Department of Zoology, Natural History Museum, London, England
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Abstract |
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Introduction |
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The issue of hydrogenosome evolution can be broken into two parts: one relating to the origin(s) of the organelle (here meaning the cell compartment) and one relating to the origin(s) of its distinctive biochemistry. As discussed previously (Müller 1993
; Embley, Horner, and Hirt 1997
), it is not necessary that all taxa have evolved hydrogenosomes in the same way, nor that organelle and biochemistry share a common origin. However, published data for hydrogenosomes in trichomonads, ciliates, and chytrid fungi are consistent with them sharing common ancestry with mitochondria (Benchimol, Durand, and Almeida 1997
; Bradley et al. 1997
; Embley, Horner, and Hirt 1997
; van der Giezen et al. 1997a, 1997b, 1998; Akhmanova et al. 1998
; Plümper, Bradley, and Johnson 1998
). This immediately poses the question of the origins of the hydrogenosomal enzymes, particularly PFO and hydrogenase.
A number of alternative hypotheses for the origins of hydrogenosomal biochemistry have been proposed. For example, in the hydrogen hypothesis for the origin of eukaryotic cells, the common eubacterial ancestor of mitochondria and hydrogenosomes brought with it all of the components of "hydrogenosomal" metabolism, including PFO and hydrogenase (Martin and Müller 1998
). The hydrogen hypothesis can thus be interpreted to infer a single common origin from an
-proteobacterium for both of these enzymes in different eukaryotes. In contrast, the syntrophic hypothesis posits an ancestral
-proteobacterium as the hydrogen-evolving partner involved in eukaryogenesis (Moreira and Lopez-Garcia 1998
). If the genes for these proteins were transferred to the host genome and retained after the initial selective pressure for hydrogen production was removed, then we could in principle test these inferences through phylogenetic analysis of PFO and hydrogenases from contemporary eukaryotes (Embley and Martin 1998
). Thus, in the simplest formulation of this scenario, each gene should trace its ancestry to the same single source related to either the
- or the
-proteobacteria. Interestingly, recent analyses suggest that PFO from phylogenetically diverse anaerobic protists is monophyletic and was therefore likely present in the common ancestor of eukaryotes, having been retained in some lineages since then (Horner, Hirt, and Embley 1999
). However, these same analyses were unable to infer a specific eubacterial sister group for eukaryotic PFO, and thus they provide no support for the predicted relationships of either the hydrogen or the syntrophic hypothesis for this particular enzyme.
Shortly after hydrogenosomes were discovered by Lindmark and Müller (1973)
, it was suggested that, because of biochemical similarities in hydrogen production, Trichomonas hydrogenosomes might have derived from strictly anaerobic endosymbiotic clostridia-like prokaryotes (Whatley, John, and Whatley 1979
; Müller 1980
). However, recent data are more consistent with the hydrogenosome organelle in Trichomonas sharing a common origin with mitochondria (Bui, Bradley, and Johnson 1996
; Germot, Philippe, and Guyader 1996
; Horner et al. 1996
; Roger, Clark, and Doolittle 1996
; Bradley et al. 1997
; Plümper, Bradley, and Johnson 1998
). Nonetheless, as mentioned above, a common origin with mitochondria need not preclude horizontal gene transfers from clostridia or other prokaryote(s) as a source of some hydrogenosome enzymes. Analyses of PFO do not provide support for a clostridial ancestry for eukaryote enzymes (Rosenthal et al. 1997
; Horner, Hirt, and Embley 1999
). However, two genes encoding iron-only ([Fe]) hydrogenases of about 50 kDa were recently reported for T. vaginalis (Bui and Johnson 1996
), and these are similar to [Fe] hydrogenases found typically in obligate anaerobic prokaryotes including clostridia and sulphate-reducing bacteria. Interestingly, there is also a report in the literature describing a partially purified 64-kDa [Fe] hydrogenase from T. vaginalis (Payne, Chapman, and Cammack 1993
).
