On the Origins of Cells

Publishedonline 5December 2002

On the originsof cells:a hypothesisfor the evolutionarytransitionsfrom abioticgeochemist ry to chemoautotrophicprokaryote s,and from prokaryotes to nucleatedcells

William Martin 1* and Michael J.Russell 2 1 Institut fu¨rBotanikIII, Heinrich-Heine UniversitaetDu ¨sseldorf,Universita ¨tsstrasse1, 40225 Du ¨sseldorf,Germany 2 ScottishUniversities EnvironmentalResearch Centre, Scottish Enterprise Technology Park, Rankine Avenue,East Kilbride, GlasgowG75 0QF, UK ([email protected] ) All life isorganized ascells. Physical compartmentation from theenvironment and self-organization of self-containedredox reactionsare themost conserved attributes ofliving things,hence inorganic matter with suchattributes wouldbe life’ s mostlikely forebear. Wepropose that life evolved in structurediron monosulphideprecipitates in aseepagesite hydrothermal moundat aredox,pH and temperature gradient betweensulphide-rich hydrothermal fluid andiron(II)-containing watersof the Hadean ocean floor. The naturally arising, three-dimensionalcompartmentation observedwithin fossilizedseepage-site metal sulphide precipitates indicatesthat theseinorganic compartmentswere the precursors of cell walls andmem- branesfound in free-living prokaryotes. The knowncapability ofFeS and NiS tocatalyse thesynthesis ofthe acetyl-methylsulphide from carbon monoxide andmethylsulphide, constituentsof hydrothermal fluid,indicates that pre-biotic synthesesoccurred at theinner surfaces of these metal-sulphide-walled compartments,which furthermore restrainedreacted products from diffusioninto the ocean, providing sufficientconcentrations of reactants to forge thetransition from geochemistry tobiochemistry. The chemistry ofwhat isknown as the RNA-world could have takenplace within thesenaturally forming, catalytic- walled compartmentsto give rise toreplicating systems.Sufficient concentrations of precursorsto support replication wouldhave beensynthesized in situ geochemically andbiogeochemically, with FeS(and NiS) centresplaying thecentral catalytic role.The universal ancestorwe infer wasnot a free-living cell,but rather wasconfined to the naturally chemiosmotic,FeS compartments within which thesynthesis of its constituentsoccurred. The first free-living cellsare suggestedto have beeneubacterial andarchaebacterial chemoautotrophsthat emergedmore than 3.8 Gyr ago from their inorganic confines.We propose that theemergence of these prokaryotic lineages from inorganic confinesoccurred independently, facilitated by theindependent origins ofmembrane-lipid biosynthesis:isoprenoid ether membranes in thearchaebacterial andfatty acid estermembranes in theeubacterial lineage. The eukaryotes,all ofwhich are ancestrally heterotrophsand possess eubacterial lipids, are suggestedto have arisen ca.2Gyr ago through symbiosis involving anautotrophic archaebacterial hostand a heterotrophic eubacterial symbiont,the commonancestor of mitochondria andhydrogenosomes. The attributes sharedby all prokaryotes are viewedas inheritances from their confineduniversal ancestor.The attributes that distinguish eubacteria andarchaebacteria, yet are uniform within thegroups, are viewedas relics oftheir phaseof differentiation after divergencefrom thenon-free-living universal ancestorand before the origin ofthe free-living chemoautotrophic lifestyle. The attributes sharedby eukaryoteswith eubacteria andarchaebacteria, respectively, are viewedas inheritances via symbiosis.The attributes uniqueto eukaryotes are viewedas inventionsspecific to their lineage. The origin ofthe eukaryotic endomembranesystem and nuclear membrane are suggestedto be the fortuitous result of the expression of genes for eubacterial membrane lipid synthesisby anarchaebacterial geneticapparatus in acompartment that wasnot fully prepared toaccom- modatesuch compounds, resulting in vesiclesof eubacterial lipids that accumulatedin thecytosol around their siteof synthesis. Under these premises, the most ancient divide in theliving worldis that between eubacteria andarchaebacteria, yet thesteepest evolutionary grade isthat betweenprokaryotes andeukaryotes. Keywords: hydrothermal vents;acetyl-CoA pathway; origin oflife; RNA-world; chemoautotrophic origins; holochirality

1. INTRODUCTION

*Authorfor correspondence ([email protected]). The Earth is4.5 billion years (Gyr) oldand the first ocean One contribution of 21to a DiscussionMeeting Issue ‘Chloroplastsand had condensedby ca.4.4 Gyr (Wilde et al. 2001). There mitochondria: functional genomics and evolution’. are goodreasons to believe that life arosehere by ca.

Phil. Trans. R.Soc.Lond. B (2003) 358, 59–85 59 Ó 2002 TheRoyal Society DOI10.1098/rstb.2002.1183 60W. Martinand M. J.Russell On theorigins of cells

3.8 Gyr,because carbon isotopedata provide evidencefor last commonancestor, its attributes canonly beaddressed biological CO 2 fixation in sedimentary rocksof that age through logical inference(Penny & Poole 1999). (Mojzsis et al. 1996; Rosing 1999; Nisbet& Sleep2001; What didthe ancestor of prokaryotes possessfor sure? Ueno et al. 2002). By 3.5 Gyr,stromatolites werepresent, It certainly possessedthe universal geneticcode, tRNA preservedmicrobial mats indicative ofdeposition by pho- (Eigen& Schuster1978; Woese et al. 1990; Eigen1992) tosyntheticprokaryotes (Walter 1983; Nisbet& Sleep andribosomes with abattery ofribosomal proteins 2001). By ca.1.5 Gyr,so-called acritarchs becamereason- (Woese2002), mostof whichare neatly encodedin acon- ably abundant,microfossils of unicellular organisms that servedsuperoperon that is presentand conserved in gene are almost certainly eukaryotes( Javaux et al. 2001) and order acrossmany genomes(Wa ¨chtersha¨user1998). Fur- probably algae becauseof an easily preservedcell wall. By thermore, theuniversal ancestorsurely possessedDNA 1.2 Gyr,spectacularly preservedmulticellular organisms (Reichard 1997; Poole et al. 2000) andDNA polymerases, appear that werevery probably redalgae (Butterfield RNA polymerases,and a battery ofaccessory translation 2000). There have beenreports ofmore ancientremains factorssuch as EF-Tu and EF-G that are presentin all claimed tobe eukaryotes, but they are equivocal. For prokaryotes (Poole et al. 1998, 1999), probably along with example Grypania isa 2.1 Gyr fossil(Han & Runnegar other more or lessubiquitous prokarytotic proteinssuch

1992), butcould just as easily bea filamentousprokaryote as F1 –F0 -typeATPases (Gogarten et al. 1989) aswell as asa filamentouseukaryote, because the cellular structure prokaryotic formsof the signal recognition particle, which ofthefossil is notpreserved. More recently, steranes were is anaccessory to translation (Brinkmann &Philippe foundin 2.7 Gyr sedimentsand were claimed toprovide 1999). Furthermore, it had tohave had all ofthepathways evidencefor theexistence of eukaryotes at that time necessaryfor efficientnucleotide and amino-acid (Brocks et al. 1999), butseveral groups ofprokaryotes biosyntheses,a corecarbon metabolism toprovide the including methanotrophic proteobacteria (seeSchouten et biosyntheticprecursors, and the cofactors required for al. 2000), myxobacteria (Kohl et al. 1983) andcyanobac- thosebiosynthetic pathways: butthis is thepoint where teria (Hai et al. 1996) make thesame kinds of compounds things get very complicated very quickly, becausealthough (for example cholesterol)claimed tobe eukaryote-specific, archaebacteria andeubacteria domany things similarly suchthat thesterane evidence for theage ofeukaryotesis whenit comesto the processing of genetic information questionableat best.Ultra-light carbon material that bears (Woese2002), they domany things fundamentally differ- theisotopic signature ofbiological consumptionof bio- ently whenit comesto central metabolism. logically producedmethane goes back to2.7 Gyr (Hayes Onesimple example for suchcore metabolic differences 1994), providing evidencefor thedistinctness of eubac- betweenthe two groups ofprokaryotes is thebreakdown teria (methanotrophs) andarchaebacteria (methanogens) ofglucose to pyruvate, theglycolytic (Embden –Meyerhof) by that time at thevery least,and furthermore indicating pathway, whichis universal among eukaryotes,but not that arguments for anorigin ofarchaebacteria only among prokaryotes. If welook among prokaryotic gen- 850 million years (Myr) ago (Cavalier-Smith 2002) must omesfor thefamiliar glycolytic enzymesthat occurin be in error. eukaryotes,we see that they have goodhomologues Thosegeological benchmarksprovide rough butrobust among awidespectrum of eubacterial genomes,but many orientation for theprocess of early evolution:the origin of ofthe corresponding homologues are missing in archaeb- prokaryotes by at least 3.5 Gyr andthe origin ofeukary- acteria (figure 1). This observation might seempuzzling otesby at least 1.5 Gyr.But biologists andgeologists alike tosome (Canback et al. 2002) butis easily attributable to wouldlike toknow a fewmore details.What happened twosimple factors.First, although archaebacteria possess during thosefirst 2.5 Gyr? Howdid life get offthe ground pathways from glucoseto pyruvate, they have several alter- tobegin with? Why didit take eukaryotesso long to native pathways that involve differentenzymatic steps, appear? Why are thetwo groups ofcontemporary prokary- hencedifferent enzymes not present in glycolysis otes(eubacteria andarchaebacteria) sogenetically coher- (Daniel &Danson1995; Scho¨nheit & Scha¨fer 1995; entyet sobiochemically different?Why are thereno Kengen et al. 1996; Selig et al. 1997). Theseinclude: (i) intermediate formsbetween prokaryotic andeukaryotic themodified Embden –Meyerhofpathway, whichemploys organization? Wheredid the nucleus come from? Here, ADP- insteadof ATP-dependent steps and a glyceral- wediscuss evolutionary processesthat may underliethe dehyde-3-phosphateoxidoreductase in place ofthe steps differencesbetween eubacteria andarchaebacteria and catalysed by GAPDHand 3-phosphoglycerate kinase;(ii) that may underliethe differences between prokaryotes and the Entner–Doudoroffpathway, whichdoes not require eukaryotesas well asthe mosaic patternsof attributes theenzymatic stepscatalysed by GPI,PFK, FBA or TPI; (Zillig 1991) sharedbetween the three. and(iii) thenon-phosphorylated Entner –Doudoroffpath- way,which shares only twoenzymatic stepsin common with glycolysis (Selig et al. 1997), thosecatalysed by enol- 2. COMMONALITIESAND DIFFERENCESLEAD ase(Hannaert et al. 2000) andpyruvate kinase(Schramm QUICKLYTO DEEPERPROBLEMS et al. 2000). Second,several archaebacteria have complete glycolytic pathways, butfor many stepsthey usetotally Understandingthe evolutionary differencesbetween differentenzymes that are unrelatedto the familiar homol- archaebacteria andeubacteria requiresa concretepicture oguesfound in eubacteria (andeukaryotes) (Scho ¨nheit & oftheir last commonancestor. This is becauseone can Scha¨fer1995; Selig et al. 1997). Theseinclude: (i) an only discussthe differentiation oftheir attributes onthe ADP-dependent(rather than ATP-dependent)hexokin- basis ofthe attributes possessedby their progenitor. But asewith nosequencesimilarity tothe eubacterial enzyme becausethere is nodirect evidence for thenature of their (Ito et al. 2001); (ii) anADP-dependent PFK that lacks

Phil.Trans. R. Soc.Lond. B (2003) On theorigins of cells W.Martinand M. J.Russell 61 m u l i i h i i

% amino acid identity in p n i o a pairwise alignments h d c s i l i o i c o k s a v _ m i

s y r 80 89 s u s i a a u m m s s b o

_ u i m r m m u c

70 79 u t u a c a h i i m m u e s r c u u b

_ s i a r t c n i i s s a b r s s 60 69 u e y i o r o c g e o t o a b l a a o s _ c t e u u i c c l l h e a c o c t o g z t

50 59 c c u c c t d m m o c o i p p c b o a i z

_ t r c o c c s o a b o y a i x o h r h o o a o n i

40 49 b y e a e c l r o o u b o h e c d e l l

_ b n a m o m f r p l n b c c a m m o e o o l i r i e u

30 39 h t o r r r o h h a o o g s o o o h c f u l t t

_ u c r r e n c e l c r l r r e e n c 20 29 y a e a h e e q a y s o g i s h a a e u y y r h h T A B L M S E C C B M A M S P

no hit or >20 A S H M M P P A T T GK/HK Arabidopsis (HK) Trypanosoma (HK) Giardia (GK) Human (HK) Saccharomyces (HK) GPI Arabidopsis Trypanosoma Spironucleus Human Saccharomyces PFK Arabidopsis (PPi) Trypanosoma (ATP) Entamoeba (PPi) Human (ATP) Saccharomyces (ATP) FBA Arabidopsis (I) Trypanosoma (I) Giardia(II) Human (I) Saccharomyces (II) TPI Arabidopsis Trypanosoma Giardia Human Saccharomyces GAP Arabidopsis Trypanosoma Giardia Human Saccharomyces PGK Arabidopsis Trypanosoma Spironucleus Human Saccharomyces PGM Arabidopsis (I) Trypanosoma (I) Spironucleus (I) Human(D) Saccharomyces (D) Enolase Arabidopsis Trypanosoma Giardia Human Saccharomyces PYK Arabidopsis Trypanosoma Giardia Human Saccharomyces

Figure 1. Global patterns of similarity between theenzymes of theglycolytic pathwaysin eukaryotes and thecorresponding enzymes from several completely sequenced eubacterial and archaebacterial genomes. Theeukaryotic amino-acid sequences were compared withall peptides from thecorresponding genome withgapped B last (Altschul et al. 1997), thepercentage amino acid identity from thepairwise alignment to thebest match was recorded and colour coded. Only such matcheswere counted asaligned over 100 amino acids or more. Note thatthe eukaryotic sequences are much more similar to their eubacterial homologues. (GK (EC2.7.1.2); HK, hexokinase (EC2.7.1.1); GPI(EC5.3.1.9); PFK, 6-phosphofructo-1-kinase (ATP–PFK, EC2.7.1.11; PPi –PFK, EC2.7.1.90); FBA (EC4.12.1.13, withclass I (Schiff-baseintermediate) and classII (endiol intermediate) types); TPI(E.C. 5.3.1.1); GAPDH (phosphorylating, EC1.2.1.12); PGK, 3-phospho- d-glycerate kinase (EC2.7.2.3); PGM,3-phospho- d-glycerate mutase(EC 5.4.2.1; 2,3-bisphospho- d-glycerate dependent (D)and independent (I) types); enolase, 2-phospho- d-glycerate hydrolase (EC4.2.1.11); PYK, pyruvate kinase(EC 2.7.1.40).)

