Advances in Microbial Physiology Volume 29

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Advances in

MICROBIAL PHYSIOLOGY

This Page Intentionally Left Blank

Advances in

MICROBIAL PHYSIOLOGY Edited by

A. H. ROSE School of Biological Sciences Bath University, UK

and

D. W. TEMPEST Department of Microbiology Uniuersity of Shefield, UK

Volume 29 1988

ACADEMIC PRESS Harcourt Brace Jovanouich. Publishers London San Diego New York Boston Sydney Tokyo Toronto

ACADEMIC PRESS LIMITED 24-28 Oval Road London NWI 7DX U . S . Edirion published by ACADEMIC PRESS INC San Diego 92101

Copyright $3 1988 by ACADEMIC PRESS LIMITED

AN Righis Reseroed

No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers

ISSN 0065-291 1

Printed in Great Britain at the Alden Press, Oxford

Contributors M. O’Brian Department of Biology, The Johns Hopkins University, Baltimore. M D 21218, USA G . A. Codd Department of Biological Sciences, University of Dundee. Dundee DDI 4HN, U K M. J. Danson Department of Biochemistry, University of Bath, Claverton Down. Bath BA2 7AY, U K W. Fischer Institut fur Biochemie, Universitat Erlangen-Nurnberg, Fahrstrasse 17, D-8520 Erlangen, FRG L. S. Frost Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 R. J. Maier Department of Biology, The Johns Hopkins University, Baltimore, M D 21218, USA W. Paranchych Department of Biochemistry, University of Alberta. Edmonton, Alberta, Canada T6G 2H7

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Contents Contributors

V

Hydrogen Metabolism in Rhizobium: Energetics, Regulation, Enzymology and Genetics M A R K R. O’BRIAN and ROBERT J. MAIER I. 11. 111. IV . V. VI.

Introduction Regulation Enzymology Energetics Genetics Acknowledgements References

2 6 13 24 38 47 47

The Physiology and Biochemistry of Pili WILLIAM PARANCHYCH and LAURA S. FROST I. 11. 111. IV. V. VI . VII .

Introduction Nomenclature Classification High-resolution studies on pilus structure Organization and expression of pilin genes Structure-function relationships of pili proteins Acknowledgements References

53 54

55 64 68 82 102 102

Carboxysomes and Ribulose Sisphosphate Carboxylase/Oxygenase G E O F F R E Y A. C O D D I. Introduction XI. Distribution and structure of carboxysomes

115 117

...

CONTENTS

Vlll

I ll . Carboxysome composition IV. Ribulose I ,5-bisphosphate carboxylase/oxygenase (RuBisCO) V. Carboxysome function VI. Further aspects of carboxysomes References

124 i32 149 155

157

Archaebacteria: The Comparative Enzymology of Their Central Metabolic Pathways MICHAEL J. DANSON

I.

Introduction

11. Archaebacterial pathways of central metabolism 111. Archaebacterial enzyme diversity

IV. Structure of archaebacterial enzymes V. Concluding remarks VI. Acknowledgements References

166 176 194 217 222 222 223

Physiology of Lipoteichoic Acids in Bacteria W . FISCHER

I.

Introduction

11. Occurrence and structure 111. Metabolism

IV. V. VI. VII.

Cellular location Biological activities Concluding remarks Acknowledgements References

Author index Subject index

233 235 247 275 277 295 296 296

303 327

Hydrogen Metabolism in Rhizobium: Energetics. Regulation. Enzymology and Genetics MARK R . O’BRIAN and ROBERT J . MAIER Department of Biology. The Johns Hopkins Uniuersity. Baltimore. M D 21218. USA

1. Introduction . . A . General background

I1 .

111.

IV . V.

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. . . . . B . Hydrogen evolution by nitrogenase . . C . Hydrogen oxidation by legume root nodules Regulation . . . . . . . A . Oxygenandcarbon . . . . B . Hydrogenase and carbon dioxide fixation C . Host control . . . . . Enzymology . . . . . . A . Purification and some properties . . B . The K,,, value for hydrogen . . . C. Electron acceptor reactivity . . . D . Oxygen lability . . . . . E . Nickel . . . . . . . F . Lipid requirement . . . . G . Kinetic mechanism of hydrogenase . . Energetics . . . . . . . . A . Physiological considerations . . . B . Electron transport . . . . . Genetics . . . . . . . . A . Mutants . . . . . . . B . Molecular genetics . . . . . Acknowledgements . . . . . . References . . . . . . . .

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ADVANCES I N MICROBIAL PHYSIOLOGY VOL ?Y ISBN 0-12-0?7729-8

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Copyright (3 1988 by Academic Press Limited All rights OF reproduclion in any Form reserved

2

M. R. O'BRIAN AND R. J . MAIER

I. Introduction A. GENERAL BACKGROUND

The term hydrogenase refers to enzymes that catalyse Hz consumption or evolution according to the reaction: (1) H2 e2 H + 2eAll hydrogenases are bidirectional to some extent in vitro, but the enzyme appears to catalyse either H2 oxidation or production in uivo. Hydrogen evolution usually occurs in anaerobic micro-organisms, and serves to get rid of excess reductant when protons are the only available oxidant (Schlegel and Schneider, 1978), whereas H2 utilization can occur in aerobic and anaerobic bacteria, and is linked to ATP-producing electron transport systems. Anaerobic bacteria oxidize H2 using sulphate, sulphur, C 0 2 , nitrate or fumarate as the terminal electron acceptor, and the photosynthetic bacteria use H2 and other compounds, rather than H20, as the reductant for COz fixation (Knaff, 1978). Aerobic Hz-oxidizing bacteria can grow with H2 and COZ as the sole energy and carbon sources, respectively. Among these bacteria, some have a soluble and a membrane-bound hydrogenase; the soluble enzyme catalyses Hz-dependent NAD+ reduction which is used for COZfixation, whereas the membrane-bound hydrogenase is linked to electron transport, and is therefore involved in energy production (Adams et al., 1981). The aerobic N2-fixing bacteria evolve and consume HI, and among this group are the rhizobia, the azotobacter and the cyanobacteria. Hydrogen evolution by these micro-organisms is catalysed by nitrogenase, and an uptake hydrogenase is responsible for H2 oxidation. Whereas the cyanobacteria have hydrogenase activity even in cells not fixing NZ(Tel-Or et al., 1977; Eisbrenner et al., 1978), hydrogenase is generally derepressed under Nz-fixing conditions in Azotobacter and in hydrogen uptake positive (Hup+)strains of Rhizobium. Autotrophic growth of R. japonicurn on H2 has been demonstrated in the laboratory (Hanus et al., 1979), and thus H2 oxidation by this bacterium, without concomitant Nz fixation, may conceivably occur in nature under some conditions. +

B. HYDROGEN EVOLUTION BY NITROGENASE

Nitrogenase catalyses the reduction of dinitrogen to ammonia according to the reaction: Nz+ 8H+ +8e-

+ 16ATP-+2NH3+16ADP+ 16Pi+ Hz

(2) As eqn. (2) shows, the physiological substrates include H + as well as N2, and

HYDROGEN METABOLISM IN RHIZOBIUM

3

H2 production is apparently obligatory to the N2 fixation reaction. What this equation does not indicate is that the ratio of Nz reduced to H2 evolved is not constant, and that this ratio can be altered in iiirro, and is variable under physiological conditions as well. Rivera-Ortiz and Burris ( 1975) demonstrated that H2 evolution by partially purified nitrogenase decreases as the N2 concentration increases. Hageman and Burris (1980) showed that the allocation of electrons to NZor H + by purified nitrogenase is dependent on the rate of electron flux through the enzyme, which in turn depends on the ATP concentration, the ratio of component I to component 11, and the concentration of reductant. In those experiments, a high electron flux favoured N2 reduction, whereas H2 production was favoured by a low flux of electrons through nitrogenase. Schubert and Evans (1976) found that only 40-60% of the electron flow to nitrogenase participates in NZreduction in various legume root nodules, and the remainder is lost through HZ evolution. These data indicate that the ratio of N2 fixed to HZproduced by nitrogenase is variable in nature, and is not merely an experimental phenomenon. Although H2 evolution by nitrogenase can be decreased by several experimental manipulations, it cannot be eliminated, and H2 production is apparently an obligatory product of biological NZ fixation. When H2 evolution by nitrogenase is plotted as a function of N2 concentration and extrapolated to infinite pNz, H2 evolution is found to occur at 13-23% of its maximal rate, implying that H2 production cannot be eliminated (RiveraOrtiz and Burris, 1975). This conclusion was confirmed by Simpson and Burris (1984), who showed that HZevolution by nitrogenase occurs at a pNz of 50 atmospheres (5.07 MPa). In those experiments, 27% of the total electron flux through nitrogenase was allocated to HZproduction, and the ratio of NZ fixed to H2 produced was about 1: 1. Thus the stoicheiometry represented in eqn. (2) is apparently an upper limit with respect to the amount of N2 that can be fixed per H2 evolved. It is not known for certain why H2 is produced during N2 fixation or why the amount produced is dependent on the rate of electron flux, but Chatt (1980) proposed a mechanism for Nz reduction by nitrogenase that could explain some of the observations discussed above. In his scheme, molybdenum is assumed to be the active site of the enzyme, and present in the trihydridic state. It is known that NZforms complexes only with transition metals in a strongly reduced state, and thus if the rate of electron flow is slow, the electrons are discharged by protons to form HZbefore the molybdenum moiety can become sufficiently reduced to bind Nz. When the electron flow to molybdenum is faster than the discharge rate to protons, the metal is capable of binding NZ and reducing it. Molybdenum in the highly reduced state can react with N2 and H + , but most of the protons will be discharged by interaction with the N2 bound to molybdenum, which they reach before they can interact with the

4

M . R . O’BRIAN AND R . J . MAIEK

metal. Under maximal N2-fixingconditions, one N2 molecule would have to displace two of the hydrides in order to bind to molybdenum, resulting in the formation of one H2 molecule. When Nz is completely reduced, it is removed to give two NH3 molecules, and two protons then bind to the metal to restore the trihydritic state. The formation of the hydride ions occurs either because there is no N? at the active site, or because it is a necessary condition for the release of NH3. This scheme can explain some features of nitrogenase enzymology, but it should be kept in mind that some of the assumptions on which the hypothesis is built have yet to be demonstrated. It is not known for certain that N? reacts with molybdenum moiety, nor is it known that the metal binds with three hydride ions in its most reduced state. It is also assumed that the molybdenum active site is in a pocket or cleft, and has limited access to protons and other substrates.

C. HYDROGEN OXIDATION BY LEGUME ROOT NODULES

Phelps and Wilson (1941) discovered that pea nodule bacteroids have hydrogen uptake activity, but cultures of R. leguminosarum do not. These findings could not be confirmed (Shug et al.. 1956), however, and the rediscovery of the uptake hydrogenase in pea nodules did not occur until 1967 (Dixon, 1967). Dixon (1972) concluded that the presence of an uptake hydrogenase increases the overall efficiency of the N2 fixation process in three ways: (i) it prevents the inhibition of nitrogenase by hydrogen gas; (ii) it consumes 0 2 and thereby protects nitrogenase from 0 2 inactivation; and (iii) it produces ATP by Hz-dependent oxidative phosphorylation. The latter two functions of hydrogenase have been demonstrated in R . japonicum (Emerich et al., 1979), some Hup+ strains of R . leguminosarum (Nelson and Salminen, 1982), and also in Azotohucter and the cyanobacteria (Adams rt a/., 1981). Hydrogen is known to inhibit nitrogenase, but it is not clear whether the intracellular H2 concentration is high enough to be inhibitory in uiuo (Robson and Postgate, 1980; Dixon et al., 1981). Schubert and Evans (1976) surveyed the magnitude of H2 evolution in leguminous and non-leguminous nodules, and found that, among the legumes, only nodules from Rhizohium spp. strain 32H1 in association with Vigna sinensis evolved very little HI. Schubert et a / . (1977) described a H:! uptake hydrogenase in a strain of R . japonicum and of R. “cowpea”, and Carter et al. (1978) reported that six out of thirty-two R . japonicum strains examined evolved little or no HZ as nodules, which was attributed to an uptake h ydrogenase. “Relative efficiency” is a parameter often used to assess the efficiency of Nz

HYDROGEN METABOLISM IN HHIZOBIC’M

5

fixation by measuring HI evolution by nodules (Schubert and Evans, 1976). I t is defined as:

-[

Rate of Hz evolution in air Rate of CzH2reductionp]

(3)

It can be seen that a low rate of H? evolution compared to C2H2 reduction will yield a high relative efficiency. In strains that lack an uptake hydrogenase (Hup-), the relative efficiency is an indication of the fraction of electrons allocated to Nz reduction, and thus the term “relative efficiency” has literal meaning. However, in strains containing hydrogenase, relative efficiency measurements are only an indication of how much of the Hz evolved by nitrogenase is consumed by hydrogenase; this parameter, in itself, does not say anything about the efficiency of N ? fixation. A relative efficiency of 1 does not mean the total electron flux through nitrogenase is directed toward NI reduction, but rather it means that all of the H? produced by nitrogenase is oxidized by hydrogenase. In this case the relative efficiency, as defined in eqn. (3), is the same regardless of whether hydrogenase actually affects the efficiency of Nz fixation. Albrecht et al. (1979) compared the effects of Hup+ and Hup- R..japonicurn strains on soybean plants grown in a greenhouse. They found that plants inoculated with Hup+ strains contained 16% more dry weight, 10% more N per total dry weight and 26% more total N. In similar experiments comparing a Hup- mutant and a Hup+ revertant, Evans et a/. (1983) found that plants inoculated with the Hup+ strain showed increases in weights of nodules, shoots, total plant material and in total N per culture. These results indicate that the Hup+ trait is beneficial to the Nz fixation process in the R .japonicirmsoybean symbiosis. However, the benefits of H7 oxidation in the R. leguminosarum-pea symbiosis are tenuous at this time, and these data are discussed later in this article. The remainder of this review will deal with selected aspects of H2 metabolism in Rhizohium. particularly R. ,japonicum and R . leguminasartmi. Although Hz metabolism and N2fixation in organisms other than the rhizobia will be discussed, it will be for comparative purposes, and the data presented pertaining to those organisms is not intended to be comprehensive. The enzymology of hydrogenase will concentrate on that of R.,japonicum since the R. leguminosarum enzyme has yet to be purified and characterized. Until very recently, different approaches have been taken in studying genetic problems concerning Hz metabolism in R. ,japonicum and R. lcguminosarum, and thus they are discussed separately.

6

M . R . O'BRIAN AND R. J. MAIER

11. Regulation

A. OXYGEN AND CARBON

Hydrogen uptake (Hup) by nodules or bacteroids of Hup+ R . japonicurn strains can be easily demonstrated, but heterotrophically-grown cells do not normally oxidize H2. The bacteroid environment is very different from that of a cultured cell, and thus there are many external factors which can potentially regulate the expression of hydrogenase activity. These factors include 0 2 , H2, carbon sources accessible to the cell, and trace elements, as well as the more elusive contributions from the plant host. Maier et al. (1978a) demonstrated that H2 uptake activity can occur in freeliving cells under reduced 0 2 and organic carbon concentrations, and in the presence of Hz. The decreased O2 tension and increased H2 concentration necessary for the derepression of hydrogenase activity may imitate the bacteroid environment, at least qualitatively, but the carbon requirement is harder to evaluate. The low carbon concentration required for good Hup activity may also be characteristic of the nodule milieu since photosynthate is believed to be limiting in the nodule (Hardy et al., 1978), but the carbon sources used by nodule bacteroids are not known with certainty, and thus it is difficult to be confident in such speculation. It is also possible that the phenotype of hydrogenase-derepressed cells is similar to that of free-living aerobic hydrogen bacteria, and that the derepression conditions do not actually simulate a nodule environment at all. The ability of R. japonicum to grow autotrophically (Hanus et al., 1979) and express ribulose 1,5-bisphosphate (RuBP) carboxylase (Simpson et al., 1979)lends credence to this notion. It was later found that hydrogenase activity can be derepressed in the complete absence of organic carbon, and is enhanced by the addition of C02 (Maier et al., 1979). Numerous carbon substrates repress hydrogenase expression, and this repression is apparently at the level of hydrogenase synthesis (Maier et al., 1979). The addition of arabinose or gluconate to derepressing cells causes the H2 uptake rates of the cells to level off, whereas the activity of these cell continues to increase in the absence of carbon. The same phenomenon is observed when O2is added to, or H2 is removed from, the derepressing cells. Since the activity of hydrogenase-derepressed cells is not inhibited by the addition of O2 or organic carbon, these substances act by repressing hydrogenase synthesis, and do not inhibit the hydrogenase enzyme. Several types of mutants of R .japonicum have been obtained that are either hypersensitive (Maier and Merberg, 1982) or insensitive (Merberg and Maier, 1983; Merberg et al., 1983) to repression by 0 2 . The 02-hypersensitive mutants were initially characterized by their ability to express Hup activity as

HYDROGEN METABOLISM IN RHlZOBlUM

7

bacteroids, but not under normal H2-derepressing conditions in culture. However, five out of seven such mutants do express Hup activity when the 0 2 tension is lowered from 2% to 0.4%. The kinetics of hydrogenase repression by 2% O2 in these mutants is similar to the repression of activity in the wild type by 20% 0 2 . The addition of 2% 0 2 to the mutants does not cause an inhibition of H2-derepressed cells, but rather it results in the cessation of derepression. This observation means that the mutants do not make a more 02-labile H2 uptake system, nor is the 0 2 sensitivity due to a general toxicity effect. Since the mutants grow heterotrophically, the mutation seems to be specific to H2 metabolism. The second class of R.japonicum mutants are insensitive to 0 2 repression of hydrogenase activity. The mutants were isolated by selecting for cells that can grow chemoautotrophically under 10% 0 2 (Merberg and Maier, 1983). The wild type cannot grow autotrophically under such high 0 2 tensions, nor can it be derepressed for Hup activity at 10% 0 2 . These 02-tolerant mutants have significantly greater 0 2 - and methylene blue-dependent H2 uptake activity as bacteroids compared with the wild type, suggesting that common factors regulate H2 oxidation in free-living cells and bacteroids. Further analyses show that these mutants are extremely interesting in several respects. Whereas the hydrogenase activity of the wild type is repressed by organic carbon, the 02-insensitive strains are considerably less sensitive to carbon repression (Merberg et al., 1983). The addition of arabinose or succinate to derepressing cells at concentrations that repress the wild type 90% or more only inhibit hydrogenase expression 30-50% in the 02-insensitive strains. These data strongly suggest that there is a common regulatory element involved in the control of hydrogenase expression by 0 2 and carbon. This assertion is supported by the observation that mutants hypersensitive to hydrogenase repression by 0 2 are also hypersensitive with respect to carbon repression (Merberg et al., 1983). The 02-tolerant strains also derepress hydrogenase in the absence of added H2 (Merberg et al., 1983), which is required for hydrogenase expression in the wild type (Maier et al., 1979). Since these mutants express hydrogenase activity in heterotrophically-grown cells, and need not be induced to express hydrogenase, they are referred to as hydrogenase-constitutive (Hup") mutants. The nature of the mutations causing the Hupc phenotype is not known, but it is apparently not due to an increase in intracellular cyclic AMP (adenosine 3',5'phosphate) concentration (Merberg et al., 1983), which has been shown to coincide with hydrogenase expression in R. japonicum (Lim and Shanmugam, 1979). It is interesting to note that all R. japonicum HupC mutants isolated thus far produce significantly more cytochrome o than does the wild type (O'Brian and Maier, 1985a). Like hydrogenase, cytochrome o is synthesized under low 0 2 conditions in many bacteria (Poole, 1983),

8

M . K. O'RRIAN A N D R . J. MAIER

suggesting that the regulatory gene altered in the Hup'mutants also affects cell systems not directly related to hydrogenase. Mutants similar to the R . juponicum Hup'strains have also been obtained in a strain of Alculigenes eutrophus (Cangelosi and Wheelis. 1984). Alculigenes eutrophus strain 17707 does not grow chemoautotrophically under 20%)0 2 . but it will grow at 4% O2and synthesize both hydrogenases. Mutants relieved of 0 2 repression (oxygen sensitivity negative, or Osec) can grow chemoautotrophically, and have soluble and membrane-bound hydrogenase activities. The Ose- mutation mobilizes with a self-transmissible plasmid that carries genes necessary for hydrogenase expression. Ose- strains can also be obtained from mutants with plasmid-borne lesions which result in the loss of soluble hydrogenase activity, showing that the Ose- phenotype is independent of soluble hydrogenase activity. Chromosomal lesions resulting in diminished soluble and membrane-bound hydrogenase activities cannot be made Ose- . These data suggest that the Ose trait may act only on the membrane-bound hydrogenase, but the inability to obtain mutants deficient only in the particulate hydrogenase makes this speculation difficult to prove. Like the Hup' R . ,juponirum mutants (Merberg et ul., 1983). the Ose- A . eutrophus mutants are relieved of hydrogenase repression by organic carbon substrates (Cangelosi and Wheelis, 1984). Since H? is known to inhibit heterotrophic growth of A . eurruphus if hydrogenase is synthesized (Schink and Schlegel, 1978; Schlesier and Freidrich, 1982), Cangelosi and Wheelis (1984) proposed that the Ose system serves to minimize this phenomenon (called the hydrogen effect). They also suggest that Hz oxidation by A . eutrophus probably occurs in a mixotrophic, rather than an autotrophic, context in nature, and the down regulation of hydrogenase activity maximizes mixotrophic growth. This idea is supported by the observation that heterotrophic growth on glycerol is inhibited by HI in the Ose- mutants, but not in the wild-type strain (Cangelosi and Wheelis, 1984). This hypothesis is reasonable from the standpoint that Hz oxidation and mixotropic growth may not be advantageous in the presence of adequate organic carbon sources, and thus hydrogenase synthesis would be repressed by organic carbon. The repression of hydrogenase by 02,however, is less obvious in terms of mixotrophic growth since there seems to be no reason to assume that a low 0 2 concentration accompanies mixotrophy in nature. However, an energy deficiency could occur under low 0 2 conditions, and thus a substrate that yields the most ATP per 02consumedwould be beneficial to the cell. Bongers (1967) found that the P/O ratio with Hz as substrate is substantially higher than with succinate or fihydroxybutyrate as substrates in A . eufrophus.This observation indicates that Hz could indeed be a good energy source when 0 2 is limiting, and would explain why hydrogenase expression is derepressed under low 01tensions. It is not known whether 0 2 and carbon regulation of hydrogenase

HYUROGI 17,000 nmol h- (mg protein)-'), and the addition of HZ to these cells increases the overall respiration rate more than eightfold (Eisbrenner et af., 1982). It is important to note that the addition of H2 increases the reduction rate of the total b- and ctype cytochrome in chemolithotrophically-grown cells (Eisbrenner et af., 1982), and thus selecting a particular cytochrome as being unique to the H2 oxidation system is arbitrary and unwarranted. (ii) The cytochrome spectra of chemolithotrophic cells presented by the Evans group shows that the H2 reduces more c-type, as well as b-type, cytochrome than does succinate (Fig. 2 of Eisbrenner and Evans, 1982b). On comparing these spectra, there appears to be no basis for concluding that only a b-type cytochrome is unique to H2 oxidation. Since succinate is a very good reductant (Appleby, 1969a,b; O'Brian and Maier, 1982), a reasonable and simple explanation for the lack of succinate-reduced cytochromes is that the spectrum was taken only 20 seconds after the addition of succinate. (iii) Eisbrenner and Evans (1982b) quantified component 559-H2by taking Hz-reduced minus oxidized difference spectra of cells in the presence of cyanide. Although the rationale for adding cyanide was not stated, it is clear that what was actually being measured, at least in some of the spectra shown (Fig. 2 of Eisbrenner and Evans, 1982b, for example), was total b-type cytochrome, and not just component 559-H2. By this quantitation method, Eisbrenner and Evans (1982b) correlated H2 uptake activity with component 559-H2 from cells grown symbiotically, heterotrophically or chemolithotrophically, and in Hup- strains. They reported a correlation coefficient of 0.890.98 and concluded that component 559-H2 is closely related to hydrogenase function or structure, and is perhaps associated with the enzyme per se. Since cytochrome expression in Rhizobium, and in other bacteria, has been shown repeatedly to be dependent on growth conditions, comparison of cells grown

'


31

under drastically different conditions is not meaningful. The correlation data also extends to Hup- strains; since component 559-H2 is, by definition, reducible only by H2, it cannot be valid to examine the extent of Hz-dependent cytochrome reduction in a strain that does not oxidize H2. Also, there is evidence that hydrogenase and cytochrome o are regulated by common factors (OBrian and Maier, 1985a), and thus a positive correlation would not be surprising. Such a correlation, however, would not warrant the conclusion that cytochrome o is structurally or functionally related to the hydrogenase enzyme in any direct way. (iv) The claim that component 559-H2 has a low redox potential, and receives electrons from hydrogenase, is based on the conclusion that this cytochrome is unique to the H2 oxidation system (Eisbrenner and Evans, 1982b). However, there are no precedents which mandate that component 559-H2 has a low potential in order to participate solely in H2 oxidation. Also, the kinetic data show that component 559-H2 is reduced more slowly than the other cytochromes (Eisbrenner et al., 1982), whereas a low potential cytochrome is expected to be more reduced when in a steady state than the components with higher potentials. Therefore, the kinetic behaviour of component 559-H2 is similar to that of cytochrome o, which is not a lowpotential cytochrome. It has been found that H2 does not reduce more 6-type cytochrome than does succinate or NADH in H2-derepressed wild type cells or in heterotrophially-grown cells that express hydrogenase constitutively (0’Brian and Maier, 1982, 1985a), showing that component 559-H~is not present in these cells. It has also been shown that there is no 6-type cytochrome which mediates electron flow from hydrogenase to ubiquinone in hydrogenase-constitutive cells (O’Brian and Maier, 1985b), ruling out a low-potential cytochromeb that accepts electrons directly from hydrogenase. Furthermore, hydrogenaseconstitutive strains express significantly more 6-type cytochrome than does the wild type, and this component is cytochrome 0,not component 559-H2. Although hydrogenase and cytochrome o may be regulated by common factors, cytochrome o is reducible by NADH and succinate, as well as by H2, and participates in substrate oxidation (O’Brian and Maier, 1985a).Cells of P . denitrificans grown autotrophically with H2 also express more cytochrome o than do heterotrophically-grown cells (Porte and Vignais, 1980), supporting the assertion that hydrogenase and cytochrome o are regulated by common factors. Despite the data showing component 559-H2 to be absent from H2oxidizing R .japonicum, the Evans group insists that this cytochrome is present in bacteroids and chemoautotrophically-grown cells, and in membranes of chemolithotrophic cells (Lambert et al., 1985b). Figure 7 shows difference spectra of wild-type bacteroid membranes using H2 or succinate as the

38

M. R. O'BRIAN AND R. J. MAIER

551.5

FIG. 7. Comparison of H2- and succinate-reduced cytochrornes in bacteroid membranes. The absorption spectra are substrate-reduced minus 02-oxidized mernbrane samples.

reductant. It is clear from these spectra that H2 reduces no more cytochrome b than does succinate, showing that component 559-Hz is not present in bacteroid membranes.

V. Genetics A. MUTANTS

The generation of mutants to study a complex and highly regulated system such as H2 oxidation is obviously desirable, but the acquisition of such strains is diffcult when the trait of interest in only expressed symbiotically, and screening mutants is cumbersome, and often impossible. Fortunately, conditions have been found in which R. japonicum expresses hydrogenase activity in free-living culture (Maier et al., 1978a), and thus mutants with lesions affecting Hz metabolism have been obtained. Hup- mutants were first selected by enriching a bacteroid suspension for cells unable to reduce PMS (phenazine methosulphonate) with HZ, and then screening for colonies that failed to reduce triphenyltetrazolium chloride in the presence of H2 (Maier et al., 1978b). Hup- mutants obtained from this screening cannot oxidize HZ


39

symbiotically or in free-living culture, and nodules formed from these mutants evolve H2, whereas the wild type does not. The triphenyltetrazolium chloride screening method has been used to isolate Hup- mutants of mungbean and urd bean Rhizobium (Pahwa and Dogra, 1981, 1983). Maier (1981) isolated over 100 ethyl methane sulphonate (EMS)-induced mutants of a USDA 122 derivative (SR) based on the inability of these mutants to grow autotrophically with H2 and C02 as the sole energy and carbon sources, respectively. The phenotypes of these mutants fall into various classes. About 70% of the mutants cannot oxidize H2, but two strains, SR106 and SR166, do have hydrogenase activity in the presence of artificial electron acceptors such as methylene blue or PMS. These mutants presumably synthesize an active hydrogenase enzyme, but may be missing some other component in the electron-transport system to 0 2 . Preliminary experiments indicate that, as bacteroids, these strains synthesize b- and c-type cytochromes similar to the wild type (M.R. O’Brian and R.J. Maier, unpublished observations), but some other component not discernible by absorption spectroscopy of fractionated cells could be missing. Four strains were isolated that have hydrogenase activity, but have no C02 uptake ability. Two of these mutants lack ribulose 1$bisphosphate (RuBP) carboxylase activity; this enzyme is the primary carboxylase functioning during autotrophic growth of R.juponicum (Lepo et al., 1980). The two carbon-fixation mutants that do not have RuBP carboxylase activity may be deficient in another enzyme in the Calvin cycle. Hydrogenase activity can be reconstituted when two EMS-induced Hupmutants are mixed together (Maier and Mutaftschiev, 1982). Soluble fractions of the Hup- mutants SRI 18 and SR146 were incubated together under 100% H2, and methylene blue-dependent HZ uptake by this mixture could be observed within two hours, with maximal activity seen after 10 hours. The kinetics of H2 uptake during the incubation period corresponded with an increase in turbidity over the same period; electron micrographs of the reconstituted system showed that membrane vesicles formed during the incubation period. Furthermore, the reconstituted vesicles could be sedimented, and the resulting pellet had Ordependent hydrogenase activity which was sensitive to cyanide. It was later found that hydrogenase activity of S R l l 8 could be partially restored by adding a chloroform-methanol extract of the wild type to crude extracts of the mutant (T.-Y. Wong and R.J. Maier, unpublished observations). It is not known yet whether a lipid or some other component of the extract is responsible for complementing the mutant. Moshiri et al. (1983) found two Hup- mutants that also lack N2 fixation ability. One of the Hup- Nif- mutants, SR143, produces green nodules, and has much less leghaemoglobin than nodules produced by the wild type. The hydrogenase activity of this mutant can be partially restored by the addition of

40

M. R. O'BRIAN AND R. J . MAIER

heme plus ATP. Apparently, SR143 makes apocytochromes, but is deficient in some aspect of heme biosynthesis. The addition of heme to crude extracts or membranes of E. coli (Haddock and Schairer, 1973) and Staphylococcus aureus (Lascelles, 1979) heme-negative mutants also restores respiratory activity in those bacteria. The failure of SR143 nodules to synthesize leghaemoglobin supports the current notion that heme for leghaemoglobin is synthesized by the bacteria rather than by the plant host (Cutting and Schulman, 1969; Avissar and Nadler, 1978). The Nif- Hup- mutant SR139 produces pink nodules, and is therefore a different mutant from SR143. Mutant SRl39 has no methylene blue-dependent Hz uptake activity, suggesting that it does not synthesize an active hydrogenase enzyme. Furthermore, nitrogenase activity in SR139 cannot be restored by the addition of components I or I1 of nitrogenase, showing that this mutant lacks both of the nitrogenase components. Since SR139 reverts to the wild type at a high frequency, the Nif- Hup- phenotype must be due to a single genetic lesion that affects the expression of three enzymes. The mutated gene may be regulatory, or it may be a structural gene that codes for a factor common to the enzymes, such as iron-sulphur clusters. It is unlikely, however, that SRl39 has a problem with general iron or sulphur metabolism since these cells grow normally under heterotrophic conditions. A 23 kb DNA fragment has been isolated that can complement SR139 (Hom et al., 1985, see Section V.B), and at least one other gene involved in the expression of hydrogenase is on this DNA segment. The gene mutated in SR139 may be unique to Nif and Hup expression since it is located near other hup genes. Several mutants have been isolated that affect the expression of hydrogenase by 02; these mutants are discussed in detail in Section 11. One class of mutants expresses hydrogenase activity in nodules, but can only be derepressed for activity in liquid culture if the 0 2 tension is reduced to 0.4%.The wild-type strain can be derepressed for activity under 2% 02, and thus these mutants seem to be hypersensitive to 0 2 repression. The very low 0 2 tension inside the nodule would explain the Hup+ phenotype of these mutants as bacteroids. The second class of mutants are insensitive to 0 2 repression, and express hydrogenase activity under atmospheric conditions. These mutants have greater methylene blue- and 02-dependent hydrogenase activities as bacteroids and free-living cells when compared with the wild type. Twodimensional polyacrylamide gels show that the 02-insensitive (Hupc) mutants synthesize at least six peptides not found in the wild-type parent strain. B. MOLECULAR GENETICS

1. Genetic Techniques Used to Study Rhizobium Rhizobium has been refractory to genetic analyses and manipulations using

HYDROGEN METABOLISM IN R H l Z O B l U M

41

standard techniques developed in E. coli. There have been no transducing phages isolated that can be used to package foreign DNA and infect Rhizobium, and although sphaeroplasts of R . japonicum have been described (Child and Sietsma, 1975; Berry and Atherly, 1984), it is difficult to transform Rhizobium efficiently. Furthermore, the standard cloning vectors used in E. coli do not replicate in Rhizohium, and finally, R . japonicum, R . lupini and Rhizohium spp. grow slowly, making genetic manipulations extremely tedious in these organisms. Despite these drawbacks, several ingenious methods have been devised to transfer DNA into Rhizobium and to create mutants using transposon and site-directed mutageneses. Plasmid RK2 is a broad host range plasmid that can be mobilized into, and replicated in, Rhizobium and other Gram-negative bacteria, but its large size (56 kb) makes it an impractical cloning vector. Ditta et al. (1980) developed a cloning system that separates the transfer and replication functions of RK2 onto separate plasmids. The actual cloning vehicle, pRK290, is 20 kb in size and contains the replication functions. The plasmid pRK20 13 contains the RK2 transfer genes cloned onto a ColEl replicon (thus it replicates in E. coli), and serves to complement pRK290 in trans for mobilization. This system can be used to mobilize relatively large DNA fragments, cloned into pRK290, into Rhizobium via conjugation. Friedman et al. (1982) refined this technique by cloning the lambda phage cos site into pRK290, making it possible to ligate 15-30 kb fragments into this cosmid vector (pLAFRl), package it into lambda phage heads and infect E. coli. The greater efficiency of DNA transfer by transduction, compared with transformation, allows the construction of much larger gene banks, and the inserts of these banks are approximately uniform in size. Ruvkun and Ausubel (1981) used pRK290 to develop a method for sitedirected mutagenesis. A Tn5-mutated nif region of R . meliloti borne on pRK290 was introduced into a R . meliloti wild-type strain. Recombination of the plasrnid-borne n i j region::Tn5 DNA with the chromosome results in a Nif- mutant if the Tn5 is inserted into a DNA region required for the expression of nitrogenase. This technique has been used to study hup genes in R.japonicum (Haugland et al., 1984). Simon et al. (1983a) modified an E. coli strain and several E. coli plasmids such as pBR325, pACYC177 and pACYC184 so that the vectors can be mobilized into Gram-negative bacteria as a means of delivering Tn5, or some other piece of foreign DNA, into the cell. This was achieved by integrating the transfer genes of plasmid RP4 into the chromosome of E. coli strain S49-20, and cloning the RP4 “mob” site into the E. coli vector. The mob site is believed to include the origin of transfer (ori T) and the recognition site for the transfer gene products. Since the vectors cannot replicate in Rhizobium, the introduced DNA can only be expressed if it is incorporated into the genome by recombination, or by transposition in the

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M. R. O'BRIAN AND R. J. MAIER

case of Tn5. It is possible, therefore, to create a population of Tn5 mutants in Rhizobium, or do site-directed mutagenesis experiments by recombining a mutated gene into the genome of a wild-type recipient. If the inserted gene is mutated by a transposon, selection for the recombinants by antibiotic resistance is straightforward.

2. Hup Genes on Indigenous Plasmids Since nif and nod genes of several Rhizobium species are found on plasmids (Nuti et al., 1979; Krol et al., 1981; Hombrecher et al., 1981; Hooykaas et al., 1981; Prakash et al., 1981), it is reasonable guess that hydrogenase genes may be on a plasmid as well. Spontaneous hydrogen oxidation mutants (Hox-) of Alcaligenes eutrophus strain TF931 lack plasmids, but they can recover the ability to express hydrogenase activity and grow autotrophically when mated with the wild-type strain HI6 (Friedrich et al., 1981b). Transfer of hydrogenase activity to the mutants occurs in the absence of a mobilizing plasmid such as RP4. Strain HI6 and the transconjugants of the strain TF93 mutants contain a large plasmid with a molecular weight of 270- lo6. These data show that A . eutrophus harbours a self-transmissible plasmid that contains genes necessary for the expression of hydrogenase activity. Further studies by the same group (Hogrefe et al., 1984; Friedrich et al., 1984) showed that genetic determinants of the membrane-bound and soluble hydrogenases are plasmidborne, but hydrogenase determinants are located on the chromosome as well. Brewin et al. (1980) investigated the possibility that hydrogen uptake genes (Hup)are on a plasmid in R . leguminosarum.Since nodgenes are known to be found on a plasmid, they transferred the nod genes of a Nod+ Hup+ strain into the Nod- Hup- strain 16015 to see if the Hup and Nod determinants were cotransferred. The Nod-containing plasmid R16JI was not self-transmissible, but could be mobilized after recombination with a transmissible R. leguminosarum plasmid. Over half of the R. leguminosarum 16015 cells that received the recombinant plasmid could nodulate peas, and all the Nod+ transconjugants had hydrogenase activity comparable to that of the wild type. They concluded that determinants of nodulation and hydrogen oxidation are genetically linked, and are cotransferred due to the Nod and Hup genes being located on the same plasmid. Attempts to locate hydrogenase genes on an indigenous plasmid in R. japonicum have not been successful, nor has any plasmid been found in a strain with substantial hydrogenase activity. Cantrell et al. (1982) looked for plasmids in Hup+ and Hup- strains, and in Hup- mutants of R . japonicum. They found that six out of eight naturally occurring Hup- strains contained plasmids, with molecular weights ranging from 44-180. lo6, and one out of seven Hup+ strains harboured a plasmid; this latter strain had very poor


43

hydrogenase activity. It was also found that some Hup- mutants of the Hup+ strain SR contained multiple plasmids. The non-revertible mutants SR 1, SR2 and SR3 contain two plasmids each, whereas the revertible mutants PJ 17, PJ18 and PJ20 do not contain any plasmid. The reason for these phenomena is not known, but several possibilities exist. The hup genes may be located on a plasmid too large to be detected by the procedures used, and a mutation causes the formation of two smaller plasmids that can be discerned. Alternatively, hup DNA may be excised from the chromosome to create plasmids and a Hup- phenotype. The latter event is possible if insertion sequencesare located near the hup locus, as is found in the nifgene region of R. japonicum (Kaluza et a f . , 1985), and thus allowing chromosomal mobilization. Either possibility would explain why SRl, SR2 and SR3 are nonrevertible. Assuming these three mutants are not siblings, the similar plasmid patterns found in them imply that the event causing the mutation is not random, although a larger population of Hup- mutants needs to be examined to verify this. This study also raises the question of whether naturally occurring Hup- strains are actually mutants of Hup+ strains, and if hup genes are contained on the plasmids of Hup- strains. Hybridization experiments using isolated hup genes would directly address this question.

