DNA Supercoiling and Temperature Adaptation: A Clue to EarlyDiversification of Life?

Purificacion Lopez-Garcıa

Institut de Ge´netique et Microbiologie, Universite´ Paris-Sud, Baˆt. 409, 91405 Orsay Cedex, France

Abstract. Cellular systems to control an appropriateDNA geometry for function probably evolved simulta-neously with DNA genomes. Such systems are basicallyDNA topoisomerases and DNA-binding proteins. There-fore, their distribution in extant organisms may be asource of information on early evolution and the natureof the last common ancestor (cenancestor). Most livingbeings need the strand-opening potential of negativeDNA supercoiling to allow transcription and other DNA-dependent processes. Mesophiles have global negativelysupercoiled DNA, essentially due to gyrase (introducingnegative supercoils) in bacteria and to DNA wrappingaround histone cores in eukaryotes. Mesophilic archaea,halophilic methanogens, and halophiles might use a gy-rase, whereas some methanogens might use histonewrapping. The existence of these two distinct mecha-nisms suggests that mesophily appeared at least twice inevolution. On the other hand, only one system which isbased on reverse gyrase (introducing positive supercoils)appears to be required for hyperthermophilic life. Ar-chaeal hyperthermophiles lacking gyrase have relaxed topositively supercoiled DNA, but hyperthermophilic bac-teria of the genusThermotoga,which have both gyraseand reverse gyrase, have negative supercoiling. This sug-gests that reverse gyrase is necessary at least locally, butwhereas these hyperthermophilic bacteria favor generalmelting potential and stability at critical active regions,hyperthermophilic archaea favor general linking excessand local melting. In this context, the existence of athermophilic (60–80°C) ancestor endowed with only re-

laxing topoisomerases is hypothesized. Such tempera-tures allow a compromise between melting potential andstability, i.e., an appropriate DNA geometry for function.Subsequent duplication and functional specialization ofexisting DNA topoisomerases would then have facili-tated adaptation to hyperthermophily and mesophily inarchaea and bacteria, respectively. If reverse gyrase is anancient character in hyperthermophilic bacteria, the ce-nancestor would have already been a hyperthermophile.Histone sequence homology and similarities of nucleo-some structural dynamics suggest that eukaryotes inher-ited this system for DNA structural homeostasis frommethanogenic euryarchaea. Some mesophilic archaeawould have improved their adaptability to mesophily byimporting gyrase from bacteria.

Key words: DNA supercoiling — DNA topoisomer-ases — Gyrase — Reverse gyrase — Hyperthermophily— Histones — Nucleosome — Cenancestor — Archaea

Introduction

The appearance of a DNA world must have been paral-leled by the development of systems to maintain theintegrity and functionality of DNA molecules. DNA to-poisomerases and DNA-binding proteins (histone andhistone-like) are the key components of such systems.Histones are found in eukaryotes and some archaea andthey allow DNA wrapping around nucleosomal cores(Kornberg 1977; Starich et al. 1996; Luger et al. 1997;Pereira et al. 1997). Histone-like proteins are widespreadin the three domains of life, but unlike true histones, theyE-mail: puri@igmors.u-psud.fr

J Mol Evol (1999) 49:439–452

© Springer-Verlag New York Inc. 1999

cannot wrap DNA. Although histone-like proteins arenot generally sequence homologues, they do share sev-eral properties, being small, generally basic, nonspecific,DNA bending, and unwinding, normally via the minorgroove (Drlica and Rouvie`re-Yaniv 1987; Schmid 1990;Laine et al. 1991; Grove et al. 1996; Robinson et al.1998). DNA topoisomerases are essential to disentangleDNA strands or duplexes, being charged to resolve to-pological problems arising from DNA-derived processes.Thus, they intervene in a high variety of cellular func-tions, ranging from transcription, recombination, andreplication (including decatenation and segregation ofchromosomes) to the regulation of DNA supercoilingand maintenance of genome stability (Wang et al. 1990;Drlica 1992; Luttinger 1995; Wang 1996). DNA topoi-somerases are mechanistically classified as type I or typeII, depending on their ability to act via a single-strand ora double-strand break. In turn, each mechanistic typegroups two distinct phylogenetic families (see Table 1)(Forterre et al. 1994, 1996; Wang 1996; Bergerat et al.1997). In Archaea, Eukarya, and Bacteria, type I and IIenzymes are indispensable and coexist in the cell, whichsuggests that both mechanistic types evolved before do-main diversification.

The hypothesis of a hot origin of life is favored nowa-days based on two kinds of evidence. Phylogenetically,the hyperthermophilic nature of deep-branching archaeaand bacteria suggest that their ancestor was also a hy-perthermophile (Woese 1987; Wa¨chtersha¨user 1988;Woese et al. 1990; Pace 1991; Stetter 1995, 1996). Inagreement, geological evidence suggests that when lifearose (3.8–4.2 Ga), Earth environments resembled hy-perthermophilic biotopes today (Baross and Hoffman1985; Nisbet 1985; Shock 1996). A hot autotrophic ori-gin appeared specially supported after the root of thephylogenetic tree of life was placed in the bacterialbranch (Woese et al. 1990; Brown and Doolittle 1995;Baldauf et al. 1996). Although the root of the tree and thehot origin of life are still controversial (Forterre 1996),some authors have indicated the need to distinguish be-tween the conditions in which the origin of life tookplace and those in which the last common ancestor orcenancestor was thriving (Miller and Lazcano 1995). Hy-perthermophilic or not, the cenancestor was already quitecomplex, as can be deduced from comparative analysesof archaeal, bacterial, and eukaryal genomes (Edgell andDoolittle 1997; Olsen and Woese 1997). A more or lesslong period of evolution in between, accompanied by the

Table 1. Classification and main features of DNA topoisomerasesa

Mechanistictype Main features

Phylogeneticsubfamily Main features Subtype

Representative/firstdescribed example(s) Reference(s)

I Catalyze the passageof one DNA strandor helix throughssDNA breaks

IA ssDNA preferentialactivity

RG-like Sulfolobusacidocaldarius

Kikuchi et al. (1984);Forterre et al. (1985);for review; see Duguet(1995)

ATP independentb Relax only negativesupercoils

I-like Topo I Escherichia coli Wang (1971); for review,see Wang (1996)

Monomericc Covalently linked to 58end of transientcleavage

III-like Topo III E. coli Srivenugopal et al.(1984); for review, seeWang (1996)

IB dsDNA preferentialbinding

Eukaryotic Topo I(Mus musculus)

Champoux and Dulbecco(1972)

Relax negative &positive supercoils

Covalently linked to 38end of cleavage

Archaeal Topo Vd

(Methanopyrusknadleri)

Kozyavkin et al. (1994)

II Catalyze the passageof a DNA helixthrough dsDNAbreaks

IIA(classic)

Relax positive &negative supercoils

G-like Topo II E. coli (gyrase,GyrA + GyrB)

Gellert et al. (1976); forreview, see Wang(1996)

ATP-dependent Catenation/decatenation58 end cleavage binding

Non-G Topo IVE. coli(ParC + ParE)

Kato et al. (1990); forreview, see Wang(1996)

Homodimer(eukaryotes) orheterotetramer(prokaryotes)

IIB Relax positive &negative supercoils

Catenation/decatenation58 end cleavage binding

Topo VI Sulfolobusshibatae

Bergerat et al. (1997)

a Topo, topoisomerase; G, gyrase; RG, reverse gyrase.b Except reverse gyrase, with an ATP-binding site in the helicase-likedomain.c ExceptM. kandlerii RG, which is split in the middle of the topoi-somerase domain (Krah et al. 1996).

d Topo V biochemically behaves like members of the IB eukaryoticfamily, but sequence analysis could confirm its inclusion in an inde-pendent family (A Slesarev, personal communication).

440

possibility of significant environmental changes, is there-fore implicit.

One of the most relevant features of hyperthermo-philes is the possession of the special DNA topoisomer-ase reverse gyrase, which has been consistently found inall bacteria and archaea growing optimally above 80°C(Duguet 1995). The discovery of this novel activity, ca-pable of creating positive supercoils in DNA molecules,not only opened up a discussion about its likely role instabilizing DNA at high temperature, but also fed someevolutionary debates. Since the gene for reverse gyraseappears to result from a fusion event between two pre-existing genes (a putative helicase + topoisomerase), theprimitiveness of hyperthermophiles was questioned (For-terre et al. 1995). A proposal for the evolution of thisenzyme in the course of a reduction process from a me-sophile to a hypothetic hyperthermophilic common an-cestor to prokaryotes was formulated (Forterre 1995).