Phil.Trans. R. Soc.Lond. B (2003) 62W. Martinand M. J.Russell On theorigins of cells sequencesimilarity toeukaryotic or eubacterial PFK but This questionleads directly toa second,even more sev- isrelated toarchaebacterial ADP-dependentGK instead ere,problem from thebottom-up perspective: given that (Tuininga et al. 1999); (iii) analdolase that sharesno life evolved from thegeochemicals, andgiven that some- sequencesimilarity with either ofthe typical eubacterial thing like anRNA-world (Gilbert 1986; Poole et al. 1998; enzymes(Siebers et al. 2001); and(iv) aTPI that shares Bartel &Unrau1999; Gesteland et al. 1999; butsee also only residual ( ca.20%) amino-acid sequencesimilarity to Orgel &Crick 1993) ever existedin which catalytic self- theeubacterial enzyme(Kohlhoff et al. 1996). Sowhat replicating systemscould get offthe ground, how on Earth didthe universal ancestorpossess in its pathway from glu- wasit possibleto attain sufficientand sustained concen- coseto pyruvate, andwhere did the glucose come from trations ofthe building blocksof life that are neededfor in thefirst place? Wewill returnto these questions shortly. any self-assemblingprebiotic systemto get tothe stage Amore striking andmuch more significant example that it couldperform asingle roundof replication? involves lipid biosynthesis.Lipid biosynthesisis important becauselipids are theconstituents of membranes andall 3. UNANSWERED,BUT NOTUNANSWERABLE, cells—with noexception —are surroundedby membranes. QUESTIONS Butarchaebacteria andeubacteria possesscompletely dif- ferentlipids (Kandler1998). Archaebacterial lipids consist The secondproblem isquite well knownin thelitera- ofisoprenoidsand various derivatives connectedto sn-gly- tureon the origin oflife andis known as the concentration cerol-1-phosphate by anether bond. Eubacterial lipids problem, asaddressed by DeDuve (1991, 1994), Mayn- consistof fatty acidsconnected to sn-glycerol-3-phosphate ard-Smith &Szathmary (1995) andothers (Cairns-Smith by anester bond (Kates 1979). The biochemical pathways 1982; Shapiro 1986). Various solutionsto this problem leading tothese two kinds of lipid couldnot differ more. have beenproposed, among whichthe currently most Their lowestcommon denominators are acetyl-CoAfor popular are probably surfacecatalysis, that is,the concen- thenon-polar componentand dihydroxyacetone phos- tration ofreactants on inorganic surfacesallowing them phate,which is non-chiral, for thebackbone. Eubacteria toreact (Bernal 1951; Weber1995; Ferris et al. 1996; synthesizetheir fatty acidsfrom acetyl-CoAby carboxylat- Huber & Wa¨chtersha¨user1998), andevaporites, that is, ing it tomalonyl-CoA whichis usedto elongate thegrow- theevaporation ofterrestrial pondscontaining organic ing chain by twocarbon unitsat atime with reduction precursorssynthesized by Miller –Urey-like reactions (Nelson 2001). Afundamentaldrawback tothe sur- ofthe3-ketoacyl intermediates.Archaebacteria synthesize et al. facecatalysis modelis that oncetwo molecules have their isoprenoidsvia thecondensation of IPP andits iso- reactedon a surface,they diffuseaway into theHadean mer, dimethylallyl diposphate,C5 unitsthat are synthe- ocean,never to react again. Afundamentaldrawback to sizedfrom acetyl-CoAvia theMVA pathway (Langworthy theevaporite modelis the sustainability ofcatalysis: any et al. 1982). Notably, eubacteria also synthesizeisopreno- pondso envisagedhas to dry upand refill withouta wash- ids,but they doso through acompletely unrelatedpath- outvery many times beforesomething like aliving system way,the DXP pathway, intermediatesof which are canarise, whichis hard toimagine given theabsence of precursorsfor thiamin diphosphate andpyridoxal diphos- continents,high andhighly frequenttides and 10 km deep phate biosynthesis(Lange et al. 2000). Conversely, oceans at ca.4Gyr (Gaffey1997; Bounama et al. 2001; archaebacteria generally donot synthesize fatty acidsat Glasby &Kasahara 2001; Kamber et al. 2001). The all. Regarding theglycerol backbone, -glycerol-1-phos- sn notionof Hadean oceans chock-full of Oparin ’s prebiotic phate and sn-glycerol-3-phosphate are stereoisomers. soupstill enjoyssome popularity (Bada &Lazcano2002), Eubacteria synthesize sn-glycerol-3-phosphate from dihy- butthe question remains ofhow a solutionat equilibrium droxyacetonephosphate with sn-glycerol-3-phosphate canstart doing chemistry. Putanother way,once auto- dehydrogenase,archaebacteria synthesizethe correspond- claved,a bowl ofchickensoup left at any temperature will ing stereoisomerwith sn-glycerol-1-phosphate dehydro- neverbring forth life. Cometsand meteorites contain genase,which sharesno sequence similarity with sn- organic compounds(Deamer 1985), asOro ´ (1961, 1994) glycerol-3-phosphate dehydrogenase(Koga et al. 1998). suggestedas a possiblesource of some of life ’s building Finally, theether and ester linkages are highly distinct blocks,but it is notobvious how to get suchcompounds chemical bonds.Of coursemany archaebacteria possess tothe necessary concentrations to generate thechemistry thederived tetraether lipids, andvarious modifications of oflife, norhow to select those few active, simple, multi- thepolar headhave long beenknown in both groups purposebuilding blocksthat make upthebasis oflife from (Langworthy et al. 1982; Zillig 1991). Addto this thefind- an interplanetary chemical supply house. ing that murein,the typical peptidoglycan oftheeubacter- The severity ofthe problem concerningthe differ- ial cell wall, ismissing in archaebacteria —one of the entnessof archaebacterial versuseubacterial membrane criteria through which thegroup wasdiscovered lipids andcell walls is,with fewexceptions (see Koga et (Kandler& Ko¨nig 1978), they possesspseudomurein and al. 1998), notas widely recognizedas it perhaps should other constituentsinstead (Kandler 1982) —andwe arrive be.What is generally recognizedas difficult is thestep to from thetop-down perspective at anexceedingly severe cellular organization (Bernal 1960; DeDuve 1991; Oro´ explanandum:given that thereis nosimilarity whatsoever 1994; Segret &Lancet2000), that is,the origin of bona in thecomponents with which archaebacteria andeubac- fide membrane-boundedcells. Coacervates or spon- teria uniformly compartmentalize their cytosolfrom the taneousself organization ofabiotically formedlipids, environment,what onEarth couldhave beenthe precur- through whatever prebiotic chemistry, isimaginable sorof their membrane andcell wall in their last com- (Deamer 1986; Oberholzer et al. 1995), butwhat is not monancestor? imaginable isputting insidethat first little lipid droplet

Phil.Trans. R. Soc.Lond. B (2003) On theorigins of cells W.Martinand M. J.Russell 63 exactly theright mixture ofself-replicating moleculespos- sessingall that is needed(a sourceof energy, for example, (a) (b) onceisolated from thesurrounding aqueous phase) to make more ofthemselves, including thelipids in which they wouldhave beenencased. Gettingthe universal ancestorinto the membranous or other cloak that it hasto have at sometime underall modelsfor theorigin oflife andthe origin ofcellsposes seemingly insurmountableproblems. In modelsfor theorigin of cells,the membranes that surroundliving systems —under whatever chemical premise (Calvin 1969) andif they are addressedat all (Deamer 1985, 1986) —justseem to come 1 from thin air, andthe ultimate contentsof cellsso derived /2 mm 10 20 mm 30 40 have toarise asa free-living cytosolthat needsto get inside,even in well-argued cases(Segret &Lancet2000). (c) 100 mm Indeed,if welook at all truly living things,which are composedof cells (that is,if weexclude viruses and the like, whichare derivedfrom cellsupon which they depend for replication), theprinciple ofsemi-permeable compart- mentationfrom theaqueous environment is even more strictly conservedthan theuniversal geneticcode, because thereare rare deviationsfrom theuniversal code(Hao et al. 2002; Srinivasan et al. 2002), butthere are noexcep- tionswhatsoever to the principle ofsemi-permeable compartmentation from theenvironment. Furthermore, an attribute oflife that all cellspossess (but viruses do not) is thecapacity toperform redox chemistry —thetransfer ofelectrons from an external donorto an available acceptor.In fact,a very simple definitionof aliving system 20 mm might be:compartments separated from their surround- (d) ings that spontaneouslymultiply with energy gleaned through self-contained,thermodynamically favourable redox reactions.If nothing about life is more conserved than compartmentalized redox chemistry, what wasthe precursorcompartment at theorigin oflife? Wehave a suggestion.

4. IRONMONOSULPHIDE: 3D CELLS, NOTJUST 2D SURFACES In 1981, 360 Myr oldhydrothermally formediron sulphide chimneyswere reported from oredeposits near the townof Silvermines, Ireland (Larter et al. 1981; Boyce et Figure 2. Iron monosulphide precipitates. ( a)A360 Myrold al. 1983). Thesestructures (figure 2 a)nowconsist of hydrothermally formed iron sulphide chimney from Silvermines, Ireland, (Boyce et al. 1983). (b) Electron mostly ofpyrite (FeS 2 ),although they probably precipi- tatedas the monosulphide (FeS) in theCarboniferous sea. micrograph of athin section of a360 Myrold pyrite Someof the chimneys contain sphalerite (ZnS).They precipitate found atthe Tynagh ore deposit, Ireland (Banks 1985). ( )Enlargement of ( ). ( )Electron micrograph of a wereformed through thehydrothermal exhalation of c b d structure formed in thelaboratory byinjecting Na S solution metal-bearing fluid,originally derivedfrom seawaterthat 2 (500 mM, representing hydrothermal fluid) into FeCl 2 had beenconvectively recycledthrough thecrust (Banks solution (500 mM, representing theiron-bearing Hadean et al. 2002). Iron,zinc and lead sulphideswere precipi- ocean) (Russell &Hall 1997). High concentrations were tatedas the hydrothermal fluid exhaled intoa 60 °C brine used to produce an examinable structure. Similar structures pool containing bacteriogenic hydrogen sulphide(Boyce are produced atsubmarine hydrothermal vents byporous et al. 1983; Russell1983; Samson& Russell1987) topro- clay(Geptner et al. 2002). ducethe ore deposits found today. These discoveries had beeninspired by themuch hotter ( ca. 380 °C) mineral- ladenblack smokerfluids emanating from chimneysin the chemically reactive ‘hatcheries’ for theorigin oflife rather easternPacific photographed by Ballard &Grassle(1979) quickly becamea popular notion(Corliss et al. 1981; (seealso Spiess et al. 1980). Although fundamentally simi- Russell et al. 1988), although notwithout staunch critics lar, thechimneys developing at thehydrothermal ventsat (Miller &Bada 1988). oceanicridge crestswere much larger than thecentimetre- Picking upona long biochemical tradition ofthoughts sizedstructures in Ireland. Nevertheless,the possible concerningthe suspectedly primitive natureof redox reac- evolutionary importance ofboth typesof spring siteas tionscatalysed by iron sulphurcentres and their possible

Phil.Trans. R. Soc.Lond. B (2003) 64W. Martinand M. J.Russell On theorigins of cells

cooler, more oxidized, more acid Hadean ocean <30 ° C, pH ca.5.5

the universal ancestor (not free-living) temperature-, redox-, and pH-gradient

DNA era

RNP era

H+ RNA era 4_ P2O7

amino acids CO2 sugarsbases 2+ 3+ organic precursors Fe >>Fe Ni2+ prebiotic chemistry FeS reduced CH3COSR carbon NiS compounds ocean crust

