- Research article
- Open Access
The [FeFe] hydrogenase of Nyctotherus ovalis has a chimeric origin
- Brigitte Boxma1, 8,
- Guenola Ricard2,
- Angela HAM van Hoek1, 7,
- Edouard Severing1,
- Seung-Yeo Moon-van der Staay1,
- Georg WM van der Staay1,
- Theo A van Alen1,
- Rob M de Graaf1,
- Geert Cremers1,
- Michiel Kwantes1,
- Neil R McEwan3,
- C Jamie Newbold3,
- Jean-Pierre Jouany4,
- Tadeusz Michalowski5,
- Peter Pristas6,
- Martijn A Huynen2Email author and
- Johannes HP Hackstein1
© Boxma et al; licensee BioMed Central Ltd. 2007
- Received: 25 May 2007
- Accepted: 16 November 2007
- Published: 16 November 2007
The hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis show how mitochondria can evolve into hydrogenosomes because they possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand. The hydrogenase permits direct reoxidation of NADH because it consists of a [FeFe] hydrogenase module that is fused to two modules, which are homologous to the 24 kDa and the 51 kDa subunits of a mitochondrial complex I.
The [FeFe] hydrogenase belongs to a clade of hydrogenases that are different from well-known eukaryotic hydrogenases. The 24 kDa and the 51 kDa modules are most closely related to homologous modules that function in bacterial [NiFe] hydrogenases. Paralogous, mitochondrial 24 kDa and 51 kDa modules function in the mitochondrial complex I in N. ovalis. The different hydrogenase modules have been fused to form a polyprotein that is targeted into the hydrogenosome.
The hydrogenase and their associated modules have most likely been acquired by independent lateral gene transfer from different sources. This scenario for a concerted lateral gene transfer is in agreement with the evolution of the hydrogenosome from a genuine ciliate mitochondrion by evolutionary tinkering.
- Codon Usage
- NADH Dehydrogenase
- Lateral Gene Transfer
Hydrogenosomes are membrane-bounded organelles of anaerobic unicellular eukaryotes that produce hydrogen and ATP. These elusive organelles were discovered in trichomonad flagellates, and eventually identified in quite a number of only distantly related unicellular anaerobes such as flagellates, amoeboflagellates, chytridiomycete fungi and ciliates [1–9]. Hydrogenosomes are phylogenetically related to both mitochondria and the various rudimentary, "mitochondrial-remnant" organelles collectively called "mitosomes" [7, 9]. The latter organelles are found in organisms previously considered devoid of mitochondria, that were once named "archaezoa" by Cavalier-Smith , although one of them, Trichomonas vaginalis, actually was already known to contain a hydrogenosome.
The hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis possess a mitochondrial genome and parts of an electron-transport chain on the one hand, and a hydrogenase on the other hand [11, 12]. Because of this combination of features they cannot be classified as being either a hydrogenosome or a mitochondrion. It is likely that this organelle evolved from a ciliate mitochondrion by the expression of a hydrogenase that enables the ciliate to use protons as electron acceptors in order to maintain its metabolic homeostasis under anaerobic conditions. A crucial aspect of this hypothesis is the evolutionary origin of the N. ovalis hydrogenase itself. This is still a matter of debate since phylogenetic analyses suffer from a lack of statistical support due to an insufficient sampling of hydrogenases [13–16].
Here we present evidence that the [FeFe] hydrogenase of N. ovalis does not belong to the clade of "ancient eukaryotic" hydrogenases that also include the non-hydrogen producing NARF's, (nuclear prelamine A recognition factors, ). The analysis of the H-cluster of 19 novel hydrogenases from rumen ciliates that were recovered in a metagenomic approach, reveals the existence of another clade of [FeFe] hydrogenases from both bacterial and eukaryotic organisms, including the one of N. ovalis, but excluding hydrogenases from other ciliates and eukaryotes.
The [FeFe] hydrogenase of N. ovalis is unique because, by a fusion with two NADH dehydrogenase subunits, it is predicted to be capable of reoxidizing NADH directly. The two accessory domains responsible for this are homologous to the 24 kDa and 51 kDa subunits of the mitochondrial NADH dehydrogenase (complex I) and to the bacterial "small hydrogenases" hoxF and hoxU [11, 16, 18]. Supporting the origin of the hydrogenase by Horizontal Gene Transfer we show here that the accessory domains are not closely related to the N. ovalis complex I subunits, but rather appear to have been acquired by lateral gene transfer from bacterial ancestors that possess a [NiFe] hydrogenase.
