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Home , Hydrogenosome, Martin Embley, Metamonad, Piromyces, Psalteriomonas

Publishedonline 12December 2002

Mitochondriaandhydrogeno somesare twoforms of the samefundamenta lorganelle

T. * ,Mark van der Giezen † ,David S.Horner ‡ , Patricia L.Dyaland Peter Foster Departmentof Zoology, The Natural History Museum, CromwellRoad, London SW75BD, UK Publisheddata suggestthat , foundin diverseanaerobic eukaryotesthat make energy andhydrogen, were once mitochondria. Ashydrogenosomes generally lack agenome,the conver- sionis probably oneway. The sourcesof thekey hydrogenosomal enzymes,pyruvate :ferredoxin oxido- reductase(PFO) andhydrogenase, are notresolved by currentphylogenetic analyses,but it is likely that both werepresent at anearly stage ofeukaryotic .Once thought toberestricted to a fewunusual anaerobic ,the proteins are intimately integrated intothe fabric ofdiverse eukaryotic cells, wherethey are targeted todifferent compartments,and not just hydrogenosomes. There isno evidence supporting theview that PFO andhydrogenase originated from themitochondrial ,as pos- itedby thehydrogen hypothesisfor eukaryogenesis.Other organelles derivedfrom mitochondria have nowbeen described in anaerobic andparasitic microbial eukaryotes,including speciesthat wereonce thought tohave diverged beforethe mitochondrial symbiosis.It thusseems possible that all eukaryotes may eventually beshown to contain an ofmitochondrial ancestry,to which different types of biochemistry canbe targeted. It remains tobe seen if, despite their obviousdifferences, this family of organelles sharesa commonfunction of importance for theeukaryotic cell,other than energy production, that might provide theunderlying selectionpressure for organelle retention. Keywords: hydrogenosomes;mitochondria; eukaryotic evolution;

1. INTRODUCTION 2. ORIGINOF THE ORGANELLECOMPARTMENT Hydrogenosomesare anaerobic organelles that make Shortly after hydrogenosomeswere discovered in Tri- energy andexcrete molecular ,aiding redox bal- trichomonasfoetus by Lindmark &Mu¨ller (1973) it was ancing,which are foundin phylogenetically diverseeukar- suggestedthat thesehydrogenosomes were descended yotes (Mu¨ller 1993; Biagini et al. 1997a).The beststudied from endosymbioticclostridia, aeubacterial group that -containinggroups are trichomonads,cili- also makeshydrogen usinghydrogenase and PFO, provid- atesand chytrid fungi butother eukaryotesliving in anaer- ing acommonorigin for organelles andbiochemistry obic habitats probably also containthem (seeBroers et al. (Whatley et al. 1979). The subsequentdemonstration of a 1993; Fenchel& Finlay 1995; Roger &Silberman 2002). doublemembrane aroundthe hydrogenosomes of T. foetus Hydrogenosomeshave thusbeen frequently invented (Benchimol &DeSouza1983), wasseen as being consist- independentlyduring eukaryotic evolution,and the ques- entwith anendosymbiotic origin. Atthat time, Tricho- tionof how we currently think eukaryoteshave achieved monas andrelated trichomonadssuch as this is thefocus of this shortreview. Two key issuesmust that composethe Parabasalia, werethought neverto have beaddressed: had any mitochondria. wereone of four protozoan groups placedby Cavalier-Smith (1983) into (i) thesource(s) of the organelle compartment housing theeukaryotic subkingdomArchezoa, the others being thehydrogen-producing biochemistry; and Archamoebae (e.g. ),Metamonada(e.g. (ii) thesource(s) of the biochemistry itself,in particular , )andMicrospora (e.g. Vairimorpha , thekey enzymes(Mu ¨ller 1993), PFO andhydro- Trachipleistophora ). weredefined as being primi- genase. tively withoutmitochondria becausethey split from other eukaryotesbefore the mitochondrial symbiosis (Cavalier- Smith 1983). The first molecular data tosuggest that *Authorfor correspondence ([email protected]). vaginalis (andby inferenceits relatives) oncecontained the † Present address: Schoolof BiologicalSciences, RoyalHolloway, Univer- mitochondrionendosymbiont, were discoveries of sityof London, Egham, Surrey TW20 0EX,UK. ‡ Present address: Dipartimentodi Fisiologia e BiochimicaGenerali, Uni- encodingmitochondrial-type heat shockproteins (Hsp70, versity of Milan,via Celoria 26,20133 Milan, Italy. Hsp60 andHsp10), on its nucleargenome (Bui et al. One contribution of 21to a DiscussionMeeting Issue ‘Chloroplastsand 1996; Germot et al. 1996; Horner et al. 1996; Roger et al. mitochondria: functional genomics and evolution’. 1996). In aerobic , theseproteins have key roles