_ HS H2CO CH3SH _ CN CH4 NH geochemistry 3 H2 CO

hot, reduced, alkaline hydrothermal solution ca.100° C, pH ca.10

Figure 3. Amodel for theorigin of life ata redox, pH and temperature gradient ata submarine hydrothermal vent. See Russell &Hall (1997) and Russell et al. (2003) for details. Theterms RNA, RNPand DNAera (instead of ‘world’) are used to emphasizethat no nucleic acid evolution ispossible without asupporting geochemistry, later biogeochemistry and finally biochemistry to provide asteadyflow of adequate concentrations of polymerizeable precursors (for examplenucleotides) and thusto underpin anysort of replication. role in early metabolism (Hall et al. 1971; Marczak et al. catalysis (Wa¨chtershaüser 1988b),this worklaid downthe 1983), butradically departing from notionsof organic casefor achemoautotrophic origin oflife, butit isnot soup,electric discharge or photocatalysis for theorigin of withoutits problems. The reductionof CO 2 with an FeS- thefirst biomolecules,Wa ¨chtershaüser (1988a) suggested H2 S/FeS2 redox couplewas shown to be a far lessener- that redox reactionsbetween iron sulphideminerals, getically favourable processthan originally suggestedby resulting in FeS 2 (pyrite) formation, couldhave provided Wa¨chtershaüser(Schoonen et al. 1999). Moreimportantly, thefree energy andelectrons needed for primordial carbon thesurface catalysis modelstill failed tosolve the problem fixation. Morefully developedas a theory ofsurface ofhow to achieve sufficientconcentrations of reactedpro-

Phil.Trans. R. Soc.Lond. B (2003) On theorigins of cells W.Martinand M. J.Russell 65 ductsto get life offthe ground. Criticizing thenotion of have formedat seepagesite hydrothermal moundsin the 2D life onsurfaces and referring tothe fate of reacted Hadean. productson surfaces,De Duve& Miller (1991, p.10 015) noted ‘These,once detached, are consideredirretriev- 5. ONTHEORIGINOF LIFE ANDCELLS ably lost’. Atthat point,geochemists studying iron sulphidepre- Foundedin geology, geochemistry andthermodyn- cipitates asthey arise andoccur in naturetook careful note amics,a modelfor the(chemoautotrophic) origin oflife ofthesedevelopments and suggested, on the basis ofgood wasproposed by Russell& Hall (1997), thesalient evidence,a different ‘…role for iron sulphide,that of elementsof which are asfollows: (i) life emergedas hot, nucleating themembrane oftheearliest cell walls ’ (Russell reduced,alkaline, sulphide-bearing submarine seepage et al. 1990, p.387). Animportant part ofthat goodevi- watersinterfaced with thecolder, more oxidized,more denceis shownin figure 2. Iron monosulphideas it nat- acid, Fe2 1 Fe31 -bearing water at deep( ca.4km) floors À urally precipitates from submarine hydrothermal exhalates ofthe Hadean ocean ca.4Gyr ago; (ii) thedifference in formsstriking structures(Russell 1983). Figure 2 b shows acidity, temperature andredox potential ofthese waters anelectron micrograph ofa thin sectionof a 360 Myr old provided agradient ofpH ( ca.fourunits), temperature pyrite precipitate foundat theTynagh oredeposit, Ireland (ca. 60 °C)andredox potential ( ca.500 mV) at theinter- (Banks1985) andreveals ahighly compartmentalized faceof these waters that wassustainable over geological innerfabric. Anenlargement offigure 2 b is shownin fig- time-scales,providing thecontinuity of conditions con- ure 2c,which showsa spectrumof internal compartments duciveto organic chemical reactionsneeded for theorigin that are incompletely separatedfrom oneanother by oflife; (iii) theexhalate waspart ofa typical hydrothermal membrane-like sheathsof iron sulphideprecipitate. systemthat brought water from theocean floor moder- Iron sulphidestructures of this type are familiar to ately deepinto the crust ( ca.5km), whereit wassubjected scientistsin thefield ofeconomic geology, butit isvery tovery high pressuresand temperatures, and thus con- worthwhile for biologists andbiochemists to have agood tainedoutgassing CO,H 2 , N2,along with reducednitro- 2 look at suchimages, becausemost biologists andchemists gen (NH3 , CN )(Schulte& Shock1995), reduced 2 wouldimagine anFeS precipitate asa mere clump ofmin- carbon (CH3 COO , H2 CO,shortalkyl sulphides),CH 4 2 eral with nointernal structure:at theextreme acubewith (Kelley 1996; Appel et al. 2001), andHS , which are hardly unreasonablepremises and which were individually six facesfor surfacechemistry. citedand justified on geochemical grounds;(iv) colloidal Naturally formed,geologically occurring 3D FeS struc- FeSprecipitate formedat theocean floor interfacewas turesof this type are extremely relevant tothe origins of inflated by theemergent exhalate toproduce structured life. Asdiscussed in great detail (Russell& Hall 1997), iron monosulphideprecipitates possibly similar in mor- they provide avery plausible solutionto the problem of phology tothose shown in figure 2; and(v) thesustained achieving concentrationsof reactantssynthesized through redox gradient ofabout half avolt acrossthe walls ofthe FeS chemistry that are sufficientto generate amolecule FeSprecipitate, which ofcourse would have contained population ofthe complexity that prebiotic chemistry NiS andsulphides of other metals presentin theHadean requires,if it isever going toevolve life. For example, iron oceanas well, provided apersistentsource of electronsfor sulphidesare knownto have high affinities for organopho- prebiotic chemistry in addition tocatalytic surfacesof sphates,cyanide (Woods 1976; Leja1982), amines great area and,most importantly, provided acontinuously (Wark &Wark 1935) andformaldehyde (Rickard et al. forming (‘growing’)networkof 3D compartmentswithin 2001). which reactantscould remain concentrated.A slightly However,the Tynagh andSilvermines chimneys(figure adaptedversion of Russell & Hall ’s(1997) modelis 2a–c)wereformed from ca. 200 °Cexhaling fluid contain- presentedin figure 3. Notably, aproton-motive force ing dissolvediron, zinc and lead, which precipitated asit foundedin anatural pHgradient existsacross the outer met cooler (ca. 60 °C)sulphide-bearing brine during the FeSmembrane. late Devonian.At theorigin oflife some4 Gyr ago, the Only fewminor modifications with respectto the orig- 2 1 conditionswere somewhat different, as Fe concen- inal modelhave beenintroduced here. One of these con- trations in theoceans were high andthe exhaling fluid cernsthe possible temperature ofthe exhalate ( ca. 70– 2 wouldhave containedhydrosulphide (HS ) (Russell & 100 °C instead of ca. 150 °C)andthe temperature ofthe Hall 1997). Simulating Hadeanconditions in thelabora- oceanfloor water ( ca. 0–30 °C versus ca. 100 °C) (Russell tory by injecting Na 2 Ssolution(approximating hydrother- et al. 2003). Modernhydrothermal exhalates canbe hot, mal fluid) intoFeCl 2 solution(approximating theiron- exceeding350 °C,or muchcooler, for example 70 °C bearing ocean)resulted in highly structurediron monosul- (Kelley et al. 2001; Geptner et al. 2002), whereby thelat- phide precipitates (Russell& Hall 1997), anelectron ter have apHof 9.8 or more.Owing tothe very poor micrograph ofwhich is shownin figure 2 d.Of course,the stability ofcritical building blocksof life suchas RNA, preciseconditions at seepagesites which existed4 Gyr temperaturescloser to 50 °Cseemmore conduciveto the ago, andwhether they wouldproduce exactly suchstruc- assembly andsustained stability ofearly organic molecules tures,is not known. But deep ocean hydrothermal convec- andself-reproducing systems (Moulton et al. 2000; tive currentsand their seepagesites should have existed Bada &Lazcano2002). Thus,it seemsmore reasonable to in theHadean (Nisbet & Sleep2001), andstrikingly struc- assumemore moderatetemperatures for thehydrothermal turedclay precipitates have beenreported at active sub- exhalate. Regarding thetemperature oftheocean, there is marine seepagesites in Iceland(Geptner et al. 2002). It is considerableuncertainty. Because the sun was much dim- hardly unreasonableto assume that metal sulphideswould mer 4Gyr ago than it is now(Newman & Rood1977;

Phil.Trans. R. Soc.Lond. B (2003) 66W. Martinand M. J.Russell On theorigins of cells

Sagan &Chyba 1997), it ispossible that theHadean night withoutpressure at 100 °Cfrom 4.5 mmol COand oceanswere intermittently frozenin global glaciation, in 100 mmol CH3 SH(Heinen& Lauwers1996) in the which casethe ocean floor temperature wouldhave been presenceof FeS and NiS, making this type ofreaction in very cold,or that they werefluid in amuchwarmer cli- FeSprecipitates extremely plausible asa prebiotic source mate dueto high levels ofgreenhouse gases such as CO 2 , ofbasic carbon building blocks.However, the experiment

CO and CH4 in theatmosphere (Nisbet& Sleep2001). producedno pyrite. CH 3COSCH3 containsa thioester The modelwould merely require theocean temperature bondand can be considered as homologous tothe busi- tobe below that ofthe exhalate. Alocal gradient oftem- nessend of acetyl-CoA. In mostorganisms that usethe peraturesexists at all submarine springs andseepages. acetyl-CoApathway today,the next step of CO 2 fixation Anotherchange involves theremoval ofthe reverse cit- is catalysed by pyruvate synthase,also called PFOR, an ric acid (TCAor Krebs)cycle as an early CO 2 fixation FeSprotein that combinesthe acetyl moiety ofacetyl-CoA pathway. Wa¨chtershaüser (1988b)madea strong casefor with CO2 andtwo electrons from ferredoxin,another thereverse TCA cyclebeing perhaps thefirst biochemical FeSprotein, to yield pyruvate (Schoenheit& Scha¨fer pathway, basedon its construeablesimilarity toFeS 1995; Yoon et al. 1999). At very high temperature and chemistry andthermodynamic attributes.But like any cyc- pressure,traces of pyruvate weresynthesized from CO lic pathway ofCO 2 fixation suchas the Calvin cycle,3- andalkyl thiols by FeScatalysts (Cody et al. 2000). In hydroxypropionate pathway or pentosemonophosphate contrastto other views(Wa ¨chtershaüser 1988b; Huber & cycle,the reverse TCA cyclerequires pre-existing, stereo- Wa¨chtershaüser1997), wesee no need for thepresence chemically correctchemical intermediatesof the cycle to of cyclic CO2 fixation pathways at this stage,because a get started.A casecan be made for theview that formol linear routeof carbon fixation wouldsuffice, given con- condensation-likereactions could have fuelledearly CO 2 - tinuousgeochemical input. fixation (Russell et al. 2003). However,the acetyl-CoA There is avast literature regarding plausible modelsfor pathway ofCO 2 fixation, better knownto many asthe prebiotic synthesesof sugars (formol condensation Wood–Ljungdahlpathway, is also very attractive asa (Quayle &Ferenci 1978; Mu¨ller et al. 1990)), bases starting point ofmetabolism becauseit is linear, using (cyanidecondensation (Oro ´ &Kimball 1962; Sanchez et electronsfrom H 2 toreduce 2 mol ofCO 2 toproduce an al. 1967)), amino acids(Hennet et al. 1992; Marshall acetyl moiety that istransferred to the thiol group ofcoen- 1994) andother molecules(Sutherland & Whitfield zyme A(Ljungdahl1994). In theCO 2-fixing direction, 1997), anda vast literature onthe broad catalytic capabili- thepathway has thenet reaction: tiesof RNA (Bartel &Unrau1999; Gesteland et al. 1999), noneof which wecandeal with adequately here.We sur- 2CO 1 8[H] 1 CoASH CH COSCoA 1 3H O, 2 ! 3 2 misethat it is conceivable andnot implausible that using with estimatedthermodynamic valuesof DGo9 suchconceptual tools one could get tosomething like an 2 1 21 = 259.2 kJ mol if 2[H] = H2 or DGo9 = 113.2 kJ mol if RNA-world(Gilbert 1986), provided that its monomeric 2[H] = NADH(Fuchs 1994). The pathway is flexible and building blocksare steadily replenishedand remain in suf- occursamong various eubacteria andvarious archaebac- ficientconcentrations, conditions that structuredFeS pre- teria. Among other things,it canbe: (i) thebasis of cipitates couldfulfil becauseof their sustainedenergy chemoautotrophy in sulphate-reducingeubacteria and inputthough thenatural redox gradient andby their com- archaebacteria, in methanogens,and in homoacetogens partmentalizing, reactant-retaining nature(Russell & such as Clostridium thermoautotrophicum whengrowing Hall 1997). chemoautotrophically (Ljungdahl1994); (ii) thesource of chemiosmoticpotential in homoacetogenswhen growing 6. HOLOCHIRALITY fermentatively (Ljungdahl1994); (iii) apathway ofacetyl-

