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Archaea form the third domain of ife, aongside theother to domains, the Bacteria and Ekarya. The dis-tinct archaea ineages that hae been identified incdethe Crenarchaeota (from the Greek crenos for spring ororigin) and the Eryarchaeota (from the Greek eryosfor diersity)1 (FIG. 1). Archaea resembe Bacteria in theirstrctra organization and metaboism, hereas theirgenetic information system (the process of transcription)shares many traits ith Ekarya.

The eary branching ineages of the archaea and bac-teria harbor manythermophilicchemolithoautotrophs, afact that as taken as an indication for a chemoitho-trophic origin of ife at high temperatres2. Most cti-

ated atotrophic archaea are either anaerobes or cantoerate or se oxygen ony at o concentrations. Theinorganic sbstrates that these organisms can oxidizeincde H

2, H

2S, S, CO, NH

3, meta sphides sch as

pyrite (FeS2), and redced meta ions. The eectron

acceptors that can be sed incde arios oxidizedinorganic componds sch as S, S

2O

32, SO

42, AsO

43,

NO3, oxidized meta ions and een CO2 (for anaerobicrespiration). The se of O

2(for aerobic respiration) is

rare and is considered a ate adaptation that refectsthe increasing oxygen content of the atmosphere after theemergence of oxygenic photosynthesis. The genera-tion of ATP foos a chemiosmotic mechanism: eec-tron fo from the redced inorganic sbstrate to theoxidized inorganic eectron acceptor is coped tothe transdction of H+ or Na+ across the cytopasmicmembrane, and the archaea H+ ATP synthase sesthe restant proton-motie force for ATP synthesis.Redcing poer for biosynthesis is aso proided bythe oxidation of redced inorganic sbstrates, athogh

the redction of NAD or NADP and ferredoxin mightreqire an energy-drien reerse eectron fo35.

In genera terms, the assimiation of CO2

into ce-ar biding bocks reqires for redcing eqiaentsand an inpt of energy. A sma organic moece s-ay seres as a CO

2acceptor, hich is inked to CO

2

by a carboxyase; hoeer, a arge coenzyme or a pros-thetic grop on an enzyme can aso fnction as a CO

2

acceptor, as is discssed beo. A CO2

acceptors msteentay be regenerated in a cyce in hich CO

2(oxi-

dation state + 4) is redced to cear carbon (aerageoxidation state 0). The energeticay nfaorabe stepsof this pathay can be drien by ATP hydroysis, andthe redction steps are drien by o-potentia redcedcoenzymes, say NADPH; occasionay, hoeer,redced ferredoxin or the redced deazafain factor 420is sed. The prodct of sch a metaboic cyce is a centracear metaboite, from hich poymer biding bockscan be deried. None of the chemoithoatotrophicarchaea seems to se the Cain cyce for CO

2fixation

(BOX 1), een thogh in some species one of the keyenzymes, ribose1,5-bisphosphate carboxyaseoxy-genase (RbisCO), is present. Instead, these organismsse dierse CO

2fixation mechanisms to generate acety-

coenzyme A (acety-CoA), from hich the biosynthesisof biding bocks can start.

This Reie discsses the atotrophic carbon fixa-tion pathays in archaea, to of hich ere discoeredony recenty. Archaea carbon fixation strategies haein common the synthesis of acety-CoA from CO

2. As

many archaea ack a fnctiona frctose 1,6-bisphosphate(FBP) adoase, hich catayses the ast, controed stepin gconeogenesis, this raises the qestion of ho

*Mikrobiologie, Fakultt

Biologie, Universitt Freiburg,

Schnzlestrasse 1, D79104

Freiburg, Germany.

Present address: WaterTechnology Center (TZW),

Karlsruher Strasse 84,

D76139 Karlsruhe,

Germany.

Present address: The Ohio

State University, Department

of Microbiology, 484 West

12th Avenue, 417A Biological

Science Building, Columbus,

Ohio 432101292, USA.

Correspondence to G.F.

email:georg.fuchs@biologie.

unifreiburg.de

doi:10.1038/nrmicro2365

Published online 10 May 2010

Thermophilic

An organism that grows best at

temperatures exceeding the

ambient temperature. Extreme

thermophiles

(hyperthermophiles) have

optimal growth temperatures

above 80 C.

Chemolithoautotroph

An organism that derives

energy from a chemical reaction

(chemotrophic) based on

inorganic substrates as electron

donors (lithotrophic), and CO2

serves as sole carbon source

(autotrophic = self-nourishing).

Autotrophic carbon fixation in archaeaIvan A. Berg*, Daniel Kockelkorn*, W. Hugo RamosVera*, Rafael F. Say*,

Jan Zarzycki*, Michael Hgler*, Birgit E. Alber* and Georg Fuchs*

Abstract | The acquisition of cellular carbon from inorganic carbon is a prerequisite for life

and marked the transition from the inorganic to the organic world. Recent theories of the

origins of life assume that chemoevolution took place in a hot volcanic flow setting through a

transition metal-catalysed, autocatalytic carbon fixation cycle. Many archaea live in volcanic

habitats under such constraints, in high temperatures with only inorganic substances and

often under anoxic conditions. In this Review, we describe the diverse carbon fixation

mechanisms that are found in archaea. These reactions differ fundamentally from those of

the well-known Calvin cycle, and their distribution mirrors the phylogenetic positions of the

archaeal lineages and the needs of the ecological niches that they occupy.

RE V I E W S

NATuRE REvIEwS |Microbiology vOluME 8 | juNE 2010 |447

20 Macmillan Publishers Limited. All rights reserved10
mailto:[email protected]:[email protected]:[email protected]:[email protected]


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HalobacterialesThermoplasmatales

Euryarchaeota

Methanosarcinales

Methanomicrobiales

Thermococcales

Methanobacteriales

Methanopyrus kandleri

SulfolobalesMethanococcales

Desulfurococcales

Thermoproteales

CrenarchaeotaMesophilic marinegroup ICrenarchaeotaN

anoa

rcha

eum

equita

ns

Ko

rarcha

eum

crypt

ofilu

m

Archaeo

globusfulgidus

1

1

1

3

2

2

1

1

1

3

gconeogenesis fnctions in these organisms. Recenty,a ne type of bifnctiona FBP adoasephosphatase

as discoered that might be the ancestra gconeo-genic enzyme. we then discss some of the reasons forthe occrrence of the obsered metaboic diersity inarchaea and the rationae behind the distribtion of theexisting mechanisms. Finay, e raise some open qes-tions that mst be addressed in ftre stdies and tochon the qestion of hether the extant atotrophic path-ays can sere as modes for an ancestra metaboism.

The reductive acetyl-CoA pathway

The redctie acety-CoA pathay is an interesting path-ay in the Eryarchaeota that may indeed be a modefor primordia CO

2fixation. Methanogenic archaea

probaby constitte a monophyetic bt dierse gropithin the Eryarchaeota (FIG. 1). They are strict anaer-

obes that derie energy mainy from to processes: theredction of CO

2sing for moeces of H

2to generate

CH4

or the disproportionation of acetate into CH4

psCO

2. The carbon assimiation pathay in these species

the redctie acety-CoA pathay rests in thefixation of to moeces of CO

2to form acety-CoA,

ith a coenzyme and an enzyme meta centre as the CO2

acceptors. It as ecidated by the aboratories of wood,lngdah, Thaer and others610 as a pathay that issed by acetogenic bacteria to synthesize acetate fromCO

2to generate ATP. This pathay aso operates in the

sphate-redcing eryarchaea generaArchaeoglobus11and Ferroglobus12.