Phil. Trans. R.Soc.Lond. B (2003) 358, 191–203 191 Ó 2002 TheRoyal Society DOI10.1098/rstb.2002.1190 192T. M.Embley andothers Evolution of hydrogenosomes in mitochondria, andphylogenetic analysis betrays their sequencesand some of these have also beenshown to be commonorigin from theendosymbiont that gave rise to cleavedduring transit (Plumper et al. 1998). For example, theorganelle (Boorstein et al. 1994; Viale &Arakaki in vitro import experiments onhydrogenosomal ferredoxin 1994). The Trichomonas Hsp60 formeda monophyletic showthat it carries acleavable eight amino-acid N-ter- group with mitochondrial Hsp60,which was most closely minal extension,that isnecessary for import intoisolated related tothe homologous protein GroELfrom a-proteo- Trichomonas hydrogenosomes(Bradley et al. 1997). The . This is thebacterial group from which themito- sametargeting sequencecan also sorta marker protein chondrial endosymbiontis thought tohave originated intoyeast mitochondria (Ha¨usler et al. 1997). Amember (Viale &Arakaki 1994; Andersson et al. 1998; Gray et al. ofthe family ofproteinshas recently 1999). The simplest interpretation ofthesedata isthat the beenisolated from Trichomonas hydrogenosomes(Dyall et for Trichomonas Hsp60 also came from themito- al. 2000). Theseproteins are foundin theinner mem- chondrial endosymbiont.The GroELfrom clostridia clus- brane ofmitochondria andare imported usingcryptic teredin aseparate part ofthe tree, well away from the internal signals (Pfanner& Geissler2001). The Tricho- Trichomonas andmitochondrial proteins(Horner et al. monas protein,which is ofunknown function, can also be 1996). The localization ofthe Hsp60 in Trichomonas imported intoyeast mitochondria, strongly suggesting that hydrogenosomes(Bui et al. 1996; Bozner1997), provided thesesignals are conserved.In thereciprocal experiment, thefirst strong data that theorganelle itself sharedcom- Trichomonas hydrogenosomescan import theyeast AAC monancestry with mitochondria. Trichomonas hydro- protein,confirming that theimport machinery required to genosomes,like mitochondria, divide by segmentationand recognizethe yeast-import signals is also presentin the partition (Benchimol et al. 1996). hydrogenosome(Dyall et al. 2000). The hypothesisthat Trichomonas Hsp60 andHsp70 At least fourphylogenetically distinctgroups ofanaer- originated from thesame symbiont that gave rise tomito- obic containhydrogenosomes (see Embley et al. chondria isthe simplest interpretation ofthese data. By 1995), suggesting that thetransition from aerobe with contrast,alternative theorieshave positedhorizontal gene mitochondria toanaerobe with hydrogenosomes,is fairly transferfrom another a-proteobacterium (Sogin 1997), easyto make within this group ofpredominantly aerobic from foodbacteria (Doolittle 1998) or from ‘more casual eukaryotes.Hydrogenosomal ciliates are ecologically sig- donors’ (Kurland& Andersson2000), toexplain thepres- nificant,providing anichefor endosymbioticmethanog- enceof Hsp genes on the of Trichomonas and ensthat usethe hydrogen andcarbon dioxide liberated by other Archezoa.However, none of these alternatives are thehydrogenosomes, to make methaneand energy foundedin phylogenetic analysesthat actually indicatean (Fenchel1993; Embley &Finlay 1994). It hasbeen alternative donorto the mitochondrial endosymbiont. argued that thefacility by whichciliates make hydro- RecentHsp60 treescontaining Trichomonas and Giardia genosomesmust result from modification ofpre-existing are consistentwith currentideas of their relationships mitochondria (Embley et al. 1995), andin somespecies, (Embley &Hirt 1998; Roger 1999), andthus with vertical for example Metopuscontortus and Cyclidium porcatum , the inheritance ofgenes from ancestorsthat containedthe hydrogenosomesstrongly resemblemitochondria in their mitochondrial endosymbiont(see Horner & Embley morphology (Finlay &Fenchel1989; Fenchel& Finlay 2001). There are nowadditional data that strongly sup- 1995). The hydrogenosomesof M.contortus also resemble port alink between Trichomonas hydrogenosomesand mitochondria physiologically, being calcium storesand mitochondria. possessinga membrane potential andan alkali lumen Trichomonas hydrogenosomeslack anassociated gen- (Biagini et al. 1997b).Atleast oneciliate, Nyctotherus ome(Clemens & Johnson2000), sotheir proteinsmust ovalis,apparently containsa mitochondrial small subunit besynthesized in thecytosol and then correctly targeted ribosomal RNAgene(Akhmanova et al. 1998). Although andimported. In mitochondria thereare twomain protein thegene product has notbeen localized tothe import pathways, eachof whichis basedupon a sophisti- hydrogenosome,such genes are normally encodedby the catedmulticomponent pathway that sharessome common mitochondrial (Gray et al. 1998). Thus,in at least proteins(Pfanner & Geissler2001). The mitochondrial onecase, there is the possibility that aciliate hydrogeno- import pathways are distinctfrom themachineries that somemay have retaineda genome.The questionof what sortproteins to other organelles suchas andper- it might encodeis an intriguing one,as mitochondrial gen- oxisomes.Mitochondrial proteinsthat are destinedfor the omestypically encodecomponents of the respiratory innermitochondrial membrane carry cryptic internal tar- chain,thought tobe absent in hydrogenosome-containing geting signals within themature proteinsthat are neces- organisms. However,the mitochondrial genomesof the sary for import. Other proteins,destined for the aerobic ciliates Paramecium and Tetrahymena are already mitochondrial matrix, are synthesizedas pre-proteins car- knownto share several openreading frames ofas yet rying apositively charged targeting sequenceat their unknownfunction (Burger et al. 2000). amino terminusthat iscleaved during import. The mito- The evolutionary origin ofchytrid fungal hydro- chondrial protein import machinery is socomplicated, and genosomeshas beenthe subject of considerable debate. correcttargeting soimportant for thehost cell, that the Reportsof a single ‘microbody-like ’ boundary membrane sameimport machinery isunlikely tohave evolved twice aroundthe hydrogenosomes of Neocallimastix (Yarlett et in differentorganelles. al. 1986; Marvin-Sikkema et al. 1993b),fuelledsugges- There is nowgood evidence, mainly from Johnsonand tionsthat fungal hydrogenosomeswere modified peroxi- co-workers,that Trichomonas hydrogenosomesimport somes(Cavalier-Smith 1987; Hackstein et al. 1998). proteinsby both mitochondrial pathways.Thus, a number Support for this hypothesiswas claimed from reports of ofhydrogenosomal proteinscarry N-terminal leader anSKL peroxisomal targeting motif, detectedusing a het-

Phil.Trans. R. Soc.Lond. B (2003) Evolution of hydrogenosomes T.M.Embley andothers 193 erologousantibody, ona putative nickel –iron–selenium second ‘cryptic internal signal ’ mitochondrial pathway for [Ni–Fe–Se]-hydrogenasefrom Neocallimastixfrontalis L2 membrane proteinscomes from recentstudies of the fun- (Marvin-Sikkema et al. 1993b).However,neither protein gal hydrogenosomal AAC(Van derGiezen et al. 2002). norgene was isolated and sequenced to confirm their This protein transportsADP andATP acrossthe hydro- identity orthepresence of the SKL motif. Acarboxyl ter- genosomemembrane; akey functionfor any energy gener- minal peroxisomal targeting motif has also beenclaimed ating organelle. It isnow clear that fungal for genesencoding chytrid hydrogenosomal adenylate kin- hydrogenosomesand yeast mitochondria usethe same ase,although thegenes themselves are apparently ofmito- protein tocarry outthis function.Thus, the hydrogenoso- chondrial ancestry(Hackstein et al. 1998). Unfortunately, mal AAChas similar properties in primary structure, neither thesedata noran associated and intriguing claim modeof transport andsensitivity towardsinhibitors asits for thepresence of elements of the peroxisomal-import mitochondrial counterpart(Van derGiezen et al. 2002; machinery in chytrids (Hackstein et al. 1998) has yet been Voncken et al. 2002a).Phylogenetic analysesdemonstrate fully published. its commonancestry with mitochondrial AACfrom aero- By contrast,two separate laboratories have published bic fungi,in atreethat is consistentwith its vertical electronmicrographs showingtwo boundary membranes inheritance from acommonmitochondrial ancestor(see around Neocallimastix hydrogenosomes(Benchimol et al. Van derGiezen et al. 2002). Crucially, whenthe hydro- 1997; Van derGiezen et al. 1997b).Thesemembranes genosomal protein is expressedin anAAC-deficient yeast, look like thetwo membranes that surround Trichomonas it is correctly imported into theyeast mitochondrial inner hydrogenosomes(Benchimol et al. 1996). Hydrogenpro- membrane andrestores its ability toexchange ATP for duction by Neocallimastix hydrogenosomesis reducedby ADP—making afunctionalmitochondrion (Van der bongkrekic acid andcarboxyatractylate, whichare inhibi- Giezen et al. 2002). torsof the mitochondrial AACin aerobic eukaryotes (Marvin-Sikkema et al. 1994). Recentattempts toconfirm 3. ORIGINOF PFO thepresence of a [Ni –Fe–Se]-hydrogenasein Neocallimas- tix have failed andit hasbeen suggested that thedata sup- Several microaerophilic microbial eukaryotesincluding porting thepresence of this enzymeand its SKLmotif are Entamoeba, Giardia and Trichomonas usethe oxygen- ambiguous (Davidson et al. 2002; Voncken et al. 2002b). sensitiveenzyme PFO todecarboxylate pyruvate toacetyl- Neocallimastix has nowbeen shown to contain an iron- CoA (Mu¨ller 1998). In Trichomonas ,PFO is localized only [Fe]-hydrogenase, atype that is distinctfrom [Ni – within hydrogenosomeswhereas in Entamoeba and Giardia Fe–Se]-(Davidson et al. 2002; Voncken et it is thought tobe cytosolic. In aerobic eukaryotesthe al. 2002b).This protein lacks any SKLmotif at its car- decarboxylation ofpyruvate iscatalysed in themitochond- boxyl terminus —in fact none ofthe published hydro- rion by thenon-homologous enzyme PDH (Kerscher& genosomal proteinscontain this motif. Other properties Oesterhelt1982). The distribution ofPFO andPDH in that fungal hydrogenosomesshare with mitochondria, but eukaryotesis sofar mutually exclusive,although some notperoxisomes, include the existence of a trans- eubacteria, for example Escherichiacoli, useboth enzymes membrane pHgradient andan alkaline lumen.Free Ca 2 1 underdifferent circumstances (Kerscher & Oesterhelt poolsand calcium phosphate precipitates have also been 1982). Whenit wasthought that Entamoeba, Giardia and detectedin fungal hydrogenosomessuggesting that, like Trichomonas wereArchezoa andthus primitively without mitochondria, they accumulatethis intracellular messen- mitochondria (Cavalier-Smith 1983), thedifferential dis- ger (Biagini et al. 1997a). tribution ofPFO andPDH wasexplained by thehypoth- As for Trichomonas hydrogenosomes,the strongest evi- esisthat PFO wasthe ancestral eukaryotic enzyme,being dencethat fungal hydrogenosomeswere once mitochon- replaced in aerobic eukaryotesby PDH from theprotomi- dria comesfrom thedemonstration that they import tochondrion(Kerscher & Oesterhelt1982). proteinsin thesame ways. Neocallimastix hydrogenosomes The story becamemore complicated whenit wasshown also lack agenome(Van derGiezen et al. 1997b), so any that all ofthe species that containPFO also containgenes proteinsthat they containmust be synthesized in thecyto- ofmitochondrial ancestry(Clark &Roger 1995; Bui et al. soland imported. Hydrogenosomal MEisencoded in the 1996; Germot et al. 1996; Horner et al. 1996; Roger et al. nucleusand targeted tothe hydrogenosome using a 1996, 1998; Horner& Embley 2001). It is also now cleavedamino-terminal targeting signal, which resembles apparent that theevidence for thesespecies branching thoseof yeast mitochondrial proteins(Van derGiezen et beforeother eukaryoteswith bona fide mitochondria is al. 1997a).Hydrogenosomal MEis also selectively weak. Entamoeba is related tothe aerobic mitochondria- imported intomitochondria, rather than peroxisomes,in containing slime mould Dictyostelium onthe basis ofmul- theheterologous host Hansenulapolymorpha, in an N-ter- tiple genetrees (Horner & Embley 2001; Bapteste et al. minal presequencedependent manner (Van derGiezen et 2002) andon morphological data (Cavalier-Smith 1998). al. 1998). Other hydrogenosomal proteins,including The hypothesisthat Giardia and Trichomonas branch hydrogenaseand b-succinylCoA synthetase, also have N- beforeother eukaryotesis based mainly upongene trees terminal extensionsresembling mitochondrial leader that wereconstructed using unrealistic assumptionsabout sequences(Brondijk et al. 1996; Davidson et al. 2002; howgenes evolve. Furthermore, theidentification ofan Voncken et al. 2002b).Thesedata suggestthat Neocallima- early branching assumesthat weknow where stix hydrogenosomesuse the classic N-terminal cleaved theroot ofthe eukaryotic treelies. Currently, there is no transit peptideimport pathway that is foundin mitochon- consensusas to where this root might be,but there are no dria. compelling publisheddata that it lies oneither the Giardia Strong evidencethat hydrogenosomesalso usethe or Trichomonas branches(Kumar &Rzhetsky1996; Hirt