3. Hup Genes in Rhizobium japonicum

A gene bank of R . japonicum was constructed into the broad host range cosmid pLAFR1, and mated en mane with Hup- recipients in order to isolate DNA fragments that complement the mutants (Cantrell et al., 1983). Hup+ transconjugant colonies were selected based on their ability to reduce methylene blue in the presence of Hz and respiratory inhibitors (Haugland et al., 1983). Hup+ colonies arose at a frequency of 6 - lop3per transconjugant when the Hup- mutant PJ17nal was the recipient, but the mutant PJ 18nal could not be complemented in trans by the gene bank. Cosmids isolated from eleven of the Hup+ transconjugants of PJ 17nal contained three common EcoRl fragments of 13, 2.9 and 2.3 kb in size. Five out of six of the Hup+ transconjugants expressed hydrogenase activity as nodules, but only at 525% of the wild-type level. One of the cosmids, pHU 1, was mutagenized with Tn5 in various regions of the insert DNA, and the mutated DNA was introduced into the wild-type genome by homologous recombination to determine the hup-specific regions of the cosmid (Haugland et al., 1984). It was found that hup-specific sequences spanned 15.5 kb as determined by the Hup phenotype of the Tn5 mutants created by the homologous recombination. The mutant PJ18nal can be complemented by fragments of pHU 1 cloned into pBR325 if the entire recombinant plasmid is integrated into the chromosome, presumably by a single cross-over event. This observation is interpreted to

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M. R . O’BRIAN AND R . J . MAIER

mean that the PJ18nal mutation site occurs within one of the subcloned fragments (several EcoRl fragments of pHUl were cloned onto one plasmid in a random order), and that the mutation is dominant. Lambert et al. (1985a) isolated additional cosmids that complement a Hup- mutant other than PJ17nal. One such cosmid, pHU52, was introduced into several naturally occurring Hup- strains of R .japonicum and into strains of R . meliloti and R . leguminosarum. These transconjugants can be derepressed for hydrogenase activity in free-living culture, and thus the authors concluded that all the determinants required for the expression of Hup activity in the free-living and symbiotic states of R. japonicum are on pHU52. Although these results are exciting, we do not agree that the data show that all the Hup determinants are on pHU52, nor is there reason to believe that pHU52 does not regulate structural genes of the host, and vice versa. Cantrell et al. (1982) provided evidence that the difference between a Hup+ and HupR.Japonicum strain is due to the rearrangement of the DNA to form multiple plasmids from the genome or from larger plasmids. If this is true, then hup genes may be present in Hup- strains which can be expressed in the presence of pHU52. Hybridization experiments are needed to see if the Hup- strains contain DNA homologous to pHU52, and further characterization of the Hup- strains is necessary to determine the nature of these strains. Rhizobium meliloti strain 102F51 has Hup activity in the free-living and symbiotic states (Ruiz-Argueso et al., 1979), and thus transconjugants of this strain must possess hup genes not borne on pHU52. Finally, R. leguminosarum strain 128C53is a Hup+ strain symbiotically, and certainly contains indigenous hup genes (Brewin et al., 1980). In fact, Tn5-induced Hup- mutants of strain 128C53 can be complemented by pHUl (Kagan and Brewin, 1985); this plasmid insert has about 25 kb in common with the pHU52 insert. It is entirely possible that pHU52 contains genes that allow the expression of hup genes in free-living culture, but it is premature to assume that the Hup+ phenotype of transconjugants harbouring pHU52 is entirely due to the expression of genes on that plasmid. Hom et al. (1985) complemented the Nif- Hup- mutant SR139 with cosmids from a R.japonicum gene bank constructed in pLAFR 1. It was found that the Hup+ transconjugants of SR139 also have nitrogenase activity both symbiotically and under conditions where nitrogenase is induced in free-living cells; SRI 39 has no nitrogenase activity under either growth condition. The nitrogenase activity of the SR139 transconjugants are much higher in freeliving cells than in nodules, relative to those of the wild type, presumably due to segregational loss of the plasmid in nodules. One of the cosmids that complements SR139 (pSH22) was tested to see if it could complement other autotrophic growth mutants. Six Hup- Nif+ mutants could be complemented by pSH22, but a carbon-fixation mutant, and a Hup- Nif- mutant different


45

from SR139 were not complemented by the plasmid. The ability of pSH22 to complement both a Hup- Nif- and a Hup- Nif+ mutant suggests that this cosmid harbours more than one gene essential for the expression of hydrogenase activity. This conclusion is strengthened by the fact that SRl39 is a revertible mutant (Moshiri et al., 1983), and one of the Hup- Nif+ mutants (SU306-47) is a Tn5-induced mutant, therefore each of these mutants almost certainly has a single lesion in a different region on the genome. These data also suggest that the niflhup gene mutated in SRl39 and the hup gene mutated in SU306-47 are located on different operons, or the hup gene is located downstream from the nif/hup gene on the same operon since the polar mutation in SU306-47 does not result in a Nif- phenotype. It should also be noted that pSH22 does not complement seven Hup- Nif+ mutants tested, thus there must be genes involved in Hup expression other than the ones discussed above. It is not known yet where on pSH22 the specific hup genes are located, but it is known that a 13.2 kb EcoRl fragment of pSH22 can complement SRl39 (P. Novak and R.J. Maier, unpublished observations). 4. Hup Genes of Rhizobium leguminosarum

As stated above, hup genes of R. leguminosarum are carried on an indigenous plasmid along with nod and nif genes. This plasmid, pRL6J1, is nontransmissible, but it can mobilize into other cells by recombining with mobilizable R.leguminosarum plasmids such as pVWJ3 1 or pVWJ51 (Brewin et al., 1980, 1982). When the recombinant plasmid pIJ1008 (recombinant of pVW5JI and pRL6JI) is introduced into Nif+ Nod+ Hup- strains, the resulting Hup+ transconjugants have superior symbiotic properties than the parent strain, as judged by plant dry weight, nitrogen content, leaf area and nitrogen concentration (DeJong et a/., 1982). One of the transconjugants, strain 3960, has better symbiotic properties than the Hup+ strain 128C53, from which the hup determinants in pRL6JI were derived. It was concluded that the increased effectiveness of strains harbouring pIJ1008 may be due to the ability of these strains to recycle the H2 evolved by nitrogenase via hydrogenase. They do not rule out, however, the possibility that some other determinant on pIJ 1008 confers superior symbiotic qualities on the transconjugants. Indeed, other experiments described below suggest that determinants other than Hup may be responsible for the symbiotic effectiveness of the Hup+ transconjugants of Hup- strains. Kagan and Brewin (1985) mutagenized a Hup+ R.leguminosarum strain to create Hup- mutants using a technique that minimizes the number of mutants that have to be screened on plants. The Hup+ strain 128C53was mutagenized with Tn5-mob, a Tn5 derivative that contains the mobilization site of plasmid RP4 (Simon et al., 1983b). The Tn5-mob insertion allows a plasmid to be

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M. R. O’BRIAN AND R. J. MAIER

mobilized, thus plasmid-linked mutations can be enriched for by mating the Tn5-mob mutants with a plasmidless recipient and selecting for kanamycinresistant (due to Tn5) transconjugants. These recipients are screened further for the presence of the symbiotic plasmid pRL6JI (strain l28C53 contains multiple plasmids) by scoring for colonies that cross-react with an antibody made against a protein encoded by pRL6JI that is expressed in free-living culture. Using this selection procedure, eight mutants were isolated that have no hydrogenase activity symbiotically. All eight mutants can be complemented with cosmid-borne R. japonicum genes that can complement a HupR.japonicum mutant (see Cantrell et al., 1983). Surprisingly, this R .japonicum DNA fragment has only weak homology with strain 128C53, whereas it is strongly homologous with several other Hup+ R. leguminosarum strains (Nelson et al., 1985, see below). Cunningham et al. (1985) used the Hup- R. leguminosarum mutants described above to study the effect of the Hup phenotype on nitrogen fixation in pea nodules. Three pea cultivars were each inoculated with one of six Hupmutants, the Hup+ parent strain, or the Hup+ parent strain with a Tn5-mob insertion on pRL6JI. It was found that, for each cultivar, at least one of the six Hup- mutants fixed as much N2 as the wild-type strain. Since previous experiments (DeJong et al., 1982) suggested that pRL6JI confers superior symbiotic properties on nodules from strains harbouring this plasmid, it was concluded that some trait encoded by pRL6J1, other than the Hup+ phenotype, is responsible for the improved symbiotic performance. At the time of writing this review, these results are only present in abstract form, and thus the data are not available for analysis. It would be interesting to examine the Hup- mutants that did not fix as much N2 as the wild type to see if these differences are significant. Since at least five of the six Hup- mutants have the Tn5-mob insertion in different sites on the plasmid (Kagan and Brewin, 1985) and have different N2-fixing capabilities, it seems that the mutation of some hup genes may affect N2 fixation despite the observation that the Hupphenotype is not always detrimental. The genetic analyses of R. leguminosarum and R.japonicum are consistent with the physiological data which show that the hydrogen oxidation systems of the two bacteria are different in several respects. There is a considerable amount of data indicating that Hz oxidation does not increase Nz fixation ability in R.leguminosarum (Cunningham et al., 1985; Nelson, 1983; Truelsen and Wyndaele, 1984),whereas most of the data for R.japonicum indicates the Hup+ phenotype confers a symbiotic advantage on the strain (Albrecht et al., 1979; Evans et al., 1983). The most obvious explanation is that the R. japonicum hydrogenase is more efficient at recycling the H2 evolved by nitrogenase than is the R. leguminosarum hydrogenase (Carter et al., 1978; Truelsen and Wyndaele, 1984). The variability in the H2 oxidation systems is

HYDROGEN METABOLlSM IN RHIZOBIUM

41

also reflected in the level of homology of the hup-specific DNA in R. leguminosarum and R.japonicum. Nelson et al. (1985) found that hup-specific DNA from R. japonicum and R. leguminosarum hybridize weakly or not at all with total and plasmid DNA from four out of twelve R. leguminosarum Hup’ strains examined. These observations not only reflect intergenic variability of hup-specific DNA, but also differences among R. leguminosarum strains. Rhizobium leguminosarum strain 128C53, the strain on which most of the genetic and mutant studies have been performed, shows only weak hybridization with hup genes from R.japonicum and from R. leguminosarum strain BIO. Interestingly, three of the four strains that have little or no homology to the hup DNA probes are known to have H2 oxidation systems coupled to ATP synthesis; most Hup+ R. leguminosarum strains seem to have Hz uptake activities not coupled to ATP synthesis (Nelson and Salminen, 1982). This is ironic since the R.japonicum strain from which the hup probes were derived has a H2 oxidation system that is coupled to ATP synthesis (Emerich et al., 1979). It is clear from these studies and from those discussed above that the regulation of hup genes in Rhizobiurn is extremely complex, and is controlled by many factors, including the host, of which almost nothing is known.

VI. Acknowledgements Work from the laboratory of Robert Maier has been supported by grants from the United States Department of Agriculture and from Allied Corporation. The authors thank Dr Robert Burris and Dr Daniel Arp for permission to reproduce their data. Thanks are also extended to Patricia Novak and Farhad Moshiri for their help with this manuscript. REFERENCES

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The Physiology and Biochemistry of Pili WILLIAM PARANCHYCH and LAURA S. FROST Department of Biochemistry. University of Alberta. Edmonton. Alberta. Canada T6G 2H7

. . . I. Introduction . . . . . . . . . I1. Nomenclature . . . . . . 111. Classification . . . . . . . . . A . Criteria for classification . . . . . . . . . B. Morphology . . . . . . . . C . Function and biochemical properties . . . . IV . High-resolution studies on pilus structure . . . . V . Organization and expression of pilin genes A . Conjugative pili . . . . . . . . B. Type 1 pili . . . . . . . . C . Pili designated Pap . . . . . . . D . Pili designated CFA/I and CFA/II (CSl, CS2 and CS3) E. Pili designated K88 and K99 . . . . . F. Pili designated NMePhe . . . . . . . . VI . Structure-function relationships of pili proteins . A . Conjugative pili . . . . . . . . . . . . B . Adhesive pili of Escherichia coli . C . Pili designated NMePhe . . . . . . VII . Acknowledgements . . . . . . . . References . . . . . . . . .

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I. Introduction Bacterial “fimbriae” or “pili” are thin (2-1 2 nm diameter) non-flagellar protein filaments found on the surfaces of many types of bacteria . They are variable in length (0.5-10 pm) and the number per cell ranges from one or two to several hundred . Most functions attributed to pili may be ascribed to their adhesive properties . These allow them to bind to other bacteria, bacteriophages. mammalian cells and inert surfaces. Piliated bacteria that adhere to ADVANCES IN MICROBIAL PHYSIOLOGY. VOL . 29 ISBN 0-12-0277294

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mammalian cells are often more pathogenic than their non-piliated counterpart because the pili enable them to become anchored to the host tissue and resist elimination by body fluids. Numerous reviews have already dealt with various aspects of the structure and function of pili (Brinton, 1965, 1967, 1971, 1977; Ottow, 1975; Tomoeda et al., 1975; Pearce and Buchanan, 1980; Gaastra and de Graaf, 1982; Jones and Isaacson, 1983; Klemm, 1985; Mooi and de Graaf, 1985). This article will attempt to update these reviews and focus on the structure and function of pili in light of recent advances employing biochemical, immunological and genetic approaches.

11. Nomenclature

Since their discovery by Anderson ( I 949) and Houwink (1949), bacterial nonflagellar filamentous appendages have been referred to as threads, filaments, bristles, cilia, fibrillae, fuzz, colonization factor antigen, adhesins, fimbriae and pili. The designation “fimbriae” (Latin for thread or fibre) was introduced by Duguid and his coworkers in about 1955 (Duguid el a/., 1955), while Brinton (1959) introduced the name “pili” (Latin for hair-like structure) four years later. Until 1964, pili were distinguished primarily on the basis of morphology, but Crawford and Gesteland (1964) found by electron microscopy that the RNA “male-specific” phage R17 adsorbed selectively to a new kind of pilus, distinct from the more numerous “type 1” (or “common”) pili. Brinton et al. (1964) confirmed this observation and named the filaments F pili after demonstrating that these filaments were required for genetic transfer as well as F-specific phage susceptibility, and that the genes for their formation were encoded by the F plasmid. Ottow (1975) subsequently suggested that the term “pili” be used for conjugative filaments encoded by self-transmissible plasmids, and the term “fimbriae” be reserved for non-conjugative filaments, many of which promote adherence to mammalian tissues. Some of the latter (K88 and K99) were initially thought to be capsular antigens (Orskov et al., 1961, 1979, while others (CFA/I, CFA/II) were described as “colonization factor antigens” (Evans et al., 1975; Evans and Evans, 1978). Structures designated CFA/II were subsequently found to consist of three different types of pili that were named “coli surface antigens” 1,2 and 3 (CSI, CS2 and CS3; Smyth, 1982). Brinton (1965) originally distinguished six types (type I-V and F) of pili, while Duguid et al. (1966) identified seven types (types 1-6 and F). However, these classifications were inconsistent with various types of pili described by other workers (Bradley, 1966; Fuerst and Hayward, 1969; Mayer, 1971;

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Schmidt, 1971; Weiss, 1971), and now only the type 1 classification is still in common use. One type of pilus was recently named Pap (pyelonephritis associated pili; Normark et al., 1983) because it was found to be prevalent among uropathogenic strains of Escherichia coli. These same pili were named P fimbriae by Kallenius et al. (1981) when they discovered that the adhesins bind to the P blood-group substance which contains the active receptor disaccharide a-D-Gal-( 1+4)-b-~-Gal. O’Hanley et al. (1985) later named these adhesins Gal-Gal pili because the synthetic analogue of this disaccharide inhibits haemagglutination by these pili. A number of other pili types were named according to the strain from which they were isolated, e.g. 978P (Isaacson et al., 1977), F41 (J. A. Morris et al., 1980), PAK (from Pseudomonas aeruginosa; Frost and Paranchych, 1977), GC (for “gonococcal”; Buchanan et al., 1973) among others. An attempt by Orskov and Orskov (1983) to introduce a new nomenclature based on serology of fimbrial antigens in which only F (fimbrial) designations would be used (Fl-Fn) has not been widely accepted. This nomenclature would lead to confusion between conjugative and non-conjugative pili, since the most widely studied conjugative pili are of the F type whose incompatibility groups (Inc) have been designated FI-FV (see p. 58). For purposes of simplicity, and because there is no general agreement regarding the nomenclature of bacterial non-flagellar surface filaments, the term pili is used throughout this review article to describe all non-flagellar surface appendages, including conjugative and non-conjugative types.

111. Classification A. CRITERIA FOR CLASSIFICATION

To date, there has been no general agreement on any specific classification scheme for pili. Three criteria that lend themselves to the classification of pili are: (1) morphology (e.g. thin flexible, thick flexible, rigid), (2) function (e.g. distinct from conjugative as adhesive pili), and (3) biochemical properties (e.g. type-related pili with free amino termini as distinct from those with NMePhe at the amino terminus). The three major groups discussed in this article are termed conjugative, adhesive and NMePhe pili, and electron microscope photographs of representative bacteria are shown in Fig. 1. B. MORPHOLOGY

A large number of electron microscope studies have described the morpho-

FIG. 1. Electron micrographs of representatives of the three pilus groups: conjugative, adhesive and NMePhe. The conjugative pili are represented by the F plasmid in Escherichiu coli HBl I and the pili have been labelled with R17 bacteriophage. The adhesive pili are represented by CFA/I pili found on Escherichiu coli H 10407 (Evans et ul., 1975). The NMePhe pili are represented by the multipiliated mutant Pseudomonar ueruginosu K/ZPfs (Bradley, 1966).

PHYSIOLOGY AND BIOCHEMISTRY OF PILl

57

logical characteristics of a bewildering array of pili types. Diameters in the range of 2-12 nm have been reported, primarily for pili from Gram-negative bacteria, although some types of filaments on Gram-positive organisms have also been described (Ottow, 1975). Bradley (1980a, b, 1983a, 1984)has published a useful system for classifying pili on the basis of electron-microscope appearance. He examined conjugative pili encoded by plasmids from 37 incompatibility groups, and found them to fall into three morphological classes: thin flexible (6-7 nm diam.), thick flexible (8-10 nm diam.), and rigid (8-1 1 nm diam.) (Table 1). In addition, they can be differentiated by the presence of tapered tips at the distal end of the pilus or knobs that represent a pilus-associated structure at the base of free pili derived from the cell surface. Conjugative pili also display a varying propensity for aggregation which aids in their identification. Many non-conjugative pili such as Type 1, CFA/I, 987P, CS 1, CS2 and Pap can also be classified within this system, since they all have the appearance of thin, rigid rods with diameters of about 7 nm (Gaastra and de Graaf, 1982; Levine et al., 1984; Klemm, 1985). Non-conjugative pili from Ps. aeruginosa (Frost and Paranchych, 1977), Moraxella sp. (Bovre and Froholm, 1972; Pedersen et al., 1972), Neisseria sp. (Salit, 1981; Stephens and McGee, 1981; Swanson et al., 1971) and Bacteroides nodosus (Every, 1979) all produce thin flexible pili (about 6 nm diam.); hence, this group can also be classified within the Bradley system. In contrast, the K88, F41 and CS3 pili are visualized by the electron microscope as very thin, flexible threads with diameters of around 2 nm (Levine et al., 1984; Klemm, 1985), while K99 pili are also flexible and quite thin (about 4.5 nm diam.; de Graaf et al., 1980). Thus, a fourth morphological class, called “very thin”, should be created to accommodate this group of pili, as well as those of 3-4 nm diameter that are produced by Bordetellapertussis (Blom et al., 1983) and B. bronchiseptica (Lee et al., 1986).

C . FUNCTION AND BIOCHEMICAL PROPERTIES

The simplest classification of pili on the basis of function is the division into two broad groups: “conjugative” and “adhesive” pili. The biochemical properties help to identify subpopulations of these pili.

I . Conjugative Pili Conjugative pili are generally encoded by self-transmissible plasmids, which are capable of passing a copy of their genetic material to a recipient bacterium through a process known as conjugation. In Gram-positive bacteria, conjugative pili have not been identified; the donor cell releases sex

TABLE 1. Morphological properties, bacteriophage sensitivity and mating type of conjugative pili

References

IncompatiRepresentative bility plasmid($ group

Escherichia coli 36,37,41,42,30 14,42,30 18,31,35,39 6.26 6.26 6.26 20.26 12.1 3,23,24,40,30,43

FI FII N HI I HI2 HI3 HI1 I1

F R1-19,R100-1 Folac.pED208 R27 R478 MIP233 pHH I457 R64

6,13,30

II + B

RIM

6.13

B

R16,R621a

6.13

K

R387

13.46

I5

pIE360

13,47

Z

pIE545

22.24.28

I2

R72 I

6,2 I 3 I ,27 6,21

I 17,30,33,48 6 6,16,30 4.8.10 32.38 5.6.25 19 2.3.5.29.32.61

X X

RAI R71 I b,R778b R391 R71"' pIN25 R753 R6K R48Y

N M U

N3 R446b RA3,pAR-32

W

sa

C D J cod T

V

Pilus morphology' Thick flexible Thick flexible Thick flexible Thick flexible Thick flexible Rigid Thick flexible Thin flexible Rigid Thin flexible Rigid Thin flexible Rigid Thin flexible Rigid Thin flexible Rigid Thin flexible Rigid Thin flexible Rigid Thick flexible Thick flexible Thick flexible Thick flexible Thick flexible Thick flexible Thick flexible Thick flexible +thin flexible Rigid Rigid Rigid Rigid

Approx. diam. Basal (nm)b knobs' 9 9 9 II II 11 9

6 II 6 II 6 6

+

+ + + + + + + -

+ + + -

6 10

9 9.5 10

9 9 9 9 9 5

II I1

++ + ? +

+ + + + + + +-

Bacteriophage sensitivity*

Distal Aggre- Serological pointsd gation' relatedness

-

+ + + + + + + + ++ + ?

+

+ + + + + + +

+

++ + + + + + + + + --? -

--

F1,FII

fl .RI 7,QB fl *,R I7*,QB* fl ,UA6,Folac pilHa pilHa pilHa pilHa Ifl,Ia,PR&IFS PR64FS

Universal Universal Universal Universal Universal Universal Surface preferred Universal' Surface obligatory Universal'

PR64FS

Universal'

PR64FS

Universal'

15,B.K,Zh PR64FS I I +B,B,K,15,Zh PR64FS

Universal'

HII,HI2.HII

IIJI +B,B~ WI +B,B,K~

121 12h CJ

Ifl ,PR64FS PR4JKe.12-2,X C-l ,C-2,J' fl,MS2*,J J

N t,tf-l

X

+

--

Mating typeg

Universal' Universal' Surface preferred Surface preferred Universal Universal Surface preferred' Universal Surface preferred Surface obligatory

IKe.PR4.X' M,X X

Surface obligatory Surface obligatory Surface obligatory

PR4.XK7

Surfaceobligatory

TABLE 1 (continued) Pseudomonos aeruginose 1.5.1 1.32.45 P- I II P-2 II P-3 I1 P-5 !I P-7 II P-8 11-34 P-9 11 P-I0 11 P-I 1 11 P-12 11 P-13

RPI R931,CAM RIP64 Rmsl63 Rms148 FP2 R2,TOL R91.5 RPI-I R716 pMG26

Rigid Rigid Thick flexible Thick flexible Rigid Thick flexible Rigid Rigid Rigid ? Thick flexible +thin flexible

8

9

+ ? ?

? ?

+ + +

+ + +

+ +

+

-

-

?

?

+

+

9 6

-

+

+ +

-

8.5-10

+ +

-+

?

IncCk

PRRl ,Pf3,1Ke,X,PR4 Surface obligatory Surface obligatory Surface obligatory Universal Surface preferred Universal PR4 Surface preferred X* Surface preferred Surface preferred Surface preferred Surface preferred

Pilus morphology describes pili according to 7.9.1 I . 'The pilus diameters are approximate values obtained from negatively stained EM samples. Knobs refers to any structure at the base of the pilus and includes plates and discs as well as round knobs. These knobs appear to contain pilus material. dOnly pili with visibly pointed tips are marked +. 'The aggregation of pili into long fibres or bundles is pilus-specific. (?) indicates that too few pili were present in a culture to determine this property. /Bacteriophages that either attach without infecting the cell or propagate but do not produce plaques are marked with an asterisk. Mating type has been defined ( I 5) as the ratio of mating ability on an agar surface as compared to that in a liquid culture. Universal has a ratio near I .O, surface preferred 45450, surface obligatory > 2000. In Pscudomonads, the mating types can be defined universal or surface preferred ( I I). There are three serotypes for the thin and thick pili of the 1 complex. The 1 complex plasmids permit universal mating when both thin and and thick pili are produced. 'The thin flexible pili of R485 have a wavy appearance with a pitch of 4.6 nm. IncP3 plasmids are the equivalent of In& plasmids in E. c o / i The plasmids Folar (IncFV), R71 (comg), TP224 and pPLS have been grouped into lncS (48) on the basis of phage sensitivity. This table is principally a compilation of the information in 6,7,9, I I , 13. The references are: Bradley, 1974b(1), 1975(2), 1976(3), 1978a(4), 1978b(5), 1980a(6), 1980b(7), 198Oc(8), 1981(9), 1982(10), 1983a(l I), 1983b(12), 1984(13), 1985(14); Bradleyeral., 1980(15), 1981a(16), 1981b(17), 1981c(18), 1982419), 1982b(20), 1982~(21).1983(22); Coetz.ee ef a/, 1980(23), 1982(24), 1983(25), 1985a(26), 1985b(27); Bradley and Coetzee, 1982(28); Bradley and Cohen, 197q29); Bradley and Fleming, 1983(30); Bradley and Meynell, 1978(3I); Bradley and Rutherford, 1975(32); Bradley and Whelan, 1985(33); Bradley and Williams, 1982(34); Armstrong et a/., 1980(35); Caro and Schnoss, 1966(36); Crawford and Gesteland, 1964(37); Dennison and Baumberg, 1975(38); Falkow and Baron, 1962(39); Feruichi et 01.. 1984(40); Lawn et a/., 1967(41);Lawn and Meynell. 197q42); Meynell and Lawn, 1968(43); Moms ef a/., 198q44); Olsen and Shipley, 1973(45); Tschape and Tietze, 198I(46). 1983(47); D.E.Bradley, personal communication (48).

60


pheromones which cause clumping of cells into mating aggregates where transfer takes place. This process has been reviewed by Clewell (1981). In Gram-negative bacteria, the transfer region of the plasmid invariably encodes a pilus, which recognizes a suitable recipient cell and brings it into contact with the donor cell in a poorly understood process (Achtman and Skurray, 1977; Willetts and Skurray, 1980, 1986; Willetts and Wilkins, 1984; Clark, 1985; Silverman, 1986; Ippen-Ihler and Minkley, 1986). Some broad host-range plasmids can transfer both inter- or intragenerically within the Enterobacteriaceae. A convenient method for cataloguing plasmids takes advantage of the fact that closely related plasmids are incapable of co-residing in the same cell, a property that is termed incompatibility (Inc) (Datta, 1975). Previous attempts to classify plasmids on the basis of pilus type (Lawn et ul., 1967), bacteriophage sensitivity and pilus serology (Meynell et al., 1968; Lawn and Meynell, 1970; Feilberg-Jorgansen et ul., 1982) or entry exclusion (Datta and Hedges, 1972) were unsatisfactory. As evidence accumulates, incompatibility appears to be the best scheme for classifying plasmids, since in general terms, plasmids within an incompatibility group exhibit similar plasmid size, similar organization of their transfer regions, similar pili in terms of morphology, serology and phage sensitivity, similar host ranges and similar genetic markers such as drug resistance, resistance to heavy metals, virulence determinants and degradative functions (Table 1). There are currently over 20 incompatibility groups for plasmids borne by E. coli (Shapiro, 1977; Bradley, 1980b). The IncP plasmids are found in Ps.ueruginosu and have been subdivided as PI,P2, etc. (Bradley, 1983b; Shapiro, 1977; Jacoby, 1977; Jacoby and Matthew, 1979). Pili associated with a particular incompatibility group can be further differentiated on the basis of serology, phage sensitivity and morphology into various pilus types. Conjugative pili show variable preferences for promoting bacterial mating in liquid or on solid media (Table 1). Flexible pili generally confer the “universal mating type” and promote mating in liquid media and on solid surfaces equally well, whereas rigid pili are usually associated with “surface preferred” (mating efficiency is higher on solid media than in liquid cultures) or “surface obligatory” (mating occurs only on solid media) mating types (Bradley, 1980b). This is illustrated by the Inci plasmids, where the presence of thick and thin pili allow these cells to become proficient at mating in broth and on plates, whereas the presence of thick pili alone allow mating only on solid surfaces. Thin I pili are thought to stabilize mating pairs formed by the thick, rigid I pili allowing mating in liquid media. Indeed, thin I pili can convert IncP and IncW mating pairs from a “surface obligatory” into a ‘‘L..iversal” system by stabilizing the fragile mating pairs formed by the rigid pili encoded by these plasmids (Bradley, 1984). IncF pili have been characterized on the basis of phage sensitivity patterns


61

(Paranchych, 1975; Willetts and Maule, 1986) and serological differences (Lawn and Meynell, 1970; Finlay et al., 1984; Worobec et al., 1983) as well as, at the molecular level, by sequencing the pilin gene. The sequence of the gene encoding F pilin revealed that the pilin subunit had a molecular weight of 7200 and that the pilin gene encoded an exceptionally long signal peptide of 51 amino-acid residues (Frost et al., 1984). The sequence of four other serotypes of F-like pilins revealed that this long signal peptide was common to all of these pilus types and that differences in serology and phage sensitivity, attributable to the pilin subunit, were localized in the N- and C-terminal regions of the pilin protein (Frost et al., 1985). Furthermore, these studies showed that in all IncF pili studied, the N-terminus was acetylated and this group was important in defining the major antigenic determinant of the pilus protein (Finlay et al., 1985;Worobec et al., 1985; Frost et al., 1985, 1986). As yet, no other pilin genes from plasmids of other incompatibility groups have been sequenced and it is not clear whether this long signal sequence, the acetylated N-terminus and the variable sequences at the N- and C-termini of the pilus proteins are characteristic of conjugative pili. 2. Adhesive Pili of Escherichia coli

Pili from E. coli that promote adherence to mammalian cells are broadly classified into mannose-sensitive (MS) and mannose-resistant (M R) classes, depending on the ability of D-mannose to inhibit binding to erythrocytes and epithelial cells. They are also distinguishable by their preference for binding to intestinal or urinary epithelial cells. Some pili that are specific for intestinal epithelial cells show preferential binding to the intestinal epithelium of humans, cattle, pigs or sheep (Gaastra and de Graaf, 1982; Klemm, 1985). Mannose-sensitive binding is mediated by Type 1 pili, which have been described as non-conjugative, chromosomally encoded thin rigid rods (around 7 nm diam.) that are plentiful (lOCr500per cell), and peritrichously arranged on the surface of the cell (Brinton, 1965; Ottow, 1975). They characteristically cause agglutination of guinea-pig erythrocytes (Salit and Gotschlich, 1977) as well as attachment to yeast cells, buccal epithelial cells, and a mannose-containing urinary glycoprotein, the Tamm-Horsfall protein (Ofek and Beachey, 1980; Orskov et al., 1980a). The function of these pili is unclear, since they are often present on both pathogenic and non-pathogenic isolates of E. coli (Orskov et al., 1980b). The majority of E. coli strains causing urinary-tract infections are able to produce both MS and MR pili (Klemm et al., 1982; Salit et al., 1983; Rhen et al., 1983a). However, virulence of uropathogenic strains of E. coli has been attributed to MR (P, Pap or Gal-Gal) pili, which bind globoseries glycolipids containing the a-D-Gal-(1+4)-P-~-Galunit of the P blood-group substance

62


(Kallenius et al., 1980; Leffler and Svanborg-Eden, 1980; O’Hanley et at., 1985). Immunological studies and amino-acid sequencing on Pap and Type 1 pili from a variety of uropathogenic isolates of E. coli have shown significant amino-acid homologies at the N-terminus of these proteins (Orskov et al., 1980b; Klemm et al., 1982; Rhen et al., 1983a; Salit et a[., 1983; Baga et a/., 1984; Klemm, 1984; van Die and Bergmans, 1984; Hanley et al., 1985). However, virtually all isolates of Pap pili were serospecific, suggesting that a wide variety of homologous pili species exist among naturally occurring strains of E. coli,and that the genes encoding these pili are polymorphic with respect to antigenic determinants. A pathogenic strain of E. coli that produces MR pili which bind specifically to human intestinal epithelium was first reported by Evans et al. (1 975). These pili were initially designated “colonization factor antigen” or CFA. However, when a second, apparently similar, antigen was discovered and designated CFA/II, the name of the first antigen was changed to CFA/I (Evans and Evans, 1978). Enterotoxigenic E. coli strains positive for CFA/I and CFA/II pili are responsible for a significant number of cases of E. coli diarrhoea in humans (Gross et al., 1978; Craviato et al., 1979). The primary structure of the 15,058 Da CFA/I pilin subunit, which contains 147 amino-acid residues (Klemm, 1982), bears no significant homology with the known sequences of other pili proteins (Klemm, 1985), although CFA/I pili are morphologically similar to Type I and Pap pili (thin rigid rods of about 7 nm diam.). The CFA/II pili originally described by Evans and Evans (1978) were subsequently shown by Smyth (1982) to consist of three distinct coli surface antigens which he named CSI, CS2 and CS3. These three antigens appear to be identical to compounds 1,2, and 3 reported earlier by Craviato et al. (1979), and have molecular weights of 16,300,15,300 and 14,800, respectively (Smyth, 1982). Pili designated CSI and CS2 are morphologically similar to Type 1 pili (thin and rigid), whereas the thin flexible CS3 pili belong to the “very thin” category (Levine et al., 1984; Mullany et al., 1983). The N-terminal aminoacid sequence of the CS2 pilus protein was recently found to be almost identical to that of the CFA/I protein, although the two antigens are immunologically distinct (Klemm, 1985). A number of animal-specific MR pili adhesins ‘have also been described. Those designated K88 and 987P bind to the gastrointestinal mucosa of piglets (Orskov et al., 1961; Sellwood et al., 1975; Isaacson et al., 1978; Isaacson and Richter, 1981; Gaastra and de Graaf, 1982), whereas K99 and F41 pili are associated with gastroenteritis in calves, lambs and newborn pigs (Smith and Linggood, 1972; Orskov et a/., 1975; de Graaf and Roorda, 1982). The K88 protein exists in several antigenic variants, three of which -K88ab, K88ac and K88ad - have been characterized immunologically (Orskov et ul., 1964) and in terms of their primary structures (Gaastra et al., 1979, 1983;

PHYSIOLOGY AND BIOCHEMISTRY OF PlLl

63

Klemm, 1981). Both K88 and F41 pilin subunits are large (around 28,OOG 29,000 Da) relative to other types of pilins, and the intact pili of both are morphologically very thin (approx. 2 nm diam.) (Gaastra and de Graaf, 1982; de Graaf and Roorda, 1982; Klemm, 1985). It is not yet known whether K88 and F41 pili are related in terms of sequence homology, although de Graaf and Roorda (1982) have shown that the two pilins are serologically distinct. It would be most interesting if the two pili types should turn out to be related, since K88 pili are plasmid encoded (Orskov and Orskov, 1966; Smith and Linggood, 1972; Shipley et al., 1978), whereas F41 pili are encoded by the chromosome (de Graaf and Roorda, 1982). Enterotoxigenic strains of E. coli positive for the K99 antigen cause severe diarrhoea in calves, lambs and piglets because the antigen facilitates bacterial adhesion to the intestinal epithelium (Burrows et al., 1976; Moon et al., 1977). The molecular weight of the K99 pilin subunit is 18,400, with PI 9.5 (de Graaf et al., 1981). Some K99-positive strains of E. coli belonging to the 0-serogroups 9 and 101 also express F41 pili, but these are chromosomally encoded and the pilin subunit is an acidic protein (PI 4.6) of 29,500 Da (de Graaf and Roorda, 1982). The nucleotide sequence of K99 pilin is known (Rosendaal et al., 1984),and has revealed that the subunit protein of these thin rigid pili (about 7 nm diam.) have extensive homology in the N- and C-terminal regions with type 1 (Klemm, 1984), Pap (Baga et al., 1984) and F72 (van Die and Bergmans, 1984) pili. The porcine small intestine can be colonized by yet another enterotoxigenic E. coli strain which expresses MR pili designated 987P (Nagy et al., 1976, 1977). These pili are chromosomally encoded, and the 20,000-Da subunit has PI 3.7 (Isaacson and Richter, 1981;de Graaf and Klaasen, 1986). Morphologically, they belong to the thin rigid group of pili, but there is no homology with other known sequences of pili proteins (Klemm, 1985). 3. N-Methylphenylalanine (NMePhe) Type Pili Adhesins

Bacteria such as Pseudomonas aeruginosa (Weiss, 1971; Bradley, 1972a), Neisseriagonorrhoeae (Jephcott et al., 1971; Swanson et al., 1971;Hermodson et al., 1978), Neisseria meningitidis (Hermodson et al., 1978), Moraxella nonliquefaciens (Froholm and Sletten, 1977), Moraxella bovis (Marrs et al., 1985), Bacteroides nodosus (Every, 1979) and Vibrio cholera (J. Mekalanos, personal communication) produce thin flexible pili, around 6 nm in diameter, which have a mainly polar distribution on the cell. These pili are involved in such processes as adhesion of the bacteria to host mucosal surfaces (Punsalang and Sawyer, 1973; Brinton, 1977; Woods et al., 1980), twitching motility (Henrichsen, 1975; Brinton, 1977; Bradley, 1980c), and bacteriophage adsorption (Bradley and Pitt, 1974). The pili are composed of identical

64


pilin subunits, which have molecular weights ranging from about 15,000 for Ps. aeruginosa (approx. 145 amino-acid residues; Watts et al., 1983a; Sastry et al., 1983) to about 18,000 for N . gonorrhoeae (approx. 160 amino-acid residues) (Robertson et al., 1977; Hermodson et al., 1978; Haas and Meyer, 1986). Pilin proteins from these bacteria show extensive amino-acid homology in the N-terminal region, which consists almost entirely of hydrophobic residues, and begin with the modified amino acid N-methylphenylalanine (NMePhe) (Frost et al., 1978; Paranchych et al., 1978; Hermodson et al., 1978; Elleman et al., 1986). For this reason, these pili are referred to as the NMePhe type. Nucleotide-sequence studies have shown that the mature protein is initially produced as a propilin with six or seven additional N-terminal residues (Elleman and Hoyne, 1984; Meyer et al., 1984; Marrs et al., 1985; Pasloske et al., 1985; Sastry et al., 1985b). Sequence similarity is extremely high within the first 30 residues, while only slight homology exists between residues 30-55 and within 50 or so residues at the C-termini (Elleman et al., 1986). There is virtually no homology in the central region of these pilin molecules, which contains the immunodominant type-specificepitope in pilins of Ps. aeruginosa (Watts et al., 1983b; Sastry et al., 1985a) and Bacteroides nodosus (McKern et al., 1985). In pilin from N . gonorrhoeae, the major serotype-specific antigenic determinant is located within a large C-terminal cysteine loop near the Cterminus (Rothbard and Schoolnik, 1985), whereas the domain that appears to be involved in adhesion of pili to mammalian cells is found in two separate regions of the central portion (residues 41-50 and 69-84; Rothbard et al., 1985). It is not yet clear how antigenic variation is achieved in Ps. aeruginosa, Moraxella sp. and Bacteroides nodosus. However, a series of studies from several laboratories have shown that antigenic variation in pilin from N . gonorrhoeae is achieved through gene rearrangement between silent genes and expression sites in the chromosome (Meyer et al., 1982; Hagblom et al., 1985; Haas and Meyer, 1986; Bergstrom et al., 1986).

IV. High-Resolution Studies on Pilus Structure The first serious attempt to examine the fine structure of pili by a combination of electron microscope and X-ray fibre diffraction techniques was that of Brinton (1965). An examination of Type 1 pili pseudocrystalline arrays revealed angle layer crystals whereby one layer of pili intersect a second layer at a constant angle of 41.5'. Amino-acid compositional analyses on purified preparations of Type 1 pili had already shown that the minimum molecular weight of Type 1 pilin was approximately 17,000 (Brinton, 1965). Having


65

determined by electron microscopy that the diameter of Type 1 pili is 7 nm. and knowing the layer angle to be 41.5", Brinton predicted that the cylindrical pilus is composed of identical subunits arranged in a helical array with a pitch distance of 2.4 nm. This prediction was tested with X-ray fibre diffraction studies on pilus fibres that had been prepared in thin-walled capillary tubes. Reasonably good diffraction patterns were obtained, and Brinton interpreted these patterns with the 17,000 Da pilin subunits arranged in a helical manner with 3.125 subunits per turn of2.3 nm pitch. It was also deduced from electron microscope observations that the cylindrical structure contained an axial hole of approximately 2 nm. Mitsui et al. (1973) carried out a similar X-ray fibre diffraction study on pili purified from a different strain of E. coli. These workers did not identify the type of pili they were studying, nor the molecular weight of the pilus subunit. However, it is likely they were also working with Type 1 pili, since their results were similar to those of Brinton (1965). The arrangement of subunits in the pili rods was found to be strictly simple-helical with 3.14 units per turn of helix and a pitch of 2.54 nm. A structural study on the pili of the non-starforming (sta-) mutant strain 3/7 of Ps.echinoides was carried out by Mayer and Schmitt (1 971) by means of direct and optical diffraction analyses of high-resolution photographs of negatively stained specimens. These pili appeared to be hollow rods of 6.5 nm outer and 2.5 nm inner diameter. The arrangement of the 8,000 Da pilin subunits was described in an array of three helices with a common axis having a pitch of about 20" and six subunits per turn. Folkhard et al. (1979) proposed a model for F pili based on their studies using fibre diffraction. They suggested that F pilli are hollow fibres with an 8.0 nm outer and a 2.0 nm inner diameter. They calculated a mass per length value of 3000 Da per angstrom, and a density of 0.77 Da per angstrom. Their original model was based on a molecular weight of 11,200. Recently, Marvin and Folkhard (1986) have re-examined their model using the correct molecular weight of 7200 for F pilin. They proposed that the subunits in F pili are related by a fivefold rotation axis around the helix axis. The helix symmetry is 25 units in two turns of a helix with a pitch of 16 nm and a crystallographic repeat of 32 nm. This parallels the structure of fd bacteriophage (Gray et al., 1981) which also has fivefold rotational symmetry and has five attachment proteins at one end of the phage (Webster and Lopez, 1985). This seems to be a recurring motif in the structure of filamentous organelles that are capable of disassociating into the bacterial membrane and has been proposed for Pf phage (Makowksi and Caspar, 1981), Pf3 phage (Day and Wiseman, 1978) and perhaps is true for the polar pili of Pseudomonus species (Folkhard et al., 1981; Watts et al., 1983a) (see p. 81). A representation of this model with respect to F pili is shown in Fig. 2(a).

320

256

(b) 2oo

150

2

ci, 100

ILI

i

50

I

0

12.8

i

0

52 A 80 A FIG. 2. Models of F and PAK pili. (a) The lattice diagram of F pili is based on information in Folkhard et al. (1979) and reinterpreted by Marvin and Folkhard (1986). The model shows a pilus diameter of 8 nm with five subunits per turn arranged with fivefold symmetry. There is a rise of 1.28 nm per subunit and the helix symmetryis 25 units in two turns of the helix with a rise of 32 nm. The sketch of the two layers of subunits illustrates the arrangement of subunits with respect to each other and the approximate surface area of a subunit exposed on the pilus surface. At present there is no information on the shape of the subunit or the packing of the subunits in the pilus. (b) The lattice diagram of PAK pili is based on information in Folkhard et a!. (1981) and Watts et al. (1983a). The pilus diameter is 5.2 nm and there are five subunits per turn but there is no fivefold rotational symmetry. There is a rise per subunit of 4.1 nm. The shape of the subunit has been calculated to be elongated with parallel alpha helices within the subunit arranged along the axis of the pilus.

