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
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N o t e d t l d e d in Proof The stiitlics of salic.ylato hydi.oxyltlse (Katagiri et c i l . , 1966) huvc>been extcnclctl (l’akemori rt n l . , 1M!)).Thc enzyme has been crystallized, and its pro1)crtics examitred cxtcnsively and related to a proposed mechanism of action. R salicylate hydroxylase isolated from another organism (White-Stevens and Kamin, 1 !170) was found to differ from that studied by Kntagiri el ul. (19U6) in so far as it catalyscd a benzoate-stimulated oxidation of NA DH,, dthough benzoate was not hydroxylated. It al)l)cv~rs that activateti oxygen, produced when benzoate was bound to tlic etizyrnc, dcconi])05c~lto form Iiydrogen peroxide ; evidently sonic
46
8. DAQLEY
structural feature of benzoate precluded hydroxylation. This “uncoupling” of oxygen activation may provide new opportunities to seek the highly reactive intermediates that probably take part in such reactions. Behaviour similar to that of benzoate is shown by 6-hydroxyriicotinate (Howell and Massey, 1970) which, although not hydroxylated, can nevertheless be bound to p-hydroxybenzoate hydroxylase and thereby facilitate both binding of NADPH, and reduction of enzyme-bonnd flavin by NADPH, (see Fig. 8, p. 21). Ribbons ( 1970) has measured oxygen consumed, NADH, oxidized and formaldehyde produced when vanillate, veratrate or m-methoxybenzoate was demethylated by a cell-free extract of Pseudomonas aeruginosa. One mole of each of the three substrates involved in a demethylation, namely oxygen, NADH, and the aromatic substrate, reacted to give one mole of each product. This is the result expected for a typical mono-oxygcnase and is in agreement with the findings of Toms and Wood (1970a)summarizedin Fig. 11 (p. 30). I have drawn attention t o the fact that catechol 1,2-oxygenases usually exhibit high substrate specificity. Another exception to this generalization appears to be provided by the enzyme present in a yeast isolated from soil by Varga and Neujahr ( 1970). The existence of a pathway of anaerobic metabolism, probably quite different from that of Fig. 1 (p. 6), is indicated by the isolation of a Pseudomonas sp. that metabolized various aromatic compounds anaerobically when nitrate was used as electron acceptor. The organism also grew aerobically with p-hydroxybenzoate using the meta-fission pathway, but these enzymes are not used in anaerobic growth (Taylor etal., 1970). It has been assumed since 1953 that bacteria convert naphthalene into a trans-dihydrodiol before ring fission occurs (p. 25). The evidence has been challenged by Jerina et al. (1971) who found that a strain of Pseudomonas accumulated a compound which they rigorously identified as cis-1,2-dihydroxy-l,2-dihydronaphthalene. Naphthalene may therefore be metabolized by reactions similar to those for benzeiia and toluene (Fig. 10 c and d, p. 24). ADDITIONAL REFERENCES Howoll, L. G . and Massey, V. (1970).Biochem. Biophya. rea. Cotnnmn. 40,887. Jerina, D. M., Daly, J. W., Joffroy, A. M. and Gibson, D. T.(1971).A r c h . Biochem. Biophya. in press. Ribbon8,D.W. (1970). FEBS’Lettera8,lOl. Takomori, S., Yasuda, H., Mihara, K . , Suzuki, K. a i d Kattigiri, M. (1909).Biochim. biophya. Actu 191,88. Taylor, R. F., Campboll, W. L. andchinoy, I. ( 1 9 7 0 ) J . Bact. 102,430. Vargtr, J. M. aiitlNerijtihr, H. Y . (1070). HUT.J . Biochem. 12,427. Whito-Stevunr;, R. H. and l
Synthesis of Enzymes During the Cell Cycle H. 0. HALVORSON, B. L. A. CARTERand P. TAURO Laboratory of Molecular Biology and Department of Bacteriology University of Wisconsin Madison, Wisconsin, U.S.A. and Department of Microbiology Haryana Agricultural University Hissar, India I. Introduction . 11. Methods for Establishing Synchronous Cultures A. PhasingMethods . B. Selection Methods . 111. Synthesis of Protein and RNA During the Cell Cycle A. Protein Synthesis B. HNA Synthesis . IV. Enzyme Synthesis During the Cell Cycle A. Introduction B. Synthosis of Enzymes in Prokaryotic Organisms Growing in a Constant Environment C. Synthosis of Enzymos 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. Acknowledgernents . Referonces .
.
. .
.
.
.
.
47 49 50 51 63
53 53 66 55 57
63 71 75 95 98 99
99 99
I. Introduction In a recent symposium on “Microbial Growth” (Meadow and Pirt, 1969),growth processes were separated into the behaviour of populations and the behaviour of single cells. This distinction reflects the growing belief that the properties of single cells cannot always be deduced from studies of random populations. In only a few cases can the biochemical 47
48
IT. 0.JIALVORSON, U . L. A . CARTER AND P. TAURO
events of the cell cycle be measured in an individual cell and therefore a major contribution to our understanding of the complex nature of the cell cycle has come from studies of synchronously growing cultures. A number of excellent reviews and monographs have appeared on various aspects of the ccll cycle (Campbell, 1957; Sclierbaum, 1960; Madrac, 1962 ; Zeuthen, 1964 ; Pirson and Lorenzen, 1966 ; Cameron and Padilla, 1966; Yadilla et al., 1969; Rusch, 1969). These bioclicmical studies 1itLvc reinforced previous cytological observations that during the cell c~y(-lc the biochemical corn1)onents of the ccll do not increase continuously i t 1 (qua1 proportions but as a series of discrete temporally organized everits. For a complete understanding of the regulation of these cvc.nts in prokaryotic and eukaryotic cells, information on the integrated synthesis of all the eellulnr components is required. A discussion of the rcgulation of all of these is bcyond the scope of this review but with the rccvnt advances in our understanding of the regulation of’ enzyme synthcsis in both prokaryotic and eukaryotic cells, mi analysis of the synt,hesis of enzymes during the cell cycle is timely. The cell cyclc was separated by Howard and Pelc ( 1 953) into G , , S, Ci2 and M periods. ‘I’liey identified the S period as the time during whirh tlic DNA o f t h e cell w a s replicated, the M period as mitosis, and (3, arid C 2 as ‘‘gt~ps” which precede and follow DNA synthesis. The relations hi^) between thc length of periods is not, constant in all organisms or even in tlie same organisni undcr different growth conditions. For example, in thc embryonic cells of sea urchins (Hinegardner et al., l!j64) mid frogs (Graliani and Morgan, 1966) a C i period is absent. A distinct S period is present in slowly growing bacterial cells but in fast growing (TIIS DNA synthesis occurs throughout t l i ~cell cycle except for a short pwiod ctt the time of fission (Lark, 1966; Helmstetter, 1987, 1068; Kubitsclick et al , 1967).In synchronized niamnialim cells, Rao and Il:ngclberg ( 1 !NO) observed that the duration of one pcriod of tlie re11 cycle as compared t o another w i ~sdcpeiident on the terri1)erature at which the cells were grown. Synchronous growth is most conveniently monitored by following the doubling of cell numbers at the end of each surcrssive cell cyclc. In :L number of orgiLnistns, liowwer, this is either inconvenient or not applicable t o tho mode of cell division. For exarnplc multiple budding c)ccurs in some yenst str;tins and in :L iiutriber of bactcria, cc4l division lcuds to chains of cells. Also in thc slimc mold, Physaru,71.polyce~halurr~, sucwssivc mitotic divisions orcur within a single plasmodiuin. In cases such as thcse, cytological and biochemical markers have been employed to defiiie the cell cycle. Mitcliisoii (1969a) has reviewed the usefulness of various cell markers in ccll cycle studies and he has emphasized the importance of a marker repeating itself over several cell cycles with a high degree of precision.
SYNTHESIS OF ENZYMES DURINQ TIIE CELL CYCLE
49
A number o f cytological indices have been employed. These include measurements of nuclei in higher organisms, cell size, appearance of buds and cell JdateS in yeast and septum formation in bacteria. I n practice, these are slow and tedious methods for monitoring a synchronous culture. A number of biochemical markers can be used t o supplement the various cytological markers The synthesis of macromolecules such as DNA it11d specific proteins during the cell cycle can be used as biochemical markers for cells growing in a constant environment. However, because microbes respond quickly t o environmental change i t is clear that, unless steady state contlitions exist for the growth of cells, it is difficult3t o rcly on biochemical markers from which tho integrated syntlicsis of c d coml)onents can bc dissociated. For example in bacteria the syntlicsis of cnzymes can continue in th e absence of DNA synthesis (Masttm and Donachic, 1966) and cell division can be uncoupled from DNA rq)lication (Rtirsc4i ant1 Van der Kamp, 1961). One of the major rcquiremc~ntsof any system employed to study the cell cycle is that it should exhibit balanced growth. This is dcfined a s the doubling of every biochemical unit witliin th e time limit of a single cell cycle without changc in the growth rate of the organisms (Campbell, 1957). ‘rhe biochemical processes should repcat in succeeding generations precisely a t tlic same time in tjhe cell cycle. Balanced growth may not be achievcd i n the cell cycle if tlie conditions t h a t operate within tlie cell are altered by tlw 1)rocess used t o obtain synchronous cells. Many systcms, cspcvially t lie sync.lironizcd vrilturw of micro-organisms that have been employed t o study the cell cycle in recent years, d o not exhibit balanccd growth. In this rcviwv we shall rcstrict our attention mainly t o cell cycle studies which are carried out under steady state conditions and display balanced growth. The precision to which th e timing o f events can be measured during the cell cycle is limited by th e degree of synchrony th a t prevails in the population. Perfect synchrony is seldom achieved. I n addition t o the heterogeneity inherent in an y method for establishing synchronous growth, random variability in generation time contributes to a decay in synchrony during subsequent cell division (Engelberg, 1964 ; Anderson and Petersen, 1964). Engelberg and Hirsch (1966) have computed th e rate of decar of synchronizaticm in cultures where the standard deviation of cell doubling times is known. 11. Methods for Establishing Synchronous Cultures “Wc cannot make 100 uniform bacterial cells divide all at the same time anymore than we can mako all sucrose molecules of a sugar solution invert at the same time” (Kahn, 1932).
50
H. 0. HALVORSON, R . L. A. CARTER A N D P. TAURO
The biochemical events which occur during the cell cycle have been examined using single cells and synchronous cultures. Only a few studies have been made with single cells because it is difficult to apply standard biochemical techniques, which are developed to study populations, to a unit as small as a single cell. The most notable exception to this has been the work of Rotman and his colleagues which will be discussed later. The development of methods of producing synchronous cultures resulting in amplification of the biochemical events which occur in a single cell has permitted extensive investigationof both the prokaryotic and eukaryotic cell cycle. Synchronous cultures have been obtained using phasing methods which synchronize a n exponential culture and by selection methods in which a synchronous population is physically separated from an exponential culture. The various methods are outlined in Table 1.
A. PHASING METHODS Campbell (1957), Scherbaum (1960), Maalae (1962) and Pirson and Lorenzen (1966) have reviewed the various phasing methods used to synchronize cultures. These methods all rely on changes in the environment (temperature, nutrients, illumination or end points of growth) t o effect synchronization of a cell population. These procedures, therefore, result in extensive metabolic disturbances t o the culture. Maabe (1962) and others have objected t o the use of such measures t o study the cell cycle because the metabolic disturbance caused by phasing methods may also result in abnormal growth. Although phasing methods are less commonly used in recent years, they are still the method of choice in some organisms ;they can provide valid synchronous cultures for examination only when it has been established that conditions of balanced growth (Campbell, 1957) arc maintained over successive cell divisions in a constant environment. A recent modification of the phasing method is the use of oscillating supply of nutrients to a chemostat population. Dawson ( 1965) found that, if nutrient was added periodically, that is once per generation time instead of continuously, t o a chemostat culture, then a synchronous culture would develop and could be maintained as long as the periodic additions of nutrient were continued. This technique has been used successfully by Von Meyenberg ( 1 969), Goodwin ( 1 969a) and Hansche (1969) to produce synchronous cultures of micro-organisms. Such chemostat populations have an advantage of making available large quantities of cells. However, when this method is uscd, cells do not grow in a constant environment because of the periodic nutrient additions and the
SYNTHESIS OF ENZYMES DURINQ THE CELL CYCLE
51
TABLE1. Methods for Establishing Synchronous Cultures Method
Organism
Reference
A . Phasing methods Endospores and stationary- Williamson and Scopes (1960); End points of growth phase bacteria, yeasts Fitz-James (1965);Sueoka and nutritional deficiencies (1966) ; Cutler and Evans (196G). in a varie1.y of organisms Inhibitors Animal cells Xeros (1962) ;Bootsma et al. (1964) ; Whitmore and Gulyas ( 1966). Light, cycles Algm, h h l g c n a SchmidtandKing(1961); Kates and Jones (1967); Patropulos (1964) Hypoxia Tetrahymenn Rasmussen (1963); Rooney and Eiler (1967). Scherbaum and Jahn (1964) ; Temperaturn Algae, Tetrahymena, Lafeber and Steenbergen change bacteria, lhglena (19G7);Scherbaum(1964); Pogo and Arce ( 1964). Chomostat Bacteria, yeast Dawson (1965);VonMeyenburg (1969);Goodwin (1969); Hansche (1969). B . Selection methods Helmstetter and Cummings Adsorption and Bacteria, animal cells (1963);Scharffand Robbins release hy (1965);BrentetaZ.(1965). growth Maruyana and Yamagita Sediniontation- Yeast, animril cells, (1956);Mitchison and velocity Tetrahymena Vincent (1965);Sinclair and Bishop (1965);Ayad et al. ( 1969);Corbett ( 1964). Sitz et al. (1970). Sedimentation- Chlorella isopycnic
biochemical events which occur may not be identical to those which occur in synchronous cultures growing in a constant environment.
B. SELECTION METHODS Synchronous cultures also have been obtained by the physical separation of physiologically identical cells from an exponential culture. The advantage of these methods is that there is minimum disturbance of the cells.
52
11. 0. HALVORSON, U . L. A . CARTER A N D P. TAUItO
hlarl1yaltrit and YtLmagitiL ( 1956) were the first to apply this prowlure. They observcd that, whcii a culture of Escherichiu coli was poured over filter paper, following a short delay a population was observed in thc filtrate that grew synchronously. Subsequently, Helmstetter and Uretz (1963) observed that the bacterial cells will bind t o and grow on the filter p a p . When the attached bacteria divide, the newly-formed daughter cells arc released from the paper and can be continuously washed off arid used to inoculate a synchronous culture. Helrnstetter and Cummings ( 1063) furthcr dcveloped this procedure to permit continuous recycling of the medium. Unfortunately this method works with only ccrtiLin strains of bacbtcria. Differential binding to a surface has also been used t o establish synchronous cultures of mammalian cells. Brent, ~t 01. ( 1 965) and Scharff itnd Robbins (1965) observed that, upon shalring rrionolaycrs of cells attacahed to glass, cells cntcring tnct:y)hase could be detaclicd. Centrifugation techniques have been used successfully t o scparate synchronous popihtions of bacteria, yeast, algae, protozoit and animal c ~ l l sfrom exponentially growing cultures. Maruyama and Yaniagita. ( 1956) observed that, bacteria can be separated by sucrosc tlensity-gradient, ccntrifrigation. Mitchison and Vincent (1965) further refined this technique so that, separation of synchronous culturcs of bacteria and yeasts could be achieved. This method has been uscd by Sinclair and Bishop (1965) t o obtain synchronous cultures of mouse 1, rrlls. Morcx rccwitly, Ayad ef nZ. (1!169), using Picloll griidierit ccwtrif‘iigation, obtained synchronous cnlturcs of mouse lymphoma cells. One disadvantage of separation methods is that a small yield is obtained, but rccently Halvorson et aZ. ( 1 970) have dcveloped a method for thc largescalc separation of synchronous yeast cells from exponential cultures using sucrosv density-gradient ccntrifugation in it zonal rotor. Whcre clear differences in density of the cell occurs during the cell cycle, equilibrium density centrifugation can be employed. Recently an isopycnio technique employing linear dcnsity gradients of Ficoll has been used by Sitz et ccl. (1970) to select synchronous cells from asynchronous cultures of CAlorella. Zonal centrifugation provides a n alternative to synchronous growth for examining the properties of cells of different age in an exponentially growing populittion. This is based on the fact that : (a)in some organisms the size distribution of an exponcntially growing population is correlated with its age' distribution (Schaechter EL al., 1962; Mitchison, 1957); and (b) the velocity of centrifugation is a function of cell volume. Thus the cell cycle should be represented across the density gradient. This procedure has been employed to study DNA (Kubitmhek et al., 1967) and RNA (Manor and Haselkorn, 1967) synthesis in E . coli and enzyme synthesis in Saccharomyces cerwisiar (B. L. A. Carter, unpublished
SYNTHESIS O F ENZYMES DURING THE CELL CYCLE
53
results). Analysis of cells directly from the gradient has the potential to avoid many of the difficulties associated with synchronously growing cultures.
III. Synthesis of Protein and RNA During the Cell Cycle A. PROTEIN SYNTHESIS Several procedures have been employed to follow thc increase of protein during the cell cycle. These include: (a) net increase of bulk protein ; (b) the incorporation of radioactive amino acids into polypeptides ; and (c) radioautographic analysis of pulse-labelled single cells. There is general agreemcwt* that protein increases continuously throughout the cell cycle of bacteria and yeasts (Abbo and Pardce, 1960 ; Cutler and Evans, 1967 ; Perrctt ti and Gray, 1968 ;Williamson and Scopes, 1961 ; Halvorson et al., 1964 ; Mitchison and Wilbur, 1962). However, this plienonienon is not g e n t ~ a lfor all eucaryotie organisms because protein synthesis is depressed just prior t o cell division of protozoa (Prescott, 1955 ; Harnburgw and Zeuthen, 1960 ; Plcsner, 1963) and maniriialian cells (Prescott and Bonder, 1962 ;Konrad, 1963; Hodge et al., 1969). The increase in protein synthesis in the cell cycle docs not mean that all individual protein s1)cwies are increasing at the same time. I n two systems, synchronous division in yeast (Halvorson et al., 1964) and outgrowth of bacterial spores ( Hoyern et al., 1968), double-labelling experirnents have shown differences in the pattern of protein synthesized at two different intervals of time.
B. RNA SYNTHESIS The synthesis of bulk RNA of both bacteria and yeast occurs throughout the cell cycle (Maruyama and Lark, 1959; Williamson and Scopes, 1960; Halvorson et al., 1964). These findings arc consistent with the observations of Mitcliison and Lark (1962) with single cells of Xchizosaccharomyces pombe. I n Chlorella ellipsoidea, RNA synthesis parallels that of protein synthesis (Iwamura, 1962). However, in other cukaryotic organisms such as Physarum polycephalum, the rate of synthesis of bulk RNA is not constant during the cell cycle. The rate of synthesis reaches a peak following the S period, declines in mid intcrphase, and again
* There are two reports to the contrary. I n synchronous cultures of Azotobacter (Lin and Wyss, 1965) and E . coli (Nishi and Kogoma, 1965) stepwise risos in brilk protein were reported. In the lattcr case, radioactive precursors wero incorporated without interruption. Fluctuatious in procursor pools, perinease systems or protein turnover could account for this apparent contradiction.
64
H. 0.HALVORSON, B. L. A. CARTER AND P. TAURO
increases during the latter half of the interphase (Mittermayer et al., 1964; Rusch, 1969). Similar results employing cultured animal cells have been reported by Crippa (1966). However, only one peak in the rate of RNA synthesis (first half of S phase) was observed in the cell cycle of HeLa cells (Pfeiffer and Tolmach, 1968). In some cases different conclusions have been reached from autoradiographic procedures. For example, Kessler (1967) reported in P.polycephulum that uridine was not incorporated during metaphase or anaphase. Where studied, ribosomal RNA (r-RNA) and transfer RNA (t-RNA) synthesis has been observed throughout the cell cycle in a number of organisms; however the rates of their synthesis may vary. In bacteria, the synthesis of RNA is continuous throughout cell cycle but the rate of r-RNA is discontinuous (Maruyama and Lark, 1959 ;Rudner et al., 1964). InSacch. cerevisiae,r-RNA and t-RNA are synthesized at a constant rate throughout the cell cycle (Tauro et aE., 1969). In cultured HeLa cells both r-RNA species as well as 4 s RNA are synthesized in a constant ratio during the cell cycle although the total rates of synthesis vary (Scharff and Robbins, 1965). Miller (1967) estimated that 20% of r-RNA was synthesized in the early stages and 6% in the later stages of the cell cycle. An understanding of the regulation of transcription of these RNA species may be complex since it is known that there are multiple r-RNA cistrons in bacteria and multiple r-RNA and t-RNA cistrons in eukaryotic cells. The pattern of synthesis of individual m-RNA species during the cell cycle is less clear. There is some evidence that pulse-labelled RNA made at two periods of the cell cycle differ in composition. In E . coli, Rudner et al. (1964) observed that the base composition of the pulselabelled RNA varied during the cell cycle. I n yeast, the isotopic profile of pulse-labelled RNA from two stages of the cell cycle differs (Halvorson et al., 1964). This is further illustrated by fluctuations in the base composition of pulse-labelled RNA during various periods of the cell cycle of a hybrid yeast (H. 0. Halvorson, unpublished observations). Similar studies have been carried out during the cell cycle in P.polycephalum. In this organism, the presence of different kinds of RNA during the growth cycle is shown by a distinct shift in the ratio of adenylic acid t o guanylic acid during the G, period (Mittermayer et al., 1964). Early in the cell cycle this ratio is higher than it is later in the cell cycle. Further, the nucleotide sequence of the newly synthesized RNA has shown that the rapidly labelled RNA during the mitotic cycle is transcribed from relatively more AT-rich regions of nuclear DNA whereas RNA transcribed later in the cycle tends t o be from regions richer in GC content (Cummins, 1969). Such variations in base composition are consistent with a model of ordered transcription during the cell cycle. However, in the absence of knowledge on possible variations in precursor pools
SYNTHESIS OF ENZYMES DURING THE CELL CYCLE
55
and rate of equilibration of these pools with exogenous RNA precursors, such conclusions are premature. In a few cases, attempts have been made to investigatethenature of the pulse-labelled RNA by DNA-RNA hybridization. Cutler and Evans (1966, 1967) were the first to attempt this approach. They isolated fragments of newly replicated DNA during the cell cycle of E . coli and hybridized each of these with RNA pulsed labelled during the cell cycle. The DNA-RNA hybridization pattern suggested a rythmic pattern of m-RNA transcription. From a detailed study of the ability of unlabelled RNA at one interval to compete with pulse-labelled RNA at another interval for sites on DNA, Hansen et al. (1970) came to a similar conclusion employing synchronous outgrowth of spores of Bacillus cereus T. On the other hand, Bello (1969), in comparing pulse labelled RNA by hybridization competition from two intervals of cultured KB cells, was unable to detect any differences. The hybridization procedure has a great potential for testing ordered transcription ; however the above experiments are limited by the fact that large segments of DNA, or the intact genome, were employed. Since these contain multiple cistrons, the resolving power of the test is low. Convincing evidence for ordered transcription has been obtained for bacteriophage development in E . coli (Bolle et al., 1968).In analogous experiments with bacteriophage h development, where it has been possible to employ fragments of the separated strands of h DNA containing one or a few cistrons for hybridization, an intricate pattern of control of transcription has emerged (for review see Szybalski et al., 1969).Although it has not yet been possible to detect changes in specific host m-RNA by hybridization during the cell cycle, in at least one case (outgrowth of bacterial spores) less direct methods measuring the capacity to synthesize enzyme after addition of actinomycin D has led to the conclusion that transcription is ordered (Steinberg and Halvorson, 1968a).
IV. Enzyme Synthesis During the Cell Cycle A. INTRODUCTION The above studies on RNA and protein synthesis during the cell cycle, although useful in outlining the gross nature of regulation in organisms, do not permit a precise understanding of the regulation of gene expression. In recent years, studies on specific enzyme formation during the cell cycle have proven more useful for investigating the regulatory mechanisms operating in dividing cells. The majority of this information has come from studies with bacteria and yeast. Recent advances in the technology of synchronization have permitted similar 3
56
H. 0. HALVORSON, B. L. A. UARTER AND P. TAURO
analyses in higher plant and animal tissue culture cclls. Provided that the limitations discussed below are considered, enzyme measurements during the cell cycle coupled with genetic analysis can provide new insights into the regulation of gene expression. One can recognize two levels of enzyme regulation: (a) regul at'ion imposed by the cxtcrnal environment ; and (b)regulation imposed by the internal environment. Prokaryotic and to a lesser cxtent unicellular eukaryotic cells have evolved to respond rapidly to changes in the environment. Since such changes can influence not only cnzyme patterns, but also gross changes in the cellular content of protein, RNA and DNA, it is essential when investigating controls imposed during the cell cycle that the environmental conditions remain constant. Because the effect of these environmental changes may be dclayed, the methods for establishing synchronous populations (as previously discussed) arc of prime importance. For example, when tempcraturr cycling or starvation procedures are employed to establish synchrony, the behaviour of early cell divisions may be markedly influenced by the previous history of the population and not be representative of later cell divisions. This problcni is of particular significance because in most systems studied only a few divisions are obtained. Some of the proccdures for establishing synchronous cultures t o minimizc these disturbances have h e n discussed earlier. Mcasurcments of specific enzyme activitics during the cell cyclc. LLI'CL valid only when basic criteria for enzymology arc? met. For cxa~nple, when enzyme measurements of intact cells are employed, it is essciitid to know whether conditions of substrate saturation are met. 111 c'its('S where the substrate is impermeable, it is common practice to deterrniiic* the specific activity of extracts of cells and, from the composition of t h e cells, calculate the enzyme level per cell. Variations of protein coritcnt during the cell cycle and differential leakage of proteins during disruption can lead to errors in estimating enzyme level per cell. Finally IniLtiy cellular metabolites are known to alter enzyme activities. Tf fluc.tnC
SYNTHESIS O F ENZYMES DURING THE CELL CYCLE
57
I n one, enzyme activity increasescontinuouslg* (linearly or exponentially) throughout the cell cycle and in the other, there is a limited period(s) (periodic) in which enzyme activity increases. If the enzyme is unstable, peak periods of cnzyme activity are observed during the cell cycle. Since prokaryotic organisms generally demonstrate continuous increases of enzyme activity while eukaryotic organisms generally demonstrate periodic enzyme increases, these will be discussed separately. Excellent reviews of enzyme synthesis during the cell cycle of prokaryotes (Donachie and Masters, 196!)) and eukaryotes (Mitchison, 1969b) have recently appeared and the reader is referred to these for further details.
B. SYNTHESIS OF ENZYMES IN PROCARYOTIC ORGANISMS GROWINGIN A CONSTANT ENVIRONMENT To follow regulation of gene expression in response to internal controls, we shall first examine the synthesis of enzymes of prokaryotic cells dividing synchronously in a constant environment. I n any given medium, some enzymes will be synthesized at basal or fully repressed levels whereas others may be fully induced in response to inducers in the medium. Under constant growth conditions the enzyme levels can change by a factor of more than 10’ (Pardeeand Beckwith, 1963).There is a third category of response in bactcria. When cells are grown in a medium lacking nutrients or other chemicals which either fully induce or repress enzymc synthesis, intermediate enzyme levels are observed. There is growing evidence that the levels of most (if not all) of the enzymes in bacteria are subject to regulation. Two classes are well documented. Biosynthetic enzymes are subject t o end-product repression and inhibition and catabolic enzymes are regulated by catabolite repression. These classes could generally be included under the term “autogenous” regulation (Donachie and Masters, 1969).Advances in our understanding of regulation of bacteria in recent years reveal that the term “autogenous” is too restrictive to include all endogenous regulatory systems. Documentation of this conclusion is beyond the scope of this review and, for a discussion of other regulatory mechanisms operative in bacteria, the reader is referred to several recent reviews in this area (e.g. Epstein
* Mitchison and Creanor (1969)have described a statistical method for determining whether a givcn set of data give a better fit t o a smooth curve or to a set of linear segments (one per cell cycle). Their method does not include analysis of multiple steps or linear segments per cell cycle which would give rise to intermediato kinctics. For those masons and tho absence of sufficient experimental points in most of the published experiments, we shall not attempt to characterize further the kinetics of crizymes which rise continuously throughout the cell cycle.
r, ?
F 4
TABLE 2. Pattern of Enzyme Synt,hesis during the Cell Cycle of Prokaryotic Organisms Condition Organism Bacillus cereusgerminating spores
Enzymea
of synthesis
Pattern
Alkaline phosphatase Manine dehydrogenase a-Glucosidase
Autoregulated Autoregulated Repressed Induced Induced
Periodic Periodic Periodic Periodic Periodic
Histidase Bacillus subtilis
Aspartate transcarbaxnoylase Autoregulated Periodic Alkaline phosphatase Dehydroquinase Histidase
Repressed Repressed Induced
Continuous Periodic Periodic
Ornithine transcarbamoylase Autoregulated Periodic Sucrase ( G )
Induced Repressed
Periodic Continuous
Reference Kobayashi et aE. (1965) Kobayashi et al. (1965) Kobayashi el al. (1965) Steinberg et al. (1965) Kobayashi et al. (1965) Steinberg et al. (1965) Masters and Pardee (1965);Donachie (1965); Masters et al. (1964). Donachie (1965) Masters and Pardee (1965) Masters and Pardee (1 965) ;Masters et al. (1964) Masters and Pardee (1965);Donachie (1965) Masters and Donachie (1966) Masters and Donachie (1966)
0
E "g m
r ?
c
z
B P
2
U
'd Y
P
c:
0 0
Escherichh coli
Repressed Autoregulated A4utoregulated Constitutive Induced
Continuous Periodic Periodic Continuous Continuous
Kuempel et al. (1965);Goodwin (1969b) Kuempel et al. (1965);Goodwin (1969b) Kuempel et al. (1965) Donachie and Masters (1969) Abbo and Pardee (1960);Cummings (1965)
Repressed Constitutive Induced Glycylglycinedipeptidase ( U ) Autoregulated
Continuous Periodic Periodic Periodic
Kiiempeletal. (1965) Goodwin (1969b) Goodwin (1969b)
Alkaline phosphatase Aspartate transcarbamoylase Dihydro-orotase /3-Galactosidase
Leucine aminopeptidase (U) Lactic dehydrogenasc Ornithine transcarbamoylase Protease (U)
Rhodopseudomom spheroides
8-Aminolaevunic acid dehydrase 8-Aminolaevunic acid synthetase (U) Alkaline phosphatase Ornithine transcarbamoylase Succinyl-CoA thiokinase
U refers to unstable enzymes.
Autoregulated Autoregulated Autoregulated Autoregulated Autoregulated
Periodic Periodic Periodic Periodic Periodic
Nishi and Hirose (1966) Kogoma and Nishi (1965) Good\%-in(1969b) Goodwin (1969b) Kogoma and Nishi ( 1965) Ferretti and Gray (1968)
Autoregulated Periodic
Ferretti and Gray (1968)
Periodic Repressed ? Autoregulated Periodic ? Autoregulated Periodic
Ferretti and Gray (1968) Ferretti and Gray (1968) Ferretti and Gray (1968)
%1: Y
z
M
E
rn 0
q
M
2 g
c3
60
11. 0. IIALVORSON,
B. L . A . CARTER A N D P. TAURO
and Beokwith, 1968 ; Martin, 1969). Until a specific cellular mechanism is shown to regulate the rate of formation of any specific enzyme, it is desirablc to consider a more general term for regulation which is self imposed by the cell. For this reason we shall call this “autoregulation”. The search for markers in the cell cycle (see Mitchison, 1969a) has revealed that the levels of various cell components (which may include enzyme-regulating agents) may fluctuate during the cell cycle and thorefore it is of interest to see whether there is a constant or discontinuous rate of synthesis of specific enzymes during the cell cycle. Table 2 summarizes
0.2
L
0 1 0 0 1
2
3
4
5-7
’12
Number of enzyme molecules
FIQ.1. Distribution of ,!I-n-galactosidasoamong glucose-growncells of Escherichia coli. Data from Rotman (1970).
the pattern of enzyme synthesis under these different conditions in bacteria. I n the fully repressed ccll, t8heenzyme level may be very low ; the best case exaniined is that of 8-galactosidase in E . coli, where this level approaches a few molecules per cell (Hogness, 1959). Rotman and his colleagues (Rotman, 1961, 1970; Ganesman arid Rotman, 1964) have investigated this situation employing a sensitive spectrofluorometric assay which enables the monitoring of enzyme distribution among individuals in the population. He found that i n repressed cultures about 50% of the individuals lacked /?-galactosidase molecules, and the rest contained various amounts ranging from one t o twenty molecules of
SYNTHESIS O F ENZYMES DURING THE CELL CYCLE
01
/I-galactosidase per cell (Fig. 1 ). Rotman suggests that periodically there is a temporary derepression of a cell caused by dissociation of the repression-operation complex for a length of time sufficient only for one m-RNA molecule t o be produced*. The enzyme produced by this temporary derepression is then diluted among the subsequent offspring and accounts for the observed distribution. As shown in Table 2, basal p-galactosidase synthesis of a population of synchronously dividing cells is continuous through the (,ell cycle. One must therefore conclude that throughout the cell cycle there is an equal (or similar) probability for any given cell to synthesize enzyme. This further leads t o the interesting conclusion that enzyme synthesis is not linked to the replication of any gene which would occur a t a fixed period in the cell cycle. Evidence to support this conclusion was reported by Overath and Stange (1966) and Fangman el al. (1967) who found that repressed /3-galactosidase synthesis in E . coli continued after the cessation of DNA synthesis. The possibility that the enzyme synthesized under these conditions is a product of the small fraction of cells which were making enzyme when DNA synthesis stopped was eliminated by the observation that the enzyme level rises uniformly in all cells (Fangman el al., 1967). At the present time, sensitive techniques are unavailable to determine the distribution of other repressed enzymes in bacterial colls. Rotman (1970) has shown t h a t , in a constitutive mutant of alkaline phosphatase in E . coli, all of the cells in the population contained high levels of enzyme. The distribution of repressed enzyme has not as yet been determined. In I’l-iosphate-repressed alkaline phosphatase synthesis in E . coli (Kuenipel et al., 1965) and B. subtilis (Donachie, 1965) and glucoserepressed sucrase synthesis in B. subtilis (Masters and Donachie, 1966), continuous increases in basal enzyme activity were observed during the cell cycle suggesting that the type of basal control observed for /3-galactosidase synthesis may be general for other basal enzymes in bacteria. Another possible cxample is the arginine-repressed synthesis of orni thine transcarbamoylase in Rhodopseudomonas spheroides (Ferretti and Gray, 1968). Donachie and Masters (1969) have interpreted these results as supporting a basal continuous synthesis throughout the cell cycle. However, closer inspection of the original data reveals that this conclusion cannot be made for two reasons. First, in the control experiment which measured the kinetics of derepressed synthesis of enzyme, conditions of balanced growth were not met. Second, from the data
* Genetic controls of basal levels of alkaline phosphatase (Jones, 1969) and /3-galactosidase (Overath, 1968) have been reported in E. coli. The results are best interpreted in ass~niiirgthat repressor activity regulates basal activity ; however, it has not been determined whether this reflects changes In amount of repressor or in the afinity of repressor for the operator.
62
H. 0. IIALVORSON, R . L. A . CARTER AND P. TAURO
presented for repressed enzyme synthesis, one cannot distinguish between continuous and stepwise increase in enzyme. Maximal (fully dcrepressed) enzyme synthesis occurs either during growth in excess concentrations of a n inducer or in constitutive mutants. The only case in which both of these have been studied in detail in the cell cycle is for ,!I-galactosidase synthesis in E . coli. I n each case, enzyme level increases continuously throughout the cell cycle (Abbo and Pardee, 1960; Cummins, 1965; Donachie and Masters, 1969) in a manner analogous to that of basal enzyme synthesis. It would be useful if additional enzymes were cxaminod under similar conditions t o establish the generality of regulation of fully derepressed synthesis during the cell cycle. Most enzymes studied during the eel1 cycle in bacteria fall into a third category. We have assumed that all enzymes which are neither fully induced or repressed are autoregulated. Table 2 summarizes the kinetics of autoregulated enzymes during the cell cycle of synchronous populations growing in a constant environment. I n most examples that have been studied, enzyme activity does not increase continuously throughout the cell cycle but periods of synthesis are followed by periods in which enzyme activity per unit volume is constant, resulting in a step-like (periodic) pattern of synthesis. However, Nishi and his colleagues (1966, 1966) showed that levels of protease a d peptidases increase and then decrease during the cell cyclc of E . coli, resulting in pcaks and troughs of activity. Decreases in enzyme activity may be caused by enzyme degradation, enzyme instability or conversion to an inactive form. The kinetics of rise of enzyme activity throughout the cell cycle for autoregulated enzyme would have one of the three general forms shown in Fig. 2 depending on the stability of the enzyme. These are highly idealized and in practice one cannot distinguish between (b) and (c). Sucrase, aspartic transcarbamoylase, glycylglycine dipeptidase, protease, leucine aminopeptide and 6-aminolaevulinic acid synthetase have all been reported as unstable during the cell cycle (Table 2). However, in no one case do the kinetics permit a decision as to whether the instability persists throughout the cell cyclc. For all the “step” and “peak” enzymes invcetigated there is a characteristic stage in the cycle for thc synthesis of those enzymes. Synthesis of different enzymes occurs a t different stages in the cell cycle but, for any one enzyme, this synthesis of t h e enzyme in a constant environment usually occurs at the same stage each cell cycle. The timing of autoregulatory steps may not be uniquely fixed in the cell cycle. Masters and 1)onachie ( 1 966) have observed that periodic synthesis of aspartate transcarbamoylase occurs at a particular stage in t h e cell cycle. This periodicity is altered in the cell cycle of B. subtilis after temporary addition of uracil. We shall return t o this matter later (Section
E. 1(b)).
SYNTHESIS OF ENZYMES DURING THE CELL CYCLE A.
I
63
I
Frociion of o generaiion
Fraction of a generation
C.
0
0.5
II
Fraction of a generaiion
FIG.2. Periodic enzyinr syntlicsis (luring the cell cycle. Periodic synthesis of (A) a stable cwzyinc; (13) an enzyme unstable for all of cell cycle; and (C) unstable for part of cell cycle.
C. SYNTHESIS OF ENZYMES i~ EUKARYOTIC ORGANISMS GROWINGIN A CONSTANTENVIRONMENT The largest body of information on enzyme synthesis during the cell cycle in eukaryotic organisms has been obtained with yeast. Since this organism is one of the simplest eukaryotes (Matile et al., 1969), it is of interest t o compare thcse dat)awith those from more complex eukaryotic organisms. l n doing so we recognize that in singlo cell cultures derived from organized tissues the prominent regulatory systems may differ from those in unicellular organisms.
64
H. 0. HALVORSON, B. L. A. CARTER AND P. TAURO
For eukaryotic cells the difference in enzyme level between repressed and fully induced cells is considerably less than that found in bacteria (Halvorson, 1964). In yeasts, growth on high concentrations of glucose reduces, by catabolic repression, the level of a number of carbohydrases to minimal (basal)levels. Growth of Sacch. cerevisiae on maltose induces a 25-fold increase of a-glucosidase above basal levels (Rudert and Halvorson, 1963). Methyl-/3-D-glucosideor low concentrations of glucose (lo-’ M ) induce 25-100-fold increases in basal j3-glucosidase in Sacch. lactis and in a Sacch. fragilis x Sacch. dobzhanskii hybrid (Herman and Halvorson, 1963 ; MacQuillan and Halvorson, 1962). In Schizosucch. pornbe, acid maltase is induced by maltose (Bostock et al., 1966) and it has been observed that lowering the glucose concentration to 0.05y0 or the phosphate concentration to 10 mg. per litre leads t o a 4.5-fold increase in sucrase (invertase) activity and an %fold increase in acid phosphatase activity (Mitchison and Creanor, 1969). Holzer et al. (1965) demonstrated that in Sacch. cerevisiae grown in ammonia the levels of NAD-glutamate dehydrogenase and threonine deaminase were reduced by 95 and 70% respectively from cultures grown in media containing amino acids as a source of nitrogen. Knutsen (1968) has shown that phosphate, in Chlorella pyrenoidosa, represses the synthesis of phosphatases by 80%. Synchronous cultures of glucose-grown yeast and phosphate-grown Chlorella thus permit an analysis of the timing of basal formation in eukaryotic organisms during the cell cycle. As summarized in Table 3, the synthesis of repressed a-glucosidase and NAD-glutamate dehydrogenase in Sacch. cerevisiae, 8-glucosidase in Sacch. lactis, and acid and alkaline phosphatases in Chlorella are all periodic during the cell cycle. One step of enzyme synthesis occurs during the cell cycle and is reproduced in successive cell cycles. Since, under the conditions of synchrony, there is no evidence for instability of these enzymes, one can conclude that there is a specific unique period of the cell cycle for repressed enzyme synthesis. These findings are in marked contrast to the pattern of fully repressed enzyme synthesis in bacteria discussed earlier. On the other hand, Bostock et al. (1966) observed continuous synthesis of maltase and sucrase throughout the cell cycle of Schizosacch. pornbe growing in glucose. These results will be discussed later. Perodic increases of repressed enzyme activities could be due either to a burst of enzyme synthesis in only a few cells of the population or to a slower rate of enzyme synthesis in all the cells of the population. To distinguish between these possibilities it is necessary to have information on the distribution of enzyme molecules in individual cells. Experiments on single eukaryotic cells similar t o those which Rotman and his colleagues developed for bacteria could provide this information. The
TABLE3. Pattern of Enzyme Synthesis during the Cell Cycle in Eukaryotic Organisms Organism
Enzyme
L-Arginase L-Ornithinecarbamoyltransferase Chlamydomones Alanine dehydrogenase reinhardtii Aspartate carbamoyltransferase Glutamate dehydrogenase Ornithine carbamoyltransferase Phosphoenolpyruvate carboxylase Chlorellapyremidosa Acid phosphatase BlastoClUdkUa emersonii
Alkalinephosphatase Aspartate carbamoyl transferase Carboxydismutaae Deoxycytidinemonophosphate deaminase Deoxythymidine monophosphate kinase (U) Dihydro-orotase Glycinamide ribotide kinosynthetaae
Condition of synthesis
Pattern
Reference
Autoregulated Autoregulated
Periodic Periodic
Domnas and Cantoni (1965) Domnas and Cantoni (1965)
Autoregulated Autoregulated
Periodic Periodic
Kates and Jones (1967) Kates and Jones ( 1967)
Autoregulated Autoregulated
Periodic Periodic
Ketes and Jones (1967) Kates and Jones (1967)
Autoregulated
Periodic
Kates and Jones (1967)
Repressed Derepreased Derepressed Autoregulated
Periodic Periodic Periodic Continuous
Knutsen (1968) Knutsen (1968) Knutsen (1968) Cole and Schmidt 1964)
Autoregulated Autoregulated
Periodic Periodic
Schmidt (1969) Shen and Schmid (1966)
Autoregulated
Periodic
Johnson and Schmidt (1966)
Autogenous Autoregulated
Continuous? Continuous?