Phil.Trans. R. Soc.Lond. B (2003) 194T. M.Embley andothers Evolution of hydrogenosomes et al. 1999). The presenceof a derivedgene fusion that example, ourtree for thesethree sequences is different affectsdihydrofolate reductaseand thymidylate synthase from that ofRotte et al. (2001). Asin previous analyses, (Philippe et al. 2000), hasrecently been used to argue that theeukaryotic sequencesare notthe nearest neighbours of theroot splits , fungi andchoanoflagellates from sequencesfrom the a- (Horner et al. 1999; other eukaryotes,including Giardia and Trichomonas Rotte et al. 2001). Thus,the currently available data pro- (Stechmann& Cavalier-Smith 2002). vide nosupport for thehypothesis (Embley et al. 1997; There are also several genetrees that suggestthat Martin & Mu¨ller 1998) that eukaryotic PFO originated Giardia (representingmetamonads) and Trichomonas from themitochondrial endosymbiont. (parabasalids) are related toeach other (Embley &Hirt There are nopublished PFO sequencesfrom chytrid 1998; Roger 1999; Henze et al. 2001; Horner& Embley fungi or from hydrogenosomal ciliates, butthere are bio- 2001). If Giardia and Trichomonas are related,and the chemical data that are consistentwith thepresence of PFO Trichomonas hydrogenosomeis a modifiedmitochondrion, in hydrogenosomesof some species. Thus, PFO and then Giardia musthave either lostmitochondria, or they hydrogenaseare enrichedin thefraction containing persistbut have notbeen recognized as such. Another for- hydrogenosomes,in extracts from theciliates Dasytricha mer archezoan,the microsporidian Trachipleistophorahom- ruminantium (Yarlett et al. 1981; Lloyd et al. 1989), inis, isnow known to contain a tiny, andpreviously Eudiplodinium and Epidinium (Yarlett et al. 1984) and Iso- overlooked,organelle ofmitochondrial ancestryand tricha (Yarlett et al. 1983). PFO isalso enrichedwith unknownfunction (Williams et al. 2002). There are also hydrogenasein hydrogenosomal fractionsfrom thechytrid suggestionsfrom theearlier literature that candidatestruc- Neocallimastixpatriciarum (Yarlett et al. 1986) and turesexist in Giardia.Cheissen(1965) describedstruc- N.frontalis L2(Marvin-Sikkema et al. 1993a).In contrast, turesthat hesuggested were ‘changedmitochondria ’ O’Fallon et al. (1991) failed todetect PFO in N.frontalis producedin responseto low-oxygen conditions.So it is EB188. WhereasAkhmanova et al. (1999) have recently probably worthlooking more closely at Giardia using suggestedthat sp. E2 and N.frontalis L2, the moderntechniques to see if it really lacks an organelle samestrain studiedby Marvin-Sikkema et al. (1993a), use (Lloyd& Harris 2002). pyruvate formate lyase,rather than PFO, asthe key pyruv- Several hypotheseshave beenproposed for theorigin of ate-metabolizing enzymein their hydrogenosomes.The PFO in anaerobic eukaryotes.Eukaryotic enzymesmore apparent conflictregarding thepresence or absenceof closely resembleeubacterial homodimeric PFO than they PFO in thehydrogenosomes of N.frontalis L2 may be due dothe multimeric PFOs that have beendescribed for in part todiffering growth conditionsof the two experi- archaebacteria (seeKletzin & Adams1996), andmost ments.For example, it has already beenshown for N. hypotheseshave positedeubacterial donors.An origin of patriciarum that PFO activity is suppressedby growth