CoAoxidation if CO 2 and H2 levels are keptextremely Anotheraddition tothe proposal ofRussell & Hall lowthrough consumptionby other microbes;(iv) apath- (1997) is that anunresolved problem —theholochirality way ofacetate disproportionation toH 2 and CO2 in ace- problem—needsto be addressed (see also Russell et al. toclastic methanogenesis;(v) apathway for the (2003) for another perspectiveon this issue).Life today assimilation ofvarious C 1 compounds;and (vi) asource consists of l-amino acidsand d-sugars,but the molecules ofelectrons for carbon fixation through oxidation ofCO at thevery beginning oflife almost certainly didnot. It to CO2 (reviewedin Fuchs1994; Ragsdale 1994; Ferry has beenargued that asymmetric surfacescould give rise 1995). tostereospecific prebiotic synthesis(Hazen et al. 2001), The key enzymeof the acetyl-CoA pathway isan butgiven thesurfaces of iron sulphide,we find it uncon- extremely intriguing protein called ACS/CODH vincing that oneor theother stereoisomerof either class (Ljungdahl1994; Ragsdale 1994; Ferry 1995; Lindahl shouldtend to be synthesized at early stages.Thus, our 2002). In theCODH domain, ACS/ CODHemploys five modelup to this point couldin principle lead toa sus- FeS clustersincluding anunusual 4FeNi5S clusterthat tainable andcompartmentalized RNA-world,but a badly binds CO2 andreduces it toCO. Aspart ofthe proposed scrambled,primitive oneof racemic sugarscoexisting with reaction mechanism,the ACS domain ofACS/ CODH aracemate ofamino acidsand everything else.A possible condensesNi-bound methyl andNi-bound CO, yielding way outof theracemic worldcould have involved theori- anNi-bound acetyl moiety that istransferred to CoASH gin ofthepeptidyl transferasereaction ofthe large riboso- yielding acetyl-CoA(Lindahl 2002). Emulating theACS/ mal subunit.Among themyriad ofmolecules that existed CODHenzymatic reaction,Huber & Wa¨chtersha¨user in theRNA-world, a population ofthem wasinvolved in (1997) showed inter alia that micromolar amountsof theorigin oftranslation, wherebyit hasbeen sensibly sug- methyl thioacetate (CH 3COSCH3 )couldbe formed over- gestedthat theoriginal functionof the ribosome wasRNA

Phil.Trans. R. Soc.Lond. B (2003) On theorigins of cells W.Martinand M. J.Russell 67 replication via triplets, nottranslation (Poole et al. 1999). might provide insights asto how biochemistry evolved, Wecan take it asa given that at somepoint aprecursor andmight evenreveal various primitive analogues ofcon- ofthe peptidyl transferasereaction (Muth et al. 2000) of temporary active centres. anRNA moleculedid evolve, and the chance stereochemistry ofthat moleculewould determine the decision of 7. THE UNIVERSALANCESTOR: THE LAST whetherit couldcatalyse thepolymerization of d- or l- COMMONANCESTOR OF PROKARYOTESONLY amino acidsinto peptides. That filter wouldbe sufficient totip theholochirality scale,because despite the presence By thetime that aribosome had evolved,a genuine ofaracemate, only amino acidsof the same a-carboncon- ribonucleoprotein world,the step to a DNAworldand the figuration wouldpreferentially endup in peptides,yielding last commonancestor would not be far off.But here we apopulation ofdistinctly handedpeptides, some of which have todepart completely from somelong-held andwidely wouldeventually needto feed back in ahypercycle-like regarded viewson the early evolutionof cells (Woese manner (Eigen& Schuster1978) intofavoured synthesis 2002), becausethe weight ofevidenceprecludes the exist- oftheir stereochemically correctpolymerizing template. enceof eukaryotes anywhere near theorigin ofcells.The Concerning d-sugars,if weassume that theFeS-cata- reasonsfor this are at least threefold. lysedsynthesis of acetyl-CoA and pyruvate waspossible First, withoutquestion, eukaryotes are ancestrally heter- asabove, then the origin ofchirality shouldbe sought at otrophs,the only autotrophy in theentire group having thethree-carbon stage. Pyruvate is notchiral, noris its beenacquired through endosymbiosisduring theorigin of phosphorylated derivative, phospho enolpyruvate. In plastids.All eukaryotesthat lack plastids andsome that organisms that usethe acetyl-CoA pathway, thenext step possessplastids are heterotrophs,and without exception ofautotrophic carbon metabolism involves thestereospec- thebackbone of their ATPsynthesisis theoxidative ific addition ofa water moleculeto the double bond in breakdownof reduced carbon compoundsthrough the phosphoenolpyruvate, yielding 2-phospho- d-glycerate. glycolytic pathway (Martin &Mu¨ller 1998). Embracing Today, that reaction is catalysed by theenzyme enolase, theview ofa chemoautotropic origin oflife, thefirst which is by far themost highly conservedof all glycolytic organisms necessarilyhad tobe chemoautotrophs enzymes(figure 1) andas far aswe are aware,is the only (Kandler1998) andheterotrophy asa microbial lifestyle enzymeof core carbon metabolism that istruly ubiquitous couldonly have arisen after autotrophshad evolved a among all free-living organisms. For thewhole remainder metabolism sufficientto support the free-living lifestyle ofcentral carbon metabolism in all organisms, thestereo- andhad subsequentlyproduced sufficient quantities of chemistry at C2 of2-phospho- d-glycerate neverchanges. reducedcarbon compoundsin theenvironment to provide This approach tothe holochirality problem implicates the heterotrophs —prokaryotic andeukaryotic —with some- peptidyl transferasereaction ofthe ribosome asthe pace- thing toeat. maker for amino-acid stereochemistryand a simpler pre- Second,all ofthe eukaryotes whose origin needsto be cursorof enolase as the pacemaker ofsugar explained (contemporary ones)seem to have possesseda stereochemistry.It is probably justcurious coincidence mitochondrionin their evolutionary past (seeEmbley & that enolaseis theonly enzymeof carbon metabolism, Hirt 1998; Roger 1999; Williams et al. 2002). The origin other than adenylate kinase,that balancesAMP, ADP and ofeukaryotes is thus hardly separable from theorigin of ATPlevels,that is encodedin thehighly conservedriboso- mitochondria (Gray et al. 1999; Lang et al. 1999). This mal protein superoperonin archaebacteria (Hannaert et wouldmean that theorigin ofeukaryotes would necessar- al. 2000), whichmight also carry afaint trace ofchiral ily post-datethe diversification ofthe a-proteobacteria origins. Atany rate, oursuggestion for theorigin ofholo- from whichthe mitochondrion is thought todescend, chirality startswith thepeptidyl transferasereaction and meaning that eukaryotesmust substantially post-datepro- isthus largely congruentwith Woese ’s(2002, p.8745) karyotes in origin. assessment ‘The evolutionof modern cells, then, had to Third, if welook beyondthe diversity ofthe genetic begin with theonset of translation ’.It wascertainly a systemand consider the diversity ofmetabolic pathways decisivestep. that prokaryotes useto synthesize the ATP that is the Oneparticularly interesting thought emergesfrom the backboneof their survival, thenwe are facedwith the foregoing, namely that theprimitive peptidessynthesized more than 150 differentredox reactionsinvolving inor- by thekind of a universal ancestorshown in figure 3 ganic donorsand acceptors used by thethermophilic pro- (particularly thesulphhydryl-containing ones)would have karyotes alonefor their coreenergy metabolism (Amend& provided achiral, proteinaceoussurface associated with Shock2001) versusthe complete diversity ofeukaryotic anFeS (or other metal sulphide)centre. This wouldhave energy metabolism, which doesnot comprise more than provided dramatic synergy for biochemical diversity, and onesingle pathway ofglucose oxidation topyruvate and shouldhave initiated anera ofbiochemical discovery,with threebasic metabolic pathways ofsubsequent pyruvate Darwinian backcoupling (via translation) tothe encoding metabolism (Martin &Mu¨ller 1998). The full diversity of nucleicacids. The very clear prediction ofthis notionis eukaryotic energy metabolism, including thediversity of that experiments ofthe type performed by Heinen& enzymescatalysing theindividual steps(figure 1), doesnot Lauwers(1996) or Huber& Wa¨chtersha¨user(1997), exceedthat ofa single,generalist a-proteobacterium. With whensupplemented with small syntheticholochiral pep- sucha narrow biochemical diversity at theroot oftheir tides(random mixtures ofvarying complexity), should ATP synthesis,it seemscompletely at oddswith common catalyse thesynthesis of a muchmore diversespectra of senseto assume that eukaryotesare anywherenearly as biologically relevant moleculesthan controlexperiments oldas prokaryotes. Prokaryotes are easily seenas hold- lacking thesynthetic peptides. Experiments of this type oversfrom thephase of evolutionwhere novel biochemical

Phil.Trans. R. Soc.Lond. B (2003) 68W. Martinand M. J.Russell On theorigins of cells routesto glean energy from theenvironment were still have usedthe naturally existing protongradient (alkaline being invented,and many suchrelics canbe found among inside,acid outside)at theseepage site. prokaryotes today.The argument by Kandler(1998), that This non-free-living universal ancestorwould have thecomplete lack ofchemoautotrophy among eukaryotes founditself at thedawn of the biochemical revolution prescribestheir origin subsequentto the origin ofprokary- wheregenes and proteins were diversifying intoa myriad otesis compelling. Butthe question of how eukaryotes offunctions, where RNA andmetal sulphidecatalysts andtheir definingfeatures might have arisen is adifferent werebeing replaced by proteins,where new pathways and matter tobe discussed in alater section. cofactorswere being inventedto augment andsubstitute There are noeukaryotesthat canlive from theelements their mineral andRNA precursors,where FeS from the alone withoutthe help ofprokaryotes (or prokaryotic mother lodewas being incorporated intoproteins as FeS endosymbionts).By contrast,there are many eubacteria clustersand where biochemistry startedto diversify into andarchaebacteria that canlive from theelements alone theforms that wereboth possibleand useful. From the (chemoautotrophs),and it is henceeminently reasonable standpointof protein structure,this age ofinvention toassume that organisms able tolive from theelements wouldhave witnessedthe origin ofbasic building blocks alone arosebefore those organisms that cannot.The con- ofbiochemical functionthat: (i) are conservedat thelevel verseargument that eukaryotesarose before prokaryotes ofa 3D structureamong archaebacteria andeubacteria; (asfor example argued in Forterre &Philippe (1999)) is and(ii) are recognizable asmodules of function in various completely unreasonablefrom thestandpoint of microbial electron-transporting proteinsas discussed by Baymann et physiology andis only tenable if onelooks at geneticsys- al. (2003). From thestandpoint of amino-acid sequence temsand genetic systems only, leaving microbial physi- conservation,this age ofinvention would have beena ology aside.But physiology isjust as important for early phaseof molecular evolutionwhere proteins were diver- evolutionas genetic systems are, becauseno genetic sys- sifying (e.g.Habenicht et al. 1994) andgetting betterat tem operateswithout a physiological systemto support it what they do,rather than notgetting worseat what they with energy in theform ofATP andprecursors for doing do,as is thought tobe thecase for theevolution of today ’s thethings that geneticsystems do. Arguing that eukary- proteins.Put another way,most modern proteins are otespredated prokaryotes, which underwentsome mys- thought toaccept amino-acid substitutionsbecause the teriousreductive process, is like arguing that land animals newamino acid doesnot significantly impair thepre-exist- predatedland plants,which nourishedthemselves from ing function(neutral or nearly neutral molecular animal excrements.The mostfundamental difference evolution).At the very beginning ofprotein evolution, betweenprokaryotic andeukaryotic physiology from the positively selectedmutations that improve thefunction of standpointof energy metabolism is illustrated in figure 4, theprotein might have beenthe rule rather than the whereonly avery small sample ofthe various redox exception,because without advantageous mutations, couplesused by prokaryotes assummarized by Amend& adaptive evolutioncannot occur (Nei 1987). Accordingly, Shock(2001) are contrastedto the entire diversity of themechanistic basis ofsequence divergence among the energy metabolism foundin eukaryotes(see also Scha¨fer mostancient proteins (intense positive selection)would et al. (1999) for anoverview ofenergy metabolism in differfundamentally from that in recently diverged pro- archaebacteria). Clearly, underour premise, embracing a teins(near neutrality). chemoautotrophic origin oflife, eukaryotessimply cannot have arisen at evenapproximately thesame time asproka- 8. FROMA NON-FREE-LIVINGUNIVERSAL ryotes.At any rate, commonsense and a bit ofbiochemi- ANCESTORTO FREE-LIVING CELLS cal reasoning dictatesthat eukaryotesmust have arisen long after prokaryotes did,as most biologists have always At somepoint, this biochemical diversification would reasonably assumedand as the microfossil recordindi- have includedthe invention of pathways that catalyse the cates. synthesisof lipids andcell wall constituents(figure 5), Our modelfor theorigin oflife tothis point hasspecifi- both ofwhich would have begunto insulate the cytosol cally addressedthe arguably mostimportant problems that from its FeSencasement, requiring anewcategory ofpro- early self-organizing andself-replicating systemsfaced, teins:membrane-associated proteins involved in redox namely: (i) wheredid the energy comefrom that isneeded chemistry. Thesewould have beenrich in FeS(and other tocontinuously synthesize the building blocksof evolving metal sulphide)centres so as to replace thefunctions orig- geneticsystems (natural redox andpH gradient); and(ii) inally associatedwith theoriginal FeS redox chemistry howdid the chemical constituentsof life manage tostay germane tothe original FeS ‘cell wall’.Asa consequence, sufficientlyconcentrated to permit any kindof replication thecatalysis performedby (Fe Ni)S (Russell et al. tooccur (structured FeS precipitates). 2003) wouldhave beengradually separatedÀ from themin- The universal ancestoras depicted in figure 3was,in eral wall andstep-by-step incorporated intoproteins, an ourview, an organism —though nota free-living one —that imprint ofwhich wouldbe reflected in theFeS centres of shouldhave possessedall ofthe attributes that are com- ancientproteins under this view,as schematically indi- monto all eubacterial andarchaebacterial chemoauto- catedin figure 6. Protein modulessuch as ferredoxins and trophs:the genetic code, the ribosome, DNA, a hydrogenaseare very commonin today ’smembrane-asso- supporting coreand intermediate metabolism (nitrogen ciatedelectron transport chains(Baymann et al. 2003). fixation, modernamino-acid, nucleic-acidand cofactor Only whena sufficientbattery ofredox carriers had biosyntheses)needed to supply theconstituents of itsrep- becomeassembled into the membrane toallow thecom- lication (doubling ofmass), compartmentation from the partments tosustain ATP synthesisvia chemiosmosisout- environment,redox chemistry andATPases, which could sidethe confines of their 3D (Russell& Hall 1997; seealso

