New comprehensive biochemistry vol 26 the biochemistry of archaea

The present volume attempts to bring together recent knowledge concerning general metabolism, bioenergetics, molecular biology and genetics, membrane lipid and cell-wall structural chemi

THE BIOCHEMISTRY OF ARCHAEA (ARCHAEBACTERIA)

New Comprehensive Biochemistry

The Biochemistry of Archaea (Archaebacteria)

Editors

M Kates

Department of Biochemistry, University of Ottawa,

Ottawa, Ont K I N 6N5, Canada

Department of Microbiology, University of Toronto,

Toronto, Ont M5S IA8, Canada

A.T Matheson

Department of Biochemistry and Microbiology, University of Victoria, Victoria, B.C V5Z 4H4, Canada

1993 ELSEVIER Amsterdam London New York Tokyo

Elsevier Science Publishers B.V

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Library of Congress Cataloging-in-Publication Data

The Biochemistry of archaea (archaebacteria) I editors, M Kates, D.J

Kushner, A.T Matheson

p cm - - (New comprehensive biochemistry ; v 26)

Includes bibliographical references and index

ISBN 0-444-8171 3-1 (acid-free paper)

ISBN 0 444 81713 1

ISBN 0 444 80303 3 (series)

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Preface

In the last ten years, considerable information has accumulated on the biochemistry of the archaea Some aspects of this subject, such as bioenergetics, molecular biology and genetics, membrane lipids, etc., have been dealt with in individual book chapters and

review articles in various treatises, but the subject as a whole has not yet been treated in

a comprehensive manner

The present volume attempts to bring together recent knowledge concerning general metabolism, bioenergetics, molecular biology and genetics, membrane lipid and cell-wall structural chemistry and evolutionary relations, of the three major groups of archaea: the extreme halophiles, the extreme thermophiles, and the methanogens We have called upon

a number of experts, all actively involved in research on the above subjects to review their specialized fields

In the Introduction, C.R Woese considers the evolutionary relationship of these microorganisms to all other living cells This is followed by a section on special metabolic features of archaea, covered in Chapters 1 through 4, respectively, by: M.J Danson on central metabolism in archaea, including carbohydrate metabolism, the citric-acid cycle, and amino-acid and lipid metabolism; VP Skulachev on bioenergetics and transport in extreme halophiles; L Daniels on the biochemistry of methanogenesis; and P Schonheit dealing with bioenergetics and transport in methanogens and related thermophilic archaea

A section on protein structural chemistry in archaea includes Chapters 5 through 7, respectively, by: D Oesterhelt on the structure and function of photoreceptor proteins in the Halobacteriaceae; J Lanyi on the structure and function of ion-transport rhodopsins

in extreme halophiles; and R Hensel on proteins of extreme thermophiles In a section

on cell envelopes (Chapters 8-10>, 0 Kandler and H Konig discuss the structure and chemistry of archaeal cell walls; M Kates reviews the chemistry and function

of membrane lipids of archaea; and L.I Hochstein covers membrane-bound proteins (enzymes) in archaea

Chapters 11 through 14 deal with aspects of molecular biology in archaea and include, respectively, DNA structure and replication by P Forterre; transcription apparatus by

W Zillig et al.; translation apparatus by R Amils et al.; and ribosomal structure and

function by A Matheson et al

The final chapters (1 5-1 7) deal with the molecular genetics of archaea: L Schalkwyk discusses halophilic genes; J Reeve and J Palmer describe genes of methanogens; and

R Garrett and J.Z Dalgaard discuss genes of extreme thermophiles

In an epilogue, W.F Doolittle presents an overview of all chapters in the larger context

of cellular evolution and our future understanding of this subject

The editors trust that this volume is sufficiently comprehensive in scope to be of use

to researchers actively engaged or interested in various aspects of the biochemistry of

archaea They hope that it will also stimulate further studes of the topics covered, and will open up new areas for investigation

M Kates D.J Kushner A.T Matheson

M Kates et al (Eds.), The Biochemiso of Archaea (Archaebacferia)

0 1993 Elsevier Science Publishers B.V All rights reserved

vii

INTRODUCTION

The archaea: Their history and significance

of the New York Times and other major newspapers, mentioned on the evening TV news programs, and even drew a quip from Johnny Carson

For the press and the public the discovery of the archaea was a highly significant event;

it had touched upon that age old basic human concern about where we come from - which interested the layman more than the promise of a brighter tomorrow through biomedical technology The biology community, on the other hand (though not scientists in general), had a decidedly different reaction For them the cloning of the somatotropin gene was

an important milestone in the ceaseless efforts of biologists to cure disease As regards the discovery of a “third form of life”, however, biologists’ attitudes generally ranged from skeptical to intensely negative Some derisively rejected the claim out of hand One well-known biologist went so far as to suggest to one of my collaborators that he publicly dissociate himself from the work Another counseled one of the three major

US news magazines not to carry the story, and it didn’t Unfortunately, very little of

this negative reaction was expressed in a scientifically proper way, i.e., in the scientific literature It would have been interesting and instructive to quote today

Why did biologists react so differently than laymen (and other scientists) to the discovery of the archaea, many even viewing the claim as bogus? Their reaction was nothing new It (the rejection, its vehemence, and the associated scorn) is a well- recognized sociological phenomenon, which Thomas Kuhn discusses extensively in his now classic work The Structure of ScientiJc Revolutions What the discovery of

the archaea had done was counter the existing paradigm, cross one of biology’s deep prejudices:

Vlll

All organisms except viruses can be assigned to one of two primary groups lprokaryotes and eukaryotes] descriptions of them can be found in the better textbooks of general biology,

a sure indication that they have acquired the status of truisms [I]

There is little doubt that biologists can accept the division of cellular life into two groupings

at the highest level expressing the encompassing characters of procaryotic and eucaryotic cellular organization [2]

To claim that a third primary group existed [3,4] was patently absurd!

As the reader might imagine, those of us involved in the discovery of the archaea had

ourselves experienced the same sense of incredulity when first confronted with the data How could there possibly be something that was neither eukaryotic nor prokaryotic? Yet that seemed the only reasonable interpretation of the data If so, then what we all took for granted regarding prokaryotes and eukaryotes must be wrong Once that

light dawned, the source of the problem was obvious: A prokaryote had originally

been defined as an organism that did not possess certain eukaryotic cellular features,

e.g., a membrane-bounded nucleus and mitochondria Defined in this purely negative way “prokaryote” could not be a phylogenetically meaningful grouping Yet, this is precisely what it had been taken to be - without any supporting evidence When, decades later, it became possible - through electron microscopy and molecular studies -

to redefine “prokaryote” (and “eukaryote”) in comparable, positive terms, and so, to test the phylogenetic validity of the prokaryote taxon, the biologist, strangely, felt no need to do so: Prokaryotes were “obviously” all related to one another; therefore, a few

representative cases would suffice As a consequence, the generalizations that came to be

known as “prokaryotic characteristics” were all based on very few examples; they were,

by and large, merely characteristics of Escherichia coli and a few of its relatives No

comprehensive characterization of prokaryotes, with the intent of testing their supposed monophyletic nature, had ever been done!