Schmidt ( 1969) Schmidt (1969)
In
;c
TAELE 3-mntiiiued Organism
Enzyme
Chinese hamster
Glucose 6-phosphate dehydrogenase Lactic dehydrogenase Thymidine kinase Thymidine kinase Alkaline phosphatase DNA polymerase Thymidine kinase
Don C cells Lily microspores Human HeLa cells
Human Henle cells
KB cells Mouse L cells
Physarum polycephalum R a t hepatoma cells
Thymidylate kinase Deoxycytidine monophosphate deaminase Alkaline phosphatase Fumarase Lactic dehydrogenase DNA polymerase (U) Deoxycytidine monophosphate deaminase (U) Ribonucleotide reductase (U) Thymidine b a s e Glucose 6-phosphate dehydrogenase Thymidine kinase Tyrosine aminotransferase Glucose 6-phosphate dehydrogenase Alcohol dehydrogenase Lactic dehydrogenase
03
m
Condition of synthesis
Pattern
Reference
Autoregulated
Periodic
Klevecz and Ruddle (1968)
Autoregulated Autoregulated Autoregulated RepreSSed Autoregulated Sutoregulated
Periodic Periodic Periodic (U) Periodic Periodic Periodic
autoregulated Autoregulated
Periodic Periodic
Klevecz and Ruddle (1968) Stubblefield and Murphree (1967) Hotta and Stern (1965) Melnykovych et al. (1967) Friedman and Mueller (1968) Brent et al. (1965); Stubblefield and Mueller (1965) Brent et al. (1965) Gelbard et al. (1969)
Repressed? Autoregulated Autoregulated Autoregulated Autoregulated
Periodic Continuous Continuous Periodic Periodic
Melnykovych et al. (1967) Bello (1869) Bello (1969) Turner et al. (1968) Mittermayer et al. (1964)
Autoregulated Autoregulated Autoregulated
Periodic Periodic Continuous
Turneretal. (1968) Littlefield (1966) Sachsenmaier and Ives (1965)
Autoregulated Autoregulated Autoregulated
Periodic Peroidic Continuous
Sachsenmaier and Ives (1965) Martinetal. (196913) Martin e t a l . (1969b)
Autoregulated Autoregulated
Continuous Continuous
Martinetal. (1969b) Martinet al. (1969b)
Smharomyeea cerevisiue
Alcohol dehydrogenase Alkaline phosphatase
Autoregulated Repressed
Periodic Periodic
a-Ammoadipic acid reductase ,4rgininosuccinase Aspartokinase Cathepsidase (U) Cytochrome oxidase DNA polymerase (U) Galactokmase a-Glucosidase
Autoregulated Autoregulated Autoregulated Autoregulated Autoregulated Autoregulated Induced Induced Autoregulated Sutorcgulated Autoregulated Autoregulated
Periodic Periodic Periodic Periodic Periodic Periodic Periodic Periodic Periodic Periodic Periodic Periodic
Eckstemetal. (1967) Tauro and Halvorson (1966); Cottrell and Avers (1970) Cox and Gilbert (1970) Tauro et al. (1968) Tauro et al. (1968) Sylv6n et al. (1959) Cottrell and Avers (1970) Eckstein et al. (1967) Cox and Gilbert (1970) Tauro and Halvorson (1966) Tauro et al. (1968) Halvorson et 01. (1966) Ton Meyenburg (1565) Von Meyenburg (1969)
Autoregulated
Periodic
Eckstein et al. (1967)
Autoregulated Autoregulated Autoregulated Autoregulated
Periodic Periodic Periodic Periodic
Von Meyenburg (1969) Tauro et al. (1968)
Autoregulated Autoregulated
Periodic Periodic
Gorman et al. (1964) Von Meyenburg (1969); Cottrell and Avers (1970) Von Meyenburg (1969) Von Meyenburg (1969)
Autoregulated
Periodic
Tairo el al. (1968)
Autoregulated Autoregulated
Periodic Periodic
Tauro el al. (1968)
Autoregulated
Periodic
Von Meyenburg (1969)
Glucose 6-phosphate dehydrogenase Glyceraldehyde 3-phosphate dehydrogenase Hexokmase Histidinol dehydrogenase Invertase Malate dehydrogenase NAD-glutamate dehydrogenase KADP-glutamate dehydrogenase Orotidine 5’-phosphate decarboxylase Peptidase (U) Phosphoribosyl-ATP pyrophosphorylase Pyruvate decarboxylase
Sylvbn et al. (1959)
2
3 1
5 rn 0
r
9 z 2! U C
ti
0 c3
IZ
d
h
:: d
2I? E
Organism Succharomycea a r e v i a h e (cont.)
Succharomycea dobzhanakii saccharonaycea dobzhanakii x frllgilis lactia Saccharomyea
s.
Schizosuccharwmyces pombe
Condition of synthesis
Pattern
Autoregulated Autoregulated Autoregulated
Periodic Periodic Periodic
Tauro et al. (1968) Tauro et al. (1968) Von Meyenburg (1969)
p-Glucosidase
Autoregulated Autoregulated Constitutive Induced Autoregulated
Periodic Periodic Periodic Periodic Periodic
Tauro et al. (1968) Kuenzi and Fiechter (1969) B. L. A. Carter (unpublished B. L. A. Carter observations) Goman et al. ( 1964)
Alkaline phosphatase a-Glucosidase p-Glucosidase p-Galactosidase p-Glucosidase p-Glucosidase Acid phosphatase Alcohol dehydrogenase Alkaline phosphatase Aspartate transcarbamoylase Homoserine dehydrogenase Maltase (acid) Ornithine transcarbamoylase Sucrase (invertase)
Autoregulated Autoregulated Autoregulated Autoregulated Autoregulated Repressed Repressed Autoregulated ? Autoregulated Autoregulated Autoregulated? Repressed? Autoregulated Repressed?
Periodic Periodic Periodic Periodic Periodic Periodic Continuous Periodic Continuous Periodic Periodic Continuous? Periodic Continuous
German et al. (1964) Gorman et al. (1964) Gorman et al. (1964) Hdvorson et al. (1964) Halvorson et al. (1964)
Tryytophan spthetase
Autoregulated?
Periodic
Enzyme
Saccharopine dehydrogenase Saccharopine reductase Succinate-cytochrome c dehydrogenase Threonine deaminase Trehalase UDP galactose 4 epimerase
Reference
1
Tingle (1967) Mitchison and Creanor (1969) Mitchison (1969b) Mitchison and Creanor (1969) Bostock et al. (1967) Mitchison (1969b) Bostock et al. (1966) Bostock et al. (1966) Bostock et al. (1966);Mitchison and Creanor (1969) Mitchison (1969b)
m 0
? 20
: 1 W F
? d c
z
3 ! i U cd
2
$0
SYNTHESIS O F ENZYMES DURING THE CELL CYCLE
69
reader is cautioned that the comparison of the kinetics of repressed enzyme synthesis in the cell cycle of prokaryotes and eukaryotes is based on relatively few examples. Further cases will have to be studied to determine whether this difference is general. There is only one example, a-glucosidase synthesis in Sacch. cerevisiae, for which the pattern of enzyme synthesis has been measured during synchronous growth under continuous repression and continuous full induction. When Sacch. cerevisiae is grown synchronously in medium containing maltose, an inducer of a-glucosidase synthesis, enzyme synthesis is periodic during the cell cycle (Tauro and Halvorson, 1966). The synthesis of induced a-glucosidases occurs at the same point in the cell cyde as that for repressed enzyme synthesis (Halvorson et al., 1966). Thus induction increases the level of enzyme synthesis but does not alter the period in which enzyme is produced. Table 3 also summarizes those cases in which maximal enzyme synthesis (induced, fully derepressed) has been measured in cells growing synchronously in a constant environment. With one exception, tyrosine aniinotransferase in rat hepatoma cclls (Martin et al., 1969b), the examples available are drawn from experiments with synchronously dividing yeasts. Galactose co-ordinately induces the synthesis of the galactose enzymes, galactokiiiase, trailsferase and epimerase in Sacch. cerevisiae (Douglas, 1961). Two of these, galactokinase and UDP galactose-4 epimerase, were observed to increase periodically in synchronous cultures growing in galactose (Cox and Gilbert, 1970; B. L. A. Carter, unpublished observations). I n an operator constitutive mutant, synthesis of epimerase also occurs periodically during the cell cycle (B. L. A. Carter, unpublished observations). The synthesis of many enzymes has been examined during the cell cycle of a variety of eukaryotes under conditions where the enzymes are neither fully induced or repressed but are autoregulated (see Table 3). I n relatively few of these cases have the mechanisms of regulation been documented. For example, aspartokinase, which is produced periodically in Sacch. cerevisiae growing synchronously in glucose (Tauro et al., 1968), is known to be repressed in this organism by threonine, homoserine (an intermediate in the threonine pathway) and by lysine (de RobichonSzulmajster and Corrivaux, 1963). Similarly, in Sacch. cerevisiae, homoserine dehydrogenase is repressed by mcthionine (Kassarevitch and de Robichon-Szulmajster, 1963) and ornithine transcarbamoylase is repressed by arginine (Bechet et al., 1962). I n glucose-grown cultures of Schizosacch. pombe both homoserine dehydrogenase and ornithine transcarbamoylase are produced periodically during the cell cycle (Mitchison, 1969b; Bostock et al., 1966). The first enzyme of the histidine pathway, phosphoribosyl-ATP pyrophosphorylase, is under end-product repression by histidine in Sacch. cerevisiae (Fink, 1964)and is produced period-
70
H. 0. HALVORSON, B. L. A. CARTER AND P. TAURO
ically in the cell cycle by cells grown in the absence of exogenous amino acids. However, periodicity during the cell cycle and end-product repression are not necessarily correlated. This is evident because two enzymes in the lysine pathway of yeast, saccharopine reductase and saccharopine dehydrogenase, are not subject to feedback repression by lysine in Sacch. cerevisiae (Jones and Broquist, 1965) but are produced periodically during the cell cycle (Tauro et al., 1968). Another interesting example is the regulation of carbohydrnsc syntheses in yeast which are subject to both substrate induction and glucose repression. Certain yeast strains, Sacch. lactis, Bncch. dobzhonskii and Sacch. fragilis, are capable of growth in succinute synthetic medium. Under these conditions intermediate levels of a-glucosidase, /3-glucosidase and p-galactosidase are produced during exponential growth (MacQuillan and Halvorson, 1962; Herman, 1963) and step-wisc synthesis is observed during the cell cycle (Gorman et al., 1964 ; Halvorsoii et al., 1964). Basic information on the mechanism of regulation of enzyme synthesis in more complex cells is meagre. Thyrnidine kinase, one of the better studied systems, has been reported t o be periodically produced during the cell cycle of human HeLa cells (Brent et al., 1965; Stubblefield and Mueller, 1965), Physarum polycephalum (Sachsenmaier and Ives, 1965), mouse L cells (Littlefield, 1966), Chinese hamster Don C cells (Stubblefield and Murphree, 1967) and Lilium microspores (Hotta nnd Stern, 1965). I n mouse fibroblasts, HeLa cells and Lilium microspores thc induction of this enzyme with thymidine has been demonstrated. However, Hotta and Stern (1965) found that the periodic increases of thymidine kinase were not accompanied by changes in the intracellular concentration of thymidine in Lilium microspores. Klevecz and Ruddle (1968) have proposed a more general regulatory mechanism to account for the periodic synthesis of glucose 6-phosphate dehydrogenase arid lactate dehydrogenase during the cell cycle of Chinese hamster cells based on parallel changes in cell volume and total protein. These authors suggested that the periodic synthesis of these enzymes is a reflection of a more general control mechanism affecting whole populations of proteins and may be the expression of an endogenous cellular rhythm. However, inspection of the data indicates that i t is not possible t o correlate chnnges in total protein with changes in the synthesis of the two enzymes with any confidence. An interesting case is that of autoregulated aspartate transcarbamoylase synthesis in Chlorella pyrenoidosn. Total enzyme activity rises continuously throughout the cell cycle (Cole and Schmidt, 1964) ; however, when the extracts are aged, a variable fraction of activity is lost (Schmidt, 1969). The unstable activity rises discontinuously through the cell cycle. R. R. Schmidt (unpublished observations) has
SYNTHESIS OF ENZYMES DURING THE CELL CYCLE
71
recently found that this activity is stabilized by UMP or a substrate of the enzyme. Thus, the variations in labile enzyme may well reflect fluctuations of ligand concentration in the extracts (and presumably concentration per cell), with the amount of ligand present determining the extent of inactivation of enzyme. The majority of studies on the synthesis of autoregulated enzymes during the cell cycle have been made on enzymes in which the mechanisms of regulation are not documented. I n some cases, such as aspartate transcarbamoylase, the regulatory mechanisms are well known in bacteria. However, it may be incorrect to assume that the same mechanisms control the synthesis of this enzyme in, for example, Chlamydomonas reinhardtii. It is clear from Table 3 that the synthesis of autoregulated enzymes with as yet undiscovered regulatory mechanisms includes examples of both periodic (most common) and continuous synthesis during the cell cycle.
CELLCYCLE Additioiial information on the synthesis of enzymes during the cell cycle has been obtained by studies of the capacity t o synthesize enzyme (induction or derepression) a t different times in the cycle. This is accomplished by removing samples at intervals during the cell cycle from a synchronous culture and incubating the cells in a medium containing inducer (or minus repressor). The rate a t which enzyme is then synthesized is a measure of the induction capacity of the culture during different intervals of cell division. The induction capacity of various enzymes from prokaryotes arid cukaryotes is shown in Table 4. Two classes of response have been observed (Fig. 3). I n the first, the ability t o synthesize enzyme at elevated rates in the presence of the inducer is observed throughout the cell cycle. This we have termed an “unrestricted” synthesis of enzyme. I n general this capacity sharply doubles during the cell cycle, presumably due t o replication of the particular gene involved. I n the second class, there are only defined periods of the cell cycle when enzyme is synthesized in response t o the addition of inducer. We have termed this class “restricted”. With the exception of germinating bacterial spores, all of the induction capacities reported in synchronous cultures of bacteria are unrestricted (Table 4). Let us consider the example of P-galactosidase synthesis in E . coli. Basal, induced and conHtitutive enzyme synthesis is continuous throughout the cell cycle (Tablc 2, p. 58) and throughout this period the induction capacity is unrestrict<ed.Using an indirect assay (capacity to support viral multiplication), Benzer (1953) observed that, in response to inducer, all the cells in an exponential culture produced P-galactosidase at the same time. A. Goldstein and M. B. Rotmaii (unpublished)
D. INDUCTION CAPACITY IN
THE
TABLE4. Induction Capacity During the Cell Cycle 4
ta
Organism A. Prokaryotes Bacillua cerew (germinating spores) Bacillus subtilia Escherichia wli
B. Eukaryotes Chlorellu pyrenoidosa
Enzyme=
a-Glucosidase Histidase Sucrase(U) Alkaline phosphatase Aspartate transcarbamoylase
Restricted Restricted Unrestricted Unrestricted Unrestricted
Dihydro-orotase p-Galactosidase
Unrestricted Unrestricted
Orotidine monophosphate pyrophosphorylase D-Serine deaminase D-Serine dehydratase Tryptophanase
Unrestricted
Acid phosphatase Alkaline phosphatase Isocitrate lyase Glutamate dehydrogenase(NADP) Nitrate reductase Lily microspores Thymidine kinase Rat hepatoma cells Tyrosine aminotransferasse Saccharom yces &is 8-Galactosidase Schizosaccharomyces pombe Sucrase
U = unstable.
Pattern
Unrestricted Unrestricted Unrestricted
Restricted Restricted Unrestricted Unrestricted Restricted? Restricted Restricted Restricted Unrestricted
References
Steinberg and Halvorson (1968a) Steinberg and Halvorson (196%) Masters and Pardee (1965) Kuempel et al. (1965) Kuempel el al. ( 1965) ; Donachie and Masters ( 1969) Donachie and Masters (1969) Donachie and Masters (1969); Donachie and Masters (1969); Nishi and Horiuchi (1966) Donachie and Masters (1969); Pato and Glaser (1968) Donachie and Masters (1966, 1969) Nishi and Horiuchi (1966) Kuempel et al. (1965); Donachie and Masters (1966, 1969) Knutsen (1968) Knutsen (1968) R. R. Schmidt (unpublished R. R. Schmidt observations) Knutsen (1968) Hotta and Stern (1965) Martin et al. (19694 J. Gorman (unpublished results) Mitchison and Creanor (1969)
I
x ?
E4 "2 m ?
?
Eel
E 5b 'd
2C
td 0
SYNTHESIS OB ENZYMES DURING THE CELL CYCLE
73
have recently confirmed this conclusion by employing more sensitive techniques which directly assay ,3-galactosidase distribution in single cells. They observed that, within several minutes after addition of inducer, p-galactosidase was detected in all of the cells in the population. Therefore, the induction capacity is unrestricted and is a measure of the maximal potential rate of enzyme synthesis in a synchronous population. One exception t o the stepwise rise in induction capacity during the cell cycle in bacteria, was observed by Nishi and Horiuchi (1966). These workers reported that, whereas in E . coli K12 Hfr H 8-galactosidase and D-serine dehydratase induction capacity rose stepwise in the cell cycle, in an F- strain of E. coli K l 2 these two enzymes rose continuously. I n an A.
B
FIG. 3. Induction capacity during the cell cycle. A. Unrestricted capacity. B. Restricted capacity.
E. coli K12 strain in which the lac gene was now carried only on an episome, /3-galactosidase capacity was stepwise whereas D-serine dehydrase capacity was still continuous. Donachie and Masters ( 1969) explained these results by suggesting that in these cultures chromosomal DNA replication was asynchronous with respect t o the cell cycle whereas replication of the episome was synchronized to the cell cycle. We agree with the remarks by Mitchison (1969b) that this explanation is a little forced and this interesting situation needs further exploration, particularly since it has been shown in Proteus mirabilis that F- factor replication occurs randomly over the bacterial cell cycle (Rownd, 1969). The unrestricted response is not characteristic of all bacterial systems. Steinberg et al. (1965) and Steinberg and Halvorson (1968a) observed that during the outgrowth of spores of B. cereus T and during the first few synchronous cell divisions there was an ordered and periodic increase
74
H. 0. IIALVORSON, B . L. A . CARTER AND P.TAURO
in a-glucosidase, L-alanine dehydrogenase, alkaline phosphatase and histidase. During outgrowth, ordered enzyme synthesis was not dependent upon DNA replication (Steinberg and Halvorson, 1968b). Of particular interest here was the observation that the induction capacity during outgrowth for a-glucosidase and for histidase was “restricted” and limited to the same intervals of time as seen in either basal or fully induced synthesis. For control of autogenous enzyme synthesis, the nietabolisni of endogenous reserves, possibly unique to the dorniarit state, could provide periodic synthesis by known mechanisms. Such explanations are, however, inadequate t o explain the periodicity in induction capacity observed for histidase and a-glucosidase. * These and other observations (Hoyem et al., 1968; Rodenberg et al., 1968; Harisen et al., 1970; Torriani and Levinthal, 1967) have strongly pointed t o outgrowth as a transition in which transcription is an ordered process (restricted). In eukaryotic cells, the induction capacity has been measured for eight different cnzymes (Table 4). Hotta and Stern (1965) first reported that thymidine kinase in the lily could be induccd by thymine only during 10% of the C1 period and close to the interval in the cell cycle whcn thc cnzynic normally appears. Several inducible enzymes have been reported in Chlorella pywnoidosa, for example, the induction of nitrite reductase by nitrite (Knutscn, 1965). Following the initiation of synchrony by light exposure, the induction capacity does not rise for several hours until DNA synthesis begins. This increase is sensitive to both chloraniphenicol and actinomycin D suggesting that transcription is initially restricted in this system. The induction capacity per cell decreases after 10 hr., just before DNA synthesis stops. Following this, multiple rapid divisions occur with the number of divisions dependcrit upon the intensity of the prior illumination (R. R. Schmidt, personal communication). In the experiments of Knutseri ( I965), 12 spores per cell were liberated after 15 hr. of illumination. Thus, in the absence of cell counts/tnl., it is difficult to evaluate the subsequent decrease in induction capacity pcr cell from his experiments. Restricted capacity to derepress acid and alkaline phosphatase activity per ml during the cell cycle in Chlorella was reported later by Knutscn (1968). R. R. Schmidt (unpublished results) has recently investigated two other inducible systems in this organism. Isocitrate lyase induction capacity per lnl. of culture is present throughout the cell cycle. The capacity doubles after each round of nuclear division. Also, NADP-dependent glutainate dehydrogenase could be induced throughout the cell cycle by NH,.
* Possible alternative mechanisms for regulating gone expression during outgrowth havo boon discussed olsowliore (Stoinborg et al., 1909).
SYNTHESIS O F ENZYMES DURINQ THE CELL CYCLE
75
Mart,in et al. (1969b)have observed that, in synchronous cultures of rat hepatoma cells, the adrenal steroid dexamethasone phosphate, an inducer of tyrosine aminotransferase, is able to induce this enzyme only during part of the G1 and throughout S. The superinduction of enzyme in the presence of actinomycin D (Thompson et al., 1966), as well as studies on the relationship between RNA and enzyme synthesis, led Martin et al. (1969a)t o concludo that the specific m-RNA was synthesized continuously but that transhtion was restricted. In Sacch. lactis, /?galactosidase can be induced either by lactose (Herman and Halvorson, 1963) or under conditions of gratuity with thiomethyl /?-D-galactoside. In synchronously dividing cells inducibility is restricted (J. Gorman, unpublished data) arialogous to that of basal or fully induced enzyme synthesis discussed earlier. An exception to the general phenomenon of restricted induction capacity in eukaryotic cells is the observation by Mitchison and Creanor (1969) that sucrase inducibility was evident throughout the cell cycle. At a given point in the cell cycle this capacity doubled. This phenomenon is not identical to the related cases in bacteria since the point in which the capacity doubled occurred one third of a generatlionafter the completion of DNA replication (G2). Thus, the gene made during the S period is not immediately available for expression.
E. SPECULATIONS ON THE MOLECULARBASISOF REGULATION IhJRING THE CELL CYCLE Since the observation by Wortman (1882) that amylase is an inducible enzyme in certain bacterial species, our understanding of the regulation of gene function in bacteria and viruses has increased dramatically. The parallel success in comparative biochemistry has during the past few years, led increasingly to the assumption that the same mechanisms govern regulation in both eukaryotic and prokaryotic cells. Elucidation of the mechanism of protein synthesis and the demonstration of m-RNA in a wide variety of organisms has strengthened this view and led to models of differentiation based on mechanisms of bacterial regulation (Jacob and Monod, 1961). Detailed examination of the field in the past two decades, based on the methods of Anderson et al. (1955), reveal an increasing susceptibility to alternate possibilities (Fig. 4). 1. Regulation by Feedback Control in Bacteria One of the most dramatic features of regulation in bacteria is the ability of a population t o respond, within a few minutes, t o changes in the environment. From detailed studies of one system a t the molecular level the following generalized picture has emerged. /?-Galactosidase synthesis in E . coli is regulated by the presence of a repressor molecule
76
€1. 0. IIALVORSON, B . L. A. CARTER AND
P. TAURO
(product of the i gene) which binds to the lac operon and prevents its transcription. When an inducer (generally a galactoside) is added, it is accumulated within the cell where i t binds t o the repressor and displaces it from DNA. The lac operon is then accessible for transcription of the /I-galactosidase m-ItNA and its subsequent translation into active enzyme. Iricrcases in /I-gslactosidase m-RNA and de now0 protein (/I-galactosidase)synthesis have both been demonstrated in this system within a few minutes after addition of the inducer. I n other systems, especially the pathways for biosyntlietic enzymes, end products of
I
I
-I 0
-0 5 Fraction of a life cycle
I
0
FIG.4. Susceptibility variation during the life cycle. Relative susceptibility ( c ) (see 4E) is defined as Pj/2 El where Pi and Ei are the individual polaritios and intensities of the population as measured by tho method of Anderson et al. (1955). c is plotted against the average functional life cycle of this population. F-lis -1.02 11. niin. For further details on the deviation a t -0.0 and -0.4, see Gale and Davies (1953)and Uinbarger (l9G1).
2
metabolism participate in the repression of the initial enzymes in a given pathway (feedback repression) and may inhibit the activities of these enzymes (feedback inhibition). For example, when histidine is available in the medium, the histidine pathway is rapidly inhibited and the relevant enzymes more slowly lost by repression and dilution by growth. Upon exhaustion of histidine below a critical threshold, derepression and enzyme synthesis occur within minutes. Feedback repression has been reported for almost all of the biosynthetic pathways (purines, pyrimidines, amino acids, vitamins) studied in bacteria and thereforc must be considered as a common b u t not universal mechanism of control (Umbarger, 1969; Martin, 1969). When a bacterial culture is growing in a glucose salts mixture, macro-
SYNTHESIS OF ENZYMES DURING THE CELL CYCLE
77
molecule synthesis will be autoregulated. A prominent, but probably not exclusive, autoregulatory control is feedback repression (autogenous control). Without prejudging the molecular basis of regulation, the model of the feedback repression has the advantage of predicting the properties of enzyme synthesis during the cell cycle. These predictions will be discussed below. (a) Fully induced or derepressed enzgme synthesis should be continuous throughout the cell cycle The data in this regard are conflicting. I n synchronous cultures of 13. suhtilis, fully induced suwase synthesis is periodic whereas in E . coli fully induced and constitutive /3-galactosidase synthesis is continuous throughout the cell cycle (Table 2). Goodwin (1969a)has re-examined the regulation of /3-galactosidase synthesis in cultures of E. coli synchronized in a chemostat by periodic phosphate feeding. He observed that the synthesis of enzyme induced by lactose or isopropylthiogalactoside, or produced in a constitutive mutant, was periodic. Also, Steinberg et al. ( 1965) observed during synchronous growth following the germination of spores that induced u-glucosidase and histidase was periodic. Although /3-galactosidase synthesis in synchronous cultures of E . coli grown in medium containing glycerol or lactose as the carbon source was in agreement with expectations of a feedback-repression model, this may not be a general phenomenon. I n the experiments of Goodwin, the periodic addition of phosphate to chemostat cultures increases the growth rate of the cells. Since the differential rate of enzyme synthesis in bacteria is known to be influenced by the growth rate, the changing growth rate may be the cause of the periodic synthesis observed. Donachie and Masters ( 1 969) have suggested that periodic sucrase synthesis in B. suhtilis is the result of catabolite repression from end products of sucrose met,abolisni. A similar explanation could be advanced to explain periodic enzyme synthesis following spore germination ; however, the finding that the induction capacity peaks at the same time as fully induced enzyme synthesis renders this hypothesis untenable. Since, as originally pointed out by McFall and Magasanik (1962), phosphate levels can influence catabolic repression, this may explain Goodwin’s results. Thus, this prediction based on an independent feedback control circuit is not met by all the available data. (b) A utoregulated enzyme synthesis should be periodic throughout the cell cycle A consequence of feedback repression is that enzyme synthesis should oscillate in individual cells. ‘L’his is evident from the following considerations. Enzyme synthesis leads to an increased rate of end-product
78
11. 0. IIALVORSON, B . L. A. CARTER AND P. TAURO
formation. When the level of these reaches a critical concentration, repression of enzyme synthesis occurs. During subsequent growth and metabolism the concent,ration of this compound(s) in the cell will decrease. When the concentration falls below the critical level, feedback repression is relieved and enzyme synthesis is renewed. For any given system, the rate of formation of end product and the rate of utilization of end product will determine the periodicity of oseillatory repression. Onc might well expect the periodicity to differ for various systems sincc not only are the concentrations of feedback repressor required to inhibit enzyme synthesis different but also the rate of formation and utilization of end product may be subject to variation. The one common feature is that the synthesis of any feedback-regulated enzyme is coupled to the activity of other cellular enzymes. I n the absence of an understanding of the integration of the regulation of these enzyme activities, one cannot, predict the frequency of these oscillations per cell cycle or whether there is a particular time in the cell cycle when they would occur. The relevant cases to examine are those in Table 2 in which enzyme synthesis has neither been demonstrated to be fully induced nor repressed. This category would include enzymes such as ornithine and aspartate transcarbamoylascs which are known to be subject to feedback repression (see Umbarger, 1969), and others for which the mechanism of regulution is unknown. It is highly significant that for all these cases enzyme synthesis is periodic and there is one period of enzyme synthesis per cell cycle when cells are grown synchronously in a constant environment. Based on the initial ideas of Goodwin (1963), mathematical models have been proposed (Purdce, 1966 ; Goodwin, 1966 ; Morales and McICay, 1967; Walter, 1969) which, given appropriate choice of constants, predict stable oscillations of enzyme synthesis during the ccll cycle. One difference between these models is that Goodwin (1966) has assumed that oscillations in enzyme synthesis can be coupled (entrained) to DNA replication. One possibility, that there is a rigid coupling between oscillations in c.nzynie synthesis and DNA replication, can be eliminated. This has been demonstrated in two systems. During outgrowth of spores of B. cereus !I'periodic enzyme synthesis is observed in the absence of net DNA synthesis (Steinberg et al., 1965). Under similar conditions using thymine-deficient spores in which there is no detectable incorporat'1011 of DNA precursors into DNA (Steinberg and Halvorson, l968b) the same results were obtained. Also in vegetative cells of B. subtilis, autoregulated synthesis of ornithine transcarbamoylase is periodic when DNA synthesis is inhibited with 8-fliiorodcoxgiiridirie (Masters and Donachie, 1966). How convincing is the evidence accumulated that periodic autoregulated enzyme synthesis is under fcedbuck control 1 It is implicit in the feedback hypothcsis that the conccntration of feedback repressor must
SYNTHESIS O F ENZYMES DURING THE CELL CYCLE
79
oscillate out of phase with enzyme synthesis in cells grown synchronously in a constant environment. Further, these oscillations should be of sufficient magnitude to account for the known kinetic parameters of feedback repression. Unfortunately such data do not exist. Possible insight into the pattern of regulation imposed by feedback control is seen from perturbation experiments in which a culture (exponential or synchronous) is briefly exposed t o a compound known t o influence feedback repression. From the experiments of Masters and Donachie (1966), Knorre (1968) and Goodwin (1969a) it is evident that such perturbations can alter the time in the cell cycle as well as the magnitude and frequency of oscillations in enzyme synthesis. However, it is remarkable that the oscillations so far observed in synchronous cultures display only one oscillation at a characteristic point in the cell cycle. It remains to be demonstrated that the primary cause of this periodicity is feedback repression. (c) Induction capacity should exist at all times during the cell cycle and
should reject gene dosage The removal of repressor whether by addition of an inducer or by derepression should lead to maximal enzyme synthesis within minutes if this synthesis is controlled solely by feedback repression. This leads to two predictions in synchronous cultures. First, it should be possible to induce at any time during the cell cycle. Second, when a particular gene doubles during DNA replication, the induction capacity should sharply double. Experiments which tested these predictions were described earlier where it was shown that, in almost all the examples examined, induction capacity exists at all times during the cell cycle and doubles sharply at a characteristic stage of the cycle. There are two exceptions to these general observations in bacteria ; that of restricted induction capacity in synchronously germinating B. cereus T spores and that of a continuous increase in induction capacity throughout the cell cycle observed in some strains of E . coli. There is evidence which suggests that the doubling of induction capacity is intimately linked t o the doubling of the structural gene of the induced enzyme. Masters and Pardee (1065) observed that the induction capacity of sucrase doubled at the same time in the cell cycle that the sucrase-transforming ability of the DNA doubles. Further indirect evidence is provided by tho finding t h a t , when DNA replication is inhibited, induction capacity remains unrestricted but no doubling of capacity is obtained (Donachie and Masters, 1966). If the doubling of induction capacity is a manifestation of the doubling of the structural gene, then in cells having one DNA replication fork per cell, this doubling should occur in the same order and reflect the spacing of the structural
80
H. 0.HALVORSON, B . L . A . CARTER AND P. TAURO
genes on the chromosome. The studies of Helmstetter (1968), Pato and Glaser (1968) and Donachie and Masters (1969) demonstrate that, at least for E . coli, this prediction is met (Fig. 5).
lpirl. 5. Correlation between the order of markers on tho gonntic map of Esoliericliirt coli, and the relative order of events during the cell cycle. Tho triangles reprrseirt. thc time of chnrigc in rate of induced synthesis of enzymes (or in rato of syiitlieHis of DNA) deterrniiiod rrlativo to cell division. The horizontal boxes enclose separatcb estimates of this tiino of chnirgc in inducibility for particular enzymes. L~IICH liavc~ been drawn from the, m i o m timu of an event to the corresponding gcnctic lociix on tho linlrago map a t the bottom. Tho lcngth of t,ho liiiltago nap hus been made equal to tho time of u cdl division cyclu. Thcrrcfore tt perfcct corrcctian woiild givo vcrticctl lines from the mean time of change in rate of induced synthesis to tho corresponding genetic locus. Tho nature of the tiniod o w n t is identified on tho right of the figure. Dashed lines indicate the continuous succession of cell cycles. (ATC, aspartate t~rnnxearbit~noyluse ; B-Gal, p-galact,oRidnso; DHO, diliydro-orotusc; Dsd, D-aerint: dearninase; OMT’, orotidine irionophospliate pyropliospliorylaae ; Tryp, tryptophanaso.) Datu from Donachic and Masters (1969).
(d) Sunimur?j I n conclusion, there is evidence that the genome of bacteria is availablct for transcription and translation at all stages in the cell cycle i ~ n dit is clear that negative feedback control of enzyme synthesis in a constant environment can cause oscillations in enzyme synthesis which will res tilt in periodic increases in the levels of enzyme as observed. However, it is difficult to explain the fact that in all examples studied there is only one period of enzyme synthesis per cell cycle at a characteristic stage of the cycle. It is unlikely, but not impossible, that a feedback control circuit independent of other control circuits in the cell should produce one
SYNTHESIS OF ENZYMES DURING THE CELL CYCLE
81
burst of enzyme synthesis per cell cycle. A more likely explanation is that in a constant environment there is an integration of many feedback control circuits; thus a periodicity in one circuit may be the result of a periodicity in another circuit which may itself be influenced by a further circuit. However, as Goodwin (1969s) points out, there must be one or more sets of autonomous (self generating) oscillators, which provide the basic periodic dynamics around which the cell cycle is organized. These self generating circuits may not involve end product repression of enzyme synthesis but they may affect such circuits and result in one period of enzyme synthesis per cell cycle. Thus it may be an oversimplification to consider t h e periodic synthesis of enzyme (observed in bacteria) in a constant environment a direct result of end product repression in a single feedback control circuit. 2 . Regulation in Eukaryotic Cells Although there is ample evidence for ordered appearance of enzymes during the cell cycle in eukaryotic organisms (Tables 3, p. 65, and 4,p. 72), the predictions of a feedback repression model are generally not fulfilled. This is not surprising since one would have expected that eukaryotic cells have evolved additional regulatory mechanisms. I n comparison with prokaryotic cells, the eukaryotic cells are larger, compartmentalized (e.g. contain a defined nucleus) and in general have larger precursor pools. Thus, the immediate response t o changes in the external environment is limited by the presence of several limiting membranes as well as a large internal buffering capavity (pools). These differences are illustrated by consideration of the maximal differential rate of expression of an individual gene in the two systems. For example, in E . coli under conditions of full induction or dfhrepression, aspartic transcarbamoylase, p-galactosidase, alkaline phosphatase or ornithine transcarbamoylase can each represent 5% or more of the total cellular protein. I n eukaryotic cells this capacity is lower by on(’to two orders of magnitude (Halvorson, 1964). One might further expect that regulatory mechanisms would diverge between eukaryotic cells which grow unicellularly (yeast, algae, protozoa) and those which usually grow as organized tissues. Since sudden changes in the extraccllular environment are uncommon in the latter category, different regulatory mechanisms may well have evolved which are not dependent upon immediacy of response to change. I n combining examples from different eukaryotic organisms, we do not mean t o imply that these are necessarily subject t o similar primary regulatory mechanisms. It can be seen from Table 3 (p. 65) that periodic enzyme synthesis during the cell cycle is characteristic not only of autoregulated enzymes in eukaryotic cells but also of induced and repressed enzyme synthesis in
82
11. 0. HALVORSON, B . L A . CARTER AND P. TAURO
these organisms. I n addition, the induction capacity is frcqucmtly restricted where tested in thew cells (Table 4, p. 72). The few exceptions to this generalization have been previously noted. l’hils, any modcl of regulation proposed for eukuryotes must account for the inability of the cell to produce active enzyme throughout the cell cycle whether induced or not. Several models for controls of enzyme synthesis in synchronous cultures have been proposed. These will be discussed each with regard to the particular systcm investigated. (a) Sequential transcription
A temporal appearance of individual enzymes has been observed during the cell cycle of eukaryotic cells. A given enzyme is synthesized once per cell cyck (periodic) at a uniquc period. We have suggested that the simplest model to provide a programmed expression of the genome is the chromosome itself (Halvorson et al., 1964). In its most explicit form this model assumes that the order of genes on the chromosome determines the programnit. for transcription and thus subsequent translation during the ccll cycle. Thus, there is a linear relationship between the time of ordered enzyme appearance and the position of genes along the chromosome. The advantage of this model is that it has a number of predictive qualities. Siricc these predictions are dependent upon knowledge of the genetics of thr organism ILR well as its behaviour during the ccll cycle, a discussion of this model will be focused on yeast. (i) The nmount hut not the time of enzyme elrpression i s under entiironnzenlal control. Basal and induced a-glurosidase synthesis have been romparctl in Sawh. ccrwisiai~(Halvorson P t aZ., 1966). Although miiltosr induccs a 50-fold increaso in the differential rate of enzyme synthesis, it does not change the period in the cell cycle during which enzyme increases are observed. I n addition, uninduced /I-galactoBidase synthesis in Sacch. Zactis oeciirs over a limited period of the cell cycle (Halvorson et al., 1964).This interval is the same as that for the expression of the capacity to induce enzyme synthesis in these organisms (J.Gorman, personal communication). Thus in these two systems removal of the repressor (by cxtracellular addition of induccr) affects only the amount of enzyme synthesized during the cell cycle. One would therefore expect that, in repressor-defective mutants (e.g. operator constitutive), enzyme synthesis should still be periodic. B. L.A. Carter (unpublished results) has tested this for UDP-gul-4-epimerase in Sacch. cerevisiae. The synthesis of this enzyme in operator constitutive and induced strains is periodic and ocrurs in the same interval of the cell cycle. (ii) For non-allelic genes there i s one period of enzyme synthesis per
SYNTHESIS O F ENZYMES DURINQ THE CELL CYCLE
83
structural gene per cell cycle. Two categories of enzymes can be distinguished in yeast. The first is illustrated by the biosynthetic enzymes in which only one structural gene per cell has been identified for any particular enzyme. As seen in Table 5 the synthesis of these enzymes during the cell cycle is periodic ; in each case one period of enzyme synthesis occurs per cell cycle. The second category is illustrated by the carbohydrascs (Mortimer and Hawthorne, 1966, 1969). In Sacch. cerevisiae, for example, six unlinked genes have been identified for a-glucosidase, six genes for sucrase (invertase), three genes for raffinase and at least five genes for a-methylglucoside fermentation. Since gene dosage is additive (Rudert and Halvorson, 1963), genetic redundancy for carbohydrases provides a selective advantage to cells whose primary function is utilization of hexoses. Yeast strains can be prepared carrying known redundancy for these genes. Employing these, the relationship between the gene dosage and the number and time of enzyme synthesis can be determined (see Table 5 ) . I n interbreeding yeast st rains, six non-allelic genes have been identified for a-glucosidase synthesis (Mortimer and Hawthorne, 1966) and the presence of any one will result in enzyme synthesis. These genes function independently and result in enzyme products which are indistinguishable (Rudert and Halvorson, 1963). Thus, it is possible, using appropriate strains, to study the synthesis of these enzymes in a common regulatory system. In diploid strains of Sacch. cwevisiap heterozygous ( M Iin I ) and homozygous ( M I M I )for one a-glucosidasc gene, only one period of enzyme synthesis was observed during the cell cycle and this occurred at the same time interval for both strains. Thus, the amount but not the time of enzyme synthesis is altered by gene dosage effects at the same locus. If additional non-allelic structural genes for a-glucosidase are added to the genome, additional periods of a-glucosidase synthesis are observed during the cell cycle (see Table 5). The number of periods of a-glucosidase synthesis in the cell cycle corresponds to the number of unlinked M genes present, each of which has a unique time of expression in the cell cycle Similarly, in Sacch. Zactis, two unlinked geiies govern /3-galactosidase synthesis. The number of steps of /3-galactosidase synthesis is dependent upon whether one or both of these genes are present (Tingle, 1967). Multiple steps of enzyme synthesis during the cell cycle are not uncommon. As shown in Table 5, multiple steps have been reported for three other enzymes in yeast and for three enzymes in Chinese hamster Don C cells. In the latter case Klevecz (1969) has reported that the three periods of ellzyme synthesis actually represent changing amounts of enzyme protein. I n the absenco of knowledge of the number of genes involved, one cannot conclude that gene redundancy is the cause of
TABLE5. Relationship between Gene Dosage and Multiple Steps per Cell Cycle Orgasism
Enzyme
Steps per cell cycle
Genotype
Q)
Reference
~~
Smhrmycea oerevkiiae
Y 53
Y 55 Y 62
Y64 Y 70
Y 84 Y 84
S m h . &b Y 123 Y 14 x Y 123
S d .dobzhanakii S m h . &&nakii
a-Glucosidase a-Glucosidase a-Glucosidase a-Glucosidase a-Glucosidase Alkaline phosphateee Invertas0
1 1 2 2 3 2 2
T a m and Halvorson (1966) T 8 W and Halvorson (1966) T a m and Halvorson (1966) M1m1M3m3 M2m2.M4m4 Halvorson et al. (1966) M,m~M~rn~M~m T a~ m and Halvorson (1966) Gorman et al. (1964) unknown unknown Gorman et al. (1964)
j3-Galactosidaw /3-Galactosidase
1 2
Gall G~lgallGallg4
MlMl MPl
F
i8
I
"8m r
j3-Glucosidase j3-Glucosidase
1
2
X
S m h . fraqdis
unknown Unknown (interspecies hybrid)
Halvorson et al. (1964) Tingle (1967)
Gorman et al. (1964) Gorman et al. (1964)
?