The other eukaryote hydrogenase for which there is currently a complete gene sequence comes from Nyctotherus ovalis, an anaerobic ciliate which contains hydrogenosomes (Akhmanova et al. 1998
). The gene encodes a putative fusion protein comprising a complete coding sequence for a 64-kDa [Fe] hydrogenase joined at its carboxyl terminus to two domains which share sequence similarity with the 24-kDa and 51-kDa subunits of mitochondrial complex I, respectively (Akhmanova et al. 1998
). The addition of this carboxyl terminus may allow the fusion protein to use NAD(P) as an electron carrier (Akhmanova et al. 1998
) rather than ferredoxins, as reported for the Trichomonas [Fe] hydrogenase(s) (Müller 1993
). Akhmanova et al. (1998)
listed a number of possible evolutionary origins for the Nyctotherus hydrogenase gene or its component parts, including lateral transfer from unknown prokaryotes and/or inheritance from the common ancestor of mitochondria and hydrogenosomes (Martin and Müller 1998
). More recently, it has been suggested that the Nyctotherus hydrogenase was assembled from mitochondrial genes for complex I fused to a [Fe] hydrogenase gene (Andersson and Kurland 1999
; Hackstein et al. 1999
). However, neither the [Fe] hydrogenase part of the fusion protein nor its carboxyl terminus was subjected to phylogenetic analysis.
Hydrogenases are a prerequisite for hydrogenosome function in diverse anaerobic eukaryotes, and they also play a central role in recent hypotheses concerning the origins of the eukaryotic cell. Here, we present a complete coding sequence for a [Fe] hydrogenase for the diplomonad Spironucleus barkhanus, and a nearly complete sequence for a [Fe] hydrogenase from Entamoeba histolytica. In addition, we isolated almost the entire coding sequence for another [Fe] hydrogenase from T. vaginalis. Phylogenetic analysis, and hypothesis testing using likelihood ratio tests and parametric bootstrapping, suggest that the [Fe] hydrogenases in anaerobic eukaryotes are not monophyletic to the exclusion of bacterial sequences. Iron-only hydrogenases from Entamoeba, Spironucleus, and Trichomonas are plausibly monophyletic, consistent with the hypothesis that a gene for [Fe] hydrogenase was present on the genome of the common ancestor of these diverse eukaryotes. The [Fe] hydrogenase from the hydrogenosomal ciliate Nyctotherus may have an origin different from that of the other eukaryotic hydrogenases. We also report the occurrence of sequences on the genomes of aerobic eukaryotes which encode small proteins of unknown function, but which contain sequence features otherwise found only in [Fe] hydrogenases (see also Barton and Worman 1999
). The implications of these findings for the evolution of eukaryotes and of hydrogenosomes, along with their relevance to the hydrogen and syntrophic hypotheses for eukaryogenesis, are discussed.
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Materials and Methods |
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Cloning of Genes Encoding Putative [Fe] Hydrogenases from E. histolytica, S. barkhanus, and T. vaginalis
As part of an Expressed Sequence Tag (EST) project on the diplomonad S. barkhanus, we characterized a cDNA with a high level of identity to characterized [Fe] hydrogenases. This clone was used as a probe to isolate a genomic DNA clone from a lambda DASHII library (Horner, Hirt, and Embley 1999
). The entire coding region for this open reading frame was sequenced directly using primer walking.
An E. histolytica EST partial sequence (AB002772) which showed similarity to the S. barkhanus hydrogenase was recovered from GenBank. We designed PCR primers EhisthydF (5'-ACATACAGTCACCGGACATGACCATAA-3') and EhisthydR (5'-GGTGGAATTTTTGGTGGTTGTTTTACTA-3') to amplify the entire 3' and most of the 5' region of the expressed sequence, respectively, in a rapid amplification of cDNA ends (RACE) experiment (Marathon System, Clontech).