However, as discussed in this report, the phylogeneticdistribution of reverse gyrase and other DNA topoisom-erase-based systems for DNA homeostasis, namely, gy-rase- and nucleosome-based systems, suggests that me-sophily appeared at least twice in evolution, possiblylater than hyperthermophily. I speculate on the existenceof a thermophilic ancestor (60–80°C) lacking supercoil-introducing topoisomerases (gyrase and reverse gyrase)with an appropriate DNA conformation for function be-ing spontaneously generated. Strategies involving spe-cialization of DNA topoisomerases, as well as histonewrapping, evolved subsequently to regulate DNA struc-ture improving the adaptability of organisms to differenttemperature niches.

The Role of DNA Supercoiling in Living Beings

DNA Supercoiling, DNA Topology, andTopoisomerase Function

DNA supercoiling is currently used to approximate thespatial structure of DNA molecules, or, more precisely,their geometry. However, the geometry of the DNA mol-eculein vivo is difficult to assess, because it is influencedby different factors, for instance, protein binding. This isthe reason why, as an easily measurable topologicalproperty, the linking number (Lk) is estimated in prac-tice. Lk represents the number of links between the twostrands of the double helix and is a constant value for acovalently closed DNA molecule (ccDNA). Lk can bedecomposed into two geometrical parameters that deter-mine the shape of the molecule in the space: Lk4 Tw+ Wr. The twist (Tw) represents the wrapping of onestrand around the other, being directly related to the pathof the helix, and the writhe (Wr) corresponds to the coil-ing of the helix’s axis in space, i.e., supercoiling (for areview, see Bates and Maxwell 1993). For a ccDNA, any

change in Tw or Wr can affect the other geometricalcomponent, but not Lk. Lk can be altered only by aprocess of cutting, passing through, and resealing DNAstrands or duplexes, i.e., a topoisomerase activity. Underthese premises, DNA supercoiling and DNA topology(Lk) are straightforwardly related. Both parameters aresometimes employed indiscriminately to describe DNAgeometry in living cells (especially in bacteria, whereDNA–protein binding is not so extensive as in eukaryoticnucleosomes; see below). The underlying basis for this isthat Tw cannot change too much at global levels (onlylocally) if general functionality is to be maintained (im-portant changes in Tw would lead to denaturation orinactive structures).

The geometry of the DNA molecule in the cell canalso be directly affected by factors other than Lk changesinduced by topoisomerase action. Among these, physicalparameters, such as salt or temperature, can make DNAan environmental sensor (McClellan et al. 1990; Higginset al. 1990). Also important are the attachment to his-tones or other DNA-binding proteins (Drlica and Rouvi-ere-Yaniv 1987; Travers 1994) and protein tracking dur-ing DNA-dependent processes (Kornberg and Lorch1992; Droge 1993; Wang and Lynch 1993; Wang 1996).None of these factors can modify the topology of accDNA moleculeper se;however, they can do it indi-rectly by attracting topoisomerases to release geometri-cally induced tensions. Most probably the primary func-tion for which topoisomerases evolved was the release oflocal torsional stress generated during transcription, rep-lication, and recombination. In addition to the resolutionof topological problems during those processes, DNAtopoisomerases are responsible, alone or with auxiliaryDNA-binding proteins, for the maintenance of globallevels of DNA supercoiling (Drlica 1992; Luttinger1995; Wang 1996).

Optimal DNA Geometry and Supercoiling Levels inPresent-Day Organisms

It has been hypothesized that a precise overall geometryof the DNA molecule exists where the appropriate rela-tive distances are maintained so that the correct interac-tion with all proteins and factors involved in DNA-dependent processes is assured (Forterre et al. 1996).This hypothesis was originally based on the differentDNA topologies found in living organisms. In all meso-philes belonging to the three domains of life, DNA isnegatively supercoiled, with specific linking differences(relative measures ofDLk) between −0.07 and −0.05 invitro (Worcel and Burgi 1972; Germond et al. 1975;Wang 1987; Sioud et al. 1988; Charbonnier and Forterre1994). On the other hand, in hyperthermophilic archaea,plasmid DNA is from relaxed to positively supercoiled,with specific linking differences from −0.006 to +0.03(Charbonnier and Forterre 1994; Lo´pez-Garcı´a and For-

441

terre 1997). A linking deficit in mesophiles would pro-vide the required activation energy for DNA-dependentprocesses, compensating for the physical effect of lowertemperatures, which would tend to increase the twist. Onthe other hand, a linking excess in hyperthermophileswould counteract the denaturing effect of high tempera-tures (Fig. 1) (Forterre et al. 1996). However, the 846-bpplasmid pRQ7 from the hyperthermophilic bacteriumThermotogasp. RQ7 has recently been shown to benegatively supercoiled (Guipaud et al. 1997), suggestingthat levels of negative supercoiling depend exclusivelyon the presence or absence of gyrase (see below). The Lkdifferences between archaeal and bacterial hyperthermo-philes suggest that a local appropriate geometry is nec-essary but also sufficient for life, provided that measuresagainst DNA denaturation or degradation exist.

Nonetheless, the hypothesis of an optimal DNA ge-ometry has to be interpreted in relative terms. Indeed, forany given organism global levels of supercoiling are un-der homeostatic control and may vary (within certainlimits) depending on, for instance, growth conditions(Balke and Gralla 1987; Jensen et al. 1995) or differentenvironmental stimuli including shifts in osmolarity(Hsieh et al. 1991b), oxygen concentration (Hsieh et al.1991a), pH (Karem and Foster 1993), and temperaturewithin the growth range of the organism (Goldstein andDrlica 1984). These environmentally induced changeshave been suggested to control overall gene expression(Higgins et al. 1990; Dorman 1995). Global supercoilingchanges are known to act locally and regulate the tran-scription of many genes having promoters sensitive tosupercoiling (Wang and Lynch 1993). In hyperthermo-philic archaea, plasmid DNA supercoiling also variesduring growth and depending on temperature and mayeven reach negative values (Lo´pez-Garcı´a and Forterre1997).

It can be concluded that a certain degree of negativesupercoiling is, at least locally, necessary for active DNAin most, if not all, organisms, since it provides the energyof activation required to open both DNA strands for tran-scription or replication (Weintraub 1985; Wang 1996).

The only possible exception would be hyperthermophilicarchaea, for which contradictory reports from in vitrotranscription systems exist. Whereas negative supercoil-ing seems to be required inPyrococcus(Soares et al.1998; C Hethke, M Thomm, and P Forterre, unpublishedresults), template topology has no influence at all at nor-mal growth temperatures inSulfolobus(Bell et al. 1998).Therefore, in these organisms high temperatures may ac-tually provide the strand-opening potential needed. How-ever, negative supercoiling is indispensable for transcrip-tion at lower temperatures (Bell et al. 1998). Thus,whereas in mesophiles global negative supercoiling isessential to facilitate opening of DNA strands, in hyper-thermophiles, the key problem is the opposite, to preventexcessive denaturation by high temperatures. Archaealhyperthermophiles generate global linking excess,whereas bacterial hyperthermophiles may avoid exces-sive local melting: i.e., whereas archaea favor generalstability and local melting, bacteria favor melting poten-tial and local stability at critical active regions (see be-low).

Systems for Homeostatic Control ofDNA Supercoiling

Gene and genome sequencing in combination with bio-chemical studies are providing a more complete and un-expected picture of the distribution of DNA-binding pro-teins and topoisomerases in the living world. I commentbriefly on the occurrence of key proteins generating therespective global levels of supercoiling in the three do-mains of life, namely, gyrase, reverse gyrase, and his-tones (summarized in Figs. 1 and 2).

Bacteria

All known bacteria have negatively supercoiled DNA.Global DNA supercoiling in bacteria is essentially due tothe concerted action of DNA topoisomerases, although

Fig. 1. Effects of temperature on theoptimal DNA geometry for function andsystems developed by living organisms forits homeostatic control. Mesophilesgenerate a linking deficit (DLk < 0), andhyperthermophilic archaea generate ageneral linking excess (DLk $ 0), whilehyperthermophilic bacteria may use reversegyrase locally (see text).

442

histone-like proteins such as HU can help to fine-tunesupercoiling levels, for instance, during thermal stress(Ogata et al. 1997; Mizushima et al. 1997). Most studiesderive from mesophilic bacteria, where control of DNAsupercoiling implies the antagonistic action of gyrase,introducing negative superturns, and a type I topoisom-erase with the opposite effect (proteinv) (Drlica 1992;Wang 1996). There are other topoisomerases in bacteria,although apparently they do not have a major role inmaintaining general levels of supercoiling. These aretype I topoisomerase III (Topo III) and type II topoisom-erase IV (Topo IV), both carrying only relaxing activities(Luttinger 1995; Wang 1996) (Table 1). Topo IV waspreviously thought to be widespread in all bacteria, but itturned out to be restricted to some branches (Fig. 2) (seebelow and Huang 1996; Kaneko and Tabata 1997; Gui-paud et al. 1997).