This was a mistake of major proportions, for, among other things, it almost certainly delayed the discovery of the archaea by at least a decade (see below) Such a logical transgression might be excused among botanists and zoologists on the grounds that they knew and cared little about prokaryotes But, for the keepers of the prokaryote, the microbiologists, never to have questioned their monophyletic nature would have

been unpardonable As it turns out, this was not actually what happened; but what

did happen, nevertheless, had the same effect Microbiologists of an earlier era were very much concerned with the phylogenetic relationships among bacteria, and were, therefore, skeptical of the idea that “prokaryote” represented a monophyletic grouping Yet, a few decades later, and for reasons that are hard even in retrospect to understand, this

critical scientific attitude was unaccountably replaced by a naive, unscientific acceptance

of “prokaryote” as a phylogenetically valid taxon We will examine this unfortunate transformation, so central to the history of the archaea, in some detail below

My reason for saying that the prokaryote-eukaryote prejudice delayed the discovery of the archaea by at least a decade is that (anecdotal) evidence for their existence had been

in the literature some time before 1977 These bits and pieces in retrospect constituted

a prima facie case that something might be wrong with the idea that prokaryote is a monophyletic taxon, and the situation cried for deeper, more comprehensive investigation

Yet, at the time no one came to this conclusion

ix

The cards were definitely stacked against the discovery of the archaea, for in addition

to the hegemony of the prokaryote concept, other factors and prejudices worked to prevent their emergence The early evidence for the existence of the archaea came in the main from an odd collection of organisms that lived in “extreme” environments

At that time conventional wisdom held that organisms living in extreme environments represent evolutionary adaptations to their environments, and such adaptations required

an organism to undergo unusual phenotypic changes Thus, not only were all bacteria

by definition “prokaryotic”; but anything that was atypical and lived in an “extreme” environment was atypical by reason of that circumstance The case for the archaea was not helped either by the fact that the key organisms in question were very unlike one another in their overall phenotypes, making it particularly difficult for even the best microbiologists to sense their relationship To make matters worse, idiosyncrasy could also be found scattered among (what turned out to be) the (eu)bacteria as well And, of course, since the natural relationships among bacteria were not understood, there was no basis for sorting any of this out

One early piece in the archaeal puzzle was methanogenesis, an unusual biochemistry that involved a variety of new coenzymes [5,6] Except for this common biochemistry, however, methanogens seemed to have little in common (morphologically, that is) with one another What did this mean? To those who took morphology as a primary indicator of relationship it meant only that patches of methanogenic metabolism existed scattered across the phylogenetic landscape, a view reflected in the seventh edition of Bergey’s Manual [7] To those who took physiology as a primary indicator of relationship, methanogens constituted a phylogenetically coherent, separate, taxon, the view that prevailed in the Manual’s eighth edition [8] Despite their highly unusual biochemistry, however, methanogens were never perceived as anything but typical “prokaryotes” in the phylogenetic sense

Another early piece of the puzzle was the unusual isoprenoid ether linked type of lipid found by Morris Kates and his colleagues in the extreme halophiles (refs [9,10]; see Chapter 9 of this volume] This sort of lipid was also to be discovered in

Thermoplasma [ 1 11 and in Sulfolobus [ 12,131 before 1977, but was not discovered in the methanogens [13a] until after the archaea were recognized as a group Yet, no phylogenetic connection among the organisms that possessed them was considered Conventional wisdom held firm; adaptation to extreme environments had somehow caused those “unrelated” lineages all to independently arrive at the same unusual lipid structure [ 121

The fact that Sulfolobus and Thennoplasma have similar lipids is of interest, but almost certainly

this can be explained by convergent evolution This hypothesis is strengthened by the fact that

Hulobacterium, another quite different organism, also has lipids similar to those of the two acidophilic thermophiles [ 141

These same organisms shared another peculiar characteristic: cell walls that did not contain peptidoglycan; and this was known to be true even for a methanogen [ 15-17] Still,

no phylogenetic interpretation followed; but in fairness it should be noted that the walls

of certain (eu)bacteria, such as the planctomyces, also contain no peptidoglycan [ 181 The extreme halophiles in addition possessed peculiar ribosomes, which contained acidic, rather than basic, proteins [ 191 While this fact alone carried no obvious

phylogenetic implications, the subsequent finding that sequences of these proteins were remarkably similar to eukaryotic ribosomal proteins - a discovery that occurred at about the same time as, but completely independent of, the rRNA characterizations - certainly did [20]

Still another clue seemed to be the discovery in llermoplasma (a wall-less

“prokaryote”) of a histone, which was interpreted to mean that this organism represented the ancestry of eukaryotes[21] Unfortunately, t h s clue turned out to be a false one, because the histone, upon later sequencing, proved to be of the bacterial, not the eukaryotic, type[22] True or not, at the time this claim might have been reason to question the prokaryote concept But, again, nothing of the sort happened - in this case perhaps because we had long been assured that the ancestor of eukaryotic cells was a

“prokaryote” that had lost its cell wall [23] It remained for Sandman et al [24] to show, much later, that an eukaryotic type of histone does indeed exist in the archaea

Despite all this tantalizing anecdotal evidence, molecular biologists and microbiologists alike (myself included) remained secure in their belief that a prokaryote is a prokaryote, and all variations from the norm have no phylogenetic significance It required more compelling evidence, evidence difficult to interpret in equivocal ways, to awaken us from our reverie And this came in the form of our rRNA oligonucleotide characterizations

[Even then, however, I was occasionally asked in early seminars about archaebacteria

why their incredibly unique rRNA sequences weren’t merely the result of adaptation to some extreme environment!]

A very few biologists actually did find the initial claim of a third form of life interesting and important Many of them were microbiologists who worked with one

or more of the archaea and found phylogenetic uniqueness a satisfying explanation for the phenotypic uniqueness they were increasingly encountering Others, such as W Zillig, who understood some particular molecular system in great detail (RNA polymerase in his case), knew immediately that an archaeal version was no typical bacterial version [25,26]; this was no “prokaryote” with which they were dealing In my experience Otto Kandler was the only biologist to understand the concept of a third “Urkingdom” (as it was then called) immediately upon encountering it Realizing the importance of this finding, he began encouraging German biologists to work on the archaea, and soon thereafter gave the field a tremendous boost by convening the first conference on “archaebacteria”, in Munich in 1981 [27]

My own involvement with the archaea came about in an unexpected way, having nothing whatever to do with any interest in unusual organisms or phylogeny per se I had developed a fascination with the genetic code in the 1960’s, and soon realized that the problem was not the purely cryptographx one that, in the days of the “comma free code”, everyone took it to be [28]