E
Y I
m
b
Z
U Cd
Y
Chinese hamster Diploid
Heteroploid
Glucose 6-phosphate dehydrogenaae Lactic dehydrogenaee
Thymidinekinaee Lactic dehydrogenase Thymidine kinase
3
unknom
Klevecz and Ruddle (1968)
3
unknown
Klevecz and Ruddle (1968) : Klevecz (1969) Klevecz (1969) Klevecz (1969) Klevecz (1969)
3
3 unknown Continuous Unknown 1 unknown
2
0
SYNTHESIS OF ENZYMES DURIN’G THE CELL OYCLE
85
multiple steps. In practice it is difficult to recognize more than three steps per cell cycle: if the gene redundancy were greater than three then it would be difficult to determine whether the rise in enzyme activity or capacity during the cell cycle was continuous or stepwise. Analysis of this situation is further complicated by the fact that the amounts of enzyme produced in response to the different M genes vary (Rudert and Halvorson, 1963) so that the rise per step would not be constant. In a related fission yeast, Schizosacch. pombe, continuous increases in maltase, sucrase and acid and alkaline phosphatases have been claimed by Mitchison and his colleagues (Bostock et al., 1966 ;Mitchison and Creanor, 1969). Since the relevant structural genes have not been determined, it is unclear whether the redundancy of carbohydrase genes inSacch. cerevisiae is also common in Schizosacch. pombe. Mitchison and Creanor (1969) reported a difference between the K , values of repressed and derepressed invertase in Schizosacch.pombe. This difference suggests the possibility that several isozymes are present as has previously been demonstrated in Sacch. cerevisiae (Gasc6n et al., 1968). However, sucrase exhibits a doubling of the induction capacity during the cell cycle which cannot be explained solely by gene redundancy. Mitchison and Creanor (1969) have proposed an alternative model which will be discussed later. (iii) Periodic synthesis of enzymes should be observed throughout the cell cycle. This hypothesis predicts that for each interval of the cell cycle a rise of one or more enzymes should be observed. As previously discussed, a continuous increase in total protein is observed throughout the cell cycle in all the yeast strains examined thus far as would be expected if steps occurred throughout the cell cycle. However, Mitchison and Creanor (1969) have recently proposed that, at a “critical point” in the cell cycle of Schizosacch. pombe, the rate of synthesis of a number of enzymes doubles. If this is a general phenomenon it should affect not only the kinetics of overall protein Synthesis but also one would not expect steps to occur throughout the cell cycle. The time in which the initial increase for a number of enzymes in Sacch. cerevisiae occurs during the cell cycle is shown in Table 5 . As can be seen, these rises are scattered throughout the cell cycle. The reproducibility of timing measurements in our experience is approximately 0.1 of a cell cycle. From these observations we agree with Mitchison and Creanor (1969) that periodic enzyme synthesis is not governed by a master “critical point”. (iv) For genes on the same chromosome, the time interval between the expression of the genes in the cell cycle should be related to their linkage distance. Some evidence supporting this hypothesis was presented earlier when it was shown that multiple allelic copies of the same gene are transcribed at the same time in the cell cycle. A corollary of this hypothesis has been observed; there is no correlation of the time of
TDLE 6. Enzymes, Structural Genes, and the Time of Expression in Saceharmyca cerevikue
Chromosome number
Structurd gene
Distance from centromeW
x
Enzyme
Approximate timing (fraction of a generation)
Reference
Galactokinase a-Amino-adipicreductaee a-Glucosidase Histidinol dehydrogenase Orotidine 5’-decarboxy1ase ASpartoldnase PR-ATP-PPase -nine deaminase a-Glucoisdase Argininoosuccinase Saccharopine dehydrogenase Saccharopine reductsse
0.45 0.3 0.1 0-3 0.1 0.25 0-3 0.45 0.5 0.7 0-45 0.65
Cox and Gilbert (1970) Cox and Gilbert (1970) Tauro and Halvorson (1966) Tauro et al. (1968) Tauro et d.(1968) Tauro et d.(1968) Tauro et d.(1968) T a m et d.(1968) Tauro and Halvorson (1966) Tauro et d.(1968) Tauro et d.(1968) Tauro et d.(1968)
a-Glucosidase a-Glucosidase
0.73 0.25
Tauro and Halvorson (1966) Tauro and Halvorson (1966)
P
r F
4 II 111
V
8R 57 R 35 R 24 L
6L 40 R
44R
VII
VIII
Ix
XTV Segments: 5
7
60 R 80 R
8R 30 R 32
R and L refer to the two sides of the centromere on the chromosome as published by Mortimer and Hawthorne (1966).
SYNTHESIS O F ENZYMES DURING THE CELL CYCLE
87
expression of genes on different chromosomes (Table 6), whether they govern the synthesis of the same enzyme (e.g. a-glucosidase) or the same pathway (e.g. saccharopine dehydrogenase, saccharopine reductase and a-amino-adipic reductase). Control of gene expression by sequential transcription also implies that the expression of closely linked structural genes should occur close together in time during the cell cycle. Evidence supporting this view has come from studies on a strain of Sacch. lactis in which /3-glucosidase is closely linked (1%recombination) to the gene for /l-galactosidase. Halvorson et al. (1966), found that, in synchronouslygrowing cultures, the initiation of synthesis of p-galactosidase and /l-glucosidase differs by only about a few percent of a generation time. I n Sacch. cerevisiae there are several examples in which the timing of linked genes has been determined in the cell cycle. Recently Cox and Gilbert (1970) observed that galactokinase and a-amino-adipicreductase, which are separated by about 50 centimorgans on chromosome 11, differ in their times of expression by 0.15 of a cell cycle. A more extensive analysis has been conducted on chromosome V where the order of expression of four different genes (ur,, thr,, hi,, i s , ) is the Same as the order of genes on the chromosome. I n the absence of further markers and lack of knowledge whether genetic distance measured by recombination is constant per nucleotide pair in DNA, it is not possible a t the moment to relate quantitatively differences in enzyme timing to genetic linkages. The results thus far, although admittedly incomplete, are consistent with the hypothesis. A testable prediction of this model is that transposition of a gene, either by translocation to another chromosome or inversion in the same chromosome, should alter the time of enzyme expression during the cell cycle. I n fact, i t is not unlikely that translocations are the cause of the accumulation of unlinked, identical genes for carbohydrases in yeasts. Cox and Gilbert (1970) have provided recently an excellent test of this prediction in yeast. As represented in Fig. 6, the genetic mapping of chromosome I1 of the Lindegren breeding stock differs significantly from the Hawthorne-Mortimer stocks. These differences could be understood if a double inversion had occurred. Of significance is that the gal, and ly, genes for galactokinase and a-amino-adipic reductase which were separated only by 50 centimorgans in the Hawthorne-Mortimer strains are separated by a greater distance in the Lindegren stock. Cox and Gilbert ( 1 970) observed that this transposition increased the difference in time of expression in the cell cycle from 0.15 to 0.6 of a cell cycle. (v) Summary. The essential feature of the sequential transcription model is that over a region of the chromosome the genes are transcribed periodically in the order of their appearance on the chromosome. This model does not define the points of origin of periodic transcription but does 4
88
H. 0.HALVORSON, B. L. A. CARTER AND P. TAURO
require that the time of their initiation is tightly coupled to the cell cycle. Sequential transcription is not a substitute for induction or repression; it limits the effective period of such regulation but not the amount of enzyme synthesized. For example, some enzymes, such as those involved in the synthesis of vitamins, are produced in very small amounts. I n these cases it is likely that the enzymes are fully repressed in addition to having a limited period of synthesis during the cell cycle. The model does not specify the number or length of regions per chromosome but could involve an entire chromosome. In this context the predictions of the model are met in the budding yeast Sacch. cerevisiae and some but not all of the observations in the fission yeast, Schizosmch. pombe. Strain -
gall
61009
Cell-cyrle _ _ map ~_
Genetic map
ac', his7
\+C-w+w
tyr,
lys;:
0
02
04
06
08
I0
I
I
I
I
I
I
?!?
H 20 centimorgans
FIG.6. Timing of synthesis of two step enzymes during the cell cycle of two strains of Sacchromyces cereviaiae; gal, and lyez are the structural genes for the enzymes galactokinase ( A ) and a-amino-adipicacid reductme ( A ) . Strain 3288C came from Drs. D. C. Hawthorne and R. K. Mortimer and strain 61009 came from Dr. C. C. Lindegren. Data from Cox and Gilbert (1070).
There are two assumptions of this model that have not been subjected to rigorous tests in yeast. These are that: (a) there is a constant interval between the transcription of a given gene and its translation and conversion to active enzyme ; and (b) the half lives of different m-RNAs are similar and are only a fraction of the generation time. The first assumption has recently been questioned by Mitchison and Creanor (1969)who observed that, following the inhibition of protein synthesis by cycloheximide, sucrase and alkaline phosphatase activities increased for another 16 to 30 min., whereas acid phosphatase activity immediately stopped. It is obvious that to test this critically the time of synthesis of
SYNTHESIS OF ENZYMES DURING “HE CELL CYCLE
89
enzyme protein as well as activity must be determined. Little is known about the half life of specificm-RNA in yeast, although it has been known for some time that yeast contains rapidly labelled RNA (Ybas and Vincent, 1960). It seems unlikely that the transcription of all RNA species in yeast can be explained on a sequential transcription model. As discussed earlier, r-RNA and t-RNA are synthesized throughout the cell cycle in Succh. cerevisiae (Tauro et al., 1969). From considerations of the amount of RNA per cell, the rate of r-RNA transcription and the known gene redundancy, the sequential transcription model for these genes seems unlikely. Continuous transcription of all of the stable RNA genes throughout the cell cycle would explain the observed accumulation of these RNA species. (b) Critical point transcriptional control This model assumes that the genome which is produced during mitosis is unavailable for transcription until some fixed period after DNA replication (critical point). On the other hand, the original genome is available for transcription throughout the cell cycle. The overall effect would be to double the rate of synthesis of a number of enzymes a t the same time in the cell cycle. It has been suggested that doublings in the rate of autoregulated (continuous) synthesis of alkaline phosphatase, acid phosphatase, and sucrase and a 60% increase in the rate of sucrase derepressed capacity occur at about the same time* in the cell cycle (Bostock et al., 1966; Mitchison and Creanor, 1969). This time (Fig. 7), termed the “critical point”, occurs about 0.36 of a cell cycle after DNA synthesis and thus is not directly related to gene dosage effects. At about the same time in the cell cycle in Schizosacch. pombe, Swann (1962) observed a temporary ten-fold increase in resistance to ultraviolet light and a doubling of the target size which was later (see Mitchison and Creanor, 1969) associated with a shift in the maximum of the action spectrum from 260 nm. (nucleic acids) to 280 nm. (proteins) and t o a resistance to 32Psuicide decay. These authors have proposed that at the critical point chromatids separate (analogous to metakinesis) and become associated with a protein backbone. I n an unspecified manner this process is related to (or responsible for) the making available of both chromatids for transcription and this leads to a sudden increase in the rate of enzyme synthesis.
* The mid point of rise in sucrase capacity is 0-38 of a cell cycle. Assuming a precursor delay for sucrase of 0.1 the mean capacity rise is 0.28. The average of this potential and the corrected rises in autoregulated synthesis (0.1 cell cycle for basal sucrase; 0.21 cell cycle for alkaline phosphatase) is 0.2 and is defined as the “oritical point”.
90
R. 0. HALVORSON, B. L. A. CARTER AND P. TAURO Nucleal divisiar
DNP synthesi! Cel plate pea
Cel divisioi
w
* i+
DN/ synthesi
Cel divisiai
Acid phosphatase
Alkaline phasphatase
FIG.7. Cell cycle maps for the rate change points of sucrase (a),alkaline phosphatase (AP) and acid phosphataso (SP). Open triangles for points and steps in the second cycle and closed triangles for the third cycle. Arrows are the means, with cross-bars giving the standard error. The stars give the points of change of rate of synthesis and of steps in true sucrase potential (allowingfor precursor delay). The bottom map gives other events in the cell cycle. (ND, nuclear division: DNA, DNA synthesis; CPP, cell plate peak: D, cell division.) The star in the square is the “critical point” a t 0.20. The length of the cycle is 145 min. Data from Mitchison and Creanor (1969).
Some related observations have been reported during the cell cycle of Smch. cerevisiae. Radiosensitivity (Bacchetti et al., 1967) and sensitivity to X-rays (Elkind and Sinclair, 1965) and ultraviolet light (Esposito, 1968) display a cyclic behaviour during the division cycle. X-ray or ultraviolet light-induced intragenic and intergenic recombination increases prior to DNA replication and declines to a minimum after the completion of replication (Esposito, 1968). Possible similarity to the “critical point” was the finding that there was no obvious polarity of recombina-
SYNTHESIS OF ENZYMES DURING THE OELL UYCLE
91
tion events as was observed for intergenic recombination in Ustilago (Holliday, 1965). Unique periods of resistance in the cell cycle have been reported in higher organisms (Clowes, 1965; Terasima and Tolmach, 1963). Evidence for the “critical point” hypothesis is based on statistical conclusions that : (a) the rate of enzyme synthesis during the cell cycle changes abruptly from one rate to another; and (b) the point at which these rates change is the same for all activities (as corrected) measured. As can be seen in Fig. 7, the individual rate changes for each enzyme vary widely over the cell cycle. Indeed the coefficient of variation is as high as 84.3% indicating considerable variation among individual samples about the mean. Considerably larger numbers of samples are required to determine : (a) if the distribution of experimentally measured rate changes for each enzyme are distributed normally about a mean; and (b) if the data from the various enzymes share a common mean (critical point). The present information does not permit a statistically valid decision on either of these questions. It may be that the rise in the rate of synthesis is a complex curve, containing multiple linear segments or steps. Alternatively, if true rate changes occur once per cell cycle, the time of their occurrence is highly variable. Periodic increases in autoregulated ornithine transcarbamoylase and aspartate transcarbamoylase synthesis occur at different periods of the cell cycle (0.3 and 0.42) than the critical period (Bostock et al., 1966). Finally, if the “critical point” regulation is general, one would expect that the rate of overall protein synthesis should double at the critical point. Published data show a smooth continuous increase of protein (Mitchison and Wilbur, 1962) and in dry mass (Mitchison et al., 1963) during the cell cycle. Thus this attractive hypothesis has yet to be proven. Histone synthesis in animal cells displays a number of formal analogies to the “critical point” factors in yeast. Histones are a family of basic proteins which accumulate during DNA replication (S phase), are metabolically unstable (Spalding et al., 1966) and are thought to play a regulatory r81e in gene expression. When the initiation of DNA synthesis is blocked in wiwo or in witro, no histone synthesis is observed (Gallwitz and Mueller, 1969). Robbins and Borun (1967) demonstrated that in synchronized HeLa cells there was a correlation between DNA synthesis and the synthesis of histones on cytoplasmic ribosomes. They subsequently provided evidence for the synthesis of histone in RNA during the S phase (Borun et al., 1967). (c) Post transcriptional control Tomkins and his colleagues (1969) have studied extensively the synthesis of tyrosine aminotransferase in asynchronous and synchronous
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€I. 0.RALVORSON, B. L. A. OARTER AND P. TAURO
cultures of rat hepatoma cells. They observed that, although this enzyme is synthesized continuously throughout the cell cycle, it can only be induced by a steroid during the S phase and part of the G1 phase of the cell cycle. They propose a post transcriptional control of enzyme synthesis, outlined below, to explain these observations. Their model states that during the inducible period of the cell cycle the structural gene and regulatory gene (for labile repressor) are continuously transcribed and translated. The repressor is assumed to bind to the molecule of m-RNA transcribed from the structural gene for the enzyme and to inhibit reversibly the translation of the messenger. The model assumes that m-RNA is degraded only when its translation is inhibited by the repressor. In the presence of inducer, the repressor is inactivated, messenger translation occurs and degradation of m-RNA ceases. If transcription is continuous, m-RNA would accumulate in the presence of inducer. During the non-inducible phase of the cell cycle, transcription of regulatory and structural genes is prevented by an unknown process. Since repressor is not synthesized, this phase is insensitive to inducer and the pre-existing m-RNA can be translated and will be stable. This model is highly speculative, but since it represents one of the few attempts to explain the behaviour during the cell cycle of higher cells, we shall discuss it in some detail. Their model makes the following predictions. (i) Synthesis of R N A is required for enzyme induction but not for the maintenance of synthesis at basal or induced rates. The model assumes that, following induction, the effective m-RNA concentration determines the rate of enzyme synthesis. This prediction is supported by observations in asynchronous cultures of rat hepatoma cells that actinomycin D addition prevented the induction of tyrosine aminotransferase but permitted enzyme synthesis and protein synthesis to continue for a number of hours (Peterkofsky and Tomkins, 1967). Cycloheximide, an effective inhibitor of protein synthesis in this system, caused decreases in enzyme activity. (ii) The decrease i n the rate of enzyme synthesis following removal of the inducer should be reversed by inhibiting R N A synthesis. This surprising prediction is based on the assumption that RNA synthesis is required for continued production of a labile protein (repressor)which inactivates or causes a degradation of m-RNA. Samuels and Tomkins (quoted in Tomkins et al., 1969) observed a rapid decline in enzyme activity following removal of inducer. When actinomycin D was added 16 min. after the removal of the steroid inducer, enzyme synthesis rapidly increased to the maximal induced level. The longer the interval between the addition of actinomycin D and the removal of inducer, the lower was the maximal enzyme activity observed. This decrease is consistent with
SYNTHESIS OF ENZYMES DURING THE CELL CYCLE
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a lability of m-RNA in the absence of inducer. From immunological precipitation experiments, the possibility that enzyme turnover was inhibited by actinomycin D could be eliminated (Martin et al., 1969b). If their explanation of superinduction is correct, then one would expect to observe a similar phenomenon in non-induced cells following actinomycin D addition. (iii) Induced cells collected in mitosis and washed free of inducer should continue to synthesize the enzyme at the fully induced rate early in the GI interval when no transcription of the structural regulatory gene occurs. Martin et al. (1969b) found that in preinduced cultures tyrosine amino transferase activity remained constant in the absence of the inducer until the initiation of transcription. Further, a t this point, enzyme activity can be stabilized by either the addition of inducer or actinomycin D. This intricate model accommodates the well known differential template survival in higher organisms. It is, however, based on the observations of only one enzyme and makes a number of assumptions, including an unitary mechanism of action of actinomycin D. (d) Post translational control I n recent years it has become increasingly clear that post translational controls play an important function in the regulation of cells of higher organisms (Pitot, 1967). Undoubtedly a number of these govern the amount and pattern of enzyme synthesis in the cell cycle of higher organisms. One of these, alluded to by the data in Table 3 (p. 65), is the instability of certain enzymes. Loss of enzyme activity could be due to loss of stabilizing ligands, loss of enzyme protein through turnover, or other causes.* The latter is particularly significant in cultured animal cells where the rate of protein turnover can approach a few percent per hour (Eagle and Pietz, 1962; Schimke et al., 1968).For turnoverper se to modulate enzyme activity in the cell cycle, heterogeneity is required. Such heterogeneity in the half lives of individual enzymes has been noted (Schimke et al., 1968)and range from about 60 min. for 6-aminolaevulinic acid synthetase (Marver et al., 1966) to 100 hr. for arginase (Schimke, 1964). Variations in the concentration of protecting ligands (Markus, 1965; Schimke et al., 1965) or the periodic synthesis of specific inactivatingsystems (Bechet and Wiame, 1965; Blobel and Potter, 1966; Ferguson et al., 1967) could explain these variations. Although periodicity in the activity of proteases has been observed in the cell cycle in both prokaryotic and eukaryotic organisms (Tables 2, p. 58, and 3, p. 65), i t has
* Numerous othermechanismscould also function.For example,in Dktyosteliurn discodeurn, decreases in cellular enzyme activity have been associated with
incorporation of enzyme into an insolublematrix (Wrightetal., 1968) or preferential release of enzyme into the extracellularmedium (Sussmanand Lougren, 1966).
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yet to be demonstrated that these have specific enzyme substrates in wivo. Therefore, although post-transcriptional controls provide attractive alternative hypotheses, their function in the cell cycle has yet to be demonstrated. (e) Other possible mechanisms of regulation in higher cells Not all of the observations on regulation during the cell cycle in higher organisms can be accommodated by the above models. One interesting example of this is evident from the observations on enzyme synthesis in Chinese hamster cells as summarized in Table 3 (p. 66). Klevecz (1969) has compared the kinetics of synthesis of lactic dehydrogenase and thymidine kinase in synchronous cultures of a pseudodiploid cell line and a heteroploid cell line of Chinese hamster cells. The pattern of enzyme and DNA synthesis differs in these two cultures. In the pseudodiploid strain, both enzymes show three periods of enzyme activity during the cell cycle which have been associated with oscillations in enzyme protein (Klevecz, 1969). In contrast, in the heteroploid strain, thymidine kinase shows one predominant peak of activity during G,. Of interest is the nearly continuous rise in lactic dehydrogenase activity throughout the cell cycles. Although differences in kinetics of DNA synthesis were observed,* it is unlikely to alter the kinetics of enzyme synthesis since inhibition of DNA synthesis reduces the amount, but not the time, of enzyme appearance. There is no obvious explanation for the differences in enzyme synthesis in these related cell lines although some possibilities could be suggested. The karyotypes and the morphology of chromosomes, especially in heteronuclear cultures such as the Chinese hamster, are known to be unstable in cultured cell lines (Krooth et al., 1968). Chromosome rearrangements based on breakage and reunion are not uncommon. In the Chinese hamster cell cultures, changes in both chromosome morphology and replication patterns have been observed.* Klevecz (1969) observed the accumulation of small metacentric chromosomes in the diploid cell lines following prolonged culturing. As judged by enzyme expression (Lyon, 1968) and evolutionary considerations (White, 1969), translocation may alter gene function. This possibility is particularly significant since it is increasingly clear in higher organisms that controls operate over groups of genes rather than individual genes. For
* Diploid cells are known to have a distinct pattern of late chromosomal replication (Hsu, 1964; Stubblefield, 1966) associated with a rapid replication of the inactive X chromosome and portions of other chromosomes late in the S phase (Gilbert et al., 1962; Seed, 1962; Kasten and Strasser, 1966; Hamilton, 1969). The heteroploid strain contains 21 Chromosomes and the pseudodiploid 22 chromosomee.
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example, based on genetic (Lyon, 1961) and biochemical measurements (Beutler et al., 1962) the hypothesis of a single active X chromosome (in mammalian female cells carrying 2X chromosomes) has been proposed. The genes on the additional X chromosome are repressed presumably in response t o a master control(s) for chromosomal function. This hypothesis has been supported by the finding that when active autosomal genes are translocated t o the X chromosome they are subject t o the “master controls” of their chromosome (Russell, 1963; Salzmann et al., 1968) with a consequent influence on the specifications of the organism (Ohno, 1969). Similar controls may be present for autosomal chromosomes (Lyon, 1968). One can thus imagine that, in such cultured cells, changes of chromosomal morphology could be associated with changes of control to different chromosome “master controls” or t o loss of one or more regulatory functions hypothesized to govern the temporal control of gene function (Britten and Davidson, 1969; and also as extended by Waddington, 1969). Klevecz and Stubblefield (1967) also pointed out that heterogeneity in the properties of heteroploid and diploid lines can also affect the synchronization of molecular events in the populations of these cells. It is difficult, therefore, to compare the behaviour of two cultivated cell lines without detailed knowledge of the two karyotypes. Summary
Discontinuous enzyme synthesis is a common feature in synchronous eukaryotic cells. Our understanding of the molecular basis of this periodicity is hampered by the lack of genetic information and by the larger multiplicity of control mechanisms in higher organisms. Based on the present information it seems unlikely that a common mechanism governs genetic expression during the cell cycle in eukaryotic cells. Evaluation of the various models discussed above is made difficult by lack of knowledge of the order of transcription, half life of individual m-RNAs and time of de novo synthesis of enzyme protein and of its conversion t o active enzyme during the cell cycle. It is reasonable t o conclude from studies on differentiation in higher organisms that segments of the genome may remain silent or unexpressed during some stages of development and active in others. With the possible exception of Sacch. cerevisiae, this type of control has not been documented in the cell cycle of eukaryotes but is a feature of several of the models proposed.
V. Why Does a Cell Divide? “When the cell has completed all the biosynthetic preparations for mitosis, mitosis will take place” (Reiner, 1968). I n this section we shall confine our discussion to bacterial cell division. Although the division of animal cells has attracted considerable interest
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in recent years (for recent reviews see Basega, 1968 and Epifanova and Terskikh, 1969), there is not yet sufficient biochemical and genetic information to discuss the relationship between events during the cell cycle and cell division. Several models have been advanced to explain the control of cell division. One of these assumes that in bacteria division occurs after the completion of a set number of random (Rahn, 1932) or sequential (Kendall, 1948) events. The clock is assumed to be reset after each cell division. Subsequent findings of a strong positive correlation between the interdivision times of a cell and its sisters* (Schaechter et al., 1962; Powell and Errington, 1963 ;Kubitschek, 1962) and the negative correlation with its ancestor (Kubitschek, 1966) rules out this type of clock mechanism. Alternatively, Koch and Schaechter (1962) proposed that cell size, rather than age, determines cell division. This model was based on previous observations (Schaechter et al., 1962) that the spread in distribution of bacterial size is less than in interdivision times. From a detailed analysis of synchronous growth in E . coli, Marr et al. (1969) concluded that one of the assumptionst in the Koch and Schaechter (1962) model was incorrect. A more likely hypothesis is that cell division is indirectly regulated by size. We shall return to this later. In this review we have seen that a number of enzyme activities vary periodically during the cell cycle in rapidly dividing populations. As previously noted, periodic synthesis in cultures growing synchronously in a constant environment (Tables 2, p. 58, and 3, p. 65) show one period per cell cycle. One might well inquire if cell division is a determinant in regulating these periodicities. If all of the processes of the cell were rigidly coupled to cell division, then one would expect the latter to determine cellular composition. In bacteria this has been shown not to be the case. As shown in Table 7 (p. 97), at different growth rates the contents per cell of RNA, protein, DNA and genome equivalents all vary. These findings do not eliminate the possibility that some of the processes are coupled to cell division. As will be discussed below, methods for inhibiting cell division but not growth have been described. One interesting test would be to determine if any of the periodicities continue in a growing cell following inhibition of cell division. Although such experiments have not been reported, two cycles of autoregulated ornithine transcarbamoylase synthesis in E . coli were reported after the inhibition of DNA synthesis (Masters and Donachie, 1966). Such behaviour would be expected for autogenous controls. Examination of the kinetics of the synthesis of enzymes known to be subject to feedback
* A topic of vigorous debate. t The model assumes that division occurs at a critical size which is independent of the size at birth.
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SYNTRESIS OF ENZYMES DURINQ THE CELL CYCLE
TABLE7. Variation in the Composition of Salmonella typhimurium With Growth Rat@
a
pb
Dry weight
RNA
2.4 1.2 0.6 0.2
0.756 0.322 0.210 0.158
0.238 0.071 0.038 0.091
pg./cell Proteinc DNA 0.515 0.238 0.162 0.132
0.023 0.01 1 0.008 0.006
Genome equiv./cell 4.5 2.4 1.7 1.4
Data from Maal0e and Kjeldgaerd (1966). p = specific growth rate (hr.-I). These values include membranes and cell wall materials.
control and of other autoregulated enzymes where DNA synthesis or cell division is inhibited would provide a basis for the evaluation of some proposed models. On the other hand one can imagine that there are periodic processes in the cell whose activities are necessary for cell division. Numerous reports of specific proteins required for cell division have appeared in the literature. Some of them act as the initiators for DNA synthesis in bacteria (Maaloe and Kjeldgaard, 1966) and in mammalian cells (Erhan et al., 1970). Others are the disulphide reductase for budding in yeast (Nickersonand Falcone, 1956), a “division protein” (possiblyoral fibres) in Tetrahymena (Williams and Zeuthen, 1966), initiator proteins for chromosome replication in Physarum (Cummins and Rusch, 1968), and inhibitors of cell division in higher organisms (Bullough, 1969). In addition, Mitchison (1 969a) noted numerous physiological stages in higher cells which : (a) affect cell division; and (b) are generally concentrated in the latter part of the cell cycle. In bacteria, a number of genes have been shown to control cell division (Hirota et at?., 1968). The process is undoubtedly complex. In addition to the requirement for the completion of DNA replication (see below), in E . coli cell division is dependent upon the ratio of putrescine to spermidine (Inouye and Pardee, 1970) and on the continued synthesis of a heat-sensitive protein (Smith and Pardee, quoted in Inouye and Pardee, 1970). Of the various elements governing cell division, the greatest attention has been directed toward DNA synthesis. A review of the regulation of DNA replication in bacteria is beyond the scope of this article and has recently been summarized by Helmstetter (1969) and Pritchard et al. (1969). For the present discussion, the following summary will suffice. There is a fixed point on the chromosome at which DNA synthesis begins. The time for a round of DNA replication is a constant independent of the growth rate, The time between the end of a round of replication and the
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H. 0. HALVORSON, B. L. A. CARTER AND P. TAURO
following cell division is also independent of growth rate. The rate of cell division is determined by the frequency of initiation of DNA replication which is itself regulated by the rate of protein synthesis. The timing of the initiation of DNA synthesis has been explained by either periodic positive or negative controls. Initiation of DNA synthesis could either begin when an initiator protein had reached a critical quantity (Cooper and Helmstetter, 1968) or when the concentration of a periodically produced inhibitor of initiation had dropped below a critical level (Pritchard et al., 1969). Let us now consider the Cooper and Helmstetter (1968)and Pritchard et al. (1969) models in light of the cell size hypothesis. In the latter case, given a periodic synthesis of an inhibitor, it is likely that the increase of cell volume would lead (by dilution below a threshold) to periodic DNA synthesis and division. The mathematical predictions of this model have yet to be critically evaluated. The model of Cooper and Helmstetter (1968) on the other hand, is dependent upon the amount (concentration?)of an initiator protein which is produced continuously during the cell cycle. When a critical amount (proportional to size) is reached, DNA synthesis commences and subsequently cell division occurs. What is not clear is how once a critical threshold per mass or per volume is achieved, initiation of DNA replication is now discontinuous. Either the hypothetical initiator protein is synthesized periodically or during initiation it is consumed. I n the latter case the assumption of continuous synthesis could be retained. The continuous synthesis of a stable initiator protein is inconsistent with the observed facts. Apart from the obvious necessity to invoke a discontinuous cellular element, the cause of bacterial cell division is unclear : the situation in eukaryotic organisms has not yet reached the speculative phase.
VI. The Importance of Temporal Order in Cells Throughout this review we have stressed, that based on our present knowledge, the pattern of enzyme regulation in synchronous cultures of both prokaryotic and eukaryotic cells is complex. This is perhaps not surprising since, in one of the simplest and the best studied systems, the regulation of bacteriophage A, at least 16;different negative and positive controls govern transcription (Szybalski et al., 1969 and W. Szybalski, personal communication). One would therefore expect that the control of the chromosome of the cell is no less complex. From the previous discussion i t seems inescapable that there are basic differences in the control of enzyme synthesis in prokaryotic and eukaryotic cells. Indeed in any organism enzyme regulation may involve, in addition to transcription, translation and post translational controls. Apparent differences in
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overall regulation may reflect differences in the integration of these various control mechanisms. An interesting example of this is the modification of the specificity of lactose synthetase of the mammary gland in rats. One protein, acting as a galactotransferase, is converted to synthesize lactose after parturition only when a second protein, u-lactalbumin, is made (Turkington et al., 1968). Such a mode of regulation is highly efficient in preserving one gene product throughout the life cycle and altering its mode of action by the level of a second effector protein. The frequent finding, especially in eukaryotic cells, of temporally organized events during the cell cycle strongly implies spacial organization. Aside from the model for sequential transcription, little attention has been directed towards this possibility. The finding that in mutants of D . discoideum in which the appearance of enzymes is temporally deranged, abnormal morphological development occurs further relates structure to ordered control. With the exception of a few observations on organelle biogenesis (Smith et al., 1968; Osumi et al., 1968; Cottrell and Avers, 1970),there is relatively little information on structural changes associated with the cell cycle. Cytological and physical chemical studies will be required to provide information on questions such as, “what are the changes occurring in the nuclear protein complex in eukaryotes during the cell cycle” and “are there compositional or conformational changes in the membrane associated with the cell cycle?”
VII.’Concluding Remarks “Begin at the beginning and go on till you come to the end :then stop”. King of Hearts.
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Microbial Formation of Methane
Department of Microbiology, University of Illinois, Urbana, lllinois 61801, U . S . A . I. An Introduction to tho E c o l ( ~ g of y Mcthnno Bacteria 11. Isolstioii of Mothane Bactrriti . A. Enrichments .
13. Tho Hungatc Techniclritr . 111. Charactoristic.; of Methane Bactcwa. . A . MorphohJgicul Types . 13. Species and Their P r o p r r t i ( ~ s . C . Hesolution of Methanohncterium nitwliamkii IV. Mass C'nlture Techniques . A. Growth on Hydrogen and Carbon Dioxide I3. Growth on Formato . C. Growth o n Methyl Alcohol . V. Biochomistry of Motliane Formation . A. Assay Systcrn . 13. Substrates C. Mcthylcobalamin as Siibstratc . D. RolcofATP . 13. C'ohnloximes as Substrritrs . 17. Role of Coonzyino M . ( i . 1iihibitoi.s of Motlitine Forinittioii . H. liotlnction of Arscriatcs 1 . Mini-Mcthane S y s t c A i n q . VI. Acknowlodgerncnts . References . .
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I. An Introduction to the Ecology of Methane Bacteria It is tny p r p o s e here to discuss certain aspects of our knowledge of the microbial formation of methane, and to view the state of the problem in 1970. The review by Barker (1956) has been a classic reference work on this subject for many years, and progress through 1966 has been reviewed by Stadtman (1867). The formation of methane is a unique biological event which is confined to a small group of 1)acteria.'l'liese organisms are poorly understood and are difficult to obtain in pure culture. Yet the biological formation of 107
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inethmc is common in nature, and methane bacteria are readily found in anaerobic environments where organic matter is being vigorously decomposed. I n these cnvironmcnts they are terminal organisms in the microbial food chain. As presented in Fig. 1, organic polymers such as cellulose lire hydrolysed by extracellular cellulases to sugars which are fermented to a variety of compounds: fatty acids, alcohols, carbon dioxide and hydrogen. Certain of these compounds, such as acetate, methanol, or c+arbondioxide and hydrogen, are the preferred substrates for methane bacteria. Methane, the simplest member of the hydrocarbon family, is poorly soluble, is essentially inert under anaerobic conditions, and is volatile. Thus, the final reduced product has ideal non-toxic. propcrties and readily escapes from the anaerobic environment. This anaerobic food chain works very well in the black muds of lakes and swnnips, where cellulose is readily undergoing decomposition. Methane found in coal mines is believed to have bcen trapped there when the coal
CELLULOSE
-
SUGARS +
FATTY ACIDS ALCOHOLS COZ HZ
f
CH4
FIG. 1. Anaerobic inicrolisl food chain by which colluloso is converted to methnne.
was formed, and the carboniferous swamps must have been an ideal environrnent for methane bacteria. Methane is commonly produced in the digestive tract of animals, where similar anaerobic food chains arc important in the degradation of food. Of all the animals which produce methane in great amounts, the cow has been most intcnsively studied. A large cow may contain 100 1. of fermenting plant products in the rumen; from this 100-1. fermentation vat, 200 or more litres of metthane may be produced per day. This gas is removed by belching ; when the belching mechanism fails due to over-ingestion of certain vegetative crops, the animal developR a condition known as bloat. For many years the formation of methane in the rumen of rattle has been considered t o be a wasteful process resulting in approximately an 8-10% energy loss. I n non-ruminants the caecum appears to be an organ in which a similar anaerobic digestion of cellulose may take placc. The types of organisms found there resemble in many instances those found in thc rumcn and the chemostat-like nature of the caecum is similar to that of the rumen. Steggerda and Dimmick (1966) have studied the formation of flatus in the human intestine and have related the c.ompositioii of the diet to the amount of gases formed. Thc dirt of certain langur nioiikeys consists of leaves, and these animals havc
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developed a pouch in the stomach in which a pH value somewhat below neutrality is maintained ; i t is here that the major decomposition of cellulose occurs, this being essentially a methanogeriic fertnentation. It might be speculated that, if the legendary dragon ever existed, it was probably a ruminant. The methane could have been stored in a special pouch and could have been forcibly ejected toward its prey ; however, the biochemistry of the ignition mechanism escapes modern-day biochemists. A dynamic example of methane fermentation is that produced in the sludge fertnenters of sewage plants. Man has designed an anaerobic environment in which the sludge from the aerobic portion of sewage treatment is allowed to digest under anaerobic conditions. A modern fermentation vat may contain 100,000 cubic feet of fermenting sludge. The vast quantities of methane produced by such a sludge-digesting facility are used to operate diesel engines, the power from which is used to pump the sewage, to run the blowers for the aerobic section of the sewage plant, and t o generate electricity. I n many such facilities the water which is used to cool the diesel engines is circulated through the fermentation vats t o maintain the temperature of the digestors at about 38”. Although hydrogen is a major product of the degradation of sugars by a variety of organisms in anaerobic environments, it is unique that quantities of hydrogen are not readily detected in anaerobic environments such as mud, intestine or rumen.
11. Isolation of Methane Bacteria A. ENRICHMENTS To enrich for hydrogen-oxidizing anaerobes the 200-ml. culture flask described by Bryant et al. (1968) is convenient. The inoculum is placed in the flask with the desired medium. A gas mixture of hydrogen and carbon dioxide (80 : 20) is passed into the flask as it is shaken on a rotary shaker. Organisms from this culture are isolated in closed tubes which contain solid medium and a hydrogen-carbon dioxide gas mixture (80 : 20) as described below. Tubes which develop a negative pressure upon incubation are likely t o contain anaerobes which oxidize hydrogen. Sewage sludge, rumen fluid, or intestinal contents may be considered as natural enrichments of methane bacteria and may be streaked directly or carried through appropriate dilutions and then streaked or “plated” in agar roll tubes as described below. Tubes with isolated colonies are tested for the presence of methane. An isolated colony from a methanepositive tube is picked t o fresh medium and streaked; after incubation, a sample of the gas atmosphere is tested for methane.
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RIICHOUIAL YOltMATION OF METHANE
111
with the needle and cotton in place. After sterilization, it is connected to a rubber tuhe which cwnveys a gas mixture which has passed through a heated copper eolunin where traces of oxygen are removed from the gas. In this m n n n e ~oxygen-free ~ gas or gas mixtures are readily passed into the flask, replacing iLir. The rntdiurn is gently hoiled ns the gas is pitsscd over the surface of the boiling medium. When the medium is fully reduced, i t s is indirated by the reduction of t u i indicator such as resazuri~i which Iias been d d c d to thc medium, it is transferred to tubes. This transfer mtty be itffwted eithw by :L mouth tube wnnected to a pipette or 1)y the use of a pipette hull) as shown it1 Pig. %A.With either 1)ipetting method, the pipette is first flushed and filled with gas above the medium and is then lowered into tlie medium so that, when the medium is transferred into tlie pipette, it is under a layer of oxygen-free gas. Another gzssing probe is placed in an empty tube, as shown in Fig. 2B. The pipette with medium is transferred quickly t o this tube, and the contents of the pipette are ex1)elled into the test tube while gassing is continued. Oxygen -free nicdiuin has now been transferred from oiic vessel to another with minirim1 contact with air. In Fig. %C,gassing is caontinued as a. solid black rubber stopper is placed in the mouth of the test tube. Removal of the gassing probe and insertion of the black stopper firmly into tlie mouth of the test tube constitute the most critical inanoeuvre of the Hurigatc trechnique. This opc>rittionmust be carried out in one smooth movement so :LS not t o t r q ) traces of air. Proficiency nt this stitge is not difficult to acquire, but the procedure does require some practice. For sterilization under steam pressure, tlie stopper must be clamped in place. Por single tubes, a clamp may be constructed of cost-hanger wire (Fig. ZD) a s suggested by W. E. ('. Moore (personal conimur~ication). Altertmtively, n rack of tubes may he clamped between two appropriate plates In this intin~ierthe tubes mtzy he steam sterilized under pressure b y conventional techniques with the solid rubber stoppers remaining i n p1itc.e during the sterilization vyc'le. M'lien sterile tubes have cooled to 50",the c l i ~ n lmay ) ~ be removed. Broth tubes may be stored for montlis at rooni te11il)ertiture.ltoll tubes of agar rnediurn are prepared by rolling the tubes under a stream of cold water from a faucet. M'ith a little practice, the agar may be solidified in an even layer around the periphery of the tube. Alternatively, the agar mity be solidified around a tube by use of R tube spinner its shown i n Fig. 21<. Agar roll tubes are stored in a vertical 1)ositiononce the agar has firmly solidified around the periphery of the tuhe. A small itrnount of water collects in the bottom of roll tubes which must not bc liandled in it horizontal position. Recently the vnlue of t h r IWII t ~ ~ hI ItW. 1 ) ~ t extcwdctl i SO thitt it I ~ O Whiis ~ I : % I of I ~ the ;L(1vkLlltagr.s of i t 1 1 ;uiwrol>icsstretbk 1)l:Ltc. To inoc*ul;~tc a stchrilr roll tube, or i~ 1)roth tube, t hc rubber stopper is f h m d briefly in the c~onvcntiorial
112
R . 8. WOLPE
manner. When it has cooled slightly, the stopper is grasped a t the very end with the fingers and i R worked loose from the opening of the tube. Prior to removal of the stopper, a syringe gassing probe is pointed a t a Bunsen flame, and the gas flow is regulated so that it definitely alters the shape of the flame when the gas jet is directed into the flame. The needle of t h e probe is then sterilized by passing it briefly through the flame. This manoeuvre is timed so that as the rubber stopper is removed from t h e tube the sterile gassing probe is immediately inserted into thc opcning of the tube. Motorized tube streakers recently have been devclol)ed, onc of these being illustrated in Fig. 3. As the tube is turned by a synchronous rriotor iLt 60 rev./min. a loopful of inoculuin is carried past the gassing probe t o the bottom of the tube. The loop is held gently AGAR MEDIUM i
INOCULATING
-
1 .
FIG.:1. Appnrnt 11st m d tochniciuo for streaking a roll tubo in tho Himgate techriiclue.
against the upper agar surface and is drawn slowly toward the mouth of t,hc tube. \\‘hen t,his has been accomplished, the rubber stopper, which has been pli~cedin i~ dust-free environment, is picked up by the largeend, is flitined briefly, and is replaced in the opening of the tube beside the gassing probe. Gas is allowed to escape for 15-20 sec. before the needle is withdrawn in one smooth motion as the sterile rubber stopper is placed firmly in the tnouth of the tube. ‘l‘he end of the tube with the rubber stopper is passed briefly through the flame and the rubber stopper is seated firmly by a twisting motion. The streaked roll tube is then incubated i n i ~ upright n position. As shown in Pig. 4, this technique has the advantage that isolated colonies occur near the mouth of the tube as the inoculurn has been diluted during streaking. The Hungate technique is beginning to attract the attention whicli it deserves as the iriost stringent of anaerobic techniques. For modifications
MICROBIAL FORMATION OF METHANE
113
of the technique, the reports by Hungate (1950), Bryant and Robinson (1961), and the Outline of Clinical Methods in Anaerobic Bacteriology should be consulted. The last source is a mimeographed, loose-leaf collection of techniques, procedures, and helpful descriptions for efficient, routine use of the Hungate method, and is prepared by the Anaerobe Laboratory, Virginia Polytechnic Institute, Blacksburg, Virginia, U.S.A. Hungate himself prefers t o use sterile hypodermic syringes in an aseptic manner for addition of inocula or removal of samples. With this
BLACK RUBBER BUNG H2: CO2
COLON I ES AGAR MEDIUM
WATER CONDENSATE FIG.4. Appearance of a streaked Hungate roll-tube after incubation. Note more isolated colonies are near the mouth of the tube.
modification the syringe needle is passed through the rubber stopper, the stopper not being removed from the tube. Bryant prefers t o sterilize the medium in a round-bottom flask with the stopper wired in place. Sterile, cooled, anaerobic medium is aseptically dispensed into sterile tubes. The procedure outlined in Figs. 2 , 3 , and 4 represents a simplified variation of the technique. In the isolation and cultivation of methane bacteria this technique is especially convenient; samples of the gas atmosphere confined within the tube may be obtained easily by aseptic insertion of a sterile syringe needle through the rubber stopper and by removal of a sample of the gas atmosphere for analysis.