In hyperthermophilic bacteria of the genusThermo-toga, where gyrase and reverse gyrase coexist, plasmidnegative supercoiling is detected (Guipaud et al. 1997).This implies that gyrase is more active or efficient thanreverse gyrase at normal growth temperatures. The func-tion of reverse gyrase in vivo is not fully understood, butits recurrent presence in hyperthermophiles suggests anessential role at high temperatures. If not for the main-tenance of relaxed or positively supercoiled overall

DNA, it may be related to resistance to increased tem-peratures or to the prevention of DNA denaturation dur-ing critical openings of the double helix (Guipaud et al.1997). Indeed, at least in hyperthermophilic archaealacking a masking gyrase activity, an increased reversegyrase activity and induction of plasmid positive super-coiling is observed during heat shock and during growthat supraoptimal temperatures (Lo´pez-Garcı´a and Forterre1997).

Eukaryotes

All eukaryotes are mesophiles or slight thermophiles andhave negatively supercoiled DNA constrained by nucleo-somes. The eukaryotic strategy to generate global levelsof supercoiling is radically different from that used bybacteria. Protein binding not only helps to adjust preciselevels of supercoiling induced by topoisomerases, but isnecessary to generate high levels of negative supercoil-ing. Eukaryotes carry only relaxing DNA topoisomerases(Table 1), and indeed, bulk chromatin is not under tor-sional stress (Sinden et al. 1980). Negative supercoilingis therefore generated, and restrained, by DNA wrappingaround the histone core, followed by relaxation of theinternucleosomal tension by topoisomerases (Saavedra

Fig. 2. Distribution of reverse gyrase, gyrase, topoisomerase IV (Topo IV), and histone genes in the three domains of life.Thick linesrepresenthyperthermophilic lineages. Archaeal and bacterial trees are based on Woese et al. (1990) and Pace (1997). Notes: a,Synechocystispossesses twogyrA genes; b, the only set of genes present inAquifexcannot be presently ascribed to a gyrase or a Topo IV by sequence similarity (see text).

443

and Huberman 1986). The final result is that, if oneremoves histones and topoisomerases, whole DNA isnegatively supercoiled to about the same extent as DNAfrom prokaryotic mesophiles (Wang 1987). Other eu-karyotic DNA-binding proteins, such as the high mobil-ity-group proteins (HMGs), exist. They are involved inDNA bending, having a function similar to that of bac-terial HU and IHF proteins (Bustin et al. 1990; Grove etal. 1996).

Archaea

In the domain Archaea, studies about the regulation ofDNA supercoiling are in their infancy, but already thesituation seems more complex. Levels of supercoilingmay vary from highly negative to positive, and the typeand content of DNA topoisomerases and DNA-bindingproteins suggest similarities to both bacterial and eukary-otic machineries (Figs. 1 and 2).

Thus, an involvement of DNA topoisomerases similarto that of mesophilic bacteria for control of supercoilingcould exist in halophilic euryarchaea, since they havehighly negatively supercoiled DNA (Mojica et al. 1994)and may carry a bacterial-like gyrase. At least, the pres-ence of bacterialgyr-like genes has been reported(Holmes and Dyall-Smith 1991), as well as sensitivity tolow doses of the gyrase inhibitor novobiocin (Sioud et al.1988).

All hyperthermophilic archaea studied so far have areverse gyrase and relaxed to positively supercoiledDNA. In crenarchaea, small DNA-binding proteins existwhich have features similar to those of bacterial (HU,etc.) or eukaryal (HMGs) histone-like proteins, includingDNA unwinding proteins, such as Sac/Sso7 and relatives(Lopez-Garcı´a et al. 1998; Mai et al. 1998; Robinson etal. 1998). In these organisms, control of supercoilingcould involve the antagonistic action of reverse gyraseand one relaxing activity (Forterre and Elie 1993; For-terre et al. 1996). Among the relaxing topoisomerasecandidates that are widespread in both archaeal king-doms, only a type II enzyme exists, topoisomerase VI(Topo VI) (Table 1). Topo VI, first purified fromSul-folobus shibatae(Bergerat et al. 1994), is the prototypeof a new phylogenetic family characteristic of archaeaand with some eukaryal protein homologues (Bergerat etal. 1997). Regarding type I topoisomerases with exclu-sively relaxing activities, Topo III-like genes have beendetected in both archaeal branches (Bult et al. 1996; Fitz-Gibbon et al. 1997), and the enzymatic activity may havebeen detected previously in archaea (Slesarev et al.1991). Also, Topo V, a biochemical member of the “eu-karyotic” IB subfamily, has been described inMethano-pyrus kandlerii (Slesarev et al. 1993) (Table 1), butwhether or not its presence is widespread in archaea isnot known.

Interestingly, early-branching euryarchaea possess

true histones (Fig. 2). These have been found in hyper-thermophilic and some mesophilic methanogens (Metha-nococcales and Methanobacteriales), as well as Thermo-coccales (Grayling et al. 1994; Sandman et al. 1994;Ronimus and Musgrave 1996; Darcy et al. 1995). How-ever, histones are present neither inThermoplasma,where a protein homologous to the bacterial HU, HTa,was described (DeLange et al. 1981; Grayling et al.1994), nor in Methanomicrobiales, where MC1 is themost abundant histone-like protein (Chartier et al. 1988,1989). In haloarchaea, a gene coding for a MC1-likeprotein was found (Darkacheva and Kagramanova 1994;A. Mankin, personal communication), but whether or nothistones occur is unknown. Histones have never beendetected in crenarchaea, despite intensive searches andthe fact that these proteins are very abundant in the cell.Archaeal histones not only exhibit sequence homologywith eukaryotic ones, but also form nucleosomal struc-tures with canonical histone folds (Starich et al. 1996).Archaeal nucleosomes have recently been found to con-strain negative supercoils at physiological conditions ofsalt and temperature (Musgrave et al. 1999). They couldhelp to provide the local melting necessary for DNA-dependent processes under a control situation, i.e., in acontext of general linking excess. Moreover, they couldhave a primordial role in mesophilic euryarchaea havinglost reverse gyrase and still devoid of gyrase. In this case,some archaea would use a “eukaryotic” strategy for ho-meostasis of DNA structure (see below).

Did Early Diversification of Life Correlate withAdaptation to Distinct Temperature Niches?

An appropriate DNA geometry for function must havebeen conserved throughout evolution, keeping the bal-ance between stability and melting potential. SinceDNA-binding proteins and topoisomerases are essentialcomponents of the machinery controlling DNA topology,their phylogenetic and environmental distribution is ba-sic to understanding their functional evolution and puta-tive role in temperature adaptation. In this section, I com-ment on these aspects, using preliminary conclusions totrace some hypothetical evolutionary pathways.

Reverse Gyrase and Hyperthermophily

An interesting example is constituted by type I topoi-somerases, in general, and reverse gyrase, in particular.Topoisomerases evolved to resolve topological problemsin nucleic acid molecules. Early replicative genomesmight have been composed of small linear nucleic acidsbefore the evolution of any topoisomerase activity. Lo-cally generated tensions might have been released spon-taneously (see below and Fig. 4, bottom). In this context,type I topoisomerases, simpler than type II enzymes and

444

ATP-independent, likely evolved first, which could con-tribute to size increase of small replicons. This mighthave occurred before the DNA era, during a putativeRNA-protein world. Moreover, type I enzymes mighthave played an essential role facilitating the transitionbetween both RNA and DNA-based worlds, particularlyTopo III-like enzymes. Not only are these ubiquitous inthe three domains of life, but also theEscherichia coliTopo III has been shown to be a true RNA topoisomerase(DiGate and Marians 1992; Wang et al. 1996). Theycould have supplied an initial segregating activity. Infact, Topo III is able in vitro to decatenate and could beinvolved in chromosomal replication or in mRNA decat-enation during transcription (DiGate and Marians 1992;Wang et al. 1996). Its absence may be lethal in somecases (Li and Wang 1998).

Once this elementary relaxing function was in place,duplication, fusion with an helicase domain, and recruit-ment of a new function (generation of positive super-coils) could have resulted in reverse gyrase and allowedhyperthermophily. Since reverse gyrase is found in bothhyperthermophilic bacteria and archaea, it may havebeen present in their common ancestor. However, thepossibility remains for a horizontal transfer from archaeato bacteria (see below).