PHYSIOLOGY AND BIOCHEMISTRY OF PiLl

67

Brinton (1971) reported that treatment of F pili with sodium pyrophosphate at 55°C and pH 4.5 caused formation of fibres of about one-third the diameter of whole pili. Incubating pili under these conditions at pH 2.0 caused irreversible formation of vesicles. Tomoeda et af. (1975) also reported the splitting of pili into thinner fibres when treated with 0.05 M HC1 at 30°C. These results interpreted the structure of F pili as ribbon-like consisting of two parallel protein rods. However, diffraction data clearly suggest that pili are helical filaments, and these results can be explained as a rearrangement of the pilin subunits within the filament. X-Ray fibre diffraction studies have also been reported on pili from Ps. aeruginosu strains PAK and P A 0 (Folkhard et al., 1981). These bacteria have polar pili which are flexible filaments of about 6 nm diameter and 2.5 jim average length (Weiss, 1971; Bradley, 1972a). Native pili consist of a single subunit of M , 15,000 (Sastry et al., 1983). Previous publications (Frost and Paranchych, 1977; Paranchych et al., 1979) had suggested a molecular weight of 18,000 based on migration of pilin in sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE) and on its amino-acid composition. This led Folkhard et af.(198 1) to interpret from X-ray fibre diffraction data that native pili consist of 4.06-4.08 subunits in a 4.1 nm turn of helix. In light of more recent information (Sastry et al., 1983,1985a) indicating a 15,000 Da subunit, this was reinterpreted as 5.06-5.08 pilin subunits per turn (Watts et al., 1983a). Native pili have the overall appearance of a hollow cylinder of 5.2 nm diameter and a central channel of 1.2 nm diameter (Folkhard et al., 1981). A representation of a model for PAK or P A 0 pili from Ps. aeruginosa is shown in Fig. 2(b). Although X-ray fibre diffraction studies have not yet been reported for other NMePhe pili (i.e. N. gonorrhoeae, N. meningitidis, M . nonliquifaciens, M . bovis and B. nodosus),these pili have a similar structure to those from Ps. aeruginosa because they all have the same diameter and share a common Nterminal sequence (Paranchych et af., 1978; Sastry et af.,1985a; Elleman et af., 1986). Although numerous attempts were made in this laboratory (T. H. Watts and W. Paranchych, unpublished work) to prepare oriented fibres of gonococcal pili, we were unable to obtain diffraction patterns of good quality. However, Deal et al. (1985) claim to have grown protein crystals of gonococcal pilin aggregates. The crystals displayed 3.0 nm spacing in one direction with 20.0 nm spacing in the second; the third direction indicated a larger aggregate. Efforts are being made to use these crystals to determine the three-dimensional structure of the pilin subunit. These workers (Deal et al., 1985) have also suggested that the secondary structure of NMePhe types of pilin consist o f a series of four antiparallel a-helix domains in the analogy to that of tobacco mosaic virus coat protein (Bloomer et al., 1978). However, secondary structure predictions in this laboratory for pilin from Ps.

68


aeruginosa (Sastry et al., 1983) suggest that the up and down 4-u-helix structural model (Weber and Salemme, 1980) may represent an oversimplification of NMePhe-type pilus structure. The structure of Bordetella perfussis pili was determined by optical density analysis of electron micrographs of paracrystalline bundles of purified pili. These pili are helical filaments with a 13 nm axial repeat containing five repeating units in two complete turns of a single start helix. The mass per unit length confirmed these results using a mass of 22,000 for serotype 2 and 21,500 for serotype 6 pili. Radial density profiles suggested that there was no axial channel and that the pilus diameter is 7.5 nm (Steven et al., 1986).

V. Organization and Expression of Pilin Genes A. CONJUGATIVE PILI

I . Plasmid-Encoded Elements Conjugative pili are specified by transfer regions of self-transmissible plasmids; the most detailed studies on bacterial conjugation have involved the IncF plasmids which have been extensively reviewed (Willetts and Skurray, 1980; Willetts and Wilkins, 1984; Clark, 1985; Silverman, 1986; Ippen-Ihler and Minkley, 1986). A transfer region generally encodes a plasmid-specific oriT sequence where the plasmid is nicked and transfer of one of the DNA strands into the recipient cell is initiated. There are several plasmid-specific transfer genes required to facilitate DNA transfer during this process. The role of the conjugative pilus is identifying a suitable recipient and bringing it into contact with the donor cell. This process is thought to involve retraction or disassembly of the pilus into the membrane. Thus, in addition to genes specifying synthesis and assembly of a pilus, there are, presumably, genes encoding pilus retraction. A third system encoded by the transfer region is surface exclusion or entry exclusion which prevents transfer of a given plasmid into cells already harbouring a related plasmid (Willetts, 1977a), and is a distinct process from that of incompatibility which prevents or slows replication of one of two plasmids within the same cell (Timmis, 1979). Surface exclusion has been shown to be a property of plasmids from the IncF complex (Willetts and Maule, 1986), P-1 (Olsen and Shipley, 1973; Barth and Grinter, 1977; Barth et al., 1978), N (Winans and Walker, 1985a), I (Meynell, 1969) and H (Taylor et al., 1985a) incompatibility groups. Other proteins encoded within the F transfer region have been identified but their function is not known. The current map of the F plasmid is reviewed by Ippen-Ihler and Minkley (1986).


69

Although the functions encoded by the transfer regions of the various plasmids remains fairly constant, the organization of the genes can vary. The transfer regions of the IncF plasmids occur as one segment of the plasmid (Sharp et al., 1973; Willetts and Skurray, 1980; Finlay et al., 1983, 1984), whereas the transfer regions of other plasmids may be divided into as many as three segments, as with the IncN plasmid pKMlOI (Winans and Walker, 1985a). A partial list of plasmids whose transfer regions have been mapped includes the IncN plasmids R46 (Brown and Willetts, 1981) and pCUl (Konarska-Kozlowska and Iyer, 1981), RP4 (IncP-I) (Barth et al., 1978), RK2 (IncP- 1) (Meyer et al., 1977), R9 1-5 (IncP- 10) (Moore and Krishnapillai, 1982a, b), R27 (IncHI) (Taylor et al., 1985b) and the Ti plasmid (Holsters et a/., 1980). The location of the pilin gene is not known for these plasmids nor is the organization of RNA transcripts required for pilus expression. Genetic analysis of the F transfer region has shown that there are at least 14 genes, (traALEKBPVWCUFQHG), contiguous with each other within the large (33 kb) tra YZ transfer operon, that are required for synthesis of pilin and assembly into an intact pilus. The organization of cistrons and control elements within the F transfer region has been reviewed extensively (Willetts and Skurray, 1980, 1986; Ippen-Ihler and Minkley, 1986). The traA gene encodes propilin (Minkley et al., 1976), a polypeptide of molecular weight 13,200 (Kennedy et al., 1977; Moore et al., 1981; Frost et al., 1984), which is processed to pilin of molecular weight 7200 (Moore et al., 1981; Frost et al., 1984). The pilin molecule is acetylated (Frost et al., 1984) and may be further modified with glucose and phosphate moieties (Brinton, 1971;J. Armstrong et al., 1981). Processing of propilin to pilin requires the traQ product, but the function of true is unclear (Moore et al., 1982). Acetylation of the pilin protein may require the N-terminal portion of the traG gene product (Laine et al., 1985). The function of the other gene products required for pilus synthesis is unknown. However, the molecular weight and cellular location of these tra gene products has been identified by cloning the F transfer region onto multicopy plasmids or transducing phages and studying the gene products in minicells or by in uitro translation (Kennedy et al., 1977; Willetts and Skurray, 1980; Laine et a/., 1985). All of the gene products involved in pilus biosynthesis are associated with the cell envelope (Kennedy et al., 1977) and several fractionate with both the inner and outer membrane, suggesting that they may be localized at Bayer's junctions or sites of fusion of the two membranes (Bayer, 1975). While the IncF plasmids share many interchangeable gene products within their transfer regions, certain genes are plasmid-specific. These genes are involved in DNA metabolism during conjugation (traMY/),surface exclusion (traS7') and control of transfer-operon expression (traJandJinOP). The genes required for pilus biosynthesis appear to be interchangeable. This homology

70

W. PARANCHYCH A N D L. S. FROST

was evident through heteroduplex analysis of several IncF plasmids (Shfrp et al., 1973) and by genetic studies recently summarized by Willetts and Maule (1986). Expression of conjugative pili is usually very low in a bacterial population (0.1-1 Yo)because of the repressed state of the transfer region. This repression is alleviated for a few generations in a daughter cell until the concentration of a repressor is restored. This phenomenon is termed HFT for High Frequency of Transfer since these cells are fully piliated and are competent for conjugation (Meynell et al., 1968). Derepressed mutants of these plasmids can be isolated (Meynell and Datta, 1967; Bradley, 1984; Willetts, 1984) and these cells usually express 1-6 pili on each cell (Frost et al., 1985). This property of conjugative plasmids has been used extensively in identification of pili on cells. The ability of one plasmid to complement the mutation in a derepressed plasmid and rest-orerepression is termed fertility inhibition e n ' ) (Meynell et al., 1968). Two genes in F-like plasmids have been identified in repression of the transfer region, namely f i n 0 and JinP, which together form the FinOP repressor system (Willetts, 1977b). Derepressed plasmids usually carry a mutation within one of these two genes (Willetts and Maule, 1986); however, in at least two cases that have been studied, derepression is due to the presence of insertion elements. The F plasmid is a naturally occurring derepressed plasmid, and this is due to the presence of an IS3 element within thefin0 gene (Clark, 1985; Willetts and Skurray, 1986). The pED208 plasmid is the derepressed form of Folac and contains an IS2 element at the beginning of the transfer operon (B.B. Finlay, L.S. Frost and W. Paranchych, unpublished work). This particular mutation results in production of approximately 20 pili on each cell. The molecular basis for the FinOP repression system has been studied in some detail, and summarized in other reviews (Willetts and Skurray, 1980, 1986; Willetts and Maule, 1986). The large traYZ operon (33 kb) is positively regulated by the traJ gene product (about 25 kDa) which is transcribed on a separate operon and is, in turn, negatively regulated by thefin0 andJinP gene products. The sequence for the three alleles of traJof F-like plasmids (Willetts and Maule, 1986) are currently available (Fowler et al., 1983; Finlay et al., 1986a; L. S . Frost, B. B. Finlay and W. Paranchych, unpublished work). The traJ-encoded protein, TraJp, previously thought to be found in the outer membrane, is a cytoplasmic protein and is insoluble and co-purifies with the membrane fraction when overproduced on high copy-number plasmids (Cuozzo and Silverman, 1986). The traJ-encoded proteins from these three alleles are very different, but two regions are conserved and one region strongly resembles a DNA binding domain (Anderson et at., 1982; Takeda et al., 1983; L. S. Frost, B. B. Finlay and W. Paranchych, unpublished work). The protein TraJp stimulates transcription from the tra YZp promoter


71

which is contained in the intergenic region between traJ and tray. There are three different known promoter sequences among F-like plasmids which match the three alleles of truJ. The exact location of this positively regulated promoter sequence has been mapped for F and R1-19 (Willetts, 19776 Gaffney et al., 1983; Fowler et al., 1983; Cram et al., 1984; Mullineaux and Willetts, 1985; Finlay et al., 1986b; Fowler and Thompson, 1986; Koronakis and Hogenauer, 1986; L. S. Frost, B. B. Finlay and W. Paranchych, unpublished results). ThefinP gene product appears to be a small anti-sense RNA molecule (Thompson and Taylor, 1982; Mullineaux and Willetts, 1985; Finlay et al., 1986b; Fee and Dempsey, 1986) which is complementary to the untranslated portion of the traJ mRNA. However, the f i n 0 gene product is absolutely required for repression of traJand, in turn, the transfer operon (Finnegan and Willetts, 1972). Thefin0 gene product may be a protein (Timmis et al., 1978; Dempsey and McIntire, 1983; Cheah et al., 1984; Willetts and Skurray, 1986) but the mechanism by which it interacts with finP and represses traJ transcription is unknown. ThefinP gene product is plasmid-specific, and six alleles among 12 plasmids studied have been identified (Willetts and Maule, 1986). The basis for this specificity is thought to reside in the loops of two stem-and-loop structures predicted from the sequence of thefinP gene (Finlay et al., 1986b). Thefin0 gene product is relatively non-specific, since thejinO mutation in the F plasmid can be complemented by a number of F-like plasmids and is the molecular mechanism of fertility inhibition (Egawa and Hirota, 1962; Meynell et al., 1968). Pilus expression can be affected by gene products from other F-like plasmids or plasmids from other incompatibility groups as well as chromosomally encoded gene products and small effector molecules such as cyclic AMP. 2. Chromosomally Encoded Control Elements At least four genetic loci have been implicated in the expression of the Ftransfer operon (Silverman, 1986). Mutations at these loci also affect the composition of the cell envelope (reviewed in Willetts and Skurray, 1986). The sfrA and cpxAB gene products affect the levels of TraJp in the cell (Beutin et al., 1981; Sambucetti et al., 1982; Silverman, 1986)although they do not act at the level of transcription initiation. The sfrA gene product may prevent premature termination (Beutin et al., 1981; Gaffney et al., 1983). They were originally thought to affect transport of TraJp to the outer membrane; however, TraJp is not an outer-membrane protein (see p. 70). The sfrB gene product is an 18,000 Da regulatory protein (Rehemtullah et al., 1986) which affects synthesis of the lipopolysaccharide core (Beutin er al., 1981;Sanderson and Stocker, 1981; Creeger et al., 1984). It also acts as a transcription

72

W. PARANCHYCH AND L. S . FROST

antiterminator of the t r a y 2 operon (Beutin et al., 1981; Gaffney et al., 1983). These chromosomally encoded proteins seem to co-ordinate transfer operon expression and the structure and composition of the cell envelope. 3 . Plasmid Encoded Control Elements

F-Like plasmids are capable of inhibiting F transfer by supplying compatible fin0 orfinP gene products (see p. 71). Many apparently unrelated plasmids encode fertility-inhibition mechanisms other than the FinOP system for inhibiting F transfer (Willetts and Paranchych, 1974; Gasson and Willetts, 1975, 1977). These systems (FinQ,U,V,W,C) act at the level of premature termination at a number of sites within the traYZ or traM (FinW) transcript or inhibit the function of one or more of the transfer gene products (Gaffney et al., 1983). This could be a general phenomenon since, for instance, F and the IncN plasmid pKMlO1 inhibit transfer of RPl (IncP-1) (Olsen and Shipley, 1975; Tanimoto and Iino, 1983; Winans and Walker, 1985b).

4 . Small Effector Molecules One small molecule known to affect pilus expression is cyclic AMP, while little is known about control of piliation by other small molecules such as ppGpp. It has been known for many years that late stationary cultures of F+ or Hfr cells acquire an F- phenotype. Piliation is maximal during the logarithmic phase of growth, and decreases as cells enter the stationary phase under aerobic but not anaerobic conditions (Ippen and Valentine, 1967; Brinton and Beer, 1967; Biebricher and Duker, 1984). Piliation is dependent on the host strain and the number of pili on each cell in stationary-phase cultures can vary from none to almost the maximum. Harwood and Meynell (1975) observed that derepressed IncI plasmids in cya crp mutants of E. coli (Perlman and Pastan, 1969), which are defective in cyclic AMP synthesis (cya) or cyclic AMP receptor protein (crp), produce many more I pili than usual and that, in cya mutants, this effect can be reversed by addition of exogenous cyclic AMP. For cells carrying F-like pili, the results were less clear. F+ cells do not seem to have this sensitivity to cyclic AMP while the derepressed plasmids R538-1, R124rd, R1-19 and R136-1 (which is almost identical to R100- 1) gave 10-fold more pili in an E. cofi cyu strain. Lawn and Meynell (1972, 1975) found that addition of anti-pilus antibodies to E. cofi carrying an IncI plasmid, or subjecting the cells to vigorous washing, caused a dramatic rise in the extent of piliation. They suggested that multipiliation due to washing or the presence of antibodies was unrelated to the cyclic AMP effect and, indeed, may be connected to the equilibrium between outgrowth and retraction rather than transcriptional


73

control by the cyclic AMP-CRP complex. Kumar and Srivastava (1983) found that Hfr(F+) strains carrying cya or crp mutations were transferdeficient, and that exogenous cyclic AMP, with the cya mutation alone, alleviated this Tra- phenotype. They speculated that transcription in this Hfr strain was controlled by cyclic AMP-CRP at the level of antitermination where this complex was required for synthesis of antiterminators which would prevent premature transcription of the long tra YZ operon or traJ gene. One interesting point in these experiments was the use as the wild-type control of E. coli CA8000 which contains the relA2 mutation and transfers at normal levels. Thus ppGpp may not be required for F pilus expression. Recently, the sequences of the promoter regions of the traJ and t r a y 2 operons for the three alleles of F-like plasmids have yielded information which helps to explain the previously noted effect of cyclic AMP-CRP (Fowler et al., 1983; Finlay et al., 1986a; L. S. Frost, B. B. Finlay and W. Paranchych, unpublished results). These two promoters are likely candidates as sites of action for the cyclic AMP-CRP complex. Using the consensus sequence for a CRP binding site (Ebright et al., 1984) which is defined as 5’-AA-TGTGA- T- - -TCA-ATA/T-3’, a potential CRP binding site was found within the promoter region of truJ in all three alleles. There was also a CRP-binding site within the R100-I tra YZpromoter region, while no strong site could be found within the F and R1-19 truYZ promoter regions. These CRP-binding sites could be involved in preventing transcription initiation at these promoters, and certainly a decrease in traJor t r a y 2 transcription would lower the level of piliation. Helmuth and Achtman (1978) reported that they added additional glucose to a culture as it entered the stationary phase of growth to encourage F-pilus production at high cell concentrations in order to facilitate F-pilus purification. This would support the idea that piliation increases under conditions where cyclic AMP production is suppressed (Adhya and Garges, 1982) for, as the concentration of glucose drops, the cyclic AMP concentration would rise and exert its effect at a number of sensitive promoters. Also, the sensitivity to arsenate of piliated cells grown in defined media containing sugars whose utilization is controlled by cyclic AMP (O’Callaghan et al., 1973b) could also involve cyclic AMP-CRP control where certain transfergene products are produced in lower amounts, emphasizing the sensitivity to the poison. Although cyclic AMP almost certainly affects expression of the various transfer operons, other factors are also involved. The effects of different oxygen concentrations and media composition on piliation cannot be wholly explained by the cyclic AMP-CRP effect, and a large number of unexplained observations concerning F pili production under various physiological conditions (for instance, temperature) are not understood.

14

W. PARANCHYCH AND L. S. FROST B. TYPE

1 PILI

The genes that encode production of Type 1 pili are located at 98 min on the E. coli linkage map (Swaney et al., 1977; Freitag and Eisenstein, 1983). Complementation studies of mutants defective in production of Type 1 pili indicated that at least three genes are required for production of these adhesins (Swaney et al., 1977).The gene encoding the structural component of Type 1 pili has now been cloned and sequenced at the nucleotide level. This gene was named JimA by Klemm (1984), and pilA by Orndorff and Falkow (1985). The two sequences were identical except that a Thr in position 140, reported by Orndorff and Falkow (1985), was absent from the sequence reported by Klemm (1984). The gene was found to specify a polypeptide 159 amino-acid residues long preceded by a 23 amino-acid residue signal peptide (Orndorff and Falkow, 1985). Klemm et al. (1985) found that the genes required for production of intact Type 1 pili are contained within a DNA segment of 8 kb. Four genes, designatedJimA, B, C and D, were found to be involved in synthesis of pili, and their order was shown to be (apparent molecular weight of the processed form of each gene product is shown in parentheses): fimB (23,000), JimA (16,50O),JimC (26,000) andJimD (89,000), organized in three transcriptional units. Orndorff and Falkow (1984a, b, 1985), Orndorff et al. (1985) and Maurer and Orndorff (1985) have also cloned the entire region responsible for Type 1 pili formation, and characterized the relevant genes. The designation and order of pili-related genes in their 9.4 kb fragment were: hyp (23,000), pilA (17,000), pilB (30,000),pilC (86,000), pilD (14,000), pilE (molecular weights not yet reported). The pilA gene encodes the structural component of Type 1 pili and corresponds to Klemm’sfimA gene product. The pilB and pilC genes correspond to Klemm’s fimC and fimD genes, respectively. Orndorff’s hyp gene is equivalent to Klemm’sJimB gene, while Klemm has not yet reported on’ the equivalent of the pilE gene. The pilE gene product has recently been shown to encode an adhesin which is distinct from the pilin subunit encoded by the pilA gene (Maurer and Orndorff, 1985). Mutants designated pilE possessed pili that were immunologically and morphologically indistinguishable from parental pili, but they were unable to cause agglutination of guinea-pig erythrocytes. It was suggested that the pilE locus specifies expression of an adhesin that becomes associated with the pilus to yield normal pili capable of haemagglutination (Maurer and Orndorff, 1985). It has been known for many years that Type 1 pili undergo “phase variation”, a metastable state in which bacteria switch back and forth between the piliated and non-piliated states at a rate of about to (Brinton, 1959, 1965; Eisenstein, 1981). The phase-switch function maps at 98 minutes,


15

adjacent to the known pi1 (orjim) genes (Freitag and Eisenstein, 1983). Orndorff et al. (1985) and Abraham et al. (1985) have shown that it is the pilA gene that is subject to this metastable control. Orndorff et al. (1985) constructed a pi1A‘-lac2 fusion, and showed that it was subject to metastable transcriptional control. The rate of switching from the Lac+ to the Lacin the opposite per cell per generation and 6.2 x phenotype was 4 x direction. Abraham et al. (1985) subcloned the switch, sequenced the DNA, and determined the molecular basis for its activity. The switch is an invertible element different from that controlling flagella in Salmonella species; it is small, consisting of 3 14 bp bounded by 9 bp inverted repeats, and it is driven by a different recombinase. It is just upstream of the pili structural gene (pilA) and contains a consensus E. coli promoter when in the “on” orientation. Klemm (1986) reported that theJimB andJimE gene products direct the phase switch into the “on” and “off” positions, respectively. These two genes were sequenced and found to encode highly homologous proteins (around 23,000 Da) that were very basic and probably capable of interacting with the DNA. Orndorff and Falkow (1984b) and Orndorff et al. (1985) have also noted that, in addition to the metastable regulation of pilA, a second type of transcriptional regulation is effected by the product of the gene hyp, adjacent to pilA. The product of the hyp gene appears to be a repressor since it inhibits piliation (Orndorff and Falkow, 1984b). It was therefore suggested that hyp may effect the metastable expression of piliation (Orndorff and Falkow, 1984b). However, when the hyp gene was inactivated by Tn5 insertion, the E. coli strain exhibited a frequency of switching from Lac+ to Lac-, and vice versa, indistinguishable from that of the parental strain. Thus, hyp does not appear to affect the metastable variation but does affect the level of transcription of the pilA gene in the O N (transcribed) mode (Orndorff et al., 1985).The hyp gene is the equivalent ofjimE, reported by Klemm (1986), who has detected activator activity, not repressor activity, for this gene product. This suggests that there are two different systems of control of pilA transcription, namely an invertible element that controls phase variation and a second element that modulates the level of expression. C. PILl DESIGNATED PAP

A majority of uropathogenic E. coli strains carry mannose-resistant haemagglutination (MRHA) activity. This is in contrast to faecal strains where only 10% of the bacteria express MRHA (Hagberg et al., 1981; Van den Bosch et al., 1980; Minshew et al., 1978). Along with other virulence determinants, these strains usually produce Pap pili which are important in adherence to uroepithelial tissues (Hull et al., 1984; Kallenius et al., 1981; Korhonen et al., 1982). The Pap gene cluster has been mapped on the chromosomes of several

76


clinical isolates of E. coli, and found to occur at a variety of locations. Also, more than one Pap gene cluster can occur on each chromosome although not all loci are necessarily expressed at once (Hull et al., 1981, 1986; Rhen et al., 1983b). The Pap gene cluster is often linked to other virulence determinants such as the chromosomal haemolysin determinant or the K1 capsular antigen. However, the intergenic region is not conserved (Low et al., 1984; Hull et al., 1986). It has been suggested that these virulence determinants are acquired by a transposition-like event since MRHA regions are bordered by a conserved sequence. This event may have occurred at some distant point in evolution since transposition of MRHA activity to a multicopy plasmid has not been possible, and the purported MRHA transposon may be degenerate (Goebel et al., 1973). The Pap gene cluster can be cloned on a plasmid vector as a single discrete entity of minimum size 10 kb, approximately, and the Pap pili, along with the associated properties of MRHA and adhesion, can be expressed by these chimeric plasmids (De Ree et al., 1985a, b; Hull et al., 1981; van Die et al., 1983, 1985; Clegg, 1982; Rhen, 1985; Rhen et al., 1983a, b, c). Hybrid chimeras containing DNA segments from several Pap-like gene clusters can express Pap pili and haemagglutination (Van Die et al., 1986a, b; Lund et al., 1985). Three Pap gene clusters cloned from separate E. coli isolates had very similar genetic organization and the genetically identifiable properties of pilin synthesis, pilin export. Pilus assembly and adhesion were trans complementable among the three gene clusters. The major differences in these gene clusters involved the central portion of the Pap pilin gene (papA) and one of the adhesin genes (papG) which, presumably, defines the antigenic variation within these proteins (Lund et al., 1985). Thus, the known gene clusters that express MRHA activity appear to be closely related to each other. This is reinforced by the protein-sequence homology among Pap pilins studied to date (Klemm, 1985; Baga et al., 1984; Rhen et al., 1985; Van Die et al., 1984a, b) as well as the highly conserved restriction maps for the Pap gene clusters currently available (Van Die et al., 1986a, b). The genetic organization of three Pap gene clusters (Pap, F71 and F72) has been described (Clegg and Pierce, 1983; Van Die et al., 1984a, b, 1985; Norgren et al., 1984; Baga et al., 1985). Synthesis of Pap pili can be separated from production of the adhesin as shown by transposon mutagenesis, isolation of mutants and recombinant-DNA manipulation. Thus, it is possible to isolate cells capable of expressing Pap pili but lacking adhesion function (Lindberg et al., 1984, 1986; Van Die et al., 1985, 1986b; Lund et al., 1985; Norgren et al., 1984; Normark et al., 1983). Normark and his coworkers have dissected the Pap system in some detail. They have identified nine genes (papZBAHCDEFG) involved in pilus


77

synthesis, expression of the adhesin and control elements. The papA gene encodes the pilin subunit termed fimbrillin (16.5 kDa) and the DNA sequence reveals the presence of a 22 amino-acid residue signal peptide (Baga et al., 1984). The papA gene was preferentially transcribed on an 800 bp mRNA. This transcription was positively regulated by two cistrons (papB and papl) upstream ofpapA . ThepapB transcript also proceeded through the papA gene to give a 1300 bp mRNA. They speculated that thepapA mRNA (800 bp) is a processed form of the papBA transcript. The papl gene precedes papB and is weakly transcribed from the opposite strand (Baga et al., 1985; Norgren et al., 1984). The presence of glucose in the medium lowers pilus expression and adhesive properties of type 1 and K99 pili (Old and Duguid, 1970; Isaacson, 1980). Similarly, Pap pili production is affected by glucose concentration (SvanborgEden and Hansson, 1978), temperature (Goransson and Uhlin, 1984) and media composition. Baga et al. (1985) have shown that papB transcription is regulated by the cyclic AMP-CRP complex and have proposed a potential CRP-binding site upstream of the papB promoter. The papEFG genes, which are on a separate operon, are not required for pilus synthesis and specify the adhesin (Lindberg et al., 1986). Genes for papFG are associated with digalactoside-specific binding while papE mutants are capable of adherence to whole cells, but purified pili from this strain do not adhere (Lindberg et al., 1984). Mutations in papA, which destroy pilus production, do not prevent cell adherence to the digalactoside receptor, presumably because of expression of the adhesin (Lindberg et al., 1984; Norgren et al., 1984; Uhlin et al., 1985).ThepapE andpapFgenes encode Pap pilin-like proteins which may represent minor components of the pili or a subpopulation of pili which define the adhesin (Lindberg et al., 1986). The papG gene product is also involved in elaboration of the adhesin and is capable of antigenic variation. However, its sequence has not been determined and its precise function is unknown. The papC (81,000) and papD (28,500) gene products are required for pilus and adhesin assembly while the function of the remaining pap gene products is unknown. Pap pili are subject to phase variation which has been demonstrated for one strain containing four fimbrial A single colony was antigens, one of which was Type 1 pili (Rhen et al., 1983~). found to contain a number of variants expressing different fimbrial antigens suggesting that the rate of phase variation in this system is high. D. PILI DESIGNATED CFA/I

AND CFA/II

(csl, cs2 AND cs3)

Pili designated CFA/I are encoded by a group of related plasmids that are between 86-93 kbp in size and also carry genes for heat-stable enterotoxin (ST enterotoxin) (Smith et al., 1982a; Willshaw et al., 1982). Smith et al. (1982b)

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W. PARANCHYCH AND L.

S . FROST

used transposon mutagenesis and complementation studies to map the genes responsible for expression of CFA/I pili. These genes were found in two regions of the plasmid separated by 37 kbp. Both regions were cloned onto compatible vectors, and it was shown that the ST genes are closely linked to one of these regions (designated region 1; Willshaw et al., 1983). Region 1 is contained within a DNA fragment of 6 kbp, while the second region (region 2) is within a 2.1 kbp fragment. Examination of the gene products of the two regions in minicells revealed that region 1 expressed the pilin subunit as well as proteins with molecular weights of 85,000, 40,000, 30,000, 28,000, 27,000, 25,000 and 15,000, whereas region 2 encoded polypeptides of 26,000, 15,000 and 14,000molecular weight (Willshaw et al., 1985). The functions of the nonpilin gene products are not yet known. The three CFA/II components (CS1, CS2 and CS3) can be distinguished serologically, and on the basis of pili morphology, pilin size and haemagglutination patterns (Smyth, 1982; Craviato et al., 1982). The genes responsible for production of CSl, CS2 and CS3 reside within the same plasmid of approximately 89 kbp size, which generally also codes for heat-labile (LT) and heat-stable (ST) enterotoxin (Penaranda et al., 1980; Mullany et al., 1983; Smith et al., 1983). Although CFA/II plasmids encode all three types of pili, expression is usually limited to one or two types. For example, the CFA/IIcontaining bacteria may produce components CS1 and CS3, CS2 and CS3, or CS2 or CS3 only. These patterns of expression appear to be related to the serotype or biotype of the host. Strains of serotype 0 6 : H16 have been shown to produce CSl or CS2. Moreover, CSl is produced only by 0 6 :H 1 6 of biotype A (rhamnose-negative), whereas CS2 is produced only by 0 6 :H 16 strains of biotype B, C and F (rhamnose-positive) (Craviato et al., 1982; Smyth, 1982).Component CS3 is produced independent of these biotypes and serotypes (Craviato et al., 1982). The mechanism underlying these phenotypic relationships is presently not understood. E. PILI DESIGNATED

K88 AND K99

Both K88 and K99 pili are encoded by plasmids that are approximately 75 kbp in size. Recombinant DNA studies on these two systems have revealed their genetic organization to be similar to that of the Pap and Type 1 pili systems, i.e. the genes are clustered, and five to nine gene products are required for pili expression (Klemm, 1985; Mooi and de Graaf, 1985). The K99 operon contains eight structural genes, at least seven of which appear to be required for K99 pilus formation. The K88 pilus system contains six structural genes, with at least five being located within a single transcriptional unit (Mooi and de Graaf, 1985). As already stated, pilin subunits encoded by the K88, K99, Pap and Type 1-related pilus systems show homology, indicating they are


79

evolutionarily related. Details of the genetics and biochemistry of these systems have been reviewed extensively (Gaastra and de Graaf, 1982; Klemm, 1985; Mooi and de Graaf, 1985). G . PILI DESIGNATED

NMePhe

The genetic organization of NMePhe pili can tentatively be divided into two groups. One group, at present consisting of gonococcal (GC) pili alone, has developed a complex system of phase switching and antigenic variation involving gene rearrangement and classical control elements at the transcriptional and post-transcriptional levels. The other group, including pili from Ps. aeruginosa and Bacteroides nodosus, shows variation in terms of serogroup and serotype as well as pilin subunit size, but these are stably maintained and phase variation and gene rearrangement are not detectable in passaged laboratory strains. Expression of GC pili has been reviewed recently (So, 1986), and is briefly summarized to afford comparison with the other system.

I . The Gonococcal Pilus System Different states of piliation on gonococcal strains were first detected as differences in colony morphology (Swanson et al., 1971; Jephcott et al., 1971). A single colony was capable of generating several colony morphologies within a few generations. For instance, piliated cells generated non-piliated variants at a rate of one in 1000 while the reverse event occurred at a rate of one in lo4lo5. This high rate of switching was thought to be due to gene rearrangement and not simple mutation. Most of the information about organization of pilin genes in G C has been derived using a single isolate, MS11, originating at the Mount Sinai School of Medicine in New York (Swanson et al., 1985). Many regions of the chromosome contain pili-related sequences (Meyer et al., 1982; Segal et al., 1986; Swanson et al., 1986). These can be divided into silent (pilS) and expressed ( p i l E ) regions. The MS11 chromosome contains two functional expression sites, pilEl and pilE2 (Meyer et al., 1984) and two silent loci encoding the constant region of the pilin molecule (residues 1-30) (So, 1986; Segal et al., 1986) as well as other silent loci containing the SV and HV sequences, one of which, pilSI, has been studied in detail. The two pilE loci are about 20 kb apart and pilSl maps about 15 kb upstream of pilEl (Haas and Meyer, 1986). Haas and Meyer (1986) sequenced thepilSl silent region and showed that it contained six tandem pilin genes which lacked the constant N-terminal region and encoded the SV and HV regions of five different pilin proteins. These pilin sequences were flanked by 39 bp repeats also found in the expression site, and these authors postulated that these sequences represent cassettes of the SV and

80


HV regions which are duplicated and transferred to the expression site(s) or other silent loci. They also suggested that the highly conserved residues interspersed within the HV and SV regions are the flanking regions of minicassettes that further alter pilin-protein sequence. However, these conserved residues may correspond to essential residues for pilus expression, mutation in which results in the P - rp + phenotype described by Bergstrom et al. (1986). The switch from the piliated ( P + ) to non-piliated (P-) phenotype can be caused by a deletion event in one or both of the expression sites, and this is thought to involve direct repeats found at these loci (Segal et af., 1985). However, this P + to P- switch is often not explainable by gene rearrangement; for instance, both pilE loci do not undergo this deletion process simultaneously. Thus, the presence of a phase switch-induced regulator which can act in trans on thepifElocus has been invoked (Segal et al., 1985; Hagblom et af.,1985; So, 1986). Other GC strains containing only one expression site, or a derivative of MSI 1 which has one expression site deleted, do not seem to express this regulator that is thought to influence transcription of functionally intact pilin genes (Bergstrom et af., 1986). Bergstrom et al. (1986) and Swanson et a f . (1986), using the primer extension technique which allows sequencing of mRNA transcripts (Hamlyn et al., 1981), have proposed that P- derivatives are the result of genetic rearrangements and mutations within the gene. They described the molecular basis of the reverting and non-reverting phenotype of P - strains described by Swanson et al. (1985). They used the GC strain with a single expression site already described, and found that non-reverting P- (Pn-) cells had a deletion at the 5’ end of the pilin gene resulting in loss of the single copy of the constant region of the pilin protein. This MS 1 1 strain had only one intact pilin gene, and the defect was not reversible by a recombination event from a second pilin gene. They then observed two types of reverting P- cells, P - rp + and P - rp - . These P-rp+ variants carried a sequence in the HV region that was identical to the copy 5 sequence from thepilSI locus described by Haas and Meyer (1986), suggesting that copy 5 encodes a non-functional pilin protein. The P-rp+ contained pilin in their membranes but did not elaborate a pilus. They found that the pilin in these P-rp+ cells was antigenically distinct from the original P cells and the sequence of the RNA transcripts showed multiple, discrete amino-acid substitutions, usually within the HV region. They concluded that these mutations affected assembly of pilin into pili since replacing the C-terminal portion of these pilin genes with DNA from P + cells restored piliation. The P - rp - cells encoded truncated pilin proteins which could be corrected by further mutation to give P + revertants. Gene rearrangement seems to depend on the general recombination pathway of the cell since the recA GC strain, constructed by M. Koomey

+


81

(personal communication), is able to undergo the P + to P- transition at normal frequencies but has a much lower rate of reversion. If the P + to Ptransition was generated by pilin mutation (Bergstrom et al., 1986), by the presence of a repressor (So, 1986) or by a specific recombinase as suggested for the Type 1 pilus system (see p. 74), then perhaps the red-dependent event would affect antigenic variation, involving recombination between the silent and expression loci. This would be an essential step in the reversion from Pto P + cells. However, it has not been shown that antigenic variation requires the cell to pass through a P- stage (So, 1986). It should be emphasized that evidence of a switching mechanism involving an invertible DNA sequence has not been detected in GC. 2. Pilus Systems in Pseudomonas aeruginosa and Bacteroides nodosus

The genetics of pili in Ps. aeruginosa and B. nodosus are not well defined. However, some comparison to the GC system can be made. The number of copies of the pilin gene in Ps. aeruginosa appears to be one (Pasloske et al., 1985, and unpublished observations). The number of serogroups of pili in Ps. aeruginosa appears to be about 10 (W. Paranchych, F. Ehftekhar, D. P. Speert, K. Volpel and B. Pasloske, unpublished observations). Moreover, the pilus serotype remains stable in laboratory strains suggesting that phase variation and gene rearrangement are not common occurrences. However, Southern blot hybridizations of the DNA and immunoblots of the total cell protein of 64 clinical isolates from cystic fibrosis patients have revealed some interesting aspects of pilus expression (W. Paranchych, Q. Sun and D. P. Speert, unpublished observations). These isolates represent samples taken from a number of patients once a year for several years. All of the isolates had a single complete gene copy of the whole pilin gene, and the restriction enzyme digest patterns fell into approximately 10 groups (unrelated to serogroup) with many of the strains resembling PAK or PAO. When a 600 bp PstI fragment which contains the conserved N-terminal region of the pilin protein, was used as a probe, homology ranged from strong to very poor. If the 1.2 kb Hind11 fragment of PAK (Pasloske et al., 1985) was used as a probe, all of the digests hybridized strongly, and preliminary evidence suggested that a region downstream of the pilin gene, bordering one of the Hind11 sites, was highly conserved in all strains. The immunoblots of cellular proteins from these strains revealed that a majority of the strains reacted with either anti-PAK or anti-PA0 pili antiserum, to varying degrees, with only a few strains being totally nonreactive and representing either completely non-homologous serogroups or strains that did not produce pili. These immunoblots made it possible to distinguish strains that carried the major epitope of PAK or P A 0 pili and

82

W. PARANCHYCH AND L. S.

FROST

those that carried minor epitopes. The molecular weight of the pilin subunits of these strains was found to vary between 13,000 and 17,000, the molecular weight of PAK and P A 0 pilin being 15,000.Thus, the pilin gene seemed to be capable of undergoing changes in molecular weight reminiscent of the Streptococcuspyogenes M protein (Hollingshead et af., 1986)and was capable of phase variation and/or antigenic variation but not in the same time scale as gonococcal strains. Currently, sequence data on a number of interesting pilin genes from these isolates are being accumulated. However, the primer extension method cannot be used because there is no conserved sequence near the 3’ end of the gene. Similar results were also reported for pili of Bacteroides nodosus (Anderson et al., 1986). These pili were found to contain two proteins, the pilin subunit (17,000 Da) and a minor protein (80,000 Da) associated with the “cap-like” structure at the base of the pilus (Mattick et al., 1984). These basal structures are reminiscent of the knobs associated with the base of conjugative pili although no large protein has been reported in preparations of conjugative pili (see Table 1). Both of the B. nodosus pilus-associated proteins were powerful immunogens. Anderson et al. (1986) found that the electrophoretic mobility of both the fimbrial (1 6,000-1 9,000) and basal (77,00&80,000) proteins varied in molecular weight between serotypes of these antigens as well as the eight defined serogroups (A-H). One interesting serogroup, H, had two pilin subunits (6,000 and 10,000 Da) which proved to be the result of proteolytic cleavage of the encoded gene product (16,000 Da) (Elleman et af., 1986). While a number of NMePhe pilin clones have been expressed in E. coli, no cloned fragment has been capable of expressing a pilus. This could be due to, the fact that the accessory or assembly genes have not yet been identified and cloned. However, the assembly process may not function in E. coli even when all of the accessory proteins are provided, since the membranes of E. coli may not support the transport and processing of proteins from Ps. aeruginosa. Hence, work is in progress to construct an appropriate Pil- Pseudomonas mutant to provide a suitable background strain for these studies.