114
R. S. WOLFE
The Hungate technique is easiest to perfect when 100% carbon dioxide or CO, + H, (!N: I ) iwe used as the gas atmosphere. Since c w h i l dioxide is Iieavier than air, replac+ementjof the rubber stopl'er without trapping air is ti less critical manoeuvrc. The technique is extremely difficult to use with 100% hydrogen since the rush of hydrogen out of the tube draws air currents into the tube. Mixtures of CO, + H, (50:50) present little difficulty, and with a little practice 80:20 mixtures are readily handled with success. For cultures which do not require hydrogen, argon (which has a density about that of air) is a convenient gas to use. Stock cultures of methane bacteria are readily maintained by the Hungate technique. Agar deeps or slants are inoculated by stabbing, and after incubation of the slant or stab, the tube is placed at -70".
111. Characteristics of Methane Bacteria A. MORPHOLOGICAL TYPES The various morphological types of bacteria are represented by methane bacteria which now are in pure culture. Jn recent years Paul H. Smith has pursued the isolation of methane bacteria from sewage sludge (Smith, 1966).Phase-contrast photomicrographs of his isolates illustrate the morphological types of methane bacteria. The coccus morphology is represented by a Methanococcus species shown in Fig. 5 ; a COCCUS in chains by Methanohacterium ruminantium, Pig. 6 ; a similar coccus but with smaller cells, Fig. 7 ; a sarcina by Methanosarcina barkeri, Pig. 8 ; rods by Methanobacterium formicicum, Fig. 10 ; a M . formicicum-type, Fig. 9 ; and Methanohacterium M.o.H., Fig. 11; a long-wave spirilluin by Methanospirillurn species, Fig. 12. The spirillum morphology of the last isolate is more apparent in Fig. 13, and the polar tufts of flagella are shown in the flagella stain presented in Fig. 14. Langenberg et al. (1968) found that organisms of the M . formicicum-type possess intracytoplasmic bodies close t o the cell membrane which are distinguished easily when cells are stained with phosphotungstic acid and examined by electron microscopy (Fig. 15). The structure and function of these bodies are unknown.
FIG.6. Phase-contrast photo~nicrographof a species of Mrthanococcus. fig^. 5-1 4 wore kindly mppliod by P. H. Smith, Univ. of Florida. h i . 6. Methanohacterium ruminantiuni. FIG.7 . Methanobacterium rwndnantium. Cells of this isolato aro about ono-hnlf the size of other strains. FIG.8. Methanosarcina barkeri.
MICROBIAL FORMATION OF METHANE
115
116 R . S. WOLFE
MICROBIAL FORMATIOX OF METHANE
117
118
R . S. \VOLPE
One of the most exciting recent events in the isolation of methane bacteria has been the re-isolation of Methanococcus vanneillii (Stadtman and Barker, 1951) by T. Stadtman (personal communication). This organism was presumed lost, since it could not be re-isolated from San Francisco Bay mud or from many other portions of the country. However, from a tube of packed, frozen cells, which had been stored in the deep freeze for over 12 years, viable cells were obtained by Stadtman. This organism is believed t o be one of the fastest-growing methane bacteria and is the only motile coccus so far isolated. Another example of motility in the methane bacteria is a motile rod, Methanobacterium, mobilis, isolated by Paynter and Hungate (1968) from the bovine rumen. Among the methane bacteria motility is not common and when observed is likely t o be sluggish.
B. SPECIES AND THEIR PROPERTIES Table 1 lists the methane bacteria which now are in pure culture as well as their habitat, morphology, nutrition, and substrates. With the exception of M . barkerii it is striking that the preferred substrates are either hydrogen and carbon dioxide or formate, the last compound being converted readily to hydrogen and carbon dioxide. Organisms which use propionate, butyrate, or higher fatty acids and alcohols are not represented in pure culture a t the present time. It is also striking that ammonium ions are the main source of nitrogen for these organisms. For certain species, an unidentified growth factor from rumen fluid is required (Payntcr and Hungate, 1968; Bryant and Nalbandov, 1S h ) . Acetate is highly stirnulatory for many species, and 2-methylbutyrute is required as a source of branch-chain carbon skeletons for certain species. Minor nutritional differences may be noticed between strains of the same species which have been isolated from different sources such a8 sewage sludge or rumen fluid. A thorough study by Tseng (1970) of the nutrition of Methanobacterium M.0.H. has shown that this organism has a more simple nutrition than previously expected. Carbon dioxide and hydrogen serve as the main carbon and energy sources. Sulphide serves as the source of sulphur. The addition of acetate, cysteine, and B-vitamins stimulate growth ;however, it should be re-emphasized that the organism grows slowly without these compounds in a defined mineral medium on hydrogen and carbon dioxide. These characteristics are those of an autotroph.
C . RESOLUTION OF Methanobacterium orneliunskii Since the culture known as MethanohuciZZus omelianskii has playcd HII important rBle in studies on the methane bacteria, an accaurlt of its
TABLE1. Types of Methane Bacteria in Pure Culture in 1970 Nutritional Requirements Organism .llethanobacterium ruminantium
Source
+
Sludge
coccus to short rod in chains As above
Methanobacillus omelianskii
Irregularly curved rod
Variable
-Methanobacte~i~iT~~ Mud, sludge formicicum J~ethanobncterizLin Rumen WkObdiS
Irregularly curved rod Short rod, motile
Variable
Methanosarcina barkeri
Mud, sludge
Sarcina
Methanococcus vannielii ethanospirillurn SP. Methanococcus sp.
Mud
Xethanobacterium strainM.0.H.
Ruinen
Morphology
Gram reaction
Sludge
Motile coccus Spirillum
Sludge
Coccrls
+
-
+ (?)
+
+
Substrates
Nitrogen
H2+C02 formate
NH3
H2+C02 formate
NH,
+ CO, (Formate not used) H2+ C02 formate H2+C02 formate
NH3
H,+C02
H,
methanol acetate H2+CO2 formate H2+CO2 formate H2+C02 formate
Vitamins
Other
Growth factor from rumen fluid Abovegrowth factor not required ; B vitamins stirnulatory B vitamins stimulatory
Acetate; 2 methylbutyrate Acetate
NH,
(?)
(?I
NH,
(?)
NH3
Growth factor from rumen fluid None
None
NH,
None
hTone
Acetate stimulatory
M
F F
0
120
R. 9. WOLFE
history and final resolution is pertinent here. To test the effect of dilute solutions of ethyl alcohol on microbes, Omelianski (1916) obtained a fermentation in a simple mineral medium which contained 1% ethyl alcohol as the substrate. This medium was inoculated with rabbit faeces from an animal that had been fed about 2% ethyl alcohol. When the gas produced by the fermentation was assayed, it was found t o contain 88% methane, 12y0 carbon dioxide, and traces of hydrogen. The culture contained large numbers of long, slightly curved rods. From an ethyl alcohol, carbonate enrichment, Barker ( I ! W i , 1940) obtained B culture from Delft canal mud which resembled that described by Omelianski ; Barker also isolated similar cultures from San Francisco Bay mud. The name Methanobacterium omelianskii was given to these cultures which formed methane in an ethyl alcohol-carbonate mineral medium. Since many cells in these cultures contained swollen areas which resembled spores, the name Methanobacillus omelianskii was suggested by Barker (1956). Stock cultures of the only culture in existence have been maintained in Barker's laboratory for over 30 years in the ethyl alcohol-carbonate medium. When colonies from this medium were inoculated into a variety of media which contained carbohydrates and organic nitrogen compounds, no growth was observed under aerobic or anaerobic conditions, a n indication that the culture was not contaminated. The carbon dioxide reduction theory of van Niel (Barker, 1956) was confirmed with cultures of M . omelianskii (Stadtman and Barker, 1949) ; the specific activity of the methane produced was equal t o the specific activity of the ''C-carbon dioxide added, when unlabelled ethyl alcohol was used as the substrate. The classical concept of the anaerobic oxidation of ethyl alcohol and the reduction of carbon dioxide by M . omelianskii is shown in Fig. 16. Two molecules of ethyl alcohol are oxidized to acetate,and the eight electrons derived from this oxidation are transferred for the reduction of carbon dioxide to methane. A number of primary and secondary alcohols also were fourid to serve as substrates. Schnellen (1947) found that cell suspensions of M . omelianskii could form methane when provided with an atmosphere of hydrogen and carbon dioxide, and Johns and Barker (1960)showed that cell suspensions oxidized ethyl alcohol to acetate and hydrogen when carbon dioxide was not available. They also observed that a hydrogen atmosphere essentially inhibited the oxidation of ethyl alcohol. Low concentrations of viologen dyes were found by Wolin et al. (1964) to inhibit methane formation by cell suspensions which were provided with ethyl alcohol and carbon dioxide. Under these conditions, hydrogen formation was much greater when methane formation was inhibited, but addition o f viologens did not stimulate hydrogen formation from ethanol in the
MICROBIAL FORMATION OF METHANE
121
absence of carbon dioxide. Thus, it was shown readily in different laboratories that molecular hydrogen was produced as well as used by cell suspensions, and that the equilibrium of the oxidation of ethyl alcohol to acetate and hydrogen by cell suspensions is easily displaced by hydrogen in the atmosphere of the reaction vessel. Experimental evidence as t o the nature of alcohol oxidation and the reduction of carbon dioxide was difficult to obtain with cell suspensions due to their extreme sensitivity t o inhibition by oxygen. The first studies of amino-acid synthesis with this strict anaerobic culture were reported by Knight et al. (1966). These were designed t o answer questions as to how cultures of M . omelianskii synthesized
7
L
8e
,
“Methunobacillw omelianakii”
1
v
amino acids from ethyl alcohol and carbonate, the only carbon compounds in a n otherwise mineral medium. Cultures were grown on 14Ccarbon dioxide and unlabelled ethyl alcohol or [l-“C], or [2-14C]ethyl alcohol and unlabelled carbon dioxide. Results of the degradation of cell protein and analysis of amino acids showed a clear pattern for synthesis. Carbon atoms from both ethyl alcohol and carbon dioxide were utilized for cell carbon, a C, C, condensation being effected for pyruvate synthesis with subsequent formation of aspartate, alanine, glycine, serine, and threonine. With the exception of glutamate and isoleucine, the labelled patterns were consistent with synthetic pathways established for Escherichia coli. I n the summer of 1965 experiments were initiated by M. P. Bryant and R. S. Wolfe to culture 111. omelianskii by the Hungate technique (see p. 110), since observation of living cells by phase microscopy indicated the possible presence of two cell types. A rich medium was used which contained rumen fluid, tryptone, yeast extract, ethyl
+
122
R . 9.WOLFE
alcohol, volatile fatty acids, cysteine-sulphide reducing agents, and mineral salts undcr a Iiydrogcn-carbon dioxide atmosphere. IJpoii serial dilution of the culture in roll tubes of t h i s medium followed by incubation, an isolated colony was picked from a high dilution tube which also yielded a positive assay for methane. When inoculated back into tubes of the same medium, t h e cells grew well and produced methane. However, inocula which were transferred to the ethyl alcohol-carbonate medium of Barker ( 1940) failed to grow. Subsequently, a culture which had incubated several months in the rich medium was tested for viability. The sealed tube exhibited a strong negative pressure, indicating that the gases had been utilized. At this time a careful series of experiments wcre
$'la. 17. Photoinicrogi.aphs of cultures of the two species isolated from Mehatao6 u c i ~ ~ uomeliwtrskii s (phase contrast) (Bryant et al., l W 7 ) . A, tho rnethanogcrnic organism grown for four days in rumen fluid modiinn with 1 : 1 H2-C02 gas phase ; B, recombined culture of t>liemethanogenic 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
THE ADAPTIVE RESPONSES O F ESCEERICEIA COLI
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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
165
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,
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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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ARTHUR L.ROOH
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
184
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
193
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
194
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.
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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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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.
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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,
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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.
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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
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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
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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
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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
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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
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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
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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
BACTERIAL FLAGELLA
236
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
BACTERIAL FLAGELLA
237
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
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-gm
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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-
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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 hlgh
indeed do contain more aspartic and glutamic acids thnii four out, o f f i v ~ thermophile flagellins, but this difference is not consistcnt and cannot, serve as the main feature responsible for the dramatic differences i n stability. Two other lines of research also led to premature conclusions. Iqirst, peptide maps of relatively less stable filaments from cells of Racilltt,~
BACTERIAL FLAQELLA
263
strain X 1 , isolated by others as a mutant of the thermophilic strain 1 0 , were so different from those of relatively heat-stable strain 10 filaments (Friedman and Koffler, 1960; Friedman, 1961) as to make a direct genetic relationship between strains X 1 and 10 most unlikely. Secondly, the earlier claim that filaments isolated from cells of B. coagulans 43p grown a t 55" were more stable than filaments from cells grown a t 26" (Friedman and Koffler, 1960; Friedman, 1961) probably was due to cytoplasmic contaminants in the preparation, since in more extensive work Holowczak (1962) was unable to demonstrate any differences in stability, and Fry (1961) could not find any differences either in aminoacid composition or the nature of the tryptic peptides obtained from the flagellins isolated from cells grown a t these two conditions. In fact, the inability of Holowczak also to show any difference in the heat stability of crude cytoplasmic isolates from cells grown a t the lower and higher ends of their growth temperatures raises some doubt regarding other reports dealing with the relative heat stability of other proteins obtained from suoh cells (Campbell, 1954, 1955). Tho amino-acid composition of flagellins from different strains of Bacillus (five mesophilic ; five thermophilic) offer a t best only indirect clues that might help explain the striking differences in stability. Each flagellin has a distinct composition (Abram and Koffler, 1962 ; D. Abram, M. Farquhar, and H. Koffler, unpublished results) and after tryptic or chymotryptic digestion yields a characteristic peptide map (M. Farquhar and H. Koffler, unpublished results; Abron, 1966). The flagellins of bacilli, as is true for all flagellins so far studied, contain either no or only a few residues of cysteine, tryptophan, tyrosine, proline, and histidine. The only consistent difference between relatively more heat-stable and less heat-stable flagellins from Bacillus strains deals with threonine. The mesophile flagellins were found to contain from 50 t o 65 threonine residues among total residues varying from 878 to 921 in 100,000 g. of protein while thermophile flagellins contain 70 to 1 1 1 out of !I02 t,o 941 total residues. If threonine, as well as lysine and arginine because of the opportunity for hydrophobic interactions that their relatively long hydrocarbon portions provide, are included among hydrophobic residues, mesophile flagellins are found to contain from 487 to 501 for 100,000 g., while thermophile flagellins contain from 512 to 554. Possibly, the relatively greater abundance of threonine or hydrophobic residues in general (selected on debatable criteria) offers greater opportunities for interactions of such residues in intramolecular stabilization. Bigelow ( 1 967) has suggested that the relative hydrophobic natures of protein molecules can be calculated from the number of nonpolar arnino acid residues and the relative hydrophobic natures of each amino acid as determined by the free energy of transfer from ethanolic
264
R . W. SMITH AND HENRY KOFFLER
t o aqueous solutions. Based on these premises, we obtained values of 821 t o 869 and 841 to 925 for the meeophile and thermophile flagellins, respectively. Since the ranges of these values overlap, the relative hydrophobic natures alone cannot explain the striking differences in stability observed, and perhaps the frequency with which relevant residues have the opportunity to react (i.e. depending on their number and location) is a more critical parameter. Of course, one should not be carried away by enthusiasm for seeking correlations since any of them may be fortuitous. As mentioned, each peptide map obtained for the mesophile and thermophile flagellin is unique (Abron, 1966; M. Farquhar and H . Koffler, unpublished), with the exception of a set of peptides that appears to behave similarly though not identically in the various flagellins. No common feature reflected by two-dimensional paper chromatography and electrophoresis (i.e. charge, solubility) can be discerned to hold true for the mesophile or thermophile flagellins as groups of proteins. So far, the data are consistent with the belief that the relative stability of a given flagellin resides in its unique sequence of amino acids. While it is plausible that all flagellins eventually will be found to share certain structural aspects, these have so far eluded observations. In the polymer, flagellin assumes a globular or ovoid shape, as observed electron microscopically (Kerridge et al., 1962 ; Abram and Kotller, 1963a; Abram et al., 1964a, 1966), but there is some indication that in solution the molecule is elongate. For example, Weibull (1948) determined the diffusion constant and frictional coefficient for P. vulgaris flagellin to be 5.2 and 1.8 respectively; the latter figure suggests an axial ratio of 1: 15for the subunit. J. Stenesh and H. Koffler (unpublished results) found the frictional coefficient for flagellins from several different strains of BacilZw to vary from 1.8 t o 2.9, and the axial ratio of a typical flagellin with an unhydrated particle molecular weight of 40,000 t o be approximately 1 :20. Since calculations of frictional coefficients depend upon a variety of corrections (for example, allowances for differences in hydration) and slight variations in the value of the coefficient greatly affect the axial ratio, the parameters regarding shape a t best represent only approximations. Clearly, however, flagellin is capable of undergoing conformational change. This was first demonstrated by Erlander et al. (l960), who, using spectropolarimetric techniques, observed a reversible coilhelix transition between p H 2.0 and 3.8. Further studies by Yaguchi et aZ. (1964) confirmed the fact that flagellins from mesophilic strains of Bacillus also undergo this coil-helix transition between p H 2 and 4 ; above pH 4 and up to 11 the rotatory dispersion properties remain essentially intact (Klein eta?., 1967,1967a, 1968).Since the most common
BACTERIAL FLAGELLA
265
procedure for obtaining purified monomeric flagellin solutions is acid treatment below pH 4 (usually pH 2 to 3) followed by high-speed centrifugation to remove acid-insoluble materials, it is fortunate that flagellin does not appear to be damaged by this treatment, since the conformationa1changes are completely reversible, and flagellin prepared in this manner is capable of assembling into normal looking filaments, as will be described later. From optical rotatory dispersion properties of Bacillus flagellin, the presence of a-helix a t pH 4 and above can be judged by the position of the trough and cross-over points a t 233 nm. respectively; the apparent helix content varies from 17 t o 34%. As the pH is lowered towards 2, the mesophile flagellins and flagellin from a strain of Bacillus with an intermediate maximum growth temperature undergo conformational transitions, and their apparent helix content drops to the range of 8 to 13%; the position of the trough is preserved while the cross-over points shift towards the far ultraviolet range, suggesting a partial loss in a-helix and a gain in random structure. The structure of flagellins from thermophile strains of Bacillus is much more stable, since they do not undergo this hydrogen ion-dependent helix-coil transition and the spectropolarimetric properties remain constant over a pH range of 2 to 11. Of course, other architectural features than the a-helix not detectable by measurements of optical rotatory dispersion properties may play significant roles in the structure of these molecules. Yarbrough et al. (1969) have obtained preliminary evidence by difference spectroscopy and solvent perturbation of tyrosine residues that flagellin of the thermophile B. stearothermophiilus 2 184 does undergo pH-dependent conformational changes between pH 2 and 4.8 which, however, are not observable when polarimetric techniques are used. This flagellin has a molecular weight of about 50,000, and contains six tyrosine residues and one tryptophan residue per molecule. The tyrosine residues were titrated spectrophotometrically. When this flagellin is unfolded in 6 M guanidine hydrochloride, all six tyrosine residues behave normally with an apparent pK of about 10. I n the absence of guanidine hydrochloride titration data suggest a t least three classes of tyrosine residues. Four can be titrated normally, one less rapidly, and the sixth not at all; the existence of such a buried tyrosine residue was confirmed by a comparison of spectra of this flagellin with those of model compounds. That this thermophile flagellin, which shows no conformational change under acid conditions as observable by optical rotatory dispersion techniques, must undergo structural changes was shown by studying pH-induced difference spectra and selective modification of flagellin with tetranitromethane (TNM), a compound that reacts only with exposed tyrosine groups. The first approach showed a typical red shift due t o the burial of one to two tyrosine residues as the pH was raised from 2 to 4.8; this
206
R. W. SMITH AND HENRY KOFFLER
indicates some unfolding a t pH 2 and some folding as the pH is increased. I n the second approach, treatment of flagellin with TNM a t p H X.5 results in the reaction of three tyrosine residues after 2 hr. At pH 10, four residues react after 1 hr.; a fifth reacts after a longer period of incubation. At pH 11.5, the fifth tyrosine residue appears to react faster than a t pH 10. This not only confirms the existence of three, probably four, classes of tyrosine residues, but suggests conformational changes as the pH is increased. Reversible helix-coil transitions, as determined by optical rotatory dispersion properties, can also be brought about by changes in temperature. Flagellins from mesophilic strains of Bncillus have midpoints ( T ,values) for such a transition near 45";flagellin from strain 194 with an intermediate tnaximum temperature has a T,value of 50" ; the therrnophile flagelliiis have values of 55" to 57". As will be discussed below, the midpoint of the temperature range during which flagellar filaments disintegrate under standardized conditions ( T, values) varies from 54" to G l " , 69", and 72" to 77" for flagella from mesophilic, intermediate, and thermophilic strains of Bacillus, respectively. Thus, the stability of flagellar filaments to heat is reflected by the relative structural stability of the protein subunits; however, no direct relationships can be found between the amount of u-helix above p H 4 and the degrec of heat stability. Flagellin-flagellin interactions appear to stabilize the structure of the filament further since thermal disintegration of the filament occurs 9-20" higher than the thermal destruction of the intramolecular helicul structure of flagellins in solution. The relative heat stability of filaments from therrnophilic organisms is also reflected by a greater stability to other disruptive agents. Urea ( 6 M ) , acetamide (10 M ) , sodium dodecyl sulphate (1.0 mg./ml.), or acid destroy filaments of mesophiles, but during similar periods of exposure have no effect on the structure of filaments from thermophiles as determined by viscosity measurements and electron microscopy (Mallett and Koffler, 1955, 1957; Mallett, 1956; Koffler et nl., 1957; Stenesh and Koffler, 1962 and unpublished results). Thermophilc filaments arc disintegrated if the concentrations are increased to 9 M (urea) and 2.5 mg/ ml. (dodecyl sulphate). Thioglycollate (0.2 M ) a t pH 8 in 5 M-urea does not affect the stability of therniophile filaments. Since this compound is capable of breaking disulphide bonds, this confirms that such bonds, as previously mentioned, are not likely involved in flagellin-flagellin interactions. Martinez and ltosenberg ( 1 9G4) followed the heat-induced disintegration of filaments of Spirillum serpens by the decrease in light scattering a t 250 nm. The optical density and temperature were recorded at B-see. intervals as the temperature was raised a t a rate of 0+5"/min.A tran.qition
BACTERIAL FLAGELLA
267
zone is seen as the temperature is increased. As a reference, the midpoint of this transition is considered to be the disintegration temperature (Td). Various salts and pH values were found to have a pronounced effect on the thermal stability of flagellar filaments. The Td increases with phosphate concentration from 46" a t 0.001 ilf to 51.5" a t 0.8 41. I n addition, the transition zone is broadened bytheadditionof phosphate. The T, is lowered to 42.5" by magnesium chloride (0.1 M ) and 38" by calcium chloride (0.1 M ) . At 0.1 M , sodium or potassium chloride has no effect. The reason for the differential effects of calcium, magnesium, sodium, potassium, and phosphate is not known. If hydrophobic bonds are involved in intermolecular attractions, salts should stabilize the filament. Sodium chloride (0.2 M ) , for example, protects purified filaments of B. purnilus from breaking during freezing and thawing (R. W. Smith and H. Koffler, unpublished results). More likely, however, interactions between subunits involve both hydrophobic and ionic bonds, and the effect of salts on the T , values of filaments from a given organism reflects the net effect, i.e. stabilization of hydrophobic bonds and destabilization of ionic bonds. The hydrogen-ion concentration has a great effect on T, values (Martinez and Rosenberg, 1964). The T, of filaments of 8. serpens decreases linearly with the pH value down to pH 5.6 below which the decrease is described as being precipitous. This destabilization of the structure of the filament may be due to conformational changes in the flagellin subunits. D. Klein, J. F. Foster, and H. Koffler (unpublished results) find that in certain pH ranges the position, size, and shape of thermal transition curves obtained from optical rotatory dispersion measurements of flagellin from various organisms depend on the pH. The value of the transitional midpoint of each curve decreases rapidly below about pH 4.5 in the case of flagellin from P. vulgaris and pH 4.0 for flagellins from B. pumilus and B. licheniformis. Many early investigators observed that flagellar filaments are disintegrated by acid. Duncan (1935) observed that the H-agglutinability of cells is destroyed by 7.8 x lop4 M-HCl. Addition of dilute acid was shown to disintegrate the structure of the filament (Weibull and Tiselius, 1945). Upon acid disintegration a t pH 3 to 4,filaments lose their characteristic viscosity and flow birefringence with no change in chemical composition (Weibull, 1948, 1949, 1950). Early workers also noted other types of phenomena that indicate disintegration of filaments by acid. Acriflavine, as well as most acridines, agglutinate motile cells to varying degrees depending on the phase and antigenic nature of the flagellin being produced (Sertic and Boulgakov, l ! M , 1930a ; Bernstcin and Lederberg, 1955). Treatment with acid a t pH 4 destroys t h e ability of a culture to undergo thisagglutination, apparently due to the disintegration
208
R. W . SMTTII AND HENRY ROFFLER
of the filaments. The above observations served as early evidence that the polymer disintegrated to smaller units. I n addition t o being more stsableto heat, flagellar filaments from tfhermophile organisms are also much more stable in 0 - ~ ) 0 M-HCl 1 than are corresponding isolates from mesophiles (Stenesh and Koffler, 1962). As the pH value of a viscous suspension of filaments is lowered, the viscosity greatly increases over the pH range 3.6 to 4.0 prior to complete solubilization (Mallett,
FIG.14. Filaments of k i U C d h 8 pumlilus flagella treated with 00(;/,ethyl alcohol, lW4 N-HCL, 4",for 9 hr. After centrifugutiori at 20,000 x y. for 1 hr.,thrpollrt was resuspended in distilled wcttor. Negatively stained with phosphotungstttte. x 117,000. *J. It. Mitchnn and H. Kofflcr, iiripiiblishcd ohscrvations.
1956). In electron micrographs of filaments treated in a comparable manner, fine fibres are observed with t>heshape 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.
BACTERIAL FLAGELLA
273
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,
BACTERIAL FLAGELLA
279
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. Burg<. ( I !)ti 1 ) constructed models for filarneiits of Proteus wulqnris hased on available X-ray diffraction data. Observations were consistent with tilainelits being composed of a number of identical interwoven strands in Iiexagoiial or pseudo-hexagonal packing. Two models were considered. The iirst consists of three strands, each being 56 A in diarncxter, and containing l!) a-helical subfibres. The alternate model is composed of scveii strands, cacIi 33 A in diameter, which are composed of scveii subfibrrs. X-Ray cliffrac-tion data correlated better with the sc:contl model altlioiigh neither could bc eliininated. I n sectioned material, filaments of P. viclpris appear t o consist of three I)arallel or loosely coiltd strands ( Forsliiid and Swanbcrk, 1!)63). X-ltity diffrwtion data, further suggested t o the authors t h a t the filament consisted of three helically wral)]icd strands (Swanbeck aiid Forslind, 1! I N ) . I>ue 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
BACTERIAL FLAGELLA
283
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.
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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
11. \\
. 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
BACTERIAL FLAQELLA
295
slight differences between native and reconstituted filaments is that completely carboxymethylated flagellin absorbs onto re-assembled but not native filaments (R.W. Smith and H. Koffler, unpublishedresults).
XI. Synthesis of the Filament Asakura et al. (1964) calculated that, if cells of Salmonella sp. accumulated all the flagellin inside the cell necessary for filament synthesis prior to polymerization, the intracellular concentration would be 5 mg./ml. Of course, much higher concentrations may be feasible if filaments are assembled in specialized structures such as those proposed by Iino and Mitani (1962)and Iino and Lederberg (1964). Polymerization of presynthesized and accumulated flagellin would appear to be necessary to explain the observation of Jacherts (1960) that synchronized cultures of Pseudomonas aeruginosa form a new polar filament within 30 sec. after cell division. Most measurements, in other systems, however, indicate a somewhat slower rate of formation. For example, Stocker and Campbell (1959) report that at 37" filaments of S. typhirnurium flagella elongate a t arateof 0.12 to 0.14 pm./min. ; at 28" the rate was 0.085 pm./min. A rate of 0.3 to 0.5 pm./min. has been reported for several organisms (Leifson, 1931 ; Murray and BirchAnderson, 1963). Cells of Spirillum serpens (Martinez and Rosenberg, 1964) and Escherichia coli (Quesnel, 1966) can generate functional filaments in 10-20 min. After deflagellation by homogenization in a blender, 30% of the cells of a culture of E . coli form new flagella within 30 min. (Novotny et al., 1969). Although some organisms may form filaments predominantly from accumulated flagellin, the rate of filament elongation appears to be of a magnitude that would permit simultaneous synthesis of the subunits. The presence of intracellular flagellin has been demonstrated in several organisms though it may not occur in all species. Internal flagellin was detected in sphaeroplasts of Proteus vulgaris (Weinstein, 1959; Weinstein et al., 1960). The amount varied from 0-02 to 0.07% of the total cell dry weight depending on the growth phase, and appeared to be largest during the late log phase of growth. McGroarty et al. (1970) found that cells ofP. vulgaris grown a t 37" contain 0-02to 0.04% of their dry weight as internal flagellin as determined by radial immunodiffusion techniques. The concentration of internal flagellin in Bacillus subtilis strains 168 and 23 has been reported to be between 0.009 and 0.033% of the dry weight (Fisher, 1963; Nasser and Koffler, 1963; Nasser, 1964). Intracellular flagellin was also detected immunologically in spheroplasts of E . coli (Vaituzis and Doetsch, 1966) and in cells of 12
296
R. W. SMITH AND HENRY ROFFLER
Spirillum serpens in which formation of flagellar filaments was inhibited by growth at 46" (Martinez and Gordee, 1966). Objections to these techniques for demonstrating the presence of internal flagellin are based on the possibility that all extracellular filaments may not have been removed prior to the disruption of the cells. This criticism was answered in the studies of Weinstein (1959) and Nasser (1964). Addition of either isolated flagella (Weinstein, 1959) or lysed sphaeroplasts carrying flagella (Nasser, 1964) to the test sample prior t o the determination did not appreciably increase the amount of intracellular flagellin detected. To check recovery, flagellin was added to sphaeroplasts prior to lysis, and it was found that about 80% of that added could be detected following the procedure used to measure intracellular flagellin (Weinstein, 1959). Nasser (1964) added a known amount of flagellin to sphaeroplasts of a mutant strain of B. subtilis that is incapable of synthesizing flagellin and also found that 80 to 100%of the added flagellin could be recovered. A functional pool of flagellin was reported in B . subtilis and S. serpens T46 (Martinez and Gordee, 1966). Deflagellated cells were able to regenerate flagellar filaments in the presence of concentrations of chloramphenicol that inhibited the incorporation of I4C-aminoacids into flagellin to the extent of 66 to 98%. I n one experiment, log-phase cells were grown for several hours in the presence of I4C-valineand 3H-lysine. After removal of flagellar filaments, the cells were placed in a growth medium containing 3H-lysine.The culture was divided into two portions, one of which received chloramphenicol. At various times of incubation, cells were removed and the 14C?H ratio in the flagellar filaments determined. If the synthesis and subsequent polymerization of flagellin continued, the ratio of 14C:3Hshould decrease; this pattern was seen in cells incubated in the absence of chloramphenicol. Although an initial decrease in this ratio was observed in the presence of chloramphenicol, the relative amounts of I4C and 3H in the filaments became stabilized after about 30 min. Thus, at least after 30 min., chloramphenicol appears to have stopped flagellin synthesis, and the filaments formed after this time were thought to have arisen from the polymerization of pre-existing intracellular flagellin. In another experiment, cells of S. serpens T46, which do not form flagellar filaments when incubated a t 45O, were placed in a growth medium containing 14C-lysineand maintained a t 45" for 3 hr. The amount of radioactivity in intracellular flagellin was then determined in a portion of the cells. The remainder of the cells were incubated a t 30" in the presence of chloramphenicol, conditions under which cells form flagellar filaments. After a period of time the amounts of radioactivity both in the intracellular flagellin and in the filament were determined. Only about 12% of the radioactivity that was in intracellular flagellin prior to incubation at 30"remained in that fraction. The decrease
BACTERIAL FLAGELLA
297
in radioactivity inside the cell was accompanied by an increase in radioactivity in the newly formed flagellar filaments. Thus, during incubation a t 45", the cells apparently synthesized flagellin, which they were unable to polymerize, and maintained it in the form of an intracellular pool. Unfortunately, it is difficult to interpret these data in terms of a "normal" cell in which filament formation is not inhibited. Another complicating factor is that treatment with chloramphenicol alone does not completely inhibit the synthesis of flagellin. More work needs to be done before the existence of a functional pool of flagellin in normal exponentially growing cells can be proven. Data indicate the absence of an intracellular pool of flagellin insalmonella typhimurium (Kerridge, 1959, 1963 ; Aamodt and Eisenstadt, 1967 ; McClatchy and Rickenberg, 1967). Kerridge (1963) observed that there was no significant lag period between the time 14C-aminoacids were added to a growing culture and the appearance of radioactivity in flagellar filaments and concluded that there was no intracellular flagellin pool. The inability of several organisms to form filaments in non-nutritive media also suggests that little or no pool exists (Roberts and Doetsch, 1966). Dimmitt et al. (1968) observed that the formation of filaments of B. subtilis is inhibited immediately by puromycin or chloramphenicol. They suggested that flagellin is polymerized into the filament immediately after synthesis and that a pool does not exist. Similar conclusions have been reached by Joys (1968). The above determinations of pool size indicate that the intracellular flagellin may amount to only 1% of the total extracellular flagellin. I n all likelihood, this small amount would not be sufficient to form a significant number, if any, of functional filaments on a deflagellated cell. It may also be argued that such an amount could have resulted from the solubilization of flagellar filaments. Other points that should be considered are that rapidly dividing cells probably do not have a significant pool of flagellin or any other protein and that the appearance of filaments is not necessarily an accurate measure of flagellin synthesis. Obviously, the question concerning the presence and function of an internal pool of flagellin is not yet settled. Early reports noted that cultures of many species are less motile or even non-motile after incubation a t the high end of their particular range of growth temperatures than at more intermediate ones (Jordan et al., 1934; Weitzenberg, 1935; Boquet, 1937; Paterson, 1939). Griffin and Robbins (1944) observed that cells of Listeria monocytogenes have proprogressively fewer flagella as the temperature is increased t o 37". Preston and Maitland (1952) found that, within 24 hr. after transfer of a culture from 22" to 37", the cells become non-motile due to the loss of flagella, which are formed again if the culture is returned to 22". Similar
298
R. W. SMI'TFI AND HENRY KOFFLER
results were reported by Bisset and Pease (1957). Bacillus megaterium KM is non-flagellated when grown at temperatures greater than 30" (Vennes and Gerhardt, 1959). Formation of flagella by E . coli is inhibited above 40" (Vaituzis and Doetsch, 1966; Adler and Templeton, 1967). Formation of flagellar filaments appears to stop immediately after transfer of a culture of P. vulgaris from 37" to 43-44' (McGroarty et al., 1970). After transfer to the higher temperature, cell division continues and existing flagella continue t o function. If a culture maintained a t 43-44' long enough to cause essentially all cells to be non-flagellated is returned to 37', approximately two t o three generation times are required before flagellar filaments can be seen. Of course,.this does not indicate when flagellin synthesis commences, or, indeed, even if synthesis is prevented a t 43-44'. We have observed, however, that the intracellular level of flagellin decreases from 0.02 to 0.04% of the dry weight of cells grown a t 37" to undetectable levels (i.e. less than 0.004%) in cells grown at 4344'. It seems likely that in P . vulgaris the synthesis of flagellin is inhibited a t 43-44' though more rapid turnover cannot be ruled out. It should also be noted that cell-wall damage apparently occurs a t 43-44' since enlarged, bulbous cells are formed. These aberrant forms do not occur when cells are grown a t 42-43', although inhibition of flagellin synthesis and formation of flagella occur also at these temperatures. Intracellular flagellin appears in cells transferred from 42-43" t o 37" after about 45 min. and after 80 min. in cells transferred from 43-44". Regeneration of flagella after transfer of a culture from 42-43' to 37' requires only about 45-50 min. compared to 120-150 min. required for cells grown a t 43-44". The longer time that 43-44' cells require may be due to the necessity of synthesizing cell-wall material before flagellin can be polymerized. I n Salmonella, both the synthesis and polymerization of flagellin can still occur a t 44', since deflagellated cells regenerate flagella a t that temperature (Kerridge, 1960, 1961). Also, cells caused t o be nonflagellated by growth in the presence of lithium chloride or phenol arc able t o regenerate flagella when placed a t 44" (Mitani, 1963). Eventually, however, the formation of new flagella is prevented and cells gradually become non-motile as the number of flagella per cell is decreased by cell division (Quadling and Stocker, 1966, 1962; Kerridge, 1960, 1961 ; Martinez and Gordee, 1966). Using a sensitive microcomplement fixation test, L. W. Aamodt and J. M. Eisenstadt (personal communication) have demonstrated that flagellin synthesis and polymerization continue for one generation then stop after transfer of a culture of Salmonella typhimurium SL 282 tryfrom 37' t o 44".Upon return of the culture t o 37", synthesis resumes after a lag period. Although tryptophan is not required for regeneration of filaments in a culture maintained at 37') it is required by a culture
BACTERIAL FLAGELLA
299
pre-incubated at 44". Apparently, the synthesis of a tryptophan-containing protein involved in the formation of flagella is inhibited at 44", but is prerequisite to the appearance of filaments after transfer to 37". This protein may be involved in the structure or regulation of synthesis of a special structure required for the formation of filaments. Inhibition of flagellin synthesis brought about by incubation a t 44" is prevented by addition of chloramphenicol (L. W. Aamodt and J. M. Eisenstadt, personal communication). Flagellin is synthesized at a faster rate after return of the cells to 37" if the incubation at 44" is performed in the presence of chloramphenicol. Apparently, inhibition of flagellin synthesis a t 44" requires the synthesis of protein. Although the function of this protein is not known, it is assumed to be active in a repression mechanism similar to that proposed for other proteins. The state of the cell wall and membrane may also affect flagellin synthesis and polymerization. While sphaeroplasts may be able to synthesize flagellin as well as normal cells, polymerization of flagellin may be inhibited (Suzuki and Iino, 1966a). Vaituzis and Doetsch (1965) suggested that the mucopeptide portion of the cell wall is required for subunit polymerization and later observed that the synthesis of flagellin appears to be inhibited in sphaeroplasts (Vaituzis and Doetsch, 1966). They suggested that the flagellin-synthesizing machinery is located in the cell membrane and is inactivated by membrane distortion. I n glycineinduced sphaeroplasts of Salmonella typhi (Sparkes et al., 1966) and Escherichin coli (Diena et al., 1968), the amount>of H antigen per cell is decreased to approximately 20% of that found in intact, normal cells. The intracellular flagellin isolated from sphaeroplnsts had a mobility in immunoelectrophoresis intermediate between the normal monomer and the completely polymerized filament. The authors suggested that in sphaeroplasts the subunits may exist in some intermediate stage of polymerization. This could easily be an artifact of the technique used for inducing the formation of sphaeroplasts. As mentioned previously, salts cause monomers to aggregate. It seems likely that ionic conditions inside the cell and/or the 1.5% glycine used for induction would preclude flagellin from existing as a monomer. Lominski and Lendrum (1942) noticed that antiseptics, chloral hydrate and copper sulphate inhibit the formation of filaments. Calcium or aluminium ions were observed t o suppress motility in Axotohncter (Tschapek and Garbosky, 1951). Weinberg and Brooks (1963, 1963a) report that 3 x lop4 M-ferrous iron or aluminium ions inhibit the formation of filaments in B. subtilis and B. megaterium. No noticeable effect is elicited by boron, magnesium, calcium, strontium, vanadium, molybdenum, cobalt, nickel, copper, zinc, or selenium. An interesting observation is that 10% of the cells of B. subtilis grown in the presence of
300
R. W. SMITH AND HENRY KOFFLER
6x M-aluminium or manganous ions are flagellated but possess filaments ten times the normal length. The reason for this phenomenon is not known. As will be discussed later, control mechanisms may operate either at the level of the synthesis of flagellin or a t the site of polymerization. Aluminium or manganous ions inhibit the formation of flagella in most (90%) of the cells in a culture; however, these ions apparently release the control that determines the length of the filaments, i.e. a t the level of polymerization, in the remaining cells. Sokolski and Stapert (1963) noted that either ferrous or ferric ions inhibit the formation of filaments in P . vulgaris, P . mirabilis, and B. cereus. Metals also inhibit motility in E . coli (Adler and Templeton, 1967). Glucose was observed to inhibit the synthesis of flagella. Sistrom and Nemser (1962) suggest that molecular oxygen may inhibit polymerization of subunits in the photosynthetic organism Rhodopseudomonm spheroides. These last observations may be explained by a n insufficient synthesis of ATP or by localized changes in the oxidation-reduction potential due t o the accumulation of oxidized cofactors (NADP) in regions where flagellin is synthesized and/or polymerized. The genetic control of synthesis has been studied in several laboratories. Both the states of flagellation and motility can be transformed by DNA. Manninger and Nogradi (1948) isolated motile encapsulated cells from culture of 23. anthracis mot- cap- that had been treated with a cell-free filtrate of B. mesentericus Flu+ cup+. Bacillus anthracis Fla- motwas transformed t o Flu+ mot+ (Tomcsik, 1950). These experiments, however, did not show conclusively that DNA was responsible for the change to flagellation. Brown et al. (1955) transformed non-motile mutants of B. anthracis to motility and found that the transforming ability was destroyed by deoxyribonuclease. As will be shown later the mot and Fla loci are genetically separable. I n this last report, it was not demonstrated that the recipient genotype was Fla- mot- instead of Flu+ mot-. Nasser and Koffler (1962) and Nasser (1964) demonstrated the transformation of B. subtilis PB-1 (a Fla- mot- try- mutant obtained from strains 168, which is Fla+ mot+ try-) to Fla+ mot+ with DNA from strain 23 (Fla+ mot+ try+).This is shown in Table 5 . The transformation is abolished by deoxyribonuclease but not by ribonuclease. Fla- DNA is incapable of enabling non-flagellated recipient cells to synthesize flagella (see Table 6). That this was not due to inactivation of the DNA in handling was shown by the fact that this preparation still possessed the ability to transform try- t o try+. Not only are the recipient cells incapable of forming flagellar filaments, but they also are unable t o synthesize flagellin as analysed by immunologic techniques (Nasser and Koffler, 1963; Nasser, 1964). DNA from Fla+ mot+ cells restores both the ability t o synthesize flagellin and t o form flagellar filaments. Stocker (1963) also transformed Fla- try-
30 1
BACTERIAL FLAGELLA
TABLE 5. Transformation for motility. 23(Ph-mol-try+)-X ~~~~~
Final dilution 10-2 10-3 10-4 10-5
PB-l(PZa-mt-try-)
~
Cells
+ DNA
32/32 95/96 47/88 7/96
Cells
+ DNase
treated with D N A 0130 31100 31100 31100
Cells 1/32 41100 0/100 2/96
Donor DNA isolated by the methods,of Marmur (1961)or Anagnostopolos and Spizizen (1961) from cells of Bacillus subtdis 23(Ph-mot-try+) wm used at a level of 8 pg./ml. The recipient cells were cultures of the non-flagellated mutant PB-l(FZa-mot-try-) obtained by treatment of strain 168 with ultraviolet radiation. The regimen for inducing competency waa essentially as described by Anagnostopolous and Spizizen (1961). The media used in this and similar experiments were as described by Spizizen (1958).DNase waa used at 50 pg./ml., MgS04 at 5 pM./ml., and the total volume of the reaction mixtures was 1.5 ml. After exposure of the recipient cells, under agitation, to DNA for 90 min. at 37O, the DNA was inactivated by the addition of DNase and Mg2+.The cell suspension was then diluted in Spizizen’s minimal medium (Spizizen, 1958)so that 0.01 ml. was expected to contain one or more motility transformants. The tubes containing the organisms to be plated were held in ice during plating; otherwise the viable count dropped. Five to seven millilitres of a semi-solid medium (agar, 5; gelatin, 10; and Difco Antibiotic Medium No. 3 in g./l,) on top of 15 to 20 ml. Difco Antibiotic Medium No. 2 served to score for motility. Any macrocolony that appeared to contain motile cells, indicated by a diffuse area of cells surrounding or leading away from the colony proper, was assumed to contain at least one transformant. The figures given are the number of motile macrocolonies t o the total number of macrocolonies scored. DNase destroys transforming ability, but RNase treatment does not (i.e. values are comparable to those in second column). Similar results were obtained when other non-flagellated recipient cells were used (PB-2 to PB-4, obtained from strain 188 by ultraviolet treatment; Pl3-5by treatment in the linear accelerator, and PB-6 by irradiation with X-rays). Fordetailssee Nasser (1964)and Fisher (1963).