Phil.Trans. R. Soc.Lond. B (2003) On theorigins of cells W.Martinand M. J.Russell 69

Figure 4. Acomparison of anarrow segment of theredox reactions involving inorganic donors and acceptors thata narrow segment of prokaryotes use for sustained ATPsynthesis(excerpted from Amend &Shock(2001)) versus theentire breadth of reactions thatnon-photosynthetic eukaryotes in toto use for sustained ATPsynthesis. Note thatthere are rare caseswhere mitochondria can operate chemotrophically, but only transiently, not for sustained ATPproduction (Doeller et al. 2001).

Koch& Schmidt 1991), naturally chemiosmotichousing have begunto look reasonably similar tomodern prokary- wouldthey have beenable tomake first attempts at life otic membranes,at least in termsof basic architecture. in thewild. With theinvention of pathways for biosynth- Notably, ourmodel for theindependent origins of esisof redox cofactors,quinones, hemes and other elec- membrane-boundedcompartmentation in archaebacteria tron-transporting centres,these early membranes might andeubacteria predictsthat theelectron- and proton-

Phil.Trans. R. Soc.Lond. B (2003) 70W. Martinand M. J.Russell On theorigins of cells

eubacteria Fe2+ H+ _ HCO3 archaebacteria free-living cells Fe3+ (chemoautotrophs)

Ni2+

escape with conservation of redox chemistry at cell surface

independent invention of cell wall biochemistry

independent invention of lipid biosynthesis

invention of most biochemistry

the universal ancestor (not free-living)

Figure 5. Amodel for theorigin of membrane-bounded prokaryotic cells from iron monosulphide compartments within which thechemoautotrophic origin of life could haveoccurred. Thedrawing implies (but does not show)very large populations of replicating systemsand unspecified physicaldistances (within, however, asingle submarine seepagesite) between diversifying replicating systems en route to free-living eubacterial and archaebacterial cells. Various kinds of genetic exchange thathave been repeatedly postulated (Woese 2002) before theorigin of atruly membrane-bounded cellular organization of prokaryotes could beeasily accommodated bythe model. Note thatiron monosulphide precipitates are initially colloidal (Russell &Hall 1997) and inflated to thegeneral typesof structure shown in figure 2byexhaling hydrothermal fluid. transporting quinonesof their independentlyevolved unrelatedbiochemical pathways in thetwo groups. Eubac- membranes shouldalso differsubstantially. That predic- teria synthesizeIPP via theDXP pathway, whichstarts tionfares quite well. The quinonesof both groups ofpro- from d-glyceraldehyde-3-phosphate andpyruvate via a karyotes possessisoprene side chains (Schu ¨tz et al. 2000; decarboxylating transketolasereaction (DXPsynthase), Berry 2002). Howeverthese isoprenes —like thelipids whereasarchaebacteria synthesizeIPP via theMVA path- themselves—are synthesizedby different,completely way,which starts with thecondensation of three molecules

Phil.Trans. R. Soc.Lond. B (2003) On theorigins of cells W.Martinand M. J.Russell 71

(1997) type conditionsin thepresence of suitable nitrogenouscompounds. Underthis view,the evolutionary basisof the attributes sharedby prokaryotes wouldbe founded in their inherit- ancefrom thelast commonancestor. The evolutionary basis for thefundamental differences that distinguish eubacteria andarchaebacteria, butwhich more or lessuni- [Fe] hydrogenase formly unitethe respective groups, could be sought in the age ofbiochemical discovery during thepersistence of pyruvate:ferredoxin oxidoreductase proto-eubacterial andprotoarchaebacterial lineages living within theconfines of their energy-ladenmineral incu- bator. Suchdifferences would include their useof many . [4Fe 4S] ferredoxin fundamentally differentcofactors such as pterin derivatives versustetrahydrofolate, coenzymeB, methanofuran, coenzymeF420, coenzymeM andcobamids in many archaebacteria (seeDiMarco et al. 1990; Thauer 1998; [2Fe.2S] ferredoxin White 2001), their useof differentpathways for thesame metabolic intermediatessuch as IPP (Lange et al. 2000) or their differencesin purinebiosynthesis (White 1997), their use(archaebacteria) or disuse(eubacteria) ofsmall nucleolar RNAsfor themodification ofribosomes (Omer et al. 2000), thedifferences between their DNAmainte- nanceand repair machineries (Tye 2000), thedifferences carbon monoxode fumarate reductase betweentheir transcriptional regulatory apparatus dehydrogenase (Thomm 1996; Bell et al. 2001) andRNA polymerases (Langer et al. 1995; Bell &Jackson1998), their quinones Figure 6. Theposition of FeS centres in some proteins that (Schu¨tz et al. 2000; Berry 2002), or,and what is probably catalyseredox reactions. Figures were sketchedfrom: [Fe]- themost important point for this paper, thedifferences hydrogenase (Peters 1999); PFOR (Chabriere et al. 1999); betweentheir membrane lipid biosynthetic pathways and [4Fe·4S] ferredoxin (Macedo-Rieiro et al. 2001); [2Fe·2S] cell-wall constituents(Kandler 1982). Manyother such ferredoxin (Kostic et al. 2002); CODH(Lindahl 2002); and differencescould be listed. The assembly ofFeS-clusters fumarate reductase, amembrane protein (Lancaster et al. 1999). Blue dots indicate active sites. intoproteins, an ancient biochemical function(Tachezy et al. 2001), wouldhave beenamong thoseattributes essentialto both lineages,a functionpossibly provided by therecently discovered SUF pathway ofFeS cluster ofacetyl-CoA (Lange et al. 2000). Notonly dothe iso- assembly (Takahashi &Tokumoto 2002). Efficientmeans preneside chain pathways differin thetwo groups, the of N2 -reduction,perhaps through primitive molybdenum – proton-and electron-transferring aromatic headsof the iron,vanadium –iron or iron-only nitrogenases(Chien et quinonesalso differin archaebacteria andeubacteria: al. 2000) wouldhave also beenprerequisite tothe tran- although somearchaebacteria possessmenaquinone and sitionto a free-living habit. ubiquinonelike eubacteria, othersuse sulphur-containing Oncethe crucial stepof membrane synthesisand a tur- heterocyclicssuch as the benzothiophenes of Sulfolobus gor-resistant cell wall had beentaken, independently, the and Acidianus (Schu¨tz et al. 2000), wherebythe methano- twokinds of prokaryotic stemlineage couldhave begun gensuse methanophenazine, which is nota quinoneat all making their attempts at thetransition tothe free-living (Berry (2002), q.v.for quinonestructural comparisons). state(independently) and begun trying their luckat life Interestingly, akey enzymeof the MVA pathway, 3-hyd- as—chemoautotrophic —free-living cells(figure 5). If one roxy-3-methylglutaryl-CoA synthaseis distantly related to cell canescape, so can the next, and the next, continu- b-ketoacyl-acyl carrier protein synthaseIII ofeubacteria ouslyuntil thewell at theseepage site ran dry,at which (Lange et al. 2000), which catalysesa similar condensation point theorigin oflife wouldhave beenover. Those free- reaction toproduce the fatty acid precursoracetoacetyl- living cellsthat possessedor inventedthe biochemical ACPfrom acetyl-ACP andmalonyl-CoA assubstrates, toolsneeded to survive outsidetheir initial confineswould indicating thepresence of an ancestral acetyl-CoAcon- have foundthemselves in avery standardkind of Darwin- densingprotein in thenon-free-living universal ancestor. ian struggle, butone with aparticular reward for ingenuity Furthermore, DXPsynthaseof the DXP isoprenoidroute at tapping newkinds of inorganic redox resourcesin an isdistantly related toPFOR, aferredoxin-dependent uninhabited,competitorless, ocean-floor world. This enzymecatalysing theC 2 C3 stepsubtending the acetyl- wouldhave beena time wheremuch of the oxygen-inde- CoApathway in many chemoautotrophs,! indicating the pendentmetabolic diversity among theprokaryotes might presenceof this primitive C 2 -transferring stepin thenon- have arisen (figure 5). The age ofbiochemical discovery free-living universal ancestor.Because both DXPsynthase wouldhave founda veritable gold mine at theoceans ’s andPFOR require TPP in their C 2 -transferring catalysis, surface,and the origin ofchlorophyll-based photosyn- theprediction clearly followsthat functionaland/ orstruc- thesisin theeubacteria emergedapparently quiteearly tural analogues ofthe TPP coenzymeshould be obtainable (Dismukes et al. 2001; Russell& Hall 2001; Xiong & from mineral catalysts underHuber & Wa¨chtersha¨user Bauer 2002). With that, theprokaryotic worldwould have

Phil.Trans. R. Soc.Lond. B (2003) 72W. Martinand M. J.Russell On theorigins of cells beenoff and running, changing thechemistry ofthe Firmly embracing thenotion of a chemoautotrophic origin oceans,and eventually supplying enoughreduced carbon oflife, thenagging matter oflacking chemoautotrophy in sedimentto allow theorigin ofheterotrophic prokaryotic eukaryotesprompted Kandlerto note (p. 25): lifestyles.At someearly time, oxygenic photosynthesis wouldhave beenproviding lowlocal concentrationsof a Thedomain Eukaryacontains neither anygenuine newand powerful electron acceptor (O 2 ),with whichearly chemolithoautotrophic nor thermophilic members grow- microbes had tocope, probably usingexactly thesame ing optimally above50 °C. […]Hence, founder groups strategies asmodern organisms: either through theuse of of thedomain Eukaryamost likely consisted of highly derived mesophilic heterotrophic pre-cells, adapted to an oxygen-detoxifying enzymes(Chapman 1999; Jenny et al. almost ‘modern’,atleast late Archaean or early Protero- et al. 1999), through theavoidance ofoxygen-containing zoic, environment of apredominantly mesophilic tem- niches,or through thegradual adaptation tooxygen, har- perature regime favourable for theevolution of nessingits utilitous oxidative poweras many oxygen- intracellular membrane systemstypical of eukaryotes. dependentmicrobes (figure 4) have done. Woese(2002, p.8742), while consideringthe immense problems ofcellular evolution,surmized: 9. EUKARYOTES:CELLS WITH SOMETHINGINSIDE Initial attemptsto frame theissue havetypically been in The foregoing modelfor theorigin ofcells (prokaryotic theclassical Darwinian mode, and thefocus to date has almost been exclusively on modeling theevolution of the ones)embraces the view ofa chemoautotrophic origin of eukaryotic cell. Thereason, of course, is clear —the life andmetabolism in general andviews organic chemis- appealof theendosymbiosis concept. Because endosym- try catalysed by FeS(and NiS) asancient and ancestral biosis hasgiven rise to thechloroplast and mitochond- in biochemistry. It accountsfor theattributes sharedby rion, whatelse could it havedone in themore remote eubacteria andarchaebacteria asinheritances from their past?Biologists havelong toyed withan endosymbiotic non-free-living commonancestor (figure 5), andaccounts (or cellular fusion) origin for theeukaryotic nucleus, and for thedifferences of eubacteria andarchaebacteria even for theentire eukaryotic cell. Theseclassical explanations havethree characteristics: they(i) invoke cells through divergencefrom that commonancestor. In this thatare basicallyfully evolved; (ii) evolve theessential section,we address the evolutionary basis for theattri- components of theeukaryotic cell well after its archaeal butes:(i) that are sharedby eubacteria andeukaryotes; (ii) and bacterial counterparts (ashas always been connoted that are sharedby archaebacteria andeukaryotes; and (iii) by the term ‘prokaryote’);and (iii) focus attention on that distinguish eukaryotesfrom prokaryotes. eukaryotic cellular evolution, whichimplies thatthe Thus,this sectionboils downto the question of the ori- evolutions of the ‘prokaryotic ’ cell types,are of adifferent character —simpler, and, it would seem, lessinterest- gin ofeukaryotes, one of biology ’smessiestproblems. To ing. [emphasisfrom theoriginal] introducethe issue as briefly aspossible, one can try to distill theessence of what Otto Kandlerand Carl Woese, Both authorsdismiss endosymbiosis as possible explana- whodiscovered the distinctness of archaebacteria from tory principles toaccounteither for thecharacters shared other then-knowncells, and who proposed the three- by eubacteria andeukaryotes, or toaccount for those domain classification oflife (Woese et al. 1990), have characters sharedby archaebacteria andeukaryotes. They recentlywritten on the topic. Considering the characters also dismissfusion models, as do we, because fusion mod- sharedby various lineages,such as those considered by elsare exactly that: they entail thefusion of two cells, each Zillig (1991), Kandler(1998, p.20) wrote: surroundedby its ownmembrane intoa single cytoplasm surroundedby onemembrane, like putting twosmaller …eachof thethree statisticallypossible pairs among the soapbubbles together toget onebigger one.Eukaryotes three domains appearsto bea sister group depending dooccasionally fusecytoplasms (for example at on thebasic phenetic characters considered. Thequasi- fertilization), butprokaryotes donot,and there is norea- random phenetic mosaicism among thethree domains sonto suspect that they ever didin thepast either. But thatthis indicated wasconfirmed atthe genomic level both authorsdo something that is very commonin the when theorthologous genes of thearchaeon Methanoc- occus jannaschii ,of theeukaryote Saccharomyces cerevisiae , literature: they lump endosymbioticmodels and fusion and of several bacterial specieswere aligned (Clayton et modelsfor theorigin ofeukaryotes into onebasket. An al. 1997). While themajority of thegenes of Methanoc- example ofan explicit fusionmodel can be found in the occus are shared witheither bacteria and/or eukaryotes, right panel offig. 3in Lopez-Garcia& Moreira (1999), only aminority of genes seemto beconfined to the wherea free-living consortiumof prokaryotes juststarts domain Archaea (Bult et al. 1996). Sucha quasi-random fusingaway, uniting uptheir cytoplasms. distribution of genes among thethree domains cannot beexplained satisfactorily byany order of dichotomous Nobodydoubts any more that theprinciple ofendosym- branching of acommon ancestral cell or bychimaerism biosisas elaborated by Mereschkowsky(1905), con- originating from fusion or engulfment among prokary- demnedby Wilson(1928) andrevived byMargulis (Sagan otes atearly evolutionary stagesas wassuggested byvari- 1967) is real andthat chloroplasts andmitochondria are ous authors (Zillig et al. 1992; Sogin 1994; Golding & descendedfrom free-living prokaryotes. Butthe endosym- Gupta 1995; Doolittle 1996; Margulis 1996). Thedistri- biontsdid not fuse with their host,rather they remained bution pattern rather calls for arevival of theprevious intact asmembrane-bounded cell compartmentswithin proposal of atripartition atan early pre-cellular stageof life (Fox et al. 1980), resulting in three dichotomously their host,a general principle in cell evolutiontermed branched separatelineages forming aphylogenetic membrane heredity (Cavalier-Smith 2000). Yetrejections ‘bush’ rather thana tree. ofthe notion that endosymbiosismight have given rise not