Gyrase and Other Type II Topoisomerases

Type II topoisomerases are more complex than type Ienzymes, often being composed of two kinds of subunitsand having ATP-dependent activity (Table 1). With theexception of gyrase, all type II enzymes can only relaxpositive and negative supercoils and are specifically in-volved in chromosome segregation and decatenationupon replication or recombination. That is the case forbacterial Topo IV, eukaryotic Topo II and, most likely,archaeal Topo VI (Luttinger 1995; Wang 1996; Bergeratet al. 1997). Chromosome segregation is an essentialfunction in increasingly larger genomes, and despite thepossible involvement of Topo III enzymes, this roleseems to be carried out essentially by type II topoisom-erases (Wang 1996). The primary function of type IIenzymes may thus have been chromosome disentanglingupon replication, although their evolution could haveprovided for a better efficiency for the removal of su-percoils as well.

As in the case of type I enzymes, one may hypoth-esize that once the primary function (chromosome decat-enation/segregation) was in place, duplication and spe-cialization in a second function, such as the activeintroduction of negative supercoils (gyrase), were al-lowed. Indeed, gyrase is a complex enzyme with at leastfour functions including maintenance of supercoiling, fa-cilitation of replication and transcription by generatingnegative supercoils in front of the respective complexes,removal of knots, and DNA bending and folding (Drlica

and Zhao 1997). In the absence of ATP, gyrase relaxesnegative supercoils. This adds regulatory possibilities tothe intracellular control of supercoiling levels as a re-sponse to environmental changes such as substrate deple-tion, osmolarity or temperature shifts (Jensen et al. 1995;Workum et al. 1996). Gyrase can also decatenate, butE.coli Topo IV is more than 100 times active for this func-tion and is more likely to carry out this role in vivo(Zechiedrich and Cozzarelli 1995). The gyration activity,which constitutes its major difference from Topo IV, isdue to its ability to wrap DNA around itself. As a matterof fact, the removal of the C-terminal end of the GyrAsubunit converts the gyrase to a relaxing Topo IV-likeenzyme (Kampranis and Maxwell 1996).

Most studies on gyrase and Topo IV have been per-formed usingE. coli as a model. However, completegenome sequencing is revealing striking differences intype II topoisomerase gene contents in bacteria. Topo IVgenes (parE and parC, homologous togyrB and gyrA,respectively) are not detected inT. maritima(Guipaud etal. 1997; http://www.tigr.org). Only one set of genes ispresent inAquifex aeolicus(Deckert et al. 1998), al-though based on gene similarity it is not clear whetherthey code for a Topo IV or a gyrase (Guipaud, personalcommunication). Also, only one set of genes can be re-trieved from the genome sequence ofDeinococcus ra-diodurans(http://www.tigr.org). In these organisms gy-rase must be the decatenase as well. The absence of TopoIV in many bacteria could well explain the failure toamplify gyrA-like sequences in the radioresistant micro-cocci andThermus,green sulfur, green nonsulfur, andPlanctomyces(Huang 1996). These organisms appear tobranch slightly earlier in the bacterial bush (Fig. 2) (Pace1997). An interesting case is the cyanobacteriumSyn-echocystissp., whose genome carries twogyrA se-quences (one more similar to its Topo IV homologueparC) but only onegyrB (Kaneko and Tabata 1997). Thissuggests that a duplication event took place just before,or during, the differentiation of cyanobacteria and that inthese organisms, GyrB could interact with both GyrA-and ParC-like proteins to perform two already individu-alized functions (GyrB + GyrA, a gyrase to controloverall levels of supercoiling, and GyrB + ParC-like, todecatenate chromosomes). Later in the bacterial branch, asecond duplication event, involvinggyrB,could have ledto the complete independence of both enzymatic func-tions and to a higher versatility for the colonization ofmeso- and psychrophilic environments. GyrA is indeed acold-shock protein (Jones et al. 1992). Interestingly,many of the bacteria with only one putative complete setof type II topoisomerases can be quite thermophilic.Thermusspp. can grow at up to 85°C, and cyanobacteriaand photosynthetic bacteria at up to 73°C (Brock 1986).

Another interesting feature on topoisomerase distri-bution is that the type II enzymes present in early-branching bacteria and archaea belong to different phy-

445

logenetic families. Topo VI is the only type IItopoisomerase detected so far in hyperthermophilic cre-narchaea and most hyperthermophilic euryarchaea (Fig.1). However, the respective B subunits of Topo VI andgyrase still exhibit some homology (Bergerat et al.1997). This could indicate that at the time of the pro-karyotic ancestor, a primitive type II topoisomerase ac-tivity existed, involving the interaction of a proto-B sub-unit with other DNA-interacting proteins able to cut and/or ligate DNA. The speciation of the domains Archaeaand Bacteria implied also the speciation of both topoi-somerase families (IIA and IIB; Table 1). This hypoth-esis would be in agreement with a primitive DNA rep-lication system at the time of the cenancestor (Edgell andDoolittle 1997; Olsen and Woese 1997).

In the euryarchaeal branch, halophiles have mostprobably imported a gyrase from bacteria (likely Grampositive). Indeed, horizontal transfer seems to have had aprofound impact in archaeal evolution (Hilario andGogarten 1993; Gogarten 1994, 1995; Doolittle 1998).Haloarchaeal gyrase genes branch in the middle of bac-terial ones (Forterre et al. 1994), and these organismsseem to have acquired other bacterial genes in this way,possibly from Gram-positive bacteria and cyanobacteria(Home et al. 1988; Altekar and Rajopalan 1990; Gogar-ten 1995; Doolittle 1998). Communities integrated bycyanobacteria and halophiles are widespread, and closefeeding interactions between them were shown to occurin cyanobacterial mats (Zviagintseva et al. 1995). Gyraseimport might have taken place earlier in euryarchaea,since Archaeoglobus fulgidusalready containsgyr ho-mologues, although the great similarity to theThermo-togagyrase (Guipaud 1998) would rather suggest an in-dependent horizontal transfer. Also, the key enzyme forits unique bacterial-like capacity to reduce sulfate seemsto have been imported from sulfate-reducing bacteria

(Doolittle 1998).Archaeoglobus, Thermotoga,and sul-fate-reducing bacterial genera are found extensively inthe same biotopes and frequently coisolated (L’Haridonet al. 1995; Hugenholtz et al. 1998).

Histone Wrapping: On the Way to Eukaryal Chromatin

As mentioned previously, a second way to generate ex-tensive negative supercoiling is DNA wrapping aroundhistone cores. This system is typically used by eukary-otes, which possess only relaxing type I and II DNAtopoisomerases (Table 1).

Eukaryotic nucleosomes are formed by an octamerbuilt upon a (H3–H4)2 tetramer plus two H2A–H2Bdimers. The roles of the tetramer and dimers in today’snucleosome are very different. Whereas the tetramer iscritical for the first interaction with DNA and to deter-mine nucleosome positioning, the H2A–H2B dimers as-semble later, having a likely role in transcriptional regu-lation. H2A and H2B probably evolved later than H3–H4, allowing further condensation of nucleosomal DNA(Ramakrishnan 1995). The tetramer (H3–H4)2 can har-bor positive and negative supercoils without affectingDNA twist (Hamiche et al. 1996). The transition betweenboth states occurs rapidly, can be triggered by thermalfluctuations, and is due to a conformational change of thetetramer to accommodate the sense of DNA coiling (Fig.3). This change takes place only in the absence of H2A–H2B dimers (their assemblage would block the left-handed structure), and a role in transcriptional regulationfor these highly dynamic transitions has been proposed(Hamiche et al. 1996).

Although archaeal histones were initially reported toform “reverse nucleosomes” by positive DNA wrapping(Musgrave et al. 1991), they apparently constrain nega-

Fig. 3. Schematic representationof the eukaryotic (H3–H4)2

conformational change toaccommodate negative and positivesupercoiling (SC) and its occurrencein archaeal nucleosomes. From leftto right, euryarchaealrepresentatives are displayedfollowing their branching order in16S rRNA trees and along athermal gradient. Hyperthermophilicspecies are indicated by anasterisk;asterisks in parenthesescorrespondto moderate thermophiles. Acommon origin of nucleosomepackaging and dynamics ineukaryotes and some methanogenicarchaea is suggested (see text).