Figure 1 | The pheet (uted) tee Ahaea. This phylogenetic tree is based on analyses of a concatamer of

nine subunits of RNA polymerase, three transcription factors and 53 ribosomal proteins from all currently finished archaeal

genomes (59 species).The numbers 1, 2, 3 and 3 refer to the presence of the reductive acetyl-coenzyme A (acetyl-CoA)

pathway (1), the dicarboxylatehydroxybutyrate cycle (2), the hydroxypropionatehydroxybutyrate cycle (3) and possibly a

modified hydroxypropionatehydroxybutyrate cycle (3). The lineages in red represent hyperthermophilic or thermophilic

archaea with an optimal growth temperature that is equal to or higher than 65C, and the lineages in blue represent archaea

with an optimal growth temperature that is lower than 65C. Autotrophic members are marked by a circle at the end of the

lineage. Note that the phylogenetic position of the Nanoarchaeota, Korarchaeota and marine group I Crenarchaeota is

currently being debated109112.

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One moece of CO2

is redced to the ee of amethy grop, hich is bond to a tetrahydropterincoenzyme. Another CO

2moece is redced to CO

bond to nicke in the reaction centre of CO dehy-drogenase (FIG. 2). CO dehydrogenase aso acts as anacety-CoA synthase. It accepts the methy gropfrom the methyated tetrahydropterin throgh a

methyated corrinoid protein, combines it ith CO toform an enzyme-bond Ni-acety grop, and reeasesthis grop ith CoA to form acety-CoA. This keyenzyme is therefore referred to as a CO dehydroge-naseacety-CoA synthase and probaby has commonroots in Bacteria and Archaea. This is in contrast tothe enzymes inoed in the formation of methytet-rahydropterin from CO

2, hich differ consideraby in

Bacteria and Archaea.The redctie acety-CoA pathay can be consid-

ered a bioogica eqiaent of the indstria Monsantoprocess, in hich acetate is prodced from CO andmethano throgh meta cataysis. There are many

ariants of the redctie acety-CoA pathay, hichdiffer in the se of coenzymes or eectron carriers.Among the atotrophic CO

2fixation pathays, the

redctie acety-CoA pathay has the oest energeticcosts, reqiring probaby ess than one ATP to makepyrate (TABLE 1). Hoeer, the demanding reqire-ments for metas, cofactors, anaerobiosis and sb-

strates ith o redcing potentia sch as H2 or COrestrict the redctie acety-CoA pathay to a imitedset of anoxic niches.

The dicarboxylatehydroxybutyrate cycle

The dicarboxyate4-hydroxybtyrate cyce (shortenedto the dicarboxyatehydroxybtyrate cyce) fnctionsin the anaerobic or microaerobic atotrophic mem-bers of the crenarchaea orders Thermoproteaes andDesfrococcaes1315(FIG. 1). Many gro as strict anaer-obes by redcing eementa sphr ith H

2to H

2S, bt

some gro nder microaerobic or denitrifying condi-tions16,17. The dicarboxyatehydroxybtyrate cyce can

Box 1 | Autotrophic carbon fixation mechanisms

Six mechanisms that assimilate CO2

into cellular material have been identified (TABLE 1). Note that the different CO2

fixation mechanisms lead to different carbon isotope fractionation values in biomass (TABLE 1).

ca e

In the CalvinBensonBassham cycle, which was discovered about 50 years ago, CO2

reacts with the five-carbon sugar

ribulose 1,5-bisphosphate to yield two carboxylic acids, 3-phosphoglycerate, from which the sugar is regenerated103. This

cycle operates in plants, algae, cyanobacteria, some aerobic or facultative anaerobic Proteobacteria, CO-oxidizing

mycobacteria and representatives of the genera Sulfobacillus (iron- and sulphur-oxidizing Firmicutes) and Oscillochloris(green sulphur bacteria). An autotrophic symbiotic cyanobacterium conferred the CO

2fixation machinery on a

eukaryotic cell giving rise to the chloroplasts of plant cells. The presence of the key enzyme, ribulose 1,5-bisphosphate

carboxylaseoxygenase (RubisCO), is often considered to be synonymous with autotrophy. Phylogenetic analysis and

general considerations denote the Calvin cycle as a late innovation72,83,84.

redute t ad e

In 1966, Arnon, Buchanan and co-workers proposed another autotrophic cycle for the green sulphur bacterium

Chlorobium limicola, the reductive citric acid cycle (also known as the ArnonBuchanan cycle)104. This cycle is less

energy-consuming than the Calvin cycle, involves enzymes that are sensitive to oxygen and is therefore found only in

anaerobes or in aerobes growing at low oxygen tensions. These include some Proteobacteria, green sulphur bacteria and

microaerophilic bacteria of the early bacterial phylum Aquificae. Initially, the reductive citric acid cycle was also

proposed to operate in certain archaea (notably Thermoproteus neutrophilus)20, but recent findings refute this proposal14.

redute aet-ezme A pathwa

At the start of the 1980s, a third autotrophic pathway was found in certain Gram-positive bacteria and methane-forming

archaea, the reductive acetyl-coenzyme A (acetyl-CoA) or WoodLjungdahl pathway610

. In these strict anaerobicorganisms that now also include some Proteobacteria, Planctomycetes, spirochaetes and Euryarchaeota, one CO

2

molecule is reduced to CO and one to a methyl group (bound to a carrier); subsequently, acetyl-CoA is synthesized from

CO and the methyl group (FIG. 2). Although this pathway is the most energetically favourable autotrophic carbon fixation

pathway (TABLE 1), it is restricted to strictly anaerobic organisms.

3-Hdxppate e

The 3-hydroxypropionate bicycle occurs in some green non-sulphur bacteria of the family Chloroflexaceae3840,43. This

seems to be a singular invention, and the pathway has not been found elsewhere. The conversion of acetyl-CoA plus two

bicarbonates to succinyl-CoA uses the same intermediates as in the hydroxypropionatehydroxybutyrate cycle, but most of

the enzymes are completely different. Furthermore, the regeneration of acetyl-CoA proceeds by the cleavage of malyl-CoA,

yielding acetyl-CoA and glyoxylate. The assimilation of glyoxylate requires a second cycle (hence the name bicycle).

Hdxppatehdxutate e

The hydroxypropionatehydroxybutyrate cycle occurs in aerobic Crenarchaeota (Sulfolobales and possibly marine

Crenarchaeota group I)25(FIG. 3b). Although some of the intermediates and the carboxylation reactions are the same

as in the 3-hydroxypropionate bicycle in Chloroflexaceae, the archaeal cycle probably has evolved independently.Daxatehdxutate eThe dicarboxylatehydroxybutyrate cycle occurs in the anaerobic crenarchaeal orders Thermoproteales and

Desulfurococcales1315. The hydroxypropionatehydroxybutyrate and dicarboxylatehydroxybutyrate cycles

are described in the main text (FIG. 3).

Monsanto processAn important method for the

manufacture of acetic acid.

The feedstock methanol is

combined catalytically with CO

to give acetic acid. The reaction

is catalysed by a metal

(rhodium) catalyst. Methanol

reacts with catalytic amounts

of HI to give methyl iodide. The

reaction cycle is completed by

the loss of CH3COI to

regenerate the metal catalyst.

The CH3COI reacts with water

to generate acetic acid and

regenerate HI.