The translation apparatus, an incredibly complex molecular aggregate that today involves of the order of 100 different molecular species, had to have evolved in stages from a far more rudimentary and inaccurate mechanism [29,30] There seemed no getting around this conclusion, and the corollary that the form of the genetic code was shaped during and by this evolution[30,31] Not only the degree and type of order in the codon catalog, but probably the actual codon assignments themselves were products of this coevolution [29-3 11 Unfortunately, it seemed that none of the crucial tell-tale interactions were evident in the translation apparatus today If so, then the code’s origin must be sought

xi

in the long gone rudimentary versions of the translation apparatus - whose nature must

be inferred somehow by means of evolutionary reconstruction

The differences between the eukaryotic and prokaryotic translation mechanisms provided an intriguing clue, for these differences appeared too profound to be trivially explained [32] A comparative dissection of the problem required a solid phylogenetic framework, and unfortunately, none existed for the prokaryotes However, Fred Sanger ’s laboratory, on their way to devising a nucleic acid sequencing technology, had come up with the oligonucleotide cataloging method for partial sequencing of RNAs, which was well suited to the problem at hand The obvious choice of molecule for comparative oligonucleotide analysis was the small subunit ribosomal RNA: Its function was not only universal but remarkably constant; rRNAs exist in the cell in high copy number, and

so, are relatively easy to isolate; and the small subunit rRNA is large enough to give a good deal of information (but not so large as to be unmanageable) What followed is now a matter of record: In screening various diverse prokaryotic (and a few eukaryotic)

rRNAs for their similarities and differences, we stumbled across Methanobacterium

thermoautotrophicum, whose rRNA oligonucleotide catalog was definitely not typical

of “prokaryotes” - but neither was it of eukaryotes [3,4,33]

2 Microbiology S changing evolutionary perspective

From a broader biological perspective the microbiology into which the archaea were born was a strange and strangely isolated biological discipline Its concerns were local, largely microbial biochemistry and physiology in general, taxonomy for purposes of species

identification, and the molecular biology of E coli It functioned as though evolution

did not exist Although it accepted the grand division of the world into eukaryotes and prokaryotes, microbiologists didn’t seek to understand the nature of the relationship between them, of their similarities and differences Neither did the macro-biologists Evolutionists in particular functioned (and still do) as though prokaryotes didn’t exist

or don’t matter: Their concerns were fossils and mathematical models of evolutionary dynamics and population flow; and prokaryotes offered precious little in the way of the first and didn’t fit neatly into the second Never mind that prokaryotes and eukaryotes shared a common ancestor, and that the question of the nature of that ancestor (answerable

or not at the time) is probably the most important question in evolutionary biology Never mind that most of evolutionary history is written in terms of microorganisms, not multicellular ones And never mind that the eukaryotes expropriated chlorophyll-based photosynthesis (probably the most important single evolutionary innovation in the history

of the organic world) and the capacity to oxidatively produce ATP via the Krebs cycle, from the bacteria (through endosymbioses) - and that without these there would be no plants, indeed no multicellular life at all I find it particularly telling that some biologists could posit that the entity that became the body of the eukaryotic cell (i.e., the host for the endosymbionts), was derived from a prokaryote that had lost its cell wa11[23], yet showed no concern that were this true, then this ancestral mycoplasma would have

to have drastically altered its biochemistry, the structure of its translation apparatus, its

transcription apparatus, etc., in the process of becoming a hll-fledged eukaryote Even today, when it is apparent that the archaea manifest many so-called “eukaryotic traits”

xii

and almost certain that the two lineages shared some sort of common ancestry [exclusive

of the (eu)bacteria], it is rare to find a microbiologist, macrobiologist, molecular biologist

or evolutionist who shows active interest in this relationship The historical roots of such attitudes cry for examination

Looking at the microbiology of a few generations ago, one does not sense an intellectual isolation from the rest of biology, nor does one see the almost total lack

of interest in evolutionary matters that later came to characterize the field To earlier microbiologists bacteria represented a primitive stage in the evolutionary flow, and their place in that flow was a matter of considerable interest:

Perhaps the designation of Schizophytae [bacteria] may recommend itself for this first and simplest division of living beings (F Cohn, 1875; quoted in translation [I])

It is my conviction that [microbial ecology] is the most necessary and fruitful direction

to guide us in organizing our knowledge of that part of nature which deals with the lowest limits of the organic world, and which constantly keeps before our minds the profound problem

of the origin of life itself (Beijerinck, 1905; quoted in translation [34])

plants and animals [may have] passed through intermediate [evolutionary] stages of increased complexity which [had] the characteristics of ‘bacteria’ [35]

Not only was there genuine concern with the place of bacteria in the natural order of

things, but the course of bacterial evolution, evolutionary relationships among the various

bacteria, and how best these could be recognized, were all central issues; as can be seen from the following:

the only truly scientific foundation of classification is to be found in appreciation of the available facts from a phylogenetic point of view Only in this way can the natural interrelationships of the various bacteria be properly understood A true reconstruction

of the course of evolution is the ideal of every taxonomist [36]

It seems acceptable that the diversity of bacterial forms is the outcome of various independent morphological evolutions which have had their startingpoint in the simplest form both existent and conceivable: the sphere [36]

it is rather naive to believe that in the distribution of their metabolic characters one can discern the trend of physiological evolution For these reasons, a phylogenetic system based solely or largely on physiological grounds seems unsound It is our belief that the greatest weight in making the major subdivisions in the Schizomycetes should be laid on morphological characters Clearly paramount is the structure of the individual vegetative cell, including such points as the nature of the cell wall, the presence and location of chromatin material, the functional structures (e.g., of locomotion), the method of cell division, and the shape of the cell [37]

Orla-Jensen regarded the chemosynthetic bacteria as the most primitive group because they can live in the complete absence of organic matter and hence are independent of other living forms This overlooks the fact that a chemosynthetic metabolism necessarily presupposes a rather highly specialized synthetic ability such as one would not expect to find in metabolically primitive forms [37]

X l l l These microbiologists understood that their natural systems were not perfect, but they also understood that, even so, the search for the true natural bacterial system should never be abandoned

inasmuch as the course of phylogeny will always remain unknown, the basis of a true phylogenetic system of classification will be very unstable indeed On the other hand it cannot be denied that the studies in comparative morphology made by botanists and zoologist have made phylogeny a reality Under these circumstances it seems appropriate to accept the phylogenetic principle also in bacteriological classification [36]

there is good reason to prefer an admittedly imperfect natural system to a purely empirical one A phylogenetic system has at least a rational basis, and can be altered and improved as new facts come to light; its very weaknesses will suggest the type of experimental work necessary for improvement On the other hand, an empirical system is largely unmodifiable because the differential characters employed are arbitrarily chosen and usually cannot be altered to any great extent without disrupting the whole system [37]

the mere fact that a particular phylogenetic scheme has been shown to be unsound by later

work is not a valid reason for total rejection of the phylogenetic approach [37]