446

tive supercoils under physiological conditions (Mus-grave et al. 1999). With the exception ofMethanopyrus,where two histone folds are fused in the same polypep-tide (Slesarev et al. 1998), a shift from negative to posi-tive wrapping can also be observed in archaeal nucleo-somes depending on salt concentration (Musgrave et al.1999). Therefore, sequence homology, structure, andnucleosomal dynamics point out to a common origin forarchaeal and eukaryal nucleosomes. In addition to theessential packaging function of eukaryotic nucleosomes,support for a more dynamic role via modification of localsupercoiling in transcriptional regulation is increasing(Wolffe and Pruss 1996; Luger et al. 1997; Luger andRichmond 1998). The same may be true for archaealnucleosomes (Musgrave et al. 1999). In fact, based onthe similarity of archaeal and eukaryotic histones to theCBF family of eukaryotic transcription factors, a parallelorigin of the nucleosome core and eukaryotic transcrip-tion from archaea has already been proposed (Ouzounisand Kyrpides 1996).

From the beginning, the physical interconversionfrom right- to left-handedness may have had a significantrole in transcriptional regulation in archaeal and eukaryalnucleosomes. Yet nucleosome formation may have alsobeen exploited to colonize mesophilic environments inarchaea devoid of gyrase, because it offers sufficientlypowerful machinery capable of generating melting po-tential at lower temperatures. In fact, looking at present-day mesophiles, they must have one of the two describedsystems to generate extensive negative supercoiling, ei-ther a gyrase (in bacteria and some archaea) or a histone-wrapping system (eukaryotes and some euryarchaea)(Figs. 1 and 2). The occurrence of these two systemsstrongly suggests an independent adaptation to mesoph-ily, i.e., mesophily appeared at least twice in evolution.

A second important conclusion is that the histone-based system used by a restricted range of methanogeniceuryarchaea possibly to package DNA, regulate tran-scription, and generate negative supercoiling may havepreceded the eukaryotic system. This constitutes a strongargument favoring recently published symbiotic hypoth-eses for the origin of eukaryotes in which an archaealmethanogenic partner is involved (Martin and Mu¨ller1998; Moreira and Lo´pez-Garcı´a 1998), in particular, ahistone-carrying methanogen having already lost reversegyrase (Fig. 2).

An evolutionary pathway in the euryarchaeal branchcan be hypothesized as follows. A gradient from hyper-thermophily to mesophily (and mesophilic halophily)can be traced along this phylogenetic lineage (Fig. 3).Hyperthermophiles possess a reverse gyrase required forpreventing general denaturation, whereas negative super-coiling (required for DNA transcription and other pro-cesses) is provided by nucleosome formation. Somemethanogens lost reverse gyrase and occupied less ther-mophilic niches. That may be the case for mesophiles

from the orders Methanococcales and Methanobacteri-ales. The closely related halophilic methanogens (Metha-nomicrobiales) and halophiles, which are mesophiles,may have imported gyrase from bacteria. Since gyraseseems to be strongly advantageous for mesophilic adap-tation, these organisms may have lost histones and sub-stituted them with histone-like proteins (like MC1 pro-teins) conferring more dynamic interactions. Thegenome as a whole would work much more in a bacte-rial-like fashion. This could also be the case forTher-moplasma,possessing the HU-like HTa. This proteinmay have a bacterial origin, and although the topoisom-erase content of Thermoplasmales is still unknown, agyrase could exist, since they are sensitive to novobiocin(Yasuda et al. 1995).Thermoplasma acidophilumwasisolated from acid coal piles, butThermoplasmarelativeshave recently been found to be much more widespreadthan thought, sharing the same environments as Metha-nomicrobiales and halophiles, for instance, in coastal saltmarshes (Munson et al. 1997). The related Methanomi-crobiales and halophiles generally thrive in salty bio-topes of this kind. Interestingly, high osmolarity and highsalt shocks are related to an increase in negative super-coiling (Hsieh et al. 1991b; Mojica et al. 1994), whichcould constitute an additional need to gain a gyrase.

A Thermophilic Ancestor

The generation of linking excess or deficit at general orlocal levels in DNA genomes may be essential for tem-perature adaptation. Hyperthermophiles utilize reversegyrase to prevent denaturation, whereas mesophiles usegyrase or histone-mediated mechanisms. In this sense,mesophily appears to be an adaptation at least as com-plex as hyperthermophily, having appeared at leasttwice. Reverse gyrase likely evolved earlier (or at thelatest at the same time) than gyrase or histone-wrappingmechanisms to control global supercoiling. One appeal-ing possibility is that the ancestor was a thermophile, stilldevoid of important supercoil-introducing activities,thriving around 60–80°C, in which the optimal DNAstructure for function was generated spontaneously (Fig.4). This configuration would be the most energeticallyfavored at those temperatures, corresponding to a com-promise between stability and melting capability. Ther-mophilic conditions would be necessary to provide theenergy of activation supplied by an excess of negativesupercoiling in mesophiles, while sufficient to preventthe risk of denaturation that higher temperatures wouldinduce. Small DNA-binding proteins likely played animportant role at this stage. They possibly stabilized thegenome, but also helped its biological activity due totheir bending/unwinding properties.

Two theoretical possibilities can be envisionned forthe further evolution of this thermophile (Fig. 4). The

447

first would imply the evolution of reverse gyrase in thearchaeal lineage, leading to a hyperthermophilic archaealancestor and a subsequent horizontal transfer to bacteria.The cenancestor would then be a thermophile. In thesecond possibility, reverse gyrase would have evolvedprior to the diversification of the prokaryotic domains,and the cenancestor would already be a hyperthermo-phile. In this case, bacterial reverse gyrase would be aremnant of early prokaryotic history.

In the hyperthermophilic lineage leading to archaea,the euryarchaeal branch developed histones serving ini-tially as stabilizing and bending/unwinding proteins,similar to crenarchaeal and bacterial histone-like pro-teins. The capacity of histones to interact and formhigher-order structures that wrap DNA was further ex-ploited for adaptation to less thermophilic environments,being the origin of the eukaryotic-like genome lifestyle.

Conclusions

Temperature and other physical parameters affect DNAconformation, which must be under homeostatic controlto allow the correct interaction with all proteins involvedin DNA-based processes. The basic components of sys-tems to regulate DNA topology in all organisms areDNA topoisomerases and DNA-binding proteins. There-fore, their phylogenetic distribution and particular func-tions are a worthy source of information about earlyevolution and temperature adaptation, especially con-cerning a putative hyperthermophilic cenancestor.

Hyperthermophilic life (optimal temperatures above80°C) requires the possession of reverse gyrase, which isprobably essential only at critical regions, since hyper-thermophilic bacteria appear to have general levels ofgyrase-induced negative supercoiling. This would be in

Fig. 4. Possibilities of prokaryoticdivergence and DNA topoisomeraseevolution from a thermophilic ancestor.The common prokaryotic ancestor waseither a thermophile (T; 60–80°C) or ahyperthermophile (HT) endowed with areverse gyrase (RG). G, gyrase.Wavyarrows indicate horizontal transfer events.Thick linescorrespond tohyperthermophilic lineages in idealizedphylogenetic trees. Shown at thebottomare putative coevolutionary steps ofgenomes and topoisomerase function.

448

agreement with the requirement of a certain degree ofnegative supercoiling to initiate transcription and otherprocesses. In hyperthermophilic archaea lacking gyrase,relaxing to positively supercoiled DNA would preventglobal denaturation, but should then provide negativesupercoiling at local regions, at least when the environ-mental temperature decreases (Bell et al. 1998). Thiscould be done in crenarchaea with the help of unwindingproteins such as the Sac/Sso7 proteins (Lo´pez-Garcı´a etal. 1998; Mai et al. 1998; Robinson et al. 1998) and ineuryarchaea by histones, which generate negative super-coiling at physiological conditions (Musgrave et al.1999). This suggests that histones originally evolved toperform a similar function as actual histone-like proteins,i.e., general stabilization, bending, and unwinding, beingused later for more complex functions.

Mesophilic life requires general levels of negativeDNA supercoiling, despite an additional local unwindingto initiate DNA activity. Those levels are generated ei-ther by gyrase (all bacteria and some archaea) or by DNAwrapping around a histone core (eukaryotes and someeuryarchaea), implying independent adaptations to lowertemperatures.

In this report, I have speculated on the existence of athermophilic ancestor thriving under thermophilic con-ditions (60–80°C), which was already endowed with re-laxing type I and primitive type II topoisomerases. Ther-mophilic temperatures supplied the energy of activationrequired for opening DNA strands without inducing ex-cessive denaturation. Hyperthermophily would have ap-peared first as reverse gyrase evolved, and the cenances-tor would have been a thermophile or already ahyperthermophile. On the bacterial branch, duplicationof gyrase genes and functional specialization (decatena-tion—control of supercoiling) led to an increased adapt-ability to mesophilic conditions. Consistent with a hy-perthermophilic archaeal ancestor devoid of gyrase,hyperthermophilic archaea seem to use proteins to aidDNA unwinding in a general context of linking excess.The DNA-binding proteins that developed in the euryar-chaeal branch, i.e., histones, turned out to be very effi-cient in generating negative supercoiling. Later, as theyhad to face less thermophilic environments, this histonepotential was exploited to create high general levels ofnegative supercoiling, which could be at the very originof the eukaryotic nucleosomal dynamics. Halophilicmethanogens and halophiles, well adapted to mesophilyand high salt, have probably acquired a gyrase from bac-teria living in the same environments, and they replacednucleosomes by a bacterial-like system of expression andgeneral chromosomal dynamics.