VI. Structure-Function Relationships of Pili Proteins Although pilus proteins are relatively small (in the range 7-28 kDa), they promote several important functions. Specific regions of the polypeptide chain are responsible for anchoring pilin to membranes, transporting pilin across membranes, interacting with various accessory proteins involved in pilus assembly, or subunit-subunit interactions. Moreover, surface-exposed regions of the pilus protein are often immunologically aWive, or function as

PHYSIOLOGY A N D BIOCHEMISTRY OF PILI

83

recognition sites for pilus-specific bacteriophages or mammalian cell receptors. However, in some systems, pili attach to their mammalian cell receptor by means of an adhesin that is distinct from the major structural component of the pilus (Norgren et al., 1984). Both genetic and biochemical approaches are being used to identify and characterize these functional domains in various types of pili. A. CONJUGATIVE PILI

1. Chemical Composition of F Pili

F pili consist of a single, repeating subunit, pilin, of molecular weight 7200 (Moore et al., 1981a; Frost et al., 1984). The presence of an acetylatedterminus has been verified for F pilin (Frost et al., 1984),ColB2 pilin (Finlay et al., 1984)and pED208 pilin (Frost et al., 1983). The presence of carbohydrate and phosphate moieties on F-like pilin remains unresolved (Brinton, 1971; Tomoeda et al., 1975; Date et al., 1977; Willetts and Skurray, 1980). Brinton reported the presence of two phosphate groups which differed in acid lability and which were alkali-stable. One of the phosphate groups was associated with a phosphoglycopeptide with the same amino-acid composition as that of the first nine amino acids in F pilin. The presence of glucose and phosphate groups on F-like pili was investigated by G. D. Armstrong et al. (1981). They analysed the phosphate and carbohydrate content of pilin purified by gel-exclusion chromatography in the presence of sodium dodecyl sulphate (SDS). Both pED208 and ColB2 pilin contained three moles of phosphate per mole of pilin. With pED208 pilin, the phosphate was completely extracted with a chloroform-methanol mixture and was found to be derived from a mixture of phosphatidylglycerol and phosphatidylethanolamine (2 : 1 molar ratio, respectively). The composition of the E. coli K12 membrane was found to be 90% phosphatidylethanolamine and 10% phosphatidylglycerol, suggesting that pili have a greater affinity for phosphatidylglycerol or that F+ cells have an altered phospholipid composition. ColB2 pilin, with a molecular weight of 7000, is almost identical

to F pilin (see Fig. 3), and contajned 0.7-0,9rnoles ofphosphateper male of pilin after chloroform-methanol extraction. Analysis by 31P-NMR of pED208 and ColB2 pilin suggested that all pilus-associated phosphate could

be accounted for as phospholipids. However, the phosphoglycopeptidefrom F pilin reported by Brinton (1971), suggests that the phosphate residue associated with ColB2 pilin may represent a covalently bound phosphate on F-like pili which is involved in an unusual linkage not detected by NMR. Similarly, preparation of pure pilin by column chromatography in SDS removed all the carbohydrate from pED208 pilin and all but 1.O mole of D-

I

1 10 20 MET ASN ALA VAL LEU SER VAL GLN GLY ALA SER ALA PRO VAL LYS LYS LYS SER PHE PHE SER LYS PHE THR

11 I11 IV V

I I1 I11

IV V

TRP] ARG

a

40 50 LEU ALA ARG ALA VAL I L E PRO ALA ALA VAL LEU MET MET PHE PHE PRO GLN LEU ALA MET ALALEU ALA ARG ALA VAL I L E PRO ALA ALA VAL LEU MET MET PHE PHE PRO GLN LEU ALA MET ALALEU ALA ARG ALA VAL I L E PRO ALA ALA VAL LEU MET MET PHE PHE PRO GLN LEU ALA MET ALA-

30 LEU ASN MET LEU ARG LEU ASN MET LEU ARG LEU ASN MET LEU ARG LEU

20

10 I I1 I11

ARG

Ac ALA GLY SER SER GLY GLN ASP LEU MET ALA SER GLY ASN THR THR VAL LYS ALA THR PHE GLY LYS ASP

IV V

I I1 I11 IV V

I I1 I11 IV V

50

PHE PHE PHE PHE m L

LEU LEU LEU LEU E U

ALA ALA ALA VAL

40 VAL LEU VAL GLY ALA VAL MET VAL LEU VAL GLY ALA VAL MET VAL LEU VAL GLY ALA VAL MET VAL GLY ALA VAL MET U W V A L GLY[ALA]MET

LYS LYS LYS LYS MET

TRP TRP TRP TRP CYS

30 VAL VAL VAL VAL ILE

VAL VAL VAL VAL ILE

LEU ALA LEU ALA LEU ALA LE ALA IL!]ALA

GLU GLU GLU GLU G L

GLY PHE GLY PHE GLY PHE GLY PHE GLY ,LEU

ALA ALA ALA ALA VAL

ILE ILE LIE ILE VAL

ILE ILE LIE ILE VAL

SER VAL SER VAL SER VAL ER VAL SLE [VAL

60 70 PHE I L E ALA VAL GLY MET ALA VAL VAL GLY LEU PHE I L E ALA VAL GLY MET ALA VAL VAL GLY LEU PHE LIE ALA VAL GLY MET ALA VAL VAL GLY VAL GLY SER PHE I L E A LEU PHE b f ? V A L GLY ASP WPFL44)

SER SER VAL VAL SER SER VAL VAL SER SER VAL VAL SER SER I L E VAL SERWQlMET

TYR MET TYR MET TYR MET TYR MET TYR-ITHR

MET MET MET MET

m k 3

THR THR THR THR

LYS LYS LYS LYS LYS

ASN ASN ASN ASN A S

VAL YS VAL YS VAL YS VAL N E

3

LEU^

FIG. 3. The amino-acid sequence of the five types of conjugative pili including the sequence of the signal peptide. The prototype plasmids encodingeachpilus type are: I, F; 11, ColB2 or R538-1; 111, R1-19; IV, R100-1;V, pED208 (Willettsand Maule, 1986; Frost er al., 1985; B.B. Finlay, L.S. Frost and W.W. Paranchych, unpublished results). The non-homologous residues (with respect to the sequence of F propilin) are boxed.


85

glucose per mole of protein from ColB2 pilin. Thus, ColB2, but not pED208 pilin, may also have a D-glucose moiety as reported for F pilin. An alternative interpretation of these observations is that the glucose and phosphate are noncovalently bound, and treatment with SDS and/or chloroform-methanol does not remove all of the phosphate and glucose from the F and ColB2 pilins. The nucleotide sequences of pilin genes from F-like pilin Types I-V showed that the major amino-acid differences occur at the N-terminus (Fig. 3). In the case of R1-19, the C-terminus contained an additional lysine residue. The sequence of the pED208 pilin gene revealed a number of conservative aminoacid substitutions in the central portion of the protein, and several substitutions involving a change in charge at the N- and C-termini. The leader sequence of pED208 propilin was significantly different from that of pilin Types I-IV, but the homology within the pilin proteins was striking. 2. The Antigenic Determinants of F-like Pili

The presence of five serotypes in F-like pili has been known for several years (Lawn and Meynell, 1970; Bradley and Meynell, 1978; Meynell, 1978). The prototypes in these studies were F, R538drd (R538-1 and ColB2 pilins are identical in sequence), R1-19drd (Rl-19), R100-ldrd (R100-1) and pED208, a derepressed derivative of FoIac (Falkow and Baron, 1962). Phenotypes R538-1 and RI-19 were stronglycross-reactive, F, R1-19, R538-1 and R100-1 were weakly cross-reactive and pED208 was serologically unique. These serotypes also correspond to the different types of F-like pili discerned by Willetts and Maule (1986) on the basis of phage-plating efficiency, which is a function of the phage attachment ability of a pilus type (Meynell, 1978). There are at least two antigenic determinants on F-like pili and the major epitope involves the N-terminus of the pilin molecule where approximately 80% of the antibodies raised against pili are directed (Worobec et al., 1983; Finlay et al., 1985). Using synthetic peptides which mimic the antigenic determinant, the acetyl group has been shown to be essential for the antigenicity of pED208 pili (Worobec et al., 1985) and F pili (Frost et al., 1986). With pED208 pili, the acetyl moiety and two leucine residues at positions 3 and 4 in the pilin protein define the major epitope of pED208 pili (Worobec et al., 1985). The nature of the secondary epitopes of F-like pili has not been chemically defined. However, the lysine residue at the C-terminus of R1-19 pilin allows these pili to be distinguished from the otherwise identical ColB2 pili, suggesting that this residue alters the secondary epitope in some way. Worobec et al. (1986) separated anti-pED208 pilus antiserum into two fractions by affinity chromatography using a synthetic peptide corresponding to the N-terminal region of pED208 pilin. Using immuno-gold labelling

86


techniques, they found that the major epitope, located at the N-terminus, was not exposed on the sides of the pilus but was exposed on the surface of knobs at the base of the pilus and, perhaps, at the pilus tip. However, the pilus tip associates readily with knobs of other pili as previously noted by Brinton (1971), and it was difficult to resolve whether the observed binding of antibodies to the pilus tip was an artifact. Pili growing out from the cell did not appear to have antibodies at their tips. These results were repeated with the F pilus system using two monoclonal antibodies to F pili (Frost et al., 1986). These monoclonal antibodies reacted with two adjacent epitopes in the Nterminal region (residues 1-1 2) of F pilin and these epitopes were also exposed on the knobs at the base of the pilus although they did not appear to be at the pilus tip. Antibodies directed against other epitopes in pED208 pili were bound to the sides of the pilus. Since F and ColB2 anti-pilus antisera do not cross-react with heterologous synthetic peptides representing the N-terminal regions of these pilins, it was possible to show, by electron microscopy, that the common antigenic determinant, a secondary epitope, between these pilus types was also located on the sides of the pili. Furthermore, the anti-F pilus antiserum failed to react with the sides of R1-19 pili under the conditions employed to prepare electron-microscope specimens. However, prolonged incubation in a mild detergent uncovered this epitope (Frost et al., 1985) suggesting that the Cterminal lysine residue in RI-19 pilin masked, but did not destroy, the common epitope. These results correspond to the findings of Lawn et al. (1971) who observed “mixed” pili resulting from F and R1-19 residing in the same cell. F and R1-19 pili were unreactive with heterologous antisera, and the “mixed” pili, containing both types of subunit, reacted with the, homologous antiserum to give a patchy appearance in the electron microscope. 3. Phage Interactions with F-like Pili

Two classes of RNA phage attach to the sides of F-like pili at distinct sites. These are typified by R17 (or MS2, f2) and QB (Crawford and Gesteland, 1964) and have been extensively reviewed by Paranchych (1975). They are known to attach to the pilus by similar but distinct attachment proteins (A protein). These attachment proteins (39,000 Da) are highly insoluble and one copy is present per virion. A domain of the A protein is exposed on the phage surface and is presumably involved in phage attachment to the pilus. Based on data in Fig. 3, this involves residues 12-20 (approximately) and the last few residues at the C-terminus. In addition, there are several physical features on the pilus surface that may define the sites of phage attachment. These include the transverse grooves between each layer of subunits (1.28 nm) as well as the small longitudinal grooves between each adjacent subunit (see Fig. 2a).


87

One of the amazing properties of F pili is their ability to cleave the R17A protein into two polypeptides to allow the phage RNA to leave the eclipsed phage particle and penetrate the cell (Krahn et al., 1972). The cleavage reaction does not occur in the cold or with free pili, suggesting that the process requires energy, presumably provided by the cell, which travels along the length of the pilus. This putative enzymic activity for F pilin is intriguing and leads to speculation as to the possible involvement of the elusive glucose and phosphate moieties (see Section VI.A.l). With regard to the interaction of filamentous DNA phage to conjugative pili, there are five copies of the attachment protein, pIII, at one end of the phage particle where the N-terminal portion forms a knob which is attached to the phage by the remainder of the molecule (Goldsmith and Konigsberg, 1977; J. Armstrong et al., 1981; Grant et al., 1981; Gray et al., 1981). If the F pilus contains five pilin subunits per turn of the helix, the five attachment proteins of f l phage could interact with the five pilin subunits at the tip and disassembly of the pilus and phage into the cell membrane may occur by similar mechanisms. For a review of fl phage structure and assembly, see Webster and Lopez (1985). 4 . F Pilus Interactions with Recipient Bacteria

During initiation of conjugation, the pilus tip recognizes a site on the recipient cell and a mating signal is transmitted to the donor cell which initiates DNA replication (Ou and Yura, 1982). The pilus is thought to retract into the donor cell, bringing the recipient cell surface into contact with the donor cell surface whereupon a fusion of the two cell envelopes is thought to occur, providing a conjugation bridge for the transfer of DNA between cells (Panicker and Minkley, 1985). Almost nothing is understood about the various steps in this process that involve the pilus, except that a functional pilus is an absolute requirement for conjugation. The F-pilus receptor on the recipient cell surface is the ompA-designated protein, a major outer membrane protein of E. coli (Manoil and Rosenbusch, 1982). This protein is also involved in structural maintenance of the cell envelope, and is a receptor for bacteriophages and colicins K and L (Lugtenberg and Van Alphen, 1983). It has a close association with lipopolysaccharide (LPS) such that mutations that affect the LPS often have a secondary effect on the stability of ompA in the outer membrane leading to conjugation-deficient (Con-) recipient cells (Schweizer and Henning, 1977; Schweizer et al., 1978; Manoil and Rosenbusch, 1982). The N-terminal half of the ompA protein traverses the outer membrane repeatedly in a cross-beta sheet conformation, exposing four regions on the cell surface (Morona et al., 1984). Two of these regions, around residues 25 and 154, are thought to be

88

W. PARANCHYCH AND L. S. FROS?

involved in defining the conjugational proficiency of F- cells by acting as the receptor site for either the F pilus tip itself or another protein in the donor cell membrane (Morona et al., 1985). Although R1-19 (pilus Type 111) and F (Type I) appear to share the same R100-1 (Type IV) apparently receptor to varying degrees on F- cells (ompA), has a different receptor which involves LPS (Havekes et al., 1977a). IncI plasmids (R144) have yet another receptor involving LPS alone (Havekes et al., 1977b). 5. F-like Pili and Surface Exclusion

Another receptor for the pilus tip is an outer-membrane protein (TraTp) which is part of the surface (or entry) exclusion system encoded by conjugative plasmids (Lederberg et al., 1952; Willetts and Maule, 1974; Minkley and Ippen-Ihler, 1977; Achtman et al., 1977; 1980). This protein, encoded by IncF plasmids, is expressed by the traT gene in the transfer operon (Kennedy et al., 1977; Manning et al., 1980). It blocks mating-pair formation between related donor cells by preventing mating-pair stabilization (Achtman et al., 1977). Protein F TraTp is thought to have intimate contact with the ompA protein, blocking the site on OmpAp, which is the receptor for the F pilus. The traT protein also has sequence homology to the OmpAp-recognizing portion of the OmpAp-specific phages (Riede and Eschbach, 1986). Protein TraTp is a highly expressed lipoprotein (Perumal and Minkley, 1984)which may interact with the tip of the pilus and thus prevent donor-recipient cell recognition (Willetts and Maule, 1974, 1986; Minkley and Willetts, 1984). The second transfer gene involved in surface exclusion, traS, which maps immediately upstream from traT(Achtman et al., 1980), encodes a gene product which is associated with the inner membrane and appears to inhibit DNA transfer (Achtman et al., 1977; Manning and Achtman, 1979). F-like plasmids are capable of expressing surface-exclusion systems which are distinct from that of the F plasmid (Alfaro and Willetts, 1972; Willetts and Maule, 1974). Willetts and M a d e (1986) defined four surface-exclusion variants which, for the most part, correspond to the different alleles for F-like pili. A fifth surface-exclusion system would be that of pED208 (Finlay and Paranchych, 1986). The sequence of the traT genes of F, pED208 (Finlay and Paranchych, 1986; E. G. Minkley, personal communication) and R100-1 (Ogata et al., 1982) are available. The homology between the three proteins is striking, and the difference between F and R100-1 TraTp lies in one aminoacid substitution (Gly->Ala) in the middle of the protein. If the pilus tip recognizes TraTp, then it reacts to very subtle changes in sequence of F and R 100-1. Another possibility is that pilus-TraTp recognition may be rather non-specific and the stringency of the surface-exclusion phenomenon may be


89

encoded by the traS gene product which appears to be unique in each case (Hansen et al., 1982; Ogata et al., 1982; Finlay and Paranchych, 1986; E. G . Minkley, personal communication). 6 . Surface Features of F-like Pili

The pilus is capable of interacting with a number of biological macromolecules. The tip of the pilus is the site of interaction with the cell surface of the recipient cell during mating-pair formation (Ou and Anderson, 1970; Achtman and Skurray, 1977) and other donor cells during the process of surface exclusion (Willetts and Maule, 1974). The tip is also the site of attachment of filamentous DNA phages M 13, fl ,fd (Caro and Schnoss, 1966) and the sides of the pilus are the sites of attachment for the spherical RNA phages R17 and QB (Crawford and Gesteland, 1964; Paranchych, 1975). In addition, antibodies are capable of recognizing epitopes on the pilus surface. One approach to elucidating pilus structure has been the identification of pilus variants which have an altered traA gene affecting phage attachment, donor ability or pili per cell. These include naturally occurring variants such as those specified by closely related IncF plasmids (Fig. 3; Paranchych, 1975; Frost et al., 1985; Willetts and Maule, 1986) and point mutants of the F plasmid itself (Silverman et al., 1967, 1968; Tomoeda et al., 1972; Orosz and Wootton, 1977; Burke et al., 1979; Willetts et al., 1980). Many F pilus variants have altered susceptibility to phages R17 or QB but retain fl sensitivity. No transfer-proficient, fl resistant variant has been isolated, suggesting that transfer, pilus outgrowth and fl attachment are closely linked. Figure 3 shows the amino-acid substitutions for four traA point mutants previously described (Willetts et ai., 1980) and summarized in Table 2. Taken together, the phage sensitivity patterns (Types I-V) and the information on the immunodominant regions of F-like pilin allow certain surface domains of the pilin subunit to be identified. The N-terminus may be exposed at the tip of F-like pili in a unique configuration and provide the specificity for pilus-related phenomena. However, electron microscopy has not clearly indicated this (see Section VI.A.2). Other types of conjugative pili have pointed tips which are clearly visible by electron microscopy and appear to be the site of attachment for a variety of filamentous phages (see Table I). These points appear to be conical arrangements of subunits instead of the tubular arrangement found in the pilus shaft. It is possible that F-like pili also have a unique configuration of pilin subunits at their tips, but that these are not clearly resolved by electron microscopy. Meynell et al. (1974) raised antisera to sheared pili in order to enrich for antibodies to the ends of pili. They found that these antibodies were unable to distinguish between F and Rl-19 pili, suggesting that a shared

W. PARANCHYCH AND

90

L. S. FROST

TABLE 2. Pilus types, efficiency of phage plating and effect of cyanide on levels of piliation of Escherichia coli strains with various F-like plasmids and their derivatives. From Willetts et al. (1980) and Frost et al. (1985). Efficiency of plating F-like plasmid

Pilus type

Inc group

F R538-1 COLB2 COLB4 RI-19 R100-I EDP208

I I1 I1 I1 111 IV V

FI F11 FII FII FII FII FV

fl

R17

QB

Pili per 100cells

100

100

90 1 3

70 86 120 60 8 0

100 95 115 117 3 3 0

143 212 360 164 180 108 1700

10

2 100

Efficiency of plating traA mutants

Donor ability

WPFL44 WPFL46 WPFL47 WPFL51

1I7

5 14 55

fl 79 R 74 1

R17

QB

Pili per 100cells

6 R R 20

R R 0.2 R

325 14 98 145

Treated with cyanide (20min) 14 37 374 80 40 22 1100 Treated with cyanide (20min) 121 6 7 0

Addition of cyanide indicates the retraction ability of that pilus type. R indicates resistance. Inc is the abbreviation for “incompatibility”.

epitope is exposed at the ends of broken pili. Experiments with two monoclonal antibodies to F pili support this hypothesis. F pili that had been sheared by sonication generated new binding sites for the monoclonal antibody (JEL92) that recognized the region near Met-9, whereas no new sites were created for the antibody (JEL93) that recognized the N-terminus (Frost et al., 1986). Jacobson (1972) made the observation that sheared pili are capable of attaching fl phage, suggesting that the N-terminal region (residues 1-12) may be exposed at the tip of an intact pilus, while the region around Met-9 is presumably exposed at the tip of broken pili and would be involved in f l attachment. In addition, the lysine residue at position 10 in R100-1 is probably partly responsible for the low phage-fl infectivity in bacteria carrying this plasmid. With the exception of pED208, a charged residue at the C-terminus (Rl-19 and WPFL44) also affects fl sensitivity suggesting that the N- and C-termini are in close apposition. If the glycophosphopeptide reported by Brinton (1971) is correct, then the glucose and phosphate moieties would be within the first nine residues of F pilin and would not be exposed on the sides of the pilus but would be exposed at the tip or buried within the structure. Since pED208 (which is glucose- and


91

phosphate-free) is fully sensitive to f l ,the phosphate and glucose would not be important in fl infection, However, they could play a role in R17 or QB infection since pED208 is completely resistant to these phage. A region roughly corresponding to residues 12-22, as well as several residues near the C-terminus, may be exposed on the lateral surface of the pilus. Mutations in these regions affect R17 and QB attachment and efficiency ofplating (see Table 2 and Fig. 3). Charged residuesat the C-terminus (RI-19, pED208 and WPFL44) lower QB sensitivity more than that of phages R 17 or fl, whereas non-conservative substitutions within residues 11-22, usually involving a change of charge, greatly affect R17 phage sensitivity with lesser effects on QB and fl (R100-1, pED208, WPFL47 and WPFL46). The mutation in WPFL46 decreases pilus number and donor ability suggesting that pilus assembly has been affected and that the region after residue 20 may be involved in holding the subunits together. The central region of the pilin molecule is highly hydrophobic and is probably involved in the quarternary structure of the pilus where at least four surfaces of the molecule take part in subunit-subunit interactions. Minkley et al. (1976) observed that whole pili could not be iodinated with Chloramine-T, suggestingthat the tyrosine residue at position 42 is inaccessible. The low PI of pili (pH 4.15; Brinton, 1971) is puzzling since the three acidic residues are outnumbered by the five lysine residues. If the phospholipid content of pili is not contributing to its acidic nature (see Section VI.A.I), then the acidic residues must be on the pilus surface while the basic residues would be buried within the structure. Although there are several reports of trace amounts of other proteins that have been reported in pilus preparations (Date et af., 1977; Helmuth and Achtman, 1978), no other protein has routinely been found associated with pili either as a tip protein or a basal protein. Recently, a small amount of protein of molecular weight 8000 has been found in preparations of F pili examined by SDS-PAGE and stained with a silver staining reagent (K. IppenIhler, personal communication). If the pilus tip recognizes all three proteins, namely the N-terminal portion of the fl phage protein (pIII) (Hill and Petersen, 1982), the sequences near residues 25 and 154 in OmpA (Chen et al., 1980) and the surface-exclusion protein TraTp (Finlay and Paranchych, 1986), then some common aminoacid sequence among these proteins might be expected to serve as an F-pilus attachment site. A comparison of these sequences was undertaken to determine whether any sequence homology between these three proteins is detectable. No strong homology was found, suggesting that either different domains on the F pilus ate involved in these interactions or other features in an F + cell are the true receptors in these processes.

92


7 . F Pilus Assembly and Retraction

The F pilus contains only a single repeating subunit, and free pili do not appear to contain other proteins arranged in a basal structure such as is found on flagella (Iino, 1969). Recently, Silverman (1987) detected a small structure in sphaeroplasts of E. coli which spans the entire cell envelope. This agrees with the proposal by a number of workers that the F pilus originates from a plasmid-encoded organelle spanning a junction of the inner and outer membranes (Bayer, 1975; Achtman and Skurray, 1977; Laine et al., 1985). The steps involved in assembly of a pilus presumably involve expression of the pilin gene product, propilin, processing of propilin to pilin, modification of the pilin subunit (acetylation and, possibly, phosphorylation and glycosylation), formation of a pilin pool in the membrane at the site of pilus synthesis, and, presumably, assembly of the various transfer-gene products and host factors required for pilus outgrowth and retraction. There may also be a site at the base of the pilus for a membrane complex involving the plasmid DNA, awaiting the signal to begin transfer after establishment of a stable mating pair. All F-like pili have an extremely long leader sequence which is twice the average length for a prokaryotic leader sequence (Michaelis and Beckwith, 1982; Perlman and Halvorson, 1983). Ippen-Ihler and her coworkers have studied the steps in processing of the pilin precursor to mature pilin and have published a review of their findings (Laine et al., 1985; Ippen-Ihler and Minkley, 1986). In short, they suggest that the 14,000-Da pilin precursor protein is processed to the mature pilin subunit of 7000 Da in the presence of the traQ gene product. There may be a two-step processing mechanism with an 8000 dalton intermediate. The 7000 Da polypeptide (Ap7) does not react with anti-F pilus antisera but a slightly more slowly migrating polypeptide called Ap7* reacts with these antibodies. Conversion of Ap7 into AP7* is facilitated by traG and may correspond to the acetylation event required for formation of antigenically competent pilin. Processing of propilin to pilin occurs in the inner membrane as does the acetylation reaction since all four species of pilin can be found at this location (Ap 14,8,7,7*) while only Ap7* is found in the outer membrane suggesting that only the mature form is polymerized into pilin. The traQ gene product is required for efficient processing to occur, even though the cleavage site at the N-terminus strongly resembles the preferred substrate for the signal peptidase of E. coli Ala-X-Ala-Ala (Perlman and Halvorson, 1983).The requirement for electrochemical potential within the membrane for protein translocation (reviewed in Wickner and Lodish, 1985) is interesting in that pilus outgrowth is also energy-requiring and the two processes may be linked. The process of F-pilus outgrowth and retraction is controversial and


93

evidence in favour of retraction is circumstantial. Several models for pilus function have been advanced (Brinton, 1971). The most widely accepted model involves pilus outgrowth and retraction being in equilibrium where energy is required for pilus assembly (Marvin and Hohn, 1969; Curtiss et al., 1969; Novotny and Fives-Taylor, 1974; O’Callaghan et al., 1978). Interfering with the energy source by addition of cyanide causes the pili to disappear. This was presumed to be evidence of pilus retraction since there was no increase in the numbers of F pili or pilin in the culture supernatant (Novotny and FivesTaylor, 1974). Similar results were obtained following addition of arsenate providing the cells were grown in minimal media on a carbon source other than glucose (OCallaghan et al., 1973b, 1978). Heating the cells to 50°C caused pilus retraction although this was thought to be by a different mechanism (Novotny and Fives-Taylor, 1978). Cooling cultures to 20°C caused pili to be shed into the medium while quick chilling to 0°C prevented this (Novotny and Lavin, 1971). Furthermore, addition of anti-pilus antibodies or RNA phages, which bind to the sides of the pilus, prevents disappearance of F pili presumably by physically blocking the retraction mechanism. These experiments would suggest that pilus outgrowth requires energy whereas retraction does not, and that conditions that block outgrowth may also cause retraction. Plasmid ColB2, an Inc FII plasmid closely related to the Inc F1 F plasmid, is insensitive to cyanide in that its pili do not retract (Frost et al., 1986). Similarly, retraction-minus mutants of F have been isolated (Burke et al., 1979), whereas ColB4 and pED208 have intermediate levels of retraction in the presence of cyanide and could represent natural mutants in the gene(s) responsible for retraction. The best evidence for F-pilus retnaction comes from experiments with filamentous bacteriophage where phage attachment to F pili has been followed by a visible shortening of the pili on the cells (Jacobson, 1972). However, Paranchych and his coworkers reported that RNA- and DNAphage infection was accompanied by an increase in pili fragments in the medium (Paranchych et al., 1971;OCallaghan et al., 1973a).Achtman and his coworkers have elaborated on the initial observations of a donor and recipient cell connected by a pilus (shown in Brinton, 1971; Ou and Anderson, 1970) and demonstrated that the mating pairs form large aggregates which are stabilized by the activity of traN and traG (Achtman et al., 1978a, b). Initial contacts between mating cells are easily disrupted by SDS which dissociates the pili while subsequent steps, when the mating cells have fused together, are SDS-resistant. Although no real evidence for pilus retraction can be proposed on the basis of these experiments, it is an attractive possibility. Whether pili retract into the donor cell or depolymerize into the recipient cell membrane is unknown. Preliminary efforts to identify transfer proteins in the recipient cell, especially pilin, were not successful (W. Paranchych, unpublished observations).

94


B. ADHESIVE PILI OF

Escherichia coli

Elucidation of the primary structures of the K99, Pap, and Type 1 pilins has revealed several common characteristics: (a) they are approximately 18 kDa in size, (b) the N-terminus is hydrophobic, (c) they show significant homology at the N- and C-termini, (d) there is a cysteine loop in the N-terminal half of the protein and (e) they have a penultimate tyrosine residue at the C-terminus (Mooi and de Graaf, 1985; Nonnark et al., 1985).Although homologies at the N- and C-termini are also present in CFA/I (14 kDa) and K88 (around 27 kDa) pilins, these proteins are significantly different in size and lack the cysteine loop (Mooi and de Graaf, 1985). It is worth noting that predictions concerning secondary structure and hydrophilicity in all of these pilins give similar profiles (Baga et al., 1984; Klemm, 1984; Roosendaal et al., 1984), suggesting that they contain analogous structural domains. Most of the above-mentioned pilins from E. coli contain highly hydrophobic amino-acid residues in the C-terminal part of the molecule, suggesting that these regions may be involved in subunit-subunit interactions (Klemm, 1985; Mooi and de Graaf, 1985). Three positions, in particular, contain highly conserved aromatic amino-acid residues. Such residues (particularly tyrosine), have been implicated in the maintenance of pilus structure in both Ps. aeruginosa (Watts et al., 1983a) and Type 1 (McMichael and Ou, 1979). Surface domains of pilus proteins in intact pili are presumably immunogenic towards the host’s immune system. Since these regions are not directly involved in maintenance of the pilus structure, they usually tolerate significant amino-acid changes, and this provides a means of evading the host’s immune system through antigenic variation. Examples of this are seen among K88 pili, which contain at least three serological subtypes, ab, ac and ad (Klemm and Mikkelsen, 1982; Dykes et af., 1985), Type 1 (Salit e l a[., 1983), and Pap pili (Nonnark et al., 1985; Mooi and de Graaf, 1985; Klemm, 1985). In all cases, the antigenic determinants were inferred on the basis of secondary structure and hydrophilicity predictions, and a comparison of amino-acid sequences in related types of pili. Only with Ps. aeruginosa (Watts et al., 1983b; Sastry et af., 1985a) and N. gonorrhoeae (Schoolnik et al., 1984, 1985) pilins have the proteins been mapped for antigenic determinants by means of proteolytic and chemical cleavage of pilin, and immunological characterization of the resulting peptides. Little is known about the localization of receptor-binding domains in adherence pili in E. coli. As already mentioned (Section III.C.2), Type 1 pili promote MS haemagglutination, whereas most other pathogenic strains promote MR haemagglutination. Pap pili bind to the a-D-Gal-(l+4))-fi-DGal unit of the P blood-group antigens of uroepithelial cells, while K88, K99, CFA/I, CFA/II and some less frequently occurring non-digalactoside-specific


95

MR pili, termed X-adhesins (Vaisanen et al., 1981), are associated with intestinal epithelium. Interaction of these pili with eukaryotic cells has long been thought to be mediated directly by pilin, since purified pili have been shown to mediate agglutination of erythrocytes and other eukaryotic cells, and to inhibit competitively binding of intact bacteria. However, recent studies have demonstrated that, in the case of Pap and Type 1 pili, the adhesin (i.e. the structure that actually mediates bacterial cell binding) is distinct from the monomer making up the pilus (Norgren et al., 1984; Maurer and Orndorff, 1985; Normark et al., 1985; Minion et al., 1986). Although the location of the adhesin molecule in the pilus has not yet been determined, there is good reason to believe that one or more minor proteins promoting the adhesin function are positioned at the pilus tip (Mooi and de Graaf, 1985; Normark et al., 1985). Thus, it is possible that one of the roles of the major structural units is extension of the adhesin molecule some distance from the cell surface to facilitate interaction with the host receptor. Two different subunit genes have also been located within the operon in the case of K88 pili (Mooi and de Graaf, 1985). The large subunit (26 kDa) constitutes the major component of K88ab pili. The existence of the small subunit (17.6 kDa) was inferred from DNA sequence data and the analysis of K88ab mutants. Although it has never been detected as a minor component of K88ab pili, mutants lacking this protein were unable to agglutinate erythrocytes or bind to intestinal epithelial cells. One interpretation for this observation was that the 17.6 kDa protein may serve as an adhesin (Mooi and de Graaf, 1985). However, Jacobs et al. (1987) have shown that two tripeptides (Ser-Leu-Phe, Ala-Ile-Phe) from the major pili subunit (p26) of K88 pili are effective in inhibiting the haemagglutinating activity of purified K88 pili and the adherence of pili to intestinal epithelial cells. Of particular interest was the fact that the region surrounding the Ser-Leu-Phe sequence showed significant homology to gonococcal pilin, suggesting that this region may also promote adherence in the case of N. gonorrhoeae pili, and that it may represent the receptor binding domain in both K88 and gonococcal pili. Nucleotide sequencing of pilin genes from E. coli, and pilin-expression studies have shown that MS and MR pilins are exported across the bacterial cytoplasmic membrane, and that they are all endowed with N-terminal signal sequences that confer the membrane translocation function (Klemm, 1985; Mooi and De Graaf, 1985; Normark et al., 1985). In order for the subunits to be processed, translocated and assembled into intact pili, various auxiliary proteins are needed, and it has been found that three to six such helper proteins exist. However, the mode of pilin interaction with these accessory proteins has not yet been elucidated (Klemm, 1985; Mooi and de Graaf, 1985; Normark et al., 1985).

96


C. PILI DESIGNATED

NMePhe

NMePhe Pili are expressed by Ps. aeruginosa, N. gonorrhoeae, N . n z @ g & f d i s , M . nonliquifaciens, M . bovis, B. nodosus and V. cholera (see Section III.C.3). To date, structure-function studies have been performed primarily on pili from Ps. aeruginosa and N . gonorrhoeae. These studies are summarized below. 1 . Pilifrom Pseudomonas aeruginosa

Pili from Ps. aeruginosa are multi-functional organelles which mediate adherence of Ps. aeruginosa to human buccal epithelial cells (Woods et al., 1980; McEachran and Irvin, 1985; Paranchych et al., 1986), human tracheal epithelial cells (Ramphal et al., 1984; Palmer et al., 1986; R. T. Irvin, unpublished observations) and human cornea (Reichert et al., 1982). The nature of the pilus receptor on human epithelial cells is not yet understood. Paranchych et al. (1985) measured pilus binding to a variety of synthetic sugars representing many di-, tri- and tetra-saccharide structures found in mammalian glycoproteins and glycolipids, and failed to reveal any significant binding to any of the sugar moieties examined. However, a wide spectrum of binding activities was observed when a variety of proteins and enzymes were used as binding substrates. Of 30 proteins tested, phosphorylase b, pyruvate kinase and aldolase showed highest pilus-binding activity, whereas proteins such as cytochrome c, trypsin and band-3 protein bound pili very poorly. It was concluded that pili may have been recognizing a polypeptide domain that resembles the true pilus receptor, suggesting that it is perhaps a protein rather than oligosaccharide in Nature. Pili from Ps. aeruginosa also promote a phenomenon known as “twitching motility” (Bradley, 1980c), and they serve as receptors for a number of pilusspecific bacteriophages for Ps. aeruginosa including RNA-containing types (Feary et al., 1964; Bradley, 1972b), DNA-containing filamentous forms (Takeya and Amako, 1966; Bradley, 1973a) and forms containing heads and long non-contractile tails (Bradley, 1973b). Both twitching motility and phage sensitivity require that pili are retractile, i.e. able to undergo a process known as “retraction” (Bradley, 1972b, c, 1974a, b, 1980~).Mutants of Ps. aeruginosa which have non-retractile pili are multipiliated, phage-resistant and unable to promote twitching motility (Bradley, 1974b, 1980~).One such mutant of strain Ps. aeruginosa K, called PAK/2Pfs (Bradley, 1974b) was instrumental in facilitating the first purification and characterization of polar pili from Ps. aeruginosa (Frost and Paranchych, 1977; Frost et al., 1978; Paranchych et al., 1978). Recently, W. Paranchych and his colleagues obtained direct evidence that pili from Ps. aeruginosa are important virulence factors in pathogenesis


97

attributable to the bacterium. Using the thermal (Stieritz and Holder, 1975), neutropenic (Cryz et al., 1983) and CaClz (Tamura and Tanaka, 1985)mouse models, the LD50 values obtained with a Pil- strain of Ps. aeruginosa P A 0 were between lo2 to lo4 higher than those obtained with the isogenic Pil+ strain. Moreover, when mice were injected with 50 p1 of anti-pilus IgG at the time of bacterial challenge, the LDsOvalues were about lo4 higher than in control mice injected with non-immune IgG. It is therefore evident that polar pili from Ps. aeruginosa represent an important virulence factor in pathogenesis. The antigenic regions of the pilus protein of Ps. aeruginosa PAK were determined by Watts et al. (1983b) and Sastry et al. (1985a). Arginine-specific cleavage was used for the initial fragmentation of the pilus protein into four large peptide fragments called cTI (1-30), cTII (31-53), cTIII (54-120) and cTIV (121-144). Fragments cTIII and cTIV were further cleaved into a variety of subfragments. The various peptide fragments were subjected to immunoblot and direct ELISA studies, as well as competition ELISA experiments. Four distinct epitopes were identified: one weak (reacting with less than 5% of the anti-plus antibodies) cross-reactive epitope in the N-terminal region (130), a moderately immunogenic epitope (reacting with 20% of the anti-pilus antibodies)in fragment 70-81, a strongly immunogenic epitope (reacting with 60% of the anti-pilus antibodies) in fragment 82-1 10, and a weak (reacting with less than 5% of the anti-pilus antibodies) epitope in fragment 128-144. The cross-reactive epitope in the N-terminal region was detectable only in denatured pili, suggesting that this region of the protein is buried at subunit interfaces in intact pili. This conclusion was supported by spectral studies performed at PAK pili by Watts et al. (1983a), who showed that Tyr-24, Tyr27 and at least one Trp are involved in subunit-subunit interactions. The epitopes in cTIII and cTIV were considered to be type-specific, since they were not cross-reactive with pilus-specific antiserum raised against the closely related Pseudomonas strain PAO. Moreover, it was evident from competition ELISA studies that the immunodominant epitopes in cTIII were at least partially conformation dependent (Sastry et al., 1985a). The fact that the antigenic determinants in cTIII and cTIV are type-specific suggests that several different pili serotypes exist among naturally occurring strains of Ps. aeruginosa. However, the extent of this serological polymorphism is presently unknown. Bradley and Pitt (1975) and Woods et al. (1982) identified at least five distinct serotypes. W. Paranchych and his colleagues (unpublished observations) screened 64 clinical isolates for the PAK and P A 0 serotypes using whole-cell immunoblot assays, and found that about 34% were PAK-specific and 20% were PAO-specific. In addition, the amino-acid sequences of four different pilus types are now known (Sastry et al., 1985b; Johnson et al., 1986b; W. Paranchych and his colleagues, unpublished observations) and are shown in Fig. 4.

PAK PA0 CD4 PA103

Met Met Met Met

-5 Lys Lys Lys Lys

+1 Ala Ala Ala Ala

Gln Gln Gln Gln

Lys Lys Lys Lys

Glu Glu Glu Glu

Gly Ala Gly A l a Gly A l a Gly Ala

Ser Ser Ser Ser

45 Thr Thr Thr Thr

Thr Thr Thr Thr

Val Val Val Val

Glu Glu Glu Glu

Leu Leu Leu Leu

Lys Lys Lys Lys

Asn Asn Asn Asn

15 le le le le

PAK PA0 CD4 PA103

10 Leu Leu Leu Leu

Met Met Met Met

I I I I

l l l l

e e e e

Val Val Val Val

Val Val Val Val

e e e e

Pro Pro Pro Pro

Gln Gln Gln Gln

Tyr Tyr Tyr Tyr

25 Gln Gln Gln Gln

Ala Ala Ala Ala

Leu Leu Leu Leu

A l Ala Ala Ala

a [ Thr Thr Thr

k i Ile Ile Ile

G l u A l a Leu G l u e r Leu G l u Ser Leu G l u Ser Leu

Ser Ser Ser Ser

Arg Arg Arg Arg

Gly Gly Gly gly

Trp Ile Ile Ile

I I I I

l l l l

30

PAK [Ser V a l L y s S e r PA0 A l a Gly S e r L y s CD4 A l a Gly Ser L y s PA103 A l a G l y S e r L y s

I I I I

Ser Ser Ser Ser

Pro Pro Pro Pro

PAK PA0 CD4 PA103

l l l l

5 Glu Glu Glu Glu

Arg Arg Arg Arg

Ala Ala Ala Ala

I I I I

e e e e

20 Ala Ala Ala Ala

Val Val Val Val

Asn Asn Asn Asn

e e e e

l l l l

e e e e

Tyr Tyr Tyr Tyr

PAK PA0 CD4 PA103

l l l l

I I I I

Leu Leu Leu Leu

Ala Ala Ala Ala

I I I I

I I I I

Thr Thr Thr Thr

Ala Ala Ala Ala

Gly Gly Gly Gly

Ala Ala Ala Ala

Phe Phe Phe Phe

Leu Leu Leu Leu

e e e e

PAK PA0 CD4 PA103

Gly Gly Gly gly

l l l l

35

55

Q

60 G l y Thr Gly Thf G l u A Ile Ile A l a Ser T h r A l a Asp I l e Leu I l e G l y T h r T h r A l a S e r T h r A l a Asp

:G: y r Va Gly T h r T h r T y r V a l Gly T h r T h r T y r Val G l y

Val Val Ile Ile

(RlamqAs; G l u P r o As Asp G l u L y s Asp G l u L y s

90 PAK I l e A l a Leu Lys P r o PA0 ~ A l a ~ \ A s p CD4 V a l A l a Val T h r I l e L y s PA103 Val A l a V a l T h r I l e L y s

~

~

Asp P r o ~ + & Asp T h r Asp T h r

80 Ala Ala Ala Ala

95 A l a As G l y G l y Asp Gly Asp

Asn Asn Asn Asn

Lys Lys Lys Lys

Leu Leu Leu Leu

Gly G l Gly Gly

85 Thr y m Thr Thr

100 G l y M A l a Asp I l e ]ASP I l e + G l y T h r Val L v s Gly Thr Ls;

(Ilel

PAK T h r a T h r Phe PA0 &Phe T h r Phe C04 Phe T h r Phe A l a T h r G l y G l n S e r S e r P r o L y s Asn A l a Gly PA103 P h e m P h e A l a T h r Gly G i n S e r Ser P r o L y s Asn A l a Gly PAK Lys I l e PA0 l y s m CD4 L s Glu PA103 & G l u PAK

I I I I

l l l l

e e e e

Cys T h r Ser

Thr Thr Thr Thr

120 Leu[ThrlArg L e u Asn A r g L e u Asn A r g L e u Asn A r g

Thr Thr Thr Thr

Ala&Asp Ala Ala Glu Ala Glu

Phe P h T h r G l n G l u G l u M e t Phe T h r G l n G l u G l u M e t Phe

130 Lys p a Thr G l y Val T r p T h r

l y m T r p aGGly Val T r Gly Val T r p

140 I l e Pro e m P r o I l e Pro I l e Pro

Lys Lys Lys Lys

Gly Gly Gly Gly

Cys Cys Cys Asn L s P r o Cys A s n h P r o

FIG. 4. The amino-acid sequence of pilins from Pesudomonas aeruginosa strains PAK, PAO, CD4 and PA103. Boxed areas represent regions of non-homologous amino-acid sequence or spaces created to obtain maximum alignment. The PAK and PA0 sequences were obtained from Sastry et al. (1985a). The sequence of CD4 (a clinical isolate of Ps. aeruginosa obtained from a patient with cystic fibrosis) represents unpublished data of W. Paranchych and his colleagues. The sequence of PA103 (unpublished data) was provided by Kit Johnson and Steve Lory (personal communication).