PFO (andhydrogenase) from clostridia waspredicted by under CO2 (Yarlett et al. 1986). Moreover,Akhmanova Whatley et al. (1979), basedupon their hypothesisof a et al. (1999) didnot actually assay for PFO activity but separate endosymbioticorigin for the Trichomonas hydro- only failed todetect PFO genesby PCR.The available genosome.Rosenthal et al. (1997) analysed eight PFO data onwhether or not N.frontalis L2 hydrogenosomes sequences,including thosefrom Entamoeba, Giardia and containPFO are thusincomplete. In theabsence of PFO Trichomonas andconcluded that therewas little evidence it has beensuggested that reducingequivalents are gener- for thecommon ancestry of theseeukaryotic PFO, prefer- atedby theoxidation ofmalate topyruvate by ME ring separate origins from differentprokaryotes, andin the (O’Fallon et al. 1991; Williams &Lloyd1993; Akh- case of Entamoeba possibly from anenterobacterium. The manova et al. 1999). The NADPH/NADHcould then be hydrogen hypothesis(Martin &Mu¨ller 1998) for thecon- coupledto hydrogen productioneither indirectly temporaneousorigin ofeukaryotes and the acquisition of (O’Fallon et al. 1991) or directly (Akhmanova et al. 1999). themitochondrial endosymbiontposits that PDH and PFO (andhydrogenase) were donated by this endosym- 4. ORIGINOF EUKARYOTICHYDROGENASE biont,a possibility also discussedby Embley et al. (1997). The mostrecent phylogenetic analysesof PFO sequences Genesencoding hydrogenases have nowbeen cloned (Horner et al. 1999; Rotte et al. 2001) recoveredeukary- andcharacterized for T.vaginalis (Bui &Johnson1996; otic sequencestogether in asingle cluster.We find the Horner et al. 2000), thehydrogenosomal ciliate N. ovalis samein figure 1, wherewe have analysed all ofthe cur- (Akhmanova et al. 1998) andthe chytrid fungi N.frontalis rently available versionsof this protein andits homologues L2 and Piromyces sp.E2 (Davidson et al. 2002; Voncken from thedatabases and genome projects. et al. 2002b).All ofthese genes encode iron-only [Fe]- Monophyly ofthe eukaryotic sequencesmake it prob- hydrogenasesof a type foundin eubacteria butnot in able that PFO wasalready presentin early eukaryotes. archaebacteria, andeach contains the distinctive [Fe –S]- However,the sparse sampling ofeukaryotic speciesand clusterthat constitutesthe active siteor ‘hydrogen’ cluster genomesmake it difficult todraw strong inferencesabout (Nicolet et al. 2000). In thehydrogenosomes of Tricho- themode of inheritance ofthis gene within theeukaryotic monas, pyruvate is oxidized by PFO andelectrons are cluster.The weakclustering ofthe andpara- transferredvia ferredoxin andhydrogenase to protons basalid enzymesis consistent with theposited relationship resulting in thegeneration ofmolecular hydrogen (Mu¨ller betweenthese two groups, and thus with vertical inherit- 1993; Kulda1999). T.vaginalis containsgenes for at least anceof their PFO genesfrom acommonancestor. The three[Fe]-hydrogenases displaying length variation at the precisebranching pattern betweenthe Entamoeba PFO N-terminus.A ‘long-form’ [Fe]-hydrogenase contains andthe N-terminal PFO domainsof thePNO fusionpro- fourputative accessory[FeS]-clusters at theN-terminus, teins of and isnot resolved; for like someeubacterial enzymes(see Horner et al. 2002).

Phil.Trans. R. Soc.Lond. B (2003) Evolution of hydrogenosomes T.M.Embley andothers 195

1.0 A eukaryotes Trichomonas vaginalis B 1.0 Giardia lamblia 0.994 Spironucleus barkhanus Euglena gracilis (PNO fusion protein) Entamoeba histolytica Cryptosporidium parvum (PNO fusion protein) 0.995 Escherichia coli Salmonella typhimurium Yersinia pestis 1.0 Erwinia chrysanthemi Vibrio cholerae Rhodospirillum rubrum (a-proteobacterium) Anabaena sp. 1 Nostoc sp. 1 Synechocystis Nostoc sp. 2 1.0 Chlorobium tepidum Methylococcus capsulatus (g-proteobacterium) 1.0 Rhodobacter capsulatus (a-proteobacterium) Chloroflexus aurantiacus Geobacter sulfurreducens 1.0 1.0 Treponema denticola A Treponema pallidum Fibrobacter succinogenes 1.0 Desulfovibrio africanus Desulfovibrio vulgaris 1.0 Clostridium pasteurianum 2 Rhodococcus albus 1.0 Campylobacter jejuni Fusobacterium nucleatum 1 1.0 Fusobacterium nucleatum 2 1.0 Porphyromonas gingivalis Bacteroides fragilis Tannerella forsythensis 1.0 Clostridium difficile 1 Clostridium difficile 2 Thermoanaerobacter tengcongensis Treponema denticola B Clostridium pasteurianum 1 Clostridium acetobutylicum A 1.0 Dehalococoides hafniense 1 Dehalococoides hafniense 2 Clostridium pasteurianum 3 Clostridium acetobutylicum 2 1.0 Clostridium perfringens 1 Clostridium perfringens 2 Enterobacter agglomerans 1.0 Klebsiella pneumoniae Erwinia chrysanthemi B Enterococcus faecalis A Enterococcus faecalis B 1.0 Lactobacilus lactis Listeria innocua Listeria monocytogenes

Figure 1. Aphylogenetic tree showing therelationships between PFOs from eukaryotes and eubacteria. For Bayesian phylogenetic analysisof thePFO and hydrogenase sequences aligned positions were recoded from theamino acid datainto the six Dayhoffgroups: C,STPAG, NDEQ,HRK, MILVand FYW. Thisis based on thenotion thatchanges within thegroups will becommon and noisy, whereaschanges between groups will berarer and so haveless saturation. Theapproach thushas similarities to themore commonly used transversion-only analysisof DNAsequences. Themodel used a6 ´ 6reversible rate matrix with14 free parameters and 5free composition parameters. Among-site rate variation wasmodelled witha free proportion of invariable sites, and afour-category discrete g-distribution for rates atvariable sites, witha free-shapeparameter. Theanalysis used theMetropolis-coupled Markovchain Monte Carlo variant (Huelsenbeck &Ronquist 2001), in software availableon request from P.G.Foster ([email protected]). All model parameters were optimized during therunning of the chain, whichwas allowed to run for 2million generations, sampledevery 500 generations, withthe first 3000 samplesbeing discarded asburn-in. Theresults showthe consensus of theremaining sampledtrees. Theposterior probabilities for some groups are shown on both trees, witha value of 1.0 representing maximum support. Thestrongly supported groups havealso been recovered in previously published analysesusing different methods, including maximum likelihood and different support measures (Horner et al. 1999, 2000; Rotte et al.2001; Voncken et al. 2002b; Davidson et al. 2002).