The culmination of microbiology’s efforts to infer a natural bacterial system came with Stanier and van Niel’s bold and imaginative global system in 1941, which concluded the following:

It is true that there are a small number of organisms of whose relationships we are still ignorant, but if it be remembered that these are mostly microbes not yet studied under laboratory conditions, it may be expected that further work will result in an elucidation of their taxonomic positions All the other bacteria can be readily subdivided into three large groups [the classes

Eubacteriae, Myxobacteriae, and Spirochaetae] [37]

It was not long, however, before doubt as to whether bacterial characteristics had any real phylogenetic significance - doubts that had always lurked in the background -

filminated What ensued was a rapid devolution, from a simple initial skepticism that a natural bacterial system could be established on the basis of currently definable bacterial characteristics; to the attitude that it will never be possible to determine a natural bacterial system; to the ultimate dismissal of a natural bacterial system as being of little value This transformation is largely captured in the following series of quotes In his famous

The only sound conclusion that seems permissible at present is that we cannot yet use physiological or biochemical characters as a sound guide for the development of a ‘natural system’ of classification of the bacteria [However] the search for a basis upon which a

‘natural system’ can be constructed must continue [38]

By 1955 the assessment took on a different tone:

What made Winogradsky (1952) grant that the systematics of plants and animals on the basis

of the Linnean system is defensible, while contending that a similar classification of bacteria

is out of the question? The answer must be obvious to those who recognize in the former an increasingly successful attempt at reconstructing a phylogenetic history of the higher plants and animals and who feel that comparable efforts in the realm of the bacteria (and bluegreen

xiv

algae) are doomed to failure because it does not appear likely that criteria of truly phylogenetic significance can be devised for these organisms [35]

And by 1962 his and Stanier's view of bacterial phylogeny was decidedly jaded:

Any good biologist finds it intellectually distressing to devote his life to the study of a group that cannot be readily and satisfactorily defined in biological terms; and the abiding intellectual scandal of bacteriology has been the absence of a clear concept of a bacterium Our first joint attempt to deal with this problem 20 years ago was framed in an elaborate taxonomic proposal, which neither of us cares any longer to defend But even though we have become skeptical about the value of developing formal taxonomic systems for bacteria , the problem

of defining these organisms as a group in terms of their biological organization is clearly still

of great importance [39]

an attitude canonized in microbiology's premier text The Microbial World:

any systematic attempt to construct a detailed scheme of natural relationships becomes the purest speculation The only possible conclusion is, accordingly, that the ultimate scientific goal of biological classification cannot be achieved in the case of bacteria [40]

the general course of evolution [for bacteria] will probably never be known, and there is simply not enough objective evidence to base their classification on phylogenetic grounds For these and other reasons, most modern taxonomists have explicitly abandoned the phylogenetic approach [41]

This retreat from a phylogenetic perspective seems to have left microbiology intellectually stunned and vulnerable Previously, microbiologists had been highly skeptical of simplistically lumping all bacteria into one grand (monophyletic) taxon

In 1941 Stanier and van Niel had said:

we believe that the three major groups among the bacteria are of polyphyletic origin [37]

And Pringsheim had warned:

The entirely negative characteristics upon which this group [prokaryotes] is based should be noted, and the possibility o f convergent evolution be seriously considered [42]

Now, in what seemed a desperate search for unifying microbial principles, microbiologists enthusiastically embraced the very notion they had earlier spurned - as the initial two quotes in this article (from Stanier and Murray [ 1,2]) and the following demonstrate:

It is now clear that among organisms there are two different organizational patterns of cells, which Chatton (1937) called, with singular prescience, the eucaryotic and procaryotic type The distinctive property of bacteria and bluegreen algae is the procaryotic nature of their cells [39]

All these organisms share the distinctive structural properties associated with the procaryotic

cell , and we can therefore safely infer a common origin for the whole group in the remote evolutionary past [40]

What caused the earlier doubting, critical attitude to be replaced by this dogmatic assertion? Granted, it had become possible in the interim to define the prokaryote, through molecular and electron microscopic characterizations, in positive, phylogenetically telling terms However, as noted above, the phylogenetically important characterizations were

confined mainly to E coli Thus, there had to have been some a priori assumption that all

prokaryotes were similar, were related, in order that (i) one or a few examples could be uncritically accepted as representative of all, and (ii) when real discrepancies did surface (see above) they were automatically taken to be the result of adaptations to unusual environments Very few complained about the danger of so superficial a characterization

of the prokaryote Here is one of the lonely exceptions:

there are remarkably few comparative studies The result is that the application of the newer adjuncts of morphology for taxonomic purposes entails generalization from limited cases [43]

As judged by the author’s later writings, it was a caution that not even he stressed, and

one that clearly was not heeded by other microbiologists or by molecular biologists

I have no real explanation for this paradigmatic shift, this uncritical turn of mind It

happened, and it happened in the context of the failure to fulfill one of microbiology’s major goals, elucidating the natural microbial system The reasons why it happened, however, appear non-scientific; and are best left to the historian The consequences certainly were most unfortunate for biology as a whole Today microbiology, fortunately, has an evolutionary dimension The question now is how long it will take microbiologists (and evolutionists) to realize this in a meaningful way, to return to the perspective of Beijerinck, Kluyver and (the young) van Niel

3 A molecular definition of the three domains

As the intellectual wave in microbiology moved away from (a natural) taxonomy and evolutionary considerations in general, an experimental undertow was pulling the field in precisely the opposite direction The first proteins were sequenced in the 1950s, and the capacity to characterize nucleic acids by oligonucleotide cataloging (as mentioned above) was developed in the 1960s, with full nucleic acid sequencing methods to follow in the 1970s In the realm of sequences one cannot avoid phylogeny, a point brought home to all

by Zuckerkandl and Pauling in their seminal 1965 publication Molecules as documents

of evolutionary history [44] Even so, microbiologists, with their counter-evolutionary

orientation, largely ignored Zuckerkandl and Pauling’s message; and none appeared to appreciate its full implications as regards a natural microbial system (Granted, a little work was done on bacterial cytochromec sequences, but it had only minor impact, especially given that those doing the work ultimately concluded, following Joyce Kilmer, that “only God can make a tree”[45], i.e., molecular sequence comparisons cannot determine bacterial relationships [4547].)