Following up the report of a partially purified 64-kDa [Fe] hydrogenase from T. vaginalis (Payne, Chapman, and Cammack 1993
), we designed primers to isolate the putative gene sequence. Published gene sequences for 64-kDa [Fe] hydrogenases from eubacteria were used to design PCR primers Tvhyd 45F (5'-TGYAGRGTYTGYCTYGTYGA-3') and Tvhyd 340R (5'-CCARGCTGGRCANRWTGTRAACATTGG-3'), which incorporate known T. vaginalis codon usage bias (McInerney 1997
). A fragment of approximately 900 bp was amplified from T. vaginalis DNA, and a RACE strategy was used to amplify the entire 3' end and most of the 5' part of the gene.
Identification of Database Sequences that Share Significant Sequence Similarity with [Fe] Hydrogenases
Fungal sequences related to [Fe] hydrogenases were recovered through BLASTP similarity searches of GenBank. Related ESTs from Danio rerio, Drosophila melanogaster, and Homo sapiens were identified through tBLASTn searches of the database of ESTs. Human genomic DNA encoding a related gene was identified though a BLAST search of the TIGR tentative human consensus database (http://www.ncbi.nlm.nih.gov/BLAST/thcblast.html). Caenorhabditis elegans genomic sequences were detected by tBLASTn searches at the Sanger Centre (www.sanger.ac.uk/Projects/C_elegans/blast_servers.html). Arabidopsis thaliana sequences were detected through tBLASTn searches at the A. thaliana database in Stanford, Calif. (genome-www2.stanford.edu/cgi-bin/AtDB/nph-blast2atdb). We also searched finished and unfinished prokaryote and eukaryote genome projects to identify [Fe] hydrogenase coding sequences, using prokaryote and eukaryote hydrogenase sequences as probes and the BLASTn, tBLASTn, and iterative (PSI) BLAST programs of the BLAST2 package (Altschul et al. 1997
).
Alignment of Inferred Protein Sequences and DNA Sequences
Inferred [Fe] hydrogenase amino acid sequences were aligned using CLUSTAL W (Thompson, Higgins, and Gibson 1994
) and adjusted manually using GDE, version 2.2 (Maidak et al. 1996
). DNA-coding regions were back-aligned to the protein alignment using PUTGAPS (J. O. McInerney, Natural History Museum, London). We excluded regions which could not be unambiguously aligned, leaving 270 aligned amino acid positions (18 sequences) for phylogenetic analyses. A second alignment accommodating the homologous sequences detected in human and yeast (102 aligned amino acids, 20 taxa) was also prepared but was considered too short for phylogenetic analysis. Alignments are available from D.S.H. (e-mail dsh@nhm.ac.uk).
Phylogenetic Analyses of [Fe] Hydrogenase Protein Sequences
To investigate potential saturation of [Fe] hydrogenase protein sequences, the numbers of inferred substitutions were calculated from a maximum-parsimony tree for each pairwise comparison and plotted against observed distances using the MUST package (Philippe 1993
).
Maximum-likelihood (ML) analyses of protein alignments were performed with PROTML in MOLPHY, version 2.3 (Adachi and Hasegawa 1996
), using the heuristic quick-add OTU search and local rearrangement (R-search) methods with the JTT-F amino acid replacement model. However, the ML models in PROTML make the assumption that all sites are equally free to vary, and when this assumption is violated, the wrong tree may be selected (Lockhart et al. 1996
; Hirt et al. 1999
). The [Fe] hydrogenase sequences contain sites which are of unvaried amino acid composition across the alignment and are thus potentially unable to vary (i.e., invariable). We therefore estimated the proportion of unvaried sites, inferred to be invariable (95%), using a two-rate model in PUZZLE (Strimmer and von Haeseler 1996
) and removed them prior to phylogenetic analyses.