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be diided into to parts: in the first part, acety-CoA, one CO

2and one bicarbonate are transformed

throgh C4

dicarboxyic acids to scciny-CoA, andin the second part, scciny-CoA is conerted throgh4-hydroxybtyrate into to moeces of acety-CoA(FIG. 3a). One acety-CoA can be sed for biosynthesisand the second seres as a CO

2acceptor for the next

rond of the cyce.The dicarboxyatehydroxybtyrate cyce starts

ith the redctie carboxyation of acety-CoA to

pyrate, a reaction that is cataysed by pyrate syn-thase (aso knon as pyrate:ferredoxin oxidoredct-ase). This oxygen-sensitie enzyme is common in strictanaerobes, bacteria and archaea. Pyrate is conertedto phosphoenopyrate (PEP), fooed by carboxy-ation of PEP to oxaoacetate, hich is cataysed byan archaea PEP carboxyase18,19. The sbseqent red-ction to scciny-CoA inoes an incompete redc-tie citric acid cyce. Originay, a compete redctiecitric acid cyce as thoght to operate20. Hoeer,

Table 1 | Pathways for autotrophic carbon fixation

Pathwa* ATPequaetthe epuate

redutat the epuate(10 [H])

co2-x

ezmeAteco

2

pee

itemedate thata e ued the

catpeatat

Ke ezme

Reductive pentosephosphate cycle(CalvinBensonBassham cycle)

Seven Five NADH orNADPH

RubisCO CO2

3-Phosphoglycerate,triose phosphatesand sugarphosphates

20 to30 113,114

RubisCO andphosphoribulokinase

Reductivecitric acid cycle(ArnonBuchanancycle)

Two Two NADHor NADPH,one unknowndonor||and twoferredoxin

2-Oxoglutaratesynthase

CO2

Acetyl-CoA,pyruvate, PEP,oxaloacetate,succinyl-CoA and2-oxoglutarate

2 to12 115,116

2-Oxoglutaratesynthase andATP-citrate lyaseIsocitrate

dehydrogenaseCO

2

Pyruvate synthase CO2

PEP carboxylase HCO3

Reductiveacetyl-CoA pathway(WoodLjungdahlpathway)

Approx. one Threeferredoxinand twoF

420H

2(in

methanogens)

Acetyl-CoAsynthaseCOdehydrogenase

CO2

Acetyl-CoA andpyruvate

< 30 83,115,117 Acetyl-CoAsynthaseCOdehydrogenase andenzymes reducingCO

2to methyltet-

rahydropterin

Formylmethanofurandehydrogenase (inmethanogens)

CO2

Pyruvate synthase HCO2

3-Hydroxypropionatebicycle

Seven Six NADH orNADPH, butone FAD isreduced

Acetyl-CoA andpropionyl-CoAcarboxylase

HCO3

Acetyl-CoA,pyruvate andsuccinyl-CoA

12.5 to13.7 118120

Malonyl-CoAreductase,propionyl-CoAsynthase andmalyl-CoA lyase

3-Hydroxypropionate4-hydroxybutyratecycle

Nine Six NADH orNADPH, butone FAD isreduced

Acetyl-CoA andpropionyl-CoAcarboxylase

HCO3

Acetyl-CoA andsuccinyl-CoA

0.2 to 3.8121 Acetyl-CoApropionyl-CoAcarboxylase,enzymes reducingmalonyl-CoA topropionyl-CoA,methylmalonyl-CoAmutase and4-hydroxybutyryl-CoA dehydratase

Dicarboxylate4-hydroxybutyratecycle

Five Two or threeferredoxin,one ortwo NADH orNADPH, andone unknowndonor

Pyruvate synthase CO2

Acetyl-CoA,pyruvate, PEP,oxaloacetate andsuccinyl-CoA

0.2 to 3.8121 4-Hydroxybutyryl-CoA dehydratasePEP carboxylase HCO

3

CoA, co-enzyme A; F420

, deazaflavin factor 420; FAD, flavin adenine dinucleotide; PEP, phosphoenolpyruvate; RubisCO, ribulose 1,5-bisphosphate carboxylaseoxygenase. *Alternative name of pathway is provided in brackets. In biological processes, when inorganic carbon is used to make organic compounds, 12C ismore weakly bonded and reacts more readily than 13C because of its lighter mass. This means that organic matter tends to become enriched in 12C (anddepleted in 13C; therefore negative sign) relative to the reservoir of inorganic carbon from which it has been drawn. Carbon stable isotopic fractionations aremeasured relative to a fossil belemnite standard (the PDB standard). Isotopic fractionations are normally small and so values are measured in parts per thousand() and expressed as d13C values as follows: d13C = [(13C/12Csample - 13C/12Cstandard) / (13C/12Cstandard)] 1000. The presence of biotin-dependent2-oxoglutarate carboxylase in, for example, Hydrogenobacter thermophilus122, can increase the energy requirements of the cycle. ||NADH in Hydrogenobacter

thermophilus123

.

Note that reduction of ferredoxin may be energy driven35

, which would increase the energy demands of the ferredoxin-dependent pathways.

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Succinyl-CoA

Succinic semialdehyde

4-Hydroxybutyrate

4-Hydroxybutyryl-CoA

Crotonyl-CoA

(S)-3-hydroxybutyryl-CoA

Acetoacetyl-CoASH

CoA

Acetyl-CoA

Acetyl-CoA

Pyruvate

PEP

Oxaloacetate

Fumarate

(S)-malate

Succinate

Acetyl-CoA

Malonyl-CoA

Malonate semialdehyde

3-Hydroxypropionate

3-Hydroxypropionyl-CoA

Propionyl-CoA

Acryloyl-CoA

(S)-methylmalonyl-CoA

Pyruvatesynthase

PEP synthase

PEPcarboxylase

Malatedehydrogenase(NADH)

Fumaratehydratase

Fumaratereductase

Succinyl-CoAsynthetase

Acetyl-CoApropionyl-CoA carboxylase

Malonyl-CoA reductase (NADPH)

Malonic semialdehydereductase (NADPH)

3-Hydroxypropionate-CoAligase

3-Hydroxypropionyl-CoAdehydratase

Acryloyl-CoAreductase (NADPH)

Acetyl-CoApropionyl-CoAcarboxylase

Methylmalonyl-CoA epimeraseMethylmalonyl-CoAmutase

Succinyl-CoAreductase

Succinic semialdehydereductase (NADPH)

4-Hydroxybutyrate-CoAligase

4-Hydroxybutyryl-CoA

dehydratase

Crotonyl-CoAhydratase

(S)-3-hydroxybutyryl-CoAdehydrogenase (NAD+)

Acetoacetyl-CoA-ketothiolase

CO2

ATP + H2O

ADP + Pi

AMP + PPi

ATP + CoASH

H2O

HCO3

H2O

H2O

Pi + AMP

HCO3

HCO3

Pi

NADH+ H+

NADPH + H+

NADPH+ H+

NADPH + H+

NADP+ + CoASH

NADP+

NADP+

ATP + CoASH

AMP + PPi

NAD(P)H + H+

NADH + H+

Fdred2

Fdox

NAD+

NAD(P)+

NAD+

2 [H]

H2O

2 [H]

CoASH

ATP

ATP

ATP + CoASH

ADP + Pi

ADP +Pi

a b

The hydroxypropionatehydroxybutyrate cycle

The hydroxypropionatehydroxybtyrate cyce fnctionsin the atotrophic crenarchaea order Sfoobaes15,2325

(FIG. 1). This grop comprises extreme thermoacidophiesfrom ocanic areas that gro best at a pH of arond2 and a temperatre of 6090 C. Most Sfoobaes cangro chemoatotrophicay on sphr, pyrite or H

2

nder microaerobic conditions26,27. The enzymes of thehydroxypropionatehydroxybtyrate cyce are oxygentoerant. One of the key enzymes, 4-hydroxybtyry-CoA dehydratase, is aso fond in fermenting costridia,in hich it pays a part in -aminobtyrate fermentation.Athogh it is inactiated by oxygen in costridia21, it issfficienty oxygen insensitie in Sfoobaes15 to oper-ate nder microoxic or een oxic conditions. Therefore,the hydroxypropionatehydroxybtyrate cyce fits e

ith the ifestye of aerobic Crenarchaeota, athogh itshod be noted that is aso present in factatie anaer-obic and een stricty anaerobic Sfoobaes species15.