Molecular sequences reveal evolutionary relationships in ways and to an extent that classical phenotypic criteria, and even molecular functions, cannot What can be seen only dimly, if at all, at higher levels of cellular organization becomes obvious in terms of molecular structures and sequences This point is nowhere more dramatically illustrated than by the present situation, where at the level of the whole-cell phenotype the archaea and bacteria are, at best, difficult to distinguish, and the differences they

do show are impossible to interpret unequivocally However, the uniqueness of the two groups is blatant and readily interpretable on the molecular level From the perspective

of ribosomal RNA sequence and structure the living world divides into three distinct classes, corresponding to three very distinct rRNA types 148,491 The same three classes

xvi

TABLE 1 Small subunit rRNA s e q u e n c e signatures defining and distinguishing the archaea, bacteria a n d

eukaryaa

base or pairb

99 I00 I00

C:G C:G

C G:C

U

Y Y:R

C:G*

U G:Y(C)

C Y(C)

C

U G:C G:C

95 I00

95 I00

98

100

98

100 I00

100

I00 I00 I00 I00 I00

100

I00

Y(C) C:G

U:A

A:U

G:C

C:G C:G

A

R:U

A'

A C:G

G:C

C

A G:C*

96 I00

98

100

96

98 I00 I00 I00

96 I00

94 I00

96

98

a Adapted from ref [50] Except where indicated, the compositions shown are invariant in each domain Analysis based upon approximately 380 bacterial, 40 archaeal, and 50 eucaryal sequences R = purine; Y =pyrimidine Those cases in which a signature composition is "pure", i.e., is not seen at all in the other two domains, are marked

complete invariance; one or a few exceptions (among bacteria) are, therefore, designated as 99% If one form dominates, it is given in parentheses

Numbering follows E coli I6SrRNA standard [76]

' R:Y as used here does not include A:C pairs

xvii

emerge from the analysis of a variety of other molecular species as well, ribosomal proteins, translation factors, RNA polymerase, etc [Unfortunately, in these latter cases the sequence collections are far smaller than those that exist for rRNAs.1

The striking differences among the archaeal, bacterial and eukaryal versions of the (small subunit) rRNA manifest themselves in several ways, in terms of homologous sequence characters, non-homologous sequence characters, and in larger secondary structural elements whose form is specific for one (or more) of the domains[50] Table 1 shows a signature, based upon homologous positions in the small subunit rRNA sequence, that defines and distinguishes the three domains[50] Fig 1 shows the positions of various non-homologous elements in the rRNA secondary structure (explained in Table 2) that do likewise[50] (These various features are discussed

in greater detail by Winker and Woese [50].) The unavoidable conclusion from these data, and from similar data from other molecular species, is that at the highest level, life on this planet is organized into three very distinct, monophyletic groupings [4,48-

501

The differences among the rRNA types are relatively profound compared to differences encountered within any given type; so much so that they in aggregate feel qualitatively different The special nature of these inter-domain differences is made all the more striking by the realization that they had to have evolved over a relatively short time period, less than one billion years; whereas intra-domain changes have been happening over the last three billion years at least [51] The evolution that transformed the most recent common ancestor of all extant life into the individual ancestors of the three domains would, then, seem of a more rapid and drastic type than that which occurred subsequently within each of the domains This would be expected if the universal ancestor had been more rudimentary than its descendants, i.e., it had a smaller genome and simpler overall organization than they did Such a rudimentary entity, the evolution of whose translation function was not yet complete, has been termed a “progenote” [32,5 1,521

Although little or nothing can be said with certainty about the universal ancestor at present, it is encouraging to know that sufficient information seems to have been retained

in the genomes and molecular structures of modem cells that in the not too distant hture biologists will be able to infer a great deal about this most important period in evolutionary history

The earlier phylogenetic studies involving rRNA sequences that produced the topology

of the universal tree did not, and could not, indicate the position of its root, i.e., the location of the universal ancestor - phylogenetic trees based upon a single molecular species are unrootable unless relative evolutionary rates can be determined However, it

is in principle possible to root the universal tree using the Dayhoff strategy [53], which

employs pairs of related (paralogous) genes whose common ancestor duplicated (and hnctionally diverged) in the ancestral lineage before the three primary lines of descent

came into being Since both members of such a pair of genes can be present in all descendant lineages, a phylogenetic tree constructed from a combined alignment (of both) will comprise two topologically equivalent halves One half serves as the outgroup for, determines the root of, the other - in this case the root of the universal tree To date, two pairs of paralogous genes, the translation factors EFTu and EFG[54] and

two related ATPase subunits [54,55], have proven useful for establishing the root of the

universal tree Both give the same result: the root lies between the (eu)bacteria and the

xviii

Fig I Secondary structural representation of the non-homologous features in 16s rRNA, i.e., individual nucleotides, pairs, and larger structures that distinguish among the archaea, bacteria and eukarya [50]; underlying secondary structure is that of E coli Arrows indicate positions of features

described in Table 2 Large structures that have a characteristic form in one or more of the domains

are shaded

xix TABLE 2

Small subunit rRNA sequence idiosyncrasies defining the various domainsa

no U:A90%

Y R

no U:A Yes C:G G:C U:A

CP or G:U

no

no

CP Yes Y:R

nob -1 A‘

Yes NCP(A:A)g 90%

R:Y(G:C) U:A 98%

NCP(YY) 90%

Yes Yes

CP 92%

no, Y R ~ ( U : A ) NCP(A:A) 92%

a Taken from ref [50]; see the text and Fig 1 for context

A bulged nucleotide occurs in a number of eukaryotic sequences at position 29.1, however Nucleotides added relative to E coli numbering are designated by decimal additions; e.g., 44.1,

In some cases position 396 is unpaired rather than position 397

Pairing in helix is largely unproven for eukaryotes; a number of NCP’s are present

“CP’ and “NCP” refer to canonical and noncanonical pairs, respectively

“ Y R does not include C:A pairs

44.2 would indicate nucleotides added between E coli positions 44 and 45

g The dominant form is given in parentheses

other two primary lines of descent (which, then, initially share a short common stem); see Fig 2

If this result is correct and if the sequences used are actually representative of the eukaryotic cell, i.e., if the basic eukaryotic cell, devoid of genetic contributions from its organelles, is not some kind of phylogenetic chimera [51], then the archaea are specific relatives of the eukaryotes [Given the importance of these deep evolutionary relationships, one would like to see a great deal more data bearing upon them.] This rooting of the universal tree places the archaea in a different perspective: From long before their discovery, the archaea had been implicitly taken to be bacteria-like, to have the bacterial “Bauplan”; a notion still prevalent [56,57] This unproductive point of view

strongly implies that since the archaea are basically bacterial in nature, there is little point

in devoting much effort to their study A far more productive, and phylogenetically proper, alternative sees the archaea not only as quite distinct from the bacteria in molecular makeup, but, in addition, as specific relatives of the eukaryotes; in which case archaea are not only worthy of significant attention in their own right, but their study should also reveal a great deal about the nature and evolution of the eukaryotic cell

x

Animals Entamoebae Slime

C yanobacteria Flavobacteria

Themtogales

Fungi Plants Ciliates

Flagellates Trichomonads

* /

used are: Mc., Methanococcus; Mb., Methanobrevibacter; M., Methanobacterium; Mt., Methano-

thermus; Mts., Methanosaeta; Mr., Methanosarcina; Mp., Methanoplanus; Ms., Methanospirillurn;

H., Haloferax; A., Archaeoglobus; Tp., Thermoplasma; Tc., Thermococcus; D., Desulfurococcus;