99

Three of the pilins described in Fig. 4 (PAO, CD4 and PA103) are serologically identical (PA0 type), whereas PAK is a unique serotype. There is about 70% homology between the PAO-specific pilins and PAK, about 80% homology between P A 0 and CD4, while CD4 and PA103 are almost identical (97% homology). The N-terminal domain (1-55) is very highly conserved and is referred to as the “constant region”. The greatest variation occurs in the central part of the molecule (56-1 10) which contains the major antigenic determinants in PAK pilin, and presumably in the P A 0 types as well. The Cterminal region (1 1 1-147) contains few significant amino-acid differences among the four proteins, and is therefore referred to as the semiconserved region. This domain contains the highly conserved cysteine loop within which is located a weak antigenic determinant (Sastry et af., 1985a). This region has also been implicated in recognition of the pilus receptor on human buccal epithelial cells (Paranchych et af.,1986; R . T. Irvin, P. Doig, W. Paranchych and P. A. Sastry, unpublished observations). The cysteine loop and the highly immunogenic epitopes in the central portion of the molecule are presumably surface-exposed regions of the intact pilus, and it is believed that the phageattachment sites of the pilus protein will also be located within these putative surface-exposed domains. Determination of the nucleotide sequence of pilin genes in Ps. aeruginosa has revealed a highly conserved, positively charged six-residue leader sequence (Pasloske et af., 1985; Sastry et af., 1985b). Although the mechanism of pilin processing is not yet understood, it is clear that it must involve removal of the six-residue leader and N-methylation of the resulting N-terminal Phe residue. Pilus assembly presumably occurs in the outer membrane or at sites where the inner and outer membrane are fused to form adhesion zones (Bayer, 1975). Watts et a f .(1982) observed approximately equal amounts of pilin in the inner and outer membranes of Ps. aeruginosa, although they did not consider the possibility that one or both of these pilin pools may be unprocessed. Finlay et al. (1986c) showed that a chimera containing the PAK pilin gene was expressed in E. coli minicells and that both unprocessed and apparently processed pilin was present in the inner membrane. More recent studies in this laboratory (B. L. Pasloske, B. B. Finlay, L. S. Frost and W. Paranchych, unpublished observation) have shown that the sixresidue leader of pilin in Ps. aeruginosa is not processed in E. coli, and that the C-terminal half of the protein interacts with the N-terminal half to stabilize the pilus protein in the inner membrane. Truncated PAK pilin mutants encoding amino-acid residues -6 to 30 (36-mer) and -6 to +59 (65-mer) were constructed using recombinant DNA techniques, and expression of these products was compared with that of normal pilin. Each of the three genes (encoding normal pilin, the 36-mer and the 65-mer) was inserted into a highexpression vector system utilizing the T7 polymerase arid its promoter (Tabor

100


and Richardson, 1985). The results of these studies showed that the hydrophobic N-terminal region (36-mer) is expressed in E. coli and inserted as a stable entity into the inner membrane. The 65-mer was also expressed and inserted into the inner membrane, but this polypeptide was gradually degraded to a product similar in size to the 36-mer. Intact pilin, which was also expressed well in this system and inserted into the inner membrane, was extremely stable and not degraded at all. These observations suggested that only the first 36 amino acids of propilin are required for membrane integration and that the C-terminal region of propilin may associate with the N-terminal region to stabilize the region between residues 3&65 against degradative processes. It is not yet known whether pilin integration into the inner membrane behaves similarly in Ps. aeruginosa, nor whether processing of propilin occurs in the inner or outer membranes. Moreover, the number of accessory gene products required for pilus assembly in Ps. aeruginosa has not yet been established.

2. Pili from Neisseria gonorrhoeae Strains of Neisseria gonorrhoeae are notable for their ability to vary the antigenic properties of their pili. Gonococcal pilins from clinical isolates are extremely heterogeneous with regard to size (Lamden et al., 1979), antigenicity (Buchanan, 1975) and amino-acid sequence (Hagblom et al., 1985; Haas and Meyer, 1986). The relationship between gonococcal virulence and a piliated phenotype was first noted by Swanson et al. (1971). Gonococcal pili were subsequently shown to be involved in haernagglutination (Buchanan and Pearce, 1976), attachment to spermatozoa (James et al., 1976), human buccal epithelial cells (Punsalang and Sawyer, 1973), epithelial cells in culture (Swanson, 1973), human endocervical cells (Schoolnik et al., 1984) and human fallopian tubes in organ culture (Ward et al., 1974; McGee et al., 1981). However, since no animal model exists for gonorrhea, it has not been possible to test directly the degree of virulence conferred by gonococcal pili. Although the pilus is apparently required for initial colonization of the genitourinary tract, a second surface antigen, the opacity protein (Op or PII) is also involved in adhesion of N. gonorrhoeae to the human mucosa (Lambden et al., 1979; Blake and Gotschlich, 1983). Thus, the role of pili as a virulence factor is inferred from the observed correlation between virulence and piliation. Application of the primer-extension nucleotide-sequencing technique to mRNA for gonococcal pilin has resulted in the determination of a large number of pilin-DNA sequences from a variety of gonococcal strains (Hagblom et al., 1985; Haas and Meyer, 1986; Bergstrom et al., 1986).

101

PHYSIOLOGY AND BIOCHEMlSTRY OF PlLl

I - - - _ _. _ _ -. _ _ _ _ j-1_ _ ._ _ I, _ _ _ - 1MePhe

48

64 68

- - - - - - -. -

86 90

- _0 _ _ - _

104110 116

,

126 121

I

CYS

_.__

144

154 151

I

CYS

Constant region Semivarioble region Hypervariable region

FIG. 5. Schematic diagram showing distribution of the constant, semivariable and hypervariable regions in gonococcal pilus proteins. Based on information in Hagblom et al. (1 989, Haas and Meyer (1986) and Bergstrom et af. ( 1 986).

Comparison of these sequences has shown that the pilin gene can be divided into constant, semivariable and hypervariable regions (Fig. 5). Nucleotide changes within the semivariable regions usually involve single-codon changes, while the hypervariable region has been shown to undergo single-codon substitutions as well as in-frame insertions and deletions of one to four codons at a time. These changes, particularly within hypervariable regions, result in the appearance of unique epitopes on the pilus (Hagblom et al., 1985; Rothbard et af., 1985; Sparling er al., 1986). Several attempts have been made to delineate the antigenic determinants in gonococcal pilins. Schoolnik et al. (1984) determined the complete amino-acid sequence of pilin isolated from gonococcal strain MSI 1 and the partial sequence of pilin from strain R10. The proteins were cleaved with CNBr to yield three fragments: CNBr-I (1-8), CNBr-2 (9-92), CNBr-3 (93-159). The pilin structure was found to have several features similar to pilin from Ps. aeruginosa, notably a homologous Nxerminal region and a cysteine loop near the C-terminus. Unlike the pseudomonad pilin, the major antigenic determinants were located in the cysteine loop near the C-terminus, whereas the central part of the molecule was found to contain a conserved receptorbinding region and possibly an immunorecessive epitope common to all gonococcal pilins (Rothbard et al., 1984, 1985). Particularly interesting was a study in which antisera were raised against each of seven synthetic peptides corresponding to constant and variable sequences of the pilin from gonococcal strain MSl1, and then tested for their ability to cross-react with intact pili from both homologous and heterologous strains, as well as their ability to inhibit bacterial adhesion to a human endometrial carcinoma1 cell line (Rothbard et al., 1985). These experiments indicated that antibodies against a semiconserved central region (69-84) were the most efficient in binding to pili from all strains tested. These antibodies also successfully inhibited a heterologous gonococcal strain from binding to the endometrial carcinoma cells, as did antibodies specific for the region 41-50. These observations suggest that the pilin is the adhesin responsible for recognizing the eukaryotic

102


receptor and that it is the conserved regions of the protein which mediate the binding function. However, Virji and Heckels (1984) reported that monoclonal antibodies directed against the variable, but not the conserved, regions of the pilus inhibit gonococcal binding to epithelial cells. One interpretation of these data is that both the conserved and variable regions of the pilus are involved in receptor recognition. Another possible interpretation is that there is another protein, distinct from the pilus structural component, which is responsible for interaction with the mammalian cell receptor. As already mentioned (Section V.F. I), antigenic variation can result from the mixing and matching of semivariable and hypervariable gene segments involving silent and expression sites on the chromosome. The total number of silent loci in the chromosome is unknown at present, but Haas and Meyer (1 986) have shown that at least one silent locus, pilS1, contains six tandem pilus-gene copies containing only the semivariable and hypervariable domains but lacking the common N-terminal regions. These copies differed from each other in the same way as the variant sequences determined by Hagblom et al. (1 985). Presumably, such minicassettes from silent regions are constantly being transferred to expression sites to generate new serologically distinct pilus types. VII. Acknowledgements We wish to thank David Bradley for help in constructing Table 1 and Don Marvin for his manuscript on F pilus structure. We also thank Karin IppenIhler, Ned Minkley and Loren Day for useful discussions. REFERENCES

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Tschape, H. and Tietze, E. (1981). Biologisches Zentralblatt 100, 353. Tschape, H. and Tietze, E. (1983). Zeitschrifi fur Allgemeine Mikrobiologie 23, 393. Uhlin, B., Norgren, M., Baga, M. and Normark, S. (1985). Proceedings of the National Academy of Sciences of the United States of America 82, 1800. Vaisanen, V., Elo, J., Tallgren, L.G., Siitonen, A., Makela, P.H., Svanborg-Eden, C., Kallenius, G., Svenson, S.B., Hultberg, H. and Korhonen, T.K. (1981). Lancet ii, 1366. Vanden Bosch, J.F., Verboom-Sohmer, U., Postma, P., de Graaf, J. and MacLaren, D.M. (1980). Infection and Immunity 29, 226. Van Die, I. and Bergmans, H. (1984). Gene 32,83. Van Die, I., van den Handel, C., Hamstra, H.J., Hoekstra, W. and Bergmans, H. (1983). FEMS Microbiology Letters 19, 77. Van Die, I., van Gefen, B., Hoekstra, W. and Bergmans, H. (1984a). Gene 34, 187. Van Die, I., van Megen, Hoekstra, W. and Bergmans, H. (1984b). Molecular General Genetics 194,528. Van Die, I., Spierings, G., van Megen, I., Zuidweg, E., Hoekstra, W. and Bergmans, H. (1985). FEMS Microbiology Letters 28,329. Van Die, I., van Megen, I., Zuidweg, E., Hoekstra, W., de Ree, H., van den Bosch, H. and Bergmans, H. (1986a). Journal of Bacteriology 167,407. Van Die, I., Zuidweg, E., Hoekstra, W. and Bergmans, H. (1986b). MicrobiulPathogenesis 1.51. Virji, M. and Heckels, J.E. (1984). Journal of General Microbiology 130, 1089. Ward, M.E., Watt, P.J. and Robertson, J.N. (1974). Journal of Infectious Diseases 129,650. Watts, T.H., Worobec, E.A. and Paranchych, W. (1982). Journal of Bacteriology 152,687. Watts, T.H., Kay, C.M. and Paranchych, W. (1983a). Biochemistry 22,3640. Watts, T.H., Sastry, P.A., Hodges, R.S. and Paranchych. W. (1983b). Infection andlmmunity 42, 113. Weber, P.C. and Salemme, F.R. (1980). Nature. London 287,82. Webster, R.E. and Lopez, J. (1985). In “Virus Structure and Assembly” (S. Casjens, ed.), pp. 236267. Jones and Bartelett, Boston. Weiss, R.L. (1971). Journal of General Microbiology 67, 135. Wickner, W. and Lodish, H. (1985). Science 230,400. Willetts, N.S. (1977a). In “R Factor: Drug Resistance Plasmid” (S. Mitsuhashi, ed.), pp. 89-107. University of Tokyo Press, Tokyo, Japan. Willetts, N.S. (1977b). Journal of Molecular Biology 112, 141. Willetts, N.S. (1984). Methods in Microbiology 17, 33. Willetts, N.S. and Maule, J. (1974). Genetical Research 24, 81. Willetts, N.S. and Maule, J. (1986). Genetical Research 47, 1. Willetts, N.S. and Paranchych, W. (1974). Journal of Bacteriology 120, 101. Willetts, N.S. and Skurray, R.S. (1980). Annual Review ofGenetics 14,41. Willetts, N.S. and Skurrary, R.S.(1986). Absrracts of the American Society for Microbiology, Washington, D.C., p. 151. Willetts, N.S. and Wilkins, B. (1984). Microbiological Reviews 48, 24. Willetts, N.S., Moore, P.M. and Paranchych, W. (1980). Journal of General Microbiology 117, 455. Williams-Smith, H. and Huggins, M.B. (1983). Journal of General Microbiology 129,2659. Willshaw, G.A., Smith, H.R., McConnell, M.M., Barclay, E.A., Kmjulac, J. and Rowe, B. (1982). Infection and Immunity 37, 858. Willshaw, G.A., Smith, H.R. and Rowe, B. (1983). FEMS Microbiology Letters 16, 101. Willshaw, G.A., Smith, H.R., McConnell, M.M. and Rowe, B. (1985). Plasmid 13, 8. Winans, S.C. and Walker, G.C. (1985a). Journal of Bacteriology 161,402. Winans, S.C. and Walker, G.C. (1985b). Journal of Bacteriology 161,425.

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Woods,D.E.,Strauss,D.C.,Johanson, W.G., Jr., Berry,V.K.andBass, J.A. (1980).Infectionand Immuniiy 29, 1146. Woods, D.E., Iglewski, B.H. and Johanson, W.G., Jr. (1982). In “Microbiology-1982” (D. Schlessinger, ed.), pp. 348-352. American Society of Microbiology, Washington, D.C. Worobec, E.A., Taneja, A.K., Hodges. R.S. and Paranchych, W. (1983). Journa/of Bacteriology 153, 955. Worobec, E.A., Paranchych, W., Parker, J.M.R., Taneja, A.K. and Hodges, R.S. (1985). Journal of Biological Chemistry 260,938. Worobec, E.A., Frost, L.S., Pieroni, P., Armstrong, G.D., Hodges, R.S., Parker, J.M.R., Finlay, B.B. and Paranchych, W. (1986). Journal of Bacteriology 167,660.

Carboxysomes and Ribulose Bisphosphate Carboxylase/Oxygenase GEOFFREY A . CODD Department of Biological Sciences. University of Dundee. Dundee DDI 4 H N . U K

I . Introduction . . . . . . . . . . . . . I1 . Distribution and structureofcarboxysomes . . . . . . . A . Chemolitho-autotrophic prokaryotes . . . . . . . . B. Photo-autotrophic prokaryotes . . . . . . . . . C . Cyanelles . . . . . . . . . . . . . 111. Carboxysome composition . . . . . . . . . . . A . Carboxysomeisolation and studies inuirro . . . . . . . B. Immuno-electronmicroscopy . . . . . . . . . IV. Ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) . . . . A . Purification and structure . . . . . . . . . . B. Activation and catalysis . . . . . . . . . . C . Specificity and regulation . . . . . . . . . . D . Genetics . . . . . . . . . . . . . V . Carboxysome function . . . . . . . . . . . A . Are carboxysomes sites of carbon dioxide fixation in uiuo? . . . . B. Do carboxysomes protect ribulose 1,5-bisphosphate carboxylase/oxygenase. C . Are carboxysomes storage bodies? . . . . . . . . VI . Further aspects of carboxysomes . . . . . . . . A . Ecological markers for autotrophy . . . . . . . . B. Man-made ribulose 1, 5-bisphosphate carboxylase/oxygenase inclusion . . . . . . . . . . . . . bodies? References . . . . . . . . . . . . . .

156 157

.

I Introduction Carboxysomes. formerly known as polyhedral bodies. occur in several groups of microbes which grow on carbon dioxide as the principle carbon source. Within these autotrophs. carboxysomes only occur among microbes that utilize the Calvin.. or reductive pentose phosphate. cycle. Carboxysomes are ADVANCES IN MICROBIAL PHYSIOLOGY. VOL. 29 ISBN 0- 12-027729-8

Copyright 0 1988. by Academic Press Limited All rights of reproduction in any form reserved

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apparently confined to prokaryotes, but not all Calvin cycle bacteria contain these inclusion bodies. Although carboxysomes were observed by electron microscopy over 25 years ago (Jensen and Bowen, 1961), insight into their biochemical nature has been more recent and has stemmed from the discovery that the organelles in the colourless sulphur-oxidizing bacterium Thiobacillus neapolitanus contain ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the COrassimilating enzyme of the Calvin cycle (Shively el al., 1973a; Shively, 1974). The Calvin cycle prokaryotes, including organisms that derive energy from the oxidation of inorganic compounds (e.g. the chemolitho-autotrophic hydrogen-, sulphur-, nitrite-, ammonia-, carbon monoxide- and iron-oxidizing bacteria) and those that do so from sunlight (e.g. the phototrophic purple bacteria, cyanobacteria and prochlorophytes), continue to attract considerable research interest from the fundamental and applied viewpoints. These organisms are responsible for the primary production of organic carbon in many aerobic and anaerobic environments on a global basis, and perform key roles in the geochemical cycling of matter (for reviews see Schlegel, 1976; Smith and Hoare, 1978; Pfennig, 1978; Stewart, 1980; Bowien and Schlegel, 1981; Kuenen and Buedeker, 1982; Gibson and Smith, 1982; Fogg, 1982; Kuenen et al., 1985; Colby et al., 1985). The RuBisCO enzymes in particular of these organisms are of interest. These enzymes facilitate autotrophic growth, and knowledge of the properties of microbial RuBisCO enzymes contributes to the understanding of their evolution, and offer prospects of modifying the RuBisCO enzymes of higher plants. In the Calvin cycle prokaryotes that contain carboxysomes, a variable and sometimes predominant proportion of the cellular RuBisCO pool is located in these inclusion bodies. For earlier reviews on microbial RuBisCO enzymes see McFadden (1973; 1980), McFadden and Tabita (1974), McFadden and Purohit (1978) and Codd (1984), and on carboxysomes see Shively (1974) and Codd and Marsden (1984). In this review, recent advances in knowledge of the occurrence, composition, properties and possible functions of carboxysomes are examined and discussed with reference to the rapidly expanding field of research on RuBisCO enzymes. It is of interest to compare the RuBisCO enzymes of prokaryotes that contain carboxysomes with those that do not. Insight into carboxysome function also may be gained by comparison with other prokaryotic inclusions and with the compartmentation of RuBisCO in chloroplasts, and these aspects are also considered. Finally, the use of carboxysomes as ecophysiological markers, and the prospects of producing RuBisCO-containing inclusion bodies in recombinant micro-organisms, are discussed.

CARBOXYSOMES AND RIBULOSE BISPHOSPHATE CARBOXYLASE/OXYGENASE

1I7

11. Distribution and Structure of Carboxysornes

Polyhedral bodies, usually surrounded by a membrane, although this is not always apparent, have been widely observed among the nitrifying bacteria, colourless sulphur-oxidizing bacteria and cyanobacteria. Their designation as carboxysomes depends on the presence of RuBisCO in the inclusion bodies. Until recently, the strict demonstration of RuBisCO in the organelles had been confined to one or two members of the nitrifiers, sulphur-oxidizers and cyanobacteria and was limited by practical problems of carboxysome isolation. Although these problems have to some extent been overcome (see Section 111), the in uitro approach to carboxysome identification and characterization is currently being complemented by the use of immunoelectronmicroscopy on sections of whole cells. This has enabled the polyhedral bodies of additional organisms to be examined and has supported earlier views (Shively, 1974; Stewart and Codd, 1975; Codd and Marsden, 1984) that in a wide range of Calvin cycle prokaryotes these organelles contain RuBisCO. A. CHEMOLITHO-AUTROPHIC PROKARYOTES

I . Nitrite- and Ammonia-Oxidizing Bacteria

The ultrastructure of these organisms has received intensive study and carboxysomes have been observed in seven out of nine strains of nitriteoxidizing bacteria (Table 1). The nitrite-oxidizing bacteria vary in their nutritional capabilities with respect to carbon compounds (Smith and Hoare, 1977) and include versatile members which can grow on organic carbon sources as well as specialist members which can only grow chemolithoautotrophically. All of the versatile strains examined possess carboxysomes and these organelles are also present in Nitrococcus mobilis, which is a specialist. They are lacking, however, from the specialists Nitrospina gracilis and Nitrospira marina (Table 1). Among the ammonia-oxidizing bacteria, the characteristic mode of carbon nutrition is specialist, and carboxysomes have not been observed in intensive studies of any of the six specialist species belonging to six different genera (Table 1). Carboxysomes have been found in only one ammonia-oxidizer to date, a marine Nitrosomonas sp. (Wullenweber et al., 1977), although whether this isolate is nutritionally versatile or a specialist has not been reported. Comparison of the presence of carboxysomes and nutritional versatility is of interest in considering possible functions for the carboxysomes. The carboxysomes of nitrifying bacteria are typically located in the central region of the cell and are not in contact with the intracellular membranes

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TABLE 1. Presence of carboxysomes in nutritionally versatile and specialist species of nitrite-oxidizing and ammonia-oxidizing bacteria

Organism

Nitriteoxidizing bacteria Nitrobacter winogradsky Nitrobacter agilis (three strains) Nitrobacter hamburgensis X 14 Nitrobacter hamburgensis Y Nitrococcus mobilis Nitrospina gracilis Nitrospira marina Ammonia-oxidizing bacteria Nitrocystis oceanus Nitrospira briensis Nitrosolobus multiformis Nitrosomonas europea Nitrosovibrio tenuis Nitrosococcus mobilis Nitrosomonas sp.

Presence of Nutritional carboxysomes capability References" V" V V V Sb

S S S S S S S S n.r.c

1-3 4 4 5 5

6

7 8 9 10 11 12 13

Versatile, i t . facultatively heterotrophic. Specialist, i.e. obligately autotrophic. n.r., not reported. References: 1, Pope et al. (1969); 2, van Gool et al. (1969); 3, Bock et al. (1974); 4, Bock et al. (1983); 5 , Watson and Waterbury (1971); 6, Watson et al. (1986); 7, Murray and Watson (1965); 8, Watson (1971); 9, Watson ct al. (1971); 10, van Gool (1972); 11, Harmser al. (1976); 12, Koops er al. (1976); 13, Wullenweber et al. (1977). (I

"

possessed by several of these organisms (e.g. Bock et al., 1983). The inclusion bodies are 100-120 nm in diameter and consist of a core surrounded by a membrane or shell (van Gool et al., 1969; Bock et al., 1974). The icosahedral shape (Peters, 1974) and appearance of the carboxysomes of Nitrobacter winogradsky and several Nitrobacter agilis isolates accounted for the organelles in nitrifying bacteria being initially described as 'phage-like particles (Bock et al., 1974; Westphal and Bock, 1974).As in sulphur-oxidizing bacteria and cyanobacteria, carboxysome numbers per cell in the nitrifying bacteria vary with cell age and environmental conditions. Between one and five carboxysomes per cell is usual (e.g. Wullenweber et al., 1977; Bock et al., 1983),although up to 200 have been observed (Watson and Waterbury, 1971). As with carboxysome composition, data of carboxysome frequency under defined growth conditions can contribute to the understanding of carboxysome function.


1 19

2. Colourless Sulphur-Oxidizing Bacteria Carboxysomes have been observed in ten out of 17 species of strains of Thiobacillus.For example, among the nutritionally specialist species, they are present in T. neapolitanus, T. thioparus and T. thiooxidans (Shively et al., 1970; Holt et al., 1974), T. kakobis (Reynolds et al., 1981) and T. albertis (Bryant et al., 1983), but not in T. denitrificans (Shively et al., 1970). Among the nutritionally versatile Thiobacillusspecies, carboxysomes are present in T. intermedius (Holt et al., 1974) and T . perometabolis (Katayama-Fujimura et al., 1984a,b), but they have not been observed in Thiobacillus versutus (formerly ThiobacillusA2; Taylor and Hoare, 1969; Shively et al., 1970), or T. novellus (Shively et al., 1970). Carboxysomes are not present in the nutritionally versatile Thiosphaera pantotropha (Robertson and Kuenen, 1983). No correlation has emerged between the presence of carboxysomes and the versatility of carbon nutrition alone of the sulphur-oxidizing bacteria (Codd and Marsden, 1984), although the occurrence of carboxysomes in the genus Thiobacillusshows good agreement when viewed against the classification system of Katayama-Fujimura et al. (1984b) based on nutritional and physiological features, fatty acid composition, their respiratory chain ubiquinones and DNA base composition (Table 2). Carboxysomes may thus be adopted as a taxonomic tool for the heterogeneous genus Thiobacillus (Katayama-Fujimura et al., 1984b), but the significance of the presence or absence of these inclusions in individual strains requires further understanding of carboxysome composition and function. Thiobacillus carboxysomes are typically located in the central region of the cell and show a polygonal (mostly ,hexagonal) profile in cell sections. Carboxysome size in vitro varies from about 70 to 500 nm in diameter, with a mean of about 110 nm (Shively et al., 1973a,b; Holt et al., 1974; Bryant et al., 1983; Katayama-Fujimura et al., 1984b). Elongated carboxysomes have also been seen in T. neapolitanus (Shively et al., 1973b). In all cases, the inclusions are of medium to high electron density under the electron microscope and are surrounded by a shell, 3.5 nm thick in T. neapolitanus (Shively et al., 1973b; Holt et al., 1974). The carboxysome shell is a single-layered structure as apparent from studies of numerous cell-thin sections (see Codd and Marsden, 1984) and the freeze-etching analysis of Thiobacillus IV-85 (Murphy et al., 1974), which revealed smooth inner and outer surfaces of the carboxysome membrane without the studded particles characteristic of bilayer membranes. The three-dimensional structure of T. neapolitanus carboxysomes has recently been studied in detail using intact and broken isolated organelles (Holthuizen, 1986; Holthuizen et al., 1986a). Mean carboxysome size was I 17 & 7 nm with a range from 97 to 132 nm. The hexagonal profiles typically seen by negative staining, freeze hydration and freeze-etching techniques are

120

G.A. CODD

TABLE 2. Presence of carboxysomes in Thiobacillus species and strains classified according to the system of Katayama-Fujmura et al. (1984b)

Organism

Source

Group 1-1 Thiobacillus versutus (A2) Thi~bucillusversutus Thiobacillus novellus Thiobacillus sp. Thiobacillus sp.

ATCC" 25364 ATCC 27793 ATCC 8093 IAMh 12816 IAM 12817

Group 1-2 Thiobacillus ucidophilus Thiobacillus acidophilus

ATCC 27807 ATCC 27977

Nutritional capability/ Respiratory Presence of optimal growth chain carboxyconditions ubiquinone somes

Versatile; prefer Or

slightly alkaline growthmedia

Q-10 Q-10 Q-I0 Q-10 Q-10

-

-

+

Versatile; prefer acidic media

Q- 10 Q-I0

Versatile

4-8 Q-8 Q-8

Specialist

4-8 Q-8

Group 111-2 Thiobacillus neapolitanus ATCC 23641

Specialist

Q-8

+

Group 111-3 Thiobacillusferrooxidans ATCC 19859

Specialist

Q-8

+

Group I1 Thiobacillus delicatus IAM 12624 Thiobacillusperometabolis ATCC 23370 Thiobacih intermedius ATCC I5466 Group 111-2 Thiobacillus denitrijicans ATCC 23642 Thiobacillus thioparus ATCC 8 158

+

+ + +

-

+

American Type Culture Collection, Rockville, Md, USA.

* Institute of Applied Microbiology Culture Collection, University of Tokyo, Japan consistent with a pentagonal dodecahedron, which was confirmed by stereomicrography. Thiobacillus carboxysomes contain arrays of 10 nm diameter particles which are often arranged in rows or circles (Shively et al., 1973a,b; Holthuizen, 1986; Holthuizen et al., 1986a,b). These doughnut-shaped particles are released when carboxysomes are ruptured, and are RuBisCO molecules.

3. Hydrogen-Oxidizing Bacteria Ultrastructural surveys of a wide range of mesophilic hydrogen-oxidizing Calvin cycle bacteria have not shown the presence of carboxysomes in


12 1

members of the genera Alcaligenes, Aquaspirillum, Pseudomonas, Corynebacterium, Xanthobacter, Azospirillum, Flavobacterium, Arthrobacter, Bacillus, Nocardia or Paracococcus (Walther-Mauruschat et al., 1977; Aragno and Schlegel, 1981; Shively et al., 1978). Thermophilic hydrogen bacteria have received little attention to date and the sole representative examined ultrastructurally, Pseudomonas thermophila K2, contains hexagonal membrane-bound bodies, 100-150 nm in diameter (Kostrikina et al., 1981). The bodies contain particles of about the same size as RuBisCO molecules and are associated with up to 80% of the extractable RuBisCO activity in vitro (Romanova et al., 1982). This strongly suggests the presence of carboxysomes in Ps. thermophila, and further studies on these organelles in thermophilic hydrogen-oxidizing bacteria will be of interest. B. PHOTO-AUTOTROPHIC PROKARYOTES

I . Cyanobacteria Although individual sections of cyanobacterial cells may fail to reveal these organelles, since they may number only one or two per cell, carboxysomes are typically present in cyanobacteria, whether growing photo-autotrophically, photoheterotrophically or chemoheterotrophically. Numerous examples can be found in the literature (e.g. Jensen and Bowen, 1961; Peat and Whitton, 1967; Gantt and Conti, 1969; Lang and Whitton, 1973; Wolk, 1973; Stewart and Codd, 1975; Stanier and Cohen-Bazire, 1977). The carboxysomes of unicellular cyanobacteria typically resemble those of the chemolitho-autotrophic prokaryotes by occurring in the centre of the cell, not in contact with the thylakoids or other inclusions; by displaying a relatively uniform size (100130 nm diameter) with a common hexagonal profile; and by being surrounded by a 3 4 nm thick membrane (Gantt and Conti, 1969; Nierzwicki-Bauer et al., 1983; Jensen and Rachlin, 1984). By contrast, a wide variation in carboxysome size, shape, number and location occurs among the filamentous cyanobacteria. Carboxysome size among this diverse assemblage varies from about 100 to 900 nm diameter and hexagonal profiles are not more frequently seen than four- or five-sided sections of the carboxysomes (e.g. Peat and Whitton, 1967;Stewart and Codd, 1975; Lang, 1977; van Eykelenburg, 1979; Jensen and Ayala, 1976a). Carboxysomes in filamentous cyanobacteria may be in the centroplasm region (Stewart and Codd, 1975) or peripherally located between the thylakoids (Lang and Whitton, 1973). The membrane surrounding the carboxysomes of filamentous strains appears as a monolayer (e.g. Jensen and Ayala, 1976a;Jensen, 1979).Close association of the carboxysomes in several Anabaena strains with microtubules has been observed (Jensen and Ayala,

I22

G.A. CODD

1976a,b). The significance of microtubules in these prokaryotes remains unclear, although this alignment of arrays of microtubules, with their longitudinal axis in close contact with a facet of the carboxysome, may be related to carboxysome assembly or mobilization of carboxysome contents in these species. Carboxysomes occur in free-living and symbiotic cyanobacteria, including species such as the Nostoc cyanobionts of Cycas revoluta root nodules, the liverwort Blasia pusilla and the lichen Peltigera canina (Stewart and Codd, 1975). The aerobic nitrogen-fixing filamentous cyanobacteria have evolved a high degree of intercellular structural differentiation. The incompatible processes of photosynthetic oxygen evolution and aerobic nitrogen fixation in the heterocystous strains are permitted to occur simultaneously by the metabolic segregation of oxygenic photosynthesis to the vegetative cells and of nitrogen fixation to the adjoining heterocysts (Fogg et al., 1973; Wolk, 1973, 1982; Stewart, 1980). The RuBisCO required to fix CO2 photosynthetically is present in the vegetative cells but lacking from mature heterocysts (Codd and Stewart, 1977a; Codd et al., 1980). Electron microscopy of large numbers of cells from six heterocystous N2-fixing cyanobacteria showed that the carboxysomes are also segregated and are not present in heterocysts (Stewart and Codd, 1975). 2. Prochlorophyta

A new division of prokaryotes has been proposed, the Prochlorophyta, to accommodate oxygenic phototrophs which contain chlorophyll a , as do cyanobacteria, but which also contain chlorophyll h, which is characteristic of green algae and higher plants but not of cyanobacteria (Lewin, 1976, 1977, 1981). Prochlorophytes lack the phycobilin pigments characteristic of cyanobacteria, but contain RuBisCO (Berhow and McFadden, 1983; Codd, 1984), consistent with their ability to assimilate CO2 via the Calvin cycle (Akazawa et al., 1978). Until recently, the only prochlorophytes known were unicellular forms living in symbiosis with marine didemnid ascidians and were assigned to the genus Prochloron. Prochloron symbionts from Diplosoma uirens and Didemnum molle contain polyhedral bodies similar to the carboxysomes in filamentous cyanobacteria (Whatley, 1977; Fisher and Trench, 1980; Cox and Dwarte, 1981). The Prochloron polyhedral bodies typically occur between the thylakoids in the peripheral cytoplasm and may be closely adpressed to the photosynthetic membranes. Biochemical investigations on the Prochloron polyhedral bodies have been constrained by the inability to grow Prochloron species in laboratory culture. However, we have demonstrated by immunoelectronmicroscopy using heterologous RuBisCO antiserum that the D . uirens Prochloron polyhedral bodies contain RuBisCO (A.M. Hawthornthwaite and G.A. Codd, unpublished work).


123

A free-living planktonic prochlorophyte has recently been found growing abundantly in Dutch freshwater lakes (Burger-Wiersma and Mur, 1985; Burger-Wiersma et al., 1986). This organism contains polyhedral bodies (Burger-Wiersma et al., 1986) and is well established in laboratory culture. Characterization of the RuBisCO and polyhedral bodies from this organism is in progress in this laboratory. C. CYANELLES

Cyanelles are blue-green prokaryotes which resemble free-living unicellular cyanobacteria morphologically, but are obligate endosymbionts in eukaryotic protists (Kies, 1980, 1984; Kies and Kremer, 1986). Differences exist between cyanelles and cyanobacteria: Cyanophora paradoxa cyanelles are surrounded by a thin lysozyme-sensitive layer, presumably prokaryotic peptidoglycan, but a complete cyanobacterial type of cell wall surrounding the cyanelle cannot be seen (Hall and Claus, 1963; Schenk, 1970). However, the genome size and copy number of the C . paradoxa cyanelle are typical of chloroplasts, rather than unicellular cyanobacteria, and this cyanelle can therefore be viewed as a photosynthetic organelle which has been derived from an endosymbiotic cyanobacterium (Herdman and Stanier, 1977). The rapid labelling kinetics of I4CO2 assimilation by cyanelles are consistent with the operation of the Calvin cycle (Kremer et al., 1979) and the RuBisCO enzymes of the cyanelles from C . paradoxa and Glaucosphaera vacuolata have been characterized (Codd and Stewart, 1977b; Codd, 1984). Polyhedral bodies have been observed in several cyanelles. Size and number of the organelles varies widely from the single 600-900 nm central polygonal body in Gloeochaete wittrockiana (Kies, 1976, 1980) to the seventy or more 130-160 nm diameter inclusions of Paulinella chromatophora (Kies, 1980, 1984). A membrane cannot be seen around the single central electron-dense body of the C . paradoxa cyanelle or the terminal, pyrenoid-like body of Glaucocystis nostochinearum, although a monolayer surrounds the bodies in Gloeochaete wittrockiana and Paulinella chromatophora (Kies, 1976, 1980, 1984). Polyhedral bodies have not been observed in the Calvin cycle cyanelles of Glaucosphaera vacuolata (Kies, 1980). The central polyhedral body of the cyanelles of C . paradoxa and the terminal body of the Glaucocystis nostochinearum cyanelles have recently been confirmed by immuno-electronmicroscopy to be carboxysomes (Mangeney and Gibbs, 1987; Mangeney et al., 1987). The transition status of cyanelles between cyanobacteria and chloroplasts confers particular interest in their carboxysomes in terms of the carboxysomes of free-living cyanobacteria and of the RuBisCO-containing pyrenoids in algal chloroplasts.

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111. Carboxysome Composition

A knowledge of carboxysome composition is necessary to provide understanding of the functions of the organelles and their contribution to the physiology of the cell. A. CARBOXYSOME ISOLATION AND STUDIES

in vitro

I . Isolation and Stability of Carboxysomes in vitro The rationale for carboxysome isolation and discussion of the various approaches used up to 1983 were detailed by Codd and Marsden (1984). Carboxysomes have been isolated from few organisms to date; from Thiobacillus neapolitanus (Shively et al., 1973a; Beudeker et al., 1980, 1981; Beudeker and Kuenen, 1981; Cannon, 1982; Cannon and Shively, 1983), Nitrobacter winogradsky (Peters, 1974; Westphal, 1977;Westphal et al., 1979); Nitrobacter agilis (KI) (Westphal and Bock, 1974; Shively et al., 1977; Westphal, 1977; Westphal et al., 1979; Biedermann and Westphal, 1979), Nitrobacter hamburgensis H XI^) (Ebert, 1982),Nirrosomonas sp. (Harms et al., 1981), Anabaena cylindrica (Codd and Stewart, 1976) and Chlorogloeopsis fritschii (Lanaras and Codd, 1981a). The stability of carboxysomes in vitro varies according to source: for example, those released from ultrasonically-disrupted cells of N . agilis and T. neapolitanus, and purified by differential or isopycnic sucrose density gradient centrifugation, or by preparative agarose electrophoresis, retain their hexagonal profile in vitro (Shively et al., 1973a, 1977; Cannon and Shively, 1983). The isolated T. neapolitanus carboxysomes can be stored for at least two months at 4°C without changing their appearance if maintained in the presence of the protease inhibitor phenylmethylsulphonyl fluoride (Holthuizen et al., 1986b). In contrast, the carboxysomes from the filamentous cyanobacteria are less stable in vitro, although carboxysome yields from Ch.fritschii were optimized by cell disruption in a French Pressure Cell and the destabilizing effect of sucrose during subsequent carboxysome purification was lowered by the use of the silicon polymer, Percoll (Lanaras and Codd, 1981a). 2. Carboxysomal Proteins In all cases, isolated carboxysomes consist mainly of protein. The presence of RuBisCO, which is the most abundant component in carboxysomes, was initially indicated by the ability of isolated carboxysomes to catalyse RuBPdependent COz assimilation (Shively et al., 1973a, 1977; Codd and Stewart,


125

1976; Harms et af., 1981; Lanaras and Codd, 1981 a). This was confirmed by the finding of immunological cross-reactivity between isolated broken carboxysomes and antibodies to the respective RuBisCO enzymes (Codd and Stewart, 1976; Beudeker et al., 1981; Holthuizen et af.,1986b). These findings are consistent with the presence within isolated carboxysomes of 1&12 nm particles which have the same dimensions and shape as RuBisCO molecules (Shively et al., 1973a, 1977; Lanaras and Codd, 1981a; Holthuizen, 1986; Holthuizen et af., 1986a,b). Differencesin the estimates of the total number of polypeptides present in isolated sodium dodecyl sulphate (SDS)-dissociated carboxysomes have been reported after SDS-polyacrylamide-gel electrophoresis (SDS-PAGE), followed by gel staining with Coomassie Blue (Table 3). It is not clear whether these totally represent real differences in carboxysome composition between species or whether they merely reflect differences in methodology between laboratories. It is possible that additional carboxysomal polypeptide components may be detected by the use of more sensitive autoradiography of SDS-PAGE gels of SDS-dissociated carboxysomes of 35S-labelled cells. Whether carboxysome composition for an individual species varies with physiological conditions during growth does not appear to have been investigated. Despite the apparent interspecific differences, the isolated carboxysomes from the different physiological groups of autotrophs are similar in the possession of two polypeptides which coincide in terms of molecular mass with the large (L) subunits and small (S) subunits of their respective RuBisCO enzymes (Biedermann and Westphal, 1979; Lanaras and Codd, 1981a,b; Cannon and Shively, 1983;Snead and Shively, 1978; Holthuizen et af.,1986b). The L and S subunit peptides of the dissociated isolated carboxysomes are present in equimolar amounts, as in the purified RuBisCO enzymes which have an 8L8S quaternary structure. The T. neapolitanus carboxysomes have recently been examined for glycoproteins and four bands have been found with M , values of 120,000, 85,000, 54,000 and 10,000 (see Table 3). Those of 120,000 and 85,000 Da are present in trace amounts. The 54,000 glycoprotein migrates during SDSPAGE close to the RuBisCO L subunit (Holthuizen et al., 1986b) and may account for earlier indications of L subunit heterogeneity in T. neapolitanus carboxysomes (Cannon and Shively, 1983). The 10,000 Da species was the most abundant glycoprotein and this, plus the other three glycoproteins, are components of the T. neapolitanus carboxysome shell (Holthuizen et al., 1986b), a finding in support of earlier proposals for the shell of the inclusions from T. neapolitanus (Cannon and Shively, 1983) and N. agilis (Biedermann and Westphal, 1979). The lack of effect of ether and chloroform on the shells of N. agilis carboxysomes and analysis of chloroform-methanol extracts of

126

G.A. CODD

TABLE 3. Comparative polypeptide composition” of isolated carboxysomes Source o f carboxysomes Nitrobacter agilish

Chlorogloeopsis Thiobacillus fritschii“ neapolitanud Polypeptide identity

120

GP

97 89 85

GP

110

74 72 69

69 67 66

56

56 54 52

LSU GP LSU

49 47 43 39 36 32 15

14 13 12 Total

7

13

8

13

ssug

10

GP

12-15

Values refer to M,in kilodaltons. Data from Biedermann and Westphal (1979). Data from Lanaras and Codd (198la). Data from Cannon and Shively (1983) and Holthuizen et al. (1986b). ‘GP, glycoprotein. LSU, large subunit of RuBisCO. 8 SSU, small subunit of RuBisCO.