These species might hae retrned to an anaerobic ife-stye hie retaining enzymes that are associated ith anaerobic enironment. The presence of genes encodingkey enzymes of the hydroxypropionatehydroxybtyratecyce in the mesophiic marine grop I Crenarchaeota25,28

(FIG. 1) sggests that these abndant marine archaea29aso se this cyce.

In the hydroxypropionatehydroxybtyrate cyce, onemoece of acety-CoA is formed from to moeces ofbicarbonate. The key carboxyating enzyme is the bifnc-tiona biotin-dependent acety-CoApropiony-CoAcarboxyase3033. In Bacteria and Ekarya, acety-CoA carboxyase catayses the first step in fatty acid

Figure 3 | Pathwa auttph co2

xat ceahaeta. The dicarboxylatehydroxybutyrate cycle

functions in Desulfurococcales and Thermoproteales (a) and the hydroxypropionatehydroxybutyrate cycle functions in

Sulfolobales (). Note that succinyl-coenzyme A (succinyl-CoA) reductase in Thermoproteales and Sulfolobales uses

NADPH14,35 and reduced methyl viologen (possibly as a substitute for reduced ferredoxin) in Desulfurococcales13,15. In

Sulfolobales, pyruvate might be derived from succinyl-CoA by C4

decarboxylation. CoASH, coenzyme A; Fdred

2, reduced

ferredoxin; Fdox

, oxidized ferredoxin; PEP, phosphoenolpyruvate.

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biosynthesis. Hoeer, Archaea do not contain fattyacids, so this enzyme obiosy has a different metaboicroe in these organisms.

The hydroxypropionatehydroxybtyrate cyce canbe diided into to parts (FIG. 3b). The first transformsacety-CoA and to bicarbonate moeces throgh3-hydroxypropionate to scciny-CoA2325, and thesecond conerts scciny-CoA throgh 4-hydroxyb-tyrate to to acety-CoA moeces25. The prodct ofthe acety-CoA carboxyase reaction, maony-CoA,is redced to maonic semiadehyde and then to3-hydroxypropionate34,35, hich is frther redctieyconerted to propiony-CoA36,37. Propiony-CoA iscarboxyated to (S)-methymaony-CoA by the samecarboxyase32,33. (S)-methymaony-CoA is isomer-ized to (R)-methymaony-CoA, fooed by carbonrearrangement to scciny-CoA by coenzyme B

12-

dependent methymaony-CoA mtase. Scciny-CoAis then conerted to 4-hydroxybtyrate and then to toacety-CoA moeces25,35; this second reaction seqenceinoing 4-hydroxybtyrate is apparenty common to

the atotrophic Crenarchaeota.Acety-CoApropiony-CoA carboxyase ses bicar-

bonate as a co-sbstrate. Pyrate is probaby formedfrom scciny-CoA throgh decarboxyation of maateor oxaoacetate, hich reqires one and a haf trns of thecyce to bid scciny-CoA from for moeces of bicar-bonate. The hydroxypropionatehydroxybtyrate cycereqires nine ATP eqiaents to make pyrate (gener-ating three moeces of pyrophosphate). Pyrophosphatemight sere as energy sorce or might be hydroysed bypyrophosphatase. Athogh the 3-hydroxypropionatepart of this cyce resembes the first part of the 3-hydroxy-propionate bicyce that fnctions in Chloroflexus auran-tiacus (a phototrophic green non-sphr bacterim)3840,the enzymes sed to synthesize propiony-CoA frommaony-CoA are not homoogos, athogh the inter-mediates are the same25,41,42. Frthermore, in C. auran-tiacus acety-CoA is regenerated by may-CoA ceaage,reqiring an additiona cyce to assimiate gyoxyate, thesecond prodct of this ceaage reaction40,43. Therefore,these pathays that sperficiay seem to be simiarmight hae eoed independenty in Sfoobaes andChorofexi.

Gluconeogenesis from acetyl-CoA

A atotrophic pathays in archaea ead to the pro-dction of acety-CoA. The biosynthesis of C

3to C

6

componds mst therefore begin ith acety-CoA.The first steps, the formation of pyrate and PEPfrom acety-CoA and CO

2, differ beteen archaea.

In strict anaerobes, the ferredoxin-dependent pyr-ate synthase catayses the redctie carboxyation ofacety-CoA, and pyrate conersion to PEP ses PEPsynthase (aso knon as pyrate:ater dikinase) orpyrate:phosphate dikinase44,45(FIG. 4). PEP carboxyasegenerates C

4componds. In the aerobic Sfoobaes,

most of the intermediate scciny-CoA is ithdranfrom the carbon fixation cyce to sere as a precrsorfor biosynthesis, and pyrate and PEP formation prob-aby proceeds throgh oxidation of sccinate to maate

or oxaoacetate. By contrast, gconeogenesis startingfrom PEP seems to be niform in different archaea.

Initiay, a the enzyme actiities and genes of theEmbdenMeyerhofParnas gconeogenic pathay,hich is necessary to form FBP from PEP, ere thoghtto be present in archaea (incding gycerate 3-phosphatekinase, gyceradehyde 3-phosphate dehydrogenase andFBP adoase). This is in contrast to the great diersitythat is seen in the archaea gycoytic pathays, hichmosty se different enzymes and intermediates (FIG. 4).Hoeer, experimentay, it proed diffict or impossi-be to detect FBP adoase actiity in archaea. In manycases this enzyme actiity cod ony be measred in thedirection of FBP formation, hereas the reerse reaction,FBP ceaage, faied46,47. This discrepancy as mysteriosas the reaction that is cataysed by FBP adoase is freeyreersibe. Hoeer, tracer stdies in seera atotrophicarchaea reeaed a hexose abeing pattern that as con-sistent ith the cassica gconeogenic rote inoingFBP adoase47,48.

Ony a sma grop of archaea contain a proen

archaea FBP adoase49, and most ack a proen FBPadoase-encoding gene. By contrast, the gene encodingan archaea type v FBP phosphatase is present in manyarchaea50. It trned ot that most archaea, except a fe(ate-eoed) mesophiic grops of the Eryarchaeaota(most Methanosarcinaes and Methanomicrobiaes ase as the (heterotrophic) Haobacteriaes (FIG. 1)), con-tain a bifnctiona FBP adoasephosphatase, hichshoed simiary high FBP adoase and FBP phos-phatase actiity51 (FIG. 4). This pace-making enzymecatayses the conersion of to triose phosphate mo-eces directy to frctose 6-phosphate and inorganicphosphate. Interestingy, this enzyme is aso presentin the deep-branching, mosty thermophiic bacte-ria ineages (Aqificae, Thermotogae, Chorofexi,DeinococcsTherms and CostridiaFirmictes),hereas it is missing in most other bacteria and inEkarya. This highy consered, heat-stabe, bifnctionaFBP adoasephosphatase might represent the ancestragconeogenic enzyme. Its distribtion pattern and ni-directiona cataytic actiity sggest that the EmbdenMeyerhofParnas pathay eoed first in the directionof gconeogenesis.