S , Sulfolobus; P., Pyrodictium; T., Thermoproteus; Tf., Thermophilum

4 Archaeal phylogenetic relationships

The archaea are known to comprise four quite distinct general phenotypes: the methanogens, the extreme halophiles, a loosely defined thermophilic (“sulfur-dependent”)

type, and thermophilic sulfate reducers [6] In that these phenotypes will be thoroughly

discussed in other chapters of this book, they will not be detailed here The four major phenotypes do not correspond to four distinct taxa of equivalent rank, however Phylogenetic relationships among the four are more complex than this, and suggest particular evolutionary relationships among the phenotypes

xxii

Fig 3 is a representative phylogenetic tree for the archaea based upon transversion distance analysis of a 16s rFWA sequence alignment [58] The position of the root of the archaeal tree has been examined in detail, and is considered firmly established [59] The salient features of the archaeal tree are these: The tree comprises two main branches, now designated the kingdoms Crenarchaeota and Euryarchaeota [48,49,60] The crenarchaeotes are relatively homogeneous phenotypically, being exclusively of the above thermophilic type The euryarchaeotes, on the other hand, comprise a potpourri of all the archaeal types The phylogenetic landscape of the euryarchaeotes

is dominated by methanogens, the three main clusters (the Methanococcales, the Methanobacteriales, and the Methanomicrobiales), plus a separate very deeply branching

lineage, represented by the genus Methanopyrus [48,58,60,61] Interspersed among

these are the extreme halophiles, the thermophilic sulfate reducing archaea, the Thermoplasmales, and the Thermococcales [48,58,60]; all known at present as relatively shallow phylogenetic clusters - whose taxonomic rank would at highest be considered

a family among the bacteria The Thermococcales (and to a lesser extent the Thermoplasmales) exhibit a general crenarchaeal phenotype A signature analysis leaves little doubt, however, that all the various non-methanogenic phenotypes grouped among the euryarchaeota are definitely more similar to the methanogens than to the crenarchaeota: The 2 1 positions in the 16s rRNA sequence listed in Table 3 distinguish the three main methanogen clusters (as a whole) from the crenarchaeotes All of the non-methanogenic euryarchaeotes show predominantly the methanogen signature, each differing from it in no more than four positions; and none shows more than two characters from the crenarchaeal signature This should be compared to the

methanogen Mp kandleri, the deepest branching of euryarchaeal lineage in Fig 3, whose

16s rRNA shows only 14 methanogen signature characters, the remaining seven being crenarchaeal[6 11

Two of the three main methanogen lineages appear phenotypically homogeneous

The third, that leading to the Methanomicrobiales, is not; see Fig 3 This lineage

has also spawned the extreme halophiles, the sulfate reducers and perhaps the Thermoplasmales [48,58,60,62] All of these relationships to the Methanomicrobiales require further justification, for they are certainly not evident at the phenotypic level [59,62]; and the extreme halophile relationship has been formally questioned [63,64] Nevertheless, this last mentioned relationship is clearly well established: A specific relationship between the extreme halophiles and the Methanomicrobiales can be demonstrated by a variety of analyses of 16s rRNA sequences - any number of variations

on evolutionary distance analysis, parsimony analysis, signature analysis [48,62] and maximum likelihood analysis (G.J Olsen, personal communication) More importantly, the relationship is also readily given by analysis of 23s rRNA sequences [62]

The placement of the sulfate-reducing archaeon Archaeoglobus fulgidus has proven

more difficult, but is now considered relatively solid As have most thermophilic rRNAs,

that of A fulgidus has a relatively high G+C content Because of this, thermophilic lineages tend to be artificially clustered by the usual analyses (The original placement of

A fulgidus in the euryarchaeal tree had been adjacent to Tc celer [65]) The placement

of A fulgidus given by transversion distance analysis (Fig 3) is deemed correct, for this

analysis is not subject to the effects of rRNA compositional variation in that total purine content of archaeal rRNAs is almost invariant [58] The placement of A fulgidus shown in

xxiii TABLE 3

Small subunit rRNA signature distinguishing the three groups of methanogens (as a whole) from the

Crenarchaeota

nucleotide

or base pa,r Methano- Crenarchae- Extreme Tp acido- A fulgidus Tc celer Mp kan-

a A dash denotes methanogen composition: cr denotes crenarchaeal composition

Y is C in all cases except some Methanomicrobiales

Y R and R Y pairs include all possibilities except A:C (C:A)

Single exception, U:U

Fig 3 has also been confirmed by maximum likelihood analysis of 16s rRNA (G.J Olsen,

personal communication), and by analyses of 23s rRNA sequences [58]

A number of the local relationships among euryarchaeal lineages remain unresolved,

however One is the relative branching order of the extreme halophile and

A fulgidus lineages from the Methanomicrobiales stem Another is the branching order among the three main methanogen lineages (a tight clustering) And, the exact position of the Thermoplasmales remains somewhat variable depending upon analytical method and makeup of the alignment The deep branching of the Thermococcales and

Mp kandleri lineages is given by all methods of analysis, however, and so, is considered

xxiv

Of the three main methanogenic lineages, the Methanomicrobiales lineage is by far the most interesting evolutionarily This is not merely because of its having spawned the extreme halophiles and sulfate-reducing archaea Even methanogenesis on this lineage shows unusual variations Whereas the other two methanogenic lines exhibit a uniform methanogenic biochemistry, varying only in the temperatures at which methanogenesis occurs, various sublines of the Methanomicrobiales produce methane from a variety of sources (acetate, methyl amines) in addition to carbon dioxide, and under a variety of conditions, e g , halophilic or alkaliphilic [6]

What evolutionary conclusions can one draw from the phylogenetic distribution of phenotypes on the archaeal tree? Unfortunately, very few It seems safe to infer that halobacterial metabolism arose from methanogenic metabolism, as did archaeal sulfate reduction When Tc celer, which has the general crenarchaeal phenotype, was thought

to be the lowest branching on the euryarchaeal side of the archaeal tree, it was argued that the general crenarchaeal phenotype was the ancestral archaeal phenotype, and so, had given rise to methanogenesis[48] However, now that a methanogen,

Mp kandleri, is known to represent the deepest euryarchaeal branching, this conclusion

is no longer defensible A far better understanding of all types of archaeal metabolism

is needed before any further conjectures are made regarding the metabolic history of the archaea

One conclusion regarding archaeal evolution does seem somewhat safe, however: The archaea have arisen from thermophilic ancestry [48] Not only are all known crenarchaeal isolates thermophilic, many of them hyperthermophiles, but the two deepest branchings

in the euryarchaeota, Thermococcus and Methanopyrus, are likewise, as are the deepest branchings within two of the three main groups of methanogens and the sulfate-reducing archaea (on the remaining methanogenic lineage, the Methanomicrobiales) Although (eu)bacterial phylogeny is not an issue here, let it be noted that the distribution of phenotypes on the bacterial tree suggests a thermophilic origin there as we11[66] The simplest assumption would then be that the universal ancestor itself lived in a hot environment, as did the ancestor common to the archaea and eukarya However, this leaves unanswered the question of why there is, then, no indication of thermophilic ancestry in the eukaryal phylogenetic tree