These hypothetical pathways of evolution can be atleast partially tested, since several predictions can bemade for present-day organisms. Some of them havealready been formulated, such as the presence of a pu-

tative gyrase in Thermoplasmales acquired by horizontaltransfer. An interesting case to study is that ofArchaeo-globus fulgidus,where reverse gyrase, gyrase, and his-tone genes occur simultaneously (Klenk et al. 1997). Inthis case, gyrase would have been acquired by horizontaltransfer from hyperthermophilic bacteria, but histoneshave not yet been lost. This would be a good candidate toverify if gyrase is the determinant of negative supercoil-ing whenever it occurs in addition to reverse gyrase. Itwould be equally interesting to analyze whether the oc-currence of two reverse gyrase genes affects the level ofDNA supercoiling inAquifex aeolicus(Deckert et al.1998) and whether its type II topoisomerase acts like agyrase or a Topo IV.

This hypothesis would also predict the existence ofnegative levels of DNA supercoiling in the meso/psychrophilic crenarchaea and euryarchaea detected byrRNA sequencing directly from the environment (Barnset al. 1996; Pace 1997; DeLong 1998). Group I psychro-philes, branching deeply into the otherwise hyperthermo-philic crenarchaeota, are probably the result of a second-ary adaptation to meso/psychrophily, as suggested by thelong branch length in the rRNA phylogenetic tree, likelythe result of a higher evolutionary rate (Preston et al.1996; Barns et al. 1996). It would now be interesting totest whether they have lost reverse gyrase and acquiredgyrase or whether they have developed an alternativemechanism to maintain general levels of negative DNAsupercoiling.

At present, molecular ecology, which now permits theidentification of many unculturable bacteria and archaea,in combination with comparative genomics and bio-chemical analysis, should help to refine the topoisomer-ase map in the living world. The role of these enzymes intemperature adaptation and evolution could then begin tobe better understood.

Acknowledgments. I wish to acknowledge Patrick Forterre for givingme inestimable scientific opportunities and Les Treilles Foundation forinviting me to the meeting on the nature of the last common ancestorin 1996. I am also grateful to Olivier Guipaud and David Musgrave forhelpful discussions and comments and to David Clarke for criticalreading of the manuscript.

References

Altekar W, Rajagopalan R (1990) Ribulose bisphosphate carboxylaseactivity in halophilic Archaebacteria. Arch Microbiol 153:169–174

Baldauf SL, Palmer JP, Doolittle, WF (1996) The root of the universaltree and the origin of eukaryotes based on elongation factor phy-logeny. Proc Natl Acad Sci USA 93:7749–7754

Balke VL, Gralla JD (1987) Changes in linking number of supercoiledDNA accompany growth transitions inEscherichia coli.J Bacteriol169:4499–4506

Barns SM, Delwiche CF, Palmer JD, Pace NR (1996) Perspectives onarchaeal diversity, thermophily and monophily from environmentalrRNA sequences. Proc Natl Acad Sci USA 93:9188–9193

449

Baross JA, Hoffman SE (1985) Submarine hydrothermal vents andassociated gradient environments as sites for the origin and evolu-tion of life. Orig Life 15:327–45

Bates AD, Maxwell A (1993) DNA supercoiling. In: Rickwood D (ed)DNA topology. “In focus” series. IRL Press, Oxford, pp. 17–55

Bell SD, Jaxel C, Nadal M, Kosa PF, Jackson SP (1998) Temperature,template topology, and factor requirements of archaeal transcrip-tion. Proc Natl Acad Sci USA 95:15218–15222

Bergerat A, Gadelle D, Forterre P (1994) Purification of a DNA to-poisomerase II from the hyperthermophilic archaeonSulfolobusshibatae.J Biol Chem 269:27663–27669

Bergerat A, de Massy B, Gadelle D, Varoutas PC, Nicolas A, ForterreP (1997) An atypical topoisomerase II from archaea with implica-tions for meiotic recombination. Nature 386:414–417

Brock TD (1986) Introduction: An overview of the thermophiles. In:Brock TD (ed) Thermophiles. Wiley–Interscience, New York, pp1–16

Brown JR, Doolittle WF (1995) Root of the universal tree of life basedon ancient aminoacyl-tRNA synthetase gene. Proc Natl Acad SciUSA 92:2441–2445

Bult, CJ, White O, Olsen GJ, et al. (1996) Complete genome sequenceof the methanogenic archaeon,Methanococcus jannaschii.Science273:1058–1073

Bustin M, Lehn DA, Landsman D (1990) Structural features of theHMG chromosomal proteins and their genes. Biochim BiophysActa 1049:231–243

Champoux JJ, Dulbecco R (1972) An activity from mammalian cellsthat untwists superhelical DNA a possible swivel for DNA re-pleation (polyoma-ethydium bromide-mouse-embryo cells-dyebinding assay). Proc Natl Acad Sci USA 69:143–46

Charbonnier F, Forterre P (1994) Comparison of plasmid DNA topol-ogy among mesophilic and thermophilic eubacteria and archaeo-bacteria. J Bacteriol 176:1251–1259

Chartier F, Laine B, Sautie`re P (1988) Characterization of the chro-mosomal protein MC1 from the thermophilic archaebacteriumMethanosarcinasp. CHTI55 and its effect on the thermal stabilityof DNA. Biochim Biophys Acta 951:149–156

Chartier F, Laine B, Belaiche D, Touzel JP, Sautie`re P (1989) Primarystructure of the chromosomal proteins MC1a, MC1b, and MC1cfrom the archaebacteriumMethanothrix soehngenii.J Biol Chem264:17006–17015

Darcy TJ, Sandman K, Reeve JN (1995)Methanobacterium formici-cum,a mesophilic methanogen, contains three HFo histones. J Bac-teriol 177:858–860

Deckert G, Warren PV, Gaasterland T, et al. (1998) The completegenome of the hyperthermophilic bacteriumAquifex aeolicus.Na-ture 392:353–358

DeLange RJ, Williams LC, Searcy D (1981) A histone-like protein(HTa) from Thermoplasma acidophilum.II. Complete amino acidsequence. J Biol Chem 256:905–911

DeLong E (1998) Archaeal means and extremes. Science 280:542–543Derkacheva NI, Kagramanova VK (1994) Archaebacterial chromo-

somes. Molek Biologiya 28:274–282DiGate RJ, Marians KJ (1992)Escherichia coli topoisomerase III-

catalyzed cleavage of RNA. J Biol Chem 267:20532–20535Doolittle RF (1998) Microbial genomes opened up. Nature 392:339–

342Dorman CJ (1995) DNA topology and the global control of bacterial

gene expression: Implications for the regulation of virulence geneexpression. Microbiology 141:1271–1280

Drlica K (1992) Control of bacterial DNA supercoiling. Mol Microbiol6:425–433

Drlica K, Rouviere-Yaniv J (1987) Histone-like proteins of bacteria.Microbiol Rev 51:301–319

Drlica K, Zhao X (1997) DNA gyrase, topoisomerase IV, and the4-quinolones. Microbiol Mol Biol Rev 61:377–392

Droge P (1993) Protein tracking-induced supercoiling of DNA: A toolto regulate DNA transactionsin vivo? BioEssays 16:91–99

Duguet M (1995) Reverse gyrase. In: Eckstein F, Lilley DMJ (eds)Nucleic acids and molecular biology, Vol. 9. Springer-Verlag, Ber-lin–Heidelberg, pp 84–114

Edgell DR, Doolittle WF (1997) Archaea and the origin(s) of DNAreplication proteins. Cell 89:995–998

Fitz-Gibbon S, Choi AJ, Miller JM, et al. (1997) A fosmid-based ge-nomic map and identification of 474 genes of the hyperthermo-philic archaeonPyrobaculum aerophilum.Extremophiles 1:36–51

Forterre P (1995) Thermoreduction, a hypothesis for the origin of pro-karyotes. CR Acad Sci Paris 318:415–422