Frthermore, FBP adoasephosphatase garanteesa nidirectiona gconeogenic pathay nder con-ditions in hich the carbon fx does not need to besitched to sgar degradation. Its combination ith

the modified EntnerDodoroff pathay (notaby thenon-phosphoryated ariant) might een ao sim-taneos and instantaneos se of groth sbstrates thatreqire either gycoysis or gconeogenesis ithot thebrden of transcriptiona regation. Its bifnctiona-ity and high sbstrate affinity ensre that heat-abietriose phosphates are qicky remoed and trapped instabe frctose 6-phosphate. Eary ife forms probabycontained itte carbohydrate (compared ith the ce-ose-containing pants, the most important primaryprodcers of carbohydrates) making sgars rare organicgroth sbstrates. The great diersity of gycoytic path-ays in heterotrophic archaea52,53 might be the rest of

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NADP+

Glucono 1,5-lactone

D-glucoseD-glucose

Glucose 6-P

NADPH + H+

ATP or ADP

ADP or AMP

Gluconate

KDG KDPG

Fructose 6-P

Fructose 1,6-P2

H2O

ATP ADP

PiATP or ADP or PP i

ADP or AMP or P i

Glyceraldehyde

Glycerate

1,3-Bisphosphoglycerate

Dihydroxyacetone-PTriose phosphateisomerase

3-Phosphoglycerate

Fdox

Fdred2-

NAD(P)+

ATP

ATP

ADP

NAD(P)+ +Pi

NAD(P)H + H+

NAD(P)+

NAD(P)H + H+

Fdox

Fdred2-

2-Phosphoglycerate

PEP

ADP

AMP + PPiADP

H2O

Pyruvate

ATP + PiATPATP + H2O

AMP + Pi

Glucose dehydrogenaseor gluconolactonase

Glucose dehydrogenase

or gluconolactonase

Glucokinase(ATP or ADP dependent)

Phosphoglucose

isomerase

Gluconatedehydratase

Phosphofructokinase(ATP-, pyrophosphate-or ADP-dependent)

Other FBPphosphatases

Archaeal FBPaldolase

FBP aldolasephosphatase

KDG kinase

Glyceraldehyde 3-P

NAD(P)H + H+

Glyceraldehydedehydrogenase(NAD(P)+)

Glycerate kinase

Aldehydeoxidoreductase(Fd)

GAPNGAPORPhospho-glyceratekinase

Phosphoglycerate mutase

Enolase

Pyruvate: phosphate dikinasePyruvatekinase

GAPDH

KD(P)G aldolase

PEP synthase

Figure 4 | ceta ahdate metam ahaea. Pyruvate and phosphoenolpyruvate (PEP) formation differ inautotrophic organisms, but gluconeogenesis starting from PEP seems to be uniform (shown by the dashed arrows). In archaea

and deep-branching bacterial lineages a bifunctional fructose 1,6-bisphosphate (FBP) aldolasephosphatase displaces the

reactions of the FBP aldolases and FBP phosphatase51. Archaea that can degrade sugars use modifications of the Embden

MeyerhofParnas (for example, Thermococcus kodakarensis; right) or EntnerDoudoroff (for example, Sulfolobus solfataricus;

left) pathways52,53. KDG (2-keto-3-deoxy-d-gluconate) and KDPG (2-keto-3-deoxy-6-phosphogluconate) are

intermediates of the non-phosphorylative or semi-phosphorylative EntnerDoudoroff pathway, respectively. As FBP is only

an intermediate of the EmbdenMeyerhofParnas pathway, the archaea that use this pathway for sugar degradation must

strictly regulate the expression of FBP aldolasephosphatase50 (or might contain a different FBP aldolase). By contrast, the

archaea that use the EntnerDoudoroff pathway can express this enzyme under both glycolytic and gluconeogenic

conditions64 without the risk of a futile FBPfructose 6-phosphate cycle. Figure is modified, with permission, from REF. 52

Blackwell Publishing(2007)andREF. 53. Elsevier (2005). Fdox

, oxidized ferredoxin; Fdred

2, reduced Fd; GAPDH, normal

NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase; GAPN, non-phosphorylating glyceraldehyde 3-phosphate

dehydrogenase; GAPOR, non-phosphorylating glyceraldehyde 3-phosphate oxidoreductase; Pi, inorganic phosphate.

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conergent eotion and een ate adaptation to sgarmetaboism54, hen arge qantities of cyanobacteriaand finay pant ce as cod sere as the main grothsbstrates. This change in goba sgar sppy might haecased the oss of the ancestra nidirectiona enzyme inheterotrophic bacteria.

Archaea generate pentose phosphates by many dif-ferent rotes. A modified oxidatie pentose phosphatepathay probaby fnctions in Halobacterium spp.55.Hoeer, the most common rote is the reersa of apathay of formadehyde assimiation that is fond insome methyotrophic bacteria. In this reerse reactionseqence, frctose 6-phosphate is conerted to pentosephosphate and formadehyde. Formadehyde can bescaenged by oxidation or by addition to tetrahydrop-terins5558. Another option (identified in Thermoplasmaspp.) is the se of transadoase and transketoase, hichcatayse the reersibe interconersion of triose phosphatesith frctose 6-phosphate to arios sgar phosphates,ths proiding pentose phosphates (as e as tetrosephosphates for aromatic amino acids)55. Note that a

different strategy for aromatic biosynthesis exists inarchaea that does not start from erythrose 4-phosphateand PEP, bt from hexoses, aspartate semiadehyde andmethygyoxa59.

Regulation of autotrophic carbon metabolism

Many atotrophic archaea are factatie atotrophs orthey can co-assimiate organic sbstrates into cearbiding bocks een thogh they do not se organicsbstrates as an energy sorce by oxidizing them to CO

2.

They often donregate the enzymes that are specifi-cay reqired for CO

2fixation hen organic sbstrates

(sch as acetate) are aaiabe. These regatory effectscan be dramatic14, and itte is knon abot the tran-scriptiona regation of those genes. Another exam-pe for the need of regation is the threatening ftiecycing of gycoysis and gconeogenesis, hich occrseen in heterotrophic archaea. Specificay, some hetero-trophic archaea form FBP adoasephosphatase consti-ttiey, hich od be deeterios dring groth onsgars if a norma EmdenMeyerhofParnas pathayere sed becase the enzyme od reconert triosephosphates into frctose 6-phosphate and inorganicphosphate. Hoeer, if gycoysis proceeds throgh ari-ants of the EntnerDodoroff pathay (especiay thenon-phosphoryated ersion, in hich triose phos-phates, frctose 6-phosphate or FBP are not inoed),

the antagonistic carbon fxes do not share commonmetaboites. under sch conditions, the anaboic andcataboic pathays might coexist ithot mta distr-bance and be formed constittiey. In other cases theyneed to be stricty regated, and ony a fe stdies haedeat ith this aspect (FIG. 4).