The 24 kDa/NuoE/hoxF – and 51 kDa/NuoF/hoxU – like regions of the [FeFe] hydrogenase polyprotein
In N. ovalis two different types of 24/51 kDa genes are found: (i) a hydrogenase variant, in which both subunits are fused with each other and with a [FeFe] hydrogenase, and (ii) a "mitochondrial" variant, in which the 24 kDa and 51 kDa genes are located on separate minichromosomes (Fig. 1b). As usual for N. ovalis and some other ciliates, the genes are located on single gene containing macronuclear minichromosomes that are capped with telomeres, making it unlikely that the genes are a contamination. Consistent with their putative function in the "mitochondrial" (hydrogenosomal) complex I (see below), these genes possess N-terminal leader sequences that likely function as a mitochondrial targeting signal. In contrast, the hydrogenase consists of a fusion of the hydrogenase, the 24 kDa and the 51 kDa subunits. Obviously, this "operon" encodes a polyprotein, since it is located on a single minichromosome, and, notably, it possesses only one (N-terminal) "mitochondrial" targeting signal (Fig 1). In contrast, both "mitochondrial" 24 kDa and 51 kDa possess their individual mitochondrial targeting signal. In addition, the "mitochondrial" 51 kDa variant contains two small introns (not shown) that are absent in the fused variant.
A multiple sequence alignment of the "mitochondrial" complex I subunits and 24 kDa/hoxF and 51 kDa/hoxU -like sequences of the hydrogenases of several N. ovalis species reveals that the hydrogenase modules are more similar to the nuoE and nuoF genes of a bacterial complex I than to a mitochondrial complex I (Supplementary Material). The 24 kDa-like module of the N. ovalis hydrogenase possesses only three of the four conserved cysteine residues that bind the [2Fe-2S] cluster N1a found in both mitochondrial 24 kDa subunits and bacterial NuoE's. The fourth cysteine residue of the hydrogenosomal [2Fe-2S] cluster has been replaced consistently by a tryptophane in all N. ovalis 24 kDa subunits sequenced (Additional File ). Stereochemical considerations and mutagenisation studies in bacterial nuoE genes have suggested that this C/W replacement most likely does not interfere with the ferredoxin-like function of the hydrogenosomal 24 kDa module . The 51 kDa-like region of both the hydrogenase domain and the putative mitochondrial complex I subunits contain a NADH binding domain with four conserved glycine residues. In addition, also a FMN binding site with its conserved glycine and proline residues, and the four conserved cysteine residues of the [4Fe-4S] cluster N3 are found in both the 51 kDa subunits of mitochondrial complex I and its bacterial NuoF homologues (Fig. 1b; supplementary material).
The H2 clade comprises all hydrogenase sequences from N. ovalis and its intestinal and free-living relatives. In addition, the H-clusters from two rumen ciliates, one amoeboflagellate, and the ciliate Trimyema sp. belong to this clade – besides sequences from the delta-proteobacterium Desulfovibrio vulgaris and the alpha-proteobacterium Rhodopseudomonas palustris. A bacterial, i.e. endo/episymbiotic origin of the sequences derived from the two rumen ciliates and the ciliate Trimyema cannot be excluded at the current state of information. The ciliate origin of the N. ovalis sequences has been confirmed by their assignment to gene-sized macronuclear chromosomes that are characteristic for Nyctotherus and its relatives. Furthermore, the codon usage is characteristic for N. ovalis (see below). The hydrogenase of the amoeboflagellate Psalteriomonas lanterna, on the other hand has recently been recovered from cDNA thereby revealing the absence of C-terminal 24/51 kDa modules (unpublished). Thus, the existence of two different eukaryotic hydrogenase clades is clearly supported, with a clustering of the N. ovalis sequence with those from Desulfovibrio vulgaris and Rhodopseudomonas palustris. The relationship of both clades to other bacterial hydrogenases remains poorly resolved, but there is no evidence for any close relationship to those bacterial taxa that are supposed to be the source for the hydrogenosomal 24/51 kDa modules.(Fig. 2, 3,), indicating an independent origin for the hydrogenase on the one hand and the 24/51 kDa modules on the other hand.
The [FeFe] hydrogenase of N. ovalis is chimeric and has been acquired by lateral gene transfer
As shown above, the 24 kDa and 51 kDa modules of the hydrogenase of N. ovalis are neither of mitochondrial nor of alpha-proteobacterial origin. Given the presence of paralogues of genuine mitochondrial descent that encode constituents of a functional mitochondrial/hydrogenosomal complex I , an acquisition of the whole hydrogenase by lateral gene transfer from different sources is very likely.
Why acquire a [FeFe]-only hydrogenase?