Forterre P (1996) A hot topic: The origin of hyperthermophiles. Cell85:789–792

Forterre P, Elie C (1993) Chromosome structure, DNA topoisomerases,and DNA polymerases in archaebacteria (archaea). In: Kates M,Kushner D, Matheson AT (eds) The biochemistry of Archaea.Elsevier, Amsterdam, pp 325–366

Forterre P, Mirambeau G, Jaxel C, Nadal M, Duguet M (1985) Highpositive supercoilingin vitro catalyzed by an ATP and polyethyleneglycol-stimulated topoisomerase fromSulfolobus acidocaldarius.EMBO J 4:2123–2128

Forterre P, Bergerat A, Gadelle D, et al. (1994) Evolution of DNAtopoisomerases and DNA polymerases: A perspective from Ar-chaea. Syst Appl Microbiol 16:746–758

Forterre P, Confalonieri F, Charbonnier F, Duguet M (1995) Specula-tions on the origin of life and thermophily: Review of availableinformation on reverse gyrase suggests that hyperthermophilic pro-cayotes are not so primitive. Orig Life Evol Biosph 25:235–249

Forterre P, Bergerat A, Lo´pez-Garcı´a P (1996) The unique DNA to-pology and DNA topoisomerases of hyperthermophilic archaea.FEMS Microbiol Rev 18:237–248

Gellert M, Mizuuchi K, O’Dea MH, Nash HA (1976) DNA gyrase: Anenzyme that introduces superhelical turns into DNA. Proc NatlAcad Sci USA 73:3872–3876

Germond JE, Hirt B, Oudet P, Gross-Bellard M, Chambon P (1975)Folding of the DNA double helix in chromatin-like structures fromsimian virus 40. Proc Natl Acad Sci USA 72:1843–1846

Gogarten P (1994) Which is the most conserved group of proteins?Homology-orthology, paralogy, xenology, and the fusion of inde-pendent lineages. J Mol Evol 39:541–543

Gogarten P (1995) The early evolution of cellular life. TREE 10:147–151

Goldstein E, Drlica K (1984) Regulation of bacterial DNA supercoil-ing: Plasmid linking numbers vary with growth temperature. ProcNatl Acad Sci USA 81:4046–4050

Grayling RA, Sandman K, Reeve JN (1994) Archaeal DNA bindingproteins and chromosome structure. Syst Appl Microbiol 16:582–590

Grayling RA, Sandman K, Reeve JN (1996) Histones and chromatinstructure in hyperthermophilic Archaea. FEMS Microbiol Rev 18:203–213

Grove A, Galeone A, Mayol L, Geiduschek EP (1996) Localized DNAflexibility contributes to target site selection by DNA-bending pro-teins. J Mol Biol 260:120–125

Guipaud O (1998) ADN gyrase et superhe´licite negative d’un elementextrachromosomique ches des bacte´ries hyperthermophile su genreThermotoga, Doctoral thesis. Universite´ Paris 7, Paris

Guipaud O, Marguet E, Knoll KM, Bouthier de la Tour C, Forterre P(1997) Both DNA gyrase and reverse gyrase are present in thehyperthermophilic bacteriumThermotoga maritima.Proc NatlAcad Sci USA 94:10606–10611

Hamiche A, Carot, V, Alilat M, et al. (1996) Interaction of the histone(H3-H4)2 tetramer of the nucleosome with positively supercoiledDNA minicircles: Potential flipping of the protein from a left- to aright-handed superhelical form. Proc Natl Acad Sci USA 93:7588–7593

Higgins CF, Dorman CJ, Ni Bhrian N (1990) Environmental influenceson DNA supercoiling: A novel mechanism for the regulation of

450

gene expression. In: Drlica K, Riley M (eds) The bacterial chro-mosome. ASM Press, Washington DC, pp 421–432

Hilario E, Gogarten JP (1993) Horizontal transfer of ATPase genes-thetree of life becomes a net of life. Biosystems 31:111–119

Holmes ML, Dyall-Smith ML (1991) Mutations in DNA gyrase resultin novobiocin resistance in halophilic archaebacteria. J Bacteriol173:642–648

Horne M, Englert C, Pfeifer F (1988) Two genes encoding gas vacuoleproteins inHalobacterium halobium.Mol Gen Genet 213:459–464

Hugenholtz P, Pitulle C, Hershberger KL, Pace NR (1998) Novel di-vision level bacterial diversity in a Yellowstone hot spring. J Bac-teriol 180:366–376

Hsieh LS, Burger RM, Drlica K (1991a) Bacterial DNA supercoilingand [ATP]/[ADP] ratio: Changes associated with a transition toanaerobic growth. J Mol Biol 219:443–450

Hsieh LS, Rouvie`re-Yaniv J, Drlica K (1991b) Bacterial DNA super-coiling and [ATP]/[ADP] ratio: Changes associated with salt shock.J Bacteriol 173:3914–3917

Huang WM (1996) Bacterial diversity based on type II DNA topoi-somerase genes. Annu Rev Genet 30:79–107

Jensen PR, Loman L, Petra B, van der Weiden C, Westerhoff, HV(1995) Energy buffering of DNA structure fails whenEscherichiacoli runs out of substrate. J Bacteriol 177:3420–3426

Jones PG, Krah R, Tafuri SR, Wolffe AP (1992) DNA gyrase, CS7.4,and the cold shock response inEscherichia coli.J Bacteriol 174:5798–5802

Kampranis SC, Maxwell A (1996) Conversion of DNA gyrase into aconventional type II topoisomerase. Proc Natl Acad Sci USA 93:14416–14421

Kaneko T, Tabata S (1997) Complete genome structure of the unicel-lular cyanobacteriumSynechocysrissp. PCC6803. Plant Cell Phys-iol 38:1171–1176

Karem K, Foster JW (1993) The influence of DNA topology on theenvironmental regulation of a pH-regulated locus inSalmonellatyphimurium.Mol Microbiol 10:75–86

Kato J, Nishimura Y, Imamura R, Niki H, Hiraga S, Suzuki H (1990)New topoisomerase essential for chromosome segregation inE.coli. Cell 63:393–404

Kikuchi A, Asai K (1984) Reverse gyrase—A topoisomerase whichintroduces positive superhelical turns into DNA. Nature 309:667–681

Klenk HP, Clayton RA, Tomb JF, et al. (1997) The complete genomesequence of the hyperthermophilic, sulphate-reducing archaeonAr-chaeoglobus fulgidus.Nature 390:364–370

Kornberg RD (1977) Structure of chromatin. Annu Rev Biochem 46:931–954

Kornberg R, Lorch Y (1992) Chromatin structure and transcription.Annu Rev Cell Biol 8:563–587

Kozyavkin A, Krah R, Gellert M, Lake JA, Stetter KO, Slesarev AI(1994) A reverse gyrase with an unusual structure. A type-I DNAtopoisomerase from the hyperthermophileMethanopyrus kandleriis a 2-subunit protein. J Biol Chem 269:11081–11089

Krah R, Kozyavkin SA, Slesarev AI, Gellert MA (1996) Two-subunittype I topoisomerase (reverse gyrase) from a hyperthermophilicmethanogen. Proc Natl Acad Sci USA 93:106–110

Laine B, Culard F, Maurizot JC, Sautie´re P (1991) The chromosomalprotein MCl from the archaebacteriumMethanosarcinasp. CHTI55 induces DNA bending and supercoiling. Nucleic Acids Res19:3041–3045

L’Haridon S, Reysenbach AL, Gle´nat P, Prieur D, Jeanthon C (1995)Hot subterranean biosphere in a continental oil reservoir. Nature377:223–224

Li W, Wang JC (1998) Mammalian DNA topoisomerase IIIa is essen-tial in early embryogenesis. Proc Natl Acad Sci USA 95:1010–1013

Lopez-Garcı´a P, Forterre P (1997) DNA topology in hyperthermophilicarchaea: Reference states and their variation with growth phase,growth temperature, and temperature stresses. Mol Microbiol 23:1267–1279

Lopez-Garcı´a P, Knapp S, Ladenstein R, Forterre P (1998)In vitroDNA binding of the archaeal protein Sso7d induces negative su-percoiling at temperatures typical for thermophilic growth. NucleicAcids Res 26:2322–2328

Luger K, Richmond TJ (1998) DNA binding within the nucleosome.Curr Opin Str Biol 8:33–40

Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997)Crystal structure of the nucleosome core particle at 2.8 A resolu-tion. Nature 389:251–260

Luttinger A (1995) The twisted “life” of DNA in the cell: bacterialtopoisomerases. Mol Microbiol 15:601–606

Mai VQ, Chem C, Hong R, Huang L (1998) Small abundant DNAbinding proteins from the thermoacidophilic archaeonSulfolobusshibataeconstrain DNA supercoils. J Bacteriol 180:2560–2563