Genomic analysis of carbon fixation

Athogh ony a imited nmber of species hae beenstdied biochemicay, approximatey 90 archaeagenomes are no aaiabe in the NCBI database, con-sideraby extending or ideas abot the distribtion ofmetaboic pathays in archaea. For exampe, athogh

a enzyme actiities of the hydroxypropionatehydroxy-btyrate cyce hae been shon ony inMetallosphaerasedula and Stygiolobus azoricus15,25, a seqencedatotrophic Sfoobaes hae the corresponding genes.Moreoer, genome seqence data inspired the recent e-cidation of the ne carbon fixation cyces inM. sedula25,60and Ignicoccus hospitalis13,61. Genome seqence data ibe highy aabe for identifying ne pathays and forthe anaysis of metaboic regation of existing path-ays6264. Sch data might aso te s hether the dis-tribtions of the pathays otined in this Reie can begeneraized, and findings from metagenomic approachesshod ao to dedce the distribtion of a particarpathay in natre6567.

The identification of a gene encoding ptatie4-hydroxybtyry-CoA dehydratase in atotrophicmembers of the gens Ferroplasma68 is of particarinterest. It seems to be expressed dring atotrophicgroth69, bt the acta pathay of CO

2assimiation

has yet to be confirmed. Genome anaysis proides anopportnity to stdy nctiated or so-groing spe-

cies. Atotrophic members of the mesophiic marinegrop I Crenarchaeota, hich incde the ammonia-oxidizing sponge symbiont Cenarchaeum spp. andfree-iing Nitrosopumilus spp., are thoght to se thehydroxypropionatehydroxybtyrate cyce. This conc-sion is based soey on the presence of the genes encodinga key enzymes of this cyce and the coincident absenceof genes encoding key enzymes of other atotrophicpathays25,28,70; frther experimenta eidence for itsoperation is necessary.

Hoeer, genomic data shod be interpretedith cation. For exampe, the sbstrate specificityof ordinary enzymes beonging to arge famiies can-not simpy be predicted from seqence comparison.One teing exampe is scciny-CoA redctase, hichis different in Sfoobaes, Thermoproteaes andDesfrococcaes1315,25,35. The presence of conseredenzymes of a pathay, hich a catayse mechanisti-cay diffict reactions, shod be sed as an indicatorfor the presence of a particar metaboic pathay, btnot as proof of its existence. Moreoer, genome anay-sis can be inconsistent ith biochemica data, as is thecase for Pyrobaculum islandicum, a cose reatie ofThermoproteus neutrophilus; genomic data (aaiabefrom the DOE joint Genome Institte ebsite) sggestthe presence of the dicarboxyatehydroxybtyrate cyce,hereas enzymatic stdies sggest the presence of a

different pathay71.On the basis of genome anaysis, areas for frther

research can be identified. If the genes encoding keyenzymes of knon atotrophic CO

2fixation pathays

are acking, it is possibe that another atotrophic path-ay exists. For exampe,Pyrobaculum arsenaticum acksthe gene encoding 4-hydroxybtyry-CoA dehydratase,bt genes coding for components of an aternatiepathay hae not been identified.

The role of RubisCO.Genome seqence data haereeaed some insights into the potentia fnct ion ofRbisCO. For forms of RbisCO hae been identified,

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ith forms IIII being tre carboxyating RbisCOenzymes. Form Iv is referred to as RbisCO-ikeprotein (RlP) and is fond in many bacteria andarchaea. Athogh strctray reated to the treRbisCOs, RlPs do not fnction as RbisCO enzymes,bt instead catayse different reactions in sphrmetaboism7274. Form III RbisCO is ony fondin archaea, and its metaboic roe is ncear 72,7580.RbisCO has been fond in many archaea that erenot reported to be abe to gro atotrophicay andaso in some Eryarchaeota that gro atotrophi-cay bt se the redctie acety-CoA pathay forCO

2fixation. Phosphoribokinase, the second key

enzyme of the Cain cyce, is aso absent in archaea.The exceptions are a fe methanogens (for exampe,Methanosaeta thermophila),Aciduliprofundum boonei and Ferroglobus placidus (genomes aaiabe from theDOE joint Genome Institte ebsite), hich containgenes encoding phosphoribokinase and form IIIRbisCO80,81. likeise, the genome ofM. thermophilaseems to contain genes encoding both sbnits of an

ATP citrate yase. It might e be that nder someconditions these archaea rn a fnctiona Cain cyce.The genome ofArchaeoglobus fulgidus (a member ofthe Eryarchaeota) contains genes encoding the keyenzymes not ony of the redctie acety-CoA pathay,bt aso of the hydroxypropionatehydroxybtyratecyce, the dicarboxyatehydroxybtyrate cyce andRbisCO11,76,82, raising the qestion of hether onyone or mtipe pathays are fnctioning in thisspecies, depending on the groth conditions.

Athogh some of the archaea that contain RbisCOmight hae other options to form ribose 1,5-bisphos-phate72,77,79, the fnction of RbisCO remains esie;hoeer, it has been proposed to hae a roe in AMPmetaboism79. A recent phyogenetic anaysis sggestedan archaea origin of both RbisCO and RlP from theform III RbisCO in methanogenic Eryarchaeota72and a ate appearance of the Cain cyce in eo-tion8385. Interestingy the genome of the atotrophicGram-positieAmmonifex degensii (aaiabe from theDOE joint Genome Institte ebsite) contains genesencoding the archaea phosphoribokinase and formIII RbisCO, sggesting atera gene transfer from theArchaea to Firmictes.

Which autotrophic pathway is used?

what are the res that goern the distribtion of

atotrophic pathays? Obiosy, hich metaboicpathay is sed depends on both the genetic pre-disposition (the phyogeny) of an organism and theconstraints of the occpied niche (the ecoogy). Thediscrete aocation of the archaea atotrophic path-ays to distinct grops mirrors these restraints. Fora discssion of some of the ecoogica determinantsof atotrophic pathays in genera, see BOX 2. withregard to phyogeny, the phyogenetic tree seems tosggest that the common ancestor of a archaea as ananaerobic thermophiic chemoithoatotroph (FIG. 1).The same concsion has been made from the anaysisof archaea genomes86. Conseqenty, anaerobic

thermophiic chemoithoatotrophic archaea shodshare some inherited metaboic traits. By contrast,aerobic, non-thermophiic or heterotrophic extantarchaea species shod represent a deried eotion-ary stage, be it aerobic sphr oxidizers, heterotrophsor haophies sing ight as additiona energy sorce.

In fact, a atotrophic pathays that are knon,except the Cain cyce, start biosynthesis from acety-CoA, hich reqires the generation (or regeneration)of acety-CoA. Anaerobic atotrophic Eryarchaeotaand Crenarchaeota hae a common heritage of car-boxyating enzymes that are essentia for acety-CoAassimiation: ferredoxin-dependent oxygen-sensitiepyrate synthase87,88 and PEP carboxyase, respec-tiey18,19. Incidentay, these to carboxyases areaso essentia for the redctie citric acid cyce. Itod seem that the primordia energy metaboismof archaea as based on either anaerobic C

1or s-

phr chemistry casing methanogens on one side andThermoproteaes and Desfrococcaes on the otherto se different methods of acety-CoA formation.