’

the T. neapolitanus organelles has indicated that lipids are not present (Biedermann and Westphal, 1979; Holthuizen et al., 1986b). Apart from RuBisCO, the identity of the remaining carboxysomal polypeptides is unknown. Activities of other enzymes of the Calvin cycle have been sought in isolated, intact and broken carboxysomes. Activities of


127

phosphoribulokinase, ribose-5-phosphate isomerase, fructose 1,6-bisphosphatase, NAD glyceraldehyde 3-phosphate dehydrogenase, or sedoheptulose 1,7-bisphosphatase were not detected in highly purified T. neapolitanus carboxysomespreparations, in contrast to cytoplasmic supernatants (Cannon and Shively, 1983; Holthuizen et al., 1986b). The possibility that phosphoribulokinase, which is metabolically contiguous with RuBisCO, may be a component of cyanobacterial carboxysomes was examined since the organelles of Ch.fritschii contain a cryptic 39,000 Da polypeptide (Lanaras and Codd, 1981a), and purification of this enzyme from Ch.fritschii showed it to consist of 40,000 Da subunits (Marsden and Codd, 1984). Only about 5% of the extractable Ch.fritschii phosphoribulokinase activity is associated with a cell-free particulate fraction (Lanaras and Codd, 198la). Sucrose density gradient centrifugation of carboxysome-containing extracts of this cyanobacterium did not result in the localization of the particulate phosphoribulokinase in the purified carboxysomes but in a thylakoid fraction (Marsden et ai., 1984). These findings have been confirmed by immuno-electronmicroscopy (see Section III.B, p. 130). Carbonic anhydrase has also been considered as a possible carboxysomal protein, since this enzyme is present at high activity in cultures of several cyanobacteria, grown in low concentrations of COZ,where it may function in inorganic carbon concentration to support the carboxylase reaction, versus the oxygenase reaction, of RuBisCO (Aizawa and Miyachi, 1986; Codd and Kuenen, 1987). The possibility also exists that carboxysomes may act as a COz-concentrating mechanism (Codd and Marsden, 1984). Carbonic anhydrase was not detected in either carboxysomal or cytoplasmic fractions of T. neapolitanus (Cannon and Shively, 1983), though the enzyme is present in high amounts in Nitrosomonas europea and Rhodospirillum rubrum which do not contain carboxysomes (Jahnke et al., 1984; Gill et al., 1984). High carbonic anhydrase activities were detected in extracts of air-grown Ch. fritschii and 90% of the activity is particulate (Lanaras et al., 1985). Although the enzyme has not been purified from Ch.fritschii, the presence of a cryptic 43,000 Da peptide in the carboxysomes purified from this source (see Table 3), along with the findings of Yagawa et al. (1984) that carbonic anhydrase from the carboxysome-containing cyanobacterium Anabaena variabilis has a M,value of 42,000 & 5,000, suggested that the Ch.fritschii carbonic anhydrase may be a carboxysomal enzyme. However, centrifugation of crude particulate Ch. fritschii extracts through sucrose density gradients yielded a sharp band of carbonic anhydrase which was well separated from the carboxysomes and thylakoids (Lanaras et al., 1985). The carbonic anhydrase of Ch. fritschii is apparently on the cell surface since high enzyme activity is exhibited by the whole cells and this is not influenced by cell disruption by ultrasonication or lyzozyme treatment (Table +

128

G.A. CODD

TABLE 4. Carbonic anhydrase activities of whole cells, broken cells and fractions of Chloroglaeopsis,fritschii

Source of enzyme"

Treatments

Specific activityh (units (mgprotein)-I)

Whole cells Whole cells Whole cells

-

Ultrasonicated Lysozyme lysate

0.3 1 0.29 0.24

Whole cells Whole cells Whole cells

+Acetozolamide (0.2 mM) +Ethoxyzolamide (20 p ~ ) Sulphanylamide (0.2 mM)

+

0.00 0.00 0.00

40,OOOg x 1 h 40,OOOgx 1 h

Pellet of broken cells Supernatant of broken cells

0.33 0.02

Purified carboxysomes Purified carboxysomes

-

0.00 0.00

Ultrasonicated

Cultures grown photo-autotrophically to mid-exponential phase on air throughout. (For comparison, specific activity ofwhole cells grown in 5% COz in air was about 0.07.) Activity was measured electrometrically: one unit of activity = (ApH/ ApH, - 1) x 10, where ApH and ApH, are the rates of pH change of enzyme-catalysed and -uncatalysed reactions (Lanaras ei al., 1985; G.A. Codd and K. Okabe, unpublished work).

4). The established carbonic anhydrase inhibitors acetozolamide, ethoxyzolamide and sulphanylamide completely inhibit whole cell enzyme activity (as with broken-cell extracts) with acetozolamide being used at concentrations that are unlikely to enter the cell. Several examples of external carbonic anhydrases exist along the green algae (Aizawa and Miyachi, 1986), but Ch. fritschii appears to be the first example of a cyanobacterium possessing an external carbonic anhydrase (Table 4). The function of the 43,000 Da Ch. fritschii carboxysome peptide remains cryptic (see Table 3).

3. Carboxysomes and Deoxyribonucleic Acid The possibility that carboxysomes may contain DNA was raised by Bock and his colleagues, since the 'phage-like inclusions of Nitrobacter spp. are of high ) and have a high 260/280 nm absorption ratio of 1.2 (Bock density ( P * ~ 1.296) et al., 1974; Westphal and Bock, 1974). This concept has been supported by the finding of a deoxyribonuclease-sensitivedouble-stranded filament, 14 pm long, with a buoyant density ( p 2 7 of 1.701 in ethidium bromide+aesium chloride gradients and an absorption maximum of 263.5 nm, associated with


129

osmotically-broken isolated N . agilis and N. winogradsky carboxysomes (Westphal, 1977; Westphal et al., 1979). Extrachromosomal DNA occurs commonly in autotrophic prokaryotes. Plasmids have been detected in purple photosynthetic bacteria, cyanobacteria, colourless sulphur-, nitrite-, ammonia-, iron- and hydrogen-oxidizing bacteria and carboxydotrophic bacteria (Herdman, 1982; Saunders, 1984; Schlegel, 1984; Friedrich, 1987). The only group in which progress has been achieved in identifying plasmid function is the hydrogenotrophic bacteria, where genes for HZoxidation, CO2 fixation, denitrification and heavy metal resistance have been located on plasmids (Schlegel, 1984; Friedrich, 1987). These organisms, with the probable exception of Pseudomonas thermophila, have not been found to contain carboxysomes (see Section 11. A). Cryptic plasmids have been reported in the following carboxysome-containing Thiobacillus spp.: T . ferrooxidans, T . neapolitanus, T . acidophilus and T . intermedius (Martin et al., 1981; Holmes et al., 1984; Toth et al., 1981; Davidson and Summers, 1983) and in Nitrobacter hamburgensis X14 (Kraft and Bock, 1984). Plasmids are also present in related strains that do not contain carboxysomes, for example Thiobacillus versutus (Gerstenberg et al., 1982) and Thiosphaera pantotropha (Chandra and Friedrich, 1986). No correlation has emerged between the possession of plasmids and of carboxysomes among the chemolitho-autotrophs. Carboxysomes are a consistent feature of cyanobacteria (Codd and Marsden, 1984) but extrachromosomal DNA has only been detected in about half of the strains examined (Herdman, 1982). We have examined three carboxysome-containing cyanobacteria for extrachromosomal DNA and specifically for the presence of DNA in the carboxysomes (Vakeria et al., 1984). Anabaena PCC7120 contains a 5 kb plasmid although this was not associated with the carboxysomes in this strain. No extrachromosomal DNA has been detected in Gloeobacter violaceus, or in Ch.fritschii using six different plasmid isolation methods. These findings preclude the possibility that Ch. fritschii and Gloeobacter violaceus carboxysomes contain extrachromosomal DNA. Gel electrophoresis of isolated intact and disrupted carboxysomes from these three cyanobacteria did not indicate the presence of plasmid DNA (Vakeria et al., 1984), although a trace of DNA was associated with carboxysomal pellets. Restriction enzyme digestion patterns of this DNA were characteristic of chromosomal DNA. We conclude that some association between the outer surface of the cyanobacterial carboxysomes and chromosomal DNA exists in broken cell preparations. Electron microscopic evidence exists for the attachment of DNA to the exterior of the putative carboxysomes of Ps. thermophila (Romanova et al., 1982)and the cyanelles of C. paradoxa (Bohnert et al., 1983). Holthuizen et al. (1986~)have recovered DNA associated with the exterior of isolated T.

130

G.A.

CODD

neapolitanus carboxysomes. This shows the same restriction enzyme digestion pattern as chromosomal DNA and hybridizes with the latter. A highly polar external surface of the carboxysome shell would facilitate the attachment of chromosomal DNA in vitro. Whether such association occurs in vivo is of interest. This has been observed in T. neapolitanus and apparently in Ps. thermophila (Holthuizen et al., 1986; Romanova et al., 1982) and raises the question of whether DNA has a functional role in carboxysome assembly. B. IMMUNO-ELECTRONMICROSCOPY

Immuno-electronmicroscopy has found valuable application in the localization of enzymes and other cell components. This approach has been used to investigate the subcellular distribution of RuBisCO using colloidal gold. Heavy labelling of the carboxysomes in cell sections of the cyanobacteria

FIG. 1. Immuno-electronmicroscopic localization of RuBisCO in Chlorogloeopsis fritschii. Cell sections were labelled with RuBisCO antibodies raised in rabbits,

followed by goat-antirabbit immunoglobulin conjugated to 20 nm colloidal particles, which may be seen covering the carboxysomesections (C). Bar marker represents 1 pm.


13 1

Chlorogloeopsis fritschii, Anacystis nidulans R2 and Anabaena cy lindrica was obtained using RuBisCO antiserum confirming the presence of RuBisCO in the carboxysomes (Fig. 1; Hawthornthwaite et al., 1985; Cossar et al., 1985). RuBisCO antiserum labelling of the cytoplasm also occurred, confirming that some of the RuBisCO pool in uiuo in the cyanobacteria is also soluble (Lanaras and Codd, 1981a,b,1982). In a group of 76 Ch. fritschii cells examined for RuBisCO gold colloid particle distribution, the RuBisCO pool appeared to be approximately equally distributed between the carboxysomes and cytoplasm (Fig. 2a). Inclusion bodies other than carboxysomes did not contain RuBisCO according to the immunogold technique, although loose association of the cytoplasmic enzyme with the thylakoids was observed in A. cylindrica vegetative cells (Cossar et al., 1985). In contrast to the A. cylindrica vegetative cells, RuBisCO antiserum labelling of the heterocysts did not occur, confirming the absence of RuBisCO and carboxysomes from these cells (Stewart and Codd, 1975; Codd and Stewart, 1977a; Cossar et al., 1985). Phosphoribulokinase antiserum-labelling of Ch. fritschii cell sections occurred in the cytoplasm, with some association with the inner surface of the cytoplasmic membrane, but no significant labelling of the carboxysomes was obtained (Fig. 2b). Gold immuno-electronmicroscopy has also recently confirmed the presence

T

50c

CA

CY

M

0

N

CA

CY

M

0

N

-"

FIG. 2. Distribution of colloidal gold particles in the localization of RuBisCO (a) and phosphoribulokinase(b) in Chlorogfoeopsisfritschii by immuno-electronmicroscopy. (a) RuBisCO antiserum: 76 cells were examined and 9345 gold particles counted; (b) phosphoribulokinase antiserum: 45 cells were examined and 28,853 gold particles counted. Gold particles associated with cell sections treated with null serum varied between 0 and 0.6% of the numbers obtained with antisera. CA, carboxysomes; CY, cytoplasm; M, cytoplasmic membrane; 0, all other discernible inclusion bodies; N, labelling on outer layers external to cytoplasmic membrane. Marker bars represent SD. From Hawthornthwaite et af. (1985).

132

G.A. CODD

of RuBisCO in the central dense body of the cyanelles of C. paradoxa and the terminal electron-dense body of Glaucocystis sp. cyanelles (Mangeney and Gibbs, 1987). As with the cyanobacterial carboxysomes, the cyanelle inclusions were not labelled with phosphoribulokinase antiserum, in contrast to the surrounding cytoplasm (Mangeney et al., 1987). Similar data using RuBisCO and phosphoribulokinase antisera have been obtained in this laboratory using sections of the symbiotic unicellular Prochloron sp. and the filamentous prochlorophyte of Burger-Wiersma et al. (1986) by A. M. Hawthornthwaite (unpublished work). Further examination of carboxysome proteins using antibodies raised against individual carboxysome components will be of interest in the comparison of carboxysomes between the different physiological groups of autotrophs and in the study of the assembly and breakdown of the organelles.

IV. Ribulose 1,ibisphosphate carboxylase/oxygenase (RuBisCO) As the COZ-assimilating enzyme of numerous major groups of autotrophic

prokaryotes (Codd, 1984), in addition to algae and higher plants, RuBisCO attracts extensive and intensive study. Research on RuBisCO has accelerated over the past decade and merits entire symposia devoted to the enzyme (e.g. Siegelman and Hind, 1978; Ellis and Gray, 1986). Earlier reviews of the structural, mechanistic, functional and regulatory aspects of RuBisCO are available (Akazawa, 1979; McFadden, 1980; Lorimer, 1981; Lorimer and Andrews, 1981; Miziorko and Lorimer, 1983; Codd, 1984; Akazawa et al., 1984). In this article, aspects of the RuBisCO enzymes of prokaryotes are reviewed, with reference where appropriate to the enzymes of plants and algae, with a view to understanding the physiology of autotrophy and carboxysome function in prokaryotes. A. PURIFICATION AND STRUCTURE

Understanding of the structural and functional properties of RuBisCO is aided by the availability of purified enzyme and RuBisCO has now been purified from over 40 prokaryotes (Akazawa, 1979; McFadden, 1980; Codd, 1984). This has been facilitated by a combination of favourable factors: RuBisCO is among the most abundant proteins in the autotrophic cell, accounting for up to 17% of total protein in Th. neapolitanus (Beudeker et al., 1981) and about 50% in Rhodospirillum rubrum (Tabita et al., 1983);many but not all RuBisCO enzymes are of high M , value ( 2500,000); enzyme activity assays are straightforward; and most enzymes are relatively stable in uitro. The main purification procedure for microbial RuBisCO involves sedimentation


133

of a high speed cell-free supernatant extract through sucrose density gradients as the principal step (e.g. Tabita and McFadden, 1974a,b; Bowien et al., 1976; Codd and Stewart, 1977~; Torres-Ruiz and McFadden, 1985). A rapid yield of purified spinach RuBisCO has recently been achieved by ion-exchange fast performance liquid chromatography (FPLC) (Salvucci et a/., 1986a). This procedure may be useful for microbial RuBisCO purification (Gutteridge et a[., 1986). All known RuBisCO enzymes from eukaryotes are proteins with high M , values (500,000-550,000), consisting of eight large subunits (L; ca. 50,00055,000) plus eight small subunits ( S ; ca. 11,000-15,000). The RuBisCO enzymes from most of the 40 or more autotrophs, examined from all of the major physiological groups of Calvin cycle prokaryotes, also have high M , values (500,000-600,000) with 8L8S quaternary structures (Table 5). Significant exceptions, however, occur among the photosynthetic purple nonsulphur bacteria. The 114,000-Da RuBisCO of R . rubrum is a 2L molecule (Tabita and McFadden, 1974b; Schloss et al., 1979).This enzyme has proved to be of outstanding value in the study of RuBisCO catalysis and function since it lacks S subunits. The RuBisCO of Rhodomicrobium uannielii may be a 6L6S enzyme (Taylor and Dow, 1980). The genus Rhodopseudomonas (Rhodobacter) is particularly interesting since out of three species examined, all have been shown to contain two RuBisCO enzymes i.e. a 6L enzyme in TABLE 5. Summary of occurrence of 8L8S ribulose 1,5-bisphosphate carboxylase/oxygenase(RuBisCO) in autotrophic prokaryotes Enzyme source Group Purple sulphur bacteria Purple non-sulphur bacteria Sulphur-oxidizing bacteria H ydrogen-oxidizing bacteria Nitrifying bacteria" Cyanobacteria Prochlorophytesd Cyanelles

No. genera No. species 3 3 1

5 1 10

1 2

Presence of 8L8S RuBisCOO

3

3

5 5 7

3h

4 I 2

2 I5 1

15 1

2

2

''

a Data condensed from Codd (1984). except for (Ebert, 1982) and (Berhow and McFadden, 1983). Absent from Rhodospirillum rubrum (2L enzyme: Tabita and McFadden, 1974a) and perhaps from Rhodomicrobium vannielii (6L6S enzyme: Taylor and Dow, 1980). RuBisCO heterogeneity exists in the remaining three species which contain 8L8S enzymes.

I34

G.A. CODD

TABLE 6. Occurrence of multiple forms of ribulose bisphosphate carboxylase/ oxygenase (RuBisCO) in autotrophic prokaryotes

Source

Designation of RuBisCO

Rhodopseudomonas sphaeroides

Form I Form I1

550,000 360,000

8L8S 6L

Gibson and Tabita (1 977a)

Rhodopseudomonas capsulata

Form I Form I1

550,000 360,000

8L8S 6L

Gibson and Tabita (1977b)

Rhodopseudomonas blastica

Form I Form I1

555,000 350,000

8L8S 6L

Sani (1985), Dow (1987)

Nitrobacter Form I hamburgensis X14 Form I1

520,000 480,000

8(L,L’)8S 8(L,L’)8S

Ebert (1982)

Molecular Quaternary mass structure

Reference

addition to the 8L8S form (Table 6). The 8L8S (Form I) and 6L (Form 11) enzymes of Rps. sphaeroides are regulated independently in response to metabolites and growth conditions. The L subunits of Forms I and I1 show considerable differences in peptide maps of tryptic digests and they are different immunologically (Gibson and Tabita, 1977a, 1985; Weaver and Tabita, 1983; Jouanneau and Tabita, 1986). Structural, immunological, kinetic and regulatory differences also occur between Forms I and 11 from Rps. capsulata (Gibson and Tabita, 1977b; Shively et al., 1984) and these findings have recently been supported by Dow ( I 987) using Rps. blastica. These differences strongly suggest that L subunits of RuBisCO enzymes Forms I and I1 are different gene products and Tabita and colleagues have shown this to be the case in Rps. sphaeroides (Gibson and Tabita, 1986;Tabita et al., 1987; Section 1V.D). L subunit heterogeneity of other microbial RuBisCO enzymes has occasionally been observed, e.g. from Rhizobiumjaponicum and N . agilis, although the possibility remains that this may be due to instability in vitro (Purohit et a/., 1982; Harrison et al., 1979). The presence of two RuBisCO forms in Nitrobacter hamburgensis soluble extracts, with different M , values (Table 6), and different L and S subunits and different responses to culture age and conditions suggests the presence of 2 RuBisCO’s in this organism. As reviewed in Section 11, 10-12 nm particles can be seen in negativelystained carboxysomes in whole cells and in vitro (Shively, 1974; Codd and Marsden, 1984). These are RuBisCO molecules of the 8L8S type, which have been most closely examined from Alcaligenes eutrophus (Bowien et al., 1976; Bowien and Mayer, 1978) which does not contain carboxysomes. The Alcaligenes model, constructed on the basis of M , values of the holoenzyme

CARBOXYSOMES AND RIBULOSE BISPHOSPHATE

CARBOXYLASE/OXYGENASE

135

and the 8L and 8s subunits plus electron microscopy and immunology, is a 13 x 13 x 10.5 nm structure with a fourfold symmetry. There are four layers perpendicular to a central hole; the top and bottom layers each contain four spherical S subunits and the central two layers each contain four L U-shaped subunits with the arms pointing outwards. The two central layers of L subunits are eclipsed. This model is supported by electron microscopic observations on the 8L8S holoenzyme from the cyanobacterium Synechococcus sp. (Andrews et af., 1981). The deduced arrangement of subunits in these prokaryotic RuBisCO enzymes differs from that of the tobacco RuBisCO barrel-shaped model which has a fourfold molecular axis running down a central pore concentric with the barrel. The barrel is 10.5 nm, 13.2 nm in diameter at its widest point, and the central aqueous pore decreases in diameter from 4.9 nm at the ends of the barrel to 0.6 nm at the mid-point. The precise arrangement of the L and S subunits within the overall smooth outline of the barrel has not been elucidated so far (Eisenberg et al., 1978; Chapman et al., 1986). Possible differences between the basic structures of microbial and plant RuBisCO enzymes with 8L8S quaternary structures would be of evolutionary significance and the unknown arrangements of the subunits in the 8L8S enzymes of the cyanelles and prochlorophytes are of particular interest in this context. The R. rubrum 2L molecule has the shape of a distorted ellipsoid about 5 x 7.2 x 10.5 nm (50 x 72 x 105 A) in size, with tight and extensive subunit interactions (Branden et af., 1986; Schneider et af.,1986). These workers have incorporated four 2L ellipsoid discs into a model, with about the same dimensions as the bacterial 8L8S enzyme (Bowien et af., 1976) which could provide the 8L basis of the microbial and plant molecule. Despite the low primary sequence homology between the R . rubrum and hexadecameric RuBisCO L subunits, high homologies exist in regions identified as catalytic and enzyme activation sites and the R . rubrum enzyme can be expected to have further value in understanding the origin and assembly of the predominantly extant 8L8S RuBisCO enzymes. B. ACTIVATION AND CATALYSIS

1 . Activation

Pioneering work by Pon et al. (1963) indicated that RuBisCO activity in vitro is enhanced by preincubation with COZ and Mg2+ before the addition of ribulose 1,5-bisphosphate (RuBP). The basis of these observations has become a fundamental principle in the understanding of RuBisCO function: namely that the enzyme, from all sources examined, can exist in vitro in an active and inactive form (Miziorko and Mildvan, 1974; Lorimer et af., 1976;

136

G.A. CODD

Laing and Christeller, 1976; Badger and Lorimer, 1976; Lorimer, 1981; Miziorko and Lorimer, 1983). RuBisCO activation is an ordered, two stage, pH-dependent process which requires the slow binding of an activating C02 molecule (AC02,not bicarbonate) followed by the rapid addition of Mg2+: slow fast E + AC02+E -AC02+ Mg2++E -AC02-Mg2' (active) (inactive)

The AC02molecule is distinct from that which is assimilated by the catalytic reaction and binding of one AC02per L subunit occurs at the activation site by reactivation with an E-lysyl residue to form a carbamate. This reaction is favoured by alkaline pH values and results in the production of a negatively charged carbonyl group from a positively charged or neutral amino group. The carbonyl group provides a binding site for a Mg2+ ion (Lorimer and Miziorko, 1980). The AC02-binding step is the rate-limiting reaction: E-lys-NH2+AC02+H (inactive)

+

+ E-IYs-NH-~CO~ +Mg2++E-lys-NH-C02-

.Mg2

+

(active)

The activation site was rigorously localized as lysine-201 of the L subunit of the spinach enzyme and lysine 191 of R . rubrum RuBisCO (Lorimer, 1981; Miziorko and Lorimer, 1983; Donnelly et al., 1983). This is most likely to be the activation site for microbial RuBisCO enzymes as a whole since the lysine201 domain is highly conserved and carbamate formation has been indicated during activation bf the enzyme from Chromatium and Thiobacillus species (see Codd, 1984). Activation of RuBisCO of Alcaligenes eutroghus resulted in a decrease in from 17.5s (inactive) to 14.3s (active) the sedimentation coefficient , S20,w1, suggesting that this enzyme may undergo conformational change during activation (Bowien and Gottschalk, 1982). However, no such changes were observed during the activation of spinach RuBisCO (Donnelly et al., 1984) and this problem is yet to be resolved.

2. Catalysis

In all autotrophs the RuBisCO is a bifunctional enzyme capable of catalysing the carboxylation or oxygenolytic cleavage of RuBP: Ribulose 1,5-bisphosphate+CO2

carboxylase .2 (3-phosphoglycerate)

or oxygenase Ribulose I ,S-bisphosphate +0 2 -3-phosphoglycerate

+2-phosphoglycollate

The oxygenase reaction, an apparently wasteful process, may impose considerable physiological and ecological constraints on all Calvin cycle

CARBOXYSOMES A N D RIEULOSE BISPHOSPHATE CARBOXYLASE/OXYGENASE

I37

autotrophs other than the anaerobes. Oxygen is a linear competitive inhibitor of the carboxylase reaction and vice versa (Lorimer, 1981; Codd, 1984). The two reactions that compete for the pentose phosphate acceptor, RuBP, occur at the same catalytic site and the oxygenase reaction requires activation to proceed via carbamate formation at L subunit lysine-201 as described for the activation of the carboxylase reaction. The carboxylase and oxygenase common catalytic site is on the L subunit. Affinity labelling studies and comparative amino acid-sequence analyses have indicated the existence of two lysine residues, distinct from that at the activation site, which are essential for catalysis. These are lysine- 166 and -329 in the R . rubrurn L subunit, and lysine-175 and -334 in the spinach L subunit (Hartman et al., 1984, 1986). Evidence for the proximity of the two essential catalytic lysine residues has been obtained using cross-linking agents. A purified peptide has been produced and sequenced by Hartman et al. (1986) and the cross-linked lysine-166 and lysine-329 residues found to be only 1.2 nm (12 A) apart. These residues occur in the same loop region of the R . rubrum L subunit according to the three-dimensional structure determined by X-ray crystallography at 0.29 nm (2.9 A) (Schneider et al., 1986). Increased knowledge of the three-dimensional structure of the active-site of RuBisCO is necessary to enable active-site mutagenesis to be applied to attempt to manipulate the relative specificities of the carboxylase and oxygenase reactions in favour of increased COZassimilation. Chemical and mechanistic details of the carboxylation and oxygenation of RuBP are emerging. A carboxylation scheme consisting of five steps has been formulated from the outstanding work of Lorimer and his coworkers (e.g. Miziorko and Lorimer, 1983; Schloss and Lorimer, 1982; Pierce ef af., 1986; Lorimer et af., 1986): (a) RuBP undergoes enolization involving deprotonation at the C-2 position to give a nucleophilic 2,3-enediol at C-2; (b) carboxylation at the 2,3-enediol produces the unstable C6 intermediate, 2’-carboxy-3-keto-~-arabinitol 1,5-bisphosphate (CKABP); (c) the C-3 ketone form of CKABP is hydrated to the gem-diol form; (d) carbon-carbon cleavage of CKABP occurs by deprotonation to produce one molecule of lower D-glycerate 3-phosphate (IPGA) plus one C-2 carbanion form of upper D-glycerate 3-phosphate (uPGA); (e) finally the C-2 carbanion derivative is stereospecifically protonated to produce uPGA. This proposed mechanism for carboxylation applies to the RuBisCO enzymes of spinach and probably Synechococcus. Understanding of the complete mechanisms of the carboxylation and oxygenation reactions (Miziorko and Lorimer, 1983; Lorimer et af., 1986) will combine with that of

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the three-dimensional structure of plant and microbial RuBisCO enzymes to enable the site-directed mutagenesis approach to be attempted by design in the goal of selectively modifying RuBisCO enzymes to favour the carboxylase at the expense of the oxygenase reaction. 3. Small Subunit Function

The available data indicate the mechanisms and site of carboxylation and oxygenation reactions are common throughout the plant and microbial 8L8S RuBisCO enzymes, and the role of the L subunits is well established. Uncertainty exists, however, about the precise function of the S subunits. Clearly, they are not necessary for activation, or catalysis of carboxylation or oxygenation by the 2L RuBisCO of R . rubrum or the 6L enzymes of the Rhodopseudomonads. Advances in the understanding of the functional role of S subunits have arisen from studies of the RuBisCO enzymes of cyanobacteria and the purple bacteria. Differences in the catalytic and regulatory properties of the Form I (8L8S) and Form I1 (6L) enzymes of Rps. sphaeroides may be due to the presence of S subunits in Form 1. On this basis, the S subunits can be inferred to: enable the requirement for Mg2+(or Mn2+) for activation and catalysis to be replaced by Ni2+or Co2+;increase the rate of C02-plus-Mg2+activation; increase the inhibition of this activation if RuBP is present; and confer a higher affinity for C02 (Gibson and Tabita, 1979). However, deduction of the role(s) of the S subunits by this means is limited since the L subunits of the Forms I and I1 RuBisCO enzymes of Rps. sphaeroides, and probably those of Rps. capsulata and Rps. blastica, themselves differ in peptide composition, physiological properties and genetic origin (see Section 1V.A). The removal of S subunits from the 8L8S RuBisCO enzymes of several microbes yields an 8L “catalytic core” whose carboxylase and oxygenase catalytic capacities are reduced equally to very low levels. The loss of RuBisCO activity in Synechococcus sp. proceeds in proportion to the degree of S subunit depletion (Andrews and Ballment, 1983) and as a corollary, the decreased activities of the 8L catalytic cores of Aphanothece halophytica and Chromatium vinosum are increased in proportion to the amounts of S subunits added (Asami et al., 1983; Incharoensakdi et al., 1985; Akazawa et al., 1984). S subunit addition to the Ap. halophytica L8 catalytic core does not affect the K, values for C02 or RuBP but increases the V,,, value for carboxylation by at least 2-3-fold (Asami et al., 1983). Akazawa and co-workers have extended this approach by comparing the effects of homologous and heterologous subunit reconstitution. The requirement for S subunits for catalysis by the 8L cores of Synechococcus sp. and Ap. halophytica can be completely satisfied by homologous S subunit addition and

CARBOXYSOMES AND RIBULOSE BISPHOSPHATE CARBOXYLASE/OXYCENASE

139

partly fulfilled by heterologous hybridization with the reciprocal cyanobacterial S subunits and isolated subunits from the purple bacterium Cr. vinosum (Incharoensakdi et al., 1985). In some instances structural differences between the RuBisCO enzymes of individual organisms may preclude successful hybridizations: S subunits from Synechococcus sp. and Ap. halophytica cannot restore the activity of the depleted C r . vinosum L8 core. However, these findings, in sum, are highly promising since they show that catalytically active hybrid 8L8S RuBisCO enzymes can be constructed in vitro by the combination of subunits from different microbes. This approach may be useful as a strategy to “improve” the catalytic properties of RuBisCO if these are influenced by S subunits. Incharoensakdi et al. (1985) have attempted this strategy using the subunits of the spinach and Ap. halophytica RuBisCO enzymes, which differ in that the apparent K , (COZ)value of the cyanobacterial enzyme is about ten times greater than that of the plant enzyme. Hydridization between the Ap. halophytica 8L core plus spinach S subunits occurred with an increase in carboxylase Vmax,but the apparent K, (C02) value of the hybrid enzyme was not significantly lower than that of the cyanobacterial holoenzyme. Heterologous hybridization has also been performed between the L subunit core of RuBisCO of Synechococcus sp. and S subunits from Prochloron sp and spinach and restoration of catalytic activity obtained (Andrews et al., 1984; Andrews and Lorimer, 1985). The heterologous S subunits bound about ten times less tightly than the homologous S subunits of Synechococcus sp. and the apparent K m (CO2) value of the heterologous construct was about twice as high (Andrews and Lorimer, 1985). Although the hybrid 8L (Synechococcus):8 s (spinach) enzyme differed in apparent K , (C02) value from either holoenzyme, the specificity factor, a measure of carboxylase versus oxygenase activity (see below) was not affected (Andrews and Lorimer, 1985). This suggests that the enzymatic partitioning between the carboxylation and oxygenation of RuBP may be specified by the L subunits only. Indeed the role for the S subunits is still little understood: although necessary for carboxylation and oxygenation by the 8L8S enzymes, the binding of COz and stable complex formation with the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate (CABP) to the enzyme of Synechococcus sp. still occurred when the enzyme was depleted by more than 90% of its S subunits (Andrews and Ballment, 1984). These findings have been supported by similar findings of RuBisCO Cr. vinosum (Jordan and Chollet, 1985). The S subunits are not therefore apparently essential for the activation process. At apparent variance with these findings is the ability of a second form of RuBisCO containing 8L subunits only, purified from Cr. vinosum, to exhibit similar carboxylase and oxygenase activities to the 8L8S form (TorresRuiz and McFadden, 1985). The catalytically competent Cr. vinosum 8L molecule was extracted and purified under protective conditions (in the

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presence of poly(ethy1ene glycol) in contrast to the acid or alkaline treatments which earlier studies have included for the removal of S subunits (Codd and Stewart, 1977c; Andrews and Ballment, 1983; Jordan and Chollet, 1985). If such methodological differences account for the disparate observations of the RuBisCO of Cr. vinosum, then it may be inferred that the S subunits are required to renature L subunits. Full understanding of the fundamental grounds for requirement of S subunits which are present in virtually all microbial RuBisCO enzymes and throughout the eukaryotes remains an important constraint in the cognition of CO2 assimilation via RuBisCO. C. SPECIFICITY AND REGULATION

2. Carbon DioxidelOxygen Specijicity

The inhibition of plant and microbial COZassimilation via the Calvin cycle by 02,as initially observed during photosynthesis by Chlorella sp. (Warburg, 1920), can be partly accounted for by the competitive inhibition of the carboxylase reaction of RuBisCO by 0 2 (Lorimer, 1981; Codd, 1984). Although the oxygenase reaction is a characteristic feature of all RuBisCO enzymes, considerable interest exists in the possibility of finding enzymes with decreased oxygenase activities ,or in the possibility of selectively reducing this activity compared with carboxylase activity. The possibility of obtaining a 50% increase in plant productivity by abolition of the oxygenase reaction is of obvious attraction to agriculture (Somerville et al., 1983). Although the oxygenase reaction may be an inevitable and unavoidable characteristic of RuBisCO (Lorimer, 1981; Codd, 1984), there are grounds for investigating selective manipulation. The carboxylase and oxygenase activities of RuBisCO from R . rubrum and Euglena gracilis are differentially affected by metal ions and temperature (Robison et al., 1979; Jordan and Ogren, 1984; Wildner and Henkel, 1978). Furthermore, a naturally occurring variation in the relative kinetic paramelers of the two competing reactions has emerged among the autotrophic microbes, algae and higher plants. This is expressed as variation in the C02/02 specificityvalues for the enzymes (Laing et al., 1974; Ogren, 1984). The interactions between photosynthesis and plant photorespiration can be expressed in terms of RuBisCO kinetics: Photosynthesis/photorespiration = u,/tuo = VcKoC/tV&O

are the carboxylation and oxygenation velocities; Vcand V, are the V,,, values of these activities; K , and KOare the K , values for the CO2 and O2 concentrations (C, 0).f is 0.5 which is the stoichiometry between the amount of O2 consumed by the oxygenase and the amount of CO2 released during photorespiration (Ogren, 1984). Absolute values for the C02/02 substrate - oc and v,


14 1

specificity of RuBisCO can be obtained by plotting the ratio of the carboxylase and oxygenase activities as a function of the ratio of the two substrates. From the above equation, the slope of the line equals VcKo/VoKc, which is the C 0 2 / 0 2substrate specificity. Values have been determined for a considerable number of the RuBisCO enzymes from C3- and Cq- (Calvin cycle and Hatch-Slack) pathways of higher plants and from one intermediary species. The substrate specificities are particularly clustered in the case of the C3-plants (Fig. 3). Fewer microbial enzymes have been characterized in this manner, although if plotted in taxonomic groups and in the likely sequence for the evolution of autotrophy, a trend can be discerned. The C02/02 specificities of the photosynthetic bacterial RuBisCO enzymes lacking S subunits are the lowest known. The Rps. sphaeroides Form I (8L8S) enzyme substrate specificity is significantly higher and the values for the cyanobacterial and microalgal RuBisCO enzymes generally lie between the photosynthetic bacterial Form I1 enzymes and the enzymes from higher plants (Fig. 3). It is likely that the low CO2/O2substrate specificities of the enzymes of the photosynthetic bacteria would not have disadvantaged those organisms that perform anoxygenic photosynthesis and show an anaerobic mode of autotrophic growth. The increase of 0 2 in the atmosphere, largely due to the

C4-plonts

0

0

C3/ C4-plants

micro-olgae

:: 0

Ic

0 . .

0

0 C 0 c

.-

-0a cyanobocterio

0

0"

0

0)

photosynthetic a 0

bacteria

0

0

I

1

I

I

20

40

60

8

0

,

FIG. 3. Carbon dioxide specificity (VcKo/Vo&) values for microbial and plant RuBisCO enzymes. From: 0 (Ogren, 1984); 0 (Kent and Tomany, 1984).

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development of oxygenic photosynthesis by the cyanobacteria, would have imposed increasing pressure on the RuBisCO enzymes of the ancestral aerobic autotrophic prokaryotes and selected for the development of RuBisCO enzymes with higher C02/02 specificities. These can be found among the cyanobacterial, microalgal and, to a greater extent, among the higher plant enzymes. Other strategies have been developed to lessen the inhibitory effect of increasing 0 2 tensions in the environment on the cyanobacteria and algae, i.e. C02- concentrating mechanisms (Aizawa and Miyachi, 1986), and in the C4-plants, namely the Hatch-Slack, or Co-dicarboxylic acid pathway (Hatch, 1977). These developments may have decreased further evolutionary pressure on the RuBisCO enzymes in these organisms. The C3-pathway higher plants, which lack these mechanisms for reducing photorespiration, remain as the group most susceptible to the inhibitory effect of 0 2 on photosynthesis. Their RuBisCO enzymes generally show among the highest substrate specificities and little interspecies variation exists (Ogren, 1984; Fig. 3). However, the variations in C02/02 substrate specificity throughout the phototrophs and in K, within the RubisCO enzymes of C3-plants (Lorimer, 1981; Miziorko and Lorimer, 1983) support the case for further attempts to manipulate specificity by recombinant DNA technology and site-directed mutagenesis. Data on the substrate specificities of RuBisCO enzymes from the aerobic chemolitho-autotrophic bacteria are lacking and it would be of interest from an evolutionary viewpoint to compare them with the range shown in Fig. 3. Similarly, insufficient information is available to compare the substrate affinities of RuBisCO enzymes from prokaryotes with and without carboxysomes (Codd and Marsden, 1984). Within the individual carboxysomecontaining species Anabaena variabilis and Thiobacillus neapolitanus, the K,,, (COZ) values of the enzyme derived in vitro from the cytoplasmic and carboxysomal pools are closely similar, e.g. 252_+7 and 2 6 9 k 9 PM for Anabaena variabilis (Badger, 1980). 2. Phosphorylated Eflectors

Numerous reports exist on the positive and negative effects of sugar phosphate intermediates of the Calvin cycle, 6-phosphogluconate (6PGLU) and nucleotides on plant and microbial RuBisCO enzymes in vitro. For example, several microbial RuBisCO enzymes, activated by preincubation with COZand Mg2+ in vitro are inhibited by 6PGLU (Tabita and McFadden, 1972; Gibson and Tabita, 1977a; Codd and Stewart, 1977c; Snead and Shively, 1978; Tabita and Colletti, 1979). On the other hand, stimulation of inactive RuBisCO by 6PGLU plus bicarbonate has been obtained with the isolated enzymes of Pseudomonas oxalaticus, cyanobacteria and the 2L enzyme of R . rubrum (Lawlis et al., 1978; Tabita and Colletti, 1979; Whitman


143

el al., 1979). The findings with R . rubrum, also obtained with fructose 1,6-

bisphosphate, 2-phosphoglycollate and NADPH, are consistent with results obtained with higher plant RuBisCO enzymes and with the conclusion that the metabolite effectors interact on the L subunit of the enzymes at the catalytic site (Lorimer, 1981). Evidence for the regulation of microbial RuBisCO in vivo by these effectors may be considered: Bassham and his colleagues found that transfer of photosynthesizing steady-state cultures of cyanobacteria from the light to the dark caused immediate cessation of I4CO2 fixation and the production of 6PGLU among the labelled sugar phosphates (Pelroy and Bassham, 1972; Pelroy et al., 1976). These workers, and Stanier and Cohen-Bazire (1977), reasoned that BPGLU, which is not a Calvin cycle intermediate but rather accumulates as an oxidative pentose phosphate pathway intermediate only in the dark in cyanobacteria, may inhibit RuBisCO in vivo. Tabita and Colletti (1979) extended this investigation by measuring cyanobacterial RuBisCO in situ using toluene-permeabilized cells. Brief treatment with toluene permits the entry of RuBP, other sugar phosphates and nucleotides. Assuming that the measurement of RuBP-dependent I4CO2incorporation into acid-stable material by toluene-treated cells gives an estimation of potential in vivo RuBisCO activity, than the inhibition of the activated enzyme by exogenous 6PGLU, fructose 6-phosphate, fructose 1,6-bisphosphate, NADPH and ATP can occur in cyanobacteria in vivo. Furthermore, stimulation of non-activated RuBisCO by these compounds was obtained with the permeabilized cells (Tabita and Colletti, 1979). The significance of 6PGLU and other metabolites on C02 fixation by plant and microbial RuBisCO enzymes in vivo is questioned, however, since the metabolites occupy the catalytic site, i.e. the RuBP-binding site (Lorimer, 1981). Badger and Lorimer (1981) have proposed that the binding of an effector to the enzyme in vitro at the catalytic site will result in a (RuBisCOCOrMg2+-effector)complex. If, on dilution of the complex in an activity assay mixture, the effector dissociates rapidly from the complex, then the enzyme would remain activated, its catalytic site would be vacant and the effector would appear in a positive mode. If the effector only dissociated slowly from the complex on dilution for assay, then the catalytic site would remain essentially occupied, RuBP binding for carboxylation would be delayed and the effector would thus appear to act negatively (Badger and Lorimer, I98 1). Although metabolites may thus increase the activation state in vitro and decrease the K,,, (CO2) value, their contribution as positive effectors in vivo is difficult to accept since they bind at the catalytic site and thus prevent substrate binding.