Phil.Trans. R. Soc.Lond. B (2003) 196T. M.Embley andothers Evolution of hydrogenosomes

Trichomonas also containstwo ‘short-form’ [Fe]-hydro- hydrogenases.The [Ni –Fe]-hydrogenase ofthe cyano- genasesthat lack theterminal [2Fe2S]-cluster andthe first bacterial endosymbiontwas apparently replaced by ahost- [4Fe4S]-cluster (Bui &Johnson1996). The functional encoded[Fe]-hydrogenase, early in theevolution of green significance ofthis structural variation for hydrogen pro- algal plastids. duction by Trichomonas is unknown,but the long-form Oneof the most surprising developmentsof recentyears hydrogenasemay correspondto the 64 kDa enzymepre- concerninghydrogenases was the finding of[Fe]-hydro- viously purified from T.vaginalis (Payne et al. 1993). genasesin non-photosyntheticeukaryotes that donot con- The N. ovalis [Fe]-hydrogenase isunique among eukar- tain hydrogenosomes.Genes encoding [Fe]-hydrogenases yotic enzymesin that it carries additional domainsat its have nowbeen discovered in Giardia (J.E.Nixon, J. carboxyl terminus(Akhmanova et al. 1998). It wasinitially Field, J.Samuelsonand M. L.Sogin,unpublished data), claimed that thesewere the 51 kDa and24 kDa subunits in itsrelative Spironucleus andin theintestinal parasite Ent- ofthe mitochondrial respiratory chain (Hackstein et al. amoebahistolytica (Horner et al. 2000). The genesfrom 1999), encouraging thesuggestion that hydrogenasesmay Entamoeba and Spironucleus are expressedat themRNA have been ‘stitchedtogether ’ more than oncefrom bits level andthey containall ofthe residues known to be andpieces of mitochondrial genes(Andersson & Kurland important tothe function of [Fe]-hydrogenases (Horner 1999). Phylogenetic analysis provides nosupport for this et al. 2000). It hasnow been shown that Giardia can make hypothesis(Horner et al. 2000). The 51 kDa subunitof hydrogen at arate that isabout tenfold less (2 nmol min –1 the Nyctotherus enzymeis not part ofthe cluster containing per 107 Giardia) than T.vaginalis (Lloyd et al. 2002a). mitochondrial 51 kDa subunitsand NuoF proteinsfrom The key toits detectionwas the development of a highly a-proteobacteria. Instead,the Nyctotherus protein clusters sensitiveand selective way ofmeasuring hydrogen (Lloyd with theNuoF componentsof the C-terminal domainsof et al. 2002a).Giventhat theorganization ofthe Entamoeba other eubacterial hydrogenases.Thus, although the and Spironucleus genesclosely resemblethat of Giardia, it Nyctotherus NuoF-like iscertainly related tomito- seemsprobable that thesespecies can also make hydrogen. chondrial proteins,it isnot of mitochondrial ancestry.It Possiblereasons for hydrogen productioninclude a role has beensuggested that the Nyctotherus enzymeuses its in redox balancing by getting rid ofany excessreducing NuoF-and 24 kDa/NuoE-likecomponents to interact equivalents(Lloyd et al. 2002b). with NADP(H) directly, asdo some eubacterial hydro- Equally remarkable are recentlydiscovered genes bear- genases(Pilkington et al. 1991; Albracht et al. 1997). ing thehallmark signaturesof [Fe]-hydrogenases onour Thus,unlike the Trichomonas enzyme, the Nyctotherus owngenome and in thegenomes of other aerobic eukary- fusionprotein may notdepend on ferredoxin asan elec- otes(Barton &Worman 1999; Horner et al. 2000). These trondonor (Akhmanova et al. 1998, 1999). genesthat are heretermed NARF-like after thefirst one The [Fe]-hydrogenase in N.patriciarum has not been tobe discovered —nuclearprelamin Arecognition factor clonedbut has beensuggested to function like the Tricho- (Barton &Worman 1999), containthe unique hydrogen monas enzyme,with both ferredoxin andPFO involved clusterthat is foundin all [Fe]-hydrogenases (Horner et (Yarlett et al. 1986; Williams &Lloyd1993). Bycontrast, al. 2000). Database searcheshave revealed that NARF- it wassuggested that chytrid hydrogenasesmight besimi- like genesare presentin thegenomes of a widevariety of lar to the Nyctotherus fusionprotein in containing access- differenteukaryotes (Horner et al. 2002), including the ory domainsthat are able tointeract directly with smallest eukaryotic genomesequenced so far, that ofthe NADP(H),as depicted in fig. 10 in Akhmanova et al. intracellular microsporidian parasite Encephalitozooncunic- (1999). The [Fe]-hydrogenase from N.frontalis L2 has uli (Katinka et al. 2001). The functionof these proteins nowbeen cloned and shown to lack accessorydomains at in differenteukaryotes, and if they canmake hydrogen,is its carboxyl terminus(Davidson et al. 2002; Voncken et unknown.Human NARF is localized tothe nucleus and al. 2002b).It thusresembles the long-form Trichomonas interactswith prenylated prelamin A,theprecursor form enzymein its primary structure,rather than the Nyctoth- oflamin involved in themaintenance of the structural erus fusionprotein. integrity ofthe metazoan nucleus(Barton &Worman It has beenknown for along time that greenalgae can 1999). Yeastapparently lacks lamin butdoes contain a producehydrogen (Gaffron& Rubin1942). The enzyme NARF-like gene,the deletion of which is lethal in ahap- is locatedin thechloroplast stroma andis linked via ferre- loid background (Winzeler et al. 2000). doxin tothe photosynthetic electron transport chain The phylogeny of[Fe]-hydrogenases isdifficult to (Happe et al. 1994). Greenalgal plastids thusmake hydro- resolve becausethe gene sequences are mutationally satu- genunder anaerobic conditionsin thelight. The genes rated,and subject to compositional biasesthat are not from Chlorellafusca , Chlamydomonasreinhardtii and Scene- fully mitigated at theamino-acid level (Horner et al. desmus obliquus codefor very short[Fe]-hydrogenases, 2000). Theseproblems translate intotrees where there is whichlack all accessoryFe –Sclustersat theN-terminus little supportfor mostrelationships. However,the tree we (Florin et al. 2001; Happe et al. 2002; Winkler et al. 2002). showhere (figure 2) andthe previously publishedones They appear tofunction satisfactorily in theabsence of (Horner et al. 2000; Davidson et al. 2002; Horner et al. theseclusters, with thecatalysis ofhydrogen production 2002; Voncken et al. 2002b),dosuggest that eukaryotic andother biochemical resultsbeing similar to Clostridium [Fe]-hydrogenases are notmonophyletic tothe exclusion pasteurianum (Florin et al. 2001). Electronsare transferred ofbacterial sequences.Simulation andlikelihood ratio directly from ferredoxin tothe catalytic domain ofthe pro- ofmonophyly have also beenused to show that thehydro- tein.The presenceof [Fe]-hydrogenases in plastids is sur- genasedata canreject the null hypothesisof eukaryotic prising, becausecyanobacteria use[Ni –Fe]-hydrogenases monophyly (Horner et al. 2000). Thus,although thecur- in their hydrogen-generating pathways andlack [Fe]- rentdata cannotexclude a commonorigin for eukaryotic

Phil.Trans. R. Soc.Lond. B (2003) Evolution of hydrogenosomes T.M.Embley andothers 197

Thermotoga maritima NADH 1.0 Neocallimastix frontalis eukaryotes Pyromyces sp. and 0.26 Nyctotherus ovalis Thermotoga Trichomonas vaginalis (long form) Trichomonas (short form) 1.0 Clamydomonas reinhardtii Scendedesmus obliquus Chlorella fusca Spironucleus barkhanus 1.0 Giardia lamblia Entamoeba histolytica 1.0 Desulfovibrio vulgaris (Hildenborough) Hyd- g Rhodopseudomonas palustris (a-proteobacterium) Megasphaera elsdenii Desulfitobacterium hafniense Shewanella putrefaciens Desulfovibrio vulgaris (Oxamicus) 1.0 Desulfovibrio vulgaris (strain Hildenborough) Desulfovibrio fructosovorans Clostridium thermocellum Thermoanaerobacter tengcongensis Ruminococcus albus Treponema denticola 1.0 Clostridium difficile Clostridium botulinum Desulfovibrio fructosovorans Eubacterium acidaminophilum Clostridium perfringens 1.0 Clostridium acetobutylicum ATCC824 Clostridium pasteurianum Clostridium acetobutylicum p262

