 1968 ; Wakabayashi P t al., I %!I). First, from the correspondence of seed particles and long filaments ultimately formed, it is clear t h a t the fragments serve as crystallization nuclei. Since nucleation is ratelimiting, the addition of crystallites promotes crystallizatioii. Second, as judged by the kinetics of reconstitution, polymerization consists of the reversible binding of monomers onto the end of existing filaments and finally the incorporation of bound monomer into the filament; only after the monomer has been incorporated can new monomers be bound. Third, the growth of filaments in vitro is unidirectional (Pye, 1!)67; Asakura et al., 1968) and addition of monomers occurs at the split end, regarded as the distal end (Abram et al., 1964a, IRBBa), of the seed particle (Asakura et n l . , 1!168). I n an excellent discussion 011 the thermodynamics and nature of the polymerization process, Oosawa and Higashi (1967) relate polarity in the filament t o configurational changes forced on the subunits upon polymerization. As mentioned previously, since the subunits in the filament probably are not arranged randomly, polarity necessarily exists at the molecular level since the topography, hence the type, location, and number of reactive regions, on one side of a protein molecule is different from t h a t on the opposite side. The morphology of reconstituted filaments depends on the nature of the constituent subunits. In Salrrionella, polymerization of flagellin monomers isolated from mutants t h a t possess flagella with an altered morphology (i.e. curly filaments) onto the end of seed fragments of wild-type filaments (and wicp versa) at high concentrations of monomer with respect t o seed results in filaments the morphology of which is determined by the monomer (Asakura rt nl., 1966). The shape of the
288
R . R . SMITH AND HENRY XOFFLER
re-assembled filaments can be altered by using a mixture of monomers isolated from different mutants, e.g. from cells having either curly or straight flagellar filaments (S. Asakura, reported in a symposium of the Third International Biophysics Congress, Cambridge, Massachusetts, 1969, and personal communication). The morphology of the reconstituted filaments is determined not only by the source of the monomers but also by the relative amounts of different monomers present in the copolymer. For example, copolymerization of flagellin from S.nbortusequi SJ 670 possessing normal filaments with flagellin from strain SJ 814, which has straight flagellar filaments, results in a t least three distinct stable morphologies with varying pitch and amplitude depending on the relative proportion of SJ 814 flagellin added. As thc proportion of SJ 81 4 flagellin is increased from zero percent t o 10, 60, or 90% of the total monomer present, the pitch and amplitude of the resultant filaments decrease. Gerber and Noguchi (1907) studied the kinetics of assembly of S. abortus-equi SL 23 flagellin by examining the volume change (dw ) upon polymerization. The change in volume at neutral pH values was dctermined to be 150 ml./mole of monomer polymerized. Between 22" and 28" the rate of assembly iricreases with temperature whereas Aw,,,, remuins constant at 157 f 4 ml./mole. Between 28" and 36", dv decreases with increasing temperature ; however, dw,,,,, increases to a maxiniurn of 306 ml./mole a t 3.5". Polymerization does not occur at temperatures greater than 35". The observed changes in volume may be mainly attributed to changes in solvent structure and not to a large change in the partial specific volume of the protein. As has been discussed, the flagellin molecule exists in different conformations depending on the temperature. Above 38", the conformation apparently is such that polymerization is not possible. Klein et al. (1967, 1907a) noticed a helixcoil transition beginning at 38" in flagellins of several species. Gerber and Noguchi ( 1 9f17) proposed t h a t flagellin might exist in two forms, an inactive conformation in which the molecule could not re-assemble (Ga) and an active one (G). The transconformation from G, to Q would then be rate limiting between 28" and 35'. Kinetic data suggest a transition state in which monomer and seed form an activated complex prior to incorporation of the monomer into the structure of the filament. The low energy of activation observed for this process further suggests t h a t the conformation of the monomer is drastically altered by this reaction. The mechanism of polymerization devised by Gerber and Noguchi (1967) is described as follows :
BACTERIAL FLAGELLA
"9