Methanogens form acety-CoA de novo from C1,and this choice is dictated by their energy metaboism.The redction of C

1to methane aos the formation of

acety-CoA by the addition of ony to more enzymes:a methy transferase ith a methy-accepting corrinoidprotein and a CO dehydrogenaseacety-CoA syn-thase. The anaerobic atotrophic Crenarchaeota, ho-eer, do not hae this C

1nit-transforming machinery

becase they are speciaized in the redction of sphrto hydrogen sphide. Instead, they se a commonanaerobic strategy to prodce C

4componds from

acety-CoA and to CO2

and generate acety-CoAfrom the C

4-compond scciny-CoA. The deried

aerobic atotrophic Crenarchaeota (specificay,Sfoobaes) sti se this acety-CoA-regeneratingmachinery. Hoeer, becase of the oxygen sensitiityof ferredoxin and pyrate synthase, they deeopedanother option to transform acety-CoA to scciny-CoA that ses acety-CoApropiony-CoA carboxy-ase. The se of carboxyphosphate as an intermediatein this reaction is aso an attractie mode for carbonfixation dring chemoeotion.

The atotrophic marine Crenarchaeota (hich areadapted to aerobic ife, mesophiic conditions andaerobic ammonia oxidation) seem to se the samemechanism of carbon fixation as the Sfoobaes.Hoeer, they might hae arried independenty at

the same rest. Amost a enzymes that are inoedin the hydroxypropionatehydroxybtyrate cyce seemto hae been recrited from different gene poos and tobeong to arge enzyme famiies that incde carboxy-ic acid-CoA igases, enoy-CoA hydratases, as e asacoho, adehyde, acy-CoA and 3-hydroxyacy-CoAdehydrogenases. This is not srprising consideringthe ease ith hich the sbstrate specificity of theseenzyme famiies can be changed by mtations. Oneexampe is maony-CoA redctase: in the Sfoobaesthis enzyme originated by dpication of the geneencoding aspartate semiadehyde dehydrogenase, anenzyme that is reqired for threonine and methionine

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biosynthesis, hereas the corresponding enzyme inChloroflexus spp. is deried from competey different

sorces. The gene encoding the postated marine cre-narchaea maony-CoA redctase has not been identi-fied yet by BlAST search and therefore seems to differfrom the knon genes.

A model for primordial metabolism?

The topic of atotrophic carbon fixation initesthe consideration of some eotionary scenarios. Themetaboism first theory assmes that ife started ina hydrotherma-ent setting in the Hadean ocean ithcataytic meta sphide srfaces89,90 or compartments91.The common ancestor of ife as probaby a chemoitho-atotrophic thermophiic anaerobe86,8991. According to

this theory, inorganic carbon fixation proceeded onmineras and as based on cataysis by transition meta

sphides. Gien the strctra and cataytic simiaritybeteen the mineras themsees and the cataytic metaor FeS-containing centres of the enzymes or cofactorsin the acety-CoA pathay, one attractie idea is thatmineras cataysed a primitie acety-CoA pathay85.There is experimenta spport for this idea; for exampe,both the thioester acety methysphide and its hydro-ysed prodct, acetate, can be prodced from CO andCH

3SH sing ony Fe and Ni sphides as cataysts92.

Seera aspects of the redctie acety-CoA path-ay are niqe, and this pathay might be cose tothe ancestra atotrophic carbon fixation pathay83.First, it ses CO, a common ocanic gas ith strong

Box 2 | Benefits of the different autotrophic pathways under different conditions

The pros and cons of the different pathways should be considered. Given that organisms that use the Calvin cycle have

come to dominate most aerobic ecosystems, they presumably have some advantages. Still, five other options exist that

obviously can pre-empt such advantages. Balancing the different requirements of the autotrophic pathways might be

what determines whether a non-Calvin-type autotrophic organism can successfully compete.

oxe, meta ad upp c1mpud

Autotrophic Euryarchaeota are strictly confined to anoxic conditions, generally specialized in metabolizing C1

compounds and/or acetate, and their energy metabolism has low ATP yields. Therefore, they need much of theC

1-transforming machinery for their energy metabolism. The reductive acetyl-coenzyme A (acetyl-CoA) pathway ideally

copes with such constraints. Also, essential metals are more available under anoxic conditions owing to the higher

solubility of the reduced forms of most metals. In Crenarchaeota, the oxygen-sensitive dicarboxylatehydroxybutyrate

cycle is restricted to the anaerobic Thermoproteales and Desulfurococcales, whereas the oxygen-insensitive hydroxypro-

pionatehydroxybutyrate cycle is restricted to the mostly aerobic Sulfolobales and possibly marine Crenarchaeota. The

two lifestyles presuppose different electron donors with different redox potentials and different oxygen sensitivity of

cofactors and enzymes. In a nutshell, energy cost-effective but oxygen-sensitive mechanisms cannot exist in aerobes

because the enzymes would be inactivated by oxygen; and not all anaerobes have C1

substrates at their disposal.

Ee demad

The different pathways require different amounts of ATP to make the cellular precursor metabolites. The costs for synthesizing

all auxiliary, CO2

fixation-related enzymes also differ, which might determine the energy costs involved. The synthesis of the

catalysts itself can require a huge amount of energy as well as nitrogen and sulphur sources, especially if the pathways involve

many auxiliary enzymes. Carboxylases with low catalytic efficiency must be synthesized in large amounts, as is the case for

ribulose 1,5-bisphosphate carboxylaseoxygenase105,106

. So, energy limitation exerts a strong selective pressure in favour ofenergy-saving mechanisms, and the energy costs are largely spent for the synthesis of autotrophy-related enzymes.

Meta uxe

In bacteria and archea, the need for sugar phosphates in the biosynthesis of cell walls is lower than in plants, which also

synthesize huge amounts of cellulose and lignin that is derived from erythrose 4-phosphate and phosphoenolpyruvate

(PEP). The main metabolic fluxes are diverted from acetyl-CoA, pyruvate, oxaloacetate and 2-oxoglutarate, and their

synthesis from 3-phosphoglycerate is partly connected with a loss of CO2. Therefore, in bacteria autotrophic pathways

directly yielding acetyl-CoA are more economical. Still, most facultative aerobic bacteria use the Calvin cycle, the

regulation of which is almost detached from the central carbon metabolism and therefore may be particularly robust.

co2

pee

As the bicarbonate (HCO3

) concentration in slightly alkaline water is much higher than the concentration of dissolved

CO2, autotrophs might profit from using bicarbonate instead of CO

2. The usage of bicarbonate is a special feature of

PEP carboxylase and biotin-dependent carboxylases (that is, acetyl-CoApropionyl-CoA carboxylase). This property

of PEP carboxylase is used in plants in crassulacean acid and C4

metabolism to increase the efficiency of photosynthesis107.

The same might be true for acetyl-CoApropionyl-CoA carboxylase, and the higher bicarbonate concentration could

potentially make up for a lower bicarbonate affinity.

c-amat a mpud

Many autotrophic bacteria and archaea living in aquatic habitats probably encounter carbon oligotrophic conditions and

grow as mixotrophs. Co-assimilation of traces of organic compounds might pay off. A complete or even a rudimentary

hydroxypropionatehydroxybutyrate cycle, for instance, allows the co-assimilation of numerous compounds. These

include fermentation products and 3-hydroxypropionate, an intermediate in the metabolism of the ubiquitous

osmoprotectant dimethylsulphoniopropionate108. It is possible that various widespread marine aerobic phototrophic

bacteria have genes encoding a rudimentary 3-hydroxypropionate cycle for that purpose43. Similarly, the dicarboxylate

hydroxybutyrate cycle allows the co-assimilation of dicarboxylic acids and substrates that are metabolized through

acetyl-CoA.