Phylogenetic Analyses of [Fe] Hydrogenase DNA Sequences
Potential mutational saturation of [Fe] hydrogenase DNA sequences and nucleotide base composition heterogeneities between sequences were investigated using plots of transitions versus transversions and "GC trees" as described previously (Lockhart et al. 1994
; Lento et al. 1995
; Charleston 1998
; Hirt et al. 1999
). Codon position 3 was potentially saturated for all comparisons and manifested large base composition variation between sequences (from 10% G+C for Clostridium perfringens to 94% G+C for Desulfovibrio fructosovorans cytochrome hydrogenase), so we excluded it from phylogenetic analyses. Nucleotide compositional heterogeneity at variable positions was observed for codon position 1 (from 44% G+C for C. acetobutylicum hydrogenase B to 68% G+C for D. vulgaris cytochrome hydrogenase) and position 2 (from 37% G+C for Thermotoga maritima B to 50% G+C for D. vulgaris cytochrome hydrogenase). In nucleotide composition "GC trees" (Lockhart et al. 1994
) for codon positions 1 + 2, the eukaryote sequences were not monophyletic.
Because we observed base composition heterogeneity at codon positions 1 and 2, we used LogDet/Paralinear distances, a phylogenetic method in PAUP*, version 4.0b4a (Swofford 1998
), which performs well in simulations even when base composition varies across the tree (Lake 1994
; Lockhart et al. 1994
). However, the LogDet/Paralinear distances method does not incorporate a correction for site-by-site rate variation, and it assumes that all sites are free to vary. We therefore used PAUP*, version 4.0b4a, to estimate the fraction of sites free to vary across our alignment using a variable/invariable-sites ML model and restricted LogDet/Paralinear distances analyses to only these estimated variable positions (427 sites for codon positions 1 + 2). We also used an ML method to investigate relationships. For these analyses, we used a general time reversible (GTR) model (Yang 1994
) with site rate heterogeneity modeled using a discrete four-category gamma distribution plus invariable sites (henceforth, GTR+
+I) (Yang 1996
). Values for all model parameters were estimated using ML over the LogDet/Paralinear distances tree and then re-estimated over the resulting ML tree. The analysis was then repeated until no further improvement in likelihood was observed. Bootstrapping used PAUP*, version 4.0b4a, with either 100 (ML) or 1,000 (LogDet/Paralinear distances) replicates.
Investigation of Relationships Among Eukaryotic [Fe] Hydrogenases Using Parametric Bootstrapping and a Likelihood Ratio Test of Monophyly
We used a likelihood ratio test proposed by Huelsenbeck, Hillis, and Nielsen (1996)
to examine relative support for monophyly of eukaryotic [Fe] hydrogenase DNA sequences. This test asks whether the difference in likelihood between the best trees estimated with and without the constraint of monophyly is significant. In the present case, the null hypothesis (H0) is monophyly of eukaryotic enzymes and the alternative, less-constrained, hypothesis (H1) is the best ML tree. The test statistic (
) is
= ln L1 - ln L0, where L1 is the likelihood of the best tree and ln L0 is the likelihood of the best monophyly tree. The null distribution of
must be generated by simulation (parametric bootstrapping) under an appropriate model. Here, it is important to note that parametric bootstrapping may not perform well for approximating a distribution if the model of DNA substitution does not match the processes which generated the original sequences (Huelsenbeck, Hillis, and Nielsen 1996
). Accordingly, we used a likelihood ratio test suggested by Goldman (1993)
to first evaluate if the GTR+
+I model and the tree could have plausibly given rise to the sequences we observe (see Results). Custom software (available on request from P.G.F.) was used for all simulations and likelihood calculations.
For the monophyly test, the distribution of the test statistic was estimated by parametric bootstrapping, by repeatedly simulating sequences based on the best monophyly tree and model. With each simulated data set, two searches were made using PAUP*, version 4.0b4a, one with the eukaryote monophyly constraint and one without that constraint. Each search began with a parsimony search to obtain a tree upon which to optimize parameters for the GTR+
+I model. Using these parameters, a search using ML was made with the parsimony tree as a starting tree for tree bisection-reconnection (TBR) swapping. Parameters were reoptimized on the best tree from that search, and using these new parameters, a second round of swapping was done using subtree pruning-regrafting (SPR, with a reconnection limit of two nodes). After a final parameter optimization, the likelihood of the best tree from each search was used to calculate the simulation statistic.