Figure 2. Therelationships between [Fe]-hydrogenases from eukaryotes and eubacteria. Seelegend to figure 1for details of analysis.

enzymes,the trees also suggestthat Thermotogamaritima enzymes(Voncken et al. 2002b).Asingle origin for the might have acquired its hydrogenasegene by horizontal threegreen algal enzymesis also strongly supported genetransfer from aeukaryote.The hydrogen hypothesis (Horner et al. 2002). Support for monophyly ofthe long- (Martin & Mu¨ller 1998) positsthat hydrogen transfer andshort-form Trichomonas enzymesis not so strong,but from themitochondrial endosymbiontto an archaebacter- thebest tree found indicates a single origin followedby ial ‘host’ wasthe first stepin eukaryogenesis,and thus it structural divergence.The positionof the enzyme from predicts an a-proteobacterial origin for eukaryotic hydro- the ciliate Nyctotherus is unstable.The strongly supported genase.Our analysesprovide nosupport for aclose relationship betweenthe two metamonad sequences, Giar- relationship betweenthe eukaryotic sequencesand the dia and Spironucleus ,andthe one from Entamoeba is sur- only a-proteobacterial [Fe]-hydrogenase sequencefrom prising, asthere are nopublished data that suggestthat Rhodopseudomonaspalustris .The latter formsa strongly thesespecies share acommonancestor exclusive ofother supportedrelationship (Davidson et al. 2002) with oneof eukaryotes.Moreover, it appears from thetree that the twodifferent [Fe]-hydrogenases (hyd- g) from Desulfovibrio sequencefrom Giardia is more closely related tothe Ent- vulgaris (strain Hildenborough),a member ofthe d-pro- sequencethan tothe other metamonad Spironu- teobacteria. It isnot clear whetherthe short NARF-like cleus sequence.One possible explanation for this topology proteins,which are notincluded in figure 2, share acom- isto posit ahorizontal genetransfer to an ancestor of Ent- monorigin with other eukaryote Fe-hydrogenases, amoeba from an ancestorof Giardia.However,the although they doappear toshare asingle commonorigin relationship between Giardia and Entamoeba was lost among themselvesand an early origin in eukaryotes whenwe used a methodthat allowed differentcompo- (Horner et al. 2002; Voncken et al. 2002b). The other sitionand rate matrices acrossthe tree (not shown). In genesencoding [Fe]-hydrogenases also showconsiderable theseanalyses Giardia and Spironucleus clusteredtogether, heterogeneity in length. The presenceof two types of with Entamoeba theoutgroup tothis pair. Further workis hydrogenasein T.vaginalis ,theloss of all accessoryFeS neededto understand the basis for this behaviour andto clustersin thealgal enzymes,and the formation ofthe investigate theevolution of eukaryotic [Fe]-hydrogenases. hydrogenasefusion protein in N. ovalis all suggestthat structural modifications are commonaspects of [Fe]- 5. CONCLUSIONS hydrogenaseevolution in eukaryotes. Within theeukaryotic clusterthere is strongsupport for Currentdata suggestthat hydrogenosomesand mito- themonophyly offungal sequencessuggesting asingle chondria share commonancestry. As there are nostrong commonorigin for the Neocallimastix and Piromyces data indicating that hydrogenosomal eukaryotesbranch

Phil.Trans. R. Soc.Lond. B (2003) 198T. M.Embley andothers Evolution of hydrogenosomes beforemitochondrial eukaryotes,it appears that all hydro- genomesindicates that theremight besome overlap in genosomesare secondaryadaptations ofmitochondria. function,perhaps related toredox sensingor balancing, For ciliates this is almost certainly true,because hydro- butit isnot known if all ofthe encoded proteins can actu- genosomal lineages are clearly descendedfrom mitochon- ally make hydrogen.The mitochondria of Euglena contain dria-containing ancestors,and the nearest relatives of apyruvate :NADP oxidoreductase,a fusionprotein con- ciliates, theapicomplexa anddinoflagellates, also contain taining PFO domainslinked toa C-terminal NADPH- mitochondria. Ashydrogenosomes lack agenome,with cytochromeP450 reductasedomain (Rotte et al. 2001). onepossible exception in Nyctotherus ,theconversion is The samefusion is foundin theunrelated apicomplexan probably oneway only.There are also lessdrastic modifi- Cryptosporidiumparvum butits cell localization is unknown cationsof mitochondrial biochemistry (Embley &Martin (Rotte et al. 2001). In Saccharomyces andother fungi,PFO 1998; Tielens& Van Hellemond1998). Theseinclude the domainsare combinedwith fragments ofredox proteins mitochondria offlatworms,which can use fumarate aster- toform fusionproteins that participate in methionine minal electronacceptor (Tielens & Van Hellemond1998), biosynthesis(Kobayashi &Yoshimoto1982; Hansen et al. andthe mitochondria ofciliates andfungi, which can use 1994; Horner et al. 1999). nitrates (Finlay et al. 1983; Kobayashi et al. 1996). The origins ofPFO andhydrogenase are notclearly Organelles ofmitochondrial ancestrybut unknown func- resolvedby currentdata andmethods of phylogenetic tionhave nowbeen described in theanaerobic amoeba analysis. Acommonorigin for eukaryotic enzymescannot Entamoebahistolytica (Mai et al. 1999; Tovar et al. 1999), berejected, but the source of the genes is unclear given anda tiny organelle has beenfound in themicrosporidian poor sampling andour inability toresolvethe roots of the parasite Trachipleistophorahominis (Williams et al. 2002). genetrees. The genescould be ancestral toeukaryotes, or The jury remains outas to whether Giardia contains an theresult of one or more early horizontal genetransfers organelle—butit doescontain genes (Roger et al. 1998; from eubacteria. Archaebacteria containdifferent kinds of Horner& Embley 2001; Morrison et al. 2001), whosepro- PFO andhydrogenase and so are lesslikely donors.There ducts(Hsp60, Hsp70) localize toan organelle in other is noevidence from currenttrees and gene sampling that eukaryotes.It ispossible that all eukaryoteswill eventually thegenes for PFO and[Fe]-hydrogenase came together befound to contain an organelle ofmitochondrial ances- from themitochondrial endosymbiont,as suggested pre- try, providing aflexible compartment towhich different viously (Embley et al. 1997) andby thehydrogen hypoth- typesof biochemistry canbe conveniently located. esisfor eukaryogenesis(Martin &Mu¨ller 1998). However, Someof these organelles canmake energy,but the thesample ofeubacteria is still small andthe trees for PFO details ofwhat elsethey cando, their detailedbiochemis- andhydrogenase already reveal avery complex picture for try, including their similarities anddifferences, are still theevolution of these enzymes. For example, thereis evi- poorly understood.For example, it isnot known if they denceof multiple copiesof genes, which cluster in differ- all carry outother essentialfunctions for thehost cell, pro- entparts ofthe tree, on the same eubacterial genome viding, despitetheir many differences,a commonselection (figures 1and2). Of potentially greater concernare claims pressureto retain theorganelle underdiverse living con- oframpant horizontal genetransfer among prokaryotes ditions.The maturation ofFe –Sclustersis reportedto be (Doolittle 1999), whichcould have beeneven more preva- an essentialfunction of yeastmitochondria (Lill &Kispal lentin thedeep past whenbarriers totransfer might have 2000). However,it iscurrently notknown if thevarious beenlower (Martin 1999). Underthis scenariothe mito- hydrogenosomes,and the other recentlydescribed chondrial endosymbiontmight have already contained organelles in anaerobesand parasites, also fulfil this func- genesoriginating from other prokaryotes (Martin 1999), tion. Trichomonas and Giardia containelements of this making thetesting of hypotheses, like thehydrogen pathway andthe Trichomonas IscSprotein containsa hypothesis,much more difficult usingphylogenetic analy- plausible hydrogenosomal targeting sequence(Tachezy et sis.However, it might still betoo early tobe so pessimistic. al. 2001). The genomeof the highly reducedmicrosporid- Claims oflarge-scale HGThave oftenbeen based upon ian parasite Encephalitazoon also containsgenes for making BLASTsimilarity scores,an approach that is potentially Fe–Sclusters,and it wason this basis that aputative flawedwhen applied crudely,or other surrogates,instead organelle with arole in Fe –Sclusterassembly wasfirst ofdetailed phylogenetic analysis (Eisen2000; Koski& predicted(Katinka et al. 2001). Anorganelle ofmitochon- Golding 2001; Ragan 2001 a,b; Salzberg et al. 2001). drial ancestryhas nowbeen found in themicrosporidian Thus,although noonecan doubt that HGTbetweenpro- Trachipleistophorahominis (Williams et al. 2002), sothis in karyotes doesoccur, its extentand effect on prokaryotic silico prediction cannow be tested. Data from theongoing genomesand phylogeny are still being determined. Entamoeba genomeproject should also provide thestarting material tobegin investigating thefunctions of its Theauthors thankR. Hirt, T.Richards and G.Walker for comments on themanuscript. M.Sogin gavepermission to organelle ofmitochondrial ancestry,the (Tovar use the Giardia hydrogenase sequence in our tree. Preliminary et al. 1999). sequence datato maketrees were obtained from microbial gen- Genesbearing thehallmark signaturesof [Fe]-hydro- ome databasesdeposited atGenBank (www.ncbi.nlm.nih.gov/ genasesand PFO are foundon thegenomes of aerobic as cgi-bin/Entrez/genom FtableFcgi).Some of thesedata were well asanaerobic eukaryotes.[Fe]-hydrogenases andPFO originally deposited byThe Institute for Genomic Research at are closely integrated intothe fabric ofdiverse eukaryotes, http://www.tigr.org. Sequencing of S.putrefaciens and T. dentic- ola wasaccomplished atTIGR withsupport from DOEand andnot just those with hydrogenosomes.This is consist- NIH-NIDCR. Datawere also obtained from theDOE Joint entwith along history for both proteinswithin theeukary- Genome Institute (JGI) athttp:/ /www.jgi.doe.gov/tempweb/ otic cell.The sofar mutually exclusive distribution of JGIFmicrobial/ and from the Clostridium difficile Sequencing NARF-like genesand longer hydrogenaseson available Group atthe Sanger Institute (ftp://ftp.sanger.ac.uk/pub/