For reaction ( 1 ) operating in the reverse direction at 30.6', the following thermodynamic parameters were calculated : dk'= 0 kcal./mole, dH = 108 kcal./mole, and ds = 367 e.u. For the activation reaction, (1),at, 25" the following were calculated: E, = 7.8 kcal./mole, d H = 7.2 kcal./mole, and d s = -10.1 e.u. As has been mentioned previously, flagellins derived from filaments by disint,egration at p H 2 largely in random coil conditions undergo conformational transitions as the pH is raised towards 4 and assume a greater helical content (Klein et al., 1968). Between pH 4 and 1 1 no additional conformational changes can be observed by spectropolarimetric methods. However, i t seems now fairly certain t h a t conformational changes occur that, cannot be detected by spectropolarimetric techniques. For example, when examined by t)hese methods, flagellin from thermophiles appears to remain constmt in conformation between pH 1 and 1 1 . I n the case of flagellin from the thermophilic organism B. stearothermophilus 1 1 8 4 , however, it can be demonstrated by analysis of difference spectra that a t least one tyrosine residue that is exposed a t pH 2 becomes buried as tJhepH is raised t o 4.8 (Yarbrough et d., l!X9). Furthermore in this case conformational changes must occur also above pH 8.4, since addit,ional previously buried t,yrosine residues become exposed between pH 8.4 and 11.5, as determined by their reactivity with tetranitromethane (Yarbrough et aH., 1969). Either these changes are not relevant to the helical portion of the molecule or are too small t o be observed by optical rotatory dispersion techniques. I n any case, all the available data indicate that there are architectural prerequisites that need t o be met before assembly can occur. The flagellin molecule is structurally versat,ile since conformational changes induced by pH., temperature, or urea are reversible even after several cycles. Under the appropriate conditions of pH, temperature, and ionic milieu, flagellin is capable of assuming the necessary conformation and/or charge to assemble. There is now reasonable evidence that additional conformational changes occur during assembly. Suggestions for this have existed since Read and his colleagues (Read et al., 1956; R.ead, 1967; Koffler, 1957) detnonstrated by quantitjat,iveprecipitin reactionst,hatflagellin binds only 16 t o 2o:;; of the antibodies directed against flagellar filaments (withthe filaments reacting 100% by definition). Furthermore, the antigennitrogen to antibody-nit,rogen ratio in the precipitat,e formed when flagellin binds antifilament, immunoglobulins differs from that of t8he homologous systetn in which flagellin binds antibodies against flagellin (6.3--7.4 and 11.7 respectively). There are ambiguities in the interpretation of these experiments since the condition of the flagellin after inject.ion into the experimental animal and in the precipitin reactions, all performed in saline solutions, is not known. Nevertheless, these data
290
R . W. SMITH AND HENRY KOFFLER
suggest diffcy-cnces in immunogenic and/or antibody-binding sites between soluble and polymeric flagellin. Whether these differences are due to different conformational states or tlic formation of additional immunogenic and antibody-binding sites by adjacent monomers cannot yet be distinguished. However, observations based on determination of' rotatory dispersion properties and circular dichroism make it likely that some conformational clianges do occur during assembly (Koffler et al., 1966; Iilein c.t al., 1967, 19ti7a, 1!)68, 1909, 1969a; Koffler and Smith, 1968 ; 8. Asaknra, personal communication). Based on the methods of' Simmons P t nl. (1961), Moflitb and Yang (1950), and Shechter and Blout (1904, 1964a), the apparent a-hclix content in flagellin or YrotPuS vulgnria, B. pumilus, B. lichPniformia, H. sp. X , and B . slPnrotherrnophilzis 2 184 was determined to double as tlie flagellin inolecule is incorporated into the structure of the filament (Klein et al., 1969, 1909s). The molecule is essentially unfolded at pH 2 arid tlie a - M i x content increases to 21 to 32 % when t h e pH is raised to about 4. Further increases in pH to 11 do catable changes in helical content. However, upon polymerization, the apparent a-helix in the protein increases from 5 0 to 70%. I n addition to tlic analyscs of the dispersion parameters and of the 433 nm. trough values, cotton effects and circular dichroism show that the increases in rotational strength upon polymerization are accompanied by shifts of the pcaks and troughs toward the red end of the spectrum. Similar results were obtained with flagellin and intact filaments of' flagella from Salriaonella typhiinurium, based on circular dicliroisni ineasnrements at 222 nm. (S. Asakura, personal communication) Flagellin a t p H 2 , or at 65" and p H 7 contained about 1 2 % a-helix. Adjustment of the p H to 7 under conditions in which assembly does riot take place, or lowering the temperature of heated solutions t o 25", results in an increase in the a-helix content t o 27 %. Upon incorporittioii into the filament, the helical content of this flagellin increases to 46 %. While all these data, are clear cut, their interpretation is not since thc aggregation of' poly-IA-glutamicacid below pH 4.5 when the molecule is from 80 to 100 "/, helical results in similar incrcases in intensity and shifts of peaks and troughs (Cassim and Yang, 1967). Since the niolcculc already is fully helical, or almost so, these changes cannot be due to increases i n helical content and must be due to aggregation. The valut~ of the Moffitt parameter, h,, normally iricreascs negatively with increases in the magnitude of tlic 233 nm. trough (i,e. a-helix content) while the value of a, increases positively. Upon aggregation of poly-L-glutamic acid, however, the value of b, either does not change a t all (Schustcr, 1965; Tomimatsu et al., 196(i) or else it bccbomes slightly less negativcl (Cassim and Taylor, 1!)65), even though large increases in rotational strength occur at the 233 nm. trough. The value of cco shows large negative