We have shown recently that the hydrogenosome of N. ovalis is a ciliate-type mitochondrion that produces hydrogen . The presence of a mitochondrial genome, mitochondrial complex I and II dependent respiratory-chain activity, in combination with a kind of fumarate-respiration identifies the N. ovalis hydrogenosome as an intermediate stage in the evolution of mitochondria to hydrogenosomes [11, 12]. But why acquire a [FeFe]hydrogenase at all? It is likely that the ancestral mitochondrion of N. ovalis oxidised NADH via an electron transport chain – as indicated by the presence of genes encoding components of mitochondrial complex I and II. An adaptation to anaerobic environments might be greatly facilitated by the acquisition of a hydrogenase, which could use NADH. It is obvious that the use of fumarate alone as endogenous electron acceptor requires a well-controlled balance between the various catabolic and anabolic reactions in the cell. Depending on the metabolic state of the cell, the NADH pool might be subject to large fluctuations. The presence of alternative oxidases in anaerobic mitochondria provides a means for the cell to cope with such fluctuations in the NADH pool . Such an alternative oxidase appears to be absent in N. ovalis, and the hydrogenase could fulfil the task to regulate the NADH pool. The chimeric [FeFe]-hydrogenase of N. ovalis is tailored for this requirement since it allows a direct re-oxidation of NADH, due to the presence of the 24 and 51 kDa modules. Other [FeFe] hydrogenases, e.g. those of Trichomonas vaginalis, require ferredoxin for hydrogen production from PFO-generated reduction equivalents, and a diaphorase to reoxidise NADH. Thus, a hydrogenase like the one found in N. ovalis, provides many advantages for an organism adapting to anaerobic environments. Since there is no evidence for the presence of hydrogenases in mitochondria/protomitochondria [30, 31], the scenario for the evolution of the hydrogenosomes of N. ovalis from ciliate mitochondria as described here, is likely to have involved complex lateral gene transfer and the fusion of functional domains. The fusion of genes of different origin has at least two advantages: guaranteeing the synthesis of all components in equimolar amounts, and facilitating the flow of electrons from NADH to H+ in a single molecule. Lastly, the evolution of a hydrogenosomal polyprotein requires only the acquisition of a single mitochondrial targeting signal, which can be acquired easily as demonstrated by the frequent retargeting of proteins in the evolution of the eukaryotic cell [31, 32].
Hydrogenases and the origin of mitochondria
The scenario depicted here for the origin of the N. ovalis hydrogenase, in which a hydrogenase was added to an aerobic mitochondrion does not necessarily hold for other hydrogenosomes, because they have evolved independently of N. ovalis and because they are metabolically less similar to mitochondria than is the N. ovalis organelle, e.g. in the way they metabolise pyruvate. N. ovalis uses a "mitochondrial" pyruvate dehydrogenase that reduces NAD, which can subsequently be reoxidized by the hydrogenase that has acquired NADH-oxidizing domains. In contrast, the hydrogenosome of Trichomonas species metabolise pyruvate via a pyruvate:ferredoxin oxidoreductase and the anaerobic chytrids metabolise it via a pyruvate formate-lyase. The proteins in the hydrogenosomes of anaerobic chytrids are phylogenetically related to the proteins in the mitochondria of aerobic fungi, suggesting also here the evolution of the hydrogenosome as a secondary adaptation to anaerobic circumstances. Trichomonas, however, appears in many-sequence based phylogenies at the root of the eukaryotic tree and does not have aerobic, mitochondria containing relatives. A scenario as depicted in the hydrogen hypothesis of Martin and Müller , in which the ancestral organelle of all mitochondria and hydrogenosomes had both a respiratory chain and a hydrogenase can therefore not be ruled out. It should thereby be noted that T. vaginalis, just like N. ovalis has the NADH oxidizing elements of complex I, but, in contrast to N. ovalis, does not have the other proteins of this complex , and is with respect to complex I more like Schizosaccharomyces pombe .
N. ovalis acquired its unique [FeFe] hydrogenase by lateral gene transfer from two different sources. Given that N. ovalis performs a kind of fumarate respiration allowing survival under anaerobic conditions, the acquisition of this peculiar [FeFe] hydrogenase allows an additional regulation of the NADH pool, which is crucial for maintaining the metabolic homeostasis under anaerobic conditions.