The archaeal ribosome and the operonal organization of rRNA genes show interesting patterns of phylogenetic variations Crenarchaeal ribosomes have relatively high ratios

of protein to RNA, while the Methanobacteriales, Methanomicrobiales and extreme

halophiles show low ratios [67] However, this cannot be taken as distinguishing the crenarchaeotes from the euryarchaeotes, for the deeply branching euryarchaeon Thermococcus celer, as well as the Thermoplasmales and the Methanococcales, all exhibit high protein:RNA ratios [67] [ProteinxRNA ratios have not been reported

for the ribosomes of the sulfate-reducing archaea or for Mp kandleri.] Other

ribosomal properties that differ between crenarchaeotes and (some) euryarchaeotes are: (i) the degree of modification of bases in rRNA (and tRNA), much higher in crenarchaeotes than in the euryarchaeotes so far characterized [68]; (ii) the presence

of a tRNA gene (for alanine) in the spacer region of the rRNA operon in all euryarchaeotes characterized, but not found so far among the crenarchaeota [69]; and (iii) a 5s rRNA gene terminally linked to the rRNA gene operon, seen in euryarchaeotes (except for Thermococcus) but not in crenarchaeotes [70]

xxv

The simplest interpretation of these rather striking differences between the crenarchaeal and euryarchaeal ribosomes is that the crenarchaeal type (and its corresponding gene organization) most closely approximates the ancestral archaeal condition On the euryarchaeal, but not the crenarchaeal branch a rather extensive evolution would then have occurred, which, at various points in euryarchaeal evolution, lowered the ancestral protein:RNA ratio in the ribosome, decreased drastically the degree of base modification

in rRNAs, and created an rRNA operon having a tRNA gene in the spacer region and a linked 5SrRNA gene The reduction in protein:RNA ratio in the ribosome would have occurred only once if those euryarchaeal groups still retaining the high ratio [67] all branched more deeply than those exhibiting the low ratio, which is the case in some phylogenetic analyses of the rRNA sequence data

5 The new microbiology

Two developments have radically transformed the science of microbiology: (i) the tech- niques that permitted the determination of a natural microbial system, and (ii) the consequent (but unanticipated) discovery of the archaea In the past, microbiology was an isolated, excessively anecdotal, and materialistic (in a molecularhiochemical sense) discipline The prokaryote/eukaryote dichotomy served as an intellectual wall that isolated microbiology from the rest of biology Now the eukaryote/prokaryote differences serve as a beacon that illumines microbiology’s distant evolutionary past, and allows the biologist to begin to glimpse the true relationships between the prokaryotes and the eukaryotes Microbial taxonomy, which had been largely a collection of scattered facts about a great variety of organisms, is becoming a totally interconnected tree of relationships, on which each species becomes the flowering tip of some particular branch Microbial metabolic pathways were, in a biological sense, superficially understood, interesting from a molecular mechanistic perspective and because of their impressive variety Now they will become parts of, and the keys to, a greater evolutionary whole, in which a simple prebiotic chemical seed grows into the myriad complex interconnected biochemical networks of modem living systems

Microbial ecology too has changed For the first time it is possible to give a complete accounting of the microbial population in any given environment Norman Pace, with bold insight, recognized that the sequencing techniques successfully used to

characterize cultured microorganisms phylogenetically, can be applied directly to any

given niche[71,72]: One can ask the question “Who’s there?”[72] of that niche, and answer it by cloning and sequencing particular genes from nucleic acid that is directly extracted from the niche[73] The construction of specific or class probes from such sequences then permits the direct microscopic identification of those organisms in the environmental sample from which the genes have come [74] Thus the microbiologist can in principle define every species in any given environment, including those that have eluded culture, and, more importantly, those that have gone completely unrecognized The combination of these methods with traditional methods of isolation should now permit the isolation, and so, detailed study of whole new groups of microorganisms Here is the microbial ecology that in the words of Beijerinck (quoted above) “ constantly keeps before our minds the profound problem of the origin of life itself.” By adding this

of the endosymbiosis hypothesis have posited [23] Given that there are two (unrelated) groups of prokaryotes and that one of them, the archaea, is more closely associated with the eukaryotes than the other, notions of the role of endosymbiosis in the origin of the eukaryotic cell are refining and changing; the possibility of endosymbioses from two very different sources now exists One wonders whether endosymbioses have been far more instrumental in shaping the eukaryotic cell than can be inferred from the (genetically) small contributions made by the mitochondria and chloroplasts Were there perhaps hosts of endosymbioses involved in this evolution, which left behind only genetic (not cytostructural) residues? Did the eukaryotic cell receive numerous infusions of smaller parts of prokaryotic chromosomes by means other than endosymbiosis? What fraction

of the eukaryotic genome has been contributed by each of the two prokaryotic types? The most extreme view here is represented by W Zillig and coworkers; based upon their experience with DNA-dependent RNA polymerase sequence analyses, they see the eukaryotic cell as nothing more than a chimeric collection of (eu)bacterial and archaeal genes ( ref [75], and W Zillig, personal communication) Let this and other possibilities

be tested through appropriate genome sequencing studies

The relatively close phylogenetic relationship between the archaea and the eukaryotes should prove of great value (especially given the small size of archaeal genomes) The archaeal RNA polymerase is closer in sequence to eukaryotic RNA polymerasesI1 and

I11 than these two are to one another[75] An even clearer and more striking situation

holds for the archaeal histone (from Methanothermus), whose sequence is closer to the

sequences of eukaryotic histones H2a, H2b, H3 and H4 than any of these four are to one another [24] Are these cases the tip of the iceberg; will a large number of individual

archaeal genes turn out to have an ancestral relationship to families of eukaryotic genes?