Martin W, Muller M (1998) The hydrogen hypothesis for the firsteukaryote. Nature 392:37–41

McClellan JA, Boublikova P, Palecek E, Lilley DMJ (1990) Superhe-lical torsion in cellular DNA responds directly to environmentaland genetic factors. Proc Natl Acad Sci USA 87:8373–8377

Miller SL, Lazcano A (1995) The origin of life-did it occur at hightemperatures? J Mol Evol 41:689–692

Mizushima T, Kataoka K, Ogata Y, Inoue R, Sekimizu K (1997) In-crease in negative supercoiling of plasmid DNA inEscherichia coliexposed to cold shock. Mol Microbiol 23:381–386

Mojica FJM, Charbonnier F, Juez G, Rodrı´guez-Valera F, Forterre P(1994) Effects of salt and temperature on plasmid topology in thehalophilic archaeonHaloferax volcanii.J Bacteriol 176:4966–4973

Moreira D, Lopez-Garcı´a P (1998) Symbiosis between methanogenicarchaea andd proteobacteria as the origin of eukaryotes: The syn-trophic hypothesis. J Mol Evol 47:517–530

Munson MA, Nedwell DB, Embley TM (1997) Phylogenetic diversityof Archaea in sediment samples from a coastal salt marsh. ApplEnviron Microbiol 63:4729–4733

Musgrave DR, Sandman KM, Reeve JN (1991) DNA binding by thearchaeal histone HMf results in positive supercoiling, Proc NatlAcad Sci USA 88:10397–10401

Musgrave D, Forterre P, Slesarev A (1999) Negative constrained su-percoiling in archaeal nucleosomes (submitted for publication)

Nisbet EG (1985) The geological setting of the earliest life forms. JMol Evol 21:289

Ogata Y, Inoue R, Mizushima T, Kano Y, Miki T, Sekimizu K (1997)Heat shock-induced excessive relaxation of DNA inEscherichiacoli mutants lacking the histone-like protein HU. Biochim BiophysActa 1353:298–306

Olsen GJ, Woese CR (1997) Archaeal genomics: An overview. Cell89:991–994

Ouzounis CA, Kyrpides NC (1996) Parallel origins of the nucleosomecore and eukaryotic transcription from Archaea. J Mol Evol 42:234–239

Pace NR (1991) Origin of life-facing up to the physical setting. Cell65:531–533

Pace NR (1997) A molecular view of microbial diversity and the bio-sphere. Science 276:734–740

Pereira SL, Grayling RA, Lurz R, Reeve JN (1997) Archaeal nucleo-somes. Proc Natl Acad Sci USA 94:12633–12637

Preston CM, Wu KY, Molinski TF, DeLong EF (1996) A psycrophiliccrenarchaeon inhabits a marine sponge:Cenarchaeum symbiosumgen. nov., sp. nov. Proc Natl Acad Sci USA 93:6241–6246

Ramakrishnan V (1995) The histone fold: Evolutionary questions. ProcNatl Acad Sci USA 92:11328–11330

Robinson H, Gao, YG, McCrary BS, Edmondson SP, Shriver JW,Wang AHJ (1998) The hyperthermophile chromosomal proteinSac7d sharply kinks DNA. Nature 392:202–205

Ronimus RS, Musgrave DR (1996) A gene,hanIA, encoding an ar-chaeal histone-like protein from theThermococcusspecies AN1:Homology with eukaryal histone consensus sequences and the im-plications for delineation of the histone fold. Biochim Biophys Acta1307:1–7

451

Saavedra RA, Huberman JA (1986) Both DNA topoisomerases I and IIrelax 2mm plasmid DNA in living yeast cells. Cell 45:65–70

Sandman K, Perler F, Reeve JN (1994) Histone-encoding genes fromPyrococcus:Evidence for members of the HMf family of archaealhistones in a non-methanogenic Archaeon Gene 150:207–208

Schmid MB (1990) More than just “histone-like” proteins. Cell 63:451–453

Shock EL (1996) Hydrothermal systems as environments for the emer-gence of life. In: Evolution of hydrothermal systems on Earth (andMars?), Ciba Foundation Symposium 202. Wiley, Chichester, pp40–60

Sinden RR, Carlson JO, Pettijohn DE (1980) Torsional tension in theDNA double helix measured with trimethylpsoralen in livingE. colicells: Analogous measurements in insect and human cells. Cell21:773–783

Sioud M, Possot O, Elie C, Sibold L, Forterre P (1988) Coumarin andquinolone action in archaebacteria: Evidence for the presence of aDNA gyrase-like enzyme. J Bacteriol 170:946–953

Slesarev AI, Zaitzev DA, Kopylov VM, Stetter KO, Kozyavkin SA(1991) DNA topoisomerase III from extremely thermophilic ar-chaebacteria: ATP-independent type I topoisomerase fromDesul-furococcus amylolyticusdrives extensive unwinding of closed cir-cular DNA at high temperature. J Biol Chem 266:12321–12328

Slesarev A, Stetter K, Lake J, Gellert M, Krah R, Kozyavkin S (1993)DNA topoisomerase V is a relative of eukaryotic topoisomerase Ifrom a hyperthermophilic prokaryote. Nature 364:735–737

Slesarev AI, Belova GI, Kozyavkin SA, Lake JA (1998) Evidence foran early prokaryotic origin of histones H2A and H4 prior to theemergence of eukaryotes. Nucleic Acids Res 26:427–430

Soares D, Dahlke I, Li WT, et al. (1998) Archaeal histone stability,DNA binding, and transcription inhibition above 90°C. Extremo-philes 2:75–81

Srivenugopal KS, Lockshon D, Morris DR (1984)Escherichia coliDNA topoisomerase III: Purification and characterization of a newtype I enzyme. Biochemistry 23:1899–1906

Starich MR, Sandman K, Reeve JN, Summers MF (1996) NMR struc-ture of HMf from the hyperthermophileMethanothermus fervidusconfirms that this archaeal protein is a histone. J Mol Biol 255:187–203

Stetter KO (1995) Microbial life in hyperthermal environments. ASMNews 61:285–290

Stetter KO (1996) Hyperthermophilic procaryotes. FEMS MicrobiolRev 18:149–158

Travers AA (1994) Chromatin structure and dynamics. BioEssays 16:657–662

Wachtersha¨user G (1988) Pyrite formation, the first energy source forlife: A hypothesis. Syst Appl Microbiol 10:207–210

Wang H, DiGate RJ, Seeman NC (1996) An RNA topoisomerase. ProcNatl Acad Sci USA 93:9477–9482

Wang JC (1971) Interaction between DNA and anEscherichia coliprotein omega. J Mol Biol 55:523–533

Wang JC (1987) Recent studies of DNA topoisomerases. BiochimBiophys Acta 909:1–9

Wang JC (1996) DNA topoisomerases. Annu Rev Biochem 65:635–692

Wang JC, Lynch AS (1993) Transcription and DNA supercoiling. CurrOpin Genet Dev 3:764–768

Wang JC, Caron PR, Kim RA (1990) The role of DNA topoisomerasesin recombination and genome stability: A double-edged sword?Cell 62:403–406

Weintraub H (1985) Assembly and propagation of repressed and de-repressed chromosomal states. Cell 42:705–711

Woese CR (1987) Bacterial evolution. Microbiol Rev 51:221–271

Woese CR, Kandler O, Wheelis ML (1990) Towards a natural systemof organisms. Proposal for the domains Archaea, Bacteria and Eu-carya. Proc Natl Acad Sci USA 87:4576–4579

Wolffe AP, Pruss D (1996) Chromatin: Hanging on to histones. CurrBiol 6:234–237

Worcel A, Burgi E (1972) On the structure of the folded chromosomeof Escherichiacoli. J Mol Biol 71:127–147

Workum M, van Dooren SJM, Oldenburg N, et al. (1996) DNA su-percoiling depends on the phosphorylation potential inEscherichiacoli. Mol Microbiol 20:351–360

Yasuda M, Oyaizu H, Yamagishi A, Oshima T (1995) Morphologicalvariation of newThermoplasma acidophilumisolates from japanesehot springs. Appl Environ Microbiol 61:3482–3485

Zechiedrich EL, Cozzarelli (1995) Roles of topoisomerase IV andDNA gyrase in DNA unlinking during replication inEscherichiacoli. Genes Dev 9:2859–2869

Zviagintseva IS, Gerasimenko LM, Kostrikina NA, Bulygina ES, Za-varzin GA (1995) Interaction of halobacteria and cyanobacteria ina halophilic cyanobacterial community. Mikrobiologiia 64:252–258

452