Isolation (and culture) of the ciliates
N. ovalis was isolated from the hindgut of the cockroaches Periplaneta americana strains Amsterdam (PA), Bayer (PB), Dar es Salaam (PD), Nijmegen(PN) and Blaberus sp. strains Düsseldorf (BD) and Amsterdam (BA) taking advantage of the unique anodic galvanotaxic behaviour of N. ovalis .Euplotes sp. was grown in Erlenmeyer flasks containing 500 ml artificial seawater (465 mM NaCl, 10 mM KCl, 53 mM MgCl2, 28 mM MgSO4, 1.0 mM CaCl2, and 0.23 mM NaHCO3). Since Euplotes sp. requires living bacteria for growth, E. coli XL1-blue was supplied at regular intervals. Alternatively, a small piece of beef-steak (approximately 1 cm3) was placed into the culture medium to allow the growth of food bacteria. Euplotes sp. cells were harvested 28 days after the start of a new culture by filtration through a 4 μm plankton gaze.
Rumen ciliates were isolated by electromigration from the rumen fluid of a grass-fed, fistulated Holstein-Friesian cow, and lysed immediately after the isolation in a 8 M solution of guanidinium chloride and stored at minus 25°C until use.
DNA isolation, total RNA isolation and cDNA synthesis
DNA of N. ovalis and Euplotes sp. was isolated according to van Hoek et al. . Total rumen ciliate DNA was prepared after purification on a hydroxyapatite column (BioRad) using standard methods. Total RNA of N. ovalis was isolated using the RNeasy Plant mini-kit (Qiagen). Adaptor-ligated cDNA was prepared according to the SMART™ RACE cDNA Amplification kit (Clontech).
Isolation of the H-cluster, the 24 kD (hoxF) and 51 kD (hoxU) modules of the hydrogenase gene, and mitochondrial-type 24 kDa and 51 kDa subunits of mitochondrial complex I
H-clusters of Fe-hydrogenases were amplified from total rumen ciliate DNA using PCR with primers described earlier . In addition, DNA from type-strain rumen ciliates, kept by the ERCULE consortium was used as template for PCR.
To isolate the (nuclear-encoded) 24 kD (hoxF) and 51 kD (hoxU) -like genes, the primer-design was based on the H-cluster and the 24 kD (hoxF) and 51 kD (hoxU) region of the hydrogenase of N. ovalis PN . Their sequences are 5'-gtnatggcntgyccngghgghtg-3' (H-cluster forward primer) and 5'-ccntcyctrcadggnacrcaytg-3' (51 kDa reverse primer 1). Sequence-specific internal primers were designed to isolate the termini of the gene-sized chromosomes in combination with a telomere-specific primer using the telomere suppression PCR method [38, 39].
To isolate the (nuclear) genes encoding the 24 kDa and 51 kDa subunits of mitochondrial complex I, respectively, primers were based on conserved amino-acid regions of mitochondrial complex I genes. Their sequences are 5'-tgyggwachachccwtg-3' (24 kDa forward primer), 5'-ccnarrcaytcdacytc-3' (24 kDa reverse primer), 5'-gmhgargghgarccwgghac-3' (51 kDa forward primer), and 5'-cangwcatytcytcytcnac-3' (51 kDa reverse primer). The ORFs were completed as described above.
The amino acid sequences of the H-cluster were aligned using Clustal × 1.81 . The program Gblocks  was used to identify regions of defined sequence conservation and exclude ambiguously aligned positions from the alignment. The phylogenetic analysis of the sequences were performed with the program MRBAYES version 3.1.2 . Markov chain Monte Carlo from a random starting tree was initiated and run for 2 million generations. In these analyses, the JTT model of amino acid substitution and four gamma distributed rates of evolution were applied. Trees were sampled every 1000th generation. The first 25% of the samples were discarded as 'burn-in', and the rest of the samples were used for inferring a Bayesian tree. Examination of the log-likelihood and the observed consistency with the similar likelihood values between the two independent runs suggest that the run reached stationarity and that these burn-in periods were sufficiently long.
The accession numbers of the sequences used to calculate this tree are Nyctotherus ovalis BA AY608627; N. ovalis PN CAA76373; the sequences from rumen ciliates have been deposited in GenBank under accession numbers AM396939 – AM396957
For the 24kD/51kD domains less sequences had to be included to delineate the evolution, allowing a "manual" sequence alignment and phylogeny approach. Alignments of representative sequences from the 24kD/51kD domains were generated with MUSCLE . Sequences were edited and the most relevant parts from the alignments were selected manually using Seaview . Phylogenies were subsequently derived using the program PHYML  using the JTT model and an estimated number of invariable sites with four substitution rate categories. 100 bootstraps were performed; they are only indicated in the tree if they are ≥ 50.