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Winkler, M.,Heil, B.&Happe, T.2002 Isolation and molecu- confusedus is that the Nyctotherus hydrogenaseappears lar characterization of the[Fe]-hydrogenase from theuni- tobe somewhat different from other publishedhydro- cellular green alga Chlorella fusca . Biochim. Biophys. Acta genases.We have recentlyfound a hydrogenasefrom Tri- 1576, 330–334. myema aciliate that iscompletely unrelatedto Nyctotherus , Winzeler, E.A., Liang, H., Shoemaker, D.D.&Davis,R. W. andthis hydrogenaseis exactly thesame type. And if you 2000 Functional analysisof theyeast genome byprecise deletion and parallel phenotypic characterization. Novartis make atree,it formsa monophyletic group with the Found.Symp. 229, 105–109. Nyctotherus hydrogenase.Now my interpretation ofthis is Yarlett, N., Lloyd, D.&Williams, A.1981 Hydrogenosomes that ciliates got their hydrogenasesat thestart ofthe radi- in therumen protozoon Dasytricha ruminantium Schuberg. ation,and have retainedthis enzyme.We are looking for Biochem. J. 200, 365–372. additional evidenceof this hydrogenasein aerobic ciliates; Yarlett, N.,Hann, A.C.&Lloyd, D.1983 Hydrogenosomes certainly the Trimyema and Nyctotherus hydrogenasesare in amixed isolate of Isotricha prostoma and Isotricha intesti- very, very similar, andin treesthey form amonophyletic nalis from ovine rumen contents. Comp.Biochem. Physiol. group. 74B, 357–364. W.Martin. Notwithstandingthe problems ofthe Ther- Yarlett, N.,Coleman, G.S., Williams, A.G.&Lloyd, D.1984 Hydrogenosomes in known speciesof rumen entodiniomor- motoga enzyme,we go back tothe hydrogenase tree, and phid protozoa. FEMS Microbiol. Lett. 21, 15–19. we look at Rhodopseudomonas ,that wasyour red.I think Yarlett, N.,Orpin, C.G., Munn, E.A., Yarlett, N.C.& that youonly have toinvoke onelateral transferfrom the Greenwood, C.A.1986 Hydrogenosomes in therumen Rhodopseudomonas lineage to Desulfovibrio whichI think fungus Neocallimastix patriciarum . Biochem. J. 236, 729–739. wasits sister,which has apparently acquired alot of hydrogenasegenes; so if youwere to take the Desulfovibrio Discussion out,then it wouldlook more like what wewereexpecting, A.G.M.Tielens( Facultyof Biology,Utrecht University, butI believe in thesetrees as little asyou do. Utrecht,The Netherlands ).Martin, thanksfor alovely talk, T.M.Embley. Yes,you can invoke asmall numberof butyou mentioned that certain ciliates containhydro- eventsand make thesetrees say whatever youwant. All I genosomes,while otherscontain mitochondria. What wouldsay is thereis nostrong positive supportfrom them couldbe the reason that thereare nociliates, norin my that candistinguish betweenany ofthesehypotheses that opinion any other organisms that containboth organelles, are outthere. But what isinteresting tome is the wide- becauseeven nowadays there will bemany environments spreaddistribution ofiron hydrogenase,which we thought that will beat onetime aerobic andat another time anaer- wasrestricted to a small numberof eukaryotes, and the obic,and there are organisms that are adjustedto that, so relationships betweenthe eukaryotic enzymes …. Giardia why notby meansof having thesetwo types of organelle? has it, as do Spironucleus and Entamoeba:what is it doing T.M.Embley. Well, Iguessthat if oneis converted in theseorganisms? from theother. Are yousuggesting that somein apopu- W.Martin. Isit knownwhat the a-proteobacterial iron lation may beconverted and others not? It isdifficult to hydrogenasedoes? Is it justa genomesequence? seehow that couldhappen. T.M.Embley. No;yes. A.G.M.Tielens.Or youcould still have theintermedi- J. Tovar (Schoolof Biological Sciences, RoyalHolloway, ate form, if youdo not want to have thetwo extremes. University ofLondon, Egham, Surrey, UK ).Howdoes the T.M.Embley. Icannotthink ofany goodreason why evolution—what weknow of the evolution of PFO —fit you cannot … butno, there are nocases. What thereare casesof, are ciliates that are aerobic that cancolonize with theevolution of the bag itself andof the hydrogenase? anaerobic environments.They change themorphology of T.M.Embley. It lookslike eukaryotic PFO ismono- their mitochondria, are more electrondense and they lose phyletic; youcannot exclude that possibility in any analy- cristae,but there is no evidence that they canmake hydro- sisthat wehave doneand that wehave published.Some gen.Bill, doyouhave asuggestionfor why wedonotget ofthe relationships within theeukaryotic cladeare rather intermediate forms? unusual,but then there are differenttypes of PFO. There W. Martin (Institute ofBotany III, Heinrich-Heine Univ- are sometypes of PFO that are fusions;yeast have taken ersita¨t, Du¨sseldorf,Du ¨sseldorf,Germany ).Well, aswe have bits ofPFO andhave madea sulphite reductaseby fusing beensaying in publications for acoupleof years now,I it todomains of other proteins,so thereis again acomplex think wedohave intermediate formsin Euglena. Euglenas history going on.The data,I think,are consistentwith an have PFO; that ispublished data. They also have pyruvate early acquisition ofPFO, with retentionin various lin- dehydrogenase,so they donot make hydrogen,which is eages.Again, wecannot get asister-grouprelationship thefunctional definition of a hydrogenosome.However, with a-proteobacteria, andhere there is better sampling they dohave oneof the key enzymesthat until nowwas of a-proteobacterial genes.I think thereare three,and specificto hydrogenosomes, namely thePFO fusion noneof them falls intoa monophyletic group with the enzyme.So I wouldagree with youthat wewould be PFO from eukaryotes.One thing Iwouldlike tosay, and it unlikely tosee both organelles in thesame cell, but it echoesback tosomething Ford Doolittle said,and which I wouldnot be surprising toseetransitions between the dif- think Bill Martin alludedto, and that is that thesampling ferentforms. is pitiful here.It wouldbe extremely surprising if wehad A.G.M.Tielens.That is what Imeant,having a actually sampled acontemporary that was secondoption. related tothe ancestor that gave rise tothis eukaryotic T.M.Embley. Ididnot show the data. Ciliates are enzyme.And even if it wasan a-proteobacterium,we have special becauseyou get nestingof hydrogenosomes with got sucha small sample that Iam very cautiouswhen this predominantly aerobic radiation, andone thing that interpreting thesetrees. They donot provide strong sup-