Phil.Trans. R. Soc.Lond. B (2003) On theorigins of cells W.Martinand M. J.Russell 73 only tothe mitochondrion but also tothe bipartite cell 8744) is definitely notan artefact, it is anincontrovertible which becamea eukaryote,are sometimesreminiscent of observation foundedin thecomparative analysis oftran- Wilson’s(1928) rejectionof thenotion that mitochondria scription andtranslation machineries.Yet, in which light wereendosymbionts to begin with: this observation shouldbe interpreted is adifferentmatter: werethe host a eukaryote or wereit an archaebacterium, More recently Wallin ( ’22) hasmaintained thatchond- wewould arrive at thesame observation. Models for the riosomes [mitochondria] maybe regarded assymbiotic origin ofeukaryotes and the nature of the host have bacteria whoseassociation withthe other cytoplasmic recentlybeen reviewed elsewhere (Martin et al. 2001). components mayhave arisen in theearliest stagesof The starting point for theremainder ofthis sectionwas evolution (p. 712). To many, no doubt, such specu- lations mayappear too fantasticfor present mention in thestudy of the phylogenies ofvarious enzymesof carbo- polite biological society; nevertheless it iswithin the hydrate metabolism in higher plants,starting with range of possibility thatthey may someday call for more GAPDH(Martin &Cerff1986; Martin et al. 1993) in the serious consideration. (p. 739). contextof their homologuesin eubacteria andarchaebacteria andextended to the complete glycolytic pathway, the The questionat theroot oftheproblem onthe origin of Calvin cycle,gluconeogenesis, the oxidative pentosephos- eukaryotes—andthe reason why biologists have tothis day phate pathway, theglyoxylate cycleand the citric acid beenunable to connect up archaebacteria, eubacteria and cycleas reviewed elsewhere (Martin &Schnarrenberger eukaryotesinto aunifiedscheme of cell evolutionthat is 1997; Martin &Herrmann 1998; Henze et al. 2001; generally acceptedby all —concernsthe host: the cell that Schnarrenberger &Martin 2002). Atthe same time, simi- acquiredthe mitochondrion. Less fundamentally prob- lar studieswere being conductedon the glycolytic lematical, butnone the less a decade-longproblem for enzymesfrom amitochondriate protists,as reviewed by endosymbiotictheory are hydrogenosomes:pyruvate- Mu¨ller (1998). For every glycolytic enzyme(except oxidizing, hydrogen- andATP-producing organelles of enolase)(Hannaert et al. 2000) thesame observation was anaerobic eukaryotesthat werediscovered by Mu¨ller scored:the eukaryotic enzymesare more similar totheir (Lindmark &Mu¨ller 1973). Longsuspected to be a third eubacterial homologuesthan they are totheir archaebact- kindof endosymbiotic organelle distinctfrom mitochon- erial homologues,as shown once more in figure 1for the dria andchloroplast andperhaps descendedfrom clostrid- glycolytic pathway, andin many casesthe archaebacteria ial ancestors(Whatley et al. 1979; Mu¨ller 1988), recent donot even possess the homologous enzyme. advanceshave shownthat they are simply ananaerobic To accountfor thefindings: (i) that eukaryotespossess manifestation ofthesame endosymbiont that gave rise to anarchaebacterial geneticapparatus; (ii) that eukaryotes mitochondria (Embley et al. 1997; Van derGiezen et al. possesseubacterial ATP-producing pathways in mito- 2002). chondria,in hydrogenosomesand in thecytosol; (iii) that Onthe bottom line,there are only twopossibilities for mitochondria andhydrogenosomes descend from acom- thenature of thehost that acquired theorganelle ancestral monancestral organelle; and(iv) that amitochondriate tomitochondria andhydrogenosomes: it waseither apro- eukaryotespossessed a mitochondrionin their evolution- karyote or aeukaryote.The long-held view that thehost ary past,a hypothesiswas formulated, which explored the wasa eukaryote drewsupport primarily from theexistence possibility that thehost might have beenan archaebacter- ofeukaryotes without mitochondria (Cavalier-Smith ium,an anaerobic andautotrophic onewith acarbon and 1987) andfrom thefinding that they brancheddeeply in energy metabolism that wasdependent upon molecular theeukaryotic branch ofthe ‘universal tree ’ ofribosomal hydrogen asan electron donor (Martin &Mu¨ller 1998). RNA(Vossbrink et al. 1987; Sogin et al. 1989). Butall of The microbial physiology ofthe model was based on themitochondrion-lacking andsuspectedly primitive phenomenaabundantly observable among modernproka- eukaryotesthat built thebasis for traditional viewson the ryotes:hydrogen transferand anaerobic syntrophy origins ofmitochondria, for example microspridians, (Fenchel& Finlay 1995). The selectivepressure associat- diplomonads,entamoebids and the like, have turnedout ing thehost to its eubacterial symbiont wasposited to have either tohave possessedmitochondria in their past or to beenthe host ’sstrict dependenceupon H 2 producedas a still possessthe organelle in reducedform (Embley &Hirt wasteproduct of the fermentative metabolism ofa facul- 1998; Roger et al. 1998; Roger 1999; Mu¨ller 2000; Phil- tatively anaerobic symbiont.This modelfor theorigin of ippe et al. 2000). They are thusnot direct descendants of mitochondria andfor theorigin ofthe heterotrophic life- an amitochondriate host.Furthermore, theseemingly style in eukaryoteswas fortuitously also amodelfor the deepbranching ofsuch organisms in the ‘universal tree ’ origin ofeukaryotes.It is shownin figure 7, which is larg- isin many if notall casesapparently justan artefact oftree ely self-explanatory. reconstruction(Embley &Hirt 1998; Philippe &Laurent Only fewminor modifications with respectto the orig- 1998). Aprime example ofthis problem are microsporidi- inal figure, butnot to the original model,have beenintro- ans,organisms that startedmany peoplebelieving that ducedhere. First, theoriginal figures (Martin &Mu¨ller mitochondrion-lacking eukaryotesare both ancientand 1998) didnot specifically indicateDNA or ribosomesin primitive onthebasis oftheir rRNA phylogeny (Vossbrick thecells (corrected here) with theeubacterial components et al. 1987). Butthe microsporidia are in facthighly shadedblue and the archaebacterial componentsshaded derivedfungi (Keeling 2001), asrevealed by genome red.There isa limit tothe amount of detail that canbe sequencing(Katinka et al. 2001), andthe seemingly deep includedin sucha figure, hencethe Gram-negative wall positionof their rRNA sequenceis a phylogenetic artefact. andlipopolysacchar idelayer ofthe symbiont are symbolized. However,the ‘great similarity ofthe archaeal andeukar- Anotherchange isthat amodelfor theorigin ofthe yal information processingsystems ’ (Woese2002, p. endomembranesystem in eukaryotes,including the

Phil.Trans. R. Soc.Lond. B (2003) 74W. Martinand M. J.Russell On theorigins of cells

Figure 7. Asummaryof amodel suggested for theorigin of eukaryotes (Martin &Mu¨ller 1998), including apossible route for theprinciple underlying theorigin of thenucleus under thatmodel asdiscussed (Martin 1999 a),where it wassuggested that theorigin of theendoplasmic reticulum endomembrane systempreceded theorigin of thenuclear compartment. Seetext.

Phil.Trans. R. Soc.Lond. B (2003) On theorigins of cells W.Martinand M. J.Russell 75 nuclearmembrane, whichis asingle foldedmembrane that wereargued tohave enteredthe eukaryotic lineage that is contiguouswith theendoplasmic reticulum, is via themitochondrial symbiont (Cavalier-Smith 1991; sketchedhere in thetop panel ofthe figure. That model Roger &Doolittle 1993). Other inventionsinclude the for theorigin ofthe nuclear membrane (thevesicular cytoskeletal componentstubulin and actin, which are model)was elaborated andcontrasted with other models probably descendedfrom their prokaryotic homologues elsewhere(Martin 1999 a);its basic tenetsare recapitu- FtsZand MreB (Erickson 2001), theprotein import lated in figure 7. apparatus ofthe two mitochondrial membranes (Truscott Anotherchange is that theeubacterial andarchaebact- et al. 2001; Pfanner& Truscott2002), themitochondrial erial lipids wereindicated somewhat more clearly although carrier family ofproteins (Van derGiezen et al. 2002; their relevancewas stated in theoriginal. The lipids are Voncken et al. 2002), andwhat-all. The cell inferred important, becauseaccording totextbook depictionsof underthis model andsketched at thetop offigure 7has cell evolution(the ‘universal tree ’),eukaryotesshould an archaebacterial geneticapparatus, eubacterial energy have archaebacterial lipids, justlike they have anarchaeb- metabolism, eubacterial lipids, theseeds of an endomem- acterial geneticapparatus: butthey donot. Eukaryotes brane systemfrom which thenucleus is inferrable and have eubacterial lipids. Fewevolutionary biologists ample geneticstarting material (twohighly divergent and addressthis issue.One suggestion to account for this is partially merged prokaryotic genomes)to evolve cyto- that thecommon ancestor of all cellswas a eubacterium, logical andgenetic traits that are specificto the eukary- andthat thesingle commonancestor of archaebacteria otic lineage. reinventedits entirelipid andcell wall chemistry sub- Thus,to deliver thegoods promised at theoutset of this sequentto the divergence from theeukaryotic lineage on section,under the premise statedhere: (i) theevolutionary anrRNA-like tree(Van Valen &Maiorana 1980; Cavalier- basis for theattributes that are sharedby eubacteria and Smith 2002). Our explanation for theorigin ofeubacterial eukaryoteslies in geneacquisition from theeubacterial lipids in eukaryotesis that thegenes for their biosynthesis commonancestor of mitochondria andhydrogenosomes; weretransferred to the host ’schromosomesand that path- (ii) theevolutionary basis for theattributes that are shared way becamefunctional there (acetyl-CoA, NAD(P)H and by archaebacteria andeukaryotes lies in thearchaebacter- dihydroxyacetone phosphate asthe precursors is in ample ial natureof the host that acquired themitochondrion; supply in all cells)and is somewhat more explicit butnot and(iii) theevolutionary basis for theattributes that dis- principally differentfrom either Zillig ’s(1991) orKoga et tinguish eukaryotesfrom prokaryotes are foundedin al.’s(1998) explanation. Anotherchange isthat thepres- noveltiesthat arosespecifically in theeukaryotic lineage enceof eubacterial andarchaebacterial genesin thecyto- subsequentto its origin, whichgiven thefossil evidence, solic chromosomesis more clearly indicatedthan in the probably occurredbetween 1.5 and2 Gyr ago. original, whereit wassimply statedin words.Note that the term ‘genetransfer ’ is notexactly correct,because gene 10. GETTINGACCUSTOMED TO THE POSSIBILITY translocation from an organelle genometo the hosts ’s OFAPROKARYOTICAND AUTOTROPHIC HOST chromosomesis always aduplicative process;in thestrict sensethe transferred genes are copied,not transferred, The modelpresented here for mitochondrial (and becausean active copy hasto remain in theorganelle until eukaryote) origins differsin several fundamentalaspects its functioncan be substitutedby thecopy residing in the from traditional models(Doolittle 1998), whichusually host’schromosomes(Allen 1993). During thephase of entail aphagocytosing eukaryote asthe host, rather than evolutionbefore the origin ofaprotein routing apparatus an H2 -dependent,autotrophic archaebacterium, andas todirect cytosolic proteins into the mitochondrion suchit has receivedwelcome criticism (Cavalier-Smith