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3. Endogenous Ribulose 1,5-Bisphosphate CarhoxylaselOxygenase Inhibitors

The binding of 6PGLU at the catalytic site of RuBisCO would clearly permit this metabolite of dark (heterotrophic) metabolism and related compounds to act as an inhibitor. Diurnal changes in RuBisCO activity in a range of higher plants have been observed (Vu et al., 1983,1984; Servaites et al., 1984; Seeman et al., 1985; Servaites, 1985).These are not explained by changes in RuBisCO protein content but by the binding of a phosphorylated compound which acts as a non-competitive inhibitor with respect to RuBP. The naturally-occurring inhibitor accumulates in the dark in the chloroplast stroma and preferentially binds to the activated form of RuBisCO. A full restoration ofcatalytic activity is rapidly achieved upon illumination (within one hour). The inhibitor binds sufficiently tightly to RuBisCO to permit it to remain associated with the enzyme purified from dark-incubated plants. Tobacco RuBisCO activity is inhibited about 90% at a ratio of 10 moles inhibitor per mole of RuBisCO active sites (Servaites, 1987). The inhibitor has been identified from garden bean and potato leaves as the pentitol monophosphate, 2'-carboxyarabinitol1-phosphate (2CAlP) (Gutteridge et al., 1987; Berry et al., 1987). Whether this or related compounds with structural similarity to the C-6 carboxylation intermediate, function to regulate microbial RuBisCO enzymes in vivo is unknown. The 2CA 1P would have the advantage over 6PGLU that it would not be needed in such high excess over the RuBisCO active sites (Servaites, 1987).

4. Ribulose 1,5-Bisphosphate CarboxylaselOxygenase Activase Further exciting advances have recently been made in the understanding of the regulation of higher plant RuBisCO in vivo.Among these is the discovery of a soluble protein needed for RuBisCO activation in vivo. The discovery of this enzyme, termed RuBisCO activase, has further helped to resolve the differences between the requirements for maximal RuBisCO activity in vitro and the optimal provision of these requirements in the whole plant (for primary references, see Ogren et al., 1986). RuBisCO activation in whole leaves at high light intensities is a widespread phenomenon and the isolation of an Arabidopsis thaliana mutant by Somerville et al. (1982), which was unable to activate RuBisCO in the light, provided the opportunity for comparative studies versus the wild type which shows light activation of the enzyme. The light activation lesion could not be accounted for by differences in the RuBisCO enzymes. Two polypeptides, M , 40-50 kDa, were present in the chloroplast stromal proteins of the wild type, but were absent from the Ar. thaliana mutant (Salvucci et al., 1985). The requirement for soluble chloroplast protein was demonstrated in a reconstituted assay system, which


145

additionally contained purified RuBisCO, thylakoids and RuBP and exhibited a light-stimulated increase in RuBisCO activity. The possibility that RuBisCO light activation may occur via the ferredoxin/ thioredoxin system, which accounts for the light activation of other Calvin cycle enzymes in plants and cyanobacteria, was discounted: the Ar. thaliana mutant was not deficient in the ability to show light activation of chloroplast fructose 1,6-bisphosphatase (FBPase) which is light-activated by the thioredoxin/ferredoxin-mediated transfer of electrons from the photosynthetic electron transport chain. Furthermore, the light activation of RuBisCO proceeds in the presence of methyl viologen which inhibits FBPase photoactivation (Salvucci et al., 1986b). The precise role of light in the activation process is not yet known. The COZrequirement for chloroplast RuBisCO activation with RuBisCO activase is about 4 PM which is significantly below the atmospheric CO2 concentration of about 10 PM (Portis et al., 1987). The two polypeptides of the RuBisCO activase of Ar. thaliana have recently been estimated to be of about 41 and 44 kDa and antibodies raised to the enzyme cross-react with leaf extracts from the homologous wild-type Arabidopsis sp., and at least 14 other diverse plants (Salvucci et al., 1987). RuBisCO activase thus appears to be widely distributed in higher plants and the immunological cross-reactivities obtained indicate that the polypeptides have been conserved. The constituent polypeptides also appear to be present in the green alga Chlamydomonas reinhardtii (Salvucci et a/., 1987). if RuBisCO activase is indeed commonly involved in the control of RuBisCO in the eukaryotic phototrophs, then a search for the origin(s) of this protein among the photosynthetic prokaryotes will be of obvious interest. D. GENETICS

Research on the genetics of microbial RuBisCO enzymes is advancing rapidly. Progress on the location, cloning, heterologous expression and regulation of the L and S subunit genes from the various physiological groups of autotrophs has been of value to microbiology. These advances are of no less interest to plant geneticists and agronomists since the prokaryotic RuBisCO genetic systems have several advantages for study and manipulation over those in eukaryotes. In almost all of the eukaryotes examined to date, the genes for the L and S subunits of RuBisCO have been located in the chloroplast and nuclear chromosomes respectively (Coen er al., 1977; Kawashima and Wildman, 1972; Akazawa et al., 1984). Detailed consideration of the genetics of RuBisCO in eukaryotes is beyond the scope of this review and recent advances can be found elsewhere (Ellis and Gray, 1986). In prokaryotes that contain 8L8S RuBisCO enzymes, the genes for both types of subunit are located on the chromosome, as are the genes of the 2L and 6L prokaryotic RuBisCOs.

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G.A. CODD

Among the photosynthetic bacteria, the RuBisCO genes have been located and cloned from R . rubrum (Nargang et al., 1984; Somerville and Somerville, 1984), Rps. sphaeroides (Fornari and Kaplan, 1983; Quivey and Tabita, 1984; Gibson and Tabita, 1986; Tabita et al., 1987) and Cr. uinosum (Viale et al., 1985; Kobayashi et al., 1987). The L and S subunit genes for the Rps. sphaeroides Form I RuBisCO are located on a single 4 kb chromosomal fragment (Gibson and Tabita, 1986). Among the cyanobacteria, the L and S subunit genes have been shown to be closely linked and have been cloned from Anacystis nidulans (Synechococcus) 630 1 (Shinozaki and Sugiura, 1983,1985; Shinozaki et al., 1983; Christeller et al., 1985; Tabita and Small, 1985; Gatenby et al., 1985; Bradley et al., 1986; Gutteridge et al., 1986), Anabaena 7120 (Nierzwicki-Bauer et al., 1984; Gurevitz et al., 1985), Spirulina platensis (Tiboni et al., 1984) and Ch.fritschii (Vakeria et al., 1986). Location of the L and S subunit genes in the cyanelles of C .paradoxa was of particular interest since these organelles are incapable of living outside of their eukaryotic host and the possibility existed that the S subunit gene may have been among the genes thought to have been transferred from the cyanelle to the host nucleus. However, as in the free-living autotrophic prokaryotes, the L and S subunit genes of C . paradoxa are both on the cyanelle chromosome (Heinhorst and Shively, 1983; Bohnert et al., 1983; Mucke et al., 1984). They are closely linked: the L subunit gene is situated 105 nucleotides upstream from the S subunit gene and the spacer sequence does not indicate the presence of a promoter sequence. Indeed, cotranscription of the L and S subunit genes from the 2500 nucleotide sequence has been demonstrated by Northern blot analysis (Starnes et al., 1985). These findings are of evolutionary interest and raise the question of when and where the segregation of the L and S subunit genes occurred in the development of eukaryotic phototrophs. Until recently, RuBisCO gene research in the eukaryotes had centred almost entirely on chlorophytic (chlorophyll a plus b ) organisms, in which, as discussed earlier, the L and S subunit genes have been invariably found to be segregated to the chloroplast and nucleus. Reith and Cattolico (1986) have chosen to examine the DNA of the non-green chromophyte alga Olisthodiscus luteus and found that the L and S subunit genes are linked on the chloroplast DNA. This may also be so in the red algae Cyanidium caldarium and Porphyridium cruentum, whose plastids are though to have originated from cyanobacterial endosymbionts (Steinmuller et al., 1983). The eukaryotic RuBisCO enzymes with linked genes may be of practical interest, in addition to evolutionary interest, since it should be possible to obtain heterologous expression and assembly of the cloned non-chlorophyte 8L8S enzyme, in contrast to the enzyme from chlorophytic higher plants (c.f. Bradley et al., 1986). Complete nucleotide sequence data for the gene coding for the R . rubrum L subunit and for the L subunit genes for the cyanobacteria Anacystis nidulans


147

6301 and Anabaena 7120 enzymes are available (Nargang et al., 1984; Shinozaki et al., 1983; Curtis and Haselkorn, 1983). The amino acid sequence ofthe R. rubrum L subunit, deduced from analysis of the enzyme purified from the organism itself (Hartman et al., 1984) and from Escherichia coli into which the L subunit gene was cloned (Somerville and Somerville, 1984; Nargang ef al., 1984) shows only 31-33% homology with the spinach L subunit. This is in contrast to the > 80% homologies between the spinach L subunit amino-acid sequence and those of other higher plants, green algae and cyanobacteria (see Hartman et al., 1984). Regions of nucleotide sequence homology are likely to be functionally important and have already been useful in confirming the activation and catalytic sites on the L subunits (Section 1V.B). Other regions sequences of high homology may be likely targets for attempts to modify RuBisCO by site-directed mutagenesis. Gutteridge et al. (1986) have investigated the effects of substituting a conserved aspartic acid residue at position 198 on the R . rubrum enzyme by glutamic acid. In this case, the mutant showed a 30% decrease in carboxylase and oxygenase activities although the apparent K,,,values for C02 and 0 2 and the activation rates with C02 and Mg2+ were unaffected. A reduction in turnover, perhaps due to a spatial need to accommodate the extra methylene group of glutamate, appears to be the sole effect in this initial attempt at site-directed mutagenesis, but systematic substitution of amino acids in the highly conserved regions of the subunits is likely to provide further mechanistic information and, it is hoped, to provide prospects for the selective manipulation of substrate specificities in favour of the carboxylase. Unlike plants that contain multiple copies of the L and S subunit genes, only one copy of the L subunit gene is present in R . rubrum (Somerville and Somerville, 1984) and single copies of each of the L and S subunit genes are present in cyanobacteria and the C . paradoxa cyanelles (Shinozaki and Sugiura, 1983; Nierzwicki-Bauer et al., 1984; Vakeria et al., 1986; Heinhorst and Shively, 1983). This indicates that the cytoplasmic and carboxysomal pools of RuBisCO are products of the same genes within each organism. Chlorogloeopsis fritschii contains only one set of L and S subunit genes (Vakeria et al., 1986) in agreement with findings with other cyanobacteria (Shinozaki et al., 1983; Nierzwicki-Bauer et al., 1984). However, Ch.fritschii, according to six plasmid isolation methods, does not appear to contain extrachromosomal DNA (Vakeria et al., 1984) and we wished to know whether extrachromosomal RuBisCO genes may be additionally present in a plasmid-containing strain. Microcystis 7820 contains 3 4 cryptic plasmids (Vakeria et al., 1985)but when these were probed under a variety of stringency conditions with a range of heterologous RuBisCO genes, no hybridization was detected. This was in contrast to positive hydridization between Microcystis chromosomal DNA and the L subunit probe from Anacystis

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nidulans (Vakeria et al., 1986). These findings indicate that if the carboxysomes of Microcystis sp. did contain extrachromosomal DNA, and there is no evidence for this in cyanobacteria (see Section III.A), then it is unlikely that such carboxysomal DNA would contain RuBisCO genes. Evidence for the presence of RuBisCO genes on extrachromosomal DNA exists, however, in the purple non-sulphur bacteria and hydrogen bacteria. The single copy of the Form I1 (6L) RuBisCO gene of Rps. sphaeroides is present on the chromosome (Fornari and Kaplan, 1983; Quivey and Tabita, 1984) but the single copy L and S genes of the Form I enzyme has been localized on an endogenous plasmid (Gibson and Tabita, 1986). The separate identity and location of the L subunit genes of the Form I and I1 enzymes confirm the observed differences in kinetic and physiological properties of the enzymes (Tabita et al., 1987) and open the possibility for unambiguous investigation of the physiological regulation of these enzymes at the genetic level. Duplicate L and S subunit genes exist in the nutritionally versatile hydrogen bacterium Alcaligenes eutrophus. The role of megaplasmids in lithoautotrophic metabolism is well established since hydrogenase genes are plasmid-encoded in several strains (Schlegel, 1984; Friedrich, 1987). For example, in Al. eutrophus HI 6 the self-transmissible 450 kb megaplasmid encodes for the two hydrogenases of this autotroph and the ability to grow chemolitho-autotrophically has been abolished by plasmid-curing of this strain (Friedrich et al., 1981). Since plasmid-cured strains are still able to grow organo-autotrophically on formate, it is clear that the essential genes of the Calvin cycle and any essential COZfixation regulatory genes must be on the chromosome (Bowien et al., 1984). However, the involvement of megaplasmids was indicated in the partial derepression of RuBisCO and phosphoribulokinase proteins upon transfer from chemoheterotrophic to autotrophic conditions. These findings were confirmed by the first demonstration of multiple phosphoribulokinase genes on the strain H 16 chromosome and megaplasmid (Klintworth et al., 1985). More recently, Bowien and colleagues have used the R . rubrum RuBisCO gene to probe for the L subunit gene and a synthetic DNA sequence constructed on the basis of the amino-terminal amino acid sequence of the homologous S subunit, to localize the S subunit gene in Al. eutrophus H 16. Both genes are present on the chromosome and the megaplasmid. In each case, the RuBisCO genes constitute an operon as evidenced by Northern blot hybridizations which indicate a 2.2 kb transcript size. In the same direction of transcription as the L and S subunit genes, but about 3.5 kb downstream and under separate transcriptional control in the chromosome and megaplasmid is the phosphoribulokinase gene (Bowien et al., 1987). Additional evidence exists for the presence of functional RuBisCO genes on the chromosome and megaplasmid of Al. eutrophus strain ATCC 17707 (Andersen and Wilke-Douglas, 1984; Andersen et al., 1986). The high


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degree of homology between the RuBisCO gene regions on the megaplasmid and chromosome and the expression of active enzyme in E. coli from the cloned plasmid genes (Bowien et al., 1987) suggest that the latter are functional and the role of the duplicate sets of Calvin cycle genes in these hydrogen bacteria requires investigation. Some preliminary generalizations concerning expression of RuBisCO genes in the prokaryotes may be ventured: although there is variation in the spacer region between the Land S subunits, cotranscription of the genes occurs in the cyanobacteria Anacystis nidulans 6301 (Shinozaki and Sugiura, 1985) and Anabaena 7120 (Nierzwicki-Bauer et al., 1984), and Al. eutrophus strains (Andersen et al., 1986; Bowien et al., 1987), as with the Cyanophora cyanelle genes (Starnes et al., 1985). Cotranscription with single promoter control is clearly one factor in the success widely achieved in the heterologous expression of structurally accurate and catalytically competent 8L8S RuBisCO from several prokaryotic sources. This is in contrast to the problems encountered in attempts to obtain expression of catalytically active RuBisCO of higher plants (see Bradley et al., 1986). Expression of microbial RuBisCO enzymes in E. coli to give at least wild-type activities has been obtained with the genes from R. rubrum (e.g. Somerville and Somerville, 1984) Rps. acidophila Forms I and I1 enzymes (Tabita et al., 1987), Cr. uinosum (Viale et al., 1985), An. nidulans (Tabita and Small, 1985; Gatenby et al., 1985), Spirulinaplatensis (Tiboni et al., 1984) and Ch.fritschii (Vakeria et al., 1986). Alcaligenes eutrophus RuBisCO genes have been expressed to produce active enzyme in Pseudomonas aeruginosa (Andersen et al., 1986) in addition to E. coli (Bowien et al., 1987). A further factor in the faithful production of the prokaryotic 8L8S enzymes in these bacterial expression systems is that the microbial RuBisCO enzymes do not require additional assembly factors to be cloned from the autotrophs. The cloned fragments containing the An. nidulans 6301 and Al. eutrophus ATCC 17707 RuBisCO genes did not code for other polypeptides (Shinozaki and Sugiura, 1983,1985; Tabita and Small, 1985; Andersen et al., 1986). The production of large quantities of microbial RuBisCO by recombinant DNA techniques will greatly facilitate study of enzyme structure and manipulation by site-directed mutagenesis and post-translational modification. This approach will be additionally useful for the production of greater quantities of potentially interesting RuBisCO enzymes from slower-growing autotrophs.

V. Carboxysome Function

Knowledge of the occurrence, structure and composition of carboxysomes has increased considerably over recent years but understanding of the

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function(s) of these organelles remains inadequate. The hypotheses proposed have all sought, justifiably, to examine carboxysome function in terms of the properties and role of RuBisCO, the most abundant carboxysomal protein component (Shively, 1974; Stewart and Codd, 1975; Beudeker et al., 1981; Lanaras and Codd, 1982; Codd and Marsden, 1984). Evidence for and against these hypotheses exist. A. ARE CARBOXYSOMES SITES OF CARBON DIOXIDE FIXATION

In Vivo?

The possibility that carboxysomes are active sites of C02 fixation in vivo is attractive since all carboxysomes contain RuBisCO. If so, then carboxysomal RuBisCO must be catalytically competent. In all cases examined, the RuBisCO extracted from carboxysomal fractions has been found to be closely similar to, or identical with, in quaternary structure, immunological, activation and catalytic properties, the enzyme from the soluble (cytoplasmic) fraction of the cell (Badger, 1980; Lanaras and Codd, 198la,b; Beudeker et al., 1981; Leadbeater, 1981; Cannon and Shively, 1983). These studies with the enzymes from several cyanobacteria and T. neapolitanus show that carboxysoma1 RuBisCO enzymes are capable of COZfixation at rates similar, on an enzyme protein basis, to their counterparts in the cytoplasm. The ability of carboxysomal RuBisCO to be activated in vivo has been suggested from the data of Cannon (1982). The transition state analogue 2-carboxyarabinitol 1,s-bisphosphate (CABP) binds irreversibly at the catalytic site of the RuBisCO L subunit and locks the activating C02 and Mg2+ onto the activation site, thereby stabilizing the otherwise labile carbamate (see Miziorko and Lorimer, 1983). When ['4C]CABP was supplied to chloroform-permeabilized T. neapolitanus cells, labelling of the cytoplasmic and carboxysomal RuBisCO was obtained (Cannon, 1982; Cannon and Shively, 1982). The possible action of chloroform in influencing the permeability of the carboxysome membrane in vivo may not have influenced these findings since the membrane lacks lipids. If the carboxysome membrane was unaffected by the chloroform permeabilization procedure then these findings indicate that carboxysomal RuBisCO is capable of being activated in vivo, and that the membrane is permeable to C02, Mg2+ and the pentose CABP. Further investigations on the possibility that carboxysomes are sites of C02 fixation have centred on measurements of carboxysome abundance and the distribution of RuBisCO between carboxysomes and cytoplasm in whole-cell studies of cultures maintained under different physiological conditions. Continuous culture of T. neapolitanus under C02-limitation resulted in a 3.5fold increase in total extractable RuBisCO activity compared with cultures grown on excess COZ (i.e. thiosulphate limitation) (Beudeker et al., 1980). Changes in total and carboxysomal RuBisCO activities in these cultures


15 I

correlated with variations in carboxysome volume density and number per cell. However, maximal COz-fixation capacity by whole cells, obtained during growth on thiosulphate plus 5% C02, varied inversely with carboxysome numbers and carboxysomal RuBisCO activities. Rates of RuBisCO activity per unit of RuBisCO protein in carboxysomal extracts from steady-state chemostat cultures of T . neupolitunus were about the same as those of the cytoplasmic enzyme in extracts of thiosulphate- or nitrogen (ammonia)limited cells. In extracts of C02-limited cells the carboxysomal RuBisCO was 4.5 times more active per unit RuBisCO protein than the cytoplasmic enzyme (Beudekeret al., 1981).These findings confirm the potential of the carboxysoma1 RuBisCO to contribute to whole-cell C02 fixation. This possibility also exists in Thiobacillus intermedius, which produces carboxysomes when RuBisCO activity enables or supports cell growth (i.e. under chemolithoautotrophic and mixotrophic growth conditions respectively) and does not produce the organelles, or RuBisCO, during chemoheterotrophic growth (Purohit et al., 1976). Carboxysomes and RuBisCO are produced in nutritionally versatile cyanobacteria irrespective of the sources of carbon and energy (Codd and Marsden, 1984) and the possibility that the organelles are active in C 0 2 fixation is open. Carboxysome numbers and levels of carboxysomal compared to cytoplasmic RuBisCO are minimal during the exponential growth phase of photo-autotrophic batch cultures of Ch. fritschii, in contrast to stationary phase cultures when almost all of the cell RuBisCO complement is located in

TABLE 7. Carboxysome abundance and photosynthetic characteristicsof Synechococcus leopoliensis under different nutrient limitations in continuous chemostat culture. From Turpin et a). (1984) Growth rate

Nutrient limitation

No. carboxysomes per cell section Reactor DIC K; DIC' pmax p (day-') % p max" ( k SE) (PM) ~