The accession numbers of the used 24 kDa subunit/NuoE/hoxF sequences are [Agrobacter tumefaciens PIR:D97514, Bos taurus Swiss-Prot:P04394, Buchnera aphidicola Swiss-Prot:P57255, Burkholderia cenocepacia AU 1054 REFSEQ:YP_621511.1, Burkholderia xenovorans LB400 REFSEQ:YP_555778.1, Dechloromonas aromatica RCB GenBank:AAZ45735.1, Deinococcus radiodurans REFSEQ:NP_295224, Drosophila melanogaster GenBank:AAL68189, Escherichia coli Swiss-Prot:P33601, Hahella chejuensis KCTC 2396 REFSEQ:YP_431454.1, Heliobacillus mobilis EMBL:CAJ44288.1, Homo sapiens REFSEQ:NP_066552, Magnetospirillum magneticum AMB-1 REFSEQ:YP_422756.1, Methanospirillum hungatei JF-1 REFSEQ:YP_502735.1, Methanothermobacter thermautotrophicus str. Delta H GenBank:AAB86022.1, Methylococcus capsulatus str. Bath REFSEQ:YP_115124.1, Mus musculus Swiss-Prot:Q9D6J6, Mycobacterium vanbaalenii PYR-1 REFSEQ:ZP_01205720.1, N. ovalis BA (24 kDa) GenBank:AY628688, N. ovalis BD GenBank:AY608628, N. ovalis PA GenBank:AY608629, N. ovalis PB GenBank:AY608630, N. ovalis PD GenBank:AY608631, N. ovalis PN GenBank:CAA76373, Neurospora crassa Swiss-Prot:P40915, Nitrosospira multiformis ATCC 25196 REFSEQ:YP_411790.1, Nitrosospira multiformis ATCC 25196 REFSEQ:YP_412360.1, Nyctotherus ovalis BA GenBank:AY608627, Paracoccus denitrificans Swiss-Prot:P29914, Paramecium tetraurelia Swiss-Prot:Q6BFW6, Psychromonas ingrahamii 37 REFSEQ:ZP_01348563.1, Ralstonia eutropha GenBank:AAC06140, Ralstonia metallidurans CH34 REFSEQ:YP_583086.1, Ralstonia solanacearum UW551 REFSEQ:ZP_00943354.1, Rattus norvegicus Swiss-Prot:P19234, Rhodobacter sphaeroides ATCC 17029 REFSEQ:ZP_00918830.1, Rhodococcus opacus Swiss-Prot:P72304, Rhodoferax ferrireducens T118 REFSEQ:YP_525088.1, Streptomyces avermitilis MA-4680 REFSEQ:NP_823011.1, Syntrophus aciditrophicus SB REFSEQ:YP_461127.1, Tetrahymena thermophila Swiss-Prot:Q23LJ5, Thermoanaerobacter tengcongensis GenBank:AAM24146, Thermococcus kodakarensis KOD1 DDBJ:BAD85803.1, Thermotoga maritima REFSEQ:NP_227828, Thermus thermophilus Swiss-Prot:Q56221, Thiobacillus denitrificans ATCC 25259 REFSEQ:YP_314904.1, uncultured archaeon GZfos26D6 GenBank:AAU83055.1]
The accession numbers of the 51 kDa subunit/NuoF/hoxU sequences are [Agrobacterium tumefaciens Swiss-Prot:Q8U6U9, Aquifex aeolicus Swiss-Prot:O66841, Arabidopsis thaliana Swiss-Prot:Q8LAL7, Aspergillus niger Swiss-Prot:Q92406, Bos taurus GenBank:AF092131, Bradyrhizobium japonicum DDBJ:BAC48402, DDBJ:BAC50177, Burkholderia xenovorans LB400 REFSEQ:YP_555778.1, Candida tropicalis Swiss-Prot:Q96UX4, Caulobacter crescentus Swiss-Prot:Q9A6X9, Chromobacterium violaceum ATCC 12472 REFSEQ:NP_900616.1, Dechloromonas aromatica RCB GenBank:AAZ45735.1, Dictyostelium discoideum REFSEQ:XP_636489.1, Eubacterium acidaminophilum EMBL:CAC39230.1, Euplotes sp. GenBank:AY608636, Hahella chejuensis KCTC 2396 REFSEQ:YP_431454.1, Heliobacillus mobilis EMBL:CAJ44288.1, Leishmania major Swiss-Prot:Q9U4M2, Magnetospirillum magneticum AMB-1 REFSEQ:YP_422756.1, Methanospirillum hungatei JF-1 REFSEQ:YP_502736.1, Methanothermobacter thermautotrophicus str. Delta H GenBank:AAB86023.1, Methylococcus capsulatus str. Bath REFSEQ:YP_115124.1, Mycobacterium tuberculosis Swiss-Prot:P95176, Mycobacterium vanbaalenii PYR-1 REFSEQ:ZP_01205720.1, N. ovalis BA (51 kDa) GenBank:AY608632, N. ovalis BD GenBank:AY608628, N. ovalis PA (51 kDa) GenBank:AY608635, N. ovalis PB GenBank:AY608630, N. ovalis PD GenBank:AY608631, N. ovalis PN GenBank:CAA76373, Neisseria gonorrhoeae FA 1090 REFSEQ:YP_208779.1, Neisseria