(Henze& Martin 2001), theprocess of gene copying from 2002). Regarding adescentof the host lineage from H 2- theorganelle tothe host, which occasional organelle death dependent,autotrophic archaebacteria, it isindeed curi- or lysis rendersinevitable (Doolittle 1999), wouldhave ousthat someproteins, such as group IIchaperonins meant acontinuousdownpour of eubacterial genesinto (Leroux et al. 1999) andhistones (Sandmann & Reeve the host’schromosomes,constantly providing newraw 1998) dolink upeukaryotes with methanogensand their geneticstarting material for theevolution of eukaryotic- relatives, butobviously genome-wideanalyses are needed specifictraits, while providing theopportunity toretain toderive further supportfor this view.Another question originally encodedfunctions as well. (The conversetrans- tosome was whether anaerobic syntrophy wouldprovide fer processfrom hostto organelle is extremely unfavour- sufficientselective pressure to select for theunsual kinds able: eventsof occasional host death or lysis donot lead ofprokaryotic morphologies required by themodel to totransfers, they lead tono progeny.) This view implicates associatean archaebacterium anda eubacterium,but truly genetransfers from theorganelle andthe integration of striking anaerobic syntrophic associationsinvolving thosegenomes within asingle cellular confines(Herrmann methanogensand hydrogen dependence(albeit by sul- 1997) asmajor geneticmechanisms underlying thegener- phate reducers)are becoming knownin thekinds of ation ofeukaryotic novelties. environmentspredicted (Boetius et al. 2000; Michaelis et Anotherchange is that histones,which are presentin al. 2002). Anothercriticism wasthat at thetime themodel methanogensand relatives (Reeve et al. 1997; Fahrner et waspresented, there was no precedence for any prokary- al.2001) aswell asin eukaryotes,are sketchedonto the oteliving insideanother prokaryote, raising questionsas host’schromosomes.Another change isthat inventions towhether it ispossible that an endosymbiosisbetween specificto the eukaryotic lineage are symbolized asgreen twoprokaryotes wouldbe possible (Doolittle 1998). In dots.These include for example spliceosomal intronsthat themeantime, aclear example has becomeknown where are probably descendedfrom eubacterial group IIintrons, oneprokaryote lives stably asan endosymbiont within

Phil.Trans. R. Soc.Lond. B (2003) 76W. Martinand M. J.Russell On theorigins of cells another (vonDohlen et al. 2001). Although themech- anaerobic or aerobic, or mostprobably all ofthe above, anism ofentry isstill unknown,that example demon- asis the case for many contemporary representativesof stratesthat it ispossible to establish anendosymbiosis thegroup, suchas Rhodobacter ,butit at least shouldhave with anon-phagocytotic,prokaryotic host,as predicted. had thephysiological potential ofa bacterium like Para- Anotherprediction wasthat mitochondria shouldbe coccus (John& Whatley 1975). The a-proteobacteria are foundthat possessand use both pyruvate dehydrogenase arather heterogeneousgroup (Woese1987): somehave andPFOR, evidencethat is forthcoming (M.Hoffmeister acquired hefty chunksof DNA from methanogens(see andC. Rotte,unpublished data). Another prediction was Chistoserdova et al. 1998) andsome have evolved very that evidencefor atrace ofglycolysis shouldbe found in elaborate mechanismsof lateral transfer,for example the mitochondria, which has beenfound (Liaud et al. 2000). genetransfer agents of Rhodobacter ,whichslice the gen- Anotherprediction was that autotrophic archaebacteria omeup into nicely transmissible 4kilobase piecesunder shouldbe able toacquire andexpress eubacterial genes, suitable conditions(Lang &Beatty 2001). Thesedays, it andthat suchaquisitions may expandthe biochemical has becomepopular toinvoke anindependent horizontal repertoire ofthe recipient, as the genome sequence genetransfer from prokaryotes toeukaryotes from anew of Methanosarcinamazei ,amethanogenwith ca. 30% anddifferent donor every time onesees a eukaryotic gene eubacterial genesin its genome,has beenconfirmed that is notarchaebacterial-like, butdoes not branch with (Deppenmeier et al. 2002). Furthermore, thebrunt of homologuesfrom a-proteobacteria (Baughn &Malamy genes that M. mazei hasacquired from eubacteria are larg- 2002), also by staunchopponents of lateral genetransfer ely associatedwith its nutritional lifestyle, in line with the (Kurland2000), andparticularly whenit suitsthe argu- predictionsof the model (but also includeproteins such ment(Canback et al. 2002). The main problem with such asGroEL and DnaK). The clearest,most easily falsifiable ad hoc useof that explanatory principle is that theeukary- (andmost often overlooked) prediction ofthe hydrogen otic genesinvolved usually share asingle commoneubac- hypothesisis that it predictsall eukaryotesto possess a terial origin (Rotte et al. 2001; Horner et al. 2002), mitochondrion(or hydrogenosome)or tohave possessed meaning that eukaryotesacquired mostof their eubacter- onein their past.Other currentmodels for theorigin of ial genesonly once,and in their commonancestor, in line eukaryotesdo not generate that prediction. with themodel set out here. Horiike et al. (2001) analysed From thestart, it wasclear that eukaryoteshad archae- theyeast genome and fulfilled thepredictions set out by bacterial ribosomes,linking thesetwo lineages. But it was Martin & Mu¨ller (1998) for theorigins offunctional also well-knownearly on(Zillig 1991) that equally numer- classesof eukaryotic genes,yet Horiike et al. (2001) did ous,possibly evenmore (Rivera et al. 1998), traits link notnotice that they had confirmedthose predictions eukaryotesand eubacteria. In someanaerobic protists, (Rotte& Martin 2001). tracesof the archaebacterial hostlineage have been There is another interesting variant oflateral genetrans- retainedin energy metabolism aswell (seeHannaert et fer asan explanatory principle asit applies toeukaryotic al. 2000; Sanchez et al. 2000; Suguri et al. 2001), clearly genes:namely totake all ofthe eubacterial genesin eukar- indicating mosaicism in eukaryotic biochemistry. Zillig yotesthat donot branch with a-proteobacterial homol- (1991) argued that eukaryotesacquired their eubacterial ogues,lump them together andattribute their origin to traits, including lipid biosynthesisand some of their glyco- onesingle hypothetical donor,usually animaginary organ- lytic genes,from aeubacterial symbiont;but because he ism (Hartman &Federov 2002; Hedges et al. 2001; thought that Giardia wasprimitively amitochondriate, he Horiike et al. 2001). Givenlateral transferand the prob- invokeda eubacterial donorthat wasdistinct from the lemsof deepphylogenies, it makes more senseto attribute mitochondrion.Today it is quiteclear that Giardia in fact suchgenes to the mitochondrial endosymbiont.Mito- didpossess a mitochondrion(Hashimoto et al. 1998; chondria aroseonly once(Gray et al. 1999; Lang et al. Roger et al. 1998; Tachezy et al. 2001). HadZillig been 1999) andlateral transferis real among free-living prokar- aware ofthat, it isdoubtful that hewould have invoked yotes(see Doolittle 1999; Jain et al. 1999; Eisen2000; theadditional endosymbiont.The hydrogen hypothesis Ochman et al. 2000). Becauselateral transferoccurs (figure 7) merely simplifies Zillig ’sschemeof cell evol- today,we shouldassume it tohave also existedin thepast. utionby oneendosymbiont, accounts for theorigin of If weincorporate that reasonable butmildly complicating hydrogenosomes,and specifies the possible ecological assumptionto our views on early evolution,it suddenly contextof symbiosis by making predictionsabout the nat- seemssilly toassume: (i) that any contemporary a-proteo- ureof the cells involved. Butit usesthe same explanatory bacterium shouldhave exactly thesame genes as it had principle ofgene acquisition from an endosymbiont,the 2Gyr ago; and(ii) that all genespossessed by thesingle commonancestor of mitochondria andhydrogenosomes, a-proteobacterium that gave rise tomitochondria should toaccount for thenumerous biochemically eubacterial befound today only among a-proteobacterial genomes traits in eukaryotesthat are toooften disregarded. (Martin 1999 b).The samerelatively insevereproblem applies tocyanobacteria (Rujan& Martin 2001) whenit comesto genes that plants acquired from plastids.Hori- 11. HORIZONTALGENE TRANSFER: zontal genetransfer was ‘apain in theneck ’ for molecular COMPLICATINGBUT NOTOBSCURINGEARLY evolutionists20 years ago (Dickerson1980) andwill still EVOLUTION bein 20 more;it has notdashed all hopesthat genome The commonancestor of mitochondria andhydro- sequenceswill help unravel evolutionary history, it has just genosomeswas a member ofthe a-proteobacteria (Yang left abit oftea in thecup, and swirling leaves are more et al. 1985), andtherefore it may have beenphotosynthetic difficult toread. After all, thedistinction between ancient or non-photosynthetic,autotrophic or heterotrophic, genetransfers and outright phylogenetic artefactsin gene

Phil.Trans. R. Soc.Lond. B (2003) On theorigins of cells W.Martinand M. J.Russell 77 treesthat span2 Gyr ormore ofevolution is anything but self-organization ofself-contained redox reactionsare the simple (Forterre &Philippe 1999 Poole et al. 1999), indi- mostconserved attributes ofliving things,hence inorganic cating that both general (pathways) andspecific (enzymes) matter with suchattributes wouldbe life ’smostlikely fore- biochemical characters in addition tobroad-scale phylo- bear. geneticcomparisons, which have revealed widespread mosaicism in eukaryotes(Brown & Doolittle 1997; We thankthe Deutsche Forschungsgemeinschaft and Bayer Doolittle 1997; Feng et al. 1997) shouldweigh in evidence AGfor support, Martin Embleyfor makingdata available ofearly cell evolution. before publication and Miklos Mu¨ ller and Allan Hall for dis- cussions.

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Wilson, E.B.1928 The cell in development and heredity , 3rd unusual,gene, whose phylogeny they cannotexplain, they edn. NewYork: Macmillan. (Reprinted in 1987 byGarland invoke anewendosymbiont. I have lookedat alot of Publishing, NewYork.) genes,and if Ihave toinvoke anewsymbiont every time Woese, C.R.1987 Bacterial evolution. Microbiol. Rev. 51, Iseea strange branch,then we would get aconglomer- 221–271. ation that justdoes not make sense.My suspicion is that Woese, C.R.2002 Ontheevolution of cells. Proc.Natl Acad. what is wrong is that ourmodels for protein evolution,in Sci. USA 99, 8742–8747. Woese, C.R., Kandler, O.&Wheelis, M.L.1990 Towards a particular for glycolytic enzymes,are sowrong that they natural systemof organisms: proposal for thedomains are producing artefactsregularly, andwe just do not Archaea, Bacteria and Eukarya. Proc.Natl Acad.Sci. USA understandhow these molecules are evolving. Sowhat we 87, 4576–4579. are doing is,rather than entertaining anotionof a new Woods, R.1976 Electrochemistry of sulfide flotation. In Flo- endosymbiosisevery time weseea newgene phylogeny — tation: A.M.Gaudin memorial volume ,vol. 1(ed. M.C. Ihave beenthere, done that —wesay what iswrong are Furstenau), pp. 298 –333. NewYork: American Institute of thegene phylogenies; andlet ustake aminimalist view Mining, Metallurgical and Petroleum Engineers Inc. wherewe have onearchaebacterial hostand one eubacter- Xiong, J.&Bauer, C.E.2002 Acytochrome b origin of photo- ial symbiont,and see how much we can explain with that. synthetic reaction centers: anevolutionary link between res- R.Blankenship ( Departmentof Chemistryand Biochemis- piration and photosynthesis. J.Mol.Biol. 322, 1025–1037. Yang, D., Oyaizu, Y., Oyaizu, H., Olsen, G.J.&Woese, C.R. try,Arizona State University, AZ,USA ).Doyou conclude 1985 Mitochondrial origins. Proc.Natl Acad.Sci. USA 82, from thefact that thearchaeal glycolytic enzymesare 4443–4447. apparently unrelatedto the eubacterial ones,that glycoly- Yoon, K.-S., Hille, R., Hemann, C.&Tabita,F. R.1999 Rub- siswas independently invented two separate times? redoxin from thegreen sulfur bacterium Chlorobium tepidum W.Martin. Yes.I embrace theview ofa chemoauto- functions asan electron acceptor for pyruvate ferredoxin oxi- trophic origin oflife, that is,we start with carbon monox- doreductase. J.Biol. Chem. 274, 29 772–29 778. ide.What doI think wasthe first pathway? Ithink the Zillig, W.1991 Comparative biochemistry of Archaea and Bac- first pathway wasthe acetyl-CoA pathway ofCO 2 fixation. teria. Curr.Opin. Genet. Dev. 1, 544–551. This isa pathway youfind among clostridia andit is wide- spreadamong archaebacteria. Itskey enzymeis carbon Discussion monoxidedehydrogenase. And what carbon monoxide