The fact that three primary groups of organisms (rather than two) exist, makes the problem of the nature of the universal ancestor a more tractable one than biologists previously thought In that nothing can be said with certainty about the ancestor at this point, my mentioning it here serves mainly to underscore its importance Perhaps the key question concerning the (most recent) universal ancestor is whether it was a progenote (see above) or a full-fledged genote[52] Given the number of known homologous genes in common between bacteria and eukaryotes, there can be little doubt that this ancestor already contained a fair number of genes However, as mentioned above, the transition from the rRNA of this ancestor to the rRNAs representative of the three primary lineages involved drastic and relatively rapid change, probably in all lineages; the simplest interpretation of this being that the universal ancestor was a simpler, more rudimentary entity than its descendants (as regards the translation function in particular), which would

xxvii

make it, by definition, a progenote [52,76] However, the biologist does not have to leave

the question of the nature of the universal ancestor in the realm of conjecture Through

a concerted program of sequencing the proper selection of genomes, a great deal can

be inferred about the nature of this important entity and its own ancestry Unfortunately,

biology today is in the throes of an internecine struggle between what I would call

“technologists”, who perceive the science as a discipline whose purpose is to solve practical problems (medical, agricultural, environmental) and “fundamentalists”, who perceive it as a basic science, one that tells us at a deep level about the nature of reality The thrust for genome sequencing (its rationale and choice of genomes to be sequenced) comes now from the technologists It is time to assert the fundamentalist perspective

Acknowledgment

The author’s work on archaea is supported by NASA grant NSG70440-18

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Contents

V

Preface

Introduction The archaea: Their history and signijicance

Carl R Woese vii

vii

I Introduction

xi

2 Microbiology’s changing evolutionary perspective

3 A molecular definition of the three domains

4 Archaeal phylogenetic relationships xxi

5 The new microbiology xxv Acknowledgment xxvii References xxvii

xv

List of contributors xxxi

Chapter 1 Central metabolism of the archaea

M.J Danson

1 I Central metabolism

1.2 Central metabolism in eukaryotes and eubacteria

2 Hexose catabolism in the archaea

2.1 The modified Entner-Doudoroff pathway of the halophiles

2.2 The non-phosphorylated pathway of the thermophiles

2.3 The glycolytic pathway of the methanogens

2.4 Gluconeogenesis

2.5 The pentose-phosphate pathway

3 Pyruvate oxidation to acety-CoA in the archaea

3.1 Pyruvate oxidoreductases

3.2 Comparison with the eukaryotic and eubacterial enzymes

3.3 Dihydrolipoamide dehydrogenase

4 The citric acid cycle in the archaea

4.1 The oxidative citric acid cycle

4.2 The reductive citric acid cycle

4.3 Partial citric acid cycles

4.4 Other pathways of acety-CoA metabolism

5 Amino acid and lipid metabolism in the archaea

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6 Evolution of central metabolism

6.1 Hexose catabolism

6.2 Pyruvate oxidation to acety-CoA

6.3 The citric acid cycle

Comparative enzymology of central metabolism

7.1 The enzymes as molecular chronometers

7.2 Citrate synthase

7.2.1

7.2.2 Archaebacterial citrate synthases

8 Conclusions and perspectives

Acknowledgements

Note added in proof

References

7

The comparative enzymology of citrate synthases

Chapter 2 Bioenergetics of extreme halophiles

l?P Skulachev

1 Introduction

2 A general scheme of energy transduction in extreme halophiles

3 Bacteriorhodopsin and halorhodopsin

3.1 Transmembrane charge displacement

4.2 Hydrogenase and non-catalytic redox proteins such as ferredoxin and cytochromes

4.2 1 The methylviologen-reducing hydrogenase (MVH)

4.2.2 Redox-active proteins: ferredoxin, cytochromes, and others

3.3 Methanogenesis from acetate

Transport of acetate into the cell

4.2.3 The F42o-reducing hydrogenase (FRH)

4.3 Alcohol dehydrogenase (ADH)

4.13 Methanol methanogenesis-related methyltransferases

4.14 Carbon monoxide dehydrogenase complex

4.15 Acetate activating enzymes

5 Key remaining physiological and enzymatic ques

2.2 Reduction of a methyl group t

3 Energetics of methanogenesis from CO2/H2

3.1 Enzymology

3.1 1 COz reduction to formyl-MFR (formate level)

3 I 2 Formyl-MFR reduction to methylene-H4MPT (formaldehyde level)

3.1.3 Methylene-H4MPT conversion to methylkoenzyme M (methanol level)

3.1.4 Methylkoenzyme M (CH3-S-CoM) reduction to methane

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3.2 Sites of energy coupling

3.2.1 General aspects

3.2.1 1 Growth yields and ATP gains

3.2.1.2 Mechanism of ATP synthesis

3.2.1.3 Thermodynamics of partial reactions

3.2.2 Methylkoenzyme M reduction to CH4 - site of primary ApH'generation

and of ATP synthesis

3.2.2.1 Heterodisulfide (CoM-S-S-HTP) reduction - coupled to primary

H' translocation 3.2.2.2 ATP synthaseiATPase

3.2.2.3 Misleading concepts of ATP synthesis

3.2.3 Methylene-H4MPT conversion to methylkoenzyme M - site of primary

AjiNa' generation

3.2.3.1 Methyl-H4MPT: coenzyme M methyltransferase - coupled to

primary Na' translocation

3.2.4 C02 reduction to methylene-H4MPT - site of primary APNa'

consumption

3.2.4.1 Formaldehyde oxidation to C02 - coupled to primary Na'

extrusion

3.2.4.2 C02 reduction to methylene-H4MPT - coupled to Na+ uptake

Role of the Na'lH' antiporter in C02 reduction to CH4

3.3.2 Role of the Na'iH' antiporter

3.3.3 Primary cycles of Nat and H'

3.3

3.3.1 Nat/H+ antiporter

Energetics of CH4 formation from formate

Energetics of CH4 formation from C02 reduction by alcohols

Energetics of C02 reduction to CH4 by methanogens versus C02 reduction to

4 Energetics of methanogenesis from methanol

4.1 Enzymology

4.2 Energetics

4.2.1, Role of the Na+/H+ antiporter

4.2.4 Growth yields

4.3 Energetics of CH4 formation from methylamines

5 Energetics of methanogenesis from acetate

5.1 Enzymology

5.2.1 CH3-S-CoM reduction to CH4

5.2.2 CO oxidation to C02

5.2.3 CH~-H~MPT:H-SSCOM methyltransferase

6 Energetics of pyruvate catabolism

6.2 Acetate formation from pyruvat the absence of methanogenesis

7 Concluding remarks on energetics

4.2.2 Role of methyltransferase

4.2.3 Role of cytochromes

5.2 Sites of energy coupling

5.3 Acetate fermentation in Methanothrix soehngenii

6.1 Methanogenesis from pyruvate

9.1 Energetics of Archaeoglobus fulgidits

9.1.1 Acetyl-CoA oxidation to C02 via a modified acetyl-CoAicarbon monox-

8.5 Phosphate

9

ide dehydrogenase pathway

9.2 Energetics of Pyrococcus furioszrs

9.2.1 Novel sugar degradation pathway in Pyrococcus furiosus

9.2.2 Sugar degradation to acetate, C02 and H2 via a novel fermentation

pathway

Acknowledgements

References

9.2.3 Open questions

Chapter 5 Signal transduction in halobacteria

D Oesterhelt and K Manvan

3.3 Identification of a switch factor

3.4 Light-induced release of fumarate

3.5 Methyl-accepting taxis proteins

3.6 Cyclic GMP

4 The photoreceptors

4.1 Spectroscopic and biochemical properties

4.2 The physiology of photoreception

3.2 Signal formation

Acknowledgement

References

Chapter 6 Ion transport rhodopsins (bacteriorhodopsin and halo-

rhodopsin): Structure and function