meningitidis Z2491 EMBL:CAB83334.1, Neurospora crassa Swiss-Prot:P24917, Nitrosococcus oceani ATCC 19707 GenBank:ABA59013.1, Nitrosomonas europaea ATCC 19718 EMBL:CAD85683.1, Nitrosomonas eutropha C71 REFSEQ:ZP_00669832.1, Nitrosospira multiformis ATCC 25196 REFSEQ:YP_411791.1, REFSEQ:YP_412360.1, Nyctotherus ovalis BA GenBank:AY608627, Paracoccus denitrificans Swiss-Prot:P29913, Psychromonas ingrahamii 37 REFSEQ:ZP_01348563.1, Ralstonia eutropha GenBank:AAC06140, Ralstonia eutropha JMP134 GenBank:AAZ60345.1, Ralstonia metallidurans CH34 REFSEQ:YP_583087.1, Ralstonia solanacearum EMBL:CAD15764.1, Ralstonia solanacearum UW551 REFSEQ:ZP_00943355.1, Rhizobium loti Swiss-Prot:Q98BW8, Swiss-Prot:Q98KR0, Rhizobium meliloti Swiss-Prot:P56912, Swiss-Prot:P56913, Rhodobacter capsulatus Swiss-Prot:O07948, Rhodococcus opacus Swiss-Prot:P72304, Rhodoferax ferrireducens T118 REFSEQ:YP_525088.1, Rickettsia prowazekii Swiss-Prot:Q9ZE33, Solanum tuberosum Swiss-Prot:Q43840, Streptomyces avermitilis MA-4680 REFSEQ:NP_823011.1, Synechococcus elongatus PCC 6301 EMBL:CAA73873.1, Syntrophus aciditrophicus SB REFSEQ:YP_461127.1, Tetrahymena thermophila SB210 GenBank:EAR96899.1, Thermococcus kodakarensis KOD1 DDBJ:BAD85803.1, Thermotoga maritima Swiss-Prot:O52682, Swiss-Prot:Q9WXM5, Swiss-Prot:Q9WY70, Thermus thermophilus Swiss-Prot:Q56222, Thiobacillus denitrificans ATCC 25259 REFSEQ:YP_314905.1, Trypanosoma brucei REFSEQ:XP_824451.1, Trypanosoma cruzi GenBank:EAN82122.1, uncultured archaeon GZfos26D6 GenBank:AAU83054.1, Yarrowia lipolytica Swiss-Prot:Q9UUU2]
Multivariate Comparative Analysis
The codon usage of the genes investigated in this study was subjected to a multivariate analysis by means of Principal Component Analysis (PCA) to visualise the genetic diversity of the ciliate species. PCA was performed using the GeneMaths XT software package (Applied Maths BVBA, Sint-Martens-Latem, Belgium ).
We thank Prof. TM Embley for providing the Trimyema sequence and the referees for their comments. The work has been supported by the EU 5th framework projects ERCULE (QLRI-CT-2000-01455) and CIMES (QLK3-2002-02151)
- Müller M: The hydrogenosome. J Gen Microbiol. 1993, 139: 2879-2889.View ArticlePubMedGoogle Scholar
- Roger AJ: Reconstructing Early Events in Eukaryotic Evolution. Am Nat. 1999, 154: S146-S163.View ArticlePubMedGoogle Scholar
- Hackstein JHP, Akhmanova A, Voncken F, van Hoek A, van Alen T, Boxma B, Moon-van der Staay SY, van der Staay G, Leunissen J, Huynen M, Rosenberg J, Veenhuis M: Hydrogenosomes: convergent adaptations of mitochondria to anaerobic environments. Zoology. 2001, 104: 290-302.View ArticlePubMedGoogle Scholar
- Martin W, Hoffmeister M, Rotte C, Henze K: An overview of endosymbiotic models for the origins of eukaryotes, their ATP-producing organelles (mitochondria and hydrogenosomes), and their heterotrophic lifestyle. Biol Chem. 2001, 382: 1521-1539.View ArticlePubMedGoogle Scholar
- Embley TM, van der Giezen M, Horner DS, Dyal PL, Bell S, Foster PG: Hydrogenosomes, mitochondria and early eukaryotic evolution. IUBMB Life. 2003, 55: 387-395.View ArticlePubMedGoogle Scholar
- Yarlett N, Hackstein JHP: Hydrogenosomes: One organelle, multiple origins. Bioscience. 2005, 55: 657-668.View ArticleGoogle Scholar
- Embley TM, Martin W: Eukaryotic evolution, changes and challenges. Nature. 2006, 440: 623-630.View ArticlePubMedGoogle Scholar
- Embley TM: Multiple secondary origins of the anaerobic lifestyle in eukaryotes. Philos Trans R Soc B-Biol Sci. 2006, 361: 1055-1067.View ArticleGoogle Scholar