D. Horner (Dipartimentodi Fisiologiae BiochimicaGen- dehydrogenasedoes, is it reducesCO 2 with molecular erali,University ofMilan, Milan, Italy ).Iwantto under- hydrogen tocarbon monoxide,takes a methyl group from standwhy thecore of glycolysis has tocome from this amethyl donor,adds that toan iron –sulphurcentre, symbiont andalso why,apart from aparsimony argument, transfersthe methyl group tothe carbon monoxide,and it isnot possible for theendosymbiosis giving rise tothe makesan acetyl group, all in onereaction. Add a sulphide mitochondrionto have happenedmore than once. in theform ofCoA, and you get twomolecules of CO 2 W.Martin. Letme take thesecond comment first. It is in,and one molecule of acetyl CoAout. It is alinear reac- justifiedand can be reworded as follows: ‘Dr Martin, if tion,so that isa goodstart, and that isexactly thereaction you’re saying this is solikely that this couldgive rise to that theHuber –Wa¨chtersha¨userexperiment modulates,so eukaryotes,how come it doesn ’thappen all thetime? ’. start with C2 .The nextreaction, in metabolism in organ- That theeukaryotes arose only once;that all eukaryotes ismsthat have theacetyl-CoA pathway, is pyruvate syn- that have beenstudied to date have amitochondrion;and thase.Pyruvate synthaseis also an iron –sulphurenzyme that all mitochondria aroseonly onceare observations.It that justadds CO 2 with electronsfrom ferredoxin,to make isan observation, it isan explanandum i.e.something that pyruvate. Sowe go uptheglycolytic pathway. OK,soyes, needsto be explained. Wehave togenerate ahypothesis Ithink that if weembrace thenotion of a chemoauto- that canexplain that observation.We have heard from trophic origin oflife, wehave toembrace thenotion that Tom [Cavalier-Smith] that secondarysymbiosis is,com- this commonancestor we infer,living in its iron sulphide pared with primary symbiosis,a very widespreadphenom- housingthat it embeddeda myriad ofways to make carbon enon.Primary endosymbiosishappened twice among backbones,not just to break them downfrom Oparin ’s countlesscells in 4billion years.It isexcruciatingly rare. organic soup.I wouldlike topoint outone very interesting Isuspectthat therareness of a free-living bacterium finding.The mostconserved enzyme of the glycolytic becoming anorganelle lies in thedifficulties of sorting out pathway is withoutdoubt enolase. Enolase catalyses the thegenetic integration ofthese systems into something first stereochemically specificreaction in carbon meta- that will survive, becausewe know of many eukaryotes bolism. It takesa water moleculeand adds it tophos- that have bacterial symbionts,but we do not know of phoenol-pyruvate, which isnot chiral, andthe product is many eukaryotesthat have acquiredindependent 2-D-phosphoglycerate, andthe stereochemistry at C 2 of organelles. It comesdown to chloroplasts andmitochon- that carbon compoundnever changes for therest of dria. So,I think,the second comment simply boils down metabolism. And,strangely, enolase,the most conserved toan interesting observation. glycolytic enzyme,and the only onethat isuniversally dis- The first questionis, why didthe core enzymes of gly- tributed,is not encoded in glycolytic operonsin archae- colysishave tocome from themitochondrial symbiont? bacteria, it sitsin thesame operon as the ribosomal That isthe application ofOccam ’srazor tosuggestions, proteins.It sitsin thesame operon as the ribosomal pro- which are commonin theliterature, that aninability to teins.And if youask me,that isthe key tothe holochirality get acitric acid cycleenzyme to branch with thealpha- problem from sugars.And the selection, at theorigin of proteobacterial homologue meansthat another endosym- translation, for incorporation ofonly L-aminoacids into biotic eventhas tobe invoked.Every time they seea new, proteinsfrom aracemate, might bea possiblesolution,

Phil.Trans. R. Soc.Lond. B (2003) 84W. Martinand M. J.Russell On theorigins of cells whichwe suggestin thepaper, for theholochirality prob- group with theleast diversity ofcharacters andmaking lem asit arisesin theamino acids.Short question,long it themost ancient and saying that therehas beenrapid answer.Yes, independent origins, theseenzymes and the evolutionof all ofthe characters that are clearly ancestral wholebiochemistry are notrelated. onbiogeochemical grounds.The kindsof chemistry we T.Cavalier-Smith ( Departmentof Zoology, University of are talking about hereare consumingpyrite or sphalerite, Oxford,Oxford, UK ).Ihave acommentand a question. or evensustained ATP productionusing carbon monox- The commentrelates tothe fact that yourightly criticize ide.These are ancientpathways. So that is howI would Carl Woesefor assumingthat eukaryotesare asold as addressthose two criticisms. eubacteria, andI have similarly criticized him for T.Cavalier-Smith. Well, OK.My questionwas that, in assuming that archaebacteria are that old,also. You have arecentpaper (Cavalier-Smith 2002), Imentioneda cou- madethat sameassumption and I think my criticisms of ple ofgene splittings, which Ithink argue against theidea him apply toyou as well …. that eukaryotescame from within archaebacteria, andI W.Martin. Iwill addressthat. OK,go ahead. wonderhow you would refute that argument. Asfor the T.Cavalier-Smith. Andyou are aware ofthe idea that carbon isotopedata, I wouldreally like tohear from any Iputforward in 1987, andsince have writtenabout more geochemistsabout thevalidity ofthese values. recently,which isthat archaebacteria andeukaryotes are W.Martin. Dr Nisbetis raising his hand. sisters,and not that eukaryotesevolved from anarchae- T.Cavalier-Smith. Good,but may Ijustmake one bacterial ancestor? point about it? The basic argument is notdirect evidence W.Martin. May Ireply? for archaebacteria; it isbased on an assumption about eco- T.Cavalier-Smith. Yes. logical interactionsbetween archaebacteria andmethylo- W.Martin. What Tom is saying isthat hehas always trophs.It isa rather complex argument basedon several placed very great importance onthe same explanandum assumptions,and I donotthink that thegeochemists have that weare addressinghere, namely that eubacteria have lookedcritically at alternative hypotheses.They have onekind of lipid, archaebacteria have adifferentkind of assumedthat archaebacteria are old,therefore it would lipid, andeukaryotes have eubacterial lipids. To explain bea reasonable assumption.Strauss et al. (1992), when that, hehas had something called theneomuran theory, reviewing this very point,do say that other explanations which,in this element,is notdifferent from Leevan are in principle possible. Valen’sandMoriana ’s1980 paper, sevenyears before,in W.Martin. OK,Iwouldlike tohave Dr Nisbetreply ,wherethey saidexactly thesame thing. Iam not Nature tothe evidence, and at thesame time recall that it is not criticizing thenovelty, but I am justsaying that this only methylotrophs that canproduce this trace.The explanandum has beenaround for along time. Zillig has associationbetween methanogens and sulphate-reducers also addressedit. Tom explains this by saying that eukary- actually causesmethanogenesis to run backwards. So otesand archaebacteria share acommonancestor and that anaerobic methanotrophy works,but it worksthrough that commonancestor was a eubacterium.Thus, he reversemethanogenesis and it producesthis trace without postulateda eubacterial commonancestor of eukaryotes methanotrophy in thestrict sense. andarchaebacteria that has many archaebacterial attri- E. Nisbet (Departmentof Geology, Royal Holloway, Uni- butesbut eubacterial lipids. This ancestoris thenargued versityof London, Egham, Surrey, UK ).Iam ageologist tohave brought forth thecommon ancestor of all eukary- andI donot know very muchbiology, butcertainly at 2.7 otesand the common ancestor of all archaebacteria, whereby thelatter are argued tohave completely rein- billion years both thecarbon andthe sulphur isotopes look ventedtheir lipid biochemical pathways prior toarchae- very comparable in many waysto what weget in themod- bacterial diversification. Idonot believe that, butwe ernEarth, with awidediversity ofcarbon pathways, cannotnecessarily prove that it iswrong. What wecan including things that fractionate carbon very substantially, prove is wrong is anelement of your mostrecent formu- andalso very substantial self-refractionation.If yougo lation ofthis model,in which youpropose that this hap- back past 3.5 billion years,the story is more complex and pened800 million years ago. Now,some geochemists are someof theoutcrops are beautiful.At 3.7 billion years,at here,and, tell meif Iam wrong,the carbon isotoperecord Issuain Greenland,there is clear evidencefor light car- for ultra-light carbon,i.e. d1 3 C values of –50‰ or –60‰ bon,but the rocks are metamorphosedso youcannot say goesback well beyond2.7 billion years;it goesright down muchmore than that therewas some sort of biological intothe same carbon levels whereSchidlowski found d1 3C process.It lookslike acetyl CoAmetabolism, butthat is apureguess. values that are typical ofpresent-day CO 2 fixation path- waysusing inorganic Cwith present-day d13 C values. T.Cavalier-Smith. That isnot specifically archaebact- This ultra-light carbon isgenerally interpreted,as there is erial. noother processthat makesit, asthe trace ofmethano- E.Nisbet.No, if youwant to say it is specifically archae- genesis.And that meansthat archaebacteria asa group bacterial, youcan say at least at 2.7 billion years.There mustbe at least threebillion years old,as common sense is also,I shouldmention, Brock ’sfinding ofchemicals that woulddictate. Another problem, Tom, with theview that look asif they are eukaryotic at 2.7 billion years.We have archaebacteria are only 800 million years old,is thethings oil at 2.7 billion years. they cando with awiderange ofinorganic donorsand T.Cavalier-Smith. Butsterols can be made by three acceptors.You can say ‘yes’,this is dueto adaptation to groups ofbacteria. extreme environments,but just looking at thebiochemical E.Nisbet.Yes, I knowthey canbe made by Archaea characters,which most people who are involved in this as well. early cell evolutionbusiness do notdo, it islike taking the W.Martin. Weagree onthat.

Phil.Trans. R. Soc.Lond. B (2003) On theorigins of cells W.Martinand M. J.Russell 85

T.Cavalier-Smith.What about thegene splitting asevi- Additional references dencefor eukaryotesbeing sisters,not derived from archaebacteria? Cavalier-Smith, T.2002 Thephagotrophic origin of eukary- W.Martin. Well, Idonot know exactly which data you otes and phylogenetic classification of Protozoa. Int. J.Syst. Evol. Microbiol. 52, 297–354. are referring to,but we have seenoperons put together Strauss, H., DesMarais, D.J., Hayes, J.M.&Summons, R.E. independentlyin many groups oforganisms. 1992 Thecarbon-isotopic record. In The Proterozoic biosphere T.Cavalier-Smith. This issplitting upgenesinto separ- (ed. J.W.Schopf& C.Klein), pp. 117 –127. Cambridge ately codedproteins. University Press. W.Martin. Well, Tom —independent?In enolasewe have seenindependent insertions at exactly thesame site. Ialso considerindependent splittings tobepossible. I was GLOSSARY hoping youwould ask why dothe eukaryotes in theribosomal RNA treebranch belowthe archaebacteria, whenour ACS:acetyl-CoA synthase modelactually saysthey shouldbranch within thearchae- CODH:carbon monoxidedehydrogenase bacteria, andthe answer is exactly thesame as for the DXP:1-deoxyxylulose-5-phosphate microsporidia. The microsporidia belong at thetop ofthe FBA: fructose-1,6-bisphosphatealdolase eukaryotic tree,but because of odd evolutionary processes GAPDH:glyceraldehyde-3-phosphate dehydrogenase in their rRNA they branch artefactually at thebottom. In GK:glucokinase themodel for theorigin ofeukaryotes that Ipresented, GPI:glucose-6-phosphate isomerase archaebacterial ribosomesare evolving in achimaeric IPP: isopentenyldiphosphate cytosol.Thus, the same thing is expectedto happen asin MVA:mevanolate thecase of themicrosporidian RNAmolecule.The ances- PFK: phosphofructokinase tral eukaryotic rRNA moleculegoes through aphaseof PFOR: pyruvate :ferredoxin oxidoreductase dramatic change,it acquiresa very long branch relative to TCA:tricarboxylic acid its archaebacterial homologues,and branches artefactually TPI: triosephosphateisomerase deepon the archaebacterial sideof the rRNA tree. TPP: thiamine pyrophosphate

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