TABLE6. Trttnsforniation for motility with D N A from non-flagellated cells. PB-ITA1 (FZa-mt-tryt)-X PB-1 (Flu-mot-try-)
Final dilution 10-2 10-3 10-4 10-5
Cells
+ DNA
0120 4/ 100 21100 0/100
+
Cells DNase treated DNA 1/20
opoo 1/100
opoo
Cells 0120 31100 o/ 100 o/ 100
The figures given are the number of macrocolonies that contained motile cells t o the total number of macrocolonies scored. DNA isolated from cells of Bacillw, aubtilk strain PB-TA1 (a transformant of strain PB-1 obtained by 23-X PB-1)was used at a level of 16 g./ml.; DNase, 50 pg./ml. MgS04,5 pM./ml. The total volume of the reaction mixture waa 1.5 ml. The procedure used was as in Table 5. For the same experiment, the transformation frequency for the try marker was 0.047%.
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mutants of H.subtilis to Flu+ try’. Motile cells were detected 3 hr. after the entrance of DNA into the recipient cells. Alteration of genotype with regard to the state of flagellation is also readily accomplished by transduction (Lederberg and Edwards, 1953; Stocker et al., 1953; Brown et al., 1955 ; Stocker, 1956 ; Spicer and Datta, 1959) and sexual recombination (Furness, 1958; Makelii, 1964). Aspects of the genetics of Salmonella have been covered in reviews by Iino and Lederberg ( 1 9 6 4 , Joys (l9c18), and lino (1969). Control of gene function and motility other than concerning the presence or absence of flagellin will be discussed later. Lederberg and Edwards ( 1953) suggested that the phase 1 and phase 2 flagellar antigens of Salmonella are determined by a series of genes a t two separable loci termed “H1” and “H2” respectively. HI and H2 are on the same chromosome but appear to be widely separated (Smith and Stocker, 1962; Iino and Lederberg, 1964). Spicer and Datta (1959), however, did observe a clone of S. typhimuriurn with an unstable transductional derivative that appeared to be diploid a t both the H1 and H2loci. If true, the H locus would have been cotransduccd although in an abortive manner. Normally they are not cotransduciblc (Iino and Lederberg, 1964). The H loci should not be confused with the flu loci. The H loci represent structural genes for the various flagellins whereas the flu genes may be involved in control of H genes, polymerization of subunits, or with special structures, for instance the postulated flagellosomes (Iino and Lederbcrg, 1964). The “Flu” designation is used t o refer to the cluster of cistrons involved in the synthesis and polymerization of flagcllin. It has been proposed, although a t present not widely accepted, that genes determining the structure and controlling the synthesis of flagellar proteins should be termed (‘hag” (Derncrec et al., 1966). As an example of current terminology, an organism that produces flagellin with antigen i in phase 1 and a different! flagellin with antigens 1 , 2 in phase 2 is designated as HI-i, H2-1,2. Makela (1964) andIino andLederbcrg (1964) have proposed that the H gene of E. coli is allelic with 151 of S. ahony. The locus in E. coli refipoiids to the gene that controls the function of H1 in Sul?nonella and the cell becomes diphasic upon introduction ofthe fI2 gene ofSalvnoiaella. I n E . coli and AS.abony the I31 locus is closely linked to the his marker (Mlikela, 1904; Iino and Lederberg, 1964). Van Alstyne et al. (1969) using cells of 12. subtilis synchronized by a burst of spore germination rnappcd the loci involved in the rate-limiting step for the formation of flagella by determining the time a t which the rate of formation doubled. They also found these loci to be located near the his A1 locus. The adjacency of the his marker and loci concerned with the formation of flagella should prove useful in the isolation of deletion mutants deficient in the formation of flagella and/or motility since one can more easily
303 select for his than for flagella mutants, and perhaps obtain cells with deletions over both loci. Iino (1961~)suggests that there is a structural homology between H I and H2. He observed a n abnormal transductant that produces the phase 1 type antigens of both the donor and recipient. The H 2 locus of the recipient had apparently been replaced with H I locus of the donor. Lederberg (1961) observed a n apparent monophasic behaviour in S. paratyphi B strain CDC-157. The 1,2 antigens were determined by an allele at the H 1 locus instead of its normal position at H2. The organism also carried a duplicate H 1 locus. Thus, the genotype is H1-b, H1-1,2 as compared to H1-b, H2-1,2 in the wild-type cells. It was suggestedthatin the course of evolution the H 2 locus originally arose as a copy of H1. This possibility was again discussed by Iino and Lederberg ( 1964). Joys and Frankel (1067) propose the existence of at-least three genetic loci concerning motility in cells of B . subtilis. These consist of a n H locus that functions as a structural gene for flagellin, a fla locus that controls the presence or absence of the filament, and a mot locus that controls the function of flagella. The three loci are non-allelic and appear to be unlinked in transformation tests. Stocker et al. (1053) reported that a mutation in any one of at least six non-homologous genetic factors can cause non-flagellation in S. typhimurium. Iino (1959) divided the g group of phase 1 antigens into eight major components. Each can mutate independently of the others. One of these components, i.e. the g subgroup, can be further divided into at least five antigenically distinguishable groups (Yamaguchi, 1965). It has recently been possible t o map these antigenic determinants within H 1 (Yamaguchi and Iino, 1966). Using pairs of flu- mutants closely linked to but with mutations on either side of H 1 , they isolated transductants that had undergone a n intra-Hl crossover event. Each antigenic determinant is located as a unit in a linear fashion within H I . Joys and Stocker (1966) found at least 13 subfactors in the determinant of phase 1 flagellin with antigen i. Many antigenic mutants were isolated that lacked one or a combination of these subfactors. Iino and coworkers havc found the use of mutants having abnormal filaments to be of great value in studying the genetics of Xalmonella. Transduction between mutant<shaving curly filaments does not result in normal filaments (Iino, 1060, 1962). A culture was described that has curly filaments in phase 1 (antigen i) and normal filaments in phase 2 (antigen I,.’). Transduction into a normal strain produces progeny with donor antigen and curly filaments in phase 1 and the recipient antigen in phase 2. The curly determinant, therefore, appears t o be closely associated with H I . Similar results were found in strains of 8. abortusequi, which are curly in phasc 2, in that the curly locus appears t o be BACTERIAL FLAQELLA
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closely associated with H2.No antigenic differences were found between curly and normal filaments. The determinant for the curly shape of the filament, therefore, is phase specific and closely linked or identical to the H loci. One tryptic peptide difference was indicated but not clearly demonstrated between flagellins of strain SL23, which has normal filaments and strain SJ30, which produces only curly filaments (Enomoto and Iino, 1966). If true, this observation would indicate that the curly locus is identical to the H locus and that a change in the sequence of amino acids in flagellin brought about by a missense mutation results in the formation of curly filaments. Iino and Mitani (1967, 1967a) examined mutants of 8.typhimurium, that have straight non-functional filaments in phase 2 (antigen 1,2) and normal filaments in phase 1 (antigen i). No antigenic differenceswere noted between normal 1,2 filaments and straight 1,2 filaments. The mutant can be transduced to normal in both phases with the donor antigen produced in phase 2 and that of the recipient in phase 1. Thc straight filaments apparently result from a mutation in the structural gene for phasc 2 flagellin, i.e. H2-1,2. Similarly, Martinez et al. (1968) isolated four mutants of B. subtilis 168, induced by different mutagenic agents, which have flagella with straight, non-functional filaments. In all cases, the mutational event involved a single identical amino-acid substitution consisting of an alanine residue being replaced by valine. Vary and Stocker (1969) identified an amber mutation in the HI gene of a non-flagellated mutant of S. typhimurium LT2. If an amber suppressor gene is introduced into the mutant cells by conjugation, the cells produce non-functional flagella that have straight filaments. In contrast to the mutants identified by Iino and Mitani (1967, 1967a), these straight filaments do not cross-react with antiserum prepared against normal filaments. Apparently, the amino-acid substitution results in a significant change in the conformation of the flagellin subunits as evidenced by the altered morphology of the filaments and the change in the nature of the antigenic determinants. These are sufficient data t o substantiate the belief that RNA and the concomitant synthesis of RNA are required for the formation of bacterial flagella, Kerridge (1960, 1961) reported that purinc and pyrimidine analogues do not affect the formation of flagella in S. typhimurium. Cells grown a t 44" are non-flagellated but do form filaments after transfer to 37". The analogues inhibit the formation of filaments when added to the culture while at 44" or within 1 hr. after transfer to 37". A "flagellasynthesizing system" was postulated, the synthesis but not the functioll of which is inhibited by either analogues or incubation a t 44". Formation of the synthesizing system appears to require the simultaneous synthesis of RNA; however, once formed, the continuous synthesis is not thought
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to be required for the formation of filaments. Possibly, the synthesizing system is housed in an autonomous structure, impermeable to the analogue, the function of which is to make and polymerize flagellin into filaments. Another possible explanation may be that the cells themselves me not permeable to the analogues at 37”. Permeability barriers may be altered by incubation a t 44” and subsequently require an hour to be re-established after transfer t o 37’. The synthesis of RNA in B . subtilis 168-15 (try- uracil-) is inhibited 80 t o SOY0 by metabolite starvation (Martinez, 1966). The rate of incorporation of I4C-leucine into flagellin appeared to be the same in both starved and non-starved cells. I n contrast, the synthesis of 8galactosidase and a-glucosidase is completely inhibited in starved cells. These observations were interpreted to indicate the existence of a stable m-RNA for the synthesis of flagellin, although it was not possible t o rule out preferential synthesis of flagellin m-RNA or the non-involvement of RNA. Stabilization could be brought about by strong binding of the m-RNA to a flagellin-synthesizing ribosome or by protection in a specialized structure. It was suggested, although now this seems highly unlikely, that this specialized structure is the hook region which may contain the “flagella-forming system” postulated by Kerridge ( 1960, 1961) and represent the hypothetical “flagellosome” of Iino and Lederberg (1964). Similarly, Aamodt (1967) found that lo-) M-8-azaguanine inhibits RNA and protein synthesis 50 to 75% in Salmonella, but does not prevent the formation of filaments. McClatchy and Rickenberg (1966, 1967) also concluded that m-RNA for the synthesis of flagellin in 8.typhimurium SL 282 (try-) is unusually stable, Tryptophan starvation or exposure of cells treated with EDTA to actinomycin D inhibits total protein, /3-galactosidase, and RNA synthesis 85 to 90% with no effect on the incorporation of I4C-leucine into flagellin. The principal objection to these several reports lies in the degree to which RNA synthesis actually was inhibited. Aamodt and Eisenstadt (1967, 1968) found that during starvation of strain SL 282 cells for tryptophan the rate of RNA synthesis is reduced to 10% of normal with no effect on the synthesis of flagellin. On the other hand, further addition of actinomycin D lowered the rate of RNA synthesis t o less than 1 yoof normal and did inhibit the synthesis of flagellin. Interestingly, the synthesis of RNA involved in the production of general cell proteins is inhibited a t lower actinoniycin D concentrations than the RNA specifically involved in the synthesis of flagellin. Mandel et a2. (1966) reports that thioguanine or mercaptopurine selectively inhibits the synthesis of flagellin in cells of B . cereus. Incorporation of 14C-lysineinto cell proteins or induction of penicillinase is not affected, even though RNA synthesis is reduced. Either compound
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inhibits, to a degree, the incorporation of diaminopimelic acid into the mucopeptide. I n view of the findings of Vaituzis and Doetsch (1965,1966) and Diena et al. ( 1968), prevention of the formation of filaments may have been a secondary effect brought about by an impaired synthesis of cellwall material. Dimmitt et al. (1968) examined the formation of filaments in uracil and tryptophan auxotrophs of B. subtilis 168. During starvation for uracil or in the presence of actinomycin D the ability to generate filaments decayed with a half-life of 5.5 min. However, cultures do continue to synthesize flagellin in the absence of tryptophan. This might be expected since tryptophan is probably not present in the flagellin of this species. Based on these observations and those of Aamodt and Eisenstadt (1967, 1068), it is unlikely that the system involved in the synthesis of flagellin differsin nature from that for other proteins. Perhaps the best available evidence concerning the mechanism of flagellin synthesis is provided by the in vitro system used in this laboratory (Gaertner, 1960; Caertner and Koffler, 1960; Koffler et al., 1966; Koffler, 1967a, b ; Sala and Koffler, 1967; Gaertner et al., 1968; Sala et al., 1968; Suzuki and Koffler, 1969, 1970). The ability of a cell-free extract of B. pumilus to incorporate 14C-labelled amino acids into flagellin is destroyed by the addition of ribonuclease but not deoxyribonuclease. Incorporation is essentially abolished by the addition of puromycin or chloramphenicol and by the omission of either amino acids, magnesium, ATP, an ATP-generating system, GTP, or ammonium chloride, as is characteristic for cell-free protein synthesis. Since flagellin, unlike most cell proteins, is soluble a t p H 2 and is capable ofself-assembly, it is possible t o isolate the newly synthesized flagellin by adjustment of the reaction mixture to p H 2, addition of unlabelled carrier flagellin to the pH 2-soluble material followed by re-assembly and a final purification and separation of the A and B proteins on a DEAE-cellulose column. Tryptic digestion of the isolated in vitro products yields peptides that appear identical to authentic peptides from flagellin when examined either by peptide mapping or by elution from an ion-excha,nge column. At least some of the flagellin molecules must be made de novo by the cellfree system since incorporation of either 35S-or ''C-methionine a t the N-terminal position can be demonstrated. A cell-free extract of a mutant non-flagellated strain of B. pumilus does not incorporate '4C-amino acids into the isolated product ; this further supports the contention that the enzymic product indeed is flagellin. More recently we have been in tlie process of resolving the flagellin-synthesizing system by using phenolsoluble RNA from cells of B. pumilus and a cell-free extract of E.coli Q13, an organism low in ribonuclease (Suzuki and Koffler, 1969, 1970, and unpublished results). Incorporation of radioactive amino acids into
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total protein occurs to the extent of about three nanomoles of leucine equivalent per mg. of RNA or 0.5 nanomoles of leucine equivalent per mg. of protein. Approximately 0.2% of the total protein synthesized can be accounted for by the synthesis of flagellin. Incorporation of labelled amino acids into flagellin A and B, under appropriate conditions, occurs in the same ratio as the synthesis of these two flagellin components in vivo. The flagellins synthesized by the E . coli enzymes and ribosomes are identical to those from a homologous B. pumilus system; flagellins A and B are synthesized in the same ratio as in vivo. Thus, in all respects thus far examined, the synthesis of flagellin appears similar t o that of other proteins. Kerridge (1959) studied the effect of amino-acid starvation on the synthesis of flagellin in auxotrophs of S. typhimurium. Three classes were defined with regard t o their requirements for exogenous amino acids. Mutants that possess a particular amino acid in their flagellin require that amino acid for the formation of filaments, presumably for the synthesis of the flagellin subunits. I n another group amino acids not present in flagellin were not required. I n a third class the synthesis of filaments was observed in the absence of amino acids that are present in small amounts in flagellin. The necessary amino acids are probably provided endogenously by the turnover of pre-existing proteins. DL-p-Fluorophenylalanine (fphe ; 0.005 M ) induces linear growth in S. typhimurium SW 1061 (Kerridge, 1959a) 1960). After 4 hr. the number of motile cells decreased from 90 to 5%. Filaments formed in the presence of fphe are non-functional and have a pitch length one-half normal. The analogue was identified in general cell proteins but could not be detected in flagellin. Thienylalanine, ethionine, norvaline, norleucine, and 5-methyltryptophan have no effect on the formation, function, or morphology of filaments. Fluorophenylalanine has no effect on the morphology or function of preformed filaments (Kerridge, 1960). Removal from the medium and replacement with phenylalanine does not permit reversion of abnormal filaments. Similar observations were made in S. typhimurium SL 448 (phe-) (Mitani and Iino, 1967). The magnitude of the effect is related to the concentration of phenylalanine relative to that of fluorophenylalanine. At weight ratios of 9:1 t o 3:7, normal, curly, and filaments with both pitch lengths are observed. Only curly filaments are present at a ratio of 2 :8 and no flagellated cells are found a t 1 :9 or 0 :1. The mechanism by which fphe acts remains obscure. At first glance, the aberrations could be caused by changes in subunit conformation induced by incorporation of fphe into the primary structure of the protein. It has not been possible, however, to demonstrate the presence of fphe in flagellin of Salmonella spp. (S. Asakura, personal communication). Fluorophenylalanine appears immediately to shut
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off the endogenous synthesis of phenylalanine in B. pumilus (R. W. Smith and H. Koffler, unpublished results). Replacement of four of the six phenylalanine residues contained in this flagellin by fphe is readily accomplished, as quantitated by amino-acid analysis of the purified modified flagellin or by incorporation of I4C-fphe of known specific activity (Koffler and Smith, 1968). Filaments with one-half the normal pitch length are only observed when the molar ratio of L-phenylalanine to DL-fphe in the growth medium is adjusted to values between 1 :26 to 1:33. At lower levels of L-phenylalanine cell division continues for a period of time depending on the concentration of the analogue. The rate of division gradually decreases resulting in elongated cells and finally stops. Motility continues for a much longer period of time. Two hours after cessation of cell division only normal filaments are observed electron microscopically on the by then greatly elongated cells. Flagella appear to tear from the cell bodies much more easily than those on control cells; intact organelles including the basal region along with portions of the cell membrane can be more frequently observed than with normal cells. When the ratio of the molar concentrations is greater than approximately 1 :26, no effect on cell division, motility, or morphology of the filaments is observed. Flagellin of B. purnilw modified by growth of cells in an analogue-containing medium so that four fphe residues are incorporated per molecule re-assembles at the same rate as normal flagellin, and the resulting filaments have a normal morphology. Therefore, incorporation per se of analogue into the primary structure of the protein does not preclude assembly into normal-appearing filaments. The filaments are slightly more stable to heat disintegration, although the modified flagellins appear normal with respect to helix content, immunodiffusion, and electrophoretic characteristics. These observations suggest that fphe alters the morphology of filaments by a,disturbance a t some point other than the structure of flagellin, perhaps at the level of polymerization, However, it is also possible that the optimal conditions for selfassembly in vitro do not adequately simulate the real environment prevailing in vivo,and that if one were able to reproduce more faithfully the natural conditions, fphe-containing flagellin might assemble into curly filaments. This is still under investigation. The non-enzymic nature of the re-assembly mechanism in vitro suggests that polymerization of flagellin in vivo is also non-enzymic and similar to crystallization (Asakura et ab., 1966). Certainly, such a process appears feasible in light of the discussion presented by Oosawa and Higashi (1967). Klug (1967) points out that self-assembly is biologically advantageous in that regulatory mechanisms can operate entirely at the level of the structural genes for flagellin,since the information for self-assembly resides in the amino-acid sequence of the molecules
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involved. This is not necessarily true, however, if polymerization occurs in a special structure, the synthesis and function of which is under genetic control. Martinez and Rosenberg (1 964) and Martinez and Gordee (1966) question whether self-assembly is operative in wivo and propose that polymerization is under the catalytic control of a “biological organizing principle”. While, a t the moment, it cannot be proven that the in wivo formation of the filament involves self-assembly, there is no need to assume a catalytic event until it is demonstrated. Moreover, even if enzymes participate in the polymerization of flagellin it would be astonishing if the basic molecular prerequisites necessary for selfassembly do not hold true also for the catalysed polymerization. However, it also would be astonishing if self-assembly occurs throughout the cell without some anatomical localization. Obviously, there are many uncertainties as to where the assembly of the filaments takes place. Intuitively, one might assume that polymerization of flagellin must occur closest to the site of its synthesis. However, the available evidence favours growth of the filament a t its distal end. For example, in vitro, monomer addition to seed fragments apparently occurs only a t the distal end (Asakura et al., 1968). On the other hand, Pye (1967) found that monomers do not polymerize onto the distal end of broken filaments still attached to the cell, and therefore, concluded that in wivo filaments elongate at the proximal end. Perhaps the presence of cells, for some unexpected reason, altered the conditions for self-assembly sufficiently to prevent the addition of monomers t o flagellar stumps. Similarly, experiments in which curly morphology brought about by the presence of fphe in the medium is used as a marker are not consistent. If cells with normal filaments are transferred to a medium containing fphe, new regions of the filament should be marked by their short pitch. Mitani and Iino (1967) observed in general that the short pitch occurs in the proximal portion of the filament when cells were grown in a medium containing a small amount of fphe in relation to phenylalanine ; 4 out of 82 observations, however, indicated the opposite. If the fphe concentration was greater than that of phenylalanine all filaments were curly. More recently, Iino (1969) has shown that in 8.typhimurium LT2, the short pitch occurs initially a t the distal end. The rate of filament elongation decreases as the length of the filament increases and reaches zero a t about five normal pitch lengths (15 pm.). The decrease in rate is thought to be due to a less efficient transport of subunits through the central core of the filament and not to a decreased synthesis of the monomer, since filaments shortened by physical agitation elongate a t the same rate as unbroken filaments of the same length. Independently, D. Kerridge (personal communication) using curliness as a label also found that the recently formed portion of the filament is
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located distally. Because of the existing ambiguities it is too early to consider the problem as settled and additional confirmation and data obtained by other approaches are needed. At first sight, the logistics of moving flagellin monomers to the distal portion of the filamont seem overwhelming, but then the ingenuity of nature dwarfs ours. The possibility that flagellin subunits are released into the medium and then deposited a t the free ends of filaments is most unlikely, since numerous factors including the concentration of flagellin, pH, and the ionic environment are limiting. It is conceivable that subunits move along the surface of the filament, but how! More likely, they move through the central hole. It is difficult to demonstrate the existence of a central hole in hooks ; but we have had indications that negative stains penetrate the centre of the hook (Abram et at., 1969). Perhaps, flagellin subunits move through the hook and are deposited a t the distal end of the hook, forming the initial layer of the filament. Subsequent subunits would have to move through the hole of t h s filament. Since we have no definitive information concerning the site of flagellin synthesis and the nature of the hook, any model for the formation of the filament needs t o be regarded as tentative. The existence of genetic mechanisms that regulate the synthesis of flagellin is well documented, but most information deals with Salmonella spp. While studying the genetics of phase variation in Salm(onella, Lederberg and Iion (1956) observed that, when the structural gene of phase 2 flagellin ( H 2 ) is active, the analogous gene for phase 1 flagellin ( H I ) is inactive or suppressed. Similarly, when H 2 is inactive, H I is active. Expression of H I depends on the state of the recipient organism in transduction experiments, whereas expression of 112 depends on the state of the donor. Activity of H I , therefore, appears to depend on the state of the H 2 locus. Iino (1958) described a controller locus termed “ah,” closely linked t o H 2 . Monophasic strains stable in phase 1 result from mutations producing the genotype ah,-. The H 2 locus does not function in these mutants. Similar controller loci werc found for H I (Iino, 1961b). Three closely linked but non-allelic regions (ah,)werc identified that regulate the function of H I . Another controller locus, vh,,was described by Iino (1961a).Mutants with the genotype vh, may be stable in either phase. Apparently a mutation in the vh, locus prevents control of the structural gene by ah,. The similarities and dissimilarities between the ah and vh loci and the operator and regulator genes have been discussed by Iino and Lederberg (1964). The ah loci may be analogous to the operator genes although it was thought unlikely that the vh loci are similar to the regulator regions. Most mutants of the&- genotype do not produce an immunologically recognizable flagellin (Iino and Lederberg, 1964). The flu loci thereforo
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may control the synthesis of a special structure responsible for flagellin synthesis or it may produce an internal inducer of flagellin synthesis (Iino and Lederberg, 1964; Iino and Enomoto, 1966). While cells of E . coli may have only onefla locus (Orskov and Orskov, 1962 ;Matsumoto and Tazaki, 1967), Iino and Enomoto (1966) described non-flagellated mutants of S. typhimurium in which the fla- loci can be separated by complementation tests into seven groups. These are designated as fla A ,fla B,f l a C, etc. Four of these correspond to four of the five groups described previously by Joys and Stocker (1963). Their fifth group was designated as a separate flu locus. Each group may correspond to a cistron. Five were found t o cotransduce with the H I gene but not with H2. Vary and Stocker (1969) examined non-motile mutants of 8. typhimurium LT2, a strain that has an amber mutation at the his C locus. Amber or ochre suppressor genes were introduced into the mutants by conjugation, and H I S + cells were selected. Twenty of 313 selected cell lines proved to be motile. Eighteen of these 20 are f h - mutants with the mutation located either in fla A ,f l a B ,f h F , or fla K . Three mutants were found that form a new complementation group designated flu M . I n S. abortus-equi, an additional locus,f l a L , was found to be most closely linkedto H 1 (YamaguchiandIino, 1969). At least two levels of control of the state of flagellation are possible. As discussed, certain genetic determinants have been described that control the functioning of the structural genes for flagellin. Second, the existence of a genetic mechanism controlling polymerization was thought to be implied by the isolation of mutants capable of synthesizing but not polymerizing flagellin (Iino and Haruna, 1960). The proteins produced by these mutants were thought to be normal in that their ability to reassemble in vitro did not appear to be impaired (Suzuki and Iino, 1966). The experimental design, however, involved the mixing of carrier control flagellin possessing the 1,2 antigens with a cell-free lysate prepared from non-flagellated mutant cells grown in the presence of I4C-algal hydrolysate. The mutants produce material that cross reacts with the flagellin e,n,x antigens (Iino and Haruna, 1960; Iino and Enomoto, 1966). The filaments isolated after re-assembly of the flagellins in the mixture proved to be radioactive. Both the 1,2 and the e,n,x flagellins were identified in the re-assembled filaments by starch gel electrophoresis. Unfortunately, one cannot exclude the possibility, in our opinion the probability, that a non-specific ooprecipitation or absorption occurred instead of a bona f i d e assembly of the mutant product into the structure of the filament. Furthermore, the lesion in flagella-negative but flagellinpositive mutants may be concerned with the synthesis of other proteins, that probably are constituents of the basal structure or hook (Vary and Stocker, 1969). I n this sense, polymerization may be prevented because
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the structures onto which the flagellin subunits need to be assembled may be missing. Apparently, a given filament can elongate for only a definite period of time, which is relatively short as compared to the functional life of the organelle (Stocker and Campbell, 1969; Kerridge, 1961 ;Wilson et al., 1966). The mechanism that controls filament length is not known. Probably this control does not involve the general cessation of flagellin synthesis everywhere in the cell, since on cells recently formed by cell division some filaments will be increasing in length whereas others will not. If the synthesis of flagellin for a given filament occurs a t or near the basal region of that filament, control could probably be most conveniently exerted at that level. While observing germinating spores of many strains of Bacillus spp., Leifson (1931) noted that flagellar filaments do not grow out simultaneously but form approximately one at a time. Similar observations were made in the case of dividing vegetative cells (Bisset and Hale, 1960). There appeared to be a gradient in the state of maturity of filaments from one pole of the cell to the other. The observations of Hoeniger (1965) on dividing cells do not support this interpretation, however. If the preceding observations were correct one would have to postulate a mechanism operating locally a t the site of polymerization of each filament. One possibility that comes to mind is that the energy required to move the flagellin monomers from inside the cell to the distal end of the filament, presumably the site of polymerization, may become limiting after the filament reaches a certain critical length. The existence of some means for controlling the synthesis of the filament can be deduced from several observations, for example, the unilinear transmission of motility (Lederberg, 1956 ; Stocker, 1956 ; Quadling, 1956). Division of a motile cell transduced from a state of non-motility, apparently by an abortive transduction, results in one motile and one non-motile daughter cell. On an agar surface, this is recognized by the production of trails or non-branching linear arrays of colonies of non-motile cells. Following cell division the motile cells leave the vicinity, while the non-motile ones continue to divide in situ, eventually forming a visible colony of non-motile cells. The determinant of this character is non-reproducing, and was termed a “motility-conferring particle”. Quadling (1 956) observed a relationship between the number of these hypothetical particles and the number of filaments per cell. Lederberg (1 956) considered them to be flagella or blepharoplasts. Stocker (1956) considered each particle to be a flagellum or “a granule which determines the production of one”. This phenomenon was also seen to occur spontaneously in 22 of 48 non-motile strains (Quadling and Stocker, 1957). Motile cells appeared in non-motile cultures at a rate of
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about 4 x per cell per generation. An intracellular event was proposed that permitted a "transient ability t o synthesize new flagella". The non-motile cells possessed the genetic information for motility but the expression of this information was prevented by some control process. Apparently, for a short period of time, control was occasionally released, and this permitted a given cell to synthesize several flagella. Control was then re-exerted and the flagella were passed in a unilinear fashion t o progeny until no cell had more than one flagellum. The average number of flagella synthesized during the period of relaxed control appears to be about 2-5 per cell. These observations were duplicated by controlling the synthesis of flagella by environmental changes ( Quadling, 1958). Fewer than 0.1% of the cells of S. paratyphi c strain SL 237 are motile after prolonged exponential growth a t 20". Occasionally motile cells arise that also transmit motility to their progeny in a unilinear fashion. The existence of a discontinuous event, i.e. the non-functioning of the control mechanism, is further supported, which permits a given cell t o synthesize no more than three flagella. Enomoto (1965) described a slow motile mutant of S. typhimurium that only produces one-third as much flagellin and has only one-third as many flagella as wild-type cells. The site of the mutation was located in the mot B cistron, and Enomoto considered it likely that the mutant gene is involved in the formation of flagellin-polymerizing organelles. Inhibition of the formation of filaments by growth a t high temperatures has also been found to have a genetic basis. Grant and Simon (1968, 1969) isolated 25 mutants of B. subtilis that are flagellated a t 37" but not when grown a t 46". These are designated as f h T S mutants. I n transduction experiments, all sites of mutation appear to be linked to the his A1 marker. Twenty strains are found to be tightly linked with the structural genes for flagellin while five loci map at two sites separable from the larger group. A third class, a t present composed of only one isolate, is only loosely linked to his A l . Iino (1961) isolated a mutant of Salmonella that remains flagellated during growth a t 44". Filaments of the mutant are antigenically similar t o those of wild-type organisms. The mutant marker was found to transfer independently of any region known to be concerned with the production of flagella. The mutant locus appears t o code for a protein involved in flagellin synthesis that is stable at 44"; the function of this protein remains unknown. It is interesting, however, that a presumably single mutational event could give rise t o a protein, perhaps a constituent of an organelle involved in flagella formation, with a n increased stability t o heat, One intriguing possibility occurs upon consideration of the findings of Aamodt and Eisenstadt who, as mentioned previously, found that protein synthesis is necessary before flagellin synthesis is inhibited
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by incubation of S. typhimuriurn a t 44". The mutant described by Iino could have resulted from a mutation in the gene coding for the protein involved in shutting off the synthesis of flagellin. A fascinating class of mutants is described in B. subtitis by Grant and Simon (1969).A mutant locus (ifm)is defined which apparently controls the degree of motility and the number of flagella per cell. The site of mutation maps near the flaTS group A mutant sites and the H locus. Mutations in the ijm locus produce cells which spread more rapidly in a semi-solid medium than do wild-type cells. The ifm locus in B. subtilis may be analogous to the region in S. typhimurium described by Iino (1961),and discussed above in that the product of these regions functions in some way either t o shut off the synthesis of flagellin or t o prevent its polymerization. Surely, mutants of these types will permit fruitful investigations into the mechanisms of flagella formation.
XII. Mechanisms for the Function of Flagella The mechanisms employed by bacterial cells to cause active co-ordinated movement of flagellar filaments and accomplish locomotion of the cell body are unknown. Surely, energy is required, but what is its source? At what site in or near the flagellum is energy utilized? Do subunits within the filament contract and, if so, how is the energy transmitted along the filament? What controls the function of flagella and coordinates their activity a t several locations on a cell? These questions also suggest many possible levels a t which the function of flagella is controlled genetically. The hydrodynamics of the movement of flagella and cell locomotion has been examined and discussed in several reports (Taylor, 1951, 1952 ; Hancock, 1953; Weibull, 1960; Holwill and Burge, 1963; Doetsch et al., 1967). Inertial forces appear t o be negligible, locomotion being accomplished when viscous forces acting on the cell body are overcome (Taylor, 1951 ; Hancock, 1953). Although initial observations on dried flagellar filaments gave the impression that cell locomotion is achieved by an undulatory movcrnent of the filaments, it now seems likely that the filaments have a spinning motion. Taylor (1952) observed that cells with spinning filaments are capable of speeds twice as great as cells which have undulating filaments. Holwill and Burge (1963) conclude that forces generated by the motion of helical filaments are sufficient to propel cells a t experimentally observed velocities, i.e. up to 50 pm./sec. The contractile nature of the filaments of bacterial flagella was suggested by Butschli (1883) and Reichert (1909). They proposed that a line of contraction runs helically along the filaments. Changes in pitch may be generated by contraction along this line. The rhythmic change from a
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straight cylindrical to a spiral structure would then propel the organism through the medium. This theory has been reconsidered by Weibull (1951) and more recently by Klug (1967). Astbury (1951, 1951a) thought that the sine waves described by filaments in dried preparations resulted from a “molecular rigor mortis” of filaments trapped in the process of contraction. We now know that this is not true since similar observations can be made on reconstituted filaments ; the spiral nature of the filament apparently is determined by the amino-acid sequence of the monomers. Weibull (1951) considered that the mechanism for locomotion should include a reversible contraction of postulated fibres within each filament. Braun (1956) observed what appeared to be fine fibres in Escherichia coli filaments and further discussed their function as contractile elements. At first, crystallographic observations were interpreted to support a contractile mechanism, and encouraged analogies between flagella and muscle (Astbury et al., 1955). However, the X-ray reflections thought to indicate contractility were later reported t o be inherent in the nature of the flagellar protein and not necessarily indicative of any mechanism of function (Beighton et al., 1958). Machin (1958) reviewed two models for flagellar motion. In the first the filaments may be considered as passive elastic fibres set in motion by some active process at their proximal end. In the second contractile elements could occur and function along the length of the filament. He concluded that the conformation assumed by filaments could only be generated by the second mechanism. However, the assumption that the morphology of filaments is determined by their motion is unfounded. Feldman and Lindstrom (1962) observed that, although completely flagellated, cells of Rhodospirillum rubrum are non-motile in a calciumfree medium. Motility is observed upon addition of calcium ions t o 10-5 M . The role of calcium in motility was correlated with its role in the function of muscle tissue. Weibull (1951) and Rinker (1957) compared the amino-acid composition of flagellin to that of myosin, actin, tropomyosin, meromyosin, wool and feather keratin, silk fibroin, human fibrinogen and epidermin, and collagen, and found no significant similarities. It seems probable, however, that the physical and chemical properties of structures formed from these proteins would be determined not by their gross composition but by the conformation of the subunits which is dictated by the sequence of amino acids in the primary structure and by forces generated on packing. Biplicity, i.e. the occurrence of filaments with two pitch lengths, one generally being one-half the normal pitch, has also been thought to indicate a contractile nature. Jarosch (1964) concluded that a periodic change in pitch could cause a motion to proceed away from the fixed end of a helix. He further suggested that torsional forces inside the
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filament were responsible for the changes in pitch. Asakura et al. (1966) also suggest that changes in pitch length as indicated by the occurrence of biplicit filaments may be important in the process of cell locomotion. I n such a mechanism, it is necessary t o postulate differentiation of the molecules within a filament into two or more different metastable states. Newton and Kerridge ( 1 966) suggest that a protein other than flagellin may be involved in the function of flagella. They further propose that internal changes in the core of the filament may be involved. So far, it has not been possible t o demonstrate another protein in the filament. However, it is likely that the hook and the basal structure contain proteins other than flagellin. Earlier, Polevitzky (1941) had suggested that “changes in pressure within the lumina of these tubular flagella induced by rhythmic contraction of the bacterial protoplasm might well account for flagellar movement and for locomotion”. Lowy et al. (1966) hypothesized that more than one element must be present if the movement of filaments is to be analogous to the contraction of muscle, and suggested that some material structurally different from flagellin may be present in the core of the filament. The presence of at least two types of flagellins in Bacillus pumilus and probably in other organisms as well needs t o be considered in this connection. Interestingly, both flagellins A and B can be re-assembled to produce spiral filaments. In order for filaments to contract each flagellin molecule must possess both oontractile and compressive properties. I n addition, all the subfibres in a given segment of filament should be in different stages of contraction a t any particular time. Newton and Kerridge (1965) suggest that either a change in the shape of the subunits or in their position relative t o one another may be the mechanism of wave transmission. A change in the diameter of subunits of less than 5% is thought to be sufficient to account for a wave transmission along the filament. I n a similar proposal Klug (1967) suggests that changes in the morphological parameters of filaments might be initiated by alterations in the basal region. He further proposes from a theoretical consideration of the crystalline nature of the filament that a helical wave could be generated a t the basal region and maintained without dampening along the length of the filament by progressive re-arrangement of the patterns of bond strains between subunits. Klug calculated that in filaments with a pitch of 2 pm. and an intersubunit distance of 50 A local deformations of about 1 A would generate a suitable helical wave. After distortion, the subunits would automatically return to the more thermodynamically favourable configuration. Mechanisms with a more passive involvement of the filament structure have been devcloped by several investigators. Brieger ( 1 963) considered that filaments do not contract, and that locomotion is controlled in some manner by the basal structure. Astbury et al. (1965) interpreted certain
BACTERIAL FLAGELLA
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crystallographic patterns as indications for the contractile nature of flagellar filaments. As noted by Champness and Lowy (1968), however, these patterns do not necessarily demonstrate the presence of both the a-helical and cross-p polypeptide configurations. No evidence was found to support a contractile mechanism or a molecular organization similar to that found in muscle (Champness and Lowy, 1968). The existence of the cross-fi configuration cannot be supported in filaments of Salmonella typhimurium or B. subtilis by infrared spectra (Burge, 1961; J. N. Champness, personal communication). Based on these observations, i t seems likely that bacterial flagella function by some mechanism unlike that operating in muscle and cilia of other organisms. Rotation of the filament with respect to the cell surface is suggested by the observations of Stocker (1956). Cells may rotate about a fixed point, as if attached to i t by their flagella, after reaction with specific antibodies against the flagella. Newton and Kerridge (1965) propose that filaments may move like rotating “cork-screws”. Cell locomotion could then be produced by the forces generated by a spinning helically-coiled filament and not by its contraction. A counter rotation of the cell body would be induced and a translational velocity as high as 50 pm./sec. could be obtained. Jarosch (1965, 1966, 1967, 1968) also considers cell locomotion to be achieved through the spinning or rotating movement of helical flagellar filaments, and has constructed models to strengthen his arguments. Doetsch (1966) concluded that filaments must be inert rigid or semirigid helices, and suggests that the filament is coupled to rotating helices within the basal structure and “. . . the part of the flagellum within the basal bulb is encircled by a rapidly contracting and relaxing ring, whose inner walls are supplied with ‘ratchets’ that engage and rotate the organelle”. Doetsch doubts, however, whether the filament structure could withstand the torsional forces placed upon it by such an operation. A similar mechanism has been described for Spirillum volutans (Metzner, 1919; Jahn and Bovee, 1965; Krieg and Tomelty, 1967). Cells of this organism possess several flagella a t each pole that appear to rotate in such a manner as to describe a cone of revolution. The filaments move a t about 40 rev./sec. while the cell body rotates in the opposite direction at about one-third that velocity. I n effect, the cell screws its way through the medium. The cause of filament rotation remains obscure. Metzner (1919) suggested that motion may originate from the distal tip of the filament but this appears unlikely a t present. Jahn and Bovee (1965) considered a model made to revolve by a contraction on one side of the filament near the point of insertion accompanied by an expansion on the opposite side. A source of excitation for the localized contraction and extension
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R. W. SMITH AND HENRY KOFFLER
would have t o travel in a circular path about the periphery of the base of the filament. Several investigators have suggested alternate possibilities that might explain the motion of flagella. Van Iterson (1963) asked whether movement could be due to an electrical gradient along the filament, secretion of electrolytes, or local changes in viscosity brought about by secretion of slime. Mitchell (1956) calculated that a potential or thermal gradient along the filament could cause streaming of water along its surface sufficient to propel the cell a t a velocity of 5 pm./sec. King (1960) suggested that all cell motion was generated by Brownian forces; in this view, the filaments function t o stabilize the cell and focus the nondirectional Brownian forces so that translational movement results. Since the contractile nature of the filament was never demonstrated, one needs to consider seriously that it may function as a propeller, and that the driving force originates from the flagellar structures more closely associated with the cell membrane. Krieg et al. (1967) suggested that an electrical impulse could be generated by a wave of depolarization in a polarized membrane. Hoyt ( 1 947) observed waves of excitation and recovery set up in the membrane of onion root cells upon application of an electric current. Resistance in the membrane broke down when a critical current was reached with a sudden discharge of potential; this was followed by a repeated build up of the rcsistance. Phenethyl alcohol (PEA) and irediamine A (IDA) affect the cell membrane of E . coli by altering its permeability to potassium ions. Motility in Proteus sp. is inhibited by PEA with no obvious effect on the morphology of flagella (Kopp et al., 1966). Both PEA and IDA are equally effective in inhibiting motility within 3 min. in E. coli andSalmonellaabortus (Wendt and Walls, 1968). Influx of potassium ions is inhibited by IDA. A net decrease in intracellular potassium ions is produced by PEA followed after 3 min. by an increased influx. This influx was presumed to restore the potential of the membrane which may have been upset by the ionic imbalance, although motility continued t o be inhibited. I n view of these latter findings, the mechanism of inhibition does not appear to be related to the disturbance of a polarized membrane brought about by the decreascd levels of potassium ions. The basal structure of Vibriofetus appears t o be attached to both the cell wall and cell membrane (Ritchie and Bryner, 1969). Conceivably any movement of the wall or membrane with respect to the other could generate motion on the filament. Final evaluation of the role of the membrane must await further advances concerning the structure and chemistry of the basal region, hook, and cell membrane, and more intesive studies should be made a t this level as new techniques evolve.