BACTERIAL FLAGELLA
29 1
changes during aggregation (Schuster, 1965). Apparently, the observed changes in intensity and positions of the peaks and troughs during aggregation of poly-L-glutamic acid do not necessarily indicate cahsnges in the a-helix content. The situation is quite different when flagellin polymerizes in agreement with the notion that conformational changes do occur. Increases in pH from 2 to 4 and above result in a large negative increase in the value of b , and, at the same time, a positive increase in the value of a, (Klein et al., 1968) as one would expect from increases in a-helix content. During aggregation of flagellin, the value of b, also becomes considerably more negative, but the value of a. changes only little (Klein et al., 1969, 1969a). The essentially unchanged value for a, during an apparent increase in a-helix content could be accounted for by a large negative change due to aggregation, as observed in the case of poly, is neutralized by a positive L-glutamic acid by Schuster ( 1 ~ 5 )which change brought about by an increase in helix content. That is, both aggregation arid an increase in helical content may contribute t o the changes observed with these techniques. While it appears plausible that the assembly of flagellin is accompanied by conformational changes, further studies are needed to establish this point with certainty. Such changes may be prerequisite to incorporation of flagellin into the filament or the consequence of flagellin-flagellin interactions. Since none of the flagellins examined contains cysteine, disulphide bonds cannot be involved in intramolecular or intermolecular interactions. Data are now being accumulated, none of them conclusive in their own right, which assign a significant role to hydrophobic bonding in such interactions. ( 1) As mentioned previously, flagellar filaments are disintegrated by urea, guanidine hydrochloride, acetamide, alcohols, dioxane, and detergents, agents that are regarded as affecting largely hydrophobic bonds. (2) Self-assembly of flagellin into “normal” filaments proceeds best near room temperature ; increases of temperature u p to a maximum that varies with the situation tend to stabilize hydrophobic bonds but labilize others. Enthalpy changes upon formation of ionic bonds are near zero whereas formation of hydrophobic bonds usually results in a positive change (Kauzmann, 1959; Scheraga, 1963). Such positive changes in enthalpy during association of molecules of flagellin have been observed by Vegotsky et al. (1965) and Gerber and Koguchi (1967). ( 3 ) The involvement of hydrophobic bonding is further suggested by the effects of salts o n flagellins. Salts strengthen hydrophobic bonds due to the decreased solubility of non-polar groups in the more polar solvent. One would predict, then, that addition of salts stabilizes the polymeric form of the protein. Flagelliri from heat- or acetone-disintegrated filaments of Salrnondla spp. reassembles only in the presence of salts
292
R.
W. SMITH A N D
HENRY KOFFLER
(Asakura ef al., 1004). Ada et nl. (1!)64) found that flagellin from aciddisintegrated filaments of Salmonella ndelaide can bc made to re-assernblc by addition of ammonium sulphate. Similarly, concentrations of fluoride, carbonate, sulphate, citrate, and phosphate greater t hail 0.3 M induce rapid and complete aggrcgation of flagellin (Wakubaynshi et al., 1969). Martinez et al. (1907) found that the flagellin from Ilncillus suhtilis and Spirillum serpens re-assemble even at pH 2 in the presenre of' 0.05 M-salt. The flagellin of Bacillus punailus (It. W. Smith aiid H. Koffler, unpublished results) and Salmonella adelaidc (Ada et ul., 1964) aggrcgate into straight structures in the prescnce of salts. In the ctlse of B. pumilus the structures form in the presence of 0.08 to 0.2 M-sodium chloride in the p H rangc of 1-4. Although salts may dampen repulsive charges that forcc the moiiorncr into a conformation unfavourable for polymerization, their principal effect probably is to cncourage hydrophobic interactions. The behaviour of myosin, the association of which is regarded to involve mainly ionic forces, is entirely different (Joscphs and Harrington, l!MS). Whercas association of flagellin molecules is favoured by salts, polymers of myosin completely disintegrate into monomers in the prescnce of 0.28 M IiCl (Joseplis and Harrington, 1 966). Also, the equilibrium constant for the association of mononicrs of myosin is independent of temperature, a property that is characteristic of reactions involving ionic forces. (4)Recently we have studied the bchavionr of tyrosine aiid methioiiinc residues on the assumption that they are likely to be located in hydrophobic regions within the molecule, and have obtained strongly suggest ivc evidence that thcy