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redction potentia, as an intermediate. Second, ithas minima energy reqirements and intimateyinks anaboism and cataboism, as the prodct ofthe pathay, acety-CoA, can be conerted to acetate acetate formation from CO

2and H

2is the energy-

yieding process in acetogenic bacteria ith theformation of ATP. Third, the pathay makes exten-sie se of coenzymes (tetrahydropterin, cobaaminand others, depending on the systematic positionof the organism), metas (Fe, Co, Ni, Mo or w) andFeS centres. Coenzymes probaby preceded the morecompex proteins as cataysts90,93. Forth, it enabes theassimiation of oatie CO, formadehyde, methano,methyamine or methymercaptane. Sch C

1nits

occr (or might hae occrred) in ocanic exhaa-tions and they react spontaneosy ith cofactors ofthe redctie acety-CoA pathay. Fifth, this pathaydepends on strict anoxic conditions as it ses o-potentia eectron donors, and some of its enzymes,notaby CO dehydrogenaseacety-CoA synthase,are inherenty highy oxygen sensitie. The reqired

metas are preferentiay ater sobe in the redcedoxidation state, hich aso reqires anoxic conditions.Sixth, the process can be simated in the aboratoryto make not ony acetythioesters, bt aso deriedprodcts by simpy incbating CO, H

2and H

2S or

methymercaptane ith Ni and Fe sats; these inor-ganic metas form mixed NiFe sphides that act ascataysts89,92. Finay, the pathay is not restricted tomethanogenic archaea, bt occrs in seera strictyanoxic grops of bacteria. It can een be reersed andsed for the oxidation of acety-CoA instead of thecitric acid cyce9496.

One can adance simiar bt ess coherent arg-ments in faor of the other pathays that are sed byanaerobes: the redctie citric acid cyce (BOX 1) andthe dicarboxyatehydroxybtyrate cyce13,84,97,98. Bothreqire FeS-containing proteins, sch as ferredoxin,and thioesters to faciitate chemica reactions. Schfeatres fit e into a simpe primordia carbon fixa-tion scheme that is postated by the iron-sphrtheory89,90. Hoeer, these cyces are restricted to eitherbacteria or archaea and might be specific (athoghancient) innoations in these grops. In addition,both grops se enzymes that are aso reqired forthe assimiation of acety-CoA regardess of ho itis made.

If e extrapoate the basic featres of contemporary

anaerobic atotrophic metaboism don to the ee ofits primitie non-enzymatic beginning, can e iden-tify the preaent prebiotic chemistry? Obiosy not,as there is no infaibe singe criterion for a primitietype of metaboism, een thogh the biochemica nitythat nderies the iing ord makes sense ony if mostof the centra metaboic intermediates and pathaysaready existed in the common ancestor. The origins ofife cannot be discoered, they mst be reinented89,99.In other ords, the phyogenetic reconstrction ofancestra metaboism reqires the separation of ifeprocesses into parts that can be expained abioticay,fooed by their reconstittion.

Progress in this endeaor reqires contribtionsfrom different discipines. Bioogists mst fooAriadnes thread of ife from the most compex formsback to the east compex and finay to the point athich ife emerged for the first time from inorganicmatter100. Microbioogists can contribte the extantmetaboic repertoire of bacteria and archaea, schas atotrophic pathays eading to the set of centrametaboites and biding bocks, from hich the eastcompex iing beings are made; not to mention theniersa coenzymes or cofactors, hich might haepreceded the more compex repicabe poymer cata-ysts (RNA and protein) for assisting atocatayticmetaboic cyces. Prebiotic inorganic carbon can asomean CO, COS, HCN, HCHO and other C

1moeces

that are party redced and more reactie than CO2.

A prereqisite for a fnctioning metaboic cyce is itsinkage to any kind of energy-proiding process and tothe generation of a primitie information-processingsystem. Ony sch a sef-reprodcing entity can enterbioogica eotion.

Bioinformaticians can shed ight on eary eo-tion by anaysing the phyogenetic origins of genes.The genomes of archaea are f of exampes in hichgene dpication has rested in to paraoges, oneof hich maintains its origina fnction and anotherthat is reieed from seection pressre and can rap-idy eoe and eentay reach another fnction52.Aternatie mechanisms of diersification based onatera gene transfer may be een more important101,102.Determining the phyogenetic roots of key enzymes ofatotrophic pathays, incding RbisCO, RlPs, ATPcitrate yase, CO dehydrogenaseacety-CoA syn-thase, acety-CoApropiony-CoA carboxyase and4-hydroxybtyry-CoA dehydratase, i be especiaychaenging. which of these genes beonged to thebasic eqipment of Archaea and Bacteria, or Archaeaony, and hich genes in Archaea ere deried fromBacteria, and ice ersa? Ansering these qestionsi eentay hep to nderstand the eary eo-tion of ife and ead to a hypothetica chronoogy ofeents that ed from chemoeotion to cear carbonmetaboism.

Earth scientists can contribte information on themost ikey physicochemica scenario for the prebi-otic geochemica enironment, detaiing bondaryconditions sch as gas composition, atmosphericpressre and temperatre. On the basis of the map

of the knon metaboic andscape and the presmedphysicochemica bondary conditions, chemists canthen test a chemicay conceiabe moecar systemsdeemed to hae a potentia for sef-assemby and sef-repication.

The bioogica options are no open for dispteand een more importanty for experimentademonstration of their potentia as candidate primor-dia reactions. Obiosy, ith six extant atotrophiccarbon fixation cyces, the map of the metaboicandscape is more dierse than preiosy thoght.Perhaps e do not yet hae a compete pictre, bt ecan begin to connect the dots.

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AcknowledgementsG. F. acknowledges the contributions of numerous doctoral orpostdoctoral students during the past 30 years: E. Stupperich,G. Eden and K. Jansen (Marburg); M. Rhlemann, S. Lnge,R. Schauder, S. Schfer and G. Strau (Ulm); and S. Herter,S. Friedmann and C. Menendez (Freiburg). Our work dependedon fruitful collaborations with W. Eisenreich, A. Bacher, H.Huber, K. Stetter, M. Mller, W. Buckel and R. Thauer. Thiswork was supported by Deutsche Forschungsgemeinschaft andEvonikDegussa. Thanks to M. Ziemski for the database analy-sis that was used as the basis for FIG. 1.

Competing interests statementThe authors declare no competing financial interests.

DATABASESEntrez Genome Project:http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj

Aciduliprofundum boonei |Ammonifex degensii |

Archaeoglobusfulgidus|Chloroflexus aurantiacus|

Ferroglobus placidus | Metallosphaera sedula | Methanosaeta

thermophila|Pyrobaculum arsenaticum | Pyrobaculum

islandicum|Thermoproteus neutrophilus

FURTHER INFORMATIONDOE Joint Genome Institute website:http://img.jgi.doe.gov

Georg Fuchs homepage:http://portal.uni-freiburg.de/

ag-fuchs

All linKs ArE AcTivE in THE onlinE PDf

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http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprjhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprjhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid379547%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid42838%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid2234%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid2234%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid2234%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid2234%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid1108%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid1108%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid1108%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid54261%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid43687%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid2224%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid2224%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid2224%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid121277%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid121277%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid2277%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid2277%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid2277%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid70771%5borgn%5dhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid70771%5borgn%5dhttp://img.jgi.doe.gov/http://portal.uni-freiburg.de/ag-fuchshttp://portal.uni-freiburg.de/ag-fuchshttp://portal.uni-freiburg.de/ag-fuchshttp://portal.uni-freiburg.de/ag-fuchshttp://portal.uni-freiburg.de/ag-fuchshttp://portal.uni-freiburg.de/ag-fuchshttp://img.jgi.doe.gov/http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj&cmd=search&term=txid70771%5borgn

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