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Prerequisite to cell locomoation is the ability to maintain an energygenerating system presumably involved in the synthesis of ATP (Clayton, 1959). Motility is inhibited in some organisms by dinitrophenol (Meynell, 1961), potassium cyanide (Raimondo et al., 1968)) and the absence of oxygen (Preston and Sherris, 1955; Sherris et al., 1957; Shoesmith and Sherris, 1960; Adler, 1966; Adler and Templeton, 1967). Cytochrome-mediated oxidative phosphorylation is reported to provide the energy for motility in Pseudomonas fluorescens (Faust and Doetsch, 1969). Translational motility is completely prevented by addition of atabrine or antimycin plus potassium ions. The effect of atabrine is neutralized by addition of FMN. Motility is also inhibited by gramicidin D and p-chloromercuribenzoate, although the inhibition by gramicidin D is overcome by addition of either potassium or ammonium ions, and cysteine reverses the effect ofp-chloromercuribenzoate. Compounds such as oligomycin, dicoumarol, 2,4-dinitrophenol, 2-n-heptyl-4-hydroxyquinoline-N-oxide, and potassium cyanide only weakly inhibit motility in P .fluorescens. Arginine (Preston and Sherris, 1955; Sherris et al., 1957; Shoesmith and Sherris, 1960) and serine (Adler, 1966; Adler and Templeton, 1967) may be metabolized anaerobically by Pseudomonas sp. and E . coli respectively to provide energy for motility. Arginine also activates motility in cells of Clostridium sporogenes (Stanbridge and Preston, 1969). Motility of obligate anaerobic organisms is inhibited by oxygen. Photosynthetic cells of Rhodospirillum spheroides are motile only when grown anaerobically in light (Sistrom and Nemser, 1962). Nulsch and Throm (1968) measured the effect of light intensity and ATP concentration on cell velocity of R. rubrum, and found that the velocity increases with increasing amounts of added ATP. Stimulation by ATP is abolished when dinitrophenol is added. Also, ATP has no effect in the presence of light presumably due to saturation brought about by the light-induced synthesis of ATP. ATP also is reported to stimulate motility in Proteus vulgaris (DeRobertis and Peluffo, 1951). Morowitz (1954) calculated the energy required per cell for locomotion and reported that cells of B. subtilis moving a t 10 pm./sec. using chemical energy with 25% efficiency require 56 electron volts/sec. per cell. Each beat of the filament was thought to be the result of a discrete chemical event, i.e. the hydrolysis of an ATP molecule. Silvester and Holwill(l965) in considering the cilia and flagella of organisms other than bacteria suggest that contraction is brought about by the dephosphorylation of a molecule of ATP. Similarly, Brokow and Benedict (1968) detected a coupling between the hydrolysis of ATP and motility in glycerinated spermatozoa. ATP but not other organic or inorganic phosphates also appeared to cause contraction of bacterial filaments (DeRobertis and Peluffo, 1951 ; DeRobertis and
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R. W. SMITH AND HENRY KOFFLER
Franchi, 1951, 1952). Other workers, however, were not able to observe contraction in the presence of ATP (Barlow and Blum, 1952, Rinker, 1957). Filaments formed by re-assembly of flagellin from heat-disintegrated filaments of Salmonella spp. are transformed to structures with one-half the normal pitch by prolonged incubation or by dialysis in the cold versus distilled water at p H 7 (Asakura et al., 1966; Oosawa et al., 1966). The normal pitch is restored upon addition of pyrophosphate or ATP. An analogy was suggested between this tramconformation and dimorphic transitions in crystals. The similarities cited by many investigators between the filaments of flagella and muscle suggested that the energy-providing system might be located in the filaments themselves. Newton and Kerridge (1966) reported the presence of a small amount of adenosine triphosphatase (ATPase) in bacterial flagella. Weibull(1950) detected no ATP in purified preparations of flagella, but warned that an energy system may be weakly bound to the surface of the filament and removed during preparation. Filaments of P . vulgaris contain only 0.03 to 0.06% phosphorus and no detectable absorption maxima characteristic of the nucleic acid bases (Weibull, 1948). Similarly, Enomoto (1966) was unable to detect ATP or ATPase in filaments of S. typhimurium. The natural Occurrence of ATP or ATPase in the filament is highly unlikely, although they may be present in preparations of filaments as contaminants. It is now generally thought that the energy for motility is derived from within the cell probably a t or near the basal region (Astbury et al., 1956; Rinker, 1957, Abram et aZ., 1966; Enomoto, 1966; Klug, 1967). The occurrence of ATPase, hence an energy-providing system, in the bacterial membrane is well documented (Mitchell, 1963; Ishikawa, 1966; Munoz et al., 1968). Thus, energy derived from the hydrolysis of ATP is readily available in or near the basal region. It seems likely that changes in the morphology of filaments induced by added ATP in vitro are due t o the charged nature of the molecule and changes in the ionic environment, and not to any special role of ATP as an energy source in the filament proper. The existence of sensory and controlling mechanisms for the functioning of flagella was first suggested by the observations of Engelmann (1881, 1882, 1882a, 1894). He noted the bacteria tended to accumulate about the chloroplasts of Spirogyra. The attractive force appeared to be the oxygen released into the medium by the functioning chloroplasts. Similarly, cells of Spirillum volutans and S. undula were attracted to illuminated algal cells (Jennings and Crosby, 1'301). Molisch [( 1907, cf. Clayton (1%3)1 observed that photosynthetic cells growing in light immediately reverse their direction of movement when the light intensity is decreased. The effectiveness of the decrease appears t o be proportional to its abruptncss. Schrammeck (1934) noticed that a de-
321 crease in light intensity of only 5 % can cause reversal of the direction of travel. Manten (1948) reported that light of wavelengths 460, 490, 530, 590, and 870 nm. was most effective in causing reversal. The wavelengths correspond to the absorption maxima of the carotenoids and bacteriochlorophyll. I n a similar study Clayton (1953) found maximum responses at 490, 510, 530, and 550 nm. ; these wavelengths also include the maxima of the pigment spirilloxanthin. No effect was noticed with an increase in light intensity. I n some way co-ordination of flagellar movement appears to be associated with the photosynthetic apparatus. Exhaustion of energy-supplying compounds seems unlikely due to the suddenness of the response. Fleming (1950) similarly observed that a small decrease in radiant heat energy can cause a reversal of direction of movement in cells of P. vulgaris. Many cells became non-motile after a heat filter had been introduced into the light path of a microscope for less than one-fifth of a second. The phenomenon of chemotaxis, i.c. the movement of cells toward or away from a given chemical environment, necessitates the existence of some sensing and co-ordinating mechanism. I n the case of the observations of Engelmann mentioned above, the attractive force appears to be the oxygen evolved from the chloroplasts. Beyernick (1893) reported that a number of species had the ability to seek optimal concentrations of oxygen. Chemotaxis t o oxygen has been described in greater detail by Baracchini and Sherris (1959) and Adler (1966, 1966a). Cells of 8. volutans are micro-aerophilic in nature, and in liquid media form bands a t levels of optimum concentrations of oxygen. Krieg et al. (1967)noticed that thesc highly motile cells remain a t the proper level by rapidly changing the direction of rotation of their polar bundles of filaments. Filaments a t both ends of the cell, which may be 50 pm.long, change their direct>ionof rotation a t the same time. The presence of a sensing device was postulated that provides a signal simultaneously t o the driving mechanism of filaments a t each end of the cell. Perhaps the most concerted study on the mechanism and control of chemotaxis is being carried out in Adler’s laboratory (Adler, 1966, 1966a; Adler and Templeton, 1967; Armstrong et al., 1967; Armstrong and Adler, 1967, 1969, 1969a). The initial question deals with the nature of the compound that activates the chemotactile mechanism. I s it the added attractant or a product formed by metabolism of the attractant! The ability of a culture to respond to an attractant does not depend on the ability of the cells to metabolize the attractant since mutants blocked in glucose metabolism or those with a deletion mutation in the galactose operon continue to be chemotactic to glucose or galactose. These mutants do not oxidize glucose or galactose and do not incorporate radioactivity from either 14C-labelled glucose or galactose. Also, the BAOTERIAL FLAGELLA
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R. W . SMITH AND HENRY KOFFLER
ability of a number of sugars, amino acids, and metabolic intermediates to elicit the chemotactic response has no relation to the ability of these compounds to support growth. Non-metabolizable analogues such as D-fucose, a-deoxy-D-glucoside, or L-sorbose are able t o get into the cells and do cause chemotaxis. Although these observations do not rule out the possibility that the compound detected by the cells is a product of the added attractant, i.e. some product of intermediary metabolism or an energy source, the results do favour the hypothesis that the attractant itself is detected by the cells. Further questions concern the existence, nature, and location of specific chemotactic receptors on the cell. Apparently specific receptors do exist, since mutants non-chemotactic to either serine or galactose still respond to other chemicals (Armstrong et al., 1967; J. Adler, personal communication). The mutants that are non-responsive to serine continue to take up and oxidize serino a t the same rate as wild-type cells, and still respond to aspartate, galactose, glucose, and ribose. Experiments designed to test for competition between the various attractants demonstrated that glucose, galactose, fucose, and ribose are recognized by a common receptor, aspartate and glutamate by another, and serine by yet another. Also, some receptors, e.g. those for aspartate, glutamate, and serine, appear to be constitutive whereas others are inducible. The postulated receptors appear t o be located in the outer layers of the cell and are distinct from the permeases, since a galactose-permeaseless mutant still responds to galactosc. Although it seems likely that the receptors will involve some type of enzyme activity, the nature of the specific recognition sites on the ccll remains unknown. Also unknown is the mechanism by which the signal from the receptor is transmitted to the flagella. At least two classes of non-chemotactic mutants have been found, one of which is non-responsive to a given single attractant yet still responds to other chemicals and another class that fails to respond to any attractant presumably due t o lesions in reactions common t o the chemotactic response (Armstrong and Adler, 1967, 1969, 1969a; J. Adler, personal communication). I n the latter class a t least three genes appear to be involved, and these may provide the information necessary for the transmission of the signal from the receptors to the flagella. One elicitation of chemotaxis is the swarming phenomenon. Many investigators feel that swarming is the result of a negative response, i.e. that a cell is attempting to avoid a certain environment (Moltke, 1929; Russ-Munzer, 1936; Lominski and Lendrum, 1947; Hughes, 1966, 1957). Moltke (1929) and Hughes (1966, 1957) observed a high mortality among swarming cells and considered the phenomenon to result from an unfavourable local environment. The obligate anaerobes Clostridiuln butyricum and Cl. sporogenes move away from air and oxygen interfaces
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formed a t the meniscus of a capillary tube filled with growth medium (Stanbridge and Preston, 1969). If unable to avoid oxygen, the cells become non-motile. Possibly, motility and the ability t o respond to a stimulus or to avoid an unfavourable environment have survival values (Smith and Doetsch, 1969). Hoeniger (1964) examined the swarming process using time-lapse photography with the phase microscope and concluded that most of the cells are normal and viable. The occurrence of highly motile cells appears to be a normal part of the life cycle of the organisms. Chemotaxis is inhibitfedby p-nitrophenylglycerol (Kopp et al., 1966) and chloramphenicol (Fleming et al., 1967; Fleming and Williams, 1968), presumably by some effect on the sensory mechanism. These observations may be interpreted as indicating the involvement of a lipoprotein membrane. p-Nitrophenylglycerol also inhibits swarming of P . mirabilis apparently without affecting normal motility or the formation of large swarm cells (Williams, 1969). Schuetze and Doetsch (1967) observed that cells of E . coli, Pseudornonas fluorescens, and Serratia marcescens are immediately rendered non-motile if the medium is adjusted to pH 9 with carbonate-bicarbonate buffer. No effects on the morphology of the filaments were observed. Motility is rapidly restored if the pH is re-adjusted to neutrality. If instead of with a carbonate buffer, the p H value is adjusted with tris buffer, sodium hydroxide, or a sodium borate buffer, motility is inhibited but not shut off immediately, and the cells retain a sluggish movement. Cells of E . coli were alternately rendered motile and nonmotile five times within a 30 min. period by adjustment of the pH value between 6.5and9.5, although the response a t pH 6.5 became progressively less vigorous. If the inhibition were due to subtle pH-dependent changes in conformation of the proteins in the filament, all the agents tested should probably have had similar effects on motility. The differential responses to the various agents used to adjust the p H and the observation that the morphology of the filaments was not altered suggests a more specific effect on the structures or reactions that either activate or drive the filaments. Certain phages attack bacterial cells by attaching to flagellar filaments (Sasaki, 1960, 1961, 1962; Yamaguchi and Iino, 1968; Raimondo et al., 1968; Betz, 1969). Although cells of B. subtilis may have 15-20 flagella per cell, attachment of a single particle of phage PBS-1 appears to inhibit activity of all the flagella (Raimondo et al., 1968).These date suggest that functioning of all filaments is under the control of one “motor”. It is difficult a t present to construct a likely mechanism whereby attachment per se of a phage particle to a filament a t a point well removed from the cell body could inhibit the functioning of other flagella. One would
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suspect that some chemical event must take place inside the cell, but the mechanism remains a mystery. Perhaps the most striking evidence for a mechanism co-ordinating the function of flagella is found in cells of Spirillum volutans. As mentioned previously, this organism has tufts of flagella a t each pole that rotate to form cones of revolution. Normally, the broad base of the cones faces away from the direction of travel. The cones, therefore, may be differentiated into head and tail types. Metzner (1919)found that chloroform, ether, or acetone placed a t the edge of a cover slip causes an inversion of the tail cone of revolution. These cells then possess the head-type pattern of rotation a t both poles. Although the filaments continue to rotate rapidly the generation of opposing forces prevents locomotion. Halogen salts cause an inversion of the head type cone and produce non-motile cells although the flagella still function. Krieg and Tomelty (1967)observed that 0.62% chloral hydrate or 0.078 to 0.166% phenol induces the formation of cells with the head-type rotation a t both ends. Cells with the tail-type movement a t each end are found in the presenco of 3.76% magnesium sulphate or 0.312% nickel sulphate. Other copper, nickel, and magnesium salts have similar effects (Krieg et aZ., 1967). Higher concentrations prevent the rotation of the filaments. The effect of chloral hydrate can be neutralized by nickel sulphate, and a t null point concentrations, the bundles of filaments continually reverse their direction of rotation. Reversal requires less than l/l6th.of a second as determined by motion pictures taken a t 16 frames/sec. The state of inco-ordination can be maintained for 30 to 60 min., a time a t which the activity of flagella stops. Krieget al. (1967)proposed that the site of action is the cell membrane. Cations could interfere with the ionic imbalance necessary to produce a polarized membrane that in some way determines the direction of rotation. It may not be necessary t o involve the cell membrane proper in such a mechanism, however, since special polar membrane linked to the cell membrane by delicate bars and surrounding the areas of insertion of flagella has been reported in sections of S. serpens (Murray and Birch-Anderson, 1962, 1963). A similar “flagellar membrane” has been described in Rhodospirillum spp. (Cohen-Bazire and London, 1967). I n Proteus vulgaris (Abram et al., 1966)and P.mirubitis (van Iterson et al., 1966)basal structures appear to be interconnected; while such a network probably represents an artifact, it possibly could function in the stimulation and co-ordination of flagellar activity. The existence of genetic determinants controlling either the synthesis or the operation of the machinery that in turn controls the movement of flagella is suggested by the isolation of flagellated non-motile mutants (Kauffmann, 1939;Edwards et al., 1946;Hirsch, 1947).These mutants
BAUTERIAL FLAQELLA 325 have a full compliment of flagella that are antigenically similar t o those of wild-type cells; the mutation, however, rendered the flagella nonfunctional. Enomoto (1962) describes a gene termed “mot” distinct from the structural genes that code for flagellin. Flagellated but paralysed cells of Salmonella apparently have a defect in the flagella-activating mechanism and not in the flagellin molecules themselves. Motile mutants of P. mirabilis that had lost the ability to swarm regained that ability upon transduction with DNA from either a flagellated or a non-flagellated culture (Coetzee, 1963). A swarming locus was postulated, which is functionally separable from the H andfla loci and is apparently involved with the sensory mechanism. In addition, a modifier gene designated as “2” was described that controls the physical appearance of a swarming culture, and that does not cotransduce with the swarming locus, Based on the observations of Hoeniger (1965) that the physical appearance of a swarming culture is influenced by the type of flagellation which varies with different stages of the growth cycle, Joys (1968) concluded that the Z locus is probably concerned with the growth cycle of the cells and not specifically with motility. The 2 locus could more specifically be involved in determing the number and possibly the location of flagella on the cell surface, i.e. be closely related in function to thefla loci. I n a n oral report T. Iino (Annual Meeting of Genetics Society, Japan, 1969) described a mutant of Pseudomonas aeruginosa that appeared motile in the light microscope but was incapable of spreading on semi-solid agar. The spr- locus was mapped and was located as follows :
leu *
-
(spr-H-flaA-fla B-fla C)
- - ade *
* * *
tryp
*
- his.
The specific function of the spr locus is not known. Another genetic determinant that may control flagella function is suggested by the observations of Vary and Stocker (1969). An amber mutant of S. typhimurium LT2 possesses functional flagella, but exhibits a n abnormal type of motility in that the cellsfrequently change their directionof travel. Vary and Stocker suggest that the locus may be involved in the chemotactic response. If so, it probably is associated with the activation mechanism as opposed to the sensing mechanism, since the phenomenon was observed in a medium of homogeneous composition. Three mot loci, mot A, mot B, and mot C, have been defined by complementation tests in 97 mutants of S. typhimurium that have a defect in the flagella-activating mechanism (Enomoto, 1966, 1966b ; Enomoto and Yamaguchi, 1966). Cotraiisduction was observed between mot A and mot B, and between mot C and H I . Mutants with deletions overlapping mot A and mot B were identified. Apparently, therefore, mot A and mot B lie next to each other, while mot C is located a t some point away from these. Mapping of the mot loci was accomplished by sexual
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R . W. SMITH AND HENRY KOBFLER
recombination between Hfr and paralysed F- strains of 8. abony (Enomoto, 1966~). The order of loci was determined to be as follows :
met-ser-HZ-his-mot G H l m o t B-nwt A-leu The location of additional loci concerned with the synthesis of flagellin and flagella and the control of locomotion has been examined in several reports (Joys, 1961; Joys and Stocker, 1963; Iino, 1964; Pearce, 1966; Iino and Enomoto, 1966; Enomoto, l966,1966a, b ; Yamaguchi and Iino, 1967). The results of these studies have been summarized by Joys ( 1968) with all loci mapping into two linkage groups as follows: f la J-f la B-f la D-f la Gf la A-mot C - H I - a h m l and HZ-ah2-vh2 T. Iino (1970, personal communication) had developed the linkage map shown below based on recombination and deletion mapping with mutants ofS. abortus-equi : cys * * * (HZ-ah2-vh2) * sur (7. * his * sup W * * fla D . fla B-(fla A , mot c)-nml-Hl-ahl-flaLfla (J,E)-fla K-mot B-mot A-fla c-fla M tre tryp * *fluf * * aroH. Armstrong et al. (1967) and Armstrong and Adler (1967) describe 40 mutants of E . coli that although motile are non-chemotactic. The mutants have lost the ability either t o detect or to respond t o a chemical gradient. Three complementation groups were suggested but not sharply defined by transduction experiments. Groups I and I1 are well defined but members of group I11 react to an intermediate degree with some members of the first two groups. Crosses between mutants with curly filaments and those with paralysed flagella result in motile cells. Apparently, the mot and H loci are not closely linked. Recently, Armstrong and Adler (1969) mapped the loci controlling chemotaxis (che) in E . coli by recombination. Three loci are defined, che A, che B, and che C , and arc located as shown. The numbers indicate minutes from thr a t 37".
-
-
-
-
0
- -
-
Interestingly many of the genes appear analogous to those described in S. typhimurium. The che loci have also been defined by abortive transduction to test for complementation (Armstrong and Adler, 1969a).
BACTERIAL FLAGELLA
327
Che A, che B, and che C can be separated as three complementation groups distinct from those designated as I, 11,and I11above. The mechanisms by which the mot and che loci control the activity of flagella are not known. Perhaps the most likely level for the exertion of this control is either a t the basal structure, cell membrane, or the hook.
XIII. Acknowledgements Current work in our laboratory has been performed with the capable technical assistance of Mrs. Florence Shen and Mrs. Willa Mae Curry. We wish t o acknowledge financial support from the United States Public Health Service in the form of grants A100685 and GM 10857 from the National Institutes of Health and GM-06329 from the National Science Foundation.
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Vary, P. S. and Stocker, B. A. D. (1969).Bact. Proc., 53. Vegotsky, A., Lim, F., Foster, J. F. and Koffler, H. (1965).Archs Biochem. Biophys. 111, 296. Vegotsky, A., Moses, H. A., Koffler, H. and Parker, H. E. (1965a).Abstr. Biophys. S O C . ,149. ~. Vennes, J. W .and Gerhardt, P. (1959).J. Bact. 77,681. Wakabayashi, K., Hotani, H. and Asakura, S. (1969). Biochim. biophys. Acta 175,195. Weibull, C. (1948).Biochim. biophys. Acta2,351. Weibull, C. (1949).Biochim. biophys. Actu 3,378. Wcibull, C . (1950).Nature, Lond. 165,482. Weibull, C. (19504. Ark. Kemi 1,573. Weibull, C. (195Ob).Actachem. scand. 4,268. Weibull, C . (19500).Ark. Kemi 1,21. Wcibull, C. (1950d).Actuchem. scuiad. 4,260. Weibull, C. (1951).Nature, Lond. 167,511. Weibull, C. (1953).J.Bact. 66,688. Weibull, C. (1953a).Actu chem. scand. 7,335. Weibull, C. (1960). In “The Bacteria”, (I. C. Gunsalus and 1%.Y. Stanior, ods.), Vol. 1, p. 153, Academic Press, New York. Weibull, C. and Tiselius, A. (1945).Ark. Kemi 20 B , no. 3. Weil, E. andFelix,A. (1917). Wien. Klin. Wschr. 30,1509. Weinberg, E . D. andBrooks, J. I. (1963).Bact. Proc. 43. Weinberg, E. D. and Brooks, J. I. (1963a).Nature, Lond. 199,717. Weinstein, D. (1959). Ph.D. Thesis : Purdue University, Lafayottr, Indiana, U.S.A. Weinstein, D., Koffler, H. andMoskowitz, M. (1960).Bact. Proc. 63. Weitzenberg, R. (1935).Zentbl. Bakt. ParasitKde 133,343. Wendt, L. W. and Walls, N. (1968).Bact. Proc. 29. Wiame, J . M., Storck, R. and Vanderwinkel, E. (1955). Biochim. biophys. Acta 18,353. Williams, F. D. (1969). Bact. Proc. 30. Williams, M . A. and Chapman, G. B. ( 1 9 6 1 ) J . Bact. 81,196. Wilson, C. E., Donati, E. J., Petrali, J. P., Vuicich, V. and Sternbcrger, L. A. (1966).Exp. molec. Path. suppl. 3, p. 44. Winebright, J. and Fitch, F. W. ( 1 9 6 2 ) J .Immun. 89,891. Yaguchi, M., Foster, J. F. and Koffler, H. (1964). Abstr. 6th. Int. Congr. Biochem., New York, p. 189. Yamaguchi, S. (1965).Rep. natn. Inst. genet., M k i m a 16,93. Yamaguchi, S . and Iino, T. (1966).Rep. natn. Inst. Genet., M k i m a 17,119. Yamaguchi, S . and Iino, T. (1967).Rep. natn. Inst. Genet., Misima 18,114. Yamaguchi, S . andIino, T. (1968).J.gen.Virol.2,187. Yamaguchi, S. and Iino, T. (1969).J.gen. Microbial. 55,59. Yamamoto, J. (1910). Zentbl. Bakt. ParasitKde 53,38. Yarbrough, L. R., Koffler,H. andFoster, J. I?. (1969).Abstr. biophys. SOC. 9, A-107. Yokota, K. (1925).Zentbl. Bakt. ParmitKde 95,261. Yuasa, A. (1936). Bot. Mug., Tokyo 50,93.
14
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AUTHOR INDEX Numbers i n italics refer to the pages on which references are listed at t ? end ~ of each article.
A
Avers, C. J., 67, 99, I00 Ayad, S. R., 51,52,100
Aamodt, L. W., 297,305,306,327 Abbo, F. E., 53, 59, 62, 99 Abbot, A., 239, 242, 244, 246, 247, 248. 249, 252, 258, 259, 286, 292, 327 Abraham, G., 219,336 A b r m , D., 224, 226, 227, 228, 230, 231, Babes, V., 223, 328 232, 233, 234, 238, 239, 244, 245, 247, Bacchetti, S., 90, 100 263, 264, 268, 275, 276, 277, 278, 279, Baker, R. F., 219, 332 280, 282, 284, 286, 287, 310, 320, 324, Balteanu, J., 251, 260, 328 Baracchini, O., 321, 328 327 Barker, H. A., 35, 44, 107, 118, 120, 122, Abrams, R., 66,106 131,145,146 Abron, H. E., 248, 249, 263, 264, 327 Ada, G. L., 239, 242, 244, 246, 247, 248, Barlow, G. H., 320, 328 249, 252, 258, 259, 260, 286, 292, 294, Barth, P. T., 97,98,104 Baserga, R., 96,100 327,335,336 Basu, D., 3, 44 Adachi, K., 2, 42, 44 Bauchop, T., 142, 145 Adelberg, E. A., 302, 329 Adler, J., 298, 300, 319, 321, 322, 326, 327, Beumann-Grace, J. B., 252,338 Bayly, R. C., 10, 12, 14, 27, 42 328 Bechet, J., 69, 93, 100 Adye, J., 260, 261, 327, 328, 333 Beckwith, J. R., 57,60,IOI, 104 Alexander, M., 3, 28, 34, 42, 44, 45 Beighton, E., 223,230, 316, 320, 328 Allam, A. M., 129, 130, 146 Bello, L.J., 55, 66, 100 Ambler, R. P., 246, 248, 328, 338 Belyavin, G., 262,328 Anagnostopoulos, C., 301,328 Bender, M. A., 53,104 Anderson, E. C., 49, 75, 76, 99 Bendigkeit, H. E., 48, 52, 103 Anderson, T. F., 224,226,252,335 Benedict, B., 319, 328 Andrewes, F. W., 253, 328 Benke, P., 95, 105 Apirion, D., 173, 217 Benzer, S., 71, 100 Arbogast, R., 181, 185, 215 Berg, P., 150, 215 Arce, A,, 51, 104 Berlin, C. M., 93, 105 Archibald, W. J., 248,328 Bernstein, A., 267, 328 Arias, I. M., 93, 106 Bernstein, K., 187,188,189,215 Arima, K., 2, 14, 45 Bessman, M. J., 150, 216 Arkwright, J. A., 220, 328 Betz, J. V., 231, 233, 323, 328 Armstrong, J. B., 321, 322, 326, 328 Beutler, E., 95, 100 Arnold, J. B., 75, 76, 99 Beyer, H. G., 251,328 Asaki, K., 226, 338 Asakura, S., 220, 229, 239, 244, 248, 260, Beyernick, M. W., 321,328 275, 284, 287, 292, 295, 308, 309, 316, Bigelow, C. C., 263, 328 Bigwood, E. J., 244,337 320,328,336,339 Astbury, W. T., 223, 230, 315, 316, 320, Bilton, R. F., 35, 42 Birch-Anderson. A., 224, 226, 227, 228, 328 295,324,335 Atkineon, D. E., 136,145 Austin, C. M., 262, 259, 260,294, 328, 336 Bird, J. A., 39, 42 341
342
AUTHOR INDEX
Birnstiel, M. L., 161, 217 Bishop, C. F., 66, 103 Bishop, D. H. L., 51,52,105 Bisset, K. A., 224, 298,312, 328 Blackman, F. F., 196, 215 Blakley, E. R., 33, 42 Blaylock, B. A,, 126, 129, 130, 133, 139, 145 Blendon, D. C., 222,328 Blobel, Q., 93, 100 Blout, E. R., 290, 337 Blum, J. J., 320, 328 Bock, R. M., 67, 69,82,84, 87,101 Boivin, A., 240, 328 Bokkenheuser, V.. 264,328 Boll, M., 93, 101 Bollag, J.-M., 28, 44 Bolle, A., 5G, 100 Boltjes, T. Y. K., 219, 328 Boniface, J., 201, 217 Booth, J.,26, 42 Bootsme, D., 51, 100 Boquet, P., 297,328 Borkenhagen, L. F.,12, 13, 46 Borun, T.W., 91, 100, 104 Bostock, C. J., 64, 68, 69, 86, 91, 100 Boulgakov, N. A., 267, 337 Bovee, E. C., 220,230,317,332 Bevre, K., 66, 98, 105 Boyland, E., 26, 42 Bradford, S.,263, 297, 306, 329 Braun, H., 220, 261, 260, 277, 316, 328 Braun, R., 54,66, 103 Hremer, H., 161, 215 Brent, T. P., 61, 52, 66, 70, 100 Ihesler, M. A., 103 Brow, K., 99, 106 Brieger, E. M., 316, 328 Brill, W. J., 129, 130, 146 Rrinton, C., Jr., 296, 336 Britten, R. J., 96, 100, 173, 178, 201, 215, 216 Rroda, P., 166, 215 Brokow, C. J., 319, 328 Bronfenbrenner, J., 185, 216 Brooks, J. I., 299, 339 Broquist, H. P., 70, 102 Brot, N., 142,145 Brown, D. G . , 143,145 Brown, D. M., 241,242,244,248,268,269, 276, 287, 292,329, 334 Brown, E. R., 300, 302, 328 Bruner, D. W., 263, 324,329,330 Bryant,M.P., 109, 110, 113,114,117,118, 122, 123, 124, 139,145 Bryner, J. H., Jr., 226, 231, 280, 282, 318, 337 Budke, L., Sl,!lOO
Buell, D. N., 100 Bui, J., 244, 248, 286, 33s Bunge, R., 223, 329 Burge, R. E., 220, 279, 314, 317, 329, 331 Buswell, A. M., 6, 45 Buswell, J. A., 29, 30, 42 Butler, J. A. V., 61, 52, 66, 70, 100 Butschli, O., 223, 277, 314, 329
C Cain, R. U., 13, 28, 33, 35, 39, 41, 42, 43 Caldwell, M. E., 297, 332 Caldwell, W. J., 222, 320 Calendar, R., 150, 215 Calvin, M., 2, 21, 44 Cameron, I. L., 48, 100, 104 Campbell, A., 48, 49, 50, 100 Campbell, A. M., 183, 215 Campbell, J. C., 220, 229, 269, 275, 295, 312,338 Campbell, L. L., 263, 329 Campbell, W. L., 46, 46 CBnovas, 5. L., 38, 39, 40, 41, 42, 42 Cantoni, E. C., 66, 100 Cardini, G. E., 26, 43 Carhart, C. R., 220, 222, 334 Carnahan, J., 295, 336 Cardrtw, H. S., 205, 215 Cartor, B. L. A., 63, 68, 69, 82, 101 Carter, C. E., 186, 216 Cartwright, N. ,J., 29, 30, 42 Cassandra, M., 90, 100 Caasim, J. Y., 290, 32:) Catelani, D., 13, 14, 42 CeMars, R., 95, 105 Chamberlain, E. M., 17, 42 Champneas, J. N., 288, 280, 317, 329 Chang, J. Y., 242, 244, 248, 329 Chapman, G. B., 226, 339 Chapman, P. J., 3, 10, 16, 17, 20, 29, 42, 43,44 Chargaff, E., 64, 105 Chatterjee, S. N., 226, 238, 329 Cherry, W. B., 300, 302, 328 Chinoy, I., 40, 4G Clark, A. J., 302, 329 Clark, F. M., 6, 42 Clark, P. G., 166, 215 Claus, D., 27, 42 Claus, G. W., 282, 329 Clayton, R. K., 319, 320, 321, 329 Clowes, F. A. L., 91, 100 Coetzee, J. N., 326, 329 Coffman, R., 162, 153, 164, 209, 215, 216
343
AUTHOR INDEX Cohen, C., 290,337 Cohen, G. N., 201, 213, 216 Cohen, S. S., 181, 185, 215 Cohen-Bazire, G., 224, 226, 227, 228, 230, 231,324, 329 Cohn, F., 219, 329 Cole, F. E., 65, 70, 100 Collins, A., 93, 103 Collins, J., 97, 98, 10.1 Collinsworth, W. L., 12, 42 Colquhoun, D. B., 220, 329 Comes, R., 239, 244, 329 Conn, H. J., 222,329 Conn, J. E., 222, 330 Cook, T. M., 314, 329 Coon, M. J., 3, 44 Cooper, R. A., 16, 42 Cooper, S., 98, 100 Corbett, J. J., 51, 100 Corrivaux, D., 69, 104 Cottrell, S. F., 67, 99, 100 Coulter, A. W., 10, 12, 42 Cox, C. D., 233, 235,335 Cox, C. G., 67, 69, 86, 87, 88, 100 Cragie, J., 252, 260, 329 Crandall, D. I., 16, 45 Crathorn, A. R., 51, 52, 66, 70, 100 Creanor, J., 57, 64, 68, 72, 75, 85, 88, 89, 90,103 Crippa, M., 54, 100 Crocker, C. G., 219,336 Crosby, J. H., 320,332 C-ings, D. .J., 51, 52, 59, 100, 10% Cummins, J. D., 97, 100 Cummins, J. E., 54, 100 Curran, J. P., 75, 106 Cutler, R. G., 51, 53, 55, 100
de Bary, F., 219, 329 De Deken-Grenson, M., 69,100 De Lange, R. J., 242,246, 331 Demerec, M., 302, 329 Deppe, C. S., 152, 153, 155, 156, 182, 183, 184, 190,216 DeRobertis, E., 220, 238, 277, 319, 329 De’Rossi, G., 239, 329 Diena, B. B., 299, 306,329, 338 Dimitrijevie’-Speth, V., 220, 329 Dimmick, J. F., 108, 146 Dimmitt, K., 235, 253, 297, 306, 329 Dintzis, H. M., 150, 215 DiPierro, J. M., 252, 329 Dodson, R. M., 10,43 Doetsch, R. N., 220, 229, 230, 252, 275, 295, 297, 298, 299, 306, 314, 317, 319, 323,329,330,337, 338 Domnas, A., 65, 100 Donachie, W. D., 49, 57, 58, 59, 61, 62, 64, 68, 69, 72, 73, 77, 78, 79, 80, 85, 89, 91, 96, 100, 103 Donati, E. J., 252, 312, 339 Dost, F. N., 144,145 Doudoroff, M., 33, 45 Douglas, D. W., 254, 329 Douglas, H. C., 69, 100 Douthet, H. A., 53, 74,102 Doyle, D., 93, 105 Drews, G., 226, 228, 338 Du Bus, R., 2,45 Duff, R . B., 29, 45 Duncan, J . T., 267, 329 Dutton, P. L., 6, 43 Duxbury, J. M., 28, 34, 45
E D Dagley, S., 3, 10, 12, 14, 16, 17, 20, 25, 26, 27, 29, 32, 41, 42, 43, 44 Dahl, M. M., 321, 322,326, 328 Dahlberg, D., 93, 106 Daly, J. W., 22, 23, 25, 26, 43, 44,46,46 Darlington, G. A., 94, 102 Darrah, J. A., 35, 42 Das, J., 226, 238, 329 Datta, N., 302, 338 Davidaon, E. H., 95, 100 navies, J. I., 25, 32, 33, 43 Davies, R., 76, 101 Davies, S. N., 254, 329 Dawson, J. E., 28, 34, 4 5 Dawson, P. S. S., 50, 51, 100 Dean,A. C. R., 187,215
Eagle. H., 93, 100 Eckstein, H., 67, 100 Edlin, G., 165,215 Edwards, P. R., 253, 254, 302, 324, 328, 329,330,333 Eguehi, G., 229, 239, 248, 260, 275, 284, 287, 292, 296, 308, 309, 316, 320, 328 Ehrenberg, C. G., 219,330 Ehrenfeld, E., 166, 215 Eiler, J. J., 51, 104 Eisenstadt, J. M., 297, 305,306, 327 Eisner, U., 9, 43 Elek, S. D., 239,252,253,280,330 Elkind, M. M., 90, 101 Ellar, D. J., 320, 335 Elliott, G. F., 220, 275, 282, 316, 334 Ellis, D., 223, 330 Elsden, 8. R., 121,145 Elvidge, J. A., 9, 43
344
AUTHOR INDEX
Emiliani, C., 76, 76, 99 Engelberg, J., 48, 49,101, 104 Engelmann, T.W., 320,330 Enomoto, M.. 230, 304, 311, 313, 320, 326, 326,330,332 Epifanova, 0. I., 96, 101 Epstein, R., 63, 64, 67, 68, 69, 70, 74, 82, 84, 86, 87, 89, 101, 104. 108 Epstein, R. H., 66,100 Epstein, W., 67, 101 Erdtman, H., 30, 43 Erhan, S., 97,101 Erikson, E. J., 262, 331 Erlsnder, S. R., 248, 264, 276, 330 Errington, F., 96, 104 Esposito, R. E., 90, 101 Evans, C. G. T., 187, 217 Evans, J. E., 51.63,66.100 Evans, W. C., 6, 14, 17, 26, 32, 33, 34, 43, 44,45
Forslind, B., 279, 282, 330, 338 Foster, J. F., 248, 264, 266, 273, 276, 288, 289, 290, 291, 292, 306, 330, 333, 339 Fowlks, W. L., 21,44 Fox, M., 61, 62,100 Franchi, C. M., 238, 277, 320, 329 Frankel, R. W., 303, 332 Franko, E. A., 97, 101 Freer, J. H., 320,335 Freudenberg, K., 28, 43 Friedman. B. L., 66,101 Friedman, S. M., 263,330 Friesen, J. D., 166, 215 Fry, K. A., 263, 330 Fuhrman, F., 223,330 Fujisctwa, H., 2, 34, 43, 44 Fukumi, H., 239,240, 264,335,338 Funess, G., 302,331
G
F Fairbanks, V. F., 96, 100 Falcone, G, 97, 104 Fangman, W. L., 61, 101, 160, 215 Farmer, V. C., 29, 45 Farqujar, M., 269, 276,330 Farr, D. R., 13.28, 3 3 , 4 1 , 4 2 , 4 3 Faust, M. A,, 319,330 Feiler, M., 261, 260, 330 Feist, C. F., 33, 41, 43 Feldman, L. A., 316, 330 Feldman, O., 48, 102 Felix, A., 220, 261, 339 Ferguson, J. J., 93, 101 Fernley, H. N., 26,43 Ferretti, J. J., 63, 69, 61, 101 Fewson, C. A., 33, 36, 43, 44 Fiandt, M., 66, 98, 105 Fiecchi, A., 13, 14, 42 Fiechter, A., 68, 103 Fife, M. A., 264, 330 Filahie, B. K., 220, 231, 337 Fina, L. R., 6, 42 Fink, 0. R., 69,101 Fisoher, A., 220,223,301, 330 Fisher, C. R., 296.330 Fisher, P. J., 222, 330 Fitoh, F. W., 262, 269, 330, 339 Fitz-James, T. C., 61, 101 Fleming, A., 321, 330 Fleming, R. W., 323, 330 Focht, D. D., 28, 43 Follett, E. A. C., 238,239, 269,330, 331 Forchhammer, J., 167,215