Phil.Trans. R. Soc.Lond. B (2003) Evolution of hydrogenosomes T.M.Embley andothers 203 port for any ofthe hypotheses, but cannot robustly reject questionof what didthe host cell dobefore it got the any ofthem either. mitochondrial endosymbiont.If it wasan archaean, didit A.E.Douglas ( Departmentof Biology,University ofYork, make iron–sulphurclusters in theway archaebacteria do? York, UK).Youquite clearly indicatedthat peroxisomes Sowhat happenedto those genes and proteins in eukary- are very unlikely tobe allied tohydrogenosomes. Do you otes? have any viewsabout theorigins ofperoxisomes? J. F. Allen (PlantBiochemistry, Lund University, Lund, T.M.Embley. Idonot think thereis any strongevi- Sweden).Why doyou rule outATP synthesis? dencefor anendosymbiotic origin ofperoxisomes. Tom T.M.Embley. Well, Idonot rule outATP synthesis, [Cavalier-Smith] has writtenabout this —is there any buthydrogenosomes do not carry outoxidative phos- strong evidencenow? phorylation; they make ATPby substrate-levelphos- T.Cavalier-Smith ( Departmentof Zoology, University of phorylation. It is finethat they cando that, butwith the Oxford,Oxford, UK ).Idonotthink so,no. I have tended Entamoeba mitosome,there is only oneper cell,so it is tohave goneaway from that tothe idea that they come unlikely tobe involved in energy metabolism. Butit is of from theendomembrane system, but I think that thereare mitochondrial ancestry. anumberof problems. W.Martin. Wasit notDavid Lloydwho recently A.E.Douglas. Andthat wouldadd circumstantial sup- showed that Giardia makeshydrogen? port toyour more directevidence that hydrogenosomes, T.M.Embley. Yes,I referredto that. evenin thechytrids, are allied tomitochondria. W.Martin. OK.I musthave missedthat. Andis there T.M.Embley. Yes,I donot think thereis any strong any evidencefor localization ofthat hydrogen production, evidenceout there that suggestschytrid hydrogenosomes compartmentalized within thecell? Doesanybody know? have elementsof peroxisomes involved. T.M.Embley. David [Lloyd] couldnot find any, and A.Barbrook ( Departmentof Biochemistry, University of people working on Giardia have lookedat thelocalization Cambridge,Cambridge, UK ).Howsensitive is your para- ofcpn60. Soltys andGupta have lookedat it, andthey metric bootstrap tothe initial treethat youmodel the founda punctatedistribution for cpn60, whichI guessis data on? consistentwith potential localization within acompart- T.M.Embley. Parametric bootstrapping is notreported ment.Andrew Roger reporteddiffuse localization, if my toperform correctly if themodel does not fit thedata, and memory servesme correctly, andDave Lloyd,in thepap- wedidtest that. The problem is that thetests that are out ersrecording hydrogen productionby Giardia, said he thereto test the fit ofthe data andthe model are fairly couldnot localize it toa particular compartment.They insensitivethemselves, so we used the Goldman test, tried anumberof antibodies, but they didnot try ananti- whichis theclassic one, and we found that thegeneral body tothe hydrogenase. time-reversible modelseemed to fit, and, along with the D.S.Horner( Dipartimentodi Fisiologiae Biochimica treethat weused, plausibly couldhave given rise tothe Generali,University ofMilan, Milan, Italy ).Asan adjunct data weobserve in frontof us. And we have also done tothat, Ihave afeeling that David publishedin aseparate someBayesian testsrecently and again it seemsto fit; the paper this year someMitotracker experiments,which data seemto fit that particular model,and really that is showedsomething —notlocalization, butsomething that all wecan think ofdoing. waspotentially abag. A.Barbrook. Ijustwondered if youhave tried changing N. Lane (Departmentof Surgery,Royal Free and Univer- thetree slightly. sity CollegeMedical School,London, UK ).Whatever mito- T.M.Embley. Yes,the parameters wouldnot change chondria startedout as, what their purposewas, all kinds very much. ofdegenerate purposes may nowbe the main one.There A.E.Douglas. Justbefore this session,you indicated wasa paper publishedfairly recently(by Kristina Peach- that perhaps wewould be able toanswer some of your man andcolleagues) but showing, I think,that eosinophils questions.Do you have any questionsthat youwish to ask have completely losttheir oxidative phosphorylation, and theaudience? exist basically for thepurpose of apoptosis now. T.M.Embley. Yes,I wantedsomeone to tell mewhat A.E.Douglas. It seemsthat thechief function of they believe wasthe core function of the mitochondrial organelles is tobe a membrane-boundcompartment. organelle. If it isnot oxidative phosphorylation, andit is T.M.Embley. Yes,you can organize all kindsof neat notproduction of hydrogen,what couldit be?Mitochon- biochemistry in there. dria doall sortsof things, haem biosynthesis,producing iron–sulphurclusters. Does anyone out there have an GLOSSARY idea? Letus suppose all eukaryoteshave got amitochon- drial organelle. Whatever it does,it isan organelle bag AAC: ADP–ATP carrier derivedfrom themitochondrial endosymbiont.What HGT:horizontal genetransfer wouldpeople predict it wouldbe doing? What wouldbe ME:malic enzyme ageneral selectionpressure for theretention of this NARF: nuclearprelamin Arecognition factor organelle? RolandLill has suggestedthat iron –sulphur PDH:pyruvate dehydrogenase clusterassembly isa key functionof yeast mitochondria, PFO: pyruvate :ferredoxin oxidoreductase sodoes anyone know if themachinery for iron –sulphur PNO: pyruvate :NADP 1 oxidoreductase clusterassembly is mitochondrial in origin? That raisesthe SKL:serine, lysine, leucine

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