- Hackstein JHP, Tjaden J, Huynen M: Mitochondria, hydrogenosomes and mitosomes: products of evolutionary tinkering!. Curr Genet. 2006, 50: 225-245.View ArticlePubMedGoogle Scholar
- Cavalier-Smith T: Kingdom protozoa and its 18 phyla. Microbiol Rev. 1993, 57: 953-994.PubMed CentralPubMedGoogle Scholar
- Boxma B, de Graaf RM, van der Staay GWM, van Alen TA, Ricard G, Gabaldon T, van Hoek AHAM, Moon-van der Staay SY, Koopman WJH, van Hellemond JJ, Tielens AGM, Friedrich T, Veenhuis M, Huynen MA, Hackstein JHP: An anaerobic mitochondrion that produces hydrogen. Nature. 2005, 434: 74-79.View ArticlePubMedGoogle Scholar
- Martin W: The missing link between hydrogenosomes and mitochondria. Trends Microbiol. 2005, 13: 457-459.View ArticlePubMedGoogle Scholar
- Horner DS, Foster PG, Embley TM: Iron hydrogenases and the evolution of anaerobic eukaryotes. Mol Biol Evol. 2000, 17: 1695-1709.View ArticlePubMedGoogle Scholar
- Horner DS, Heil B, Happe T, Embley TM: Iron hydrogenases–ancient enzymes in modern eukaryotes. Trends Biochem Sci. 2002, 27: 148-153.View ArticlePubMedGoogle Scholar
- Voncken FGJ, Boxma B, van Hoek AHAM, Akhmanova AS, Vogels GD, Huynen M, Veenhuis M, Hackstein JHP: A hydrogenosomal [Fe]-hydrogenase from the anaerobic chytrid Neocallimastix sp. L2. Gene. 2002, 284: 103-112.View ArticlePubMedGoogle Scholar
- Hackstein JHP: Eukaryotic Fe-hydrogenases – old eukaryotic heritage or adaptive acquisitions?. Trans Biochem Soc. 2005, 33: 47-50.View ArticleGoogle Scholar
- Barton RM, Worman HJ: Prenylated prelamin A interacts with Narf, a novel nuclear protein. J Biol Chem. 1999, 274: 30008-30018.View ArticlePubMedGoogle Scholar
- Akhmanova AS, Voncken FGJ, van Alen TA, van Hoek AHAM, Boxma B, Vogels GD, Veenhuis M, Hackstein JHP: A hydrogenosome with a genome. Nature. 1998, 396: 527-528.View ArticlePubMedGoogle Scholar
- Vignais PM, Billoud B, Meyer J: Classification and phylogeny of hydrogenases. FEMS Microbiol Rev. 2001, 25: 455-501.View ArticlePubMedGoogle Scholar
- Friedrich T, Böttcher B: The gross structure of the respiratory complex I: a Lego system. Biochim Biophys Acta. 2004, 1608: 1-9.View ArticlePubMedGoogle Scholar
- Gabaldon T, Rainey D, Huynen MA: Tracing the evolution of a large protein complex in the eukaryotes, NADH : Ubiquinone oxidoreductase (Complex I). J Mol Biol. 2005, 348: 857-870.View ArticlePubMedGoogle Scholar
- Preis D, Weidner U, Conzen C, Azevedo JE, Nehls U, Röhlen D, Vander Pas J, Sackmann U, Schneider R, Werner S, Weiss H: Primary structures of two subunits of NADH:ubiquinonereductase from Neurospora crassa concerned with NADH-oxidation. Relationship to a soluble NAD-reducing hydrogenase of Alcaligenes eutrophus. Biochim Biophys Acta. 1991, 1190: 133-138.View ArticleGoogle Scholar
- Smith MA, Finel M, Korolik V, Mendz GL: Characteristics of the aerobic respiratory chains of the microaerophiles Campylobacter jejuni and Helicobacter pylori. Arch Microbiol. 2000, 174: 1-10.View ArticlePubMedGoogle Scholar
- Meyer J: Ferredoxins of the third kind. FEBS Lett. 2001, 509: 1-5.View ArticlePubMedGoogle Scholar
- Williams AG, Coleman GS:The rumen protozoa. 1992, New York: Springer Verlag,View ArticleGoogle Scholar
- Regensbogenova M, Pristas P, Javorsky P, Moon-van der Staay SY, van der Staay GWM, Hackstein JHP, Newbold CJ, McEwan NR: Assessment of ciliates in the sheep rumen by DGGE. Lett Appl Microbiol. 2004, 39: 144-147.View ArticlePubMedGoogle Scholar