Caertner, F. H., 244, 290, 306, 331, 333, 337 Gaffield, W., 290, 338 Gafford, R. S., 9, 43 Gale, E. F., 76, 101 Gall, J., 161, 215 Galli, E., 13, 14, 42 Gallwitz, D., 91, 101 Canesman, A. K., 60, 101 Ganguli, B. N., 3, 44 Gsnsohow, R., 93,105 Carbosky, A. J., 299,338 Gerd, S., 239, 261, 252, 331 Gascbn, S., 86, 101 Ceary, P. J., 20, 43 Geidusohek, E. P., 66, 100, 161, 152, 165, 215 Gelbard, A. S., 66, 101 Gelehrter, T. D., 91, 92, 106 Gerber, B. R., 288, 291, 331 Gerhardt, P., 262, 298, 339 Gibson, D. T., 10, 14, 20, 26, 27, 29, 41, 42, 43,46,46 Giesbrecht, P., 262.331 Gilbert, C. W., 94, 101 Gilbert, J. B., 07, 69, 86, 87, 88, 100 Gillert, I<. E., 262, 331 Gillette, J. R., 26, 44 Glaaer, D. A., 72,80,104 Glauert, A. M., 220, 231, 233, 238, 260, 264,276,279,282,331,333 Glazer, A. N., 241, 242, 244, 246, 248, 268, 269, 276, 287, 292, 329, 331, 334 Gnosspelius, A,, 264,331 Goldberg, H. S., 222, 328 Goldenberg, H., 206,206,215
AUTHOR INDEX
Goldin, M., 262, 335 Goodman, D., 161,216 Goodwin, B. C., 60, 61, 69, 77, 78, 79, 81, I01 Gordee, E. Z., 296, 298, 309, 334 Gordon, J., 238,239,269,330,331 Gordon, M. A., 300, 302,328 Gorman, J., 63,64, 67, 68, 70, 82, 84, 101 Goto, S., 262, 263, 331 Grace, J. B., 226,331 Graham, C., 48, 101 Granner, D. K., 66, 69, 72, 76, 91, 92, 93, 103,106 Grant, D. J. W., 36,43,44 Grant, G. F., 268, 302, 313, 314, 331, 338 Gray, E. D., 63,69,61, I 0 1 Gray, P. H . H., 222, 331 Greenberg, L., 299, 306, 329, 338 Greenbury, C. L., 262,331 Griffin, A. M., 297, 331 Griffiths, B. W., 299,338 Griffiths, E., 26, 33, 43 Gross, C., 61, 101 Gross, S. R., 9, 43 Gruachka, T., 220,261, 260, 331 Guha, A., 66, 98,105 Gulyas, S., 61,106 Gunsalus, I. C., 2, 3, 43, 44, 45 Guroff, G., 22, 26, 43 Guyer, M., 7, 43
H Hageage, 0. J., 220, 329 Hale, C. M. F., 224, 312, 328 Hall, B. D., 166, 217 Halvorson, H., 33, 42 Halvorson, H. O., 62, 63,64, 66, 68, 64, 67, 68, 69, 70, 72, 73, 74, 76, 77, 78, 81,82, 83, 84, 86, 86, 87, 89, 99, 101, 102, 103,104, 105,106 Hamburger, K., 63, 101 Hamilton, A. I., 94, 101 Hancock, G. J., 314,331 Hansche, P. E., 60,61, 101 Hansen, J. N., 66, 74,101 Hanson, J., 220, 238, 239, 269, 275, 278, 279, 280, 282,316.334 Happold, F. C., 33,43 Harrington, W. F., 292,332 Hartmen, P. E., 302,329 Haruna, I., 311, 332 Hasellcorn, R., 62, 103, 161, 162, 166, 215 Heskill, J. S., 260, 336 Hatani, S., 220. 229, 244, 287, 320, 336 Hawkins, C., 91,103 Hawthorne, D. C., 83,86,104
345
Hayaishi, O., 2, 3, 4, 13, 20, 21, 22, 26, 27, 33, 34, 36, 43, 44, 45 Hedegmrd, J., 3, 43 Hegeman, G. D., 6, 7, 20, 33, 36, 41, 43, 44 Heller, L., 261, 262, 331 Helmstetter, C. E., 48, 61, 62, 80, 97, 98, 100,101,102 Henderson, D. W., 261, 331 Henrici, A. T., 186, 215 Hensley, M., 26, 27, 4 3 Herman, A., 64, 70, 76, 101, 102 Hershey, A. D., 186, 186, 215, 216 Hesp, B., 2, 21, 44 Heumann, W., 233,239, 277, 334 Hierholzer, G., 64, 102 Higa, A., 68, 102 Higashi, H., 2, 45 Higashi, S., 287,308,336 Highman, W., 239, 262, 263, 280, 330 Hijkamp, H. J. J., 66.98, 105 Hill, R. L., 99, 106 Hilz, H., 67,100 Hinegardner, R., 48,102 Hinshelwood, C., 183, 187,215,226 Hinterbergor, A., 223,331 Hirose, S., 69, 82, 104 Hirota, Y., 97,102 Hirsch, H. R., 49,101 Hirsch, W., 324,331 Hodge, L. D., 63,102 Hoeniger, J. F. M., 224, 227, 229, 230,231, 233, 269, 282, 312, 323, 324, 326, 331, 338 Hofer, A. W., 222,331 Hoffman, A., 262,335 Hofman, S., 262,331 Hognees, D. S., 60, I02 Holliday, R., 91, 102 Holowczak, M. A. K., 263, 331 Holt, S. C., 233, 236, 335 Holtzman, J., 26, 44 Holwill, M. E. J., 220, 314, 319, 329, 331, 337 Holzer, H., 64, 93, 101, 102 Homma, J. Y.. 262,263. 331 Hood, J. R., 62,96, 105 Hopkins, H. A., 61, 62, 105 Hopper, D. J., 3, 16, 42, 44 Horiuchi, T., 62, 72, 73, 104, 192, 216 Horne, R. W., 224, 226, 231. 233, 238, 260, 264, 276, 279, 282. 331, 333, 337, 338 Hosokawa, K., 2, 21.44 Hotani, H., 287,292,339 Hotta, Y., 66. 70, 72, 74, 102 Houwink, A. L., 219, 220, 222, 224, 226, 231, 331
346
AUTHOR INDEX
Joos, A., 261, 332 Jordan, E. O., 297, 332 Josephs, R., 292,332 Joys, T. M., 220, 229, 244, 297, 302, 303, 311,326,326, 332 Juempel, P. L., 68,103 Julien, J., 166, 216, 217
Howard, A., 48, 102 Howell, L. G.. 46, 46 Hoyem, T., 63, 74, 102 Hoyt, It. C., 318, 331 Hradecna, Z., 66, 98, 105 Hsu, T. C., 94, 102 Hueppe. F., 219,331 Huoy, C. R..264, 330 Hugh, R., 222, 264, 334 Hughes, D. E., 27, 44 Hughes, W. H., 322, 332 Hungate.R. E., 110, 113, 118, 146 Hunt, A. L., 27, 44 Hurwitz, J., 160, 216
K
I Ichiki, A. T., 268, 2G9, 279, 294, 334 Ichiyama, A., 20, 44 Idriss, J., 74, 105 Iino, T., 220, 223, 229, 239, 248, 276, 278, 284, 287, 292, 296, 303, 304, 306, 307, 308, 309, 313, 314, 316, 320, 323, 326, 331, 332, 333, 335, 338, 339 Ingraham, J. L., 9, 33 Inoue, H., 34, 44 Inouye, M., 97, 102 Irion, E., 133, 145 Ishikawa, S., 320, 332 Itada, N., 27, 44 Ivos, D. H., 66, 70, 105 Iwanura, T., 63, 102 Iwayama, Y., 2, 42
304, 331,
253. 299, 310, 328,
260, 302, 311, 330,
J Jacherts, D., 295, 332 Jacob, F., 76, 97, 102 Jaegor, J. C., 206, 215, 216 .Jahn,T. L., 61, 105, 220, 230, 317, 332 Jarosch, R., 222, 316, 317, 332 Jeffrey, A.M., 46,46 Jenkins, C. E.. 262, 332 Jennings, H. S., 320, 332 Jerina, D. M., 22, 23, 26, 26, 43, 44, 46, 46 Joetten, K. W., 261, 260, 332 Johns, A. T., 120, 145 Johnson, B. F., 39, 41, 42 Johnson, F. H., 219, 224, 332 Johnson, P. A., 26, 27, 43 Johnson, R. A., 66. 10% Johnston. W. H., 76, 76, 99 Jones, E. E., 70, 102 Jones, R. F., 61, 66, 102 Jones, T. C., 61,102
Kabat, E. A., 266,332 Kagamiyama, H., 2, 13, 33. 44 Kajiwara, K., 91, 105 Kallio, R. E., 26, 43 Kamath, S. A., 97, 101 Kamimoto, M., 2, 44 Kamin, H., 46, 46 Kanetsuna, F., 34, 44 Karlsson, J. L., 36, 44 Katsai, M., 220, 229, 244, 287, 320, 336 Kassarevitch, Y., 69, 102 Kasten, R. H., 94, 102 Katagiri, M., 3, 27, 44, 45, 46, 46 Kates, J. R ., 61, 65, 102 Kato, I., 264, 330 Kauffinann, F., 220, 263, 254, 324, 330, 332 Kaufman, S., 22, 44 Kauzmann, W., 291,332 Keeler, R. F., 226, 231, 280, 282, 337 Kelley, W. S., 160, 216 Kendall, D. G., 96, 102 Kennedy, F. S., 142, 143, 144, 146 Kennedy, S. I. T., 36, 44 Kennell, D., 168, 169, 216 Kent, A. B., 61,62, 105 Kepes, A., 167, 201, 213, 216 Kerridge, D., 220, 226, 229, 231, 233, 238, 246, 260, 264, 276, 279, 282, 297, 298, 304, 306, 307, 312, 316, 317, 320, 331, 332, 333,335 Kosslor, D., 64, 102 Key, A., 33, 43 Keynan, A., 68, 73,77, 78, I06 Kim, J. H., 66, 101 Kim, S., 247, 333 King, A. L., 318,333 King, K. W., 61, 105 Kinghorn, M. L., 91, 103 Kingsley Smith, B. V., 239, 262, 263, 280, 330 Kirkpatrick, J., 220, 329 Kits, H., 2, 44 Kitagawa, M.,27, 44 Kjeldgaard, N. O., 97, 103, 162, 167, 168, 167, 190,215,216,217 Kliirner, F. G., 27, 45
347
AUTHOR INDEX
Klein, D., 264, 273, 288, 289, 290, 291, 306,333 Klevecz, R. R., 66, 70, 83, 84, 94, 95, 102 Klug, A., 220, 230, 282, 308, 315, 316, 320, 333 Knight, M., 121, 145 Knorre, W. A., 79,102 Knutson, G., 64, 65, 72, 74, 102 Kobayashi, S., 27, 44, 58,102 Kobayashi, T., 239, 240, 244, 247, 249, 275,333,337 Koch, A., 52, 96, 102, 105 Koch, A. L., 152, 153, 154, 155, 156, 157, 164, 166, 167, 168, 169, 171, 173, 174, 175, 176, 177, 182, 183, 184, 189, 190, 194, 195, 201, 203, 208, 209, 210, 215, 216,217 Koch, F. E., 220,333 Koch, J. R., 26, 43 Koch, R., 219, 333 Koffler, H., 220, 224, 226, 227, 228, 230, 231, 232, 233, 234, 237, 238, 239, 240, 244, 245, 247, 248, 249, 252, 256, 260, 261, 263, 264, 265, 266, 268, 269, 273, 275, 277, 278, 279, 280, 282, 284, 286, 287, 288, 289, 290, 291, 292, 293, 295, 298, 300, 306, 308, 320, 324, 327, 328, 330, 331, 333 334, 335, 337, 338, 339 Kogoma, T., 53, 59, 62, 102, 104 Kojima, Y., 34, 44 Konrad, C. G., 53,102 Kopp, R., 275, 318, 323,333 Kornberg, A., 150, 216 Kornberg, H. L., 16, 42 Kossel, H., 220, 333 Kramer, E., 220, 333 Krieg,N. R., 317, 318, 321, 324,333 Krishnamurty, H. G., 4, 5, 44 Krooth, R. S., 94, 102 Kubitschek, H.. 48, 52, 96, 102 Kubitschek, H. E., 159, 103, 216 Kuempel, P. L., 59, 61, 72, 103 Kuenzi, M. T., 68, 103 Kumar, S., 55, 98, 105 Kuno, S., 27, 44 Kurland, C. G., 165, 216 Kurth, H., 219,333 Kurz, W., 33, 42 Kushner, D. J., 220, 333
Lacroute, F., 150, 152, 216 Lafeber, A., 51,103 Lajtha, L. G., 94, 101 Lampen, J. O., 85,101 Langenberg, K. F., 114, 117,145 Lark, K. G., 48, 53, 54,103 Larway, P., 17, 44 Lasseur, P., 223,333 Latimer, R. G., 305, 334 Lawn, A. M., 235,252,333 Lazzarini, R. A., 151, 165, 217 Lederberg, J., 220, 267, 295, 302, 303, 305, 310, 311, 312, 328, 332, 333, 338 Lee, S. S., 10, 45 Leene, W., 224, 230, 338 Lehman, I. R., 150,216 Leiberman, I., 66, 106 Leifson, E., 220, 222, 264, 269, 295, 312, 333,334 Le Minor, L., 254, 330 Lcmme, R., 318, 323, 333 Lendrum, A. C., 275, 299, 322,334 Levinthal, C., 58, 74, 102, 106 Lezius, A., 131, 145 Liebig, J., 195, 216 Lijungdahl, L., 133, 145 Lim, F., 273, 291,339 Lin, L., 53,103 Lind, P. E., 259. 260, 334 Lindstead, R. P., 9, 43 Lindstrom, E. W., 315, 330 Littlefield, J. W., 66, 70, 103 Ljachowetzky, M., 220, 334 Loeffler, F., 222,334 Lofgren, R., 224, 226, 334 Loken. M. R., 48, 52,103 Lominski, I., 275, 299, 322, 334 London J., 224, 226, 227, 228, 230, 231, 324,329 Loos, M. A., 28, 44 Lorenzen, H., 48, 50, 104 Lougren, N., 93,105 Lowenstein, J. M., 27, 44 Lowenstein, P., 220, 328 Lowy, J., 220, 222, 223, 233, 238, 239, 244. 248, 268, 269, 275, 278, 279, 280, 282, 294,316,317,329,334 Loeeron, H. A., 55, 98, 105 Lundh, N. P., 279,282,304,319,323,334, 336 Lyon, M. F., 94, 95,103
L Labaw. L. W.. 277.333 Laberge, M., 63, 54, 67, 68, 69, 70, 82, 84, 87,101 Lacey,B. W., 260,333 Lack, L., 14,16,44
M Maal~e,O., 48, 50, 97, 103, 152, 156. 157, 158, 165, 190, 216, 217 Mabry, T. J., 26, 27, 43
348
AUTHOR INDEX
MacDonald, D. L., 9, 44 MacDonald, F., 220, 222, 334 Machin, K. E., 316, 334 MacQuillan, A. M., 64, 70, 103 Maeno, H., 3, 45 Magamnik, B., 77, 103 Mahr, Sr. V. M., 56, 98, 105 Maitland. H. B., 297, 336 Maitra, U., 160, 216 MBkelil, P. H., 262, 302, 334 Maki, Y., 2, 44 Mallett, G. E., 220, 244, 260, 261, 262, 286, 268, 276, 328, 333, 334 Malmgren, H., 67, 105 Malvoz, E., 261, 334 Mandel, H. G., 305,334 Manninger, R., 300, 334 Manor, H., 52, 103, 161,216 Manten, A., 321,334 Markus, G., 93, 103 Marmur, J., 301, 334 Marr, A. G., 96, 103 Martin, D. W., 66, 69, 72, 76, 91, 92, 94, 103, I06 Martin, R. G., 60, 76, 103 Martincz, R. J., 229, 237, 239, 241, 242, 244, 246, 247, 248, 262, 268, 269, 260, 266, 267, 269, 276, 279, 282, 287, 292, 294, 296, 296, 298, 304, 306, 309, 319, 323, 331, 334, 336 Martius. C., 18, 44 61, 62, 63, 64, 103 Maruyama, Y., Marver, H. S., 93, 103 M a n , R., 233,239,277,334 Maseles, F. C., 26, 43 Mason, H. S., 21, 44 Massey, V., 4 6 , 4 6 Masters, M., 49, 67, 68, 69, 61, 62, 84, 68, 69, 72, 73, 77, 78, 79, 80, 86, 89, 91, 96, 100, 103 Masuxawa, E., 99, 104 Matile, Ph., 63, 103 Matsumoto, H., 311, 334 Matzuahita, T., 220, 331 Mauro, F., 90,100 Mayer, M. M., 266,332 McBride, €3. C., 109, 124, 129, 136, 137, 138, 143, 144, 145 McCarthy, B. J. 173, 178, 215, 816 McClatchy, J. K., 229, 238, 297, 306, 334 McClure, F. T., 201, 215 McCoy, E., 260,261,334 McDonough, M. W., 220, 240, 242, 243, 246, 247, 248, 264, 276, 282, 294, 316, 334,335,338 McFall, E., 77, 103 McOroarty, E., 296,298,335 McKay. D., 78,103
McQuillen, K., 160, 216 McWharter, A. C., 264, 329, 330 Meadow, P., 47,103 Melnykovych, G., 66, 103 Menolasino, N. J., 262, 335 Menten, M. L., 194, 195, 216 Mesrobeanu, L., 240, 328 Metzner, P., 317, 324, 335 Meyer, A., 223, 335 Meynell, E. W., 319, 335 Michaelis, L., 194, 195, 216 Migula, W., 220, 335 Mihara, K., 46, 46 Milos, A. A,, 262, 328 Miles, E. M., 262, 388 Miller, A. 0. A., 64, 103 Miller, 5. F. A. P., 260, 336 Miller, R. L., 76, 76, 99 Millman, B. M., 220,276,282, 316,334 Milno, G. W., 26, 44 Mironesco, T. G., 220,335 Mitani, M., 220, 223, 229, 278, 296, 298, 304, 307, 309, 332, 335 Mitchell, 0 .F., 260, 336 Mitchell, J., 260, 335 Mitchell, P., 318, 320, 335 Mitchen, J.R., 231,232,234,237,284,275, 327,335 Mitchison, J. M., 48, 61, 62, 63, 66, 67, 60, 64, 68, 69, 72, 73, 76, 86, 88, 89, 90, 91, 97, 100, 103 Mitsui, C., 220, 263, 332 Mittermayer, C., 64, 66, 103 Moffitt, W., 290, 335 Moltke, O., 220, 322, 335 Monier, R., 166, 216, 217 Monod, J., 76, 102, 169, 201, 210, 216 Moody, M. D., 300, 302,328 Moor, H., 63, 103 Moore, D. H., 262, 331 Moore, S., 244, 335, 337 Morales, M., 78, 103 Moran, A. B., 263, 324,329, 330 Morgan, R., 48, 101 Morowitz, H. J., 319, 335 Morrison, R. B., 229, 335 Morse, L., 186,216 Mortimer, R. K., 83,86, I01 Moses, H. A., 247,339 Moskowitz, M., 262, 266, 289, 296, 337, 339 Mosley, V. M., 277, 333 Moss, P., 34, 43 Mudd, S., 224,226,262,263,331,335 Mueller. a. C., 66, 70, 91, 101, 105 Muir, R. D., 10, 4 3 Muldal, S., 94, 101 Muller, J., 276, 318, 323, 333
349
AUTHOR INDEX Munoz, E., 320,335 Murata, M., 264,330 Murphree, S., 66, 70, 105 Murray, R. G. E., 224, 226, 227, 228, 295, 324,335
N
,
Nakagawa, H., 34,44 Nakamura, S., 20,44 Nakaya, R., 264,335 Nakazawa, A., 34,44 Ntlkazawa, T., 34,44 Nalbandov, O., 118, 139, 145 Narasimhachari, N., 4, 45 Nass, G., 160, 215 Nasser, D. S., 295, 296, 300, 301, 335 Nath, K., 164, 164, 182, 216 Nauman, R. K., 226, 233, 235, 238, 335 Neidhardt, F. C., 160, 166, 215, 216 Neish, A. C., 28, 43 Nelson, .J. B., 261, 260, 335 Nemser, S., 300, 319,337 Neser. M. L., 219,336 46,46 Neujahr, H. Y., Neuman, F., 220,335 Neumann, N. P., 86,101 Neustadtl, R., 220, 335 Newton, B. A,, 220, 316, 317,320, 335 Nickerson, K., 74,104 Nickerson, W. J., 97, 104 Nicolle, C., 220, 335 Nierlich, D. P., 166, 176, 216 Nilson, E. H., 96,103 Niehi, A., 63, 69, 62, 72, 73, 102, 104 Nishizuka, Y., 20,44 Nogradi, A., 300, 334 Noguchi, H., 288,291,331 Norris, T. E., 162, 164, 160, 164, 167, 168, 169, 180, 215,216 Nossal, G. J. V., 239, 242, 244, 246, 247. 248, 249, 262, 268, 269, 260, 286, 292. 294, 327, 328,334, 335,336, 337 Novick,A., 61,101, 169, 162,192,216,217 Novotny, C., 295,336 Nozaki, M., 2, 4, 13, 33, 34, 43, 44 Nozawa, M., 264,330 Nulsch, W., 319,336
0 Oda. Y., 36, 45 Ogiuti, K., 220, 336 Ohno, S., 96, 104 Ohoson, R.. 67,105
Omelianski, W. L., 120, 145 Oosawa, F., 220, 229, 244, 287, 308, 320, 336 Orcutt, M. L., 239, 261, 336 Ornston, L. N., 7, 9, 10, 36, 38, 39, 41, 42, 42,44 0rskov, F., 219,311,336 Orakov, I., 311,336 Osumi, M., 99, 104 Overath, P., 61, 104 Overbech, J., 220, 333 Oxender, D. L., 201,217
P Padilla, G. M., 48, 100, 104 Paduch, V., 67, 100 Paik, W. K., 247, 333 Painter, R. R., 96,103 Palen, M. E., 220,222,334 Palleroni, N. J., 14, 33, 45 Pardee, A. H., 53, 57,58, 69, 61, G2, 74, 78, 79, 97, 99, 103, 104 Parish, C. R., 242, 244, 246, 248,249, 268, 269, 260,327,336 Parker, H. E., 247, 339 Patel, J. C., 36, 43,44 Patel, M. D., 3, 43 Pateraon, J. S., 264, 297,336 Pato, M. L., 72, 80, 104 Paynter, M. J. B., 118, 145 Pearce, U. B., 326,336 Pease, P., 226,298,328,336 Pelc, 6. R., 48, 102 P e l d o , C. A., 220,319,329 Penley, M. W., 143, 145 Perez, A. G., 66, 101 Peterkofeky, B., 92, 104 Petersen, D. F., 49, 99 Peterson, E. J., 21, 44 Peterson, J. A., 3, 44 Peterson, R. N., 201,217 Peterson, W. D., Jr., 222, 32.9 Petrali, J. P., 262, 312, 339 Petropulos, S., 61, 104 Pfeiffer, S. E., 64, 104 Pietschmann, K., 220, 222,336 Pietz, K. A., 93,100 Pijper, A., 219, 220, 223. 224, 240, 262, 336 Piper, J., 74, 104 Piperno, J. R., 201, 217 Pirson, H., 48, 60, 104 Pirt, S. J., 47,103 Pitot, H., 93, 104 Plesner, P., 63, 104 Pogo, A. O., 61,104
350
AUTHOR INDEX
Polevitzky, K., 224,226,282,316,335,336 Porter, A. M., 230, 316, 328 Postgate, J. R., 144, 145 Potter, V. R., 93, 100 Powell, E., 90, 104 Powell, E. D., 187, 217 Premkumar, R.,14, 45 Prenant, A., 223, 336 Proscott, D. M., 53, 104 Preston, N. W., 297, 319, 323, 336, 337, 338 Preusser, H. J., 224, 277, 336 Pritchard, R. H., 97, 98, 104 Proctor, M. H., 6, 44 Prokop-Schneider, D., 54, 105 Pye, J., 239, 242, 244, 247, 262, 268, 286, 287, 292, 309, 327, 336
0 Quadling, C., 298, 312, 313, 33ti QI1CsI101, I,., 285, 336
R Rahn, O., 49, 96, 104 Raimondo, L. M., 282,319, 323,336 Rao, B., 48,102 Rao, P. N., 48, 104 Hasmussen, L., 61, 104 Ravdin, R. G., 16, 45 Read, K. S., 262, 256, 289, 336, 337 Reagh, A. L., 261, 328.338 Rechcigl, M., 93, 103 Reed, D. J., 144, 145 Rees, M. W., 246, 328 Reichort. K., 220, 277, 314, 337 Reiner, J. M., 96, 104 Reisher, S., 97, 101 Reiter, D., 297, 332 Reitman, C., 223, 331 Remaen, C. C., 226,228,337 Renson, J., 22. 26, 43 Rhoads, C. A., 22, 43 Rhodes, M. E., 220, 222,337 Ribbons, D. W., 4, 7, 14, 26, 32, 45, 46,46 Rickenborg, H. V., 229, 238,297, 306,334 Riis, M., 306, 334 Rinker, J. N., 220, 224, 230, 231, 239, 240, 244, 247, 249, 269, 276, 315, 320, 333, 334,337 Ritchie, A. E., 226, 231, 280, 282, 318, 337 Robbins, E., 61, 62, 63, 64, 91. 100, 102, 104, 105 Robbins, M. L., 297, 331 Robertson, A.M., 126, 126, 134, 136, 139, 140, 141,145
Roberts, F. F., Jr., 229, 275, 297, 337 Roberts, R. B., 173, 226 de Robichon-Szulmajster, H., 09, 102, 104 Robinow, C., 220, 337 Robinow, C. F., 63, 103 Robinson, I. M., 110, 113, 145 Rodenburg, S., 63, 74, 102, 104, 105 Rodrigues, D., 33, 43 Rogers, 0. E., 220,231, 3J7 Rooney, D. W., 61, 104 Rorsch, A., 49,104 Roaen, C. G., 144,146 ltosenberg, E., 260, 260, 207, 209, 276, 296, 309,334 Roaaet, It., 166,216,217 Roth, L. E., 282,329 Rotman, M. B., 00, 61, 101, 104 Rowley, J., 94,101 Rownd, R., 73,104 Ruddle, F. H., 66, 70, 84, I02 Rudert, R., 64, 83, 86, 105 Rudner, It., 54, 105 R w h , H. P., 48, 54, 60, 97, 100, 1 0 3 , 106 ltussell, L. B., 95, 105 Ruaa-Munzer, A,, 220,322,337 Rutman, R. J., 97, 101 ltyter, A., 97, 102
s Sachaenmaier, W., 66, 70, I05 Sakazaki, R., 254, 330 &la, F., 244, 306, 331, 337 Salaer, W., 55, 100 Salton, M. R. J., 224, 320, 335, 337 Salzrnann, J., 96, 105 Samuels, H. H., 91, 92, I06 Sanarelli, G., 220, 337 Sando, N., 99,104 Sasaki, I., 323, 337 Sato, T., 26, 42 Savage, N., 219,336 Schaechter, M., 62, 96, 102, 105, 150, 168, 190, 194, 216, 217 Schaffer, H.. 261.200. 328 Scharff, M. D., 61, 62; 63, 64, 91, 100, 102, 105 Schechter, E., 290,337 Scher, S.,6, 44 Scheraga, H. A., 291, 337 Scherbaum, 0. H., 48,60,61, 105 Schimke, R. T., 93, 105 Schleaainger, D., 173, 217 Schmidt, R. R.,61, 62, 65, 70, 72, 74, 100, 102,105 Schnellen, C. G. T. P., 120, 145 Schoenheimer, R., 171, 217 Schram, E., 244,337
351
AUTHOR INDEX
Schrammeck, J., 320, 337 Schrauzer, G. N., 136, 137,145 Schuster, T. M., 290, 291, 337 Schuetze, P. L., 323, 337 Schutze, H., 262, 337 Schweitzer, E., 54, 89, 99, 105, 106 Scopes, A. W., 51,53,106 Seed, J., 94, 105 Seidler, R. J., 238, 337 Seidman, M. M., 29, 33, 34, 45 Seiffert, W., 220,337 Senior, P. J., 14, 45 Senoh, S., 2, 44 Sertic, V., 267, 337 Shakespeare, William, 181, 217 Shaw, D. W., 12,13,45 Shellam, G. R., 260, 337 Shen, S . It., 65, 105 Sherris, J. C., 319, 321, 328, 336, 337 Shilo, M., 238, 327 Shoesmith, J. G., 319,337 Shortman, K. D., 260, 336 Shunk, I . V., 222, 337 Sibert, J . W., 136, 137, 145 Sih, C. J., 10, 43, 45 Silvester, N. R., 319, 337 Simmons, N. S., 290,337 Simms, E. S., 160,216 Simon, M. I., 235, 253, 258, 297, 302, 306, 313,314, 329,331, 338 Simpson, F. J., 4,6,33, 42,44,45 Sims, P., 25, 42 Sinclair, R., 51, 52, 105 Sinclair, W. K., 90, 101 Sistrom, W. R., 300, 319, 337 Sitz, T. O., 51, 62, 105 Smith, A. It. W., 29, 30, 42 Smith, D., 99, 105 Smith, J. L., 323, 337 Smith, P. H., 114,145 Smith, R. W., 234, 237, 244, 245, 286, 290, 293, 295, 298, 308, 333, 335, 337, 338 Smith, S. M., 302, 338 Smith, T., 220, 251, 338 Smoluchowski, M. von, 197,217 Sneath, P. H. A., 226,265,338 Sokolski, W. T., 300, 338 Spalding, G., 91,105 Sparkcs, B. G., 299,338 Spencer, M., 220, 222, 223, 244, 278, 316, 334 Spicer, C. C., 302, 338 Spiegelman, G., 66,74, 101,161,217 Spizizen, J., 301,328,338 Sreeleela, N. S., 14, 45 Stadtman, T. C., 107, 118, 120, 126, 129, 130, 133, 145,146
Stanbridge, T. N., 319,323,338 Stange, H., 61, 104 Stanier, R. Y., 2, 7,9, 14, 33,35,38, 39,40, 41, 42, 42, 44, 45 Stapert, E. M., 300, 338 stam, M. P., 238,277,337,338 Steele, W. J., 161, 217 Steenbergen, C. L.M., 51, 103 Steggerda, F. R., 108,146 Steinberg, W., 55, 68, 72, 73, 74, 77, 78, 102,104,105 Stenesh, J., 260,266,268,275, 338 Stent, G. S., 150, 161, 162, 216 Stern, H., 66, 70, 72, 74, 102 Sternberger, L. A., 262, 312, 339 Stocker, B. A. D., 220, 229, 230, 246, 269, 275, 296, 298, 300, 302, 303, 304, 311, 312, 315, 317, 325, 326, 328, 332, 336, 338,339 Stopher, D. A., 32, 43 Storck, R., 224, 339 Strange, R. E., 187, 217 Strasser, F. F., 94, 102 Stubblefield, E., 66, 70, 94, 95, 102, 105 Stubba, J. D., 166,217 Stulberg, C. S., 222, 329 Subba Rao, P. V., 14,45 Suda, M., 36,45 Sueoka, N., 61,105 Sueae, H. E., 75, 76,99 Sugiyama, S., 14, 45 Sullivan, A,, 244, 246, 286, 338 Summers, W. C., 66, 98, 105 Sunakawa, S., 239,240, 338 Sussman, M., 93,105 Sutton, W. B., 2, 45 Suzuki, H., 244, 246, 286, 299, 306, 311, 338 Suzuki, K., 27, 44, 46, 46 Swanbeck, G., 279,282, 330, 338 Swann, M. M., 89,105 Swayze, A. B., 66,103 Sweeney, E. W., 93,105 SylvBn, G., 67, 105 Szent-Gyorgyi, A. C., 290,337 Szilard, L., 169, 162, 192, 217 Szybalski, W., 66,98,105
T Takagi, A., 226, 338 Takamori, S., 27,44 Takeda, H., 2,45 Takeda, Y., 2, 34, 42, 44 Takemori, S., 46,46
352
AUTHOR INDEX
Talalay, P., 10, 12, 13, 42, 45 Tanaka, H., 14,45 Tanioka, H., 2, 42 Teniuchi. H., 26, 34, 44, 45 Tarvin, D., 6,45 Tatum, E. L., 9 , 4 3 Tauro, P., 62, 63, 64, 67, 68, 69, 70, 82, 84, 86, 87, 89, 99, 101, 105, 106 Tauschel, H.-D., 226, 228, 338 Tawera, J., 226,338 Taylor, B. F., 46, 46 Taylor, E. W., 290, 329 Taylor, G. I., 314, 338 Taylor, K., 66, 98, 105 Tazaki, H., 311,334 Telegdi, V. L., 76, 76, 99 Tempest, D. W., 187, 217 Templeton, B., 298, 300, 319, 321, 327 Tenbroeck, C., 220, 261, 338 Terasima. T., 91, 106 Terskikh, V. V., 96,101 Tesdal, M., 264, 332 Thompson, E. B., 76,91.92, 106 Thorell, B., 67, 105 Thornley. M. J., 226,338 Throm, G., 319,336 Tiedje, J. M., 28, 34, 45 Tingle, M., 68, 83, 84, 106 Tiselius, A., 267,339 Tobias, C. A,, 67, 105 Tolmach, L. J., 64, 91, 104, 106 Tomcsik, J., 262, 300,338 Tomelty, J. P., 317, 318, 321, 324, 333 Tominatsu, Y.. 290, 338 Tomizewa, J., 192, 216 Tomkins, G. M., 66, 69, 72, 76, 91, 92, 93, 103,104,106 T o m , A., 28, 29, 30, 32, 33, 34, 46, 46 Torriani, A., 74,106 Trenel, M., 220, 335 Tronick, 8 . R., 279,304, 334 Trudgill, P.w., 2 , 2 7 , 4 5 Truper, H. G., 226,228,337 Tschepek, M., 299,338 Tschudy, D. P., 93,103 Tseng, S. F., 118, 146 Tsong, Y. Y., 10, 45 Turkington, R. W.. 99,106 Turner, M. K., 66, 106
U Uchida, H.. 239,240,264,335,338 Udenfriend, S., 22, 26, 26, 43, 44 Umbarger, H. E., 76,78, 106 Uretz, R. B., 62, 102
V Vaidyanathan, C. S., 14,45 Vaituzis, Z., 296, 298, 299, 306, 314, 329, 338 Valentino. L., 244, 329 Van Alstyne, D.. 302,338 Vanaman, T. C., 99, 106 Van dsr Kamp, C., 49, 104 Van der Walt, J. P., 219, 336 Vanderwinkel, E., 224,338 van Iterson, W., 219, 220, 222, 224, 226, 227, 230, 231,238, 318, 324, 331, 338 van Tieghem, P., 219,338 van Zenten, E. N., 224,227,230,231,324, 331,338 Varga, J. M., 46, 46 Vary, J., 74, 104 Vary, P. S., 304, 311,326,330 Vatter, A. E., 224, 226, 227, 228, 230, 231, 232, 233, 234, 238, 264, 268, 276, 277, 278, 280, 282, 284, 287, 320, 327 Vegotsky, A,, 247,273, 291,339 Velnzquez, A. A,, 94,102 Vennes, J. W., 262,298, 339 Verneir, P., 223, 333 Vincent, W. S., 61, 62, 89, 103, 106 Vitello, L., 290, 338 Vogel, E., 27, 45 Vogel, H. J., 41, 45 Von Meyenburg, K., 60,61,67,68,106 Vos, O., 61,100 Vuicich, V., 262, 312, 339
W Waddington, C. H., 96, 106 Wailes, K. A., 323, 330 Wakebayashi, K., 287,292,339 Walker, N., 14, 26, 27, 42, 45 Wallace, H., 161, 217 Wallace, R., 299, 306, 329 Walls, N., 318, 339 Walter, C., 78, 106 Walton, G. M., 138, 145 Wang, K. C., 10,43, 45 Ward, C., 93, 106 Warren, G., 224,332 Waterbury, J. B.,226,228,337 Watson, S. W., 226, 228, 337 Webley, D. M., 29, 45 Weibull, C., 220, 223, 224, 230, 239, 240, 244, 247, 248, 261, 262, 264, 267, 269, 273, 276, 314,316, 320,328, 331, 339 Wed, A. J., 220,328
353
AUTHOR INDEX
Weil, E., 220, 251, 339 Weinberg, E., 58, 73, 77, 78, 105 Weinberg, E. D., 299,339 Weinstein, D., 252, 295, 296, 339 Weissbach, H., 142, 145 Weitzenberg, R., 297, 339 Wells, J. S., Jr., 318, 321, 324, 333 Wendt, L. W., 318,339 West, M. C., 253, 330 Westlake, D. W., 4, 45 Wetlaufer, D. B., 290, 337 Wheelis, M. L., 14, 39, 40, 42, 45 White, M. J. D., 94, 106 White-Stevens, R. H., 45, 46 Whitlock, H., Jr., 10, 43 Whitmore, G. F., 51,106 Whitson, G. L., 48, 104 Wiame, J., 69, 93, 100 Wiame, J. M., 224, 339 Wilbur, K. M., 53, 91, 103 Williams, F. D., 28, 43, 323, 330, 339 Williams, M. A., 226, 339 Williams, N. E., 97, 106 Williams, R. C., 277, 338 Williamson, D. H., 51, 53, 106 Williamson, J. P., 62, 96, 105 Wilson, C. E., 252, 312, 339 Wilson, J. K., 222, 332 Wiltshire, G. H., 26,45 Windgassen, R. T., 136,145 Winebright, J., 252, 259, 330, 339 Winslow, R. M., 151, 165, 217 Winstanley, D., 51, 52, 100 Wistar, R., 259, 336 Witkop, B., 22, 23, 25, 26, 43, 44 Witt, I., 64, 102 Wolfe, G. E., 222, 329 Wolfe, R. S., 109, 114, 117, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 138, 140, 141, 142, 143, 145,146 Wolin, E. A., 120, 122, 123, 127, 128, 131, 145,146
Wolin, M. J., 120, 122, 123, 127, 128, 131, 132,133,145, 146 Wood, H. G., 133,145 Wood, J. M., 10, 20, 28, 29, 30, 32, 33, 34, 43, 45,46, 129, 130, 132, 133, 134, 136, 137, 142, 143, 144,145,116 Wortman, J., 76,106 Wright, B. E., 93, 106 Wyas, O., 63, I03
X Xeros, N., 61, 106
Y Yaguchi, M., 264, 273, 289, 290, 291, 333, 339 Yamagita, T., 51, 62, 103 Yamaguchi, S., 263, 303, 311, 323, 325, 326,330,339 Yamamoto, J., 223, 339 Yamamoto, s.,2, 3, 44, 45 Yang, J. T., 290,329,335 Yankofsky, S. A,, 161, 217 Yano, K., 2, 14, 45 Yarbrough, L. R., 265, 289, 292, 293, 338, 339 Yasuda, H., 27, 44.46, 46 Ydas, M., 89, 106 Yeh, M., 95,100 Yokota, K., 220,261,260,339 Yoshioka, H., 26, 27, 4 3 Yuan, D., 161,215 Yuasa, A., 223, 339
Z Zaltzman-Nirenberg, P., 25, 26, 44 Zeuthen, E., 48,63,97, 101,106 Zinder, N. D., 302,303, 338 Zworykin, N., 224,332
SUBJECTINDEX A Acetamide, effect of, on bacterial flagella, 266
Acetate, stimulatory effect of, on methane bacteria, 118 Achrmobacter sp., metabolism of toluene by, 27 Acid phosphatase synthesis, cell cycle maps for rate points in, 90 AcriAavine, effect of, on bacterial flagella, 267
Actinomycin D, effect of, on enzyme synthesis, 92 Activating enzymes for protein synthesis, cost of synthesis by cell, 150 Active transport in bacteria from low external concentrations, 192 Adaptive responses of Escherichia coli to a feast and famine existence, 147 Adenosine, kinetics of uptake of, by Escherichia coli, 201 Adenosine triphosphatase, activity of, in methane bacteria, 134 possible role of, in flagellar motility in bacteria, 320 Adenosine triphosphate, role of, in methane formation, 134 Aerobacter spp., attachment of flagella to, 226
Agglutination of flagellins and antieera, 252
Alanine dehydrogenase, synthesis of, in the cell cycle, 58 Alcohol dehydrogenase, synthesis of, in the cell cycle, 66 Alcohols, effect of, on bacterial flagella, 276 Algae, synchronous cultures of, 51 Alkaline phosphatase, induction capacity for, in the cell cycle, 72 Alkaline phosphatase, synthesis, cell cycle maps for rate change points in, 90 synthesis of, during the cell cycle, 58 in yeast in relation to gene dosage, 84 Alkylbenzene sulphonates, degradation of, 28
Aluminium ions, inhibitory effect of, on flbgellin synthesis, 299 355
Amino-acid composition of flagellins, 240 Amino-acid starvation, and nucleic acid synthesis in bacteria, 164 effect of, on flagellin synthesis, 307 Amino-acid synthesis in methane bacteria, 121
Amino acids, kinetics of uptake by Escherichia coli, 201 a-Amino-adipic acid reductase, genetics and time of expression of, in the yeast cell cycle, 86 synthesis of, in the yeast cell cycle, 67 8-Aminolaevulinic acid dehydraae, synthesis of, in the cell cycle, 69 Anaerobic breakdown of aromatic compounds, 5 Anaerobic environments and methane bacteria, 108 Anchoring element, b a a 1 structure functioning as an, 230 Animal cells, synchronous culture of, 51 Animals, digestive tracts of, and methane production, 108 Anthracene, degradation of, 25 Antibiotic limitation of growth of Eecherichia coli in the gut, 200 Antiseptics, inhibitory effect of, on synthesis of flagellin, 299 Antisera against flagellins, 251 Antiserum, uae of, to differentiate flagellar hooks and filaments, 235 Aquomethylcobinamide, structural formula of, 132 Architecture of bacterial flagella, 276 Arene oxides as intermediates in degradation of aromatic hydrocarbons, 25 Arginase, synthesis of, in the cell cycle, 65 Argininosuccinase, genetics and time of expression of, in the yeast cell cycle, 86
synthesis of, in the yeast cell cycle, 67 Aromatic cleavage mechanisms, physiological significance of, 32 Aromatic compounds, catabolism of, by micro-organiems, 1 made by man, catabolism of, 3 Aromatic hydrocarbons, oxidation of, to catechols, 25
356
SUBJECT INDEX
Arrangements of protein subunits in flagellins, 276 Arsenate, reduction of, by methane bacteria, 143 Artifact, possibility that extra RNA in bacteria is an, 161 Aspartate transcarbamoylase, induction capacity for, in the cell cycle, 72 synthesis of, in the cell cycle, 68 Aspartokinase, genetics and time of expression of, in the yeast cell cycle, 86 synthesis of, in the yeast cell cycle, 67 A apergillua f a m e , quercetinase production by, 4 h s a y for methane formation, 127 Autogenoua regulation of enzyme synthesis, 67 Azotobacter spp., protein synthesis in cell cycle of, 63
Benzenesulphonate, degradation of, 28 Renzenoid compounds, metabolism of, by Rhodopseudomoms priluetria, 6 Benzoate degradation by bacteria, 38 la Benzoate pathway in M o r a ~ ~ l calcoocetica, 40 Benzoie acid, anaerobic breakdown of, 6 Biochemical markers of synchronous growth, 49 Biochemistry of methane formation, 127 Biological clocks in bacteria, 96 Biplicity in bacterial flagella, 316 R~nstocladielkr emeraonii, synthesis of enzymes in cell cycle of, 66 Breakage of bacterial filaments. 274 Brevibacteriumfuscum, oxidation of methyl catechols by, 34 Brownian movement, effects of, on solute uptake by bacteria, 203 Bursts of enzyme synthesis in the cell cycle, 64
B Bacilli, molecular weight of flagellins from, 248 Bacillus cereus, nucleic acid synthesis in outgrowth of spores of, 66 synthesis of enzymes in germinating spores of, 68 core peptide in flagellin of, 260 effect of ethanol on flagella from, 270 electron micrograph of a flagellar preparation from, 232 Bacillus spp., amino-acid composition of flagellins from, 241 flagellar hook in, 231 B. atearothernophiha, flagellar hook in, 231 B. subtiha, enzyme synthesis in synchronized cultures of, 77 synthesis of enzymes in cell cycle of, 68 Bacteria, regulation by feedback control in, 76 slowly growing, extra RNA in, 162 RNA synthesis in, 164 synchronous cultures of, 61 Bacterial cell division, reasons for, 96 Bacterial degradation of thymol, 17 Bacterial flagella, 219 Bacterial growth cycle, 181 Bacterial growth limited by solute transport, 196 Bacterial metabolism of gentisates, 14 Balanced growth in microbes, 49 Basal dim, attachment of flagella to, 226 Basal material on bacterial flagella, 223 Basal rates of enzyme synthesis, 92 Benzene, metabolism of, 24
C Caffeic acid, degradation of, 28 Calcium content of flagellin, 248 Calcium ions, inhibitory effect of, on flagellin synthesis, 299 Calculations of rate of formation of flagella, 296 Camphor, catabolism of, by micro-organisms, 3 Carbohydrases, multiple copies of genes for, in eukaryotes, 83 regulation of synthesis of, in the yeast cell cycle, 64 Carbohydrate in flagellins, 247 Carbohydrates, kinetics of uptake by Escherichin coli, 201 Carbon dioxide requirement for methane bacteria, 118 Carbon tetrachloride, effect of, on methane formation, 142 fl-Carboxy-ci8,ciamuconate in the degradation of catechol in Neurospora craeaa, 9 Carboxydismutm, synthesis of, in the cell cycle, 66 Catabolic sequences, regulation of, in micro-organisms, 32 Catabolism of aromatic compounds by micro-organisms, 1 Catabolite repression, in prokaryotes, 67 of sucrase synthesis in Bacillus subtilis, 77 Catechol, ortho-fission pathway for degradation of, 7 regulation of metn-fissionpathway for, 41
SUBJECT INDEX
Catechols, degradation of, 18 meta-fission pathways for, 10 Cathepsidase, synthesis of, in the yeast cell cycle, 67 Cell cycle, induction capacity in, 71 maps for rate-change points for sucram synthesis, 90 synthesis of enzymes during, 47 Cell cycles, enzyme synthesis during, 66 Cell division, in the bacterial growth cycle, 182 nature of trigger in, 194 reasons for, 96 specific proteins, 97 Cell extracts, need for ATP in, to produce methane, 134 production of methane by, 128 Cell-free system for flagellin synthesis, 304 Cell plate count as an index of synchronous growth, 49 Cell size, as a factor in control of cell division, 96 aa an index of synchronous growth, 49 of enteric bacteria, 194 Cell viability in the bacterial growth cycle, 182 Cell volume, importance of, in production of synchronous cultures, 62 Centrifugation techniques for producing synchronous cultures, 62 Cessation of DNA synthesis and repression of /3-galactosidase synthesis, 61 Cetylpyridinium chloride, effect of, on bacterial flagella, 276 Characteristics of methane bacteria, 114 Chemostat cultures, synchronous growth in, 60 Chemostats, description and operation of, 169 minimum rate of addition of medium to, 161 Chemotaxis, genetical control of, in bacteria, 326 mechanisms for, 321 Chinese hamster cells, enzyme regulation in pseudodiploid cell lines of, 94 Chinese hamster, synthesis of enzymes in cell cycle of, 66 Chlarnydmonaa reinhardtii, synthesis of enzymes in cell cycle of, 66 Chloramphenicol, effect of, on flagella production by bacteria, 229 effect of, on incorporation of amino acids into flagellin, 306 Chlwella ellipsoiden, ribonucleic acid synthesis in cell cycle of, 63 Chlorella pyrenoidosa, induction capacity in the cell cycle of, 72
367
Uhwdla pyrenoidoaa-continued synthesis of enzymes in cell cycle of, 64 Chlorella spp., synchronous cultures of, 61 Chlorinated hydrocarbons, effect of, on methane formation, 142 4-Chlorocatechol, degradation of, 28 Chloroform, effect of, on methane formation, 142 Chloromuconic acids.-dearadation of. 34 Chromatographic separation of flagellins, 246 Chrmobacteriurn spp., attachment of flagella to, 226 Chromosome replication, initiator proteins for, 97 Chymotrypsin, effect of, on bacterial flagella, 276 Cinnamic acid, anaerobic breakdown of, 6 degradation of, 28 Clocks, biological, in cells, 96 Clostrirliumpastcurianum, methano formation by, 144 Clostridium spp., flagellar hook in, 231 Coal mines and production of methane by microbes, 108 Cobaloximes as substrates for methane bacteria, 136 Cobamide protein, involvement in methyltransfer reactions, 133 Coenzyme M, in methane formation, 138 properties of, 138 Composition of Salmonella typhimurium, effect of growth rate on, 97 Computer treatment of lag phase of bacterial growth, 191 Concentration of external solute, effect of, on rate of active uptake, 192 Concentration profiles round a spherical cell, I98 Conditions required for re-assembly of protein subunits of flagella, 286 Conformational changes, during re-assembly of bacterial flagella, 289 in bacterial flagellins, 266 Conidendrin, degradation of, 30 Coniferyl alcohol, degradation of, 28 Constant efficiency hypothesis in bacterial protein synthesis, 152 Continuous increase in enzyme activity in the cell cycle, 67 Contractile processes in bacterial flagella, 316 Core peptide in flagellin of Bacillua purnilus, 260 Cork-screw movements of bacterial flagella, 317 Correlation of the order of genetic markers and events in the cell cycle, 80
358
SUBJECT INDEX
Corrinoid protein, role of, in methyl transfer reactions, 134 Costs of protein synthesis for the bacterial cell, 160 Counting of nuclei as an index of synchron o w growth, 49 Coupling of transcription and translation in bacterial protein synthesis in bacteria, 161 Cresol, degradation by pseudomonads, 10 metabolism of, by pseudomonads, 27 Critical point, in enzyme synthesis in the cell cycle, 86 transcriptional control, 89 Curly bacterial filarnents, genetics of, 303 Cyanogen bromide, effect of, on flagellins, 269 Cyclohex-1-me-1-carboxylate, production of, by Rhodopseudomonaapalustria, 6 Cylindrical ccll, effects of motility on solute uptake by, 204 Cylindrical cells, solute concentration profiles around, 206 Cysteine residues, absence of, from flagellins, 240 Cytidine, kinetics of uptake of, by Eacherichia coli, 201 Cytochrome oxidase, synthesis of, in the yeast cell cyclc, 67 Cytological indices of synchronous growth, 49 Cytoplasmic membrane of bacteria, attachment of flagella to, 224