are irivolvcd in intermolecular interactions. I''or example, as judged by its reactivity a t pH 8.5, flagellin of Iz. stenrotherrrrophilus 2184 in polymeric form has one out of six tyrosine residues exposed, while in monomeric form undcr these conditions three are exposed (Yarbrough et nl., 1089 and unpublished results). One of tliesc three tyrosines is probably the same as the one residue exposed in polytneric form. When filaments in which one tyrosine per flagellin molecule already has been nitrated are disintegrated a t pH 2, and the released monomers containing one modified tyrosine are treated further with tetranitromethane, only two additional tyrosine residues are react ive. The modified flagellin obtained by acid disintegration of the modified polymer is capable of assembling a t pH 5.8 as well as a t pH 0.0, a pH a t which the single exposed modified tyrosine residue is ionized. Since this residue is exposed in filaments and therefore probably not involved in assembly, its electrical charge docs not seem to be significant. When monomeric flagellin is modified a t pH 8.5, two derivatives can be separated, one being the monomer with three of the tyrosine residues modified. The other is a dimer apparently covalently linked through two modified
BACTERIAL FLAGELLA
293
tyrosine residues one furnished b y each monomer. Not only do the isolated monomers assemble into filaments b u t so do the purified diiners, suggesting t h a t tyrosiiie-tyrosine iiiteractioiis (more likely interactions of the hydrophobic regions in which the tyrosine residues are located) may be involved in assembly. Unlike monomers in wliicli only one tyrosine reHidue has been modified, the monomers aiid dimers with three modifications per monomer assemble a t p H 5.8 but not a t pH !).o. This confirms the idea that the condition of one or two tyrosine residues is critical t o assembly, since assembly does not occur when they are ionized. The hydrophobic* nature of intermolecular bonds has also been investigated b y cherniral modification of methionine residues (Smith and Koffler, 1067, 1!lOX, I !)ti!), and unpublished results ; Koffler and Smith, 1!168; Smith et nl., 1!)68). Flagellin of B. pziwii2u.s has a molecular weight of about 32,000 and Contains nine methioniiie residues per molecule. No cysteine, tjrrosinc, or tryptophan has been detected. As mentioned, methionine residues w e thought t o reside primarily in hydrophobic regions ; modification of these residues with iodoacetic acid t o form the carboxyrnet hyl sulphonium salt should decrease the hydrophobic nature of nietliioiiine-coritaiiiirig regions. I n the intact filament, methionine residues are riot exposed t o the reagent since carboxymethylation does not occiir a t pH 5.5 t o 6 . 5 . A t pH 2 . 2 carboxymethylation of methionine residues follows pseudo-first order kinetics (Smith and Koffler, l!MiX, 1!%!)). All residues react and do so at the same rate supporting the assumption t h a t they exist in similar chemical environments. This is expected since flagellin of B. pzcrrLiZus exists essentially as a random coil at this p H aiid all residues should be equally exposed t o the solvent. Identical kinetics are found regardless of whether naturally occurring mixtures of the A and B proteins or t h e A and B proteins separately are modified. Destruction of the ability t o re-assemble during earboxymethylation also follows pseudo-first order kinetics. Modification of an average of 15 t o 20 of the mrthionine residues completely destroys the ability of a population of molecules t o rc-assemble. I n samples modified t o the average extent of less than one residue per molecule, some of the inore lightly modified molecules retain the ability t o re-assemble. Iteassembled filaments cvmtaining modified molec*ules are slightly, but reproducibly, less heat stable, having a Td value of 58" as compared t o 60" for filaments re-assembled from unmodified flagellin. The inability of the modified molecules t o re-assemble cannot be explained by the destruction of intramolecular helix structures, as determined by optical rotatory dispersion measurements. Furthermore, the stability of the helix t o heat i n modified monomers is identical t o t h a t in coiitrol monomers. A comparison oftryptic peptides of completely carboxymethylated
294
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. SMITII A N D IIENItY
KOBNLER
flngellin with those from slightly modified molecules that retain tlic tibility to rcwLsscnible iiitlicatcs flitit k t t leitst one specific tnetliioiiine residue exists in flagellin A and anothcr in 13 tlir modifiration of which cwin1)letclytlcstroys the ;hility to re-assemble. 'i'liese data iitdicatc that association of flagellin molecules to form thc filament structiirc is sciisitivc to altrrations in hydrophobic regions of the molecule. M'ithout romrnenting on tlie ])ossiI)le mechanism, Ichiki and Martinez ( 1 !)(is) report that treat tnent of flagellin of 13. subtilis with sodium periodatr c*ornpletelydestroys the ability to re-assemble. Likewise, flagelliii of H. piirnilis fails t o re-assemble following tretitriient with periodiLfc* (1%. 1V. Smith and H . I