- Davidson EA, van der Giezen M, Horner DS, Embley TM, Howe CJ: An [Fe] hydrogenase from the anaerobic hydrogenosome-containing. fungus Neocallimastix frontalis. 2002, 296: 45-52.Google Scholar
- Bleijlevens B, Buhrke T, van der Linden E, Friedrich B, Albracht SPJ: The auxiliary protein HypX provides oxygen tolerance to the soluble [NiFe]-hydrogenase of Ralstonia eutropha H16 by way of a cyanide ligand to nickel. J Biol Chem. 2004, 279: 46686-46691.View ArticlePubMedGoogle Scholar
- Tielens AGM, van Hellemond JJ: Anaerobic mitochondria: properties and origins. Origin of mitochondria and hydrogenosomes. Edited by: Martin WF, Müller M. 2007, Berlin Heidelberg: Springer Verlag, 85-103.View ArticleGoogle Scholar
- Gabaldon T, Huynen MA: Reconstruction of the proto-mitochondrial metabolism. Science. 2003, 301: 609-609.View ArticlePubMedGoogle Scholar
- Gabaldon T, Huynen MA: Shaping the mitochondrial proteome. Biochim Biophys Acta-Bioenerg. 2004, 1659: 212-220. Sp. Iss. SIView ArticleGoogle Scholar
- Esser C, Ahmadinejad N, Wiegand C, Rotte C, Sebastiani F, Gelius-Dietrich G, Henze K, Kretschmann E, Richly E, Leister D, Bryant D, Steel MA, Lockhart PJ, Penny D, Martin W: A genome phylogeny for mitochondria among alpha-proteobacteria and a predominantly eubacterial ancestry of yeast nuclear genes. Mol Biol Evol. 2004, 21: 1643-1660.View ArticlePubMedGoogle Scholar
- Boxma B, Voncken F, Jannink S, van Alen T, Akhmanova A, van Weelden SW, van Hellemond JJ, Ricard G, Huynen M, Tielens AG, Hackstein JH: The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E. Mol Microbiol. 2004, 51: 1389-1399.View ArticlePubMedGoogle Scholar
- Martin W, Müller M: The hydrogen hypothesis for the first eukaryote. Nature. 1998, 392: 37-41.View ArticlePubMedGoogle Scholar
- Hrdy I, Hirt RP, Dolezal P, Bardonova L, Foster PG, Tachezy J, Embley TM: Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I. Nature. 2004, 432: 618-622.View ArticlePubMedGoogle Scholar
- van Hoek AHAM, Sprakel VS, van Alen TA, Theuvenet AP, Vogels GD, Hackstein JHP: Voltage-dependent reversal of anodicgalvanotaxis in Nyctotherus ovalis. J Eukaryot Microbiol. 1999, 46: 427-433.View ArticlePubMedGoogle Scholar
- van Hoek AHAM, van Alen TA, Sprakel VSI, Hackstein JHP, Vogels GD: Evolution of anaerobic ciliates from the gastrointestinal tract: Phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives. Mol Biol Evol. 1998, 15: 1195-1206.View ArticlePubMedGoogle Scholar
- Curtis EA, Landweber LF: Evolution of gene scrambling in ciliate micronuclear genes. Ann NY Acad Sci. 1999, 870: 349-350.View ArticlePubMedGoogle Scholar
- Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA: An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res. 1995, 23: 1087-1088.PubMed CentralView ArticlePubMedGoogle Scholar
- Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ: Multiple sequence alignment with Clustal X. Trends Biochem Sci. 1998, 23: 403-405.View ArticlePubMedGoogle Scholar
- Castresana J: Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000, 17: 540-552.View ArticlePubMedGoogle Scholar
- Ronquist F, Huelsenbeck JP: MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003, 19: 1572-1574.View ArticlePubMedGoogle Scholar
- Edgar RC: MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004, 5: 1-19.View ArticleGoogle Scholar
- Galtier N, Gouy M, Gautier C: SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci. 1996, 12: 543-548.PubMedGoogle Scholar
- Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003, 52: 696-704.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.