D Degradation, of conidendrin, 30 of ribosomal-RNA in bacteria, 166 of trihydric phenols, 17 Degree of synchronous growth, 49 Dehydroquinase, synthesis of, in the cell cycle, 68 Demethylation of methyl coenzyme-M, mochanisni of, 139 Density-gradient centrifugation, uae of, to produce synchronous cultures, 62 Deoxycytidine monophosphate deaminase, synthesis of, in the cell cycle, 66 Deoxyribonucleic acid content as an index of synchronous growth, 49 Deoxyribonucleic acid, cost of synthesis by the cell, 160 Deoxyribonucleic acid polymerase, cost of synthesis by cell, 160 syntliesia of, in the cell cycle, 66 Deoxyribonucleic acid synthesis and regulation of cell division, 97
Deoxythymidine monophosphate kinase, synthesis of, in the cell cycle, 66 Derepression of enzyme synthesis in degradation of aromatic compounds, 36 Description and operation of chemostats, 169 Design features of chemostats, 169 Deaulfotomaculum spp., methane formation by, 144 DesuEfovibrio spp., methane formation by, 144 Detergents, catabolism of, 3 Dexamethasone phosphate, induction by. in hepatoma cells, 76 Dichlorocatechol, degradation of, 28 2,4-Dichlorophenolindophenol, dcgradation of, 28 Diffusion-limited uptake of solutes, effect of motility on, 204 Diffusion, rate of, by nutrionts around a spherical roll, 196 Digestive tracts of animals and methano production, 108 Dihydro-orotaae, synthesis of, in the cell cycle, 69 2,3-Dihydroxybenzoate, degradation of, 14 Dihydroxyphenols, enzymio degradation of, 7 2,3-Dihydroxy-p-toluate, degradation of, 14 Dimethyllysine in flagellin, 246 2,4-Dinitrophenol, inhibitory effect of, on methane formation, 141 Dioxane, effect of, on bacterial flagella, 276 Di-oxygenases, in catabolism of aromatic compounds by micro-organisms, 2 in micro-organisms, 4 Discs, basal, attachment of flagella to, 226 Disruption of methane bacteria, 128 Distribution of 8-galactosidase among glucose-grown cells of Escherichin coli, 60 Don C cells, synthesis of enzymes in cell cycle of, 66 Doubling times of Escherichia coli, 148 Dragons, biochemistry of, 109
E Ecology of methane bactoria, 107 Economy of Nature, inert compounds in the, 2 Ectothiorhodospira mobilis, attachment of flagella to, 226
369
SUBJECT INDEX
Efficiency, of ribosome synthesis in bacteria, 152 of solute uptake by Escherichia coli, 201 Electron microscopy, and architecture of bacterial flagella, 276 of bacterial flagella, 220 Energy charge and metabolism of methane bacteria, 136 Energy-generating systems and motility of bacterial flagella, 319 Energy involvement in flagellar movement, 314 Energy source, function of basal structure as an, in flagella synthesis, 230 Enrichment cultures for methane bacteria, 109 Enteric bacteria, cell size in, 194 Environment changes to produce synchron o w growth, 50 Enzyme action, regulation of, in breakdown of aromatic compounds, 4 Enzyme induction and RNA synthcsis, 92 Enzyme regulation in catabolism of aromatic compounds, 4 Enzyme synthesis, during cell cycles, 55, 56 Enzymes, synthesis of, during the cell cycle, 47 Eosomal-RNA, kinetics of synthesis of, in bacteria, 174 Epoxides as intermediates in degradation of aromatic hydrocarbons, 25 EscheTichiu coli, adaptive responses of, to a feast and famine existence, 147 capacity for enzyme induction in cell cycle of, 72 correlation between genetic markers and events in the cell cycle of, 80 doubling times of, 148 effect of temperature on flagella formation by, 229 efficiency of solute uptake by, 201 evolution of transport systems in, 196 extra RNA in, 153 /3-galactosidam synthesis in a glucoselimited culture of, 163 motility as a factor in solute uptake by, 203 rate of protein synthesis in, 153 synchronous cultures of, 52 synthesis of enzymes in cell cycle of, 59 uptake of nucleosides from the gut by, 200 viscosity effects on solute uptake by, 202 Ethanol, as a substrate for Methanobacterium cmzeliunskii, 121 effect of, on bacteria flagella, 270 Euglena spp., synchronoue cultures of, 51
Eukaryotic cells, induction capacity in the cell cycle of, 74 regulation of enzyme synthesis in, 81 Eukaryotic organisms, synthesis of enzymes in the cell cycle of, 63 Evolution, of transport systems in Esclierichia coli, 196 microbiological, studied in the laboratory, 193 Evolutionary significance of regulatory mechanisms in microbes, 41 Experimental determination of uptake parameters by growth studies, 210 Exponential increase in enzyme activity in the cell cycle, 57 External concentrations of solute, effect of, on uptake by bacteria, 192 Extra RNA in slowly growing bacteria, 152
F Factor B, nature of, in methane formation, 132 Femt and famine existence, adaptation of Escherichia coli to, 147 Feedback control, regulation by, in bacteria, 75 Ferredoxin, role of, in methyl transfer reactions, 134 Ferulic acid, degradation of, 28 Filament elongation, regulation of, in bacterial flagella, 312 Filament rotation in bacterial flagella, 317 Filaments, flagellar, isolation and purification of, 239 flagellar, stability of, 261 of flagellin, synthesis of, 295 Flagellar, antigens, 251 attachment of, to cell, 223 filament, protein nature of, 240 filaments, isolation and purification of, 239 precipitin reaction of, with serum, 255 formation in the absence of RNA synthesis, 229 genes, 302 mechanisms for the function of, 314 phages in bacteria, 323 Flagellin, composition of, 240 filaments, synthesis of, 296 intracellular, detection of, 295 lysine content of, 246 nature of, 240 purification of, 240 synthesis, cell-free system for, 304 inhibition of, 299 site of, in cells, 296
3 60
SUBJECT INDEX
Flagellins, and immunoglobulins, 269 immunology of, 261 shape of, 264 stability of, 280 Flatus, methane content of, in humans, 108 Fluorophenylalanine, effect of, on flagellin synthesis, 307 Flux of solute &crow tho surface of the bacterial ccll, 196 Folate, as a substrate for methane bacteria, 129 Formaldehyde, as a substrate for methane bacteria, 129 Formate, as a substrate for methane bacteria, 129 growth of methane bacteria on, 126 Formation of methane from methyl Factor 111, 132 Fumarase, synthesis of, in the cell cycle, 68 Function of bacterial flagella, 219 Function of flagella, rnechaniarns for, 314 Fungi, metabolism of aromatic acids by, 36 l k o i c acid, dogradation of, 27
G Galactokinase, genetics and time of expression in the yeast cell cycle, 86 synthesis of, in the yeast cell cycle, 67 8-Galactosidaae message in Escherichia coli, life span of, 168 /I-Galactosidase synthesis in a glucoselimited culture of Escherichia coli, 183 8-Galactosidase synthesis in Escherichia coli, kinetics of, 164 8-Galactosidase, synthesis of, in the cell cycle, 69 synthesis of, in yeast in relation to gene dosage, 84 Gallic acid, metabolism of, 18 Gas atmosphere, use of, in the Hungate technique, 114 Gas chromatography, assay of methane by, 127 Gene dosage and induction capacity, 79 Gene expression and gene linkage in eukaryotes, 85 Generation of ATP in methane bacteria, 134 Genes, flagellar, 302 for chemotaxis in bacteria, 326 Genetical control of chemotaxis in bacteria, 326 Genetic control of flagellin synthesis, 300 Genetic markers, correlation of, with events in the cell cycle, 80
Genetic regulation of flagella synthesis, 310 Genetics of curly bacterial filaments, 303 Gentisates, bacterial metabolism of, 14 Glucose, kinetics of, uptakc by Eechorich~o coli, 201 Glucose-limited culture of Escherichia coli, 8-galactosidase synthesis in, 163 Glucose 6-phosphate dehydrogenase, synthesis of, in the cell cycle, 66 a-Glucosidaae, genetics and time of expression of, in the yeast cell cycle, 86 induction capacity for. in the cell cycle, 72 synthesis in Succhurmyces cerevieiae, 82 synthesis of, in the yeast cell cycle, 64 in the cell cycle, 68 in yoast in rclation to gene dosage, 84 /%Glucosidasc, synthesis of, in the yeast cell cycle, 64 Glutamate dehydrogenase, induction Capacity for, in the cell cycle, 72 synthesiB of, in tho yeast cell cycle, 64 Glyceraldohyde 3-phosphate dehydrogen-8, synthesis of, in the yeast cell cyclo, 67 Glycinamide ribotide kinosynthetase, synthesis of, in the cell cycle, 66 Glycoproteins, flagellins as, 247 Glycylglycine dipeptidase, synthesis of, in cell cycle, 69 Growth cycle, bacterial, model for, 186 discussion of, for bacteria, 181 Growth of methane bacteria, on carbon dioxide, 124 on formate, 126 on hydrogen, 124 Growth-rate constant, RNA contents in bacteria as a function of, 167 Growth rate, effect of, on composition of Salmonella typhimuriuna, 100 effect of, on life span of 8-galaotosidnso messclge in bacteria, 168 Growth studies, experimental determination of uptake parameters by, 210 Guaiacol, formation during degradation of conidendrin, 32 Guanine, incorporation of, into bacterial nucleic acids, 180 Gut, uptake of nucleosides from the, by Eacherichia coli, 200
H Half life of messenger-RNA in bacteria, 167 Half lives of enzymes in relation to tho cell cycle, 93
36 1
SUBJECT INDEX
Halogenated benzenoid compounds, degradation of, 24 Hauch antigens, 251 Heat, effect of, on bacterial flagella, 260 Helicity, changes in, during re-assembly of bacterial flagella, 290 in bacterial flagella, 220 in bacterial flagellins, 265 Helmstetter and Cummings technique for producing synchronous cultures of bacteria, 62 Hexokinase, synthcsis of, in the yeast cell cycle, 67 Histidase, induction capacity for, in the cell cycle, 72 synthesis of, in the coll cycle, 58 Histidine, limitation of bacterial growth, 189 residues, absence of, from flagellins, 240 Histidinol dehydrogenase, genetics and time of expression of, in the yeast cell cycle, 86 synthesis of, in the yeast cell cycle, 67 Histone synthesis in animal cells, 91 History of bacterial flagella, 219 Hollow-tubc structure of bacterial flagella, 278 Homogentisate, degradation of, 14 oxygenase in micro-organisms, 2 Homoprotocatechuate-2,3-oxygenase in micro-organisms, 2 Homoserine, as a substrate for inethane bacteria, 129 dehydrogenase, synthesis of, in the yeast cell cycle, 68 Hook, in flagella formation, 230 proteins attached to bacterial flagella, 236 Hooks, attachment of flagella to, 228 flagellar, composition of, 234 dimensions of, 234 fine structure of, 233 function of, 237 subunits in, 232 Human HeLa cells, synthesis of enzymes in cell cycle of, 66 Human Henle cells, synthesis of cnzymes in cell cycle of, 66 Hungate technique, 110 Hydrocinnamic acid, anaerobic breakdown of, 5 degradation of, 28 Hydrodynamics of flagellar movement, 314 Hydrogen, as a substrate for methane bacteria, 118 ions, effect of, on bacterial flagella, 267
Hydrophobic bonding interactions, importance of, during re-assembly of bacterial flagella, 291 m-Hydroxybenzoate, degradation of, 14 2-Hydroxycyclohexanecarboxylate, production of, from benzoate by Rhodop8eUdO?nO?Uh4 palUStti8,
6
Hydroxylation, of phenylalanine, 22 of p-hydroxybenzoate, 21 Hydroxylations in aromatic metabolism, 20 Hypoxia, use of, to produce synchronous cultures, 51
I Idealized bacterial growth curves, 185 Illumination, changes in, to produce synchronous cultures, 60 Immunoglobulins and flagellins, 259 Immunology of flagellins, 251 Incorporation of amino acids into flagellin, 306 Induction capacity, and gene dosage, 79 in the cell cycle, 71 Induction of /l-galactosidase synthesis in bacteria, 76 Inert compounds in the economy of Nature, 2 Inhibition of flagellin synthesis, 299 Inhibitors, of methane formation, 139 use of, to produce synchronous cultures, 61 Initiator proteins for protein synthesis, 97 Integration of syntheses in the cell cycle, 47 Intestinal contents as a source of methano bacteria, 109 Intestinal flora, basis for selection of bacteria in, 191 Intestines, growth of Eschstichin coli in, 147 Intracellular flagellin, detection of, 295 Invertase, multiple copies of genes for, in yeast, 83 synthesis of, in relation to gene dosage in yeast, 84 in the yeast cell cycle, 67 Isocitrate lyase, induction capacity for, in the cell cycle, 72 Isolation, of flagellar filaments, 239 of methane bacteria, 109 3-1sopropy1catecho1,degradation of, 18 Isopycnic technique, use of, t o produce synchronous cultures, 52 Isotope turnover in bacteria, 181
362
SUBJECT INDEX
K KB cells, synthesis of enzymes in cell cycle of, 66 fi-Ketoadipate enol lactone in catechol degradation, 7 fi-Ketoadipate, role as a repressor of enzyme synthesis, 38 Kinetics, of 8-galactosidase Synthesis in Escherichia coli, 164 of methane formation from serine, 130 of protein synthosis in the cell cycle, 63 of re-assembly of bacterial flagella, 288 Klebsiclla asrogenee, degradation of p hydroxybenzoato by, 36
L Lac operon in Escherichk coli, 76 Lactic dehydrogeanse, synthesis of, in relation to gene dosage in eukaryotes, 84 synthesis of, in the cell cycle, 69 Lactonizing enzymos in catabolism of cahchol, 9 Lactose, kinetics of uptake by Escherichia coli, 201 utilization by Ewherichia coli and the Monod equation, 212 Lag phase, of bacterial growth, coniputcr treatment of, 191 of growth in bacterial cultures, 190 Laminar flow round a micro-organism, 207 Length of bactorial flagella, 222 Leptospira spp., attachment of flagella to, 226 flagellar sheaths on, 238 Leucine aminopeptidase, synthesis of, in cell cycle, 69 Leucine, kinetics of uptake by Escherichia coli, 201 Life span of the 8-galactosidase message in Escherichk coli, 168 Light cycles, w e of, to produce synchronous cultures, 61 Lignin, degradation of compounds dorivcd from, 28 Lily microspores, synthesis of enzymes in cell cycle of, 66 Lysine, incorporation of, into flagellin, 246 in flagellin, 246 pathway in yeast, synthesis of enzymes for, in the cell cycle, 70
M Macroglobulins in flagellins, 269
Macromolecular synthesis, in the bacterial growth cycle, 182 speed of, in bacteria, 149 Magnesium content of flagellin, 248 Malate dehydrogenwe, synthesis of, in the yeast cell cycle, 67 Maleylacetoacetate isomerase in pseudomonads, 14 Maleylpyruvate, isomerization of, by pseudomonads, 16 Malt-, synthesis of, in the cell cyclc of Schizosaccharomycea pombc, 64 Mammalian intostine, selection of bacteria in, 193 Mammalian tissues, methane formation by, 144 Mandelic acid, degradation of, by Pseudomonas putida, 36 Man-made aromatic compounds, cetabolism of, 3 Mapping of flagellin genes, 302 Mass culture of methane bacteria, 124 Master controls of the chromosome, 96 Mechanisms for the function of flagella, 314 Mercaptopurine, selective effect on flagellin synthesis, 306 Mesophile flagellar filaments, effect of heat on, 261 Mesophile flagellins, amino-acid composition of, 262 Mesophiles, peptide maps of flagcllins from, 249 Messenger-RNA, cost of synthesis by cell, 160 for flagella synthesis, 229 for flagellin synthesis, 306 kinetics of synthesis of, in cell cycle, 64 Synthesis in stringent strains of Escherichia coli, 167 Messenger synthesis, rate of, in bacteria, 161 Metabolism, of gallic acid, 18 of thymol, 17 Metacentric ohromosomcs, accumulation of, in the cell cycle, 94 Mcta-fission pathways for catechols, 10, 41 Metal contents of flagellins, 247 Metapyrocatechase, microbial, 2 Methane, assay system for, 127 bacteria, characteristics of, 114 cobaloximes as substrates for, 136 ecology of, 107 growth of, on hydrogen and carbon dioxide, 124 on methanol, 126 in pure culture, 119
SUBJECT INDEX
Methane-continued bacteri-ontinued isolation of, 109 -8 culture of, 124 morphological types of, 114 pools of ATP in, 136 reduction of arsenate by, 143 formation, biochemistry of, 127 by sulphate-reducing bacteria, 144 inhibitors of, 139 role of ATP in, 134 role of coenzyme-M in, 138 microbial formation of, 107 production by cell extracts, 128 Methanobacteriumfomnicicum, morphology of, 114 M. molilis, isolation of, 1 18 M. omelianskii, kinetics of methane formation by, 130 resolution of, 118 M. rumirrnntzum, morphology of, 114 problems of breaking, 128 Methanobacteriumspp., reduction of amenate by, 143 Methunococcus sp., morphology of, 114 Mrthanococcus vanneillii, isolation of, 1 18 Methanogenic fermentations, 109 Methanol, growth of methane bacteria on, 126 Msthanosarcina, methylcobalamin as a substrate for, 130 Methanosarcina barkeri, growth of, on methanol, 126 methyl-transfer reactions in, 133 morphology of, 114 Methanospirillurn spp., morphology of, 114 Methods, for establishing synchronous cultures, 49 used to investigate regulation of metabolism, 39 Methyl alcohol, growth of methane bacteria on, 126 Methylarsines, structural formulae of, 143 Methylation, of coenzyme-M, mechanism of, 139 of lysine in flagellin, 246 2-Methylbutyrate, as a substrate for methane bacteria, 118 3-Methylcatechol, degradation of, 13, 18 4-Methylcatechol, degradation of, 13 Methylcobalamin, as a substrate for methane bacteria, 130 structural formula of, 131 Methylene chloride, effect of, on methane formation, 142 Methyl Factor 111,nature of, 132 N-Methyllysine in flagellin, 246
363
a-Methylserine as a substrate for methane bacteria, 129 Methyl-transfer reactions in Methanosarcina barkeri, 133 Michaelis constants for solute uptake by Escherichia coli, 201 Microbial fallibility, principle of, 3 Microbial formation of methane, 107 Microbiological evolution studied in the laboratory, 193 Mini-methane systems, 144 Minimum rate of addition of medium to chemostats, 161 Mitosis period in the cell cycle, 48 Mixed-function oxidases in metabolism of aromatic compounds, 21 Mixing efficiency and movement in microbial cultures, 207 Model for bacterial growth cycle, 186 Modification of substituent groups before ring cleavage by microbes, 27 Molecular basis of regulation in the cell cycle, speculations on, 76 Molecular recalcitrance, problems of, 3 Molecular weight of flagellins, 248 Monkeys, methane production by the intestinal microfloras of, 109 Monod plot for slowly growing bacteria, 179 Monod plots, nature of, 176 Mono-oxygenases, in metabolism of aromatic compounds, 21 in micro-organisms, 2 Moraxella calcoacetica, benzoate pathway in, 42 degradation of catechol by, 33 methods used to study enzyme regulation in, 39 Morphological types of methane bacteria, 114 Morphology, of bacterial flagella, 220 of re-assembled bacterial filaments, 287 Motility, and bacterial flagella. 220 as a factor in solute uptake by Escherichia coli, 203 genetic loci for, 303 transformation for, 301 Motion of flagella, possible explanation for, 318 Motionless spherical cell, solute uptake by, 196 Moue L cells, synthesis of enzymes in cell cycle of, 66 Movement and mixing efficiency in microbial cultures, 207 Moving spherical cells, solute uptake by, 203 Muconate blockin Moraxella calcoaceticu,40
364
SUBJECT INDEX
Multiple copies of gencs in eukaryotes, 83 Mutants, flagellar, 310
N Naphthalene, degradation of, by soil micro-organisms, 32 metabolism of, 24 Natural assembly of flagellar subunits, 310 Nature of bacterial flagella, 219 Nsuroepora c r a r y ~degradation ~, of catechol by, 9 Nicotinic acid, degradation of, 27 NIH shift, 22 Nitrate reductam, induction capacity for, in the cell cycle, 72 Nocardia poaca, degradation of hydrocinnamic acid by, 29 N . restrictus, degradation of steroid8 by, 10 Non-alleling genes, expreeaion of, in yeast, 82 Non-functional bacterial filaments, 304 Nucleic acid synthesis, step time for addition of a nucleotide in bacteria, 161 Nucleic acids, bacterial, incorporation of w i n e into, 180 Nucleosides, kinetics of uptake by Escherichia coli, 201 Nucleosides, kinetics of uptake of, by E’scherichia coli, 200 Nutrient limitation, use of, to produce synchronous cultures, 60
0 Ohne antigens, 261 0-Methylserine aa a substrate for methane bacteria, 129 Operation and description of chemostats, 169 Ordered appearance of enzymes in the cell cycle in eukaryotes, 81 Ornithine transcarbtlmoylase, synthesis of, in the cell cycle, 68 Orotidine monophosphatc pyrophosphoryl&BB, induction capacity for, in the cell cycle, 72 Orotidine 6’-phosphate decarboxylase, synthesis of, in the yeaet cell cycle, 67 Ortho-Fission pathway, of degradation, 7 regulation of, 36 Oscillating supply of nutrients to produce synchronous cultures, 60
Oscillations in enzyme synthesis in the cell cycle, 78 Outgrowth of bacterial spores, protein synthesis during, 63 Oxidation, of aromatic hydrocarbons to catechols, 25 of phenylpropanoid structures arising from lignin, 28 2-Oxocyclohexanecarboxylate,produrtion of, from benzoate by Rhodopeszidom o m s paluetris, 6 0x0-enoic acids, production of, by degradation of phenol, 12 Oxygen, aa an inhibitor of methane formation, 140 inhibitory effect of, on benzoate metabolism by Rhodopseudomoms pnlustris, 6 Oxygenases in catabolism of aromatic ronipounds by micro-organisms, 2
P Pura-Hydroxybenzoate, degradation of, by Klebsiella nerogenes, 36 hydroxylase, 20 Parallel strands, arrangement of flagollin molecules as in flagella, 280 PenicilZium, degradation of aromatic acids by, 36 Pentachlorophenol, effect of on formation, 142 Peptide maps from flagellins, 249 Periodic autoregulated synthesis of en. zymes in the cell cycle, 78 Periodicity in synthesis of enzymes in the cell cycle, 62 Periodic synthesis of enzymes in the cell cycle, 67 Periods in the cell cycle, 48 Pormeability mutants in degradation o f aromatic compounds, 39 Permeaae synthosis in bacteria, 192 Permeases, number of, on the bacterial surface, 194 Peroxides, as intermediates in degradation of aromatic hydrocarbons, 20 Pesticides, catabolism of, 3 Phase variation of Salmon.elh, flagellins and, 246 Phaaes of growth in bacterial cultures, 182 Phasing methods t o produce synchronous cultures, 60 Phenanthrene, degradation of, 26 Phenol degradation by pseudomonads, 10
SUBJEUT INDEX
Phenols, trihydric, degradation of, 17 Phenylacetate, degradation of, by a pseudomonad, 33 Phenylacetic acid, anaerobic breakdown of, 6 Phenylalanine hydroxylase, 22 Phenylpropmoid structures, degradation of, 28 Phosphate limitation, extra R N A synthesis in Escherichia co2i under conditions of, 168 Phosphoenolpyruvate carboxylase, synthesis of, in the cell cycle, 66 Phosphoserine as a substrate for methane bacteria, 129 Photo-assimilation of benzoate by Rhodopseudomoms palustris, 6 Photomicrographs of Methanobacterium omelianskii, 122 Phthalic acid, anaerobic breakdown of, 5 pH Value, effect of, on bacterial flagella, 267 Physaruna polycephalum, ribonucleic acid synthesis in cell cycle of, 53 synthesis of enzymes in cell cycle of, 66 use of, in studies on the cell cycle, 48 Physical separation of cells, use of, to produce synchronous cultures, 61 Physiological functions of pathways in micro-organisms, 32 Picolinic acid, degradation of, 27 Pimelate, production of, from benzoate by Rhodopseudomoms palustria, 6 Pitch length in bacterial flagella, 269 Plants, synthesis of inert aromatic compounds by, 2 Polymerization of flagellar proteins, site of, 229 Pool constituents in bacteria, turnover of, 174 Pool, intracellular, of flagellin, 296 Pools of ATP and ADP in methane bacteria, 136 Post-transcriptional control in the cell cycle, 91 Post-translational control in the cell cycle, 93 Precipitin reaction of flagellar filaments with serum, 265 Prokaryotes, synthesis of enzymes in cell cycle in, 67 Proline, kinetics of uptake by Escherichia coli, 201 residues, absence of, from flagellins, 240 Propeller, possible action of bacterial flagella as a, 3 18 Properties of methane bacteria, 118 Protease, synthesis of, in the cell cycle, 69
365
Protecting ligands and the stability of enzymes, 93 Protein content of Salmonella typhimurium as affected by growth rate, 97 Protein nature of the bacterial flagellar filament, 240 Protein of bacterial flagella, nature of, 223 Protein subunits, arrangements of, in flagellins, 276 Protein, synthesis of, in the cell cycle, 53 synthesis, rate of, in Eecherichia coli, 163 Proteins, hook, in bacterial flagellar, 236 Proteolytic enzymes, effect of, on bacterial flagella, 275 Proteus, attachment of flagella to, 224 Proteus spp., amino-acid composition of flagellins from, 242 flagellar hook in, 23 1 P . vulgaris, electron micrograph of flagella on, 221 flagellar sheaths on, 238 Protocatechuate-3,4-dioxygonasein microorganisms, 2 Protocatechuate metabolism by Rhodopseudomoms palustria, 6 Protocatechuate, ortho-fission pathway for degradation of, 7 Protocatechuic acid, degradation of, 19 Pseudodiploid cell lines and enzyme regulation in Chinese hamster cells, 94 Pseudomonas acidovorans, degradation of ferulic acid by, 29 P . desmolylicn, catabolism of aromatic compounds by, 2 1’. Juwescem, degraclation of 2,3-dihydroxybenzoate by, 14 1’. olevwam, catabolism of aromatic compounds by, 3 1’. putida, catabolism of aromatic compounds by, 2 catechol degradation by, 7 degradation of aromatic compounds by, 33 degradation of mandelic acid by, 36 degradation of thymol by, 17 P . teatosteroni, catabolism of aromatic compounds by, 3 degradation of steroids by, 10 Pseudomoms spp., flagellar sheaths on, 238 Pure cultures of methane bacteria, 108,119 Purification of flagellar filaments, 239 Puromycin, effect of, on amino-acid incorporation into flagellin, 306 Pyruvate as a substrate for methane formation, 128 Pyruvate decarboxylase, synthesis of, in the yeast cell cycle, 67
366
SUBJECT INDEX
Q Quercetin, ring cleavage in, 4 Quercetinam production by Aspergillue flaws, 4
R Radioactivity, incorporation of, into bacterial nnclcic acids, 177 Radiosensitivity in Gaccharornyces cereuisiae, 90 Raffinese, multiple copies of genes for, in yeast, 83 Rat hepatoma cells, synthesis of enzymes in ccll cycle of, 66 synthesis of tyrosine aminotransferase by, 91, 92 Rate of formation of flagella, calrulations on, 296 Rato of protein synthesis in Ihcherichia coli, 163 Rats, tolerance to flagcllins in, 260 Reactions converting aromatic compounds into ring-fission substrates, 20 Reasons for cell division, 95 Re-assembled bacterial flagolla, olectron microscopy of, 294 Re-aasembly of protcin subunits in flagella, 284 Rcduction of arsenate by mothano bacteria, 143 Rcdundancy, i n bacteria, physiological considerations of, 193 in the yeast gcnomo, 83 Regulation, by feedback control in bacteria, 75 during the cell cycle, speculations on the molecular basis of, 76 of catabolic sequences in micro-organisms, 32 of enzyme action in breakdown of aromatic compounds, 4 of enzyme synthesis in eukaryotic cclls, 81 of gene expression during the cell cycle, 65 of' metabolism, methods used to investigate, 39 of meta-fission pathway for catechol, 41 Regulatory mechanisms, evolutionary s i g nificance of, 41 Replication of genes, rolation of, to enzyme Synthesis, 61 Resolution of Methanobacterium omelian8kii, 118
Resorcinol, degradation of, 17 Restricted synthesis of enzymes in the cell cycle, 71 Rhodopssdomoms palustriu, attachrncnt of flagella to, 226 degradation of p-hydroxybenzoate by, 20 metabolism of benzonoid compounds by, 6 Ilh. spheroidos, synthesis of enzymes in cell cycle of, 5 9 Rhodospirillurn spp., attachment of flag. ella to, 226 flagellar hook in, 231 Ribonucleic acid contents of bacteria as a function of growth-rate constant,, 167 Ribonucleic acid, extra, in slowly growing bacteria, 152 involvemont of, in flagellin Synthesis, 304 possible presence of, in flagellar hooks, 237 synthesis of, in the cell cycle, 53 Ribonucleic acid polymerase in bacteria, 150 Ribonucleic acid synthesis, and cnzymc induction, 92 flagolla synthesis in the absonrc of, 229 in slowly growing bacteria, 164 Ribonucleotide reductaso, synthesis of, in the cell cycle, 66 Ribosomal efficiency in bacterial protein synthesis, 190 Ribosomal protcin, cost of synthesis by cell, 150 Ribosomal-RNA, rost of synthesis by cell, 150 degradation of, in bacteria, 166 kinetics of synthosis of, in cell cycle, 54 Ribosomes per genorne, number of, in bactoria, 162 Rod-shaped cells, solutc uptake by, 205 Roll tubos, use of, in isolation of mothano bacteria, 111 Rotating helices, possible operation of, in bacterial flagella, 317 Rotation of bacterial flagellin filanionts, 317 Rumen fluid as a sourco of mothano bacteria, 109 Ruminants and mothane production, 108
s Saccharomyces cerevisiae, radiosensitivity in, 90 synthesis of carbohydrasos in the cell cycle of, 64
SUBJECT INDEX
Saccharomyces cerevisiae-continued synthesis of enzymes in the cell cycle of, 67 X-ray sensitivity of, 90 S. lnetis, induction capacity in the cell cycle of, 72 synthesis of enzymes in tho cell cycle of, 64 Saccharopine dehydrogenase, synthesis of, in the yeast cell cycle, 68 Saccharopine reductaee, genetics and time of expression of, in the yeast cell cycle, 86 synthesis of, in the yeast cell cycle, 68 Salicylate hydroxylase, 27 Salicylic acid, anaerobic breakdown of, 5 Salmonella spp., amino-acid composition of flagellin from, 242 effoct of shift-up experimonts on cultures of, 190 flagollar hook in, 231 Srrlmonella typliimurium, effect of growth rate on composition of, 97 Schizosncchnromyces pombe, induction capacity in the cell cycle of, 72 ribonucleic acid synthesis in cell cycle of, 53 synthcsis of maltase in cell cycle of, 64 Secophenols, production of, by degradation of steroids, 10 Seeding, need for, in re-assembly of bacterial flagella, 287 Selection in Escherichin coli, 148 Selection in tryptophan-limited cultures of bacteria, 192 Selection methods, use of, to produce synchronous cultures, 51 Selection of bacteria in natural rnvironments, basis of, 191 Selective advantage of cxtra RNA in bacteria, 156 Sequential transcription in eukaryotes, 82 Serine as a substrate for methane bacteria, 139 Serine deaminasc, induction capacity for, in the cell cycle, 72 Serine rlehydratase, induction capacity for, in tho cell cycle, 72 Sewage sludge a source of methane bacteria, 109 Sheath-like structures associated with bacterial flagella, 238 Sheaths, flagellar, nature of, 238 Shift-up experiments with Escherichia coli, 153 Simultaneous adaptation in micro-organisms, 35 Site of attachment of bacterial flagella, 223
367
Site of flagellin synthesis in cells, 296 Site of polymerization of flagellar proteins, 229 Size distribution of cells, importance in preparation of synchronous cultures, 62 Size of organisms, effect of, on rates of active uptake, 192 Slowly growing bacteria, extra RNA in, 152 Monod plots for, 179 RNA synthesis in, 164 Sludge fermenters and methane production, 109 Small size of micro-organisms, physiological reasons for, 203 Sodium dodecyl sulphate, effect of, on bacterial flagella, 266 Soluble flagellin, nature of, 256 Solute uptake, by a motionless spherical cell, 196 by Escherichia coli, efficiency of, 201 by rod-shaped cells, 205 by spherical moving cells, 203 Sonication, effect of, on bacterial flagella, 275 Specific enzyme activities, variation of, during the cell cycle, 55 Specificity of permeases, possible alteration of, 194 Speculations on the molecular basis of regulation in the cell cycle, 75 Speed of macromolecular synthesis in bacteria, 149 Spherical cell, motionless, solute uptake by, 196 Spherical moving cells, solute uptake by, 203 Spirillum serpens, glycoprotein in flagellins of, 247 Spirillum spp., attachment of flagella to, 226 flagellar hook in, 231 Spirillum volutam, mechanisms of motility in, 324 Stability, of enzymes and the cell cycle, 93 of flagellins, 260 Staining of bacterial flagella, 222 Starvation for amino acids, effect of, on flagellin synthesis, 307 Steady-state uptake of nutrients by a spherical cell, 197 Step-like pattern of synthesis of enzymes in the cell cycle, 62 Step time for addition of nucleotides in bacterial nucleic acid synthesis, 151 Stereochemistry of degradation of catechols, 12
368
SUBJECT INDEX
Steroids, degradation of, 10 Stoichiometry of hydrogen oxidation by methane bacteria, 126 Stringent strains of Eecherichia coli and nucleic acid synthesis, 166 Substrates, for methane formation by bacteria, 128 used by methane bacteria, 118 Subunit structure of flagellins, 249 Subunits, protein, arrangement of, in bactorial flagella, 276 Succinate-limited cultures of Eecherichin coli, induction of /3-gttlactosidase synthosis during, 166 Succinyl-CoA thiokinase, synthesis of, in the cell cyclo, 69 Sucrase, induction capacity for, in the cell cycle, 72 periodic synthesis of, in Bacillua aubtilie, 77 synthesis, cell cycle maps for rate change points in, 90 synthesis of, in the cell cycle, 68 Sulphate-reducing bacteria, mcthane formation by, 144 Sulphur-limited growth of Escherichia coli, protein synthesis in, 164 Symbiosis in rolation to Methanobacterium ondianabii, 123 Synchronous cultures, methods for establishing, 49 Synchronous growth, in chemostat cultures, 60 in studies on tho cell cycle, 48 Synthesis, of enzymes during the cell cyclo, 47 of flagellin filaments, 296 of protein and ribonucloic acid in the cell cyclo, 63 period in tho cell oyclo, 48 Syringic acid, degradation of, 18
T Temperature, effect of, on flagella formation by bacteria, 229 effect of, on synthesis of flagellin, 297 shifts, use of, to produce synchronous cultures, 60 Temporal order in cells, importance of, 98 Temporally organized events in the cell cycle, 47 N-Terminal residues of bacterial flagellins, 244 Tetrahymenu spp., synchronous cultures of, 61
Timing of expression of linked genes in yeast, 87 Tolerance t o flagellins in rats, 260 Toluene, metabolism of, 24 Toluene-p-sulphonate, degradation of, 28 Topography of bacterial flagella, 282 Torsional forces in a bacterial filament, 316 Total RNA synthesis in bacteria, Monod plots for, 178 Theoretical growth curves for bacteria, 186 Thermal disintegration of bacterial flagolla, 260 Thermal gradients along bacterial flagella, 318 Thermolability of flagella, 260 Thermophile flagella filaments, effect of heat on, 261 Thermophile flagollins, amino-acid composition of, 262 Thennophilos, peptide maps of flagollins from, 249 Therrnophilic strains of bacilli, amino-acid composition of flagellins from, 241 Thioglycolate, effect of, on bacterial flagella, 266 Thioguanine, selective effect on flagollin synthesis, 306 Thiomethylgalactoside, kinetics of, uptake by Eacherichia coli, 201 Threonine deaminase, genetics and time of expression of, in the yeast coll cycle, 86 synthesis of, in the yeast cell oycle, 64 Thymidine kinase, synthesis of, in relation to gene dosage in eukaryotes, 84 synthesis of, in the cell cycle, 66 Thymidylate kinase, synthesis of, in the coll cycle, 66 Thymol, metabolism of, 17 Tracer kinetics in study of nucloic acid synthesis in bacteria, 169 Transcription in protein synthesis, speed of, in bacteria, 160 Transcriptional control, critical point in, with yeast, 89 Transfer-RNA, cost of synthesis by cell, 150 synthesis in stringent strains of Eacherichia coli, 167 Transformation of gene for flagellin synthesis, 300 Tramport, active. by bacteria from very low external concentrations, 192 limitations of bacterial growth, 195 machinery in baoteria, redundancy in, 193
369
SUBJEOT INDEX
Trehslase, synthesis of, in the yeast cell cycle, 88 Trigger, nature of, in cell division, 194 Trihydric phenols, degradation of, 17 Trihydroxyphenols, enzymic degradation of, 7 Trypsin-digestible sheaths on bacterial flagella, 238 Tryptic peptides, from flagellar filaments, chromatography of, 236 Tryptophan-limited growth of bacteria, 192 Tryptophan residues, absence of, from flagellins, 240 Tryptophan synthetase, synthesis of, in the yeast cell cycle, 68 Tryptophanase, induction capacity for, in the cell cycle, 72 Tyrosine, anaerobic breakdown of, 5 aminotransferase, induction capacity for, in the cell cycle, 72 synthesis in the cell cycle, 66, 91 Tyrosine residues, absence of, from flagellins, 240
U Ultraviolet radiation, sensitivity of, t o Saccharonayces cereviaiae, 90 Unrestricted synthesis of enzymes in the cell cycle, 71 Unwinding of DNA a8 a rate-limiting process in bacterial protein synthesis, 161 Uptake parameters, experimental determination of, by growth studies, 210 Urea, effect of, on bacterial flagella, 266 Uridine, kinetics of uptake of, by Escherichia coEi, 201
Ustilago sp., intergenic recombination in, 91
V Valine. kinetics of uptake by Escherichia coli, 201 Vanillic acid, degradation of, 29 Vibrio spp., attachment of flagella to, 226 flagellar hook in, 231 Viscosity, effect of, on solute uptake by Escherichia coli, 202 Vitamin B,, in methane formation, 136
W Wasteful nature of methane production in the rumen, 123 Wavy appearance of bacterial flagella, 277
X X-Ray diffraction studies and architecture of bacterial flagella, 276 X-Ray sensitivity of Saccharomyces cereuisiae, 90
Y Yeast, synthesis of enzymes in the cell cycle of, 63 Yeasts, synchronous cultures of, 51
Z Zonal centrifugation, use of, t o produce synchronous cultures, 62
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