DICb Phosphate Nitrate

0.25 1.71 0.27 1.56 0.27 1.56

13 85 13 80 13 80

3.40f0.30 0.57 f0.09 0.41fO.10 0.34f0.10 0.40f0.10 0.40 f0.10

~~~

4.4 1700 6000 4090 5500 2800

109 308 485 399 ndd nd

Approximate estimate of per cent of maximum specific growth rate 01). Dissolved inorganic carbon concentration in reactor vessel. Half-saturation constant for photosynthesis for dissolved inorganic carbon (DIC). Not determined.

1.9 1600 1250 1500 nd nd

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the carboxysomes (Lanaras and Codd, 1982). These findings suggest that the carboxysomes have a principal role other than in C02 fixation in these cultures but the latter possibility is not excluded. Turpin et al. (1984) have studied carboxysome abundance versus photosynthetic characteristics of the cyanobacterium Synechococcus leopoliensis in chemostat culture (Table 7). This valuable study shows that carboxysome number per cell section under carbon limitation at 85% of maximum specific growth rate is marginally, but significantly, less than in phosphate- and nitrogen-limited cells. Under severe inorganic carbon limitation, at only 15% of pmax,carboxysome sections per cell section increased to exceed carboxysome abundance in phosphate- and nitrogen-limited cells by between six and ten times (Table 7). This increase was accompanied by a decrease of three orders of magnitude in the half-saturation constant of photosynthesis for dissolved inorganic carbon. These findings suggest strongly that under air-saturated levels of 02, inorganic carbon supply has a major effect on carboxysome numbers in S. leopoliensis and that the carboxysomes are functional in this organism under severe inorganic carbon limitation. The ability of the carboxysomes to protect RuBisCO from inhibition by 0 2 under these conditions is considered in Section V.B. If carboxysomal RuBisCO is active in C02 fixation in uiuo, then it is essential that the carboxysome membrane is permeable to RuBP. Phosphoribulokinase, which produces RuBP by phosphorylating ribulose 5-phosphate, is not present in the carboxysomes of Ch. fritschii, Synechococcus sp., T. neapolitanus or the Cyanophora or Glaucocystis cyanelles (Lanaras and Codd, 1981a; Cannon and Shively, 1983; Hawthornthwaite et al., 1985; Holthuizen et al., 1986b; Mangeney et al., 1987). Carboxysomes d o not contain Calvin cycle enzymes other than RuBisCO (Cannon and Shively, 1983; Holthuizen et a f . , 1986b), with the further implication that if the carboxysomal enzyme fixes C02 in vivo, then the carboxysome membrane must be permeable to the products of RuBisCO, 3-phosphoglyceric acid and 2-phosphoglycolate if carboxysomal RuBP oxygenation occurs, in addition to RuBP. If carboxysomes are a site of C02 fixation in viuo, then it is further possible that they act as a CO2-concentrating mechanism to favour the carboxylase reaction and help to account for the lack of photorespiration in cyanobacteria. This prospect has been discussed in detail previously and the existence of a inorganic carbon transport and concentration system at the cyanobacterial cell membrane is well documented. Carbonic anhydrase is also involved in inorganic carbon concentration (Aizawa and Miyachi, 1986), although the enzyme is lacking from the carboxysomes of T. neapolitanus and Ch.fritschii (Cannon and Shively, 1983; Lanaras et al., 1985; Holthuizen et al., 1986b).


153

B. DO CARBOXYSOMES PROTECT RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE/ OXYGENASE

Ribulose 1,5-bisphosphate carboxylase/oxygenases are inhibited by several chemical and physical agents. Although the physiological significance of the inhibitory effects of metabolic intermediates is questionable, the inhibitory effect of 0 2 on CO2 fixation is an undoubted and indeed universal feature of RuBisCO enzymes (see Sections IV. B and IV. C). If carboxysomes served principally to protect RuBisCO from inhibition by 0 2 , then this would imply that the carboxysomal enzyme was active in COZfixation in vivo, and that carboxysomes were a characteristic feature of aerobic autotrophs. Speculation about carboxysomal functions in the different groups of autotrophs should not overlook the possibility that the organelles may play different roles in different physiological groups. However, in the context of protection from 02,it is worthy of note that carboxysomes do not occur in the anaerobic purple sulphur- and purple non-sulphur bacteria. Within the aerobic autotrophs, however, the only group in which they consistently occur are the oxygenic cyanobacteria. Although the organelles are also present in the three prochlorophytes examined, they d o not occur in all cyanelles (see Sections 11. B and 11. C). A protective role against 0 2 would also be consistent with the presence of carboxysomes in the aerobic chemolitho-autotrophs. Although carboxysomes commonly occur among the sulphur-oxidizers and nitrifiers, their distribution is variable (see Tables 1 and 2) and of the many aerobic hydrogen-oxidizing bacteria examined, carboxysomes have apparently only been found in one (thermophilic) strain, Pseudomonas thermophila K2 (Kostrikina et al., 1981; Romanova et al., 1982). If carboxysomes do function to protect RuBisCO from inhibition by 0 2 , then variations in carboxysomes abundance and in the subcellular distribution of RuBisCO between the organelles and the cytoplasm may be observed under changing 0 2 tensions. The anthropocentric rationale supposes that the carboxysome-producing prokaryote would respond by forming more of the organelles to contain a greater proportion of the cell’s RuBisCO complement under adverse high O~/lowCOZconditions, further assuming that it possessed the ability to do so. The distribution of RuBisCO protein between the cytoplasm and carboxysomes in continuous cultures of T . neapolitanus was not influenced by varying 0 2 tensions, providing no support for the 0 2 protection hypothesis (Beudeker et al., 198I). The potential contribution of an O2 protection mechanism for RuBisCO of S. leopoliensis has been estimated by Turpin et al. (1984) at various ratios of internal to external dissolved inorganic carbon (DIC) ratios. The protection of RuBisCO from 0 2 may increase net photosynthesis by 100% and 18% at cyanobacterial DIC ratios of 100 and 1000 respectively. These observations leave unanswered the question

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of whether the carboxysomes confer 0 2 protection to RuBisCO. However, with a major increase in carboxysome numbers in S. leopoliensis in response to carbon limitation (Table 7), an active role of carboxysomes in cyanobacterial photosynthesis appears to be likely. Other agents that inhibit higher plant RuBisCO enzymes include sulphite, sulphate and hydrogen fluoride gas at concentrations present in industriallypolluted environments (Khan and Malhotra, 1982; Parry and Whittingham, 1983; Buckenham et al., 1982). The effect of these compounds on microbial RuBisCO enzymes has not received attention. However, the success of Calvin cycle prokaryotes in sulphur-containing environments (van Gemerden and de Wit, 1986)merits study of the effects of toxic sulphur compounds on microbial RuBisCO enzymes. The RuBisCO enzymes from freshwater and halophilic cyanobacteria are inhibited by NaCl in vitro (Cook, 1980; Incharoensakdi et al., 1986). Although intracellular osmotica, particularly glycine betaine, relieve the inhibition by NaCl, the relative susceptibilities of the carboxysomal RuBisCO pool compared to the cytoplasmic enzyme to in uitro and in uiuo treatments has not been determined. The possibility that carboxysomes may confer protection on RuBisCO in uiuo against adverse environmental parameters is under current study in this laboratory.

c. ARE CARBOXYSOMES STORAGE BODIES? The possibility that carboxysomes may serve as deposits for the storage of RuBisCO was proposed by Shively (1974) and Stewart and Codd (1975). Several observations support, but do not prove, this possibility. Carboxysome numbers per cell increase in old stationary phase cultures of Nitrobacter winogradskyi and decrease during reactivation of the cells by the supply of fresh nutrients (Bock and Heinrich, 1971). In nutritionally versatile cyanobacteria, RuBisCO and carboxysomes continue to be produced during chemoheterotrophic growth, although the enzyme is not required catalytically under these conditions. Levels of RuBisCO protein in photo-autotrophic and chemoheterotrophic Nostoc 6720 cultures are similar. In nitrate-grown chemoheterotrophic mid-exponential phase cells, most of the enzyme is present in the carboxysomes; in mid log-phase photo-autotrophic cells most of the enzyme is cytoplasmic (Leadbeater, 1981; Codd and Marsden, 1984). Most of the RuBisCO in this versatile autotroph is thus sequestered in the carboxysomes when it is not active, or required, for growth and a storage function for the organelles under these conditions can be inferred. However, in other versatile autotrophs, neither carboxysomes nor RuBisCO are produced during chemoheterotrophic growth, e.g. Thiobacillus intermedius (Purohit et al., 1976). Chemostat cultures have also been used to address the storage hypothesis.


155

Carboxysomal RuBisCO protein levels were minimal during the growth of T.neapolitanus under nitrogen limitation, consistent with this hypothesis (Beudeker et al., 1981). In addition, a rapid doubling of carboxysomal RuBisCO protein levels was obtained following the addition of ammonia to nitrogen-limited cultures. However, the transfer of nitrogen-limited steadystate cultures to nitrogen starvation conditions did not result in a breakdown ofcarboxysomal or cytoplasmic RuBisCO, suggesting that the T. neapolitanus carboxysomes do not act as general protein reserves (Beudeker et al., 1981). The lack of effect of growth of S. leopoliensis under nitrogen (nitrate) limitation (Table 7) does not support the storage hypothesis in this nutritionally specialist cyanobacterium (Turpin et a/., 1984). The likelihood that carboxysomes serve as general protein reserves in cyanobacteria should be viewed against the presence of two other major nitrogen reserves in these organisms: the phycobilisomes, which serve as protein reserves in addition to accommodating the accessory pigments for photosynthesis, and the cyanophycin granules (Stanier and Cohen-Bazire, 1977). The cyanophycin polypeptide of the latter bodies consists of a copolymer of equimolar quantities of arginine and aspartic acid. Cyanophycin is not synthesized on ribosomes and presents simpler requirements as a general nitrogen reserve than the transcription and translation of RuBisCO genes and the assembly of their products into the 8L8S enzyme for its sequestration into membrane-bound carboxysomes. This argument does not militate against a role for carboxysomes in the specific storage of the RuBisCO enzyme under specific conditions.

VI. Further Aspects of Carboxysomes A. ECOLOGICAL MARKERS FOR AUTOTROPHY

Since carboxysomes only occur in prokaryotes that contain RuBisCO and are capable of assimilating CO2 via the Calvin cycle, then their easily recognized appearance may be useful as a structural marker for autotrophy in microbial ecology (Codd and Marsden, 1984). Unfortunately for this purpose, carboxysomes are not a feature of all Calvin cycle autotrophs although they are confined to autotrophs. The remarkable discovery of the dense growths of microbes and animals around deep-sea hydrothermal vents has been among the most exciting microbial ecophysiological advances of recent years (see Jannasch and Mottl, 1985). Primary production in these totally dark environments is performed by chemolitho-autotrophic bacteria, including free-living species of the sulphur-oxidizers Thiobacillus and Thiomicrospira and a range of symbiotic bacteria, living in vestimentiferan and pogarophoran

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CODD

worms and in molluscs (Cavanaugh, 1983; 1985). Several of these bacterial symbionts contain Calvin cycle enzymes but apparently no carboxysomes. However, other bacterial symbionts present in the gills of clams from sulphide vent environments have not been characterized enzymically, but they contain polyhedral electron-dense bodies which may be carboxysomes (Giere, 1985). Until recently, the marine non-heterocystous filamentous Nz-fixing cyanobacterium Oscillatoria (Trichodesmium) erythraea could not be grown in laboratory culture. This has been a constraint on research into the mechanism of N2-fixation in this major bloom-forming species and in particular on how the organism may enable the physiologically-incompatible processes of N2 fixation and oxygenic photosynthesis to occur in the same filament. A clear morphological differentiation at the subcellular level may, however, be seen along the filaments. Carboxysomes, indicating the presence of RuBisCO and photosynthetic capacity, are abundant in the terminal cells a t each end of the filaments and numbers of the organelles decrease toward the central carboxysome-free cells in the central region (Bryceson and Fay, 1981). The central cells are thought, from the absence of carboxysomes, not to perform photosynthesis. If these cells lack Photosystem I1 in addition to carboxysomes and RuBisCO, then the central cells may enable the 02-sensitive nitrogenase, if present, to function in an environment of reduced 0 2 tension, as in heterocystous cyanobacteria. B. MAN-MADE RIBULOSE 1 ,j-BISPHOSPHATE CARBOXYLASE/OXYGENASE INCLUSION BODIES?

The production of large amounts of microbial RuBisCO in foreign recombinant heterotrophic bacteria is now feasible (see Section 1V.D). For example, photosynthetic bacterial and cyanobacterial RuBisCO protein, when expressed in the presence of the gratuitous inducer isopropyl P-D-thiogalactopyranoside by E. coli, may account for up to 3-15% ofcell protein (Somerville and Somerville, 1984; Viale et al., 1985; Gatenby et al., 1985; Tabita and Small, 1985). It is likely that considerably higher levels of RuBisCO will be made by recombinant DNA techniques. At least 20 eukaryotic polypeptides, expressed in E. coli as fusion proteins or directly, accumulate as inclusion bodies (see Harris, 1983; Marston, 1986). Examples include insulin A and B chains and calf prochymosin. When produced in the original eukaryotic cells, these proteins are in soluble form. The man-made inclusion bodies are amorphous aggregates and are not surrounded by, or are in close contact with, membranes (Schoemaker et al., 1985; Schoener et al., 1985). Examples of normal E. coli proteins expressed to high levels using recombinant DNA methods that accumulate as inclusion bodies are also known (Cheng, 1983). The bacterial 8L8S RuBisCO is one of


1 57

themost complex proteins to have been cloned and expressed in foreign hosts. Results so far indicate that most of the cloned RuBisCO is soluble; it will be of interest and value for comparison with authentic carboxysomes to see if inclusion bodies consisting of RuBisCO are produced in recombinant hosts. REFERENCES

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van Eykelenburg, C. (1979). Antonie van Leeuwenhoek 45,369. van Gemerden, H. and de Wit, R. (1986). In “Microbes in Extreme Environments” (R.A. Herbert and G.A. Codd, eds), pp. I 1 1-127. Academic Press, London. van Cool, A.P. (1972). Archiv. fur Mikrobiologie 82, 120. van Cool, A.P., Lambert, R. and Laudelout, H. (1969). Archiv. fur Mikrobiologie 69,281. Viale, A.M., Kobayashi, H., Takabe, T. and Akazawa, T. (1985). FEBS Letters 192,283. Vu, C.V., Allen, L.H. and Bowes, G. (1983). Plant Physiology 73, 729. Vu, C.V., Allen, L.H. and Bowes, G. (1984). PIanf Physiology 76, 843. Walther-Mauruschat, A., Aragno, M., Mayer, F. and Schlegel, H.G. (1977). Archives of Microbiology 114, 101. Warburg, 0. (1920). Biochemische Zeitschriji 100, 188. Watson, S.W. (1971). Archiv fur Mikrobiologie 75, 179. Watson, S.W., Graham, L.B., Remsen, C.C. and Valois, F.W. (1971). Archiv fur Mikrobiologie 76, 183. Watson, S.W. and Waterbury, J.B. (1971). Archiv fur Mikrobiologie 77, 203. Watson, S.W., Bock, E., Valois, F.W., Waterbury, J.B. and Schlosser, U. (1986). Archives of Microbiology 144, 1. Weaver, K.E.and Tabita, F.R. (1983). Journal ofBacteriology 156,507. Westphal, K. (1977). Ph.D. Thesis, University of Hamburg, W. Germany. Westphal, K. and Bock, E. (1974). Archives ofMicrobiology 101, 121. Westphal, K.,Bock, E., Cannon, G.C. and Shively,J.M. (1979). Journalof Bacteriology 140,285. Whatley, J.M. (1977). New Phytologist 79, 309. Whitman, W.B., Martin, M.N. and Tabita, F.R. (1979). Journal of Biological Chemistry 254, 10184. Wildner, G.F. and Henkel, J. (1978). PIanta 146, 223. Wolk, C.P. (1973). Bacteriological Reviews 37, 32. Wolk, C.P. (1982). In “The Biology of Cyanobacteria” (N.G. Carr and B.A. Whitton, eds), pp. 359-386. Blackwells Scientific, Oxford. Wullenweber, M., Koops, H-P. and Harms, H. (1977). Archives of Microbiology 112, 69. Yagawa, Y., Shiraiwa, Y. and Miyachi, S. (1984). Plant and Cell Physiology 25, 775.

Archaebacteria: The Comparative Enzymology of Their Central Metabolic Pathways MICHAEL J . DANSON Department of Biochemistry. University of Bath. Clauerton Down. Bath BA2 7 A Y . England

I. Introduction

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A . Archaebacteria . . . . . . . . . B . Central metabolism of eubacteria and eukaryotes . . I1. Archaebacterial pathways of central metabolism. . . . A . Hexose catabolism . . . . . . . . B. Gluconeogenesis . . . . . . . . . C . Glycerol synthesis . . . . . . . . D. The citric acid cycle . . . . . . . . E . Patterns of the archaebacterial central metabolic pathways . 111. Archaebacterial enzyme diversity . . . . . . A . Dehydrogenaseswithdualcofactor specificity . . . B . 2-0xoacid:ferredoxin oxidoreductases . . . . C . Dihydrolipoamide dehydrogenase . . . . . D . Citratesynthaseandsuccinate thiokinase . . . . E . Comparative enzymology . . . . . . . IV . Structure of archaebacterial enzymes . . . . . . A . Halophilic enzymes . . . . . . . . B . Thermophilic enzymes. . . . . . . . V . Concluding remarks . . . . . . . . . VI . Acknowledgements . . . . . . . . . . . . . . . . . . . References .

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MICHAEL J . DANSON

I. Introduction A. ARCHAEBACTERIA

I . The Archaebacteria The majority of microbiologists, and many biochemists and molecular biologists, will be familiar with the concept of the archaebacteria. To such readers, recognition of the archaebacteria as a phylogenetically distinct group of organisms, that are as distinct from the eubacteria as they are from the eukaryotes, needs neither explanation nor justification. However, to those unfamiliar with the idea that there are at least three major groups (Kingdoms) of living organisms, a brief discussion of the archaebacteria might be helpful and may set in context the value of the comparative enzymology with which this review is primarily concerned. This discussion is not intended to provide a comprehensive review of the archaebacteria; for such information the reader is directed towards a number of excellent articles and books recently published (Fewson, 1986; Woese and Olsen, 1986; Kandler and Zillig, 1986; Woese and Wolfe, 1985; Kandler, 1982; Woese, 1981, 1987). The long-held view that all organisms can be divided into two Kingdoms, the prokaryotes and the eukaryotes, has been seriously questioned in recent years. Carle Woese and George Fox have demonstrated that 16s and 18s ribosomal RNA (rRNA) molecules are excellent molecular chronometers for measuring phylogenetic relationships among organisms: these rRNA species are constant in function, universal in distribution, easily isolated, are relatively large molecules and have both moderately and highly conserved parts of their sequence across large phylogenetic distances (Woese, 1985, 1987). Therefore, it is argued that rRNA sequence comparisons can accurately measure both small and large phylogenetic distances. The method of comparison is that of oligonucleotide cataloguing (Sanger et al., 1965; Uchida et al., 1974; Woese et al., 1976) whereby 16S/18S rRNA is digested with ribonuclease T1 and the resulting oligonucleotides are separated by twodimensional paper electrophoresis and sequenced. The catalogue so generated is characteristic of an organism, and can be quantitatively compared with that of any other organism by means of a binary association coefficient which takes into account oligonucleotide sequences of six bases or longer (Fox et al., 1977). The coefficient measures the fraction of bases in any two catalogues found in oligonucleotides common to the two catalogues, and is related to the actual number of nucleotide differences between the rRNA sequences, but in an unknown and non-linear way. Phylogenetic analysis by oligonucleotide cataloguing (Woese and Fox, 1977; Fox et al., 1980; Woese, 1981) has led to the proposal that there are at

CENTRAL METABOLIC PATHWAYS OF ARCHAEBACTERIA

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least three Kingdoms of living organisms: (a) the Eubacteria (the true bacteria); (b) the Eukaryotes; and (c) the Archaebacteria. Although eubacteria and archaebacteria may represent primary evolutionary lineages, the eukaryotic cell is now thought to be a genetic chimera; that is, the cytoplasm and organelles may be of different descents, evidence having been presented for the eubacterial origin of chloroplasts and mitochondria (Gray, 1982; Gray and Doolittle, 1982; Spencer et al., 1984). Archaebacteria encompass three basic phenotypes, namely methanogenic, halophilic and sulphur-dependent (reviewed by Woese and Olsen, 1986; Fewson, 1986; Woese, 1987). The methanogens are obligate anaerobes that reduce carbon dioxide to methane. Methanogenesis is obligatory and no secondary energy sources have been identified (Balch et al., 1979; Whitman, 1985). Halophiles need high concentrations of sodium chloride with some members growing in saturated (5.2 M) salt solutions and others having the additional requirement of high pH optima (pH 9-10) (Kushner, 1985). Sulphur-dependent archaebacteria (Stetter and Zillig, 1985) are found in thermophilic environments (55-lOO0C)and can either reduce or oxidize sulphur to produce energy. Aerobic and anaerobic species are known, so too are autotrophs and heterotrophs. Many sulphur-dependent archaebacteria are thermoacidophiles, growing at acidities as low as pH 1.0 and, for this reason, the thermoacidophilic archaebacterium Thermoplasma acidophilum is often grouped phenotypically with these organisms even though it is an obligate heterotroph with no dependence on sulphur. These extreme conditions in which archaebacteria are found impose phenotypes apparently well suited to the type of environment thought to exist during early life on earth, 3-4- lo9 years ago. From such observations the name archaebacteria was tentatively suggested (Woese and Fox, 1977), although the question of their “primitive nature” is still a matter of debate.

2. Archaebacterial Phylogeny The concept of the archaebacteria as a phylogenetically distinct group, originally formulated from the rRNA oligonucleotide catalogues, has been strengthened by analysis of the complete sequences. The 16S/18S rRNA sequences from various methanogenic, halophilic, thermoacidophilic and sulphur-dependent archaebacteria and from a number of eubacteria and eukaryotes have been determined (referenced in Woese and Olsen, 1986) and the data support a clear distinction between the archaebacterial kingdom and those of the eukaryotes and the eubacteria (Fig. I). Similarly, the complete sequence data are consistent with the archaebacteria comprising two main divisions (Fig. 2); namely, ( I ) the thermophilic sulphur-dependent archaebac-

ARC HAEBACTERIA sulldobw solfatwicus

m e r w o t w s tmax 0.1

Hdobacterium volcmii Mathmspiriiium hunqalai &thanobacterium formicicum Mathamcoccus vannieilii ?kmococcus cder

EUBACTERIA

EUKARYOTES

FIG. 1. Unrooted phylogenetic tree constructed from 16s (or 18s) rRNA sequences. Complete rRNA sequences were aligned and estimates of sequence divergence (mutations fixed per sequence position) were calculated and used by Woese and Olsen (1986) to infer the phylogenetic tree. The scale bar corresponds to a tree branch length of 0.1 mutations fixed per sequence position. Reproduced with the permission of Woese and Olsen (1986).

0.01

Methanococcus vannielii

-

Methanobacterium formicicum Thermoplasme acidophilum Methanospirillum hungatei Halococcus morrhuae Halobacterium cutirubrum lhrmococcus celer

Sulfoobus solfataricus Thermproteus tenax

FIG. 2. Unrooted phylogenetic tree for the archaebacteria based on 16s rRNA sequences. The tree was constructed by Woese and Olsen (1986) from alignment of complete rRNA sequences as described in the legend to Fig. 1. The scale bar corresponds to a tree branch length of 0.01 mutations fixed per sequence position. Reproduced with the permission of Woese and Olsen (1986).


169

teria, but not including Thermococcus celer and Tp. acidophilum; and (2) the methanogens and extreme halophiles, plus Tc. celer and Tp. acidophilum. This conclusion has recently been supported by measurements of total rRNA hybridization homologies (Klenk et al., 1986). The rRNAs of 17 species of archaebacteria were hybridized to corresponding and noncorresponding nitrocellulose-bound DNAs. Hybridization homologies were calculated from hybridization yields, corrected for different genome lengths and numbers of rRNA operons per genome, and from them a phylogenetic tree was constructed (Fig. 3). This tree resembles that obtained by comparison of the total sequences of 16s rRNAs (Fig. 2), except in a few details of the precise branching orders. Most significantly, it suggests that Thermococcales represent a third branch of the archaebacterial kingdom beside the branch of the methanogens with halophiles and that of the Sulfolobales with Thermoproteales. The thermophilic phenotype appears in all three of the major branches of archaebacteria, and Woese and Olsen (1986) have therefore raised the possibility of this being the ancestral archaebacterial phenotype. Criteria other than the rRNA sequences have also been used to deduce phylogenetic relationships. These include 5 s rRNA secondary structural features (Fox et al., 1982; Wolters and Erdmann, 1986), the structure of DNAdependent RNA polymerases (Zillig et al., 1985) and ribosome morphology THERMOCOCCALES Cdduplex woesei METHANOMICROBIALES THERMOPLASMALES

Methonolobus findorius

Thermaplosmo ocidophilum

SULFOLOBALES

METHANOCOCCALES Methonococcus vannielii

hfethanofhermus fervidus METHANOBACTERIALES Mefhombacleriumfhermooufotrophicum

fenax THERMOPROTEALES Dewlfurococcus mucows

FIG. 3. Phylogenetic tree of the archaebacteria based on hybridization homologies. The tree was constructed by Klenk et al. (1 986) from hybridization homologies between rRNAs and nitrocellulose-bound DNAs (see text for details). The organisms on both sides of the tree have the same mean distance to the vortex at the centre of the tree. Reproduced with the permission of Klenk et al. (1986).

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MICHAEL J . DANSON

(Lake et al., 1984, 1985). In all three cases, a specific relationship between the sulphur-dependent archaebacteria and the eukaryotes is inferred, with the eubacteria arising from the halophilic-methanogenic branch. Moreover, Lake et al. (1984, 1985) and Lake (1986) propose that sulphur-dependent archaebacteria form a kingdom called “eocytes”, that halophilic archaebacteria and the eubacteria form another called the “photocytes” and that the term “archaebacteria” is reserved to cover only the methanogens. These other proposed branching orders have been critically reviewed by Woese and Olsen (1986) and by Woese (1987). Whatever one’s position, however, it is clear that archaebacterial phylogeny is still a matter of contention, and it is a priority to make further comparable measurements of sequence homologies.

3. Biochemistry of the Archaebacteria Many biochemical features of the archaebacteria serve to reinforce their distinct phylogenetic position (reviewed in Fewson, 1986) and a number of these are outlined in this section. In contrast to the straight-chain fatty acyl ester-linked glycerolipids (with sn-1,2-glycerol) of eubacterial and eukaryotic membranes, archaebacterial lipids are isopranyl ether-linked glycerolipids with an sn-2,3-glycerol configuration (Langworthy, 1985). The thermoacidophilic and some methanogenic glycerolipids contain tetra-ethers, allowing formation of lipid “monolayer” membranes, with the additional feature in thermoacidophiles that the biphytanyl chains may contain from one to four cyclopentyl rings (Langworthy, 1985). In addition, the archaebacterial cell-envelope is distinctive in its lack of the characteristic eubacterial murein (Kandler and Konig, 1985).In its place are found residues of N-acetylated glucosamine or galactosamine, Llysine, L-glutamate, L-alanine or L-threonine and N-acetyl-L-talosaminuronic acid. Finally, the base modifications of transfer RNA (tRNA) are characteristic of archaebacterial species (Gupta, 1985). Interestingly, although the above features are unique to archaebacteria, certain others may be typically eubacterial or eukaryotic. Thus, on the eubacterial side, archaebacteria are distinctly prokaryotic in organization and morphology. Also, the rRNA components of the archaebacterial ribosome have a number of eubacterial features (Matheson, 1985); the 70s ribosome contains one molecule of 5S, 16s and 23s rRNA (Viscentin et al., 1972) and the organization of these rRNA genes is similar to that in eubacteria (Hofman et al., 1979). Furthermore, a putative Shine-Dalgarno sequence has been found in a halophilic 16s rRNA (Kagramanova et al., 1982) and its secondary structure more closely resembles that of eubacterial 16s rRNA than that of the eukaryotic 18s molecule (Kagramanova et al., 1982; Gupta et al., 1983).


171

Further morphological aspects of the ribosome are eubacterial in nature although, at the level of the individual ribosomal components, the structural features are significantly closer to eukaryotic molecules than to those of the eubacteria (Matheson, 1985). However, as indicated previously, Lake et al. (1984, 1985) consider the morphology of the archaebacterial ribosome to be distinctive and have used it to deduce phylogenetic relationships. Archaebacteria also have many typically eukaryotic characteristics. (a) Introns have been found in the genes for tRNAL""and tRNASerof Suvolobus solfataricus (Kaine et al., 1983), for tRNA'F of Halobacterium volcanii (Daniels et al., 1985) and for 23s rRNA of Desulfurococcus mobilis (Kjems and Garrett, 1985). (b) Basic histone-like proteins associated with DNA have been found in thermoacidophilic (Searcy and Stein, 1980; Green et al., 1983) and methanogenic (Thomm et al., 1982) archaebacteria. Partial sequence analysis of the HTa protein from Tp. acidophilum shows it is more homologous to eukaryotic nuclear DNA-binding proteins than it is to counterparts from eubacteria (Searcy and DeLange, 1980). (c) Some mRNA molecules have long polyadenylated sequences at the 3'-termini, similar to those in eukaryotes (Ohba and Oshima, 1983; Oshima el al., 1984) and initiation of protein synthesis from mRNA seems to occur in archaebacteria with methionyl-tRNA as in eukaryotes rather than with N-formylmethionyltRNA as in eubacteria (Gupta, 1985). (d) All archaebacteria possess an elongation factor (EF-2) which is ADP-ribosylated by diphtheria toxin, as are eukaryotic EF-2s (Klink, 1985). (e) The transcriptional apparatus of archaebacteria shows a uniform and "eukaryotic-type" antibiotic sensitivity although that of the translational system is heterogeneous (Bock and Kandler, 1985).

4 . Enzymology of the Archaebacteria It is clear that, for the purposes of establishing the phylogenetic status of the archaebacteria, much emphasis has been placed on the molecular biology of these organisms and on the chemical nature of their cell walls and membranes. However, it is also becoming clear that the pathways of metabolism in archaebacteria and their constituent enzymes are equally fruitful areas for investigation. It is the purpose of this review to concentrate on the enzymology of archaebacteria, not in isolation but in comparison with that of eubacteria and eukaryotes. Therefore, the enzymes of the central metabolic pathways have been chosen for review because, as explained in the following section, not only are these pathways thought to be some of the first cellularly established metabolic routes but also because they are the most studied and well-characterized systems in non-archaebacterial species.

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MICHAEL J . DANSON

B. CENTRAL METABOLISM OF EUBACTERIA A N D EUKARYOTES

It has been argued that the universal occurrence of sugars and the use of their derivatives for biosynthesis might suggest that development of a sugar-based biochemistry was an early evolutionary innovation (Gest and Schopf, 1983). Indeed, D-glucose is now the most abundant compound in the biosphere and its catabolism may have been one of the first energy-conversion processes to have been successfully exploited by living cells. Such reasoning has led to an investigation of the pathways of hexose metabolism in archaebacteria but, before this work is described, it would seem pertinent to discuss briefly those catabolic routes in eubacteria and eukaryotes. I . Pathways of Glucose to Pyruvate

The pathways of sugar catabolism in eubacteria have been comprehensively reviewed by Payton and Haddock (1985) and by Cooper (1986) and are summarized, with those of eukaryotic organisms, in Fig. 4. The following summary indicates the salient features of these pathways from which a comparison with those in archaebacteria can best be appreciated. The Embden-Meyerhof glycolytic pathway is characteristic of eukaryotic cells and a large number of anaerobic and facultatively anaerobic eubacteria. In glycolysis, glucose is phosphorylated to fructose 1,6-bisphosphate which in turn undergoes aldol cleavage to two interconvertible three-carbon sugars, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The two trioses enter a common route of catabolism to pyruvate (Fig. 4). A key enzyme of the glycolytic sequence, 6-phosphofructokinase, is absent from many strictly aerobic eubacteria, implying that this pathway is inoperative in these organisms. Instead, glucose can be catabolized via the Entner-Doudoroff pathway (Entner and Doudoroff, 1952); glucose 6-phosphate is oxidized to 6-phosphogluconate before being dehydrated to 2-keto-3-deoxy-6-phosphogluconateand undergoing subsequent aldol cleavage to yield glyceraldehyde 3-phosphate and pyruvate (Fig. 4). Glyceraldehyde 3-phosphate is metabolized to give a second molecule of pyruvate via the same sequence of reactions as in the glycolytic sequence. Consequently, only one molecule of ATP is generated for each molecule of glucose fermented, as opposed to two ATP molecules via glycolysis. There is a third route for the catabolism of glucose (Fig. 4), namely the hexose-monophosphate pathway (pentose-phosphate pathway; see Racker, 1948, 1957). It is probable that the reactions of this pathway do not operate generally as a cycle for glucose oxidation, but serve to provide the cell with NADPH and pentose and tetrose sugars. Further oxidation of these sugars to glyceraldehyde 3-phosphate is possible, the metabolic fate of which is the same as in the Embden-Meyerhof and Entner-Duodoroff pathways.

Glucose ATP

Ht NADPH

a

NADP'

NADP*

6-Phosphogluconate + - 4

-

'

NADPHt Ht

lH2

6- Phosphogluconate

- Glucose 6-phosphate

I

t (Ribulose 5-phosphate Ribose 5-phosphate

Fructose 6-phosphote

6-phosphogluconate

Erythrose 4-phosphate) I

Fructose 1,6-bisphosphate

I I

A

I I

I I

Dihydroxyocetonephosphate

I I

I

. '--// *

\ \

0.90 0.88 Enterococcusjaecalis, NCIB 39 0.41 a-Glcl.2‘ > 0.90 Lactococcus lactis, various strains 0.10-0.50 a-Gal 0.174.60 Listeria monozytogenes, ATCC 153 I3 0.17 0.31 a-Gal Listeria, various species 0.21-0.36 a-Gal 0.114.34 Bacillus lichenijormis, DSM 13 0.51 a-Gal, a-GlcNAc 0.02,0.18 Bacillus lichenformis, AHU 1371 0.69 a-GlcNAc 0.15 Bacillus subtilis, W 23 0.40 a-Glc, a-GlcNAc 0.20,0.21 Bacillus subtilis, various strains 0.35-0.55 a,fi-Glc, a-GlcNAd‘ 0.04,0.214.43

19k 1 19k I n.d.‘ n.d. 19-26 23 16-33 27 28 24 25-33

2 1, 5

5 13 13 I , 6.9 14, 15

14 5

2 16 2

“Glycosyl residues, as so far studied, belong to the D-series and are in the pyranose form. *Absent from Staphylococcusaureus H gol-’ aR(W. Fischer, unpublished work), a mutant lacking the usual Nacetylglucosaminylsubstituents on the wall teichoic acid (Heckels et al., 1975). CStructures: a-Glc(1-; a-Glc( I-Z)a-Glc(I-; a-Glc(l-2)a-Glc(l-2)a-Glc( I-; a-Glc(l-2)a-Glc(l-2)a-Glc(l-2)aGlc( 1-. dNot present in all strains, while the anomeric form is strain-specific. References: I , Fischer et al. (1981);2, lwasaki et ul. (1986);3 , Kelemen and Baddiley (1961);4. Nakano and Fischer (1978);5, W. Fischer (unpublished work); 6,Fischer et a / . (1980b); 7, Fischer et a / . (1980a);8, McCarty (1964);9.Schleiferet al. (1985);10, Rajbhandari and Baddiley (1963);I I . Fischer and Rose1(1980);12,Cabacungdn and Pieringer (1985);13. Wicken and Baddiley. (1963);14,Ruhland and Fiedler (1987);15, Uchikawa el u/. (1986); 16. Fischer and Koch (1981).

242

W. FISCHER

each substituent. In a few bacteria the glycosyl substituents are group antigens (for a review see Wicken and Knox, 1975a). The presence of non-substituted glycerol residues in almost all lipoteichoic acids (Tables 3 and 4) raises the question as to whether these lipoteichoic acids are mixtures of substituted and non-substituted chains or whether all chains are partially substituted. Anion-exchange chromatography on columns of DEAE-Sephacel separates molecular species of lipoteichoic acid in the order of increasing negative charge, i.e. in the order of decreasing alanine-ester content (Fischer and Rosel, 1980). Using this procedure, it could be shown that unsubstituted species are absent from the lipoteichoic acid of Staph. aureus and that all molecular species are alanylated within a narrow range (Fischer and Rosel, 1980). Lectin-affinity chromatography specific for a-D-galactopyranosyl residues likewise demonstrated the absence of nongalactosylated species from the lipoteichoic acid of Lactococcus lactis (Wicken and Knox, 1975b). A means to study the distribution of alanyl residues along the chain was provided by the discovery of a phosphodiesterase (Schneider and Kennedy, 1978) which, along with a phosphomonoesterase, degrades Dalanyl lipoteichoic acid stepwise from the terminus distal to the lipid moiety (Childs and Neuhaus, 1980; Fischer et al., 1980b). Using this procedure with the lipoteichoic acid from Staph. aureus, it was shown that each third of the chain had the same D-alanine-phosphate ratio. This revealed a homogeneous distribution of alanyl ester substituents along the chain suggesting either a regular or a random arrangement (Fischer et al., 1980b). "P NMR Spectroscopy which, as recently discovered, provides a considerable amount of information on chain substitution (Batley et al., 1987) indicates a random rather than a regular distribution of alanine ester substituents in the lipoteichoic acid of Staph. aureus (W. Fischer and W. Bauer, unpublished TABLE 4. Chain composition of substituted poly(g1ycerophosphate) lipoteichoic acids".From Fischer and Koch (1981) Species: Bacillus subtilis

Grob AlaGro GlcGro GlcNAcGro

Lactococcus lactis

Enterococcus faecalis

W-23

Marburg

NCDO 712

Kiel27738

0.23 0.42 0.20 0.21

0.28 0.38 0.17 0.18

0.31 Grob 0.21 AlaGro GalGro 0.39 AlaGalGro 0.09

Grob AlaGro GlczGro AlaGlc2Gro

0.23 0.29 0.29 0.19

'Measurement after hydrolysis with 40% (w/w) aqueous hydrogen fluoride (Fischer et al. 1980b, 1981); values quoted are molar ratios to phosphorus. 'Non-substituted glycerol.

PHYSIOLOGY OF LIPOTElCHOIC ACIDS IN BACTERIA

243

observations) and Lactobacillus fermentum (Batley et al., 1987). The glycosyl substituents in the lipoteichoic acid of Ent. faecalis are also randomly distributed. Two or three different substituents on one lipoteichoic acid (Table 3) may be linked, as shown in Table 4, either individually to separate glycerol residues or, as with alanine ester residues, be in part attached to glycosyl substituents. The high degree of glycosylation of the lipoteichoic acids from Ent. faecalis strains NCIB 8191 and NCIB 39 suggests that most of the alanine ester is bonded to glycosyl substituents (Table 3). That hexosyl and alanyl substituents occur on the same rather than on separate chains was demonstrated with the lipoteichoic acid from L. lactis. It did not separate on DEAESephacel into species containing either D-alanyl ester or galactosyl residues and, on rechromatography, after removal of the alanyl substituents, unsubstituted species did not appear (W. Fischer, unpublished observations). B. POLY(DIGALACTOSYL,GALACTOSYLGLYCEROPHOSPHATE)LIPOTEICHOIC ACID

An unusual structure was detected in the lipoteichoic acid from Lactococcus garvieae (Fig. 4). Digalactosyl residues are intercalated between the glycerophosphate residues, while the glycerophosphate residues are consistently substituted at C-2 with monogalactosyl residues (Koch and Fischer, 1978; Schleifer et al., 1985). As in other species of Lactococcus the lipid anchor is Glc(cr 1-2)Glc(a1-3)acyl2Gro and the 6-0-acylated derivative thereof (see Table 1). The chain structure is reminiscent of poly(hexosy1 glycerophosphate) wall teichoic acids (Archibald, 1974) but differs from them by containing sn-glycero-1-phosphate residues (Koch and Fischer, 1978). C. GLYCEROPHOSPHATE-CONTAINING LIPOGLYCAN

The lipoteichoic acid from Bijidobacterium bifidum was originally reported to consist of two 1,2-linked poly(g1ycerophosphate) species, one carrying a glucan, the other a galactofuranan on the glycerol terminus of the poly(g1ycerophosphate) chain distal to the lipid moiety (Op den Kamp et al., 1984). Recent studies suggested the structure depicted in Fig. 5 (Fischer, 1987; Fischer et al., 1987). In this structure, the amphiphilic chain is a linear lipoglucogalactofuranan and the glycerophosphate residues are not intercalated into the chain but are attached as monomeric side branches to the galactofuranosyl residues. As in poly(g1ycerophosphate) lipoteichoic acids, the glycerophosphate residues have the sn- 1-configuration and are in part substituted by alanine esters which, however, have the L-configuration in contrast to the D-alanyl residues of poly(g1ycerophosphate) lipoteichoic acids. The lipid anchor was tentatively identified as /?-~-Galp(1-3)acylzGro and may

245

PHYSIOLOGY OF LIPOTEICHOIC ACIDS IN BACTERIA

CH20-X

I

HbCIOH

I

9

H 2 C-0-7-0-

- X = Ala ( 2 0 - S o 0 / ~ )- ,H

[

‘i

0-H- -0-C-H uH2] ~ - ~ - - @ 3 H 2

3 -0-C-R H2 -0-5-R

0 FIG. 5. Structure of a lipoglycan from Bijidobacterium bijidurn DSM 20239 bearing sn-glycero-1-phosphate side chains partially substituted with L-alanyl ester. n, 7-1 0; m,8-1 5.

therefore be derived from one of the membrane galactoglycolipids from this organism (Veerkamp, 1972). In contrast to poly(g1ycerophosphate) lipoteichoic acids, in this novel amphiphile the glycerophosphate residues are kept a certain distance from the membrane by the glucan moiety. Actinomycetes and Streptococcus sanguis biotype B lack serologically detectable poly(g1ycerophosphate) lipoteichoic acids (Hamada et al., 1976, 1980). In place of them they seem to contain amphiphilic heteropolysaccharides whose structures have not yet been unravelled (Wicken et al., 1978; Yamamoto et al., 1985). Small amounts of possibly monomeric glycerophosphate residues may be present. D. SUCCINYLATED LIPOMANNAN

Micrococcus luteus, MicrococcusJlavus and Micrococcus sodonensis also lack serologically detectable lipoteichoic acids, but instead possess a succinylated lipomannan (Powell et al., 1974, 1975; Owen and Salton, 1975a; Pless el al., 1975; Fig. 6). The hydrophilic moiety contains 50-70 (1-2)-, (1-3)-, and (1 -6)linked a-D-mannopyranosyl residues and two 2,4-substituted branch points. Between 10 and 25% of the mannosyl residues are substituted with ester-linked succinate. A neutral lipomannan lacking succinyl residues has been isolated from Micrococcus agilis (Lim and Salton, 1985). As shown with the lipomannan from M . luteus, the lipid moiety is diacylglycerol (Powell et al., 1975), possibly as part of the sequence Man(crl-3)Man(crI-3)acyl~Gro which is the major membrane glycolipid in M . luteus (Lennarz and Talamo, 1966). A functional analogy of lipomannan to lipoteichoic acids has been proposed in view of their amphiphilic nature, membrane localization, net negative charge and similar Mg2+-binding properties (Powell et al., 1975; Wicken and Knox, 1980). The glycerophosphate-containing lipoglycan of BiJidobacterium bifidum (Section I1.C) may be considered a structural link between lipomannans and lipoteichoic acids.

246

( k)

Ma nnosylfmannosyl A I

T

W . FISCHER

(

H

CH20H

w d 0 - C H 2

/\ 0 0- -10

I HC-0-CO- R

I

H,C-0-CO-R

FIG. 6. Structure of the succinylated lipomannan from Micrococcus luteus. E. “LIPOTEICHOIC ACID” FROM

Streptococcus pneumoniae

Streptococcus pneumoniae strains possess in place of a poly(g1ycerophosphate)-type lipoteichoic acid a unique macroamphiphile, the pneumococcal Forssman antigen, also called pneumococcal lipoteichoic acid. Although known since 1943 as “lipocarbohydrate” (Goebel et al., 1943) and during the last decade intensively studied for biological activities (Section V.B), the structure of this polymer has not been unravelled. It contains fatty-acyl residues (5.7-6.5%), ribitol phosphate, galactosamine, glucose and choline phosphate (Goebel et al., 1943; Fujiwara, 1967; Brundish and Baddiley, 1968; Briles and Tomasz, 1973). With the exception of the fatty-acyl residues the composition is similar to that of the pneumococcal wall teichoic acid whose structure has been established as shown in Fig. 7. In spite of the similarity in

iH A C

FIG. 7. Structure of the repeating unit of the wall teichoic acid from Sfreptococcus pneumoniae (Jennings et al., 1980).The pneumococcal Forssman antigen (pneumococcal “lipoteichoic acid”) has a similar composition but, in addition, contains fatty-acyl esters on a non-identified lipid moiety.

PHYSIOLOGY OF LlPOTElCHOlC ACIDS IN BACTERIA

241

composition, lipoteichoic acid is not a precursor of this wall teichoic acid as was deduced from pulse-chase experiments using radiolabelled choline (Briles and Tomasz, 1975). F. QUANTITATIVE ASPECTS

Earlier estimates of the cellular content of lipoteichoic acids may have yielded too low values, because difficulties in quantitative extraction of lipoteichoic acids from bacteria have not been recognized until recently (Fischer and Koch, 1981; Huff, 1982; Fischer et al., 1983). Values between 1 and 3% of the cell dry weight have been reported, and about 2-3% may be calculated from the estimate of 120 pmol lipoteichoic acid phosphorus or glycerol per gram dry weight (Wicken et al., 1973; Fischer et al., 1983). For certain bacteria the content may vary depending on growth conditions as will be discussed in Section III.F.5. In Staph. aureus, grown in batch culture, lipoteichoic acid, teichoic acid and nucleic acids contribute 13,29 and 58%, respectively, to total polymer phosphorus (Fischer et al., 1983). Taking the different chain lengths of teichoic acid and lipoteichoic acid into account, their molar ratio is approximately 1.5. Estimates of lipoteichoic acids and membrane lipids suggest that, in the membrane of L. lactis and Staph. aureus, lipoteichoic acids represent every tenth and twentieth lipid amphiphile molecule, respectively (Fischer, 1981; Koch et al., 1984). Having long chains, lipoteichoic acids nevertheless represent approximately 50% of the membrane amphiphile glycerol in Staph. aureus (Koch et al., 1984), 40% in spores of B. megaterium (Johnstone et al., 1982) and 30% in Ent. faecalis (Carson et al., 1979; Shungu et al., 1980).

111. Metabolism A. BIOSYNTHESISOF POLY(GLYCEROPHOSPHATE) LIPOTEICHOICACIDS

In 1974, in vivo pulse-chase experiments by Glaser and Lindsay and by Emdur and Chiu provided suggestive evidence that, in Staph. aureus and Strep. sanguis, the glycerophosphate required for lipoteichoic acid biosynthesis is derived from phosphatidylglycerol. Support for the donor function of phosphatidylglycerol was obtained by in vitro experiments using membrane preparations of Strep. sanguis (Emdur and Chiu, 1975; Mancuso et al., 1979), Ent. faecalis (Ganfield and Pieringer, 1980) and toluene-treated cells of Lactobacillus casei (Childs and Neuhaus, 1980). CDP-Glycerol containing snglycero-3-phosphate could not substitute for phosphatidylglycerol, as was shown with particulate enzyme preparations from Enr. faecalis (Pieringer et

248

W. FISCHER

al., 1981). In experiments with B. subtilis, inhibition of biosynthesis of phosphatidylglycerol with 3,4-dihydroxy-butyl- 1-phosphonate, an analogue of sn-glycero-3-phosphate, resulted, as expected, in a block in lipoteichoic acid synthesis (Deutsch et al., 1980). In pulse-chase experiments in which growing cells of Staph.aureus (Koch et al., 1984)and B. subtilis (Koga et al., 1984)were labelled with [3H]glycerol, phosphatidylglycerol was isolated and, after hydrolysis with moist acetic acid or phospholipase C, the labelling pattern of the diacylglycerol and glycerophosphate moieties was studied separately (Fig. 8). A rapid and virtually complete turnover of the non-acylated glycerol moiety into lipoteichoic acid was observed and, when the phosphate group was labelled, it showed an analogous behaviour (Koga et al., 1984). A promising novel system for studying details of lipoteichoic acid synthesis is afforded by membrane vesicles released from L. casei by treatment with penicillin (Ntamere and Neuhaus, 1987). These vesicles catalyse incorporation of label from ['4C]glycero-3-phosphate and UDP-['4C]glucose into poly(g1ycerophosphate) and glycolipid. The mode of chain elongation was elucidated by differential radioisotope

FIG. 8. Turnover of the non-acylated glycerophosphate moiety of phosphatidylglycerol into lipoteichoic acid in growing Staphy[ococcus aureus in a pulse-chase experiment using [2-3H]glycerol; the arrow indicates the onset of the chase. Symbols: 0, lipoteichoic acid; A , glycerophosphate moiety and 0 , diacylglycerol moiety of phosphatidylglycerol released by treatment with phospholipase C and separated by phase partition (Koch et al., 1984).


249

labelling techniques. In Ent.faecalis, lipoteichoic acid was pre-labelled in vivo with ['4C]glycerol and subsequently elongated in vitro using particulate (Cabacunenzyme preparations and [I (3)-gly~erol-~H]phosphatidylglycerol gan and Pieringer, 198I). In Lb. casei, [2-gly~erol-~H]lipoteichoic acid, formed in growing bacteria, was elongated in toluene-treated cells using ['4C]glycero3-phosphate as the second label (Taron et al., 1983). The lipoteichoic acids were isolated and degraded stepwise from the glycerol terminus distal to the lipid moiety by the joint action of phosphodiesterase and phosphomonoesterase from Aspergillus niger. In both polymers the ratio of the two isotopes in the glycerol released indicated chain growth distal to the lipid anchor (Fig. 9). This mode of chain growth is identical with that for wall teichoic acids (Burger and Glaser, 1964; Kennedy and Shaw, 1968) and differs from chain-extension of peptidoglycan which is elongated by transfer of the growing chain to the next repeating unit nearest to the lipid carrier (Ward and Perkins, 1973). Growth distal to the lipid carrier, as in lipoteichoic acid synthesis, allows the whole chain to be synthesized linked to the definitive lipid anchor. It requires,

U

100

200

300

LOO

500

T I M E (h) FIG. 9. Data showing mode of lipoteichoic acid chain extension in Lactobacillus casei. Lipoteichoic acid in bacteria was labelled in succession with [2-3H]glycerol and ['4C]glycerol,isolated, and from the glycerol terminus degraded stepwise by the joint action of phosphodiesterase and phosphomonoesterase from Aspergillus niger. At time intervals the glycerol released was analysed for 14C-and 3H-radioactivity. From Taron et al. (1983).

250

W. FISCHER

on the other hand, that during the synthesis either the chain or the growing terminus of the chain remains in contact with the membrane. Synthesis of glycolipids has been studied with crude enzyme preparations from various Gram-positive bacteria. Generally, 1,2-di-O-acyl-sn-glycerolis the initial lipid substrate to which the monosaccharide residues are sequentially transferred from nucleotide-linked hexosyl substrates (Kaufman et al., 1965; Lennarz and Talamo, 1966; Pieringer, 1968; Veerkamp, 1974). The diacylglycerol residue had been suggested to originate from phosphatidic acid through the action of a phosphatidic acid phosphatase (Krag et al., 1974) before it became evident that amounts of diacylglycerol, far greater than needed for glycolipid synthesis, result from lipoteichoic acid synthesis (see Section 1II.C). In lactococci (Fischer et al., 1978a; Schleifer et a!., 1985), enterococci (Fischer et al., 1973b, 1978b) and lactobacilli (Fischer et al., 1978b; Nakano and Fischer, 1977,1978) phosphatidyl and fatty-acyl diglycosyldiacylglycerolipids that occur as lipid moieties in lipoteichoic acids are frequently also present in the free state among membrane lipids. These glycolipid derivatives may therefore be used as acceptor substrates in lipoteichoic acid synthesis rather than be formed by acylation or phosphatidylation of the completed polymer. The additional finding of sn-glycero-1-phosphate-bearing derivatives of these acyl- and phosphatidylglycosyldiacylglycerolipids supports this idea and leads, in Ent. faecalis for example, to the putative biosynthetic sequence depicted in Fig. 10. Biosynthesis of phosphatidylglucosyldiacylglycerol has been demonstrated with particulate enzyme preparations from Ent. faecalis which catalysed phosphatidyl transfer from bisphosphatidylglycerol and phosphatidylglycerol to the glycolipid. Since these membrane preparations also catalysed conversion of phosphatidylglycerol to bisphosphatidylglycerol and the latter was more active in phosphoglycolipid synthesis, the following sequence of reactions has been suggested to occur (Pieringer, 1972): Glc(a1 -2)Glc(a1 -3)acyl2Gro PtdlGro Gro

‘PtdGro

With particulate membrane preparations from the same bacterium, it could further be demonstrated that radiolabelled phosphatidyldiglucosyldiacylglycerol was used as an acceptor substrate in lipoteichoic acid synthesis which proceeded from endogenous phosphatidylglycerol (Ganfield and Pieringer, 1980). The radiolabel appeared in a water-soluble product which seemed to increase in length on longer incubation times as indicated by increasing

FIG. 10. Structural and possible biosynthetic relationships between glycolipid, phosphoglycolipids and the two molecular species of lipoteichoic acid in Enterococcus fueculis. For structures of phosphoglycolipids (Fischer et ul., 1973a, b; Fischer and Landgraf, 1975) and lipoteichoic acid (Toon et ul., 1972; Ganfield and Pieringer, 1975; Fischer et ul., 1981) see the references given; for chain substituents of completed lipoteichoic acids (X)see Table 3.

252

W. FISCHER

T I M E (h) FIG. 1 1. Time course of incorporation of [g~uc~se-’~C]phosphatidyldiglucosyldiacylglycerol (nmol (mg protein)-’) into saline extractable polymer from a chlorofommethanol-water (1 : 1 :0.125, by vol.) supernatant ( 0 ) and an insoluble proteincontaining pellet fraction (0)by membrane preparations of Streptococcus fuecalis. From Ganfield and Pieringer (1980).

insolubility in chloroform-methanol-water (Fig. 1 1). The water-soluble product was characterized as micellar non-substituted poly(g1ycerophosphate) by column chromatography and analysis. Short-chain homologues of lipoteichoic acids were also synthesized in toluene-treated cells of Lb. casei and, under appropriate conditions, elongated to water-soluble polymers (Brautigan et al., 1981; Taron et al., 1983). On Bligh-Dyer phase partition, these short-chain homologues separated into the chloroform layer and might therefore be earlier intermediates in assembly of lipoteichoic acid than are the short-chain products obtained from Ent.faecalis which partitioned into the aqueous layer (Fig. 11). In pulseechase experiments in which the fatty-acyl residues of growing Staph. aureus were labelled with [I4C]acetate, the I4C-label appeared in and liposuccession in Glc(B1-3)acy12Gro, G l c ( ~ l - 6 ) G l c1(-3)acylzGro ~ teichoic acid (Fig. 12). The glycerophosphoglycolipid that had been isolated and characterized earlier from this organism (Fischer et al., 197%) has been shown to be an intermediate in this sequence by the pulse-chase experiment with [3H]glycerolin Fig. 13 (Koch et al., 1984): Glc2acyl2Gro + PtdGro-+ GroP-Glc2acyl2Gro+ acylzGro +24 acyl2Gro. GroP-Glc2acyl2Gro+ 24 PtdGro+(GroP)2~-Glc~acyl2Gro

As shown in Figure 13, the glycerophosphate moiety of the glycerophosphoglycolipid gained and lost radioactivity very rapidly, which is expected if the small pool of glycerophosphoglycolipid turns over to the large pool of lipoteichoic acid (see Table 5). On the other hand, the radiolabel of the


253

-a lo5

-U I

45

75

105 135 165 195

t T I M E imin) FIG. 12. Pulse-chase kinetics of lipid amphiphiles in growing Staphylococcus aureus on labelling of fatty acids with [14C]acetate.The arrow indicates onset of the chase. Symbols: A , phosphatidylglycerol; 0 , diacylglycerol; 0 , lipoteichoic acid; 0 , diglucosyldiacylglycerol; 0 , glucosyldiacylglycerol; 0 , phosphatidic acid. From Koch et al. (1984).

glycerol residue in the glycolipid moiety increased continuously throughout the chase period (Fig. 13), gaining radioactivity through glycolipid synthesis from the long-lasting label in the diacylglycerol pool (Fig. 12). Two chemically different linkages are formed in lipoteichoic acid synthesis; the first is the linkage of glycerophosphate to the glycolipid, the second is that of glycerophosphate units to each other. The discovery of glycerophosphoglycolipids in a wide range of Gram-positive bacteria, together with the usual absence of higher homologues (Fischer et al., 1978b) suggests that two glycerophosphate transferases might be involved. One would recognize the glycolipid substrate, thereby forming the glycerophosphoglycolipid intermediate, and a second would polymerize the chain. The initial transferase seems to be highly specific since, in some bacteria, such as in Lb. cusei, certain glycolipids are strictly selected (Fig. 14), and in all instances studied the glycerophosphate residue is exclusively linked to C-6 of the non-reducing hexosyl terminus of the glycolipid moiety. Non-glycosylated diacylglycerol

254

W.FlSCHER

T I M E (rnin) FIG. 13. Pulse-chase kinetics of the two glycerol moieties of GroP-+6Glc(B1-6) GIc(B1-3)acylzGro,the glycerophosphoglycolipidof Staphylococcus aureus, on labelling growing cells with [2-3H]glycerol. Symbols: diglucosyldiacylglycerol moiety; A , glycerophosphate moiety; A, glycerophosphate-glycolipid which escaped conversion into lipoteichoic acid. From Koch e f al. (1984) where details may be found.

.,

found as the lipid moiety in the lipoteichoicacid of certain bacteria (see Table 1) suggests that in these bacteria diacylglycerol or phosphatidylglycerolserves as the initial acceptor substrate. B. BIOSYNTHESIS OF RELATED MACROAMPHIPHILES

Although biosynthesis of the poly(glycosylg1ycerophosphate) lipoteichoic acid from Lactococcusgarvieae has not been studied, the sn-1-configuration of its glycerophosphate residues (Koch and Fischer, 1978; Fischer et al., 1982) provides strong evidence for phosphatidylglycerol being the glycerophosphate donor. A set of galactosylated sn-glycero-1-phosphoglycolipids, detected in this organism (Fischer et al., 1979), can be incorporated into a putative biosynthetic sequence leading to a compound that carries Gal(a16)Gal(al-3),Gal(a 1-2)Gro-1-phosphate, the complete repeating unit of the

H

V

W

-

R

y

2

HCUCO-R

1

H2C-0-CO-R

E

H -KO-R H2 -0-CO-R

'Q

w ~ I ~ - C O - R

H -0-CO-R

FIG. 14. Structures of glycolipids, glycerophosphoglycolipids and lipoteichoic acid from Lactobacillus casei. For details, see Fischer et a[.(1978~)and Nakano and Fischer (1977, 1978), for substitution of lipoteichoic acid (X) see Table 3.

258

W. FISCHER

lipoteichoic acid, on the definitive glycolipid moiety (Fig. 15). One might therefore suggest an assembly of the repeating unit on the growing chain by successive transfer of glycerophosphate and individual galactosyl residues. The sn-glycero- 1-phosphate side chains of the lipoglycan from Bifidobacterium bijidum (for its structure see Fig. 5) have been shown to be derived from phosphatidylglycerol. Membrane preparations catalysed transfer of radiobut not from CDP-[“’C]glycerolto a activity from ph~sphatidyl[’~C]glycerol product having the properties of the macroamphiphile of this organism (Op den Kamp et al., 1985a). Since, in these experiments, incorporation of radioactivity into the polymer from UDP-[14C]glucoseand UDP-[’4C]galactose was not observed, the glycerophosphate was apparently transferred to preformed lipoglycan. That galactofuranosyl and glycerophosphate residues may be transferred separately to the completed lipoglucan is inferred from the finding of the molecular species in the native amphiphile that have a smaller number of galactofuranosyl residues incompletely substituted with glycerophosphate (Fischer, 1987). Whether synthesis of the glucogalactofuranan moiety requires hexosyl derivatives of phosphoryl undecaprenol as glycosyl donors remains to be studied. In Micrococcus luteus, cr-D-mannopyranosyl-I -phosphoryl undecaprenol is the substrate for synthesis of the lipomannan (Scher et al., 1968; Scher and Lennarz, 1969). It is formed from GDP-a-D-mannose which, on the other hand, is directly used for synthesis of mono- and dimannosyldiacylglycerol in this organism (Lennarz and Talamo, 1966). If the synthesis of lipomannan starts from dimannosyldiacylglycerol,both activated mannosyl donors will be required for total synthesis. C . INFLUENCE OF LIPOTEICHOIC ACID BIOSYNTHESIS ON THE TURNOVER OF MEMBRANE LIPIDS

In Gram-positive bacteria a rapid turnover of membrane lipids, particularly of phosphatidylglycerol, was recognized I5 years ago (Short and White, 1970, 1971), but it was not until recently that the driving force for this process was detected in lipoteichoic acid biosynthesis (Koga et al., 1984; Koch et al., 1984). The dynamics involved for example in Staph. aureus can be deduced from the data in Table 5 . Lipoteichoic acid, although present at not more than 6 mol% contains approximately 50% of the total amphiphile glycerol and three times the amount of the non-acylated glycerol moiety of phosphatidylglycerol. The total membrane phosphatidylglycerol must therefore turn over three times for biosynthesis of lipoteichoic acid in one bacterial doubling. The diacylglycerol formed concomitantly is six times the amount present in the diacylglycerol pool. If, in the chase after labelling with [2-3H]glycerol,radioactivity in the diacylglycerol moieties of all of the lipid amphiphiles is balanced, it becomes

259


TABLE 5. Composition of lipid amphiphiles in logarithmically growing Stuphylococcus aureus. From Koch et al. (1 984) Composition Amphiphile

(per cent of amphiphile glycerol)

(mol Yo)

33.0 6.5 1.1

Glc(~l-6)Glc(B1-3)acyl~Gro GroP-6Glc(~1-6)Glc(~1-3)acyl~Gro

50.4 9.9 1.1

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