Phylogenetic Analysis of the Carboxyl Terminus of the Nyctotherus Fusion Protein DNA and Inferred Protein Sequences
An amino acid alignment of mitochondrial complex I 51-kDa components and their prokaryote homologs (bacterial NADH dehydrogenase NuoF subunits and soluble hydrogenase components) was prepared as described above. Regions which could not be unambiguously aligned were excluded from phylogenetic analysis, as were inferred invariable sites. ML analysis was performed as described for a data set of 25 taxa and 145 variable positions.
For DNA analyses, we used the same strategy involving LogDet/Paralinear distances analyses and ML analyses as described above for the [Fe] hydrogenase sequences. For these analyses, we excluded codon position 3, leaving 370 codon 1 + 2 positions, of which 98 were constant across the alignment. ML analyses indicated that approximately 3% of the latter were potentially variable, so this proportion of observed constant sites was included in our analyses of variable positions (a total of 275 sequence positions).
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Results |
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We recovered a weak match from an EST survey of E. histolytica (Tanaka, Tanaka, and Mitsui 1997
) to the new Spironucleus sequence. This clone (AB002772) had been designated as an unusual ferredoxin and contains conserved cysteines corresponding to potential [4Fe-4S] clusters. Alignment of the conceptually translated product (466 amino acids) from the almost-full-length sequence revealed the highest level of identity (35%) to the S. barkhanus [Fe] hydrogenase (fig. 1
).
Sequences Related to [Fe] Hydrogenases on the Genomes of "Higher" Eukaryotes
Searches of GenBank using the C. pasteurianum hydrogenase 1 amino acid sequence as a probe revealed significant matches (S = 99115, E = 5e-205e-24, 23%28% identity over a 300-aa overlap) on hypothetical open reading frames from Saccharomyces cerevisiae (YNYO_YEAST accession number P23503), Shizosaccharomyces pombe (SPCC1450.10c accession number CAB40177), and Kluveromyces lactis (LET1 accession number X70373) and H. sapiens (Narf accession number AF128406). The encoded proteins are broadly colinear with the TvhydA gene product, as well as with the [Fe] hydrogenase sequences detected in S. barkhanus and E. histolytica (fig. 1
). Like these proteins, the fungal and human gene products lack 120140 amino acids with respect to the N-terminal region of many bacterial [Fe] hydrogenases, the N. ovalis [Fe] hydrogenase, and the new T. vaginalis [Fe] hydrogenase reported here. In reciprocated BLAST searches, these proteins match only themselves and [Fe] hydrogenases. We also recovered highly significant matches from D. melanogaster (EST accession number AA439709) and D. rerio cDNA (accession number AA495126). Closely related sequences were also detected on the genomes of C. elegans (clone Y54H5A) and A. thaliana (CAB10420). While none of these sequences represent complete open reading frames, the finding that most are expressed is consistent with the notion that they represent parts of functional genes.
To gain some insight into the possible functionality of our new sequences and those from the databases, we investigated the distribution of residues which are considered important for [Fe] hydrogenases based on the crystal structure of the C. pasteurianum [Fe] hydrogenase 1 and the D. desulfuricans periplasmic [Fe] hydrogenase (Peters et al. 1998
; Nicolet et al. 1999
). The new T. vaginalis [Fe] hydrogenase contains all of the [Fe-S] center coordinating residues, including those implicated in the coordination of FS2, FS4c, FS4b, FS4a, and the hydrogenase catalytic cluster (HC) (fig. 1
), which have been demonstrated for the C. pasteurianum hydrogenase 1. The S. barkhanus and E. histolytica homologs, along with TvhydA and TvhydB and the dimeric hydrogenase of Desulfovibrio sp., share conserved cysteines implicated in FS4b, FS4a, and HC coordination, while the fungal, human, and Drosophila sequences specify the expected arrangement of cysteines for coordination of FS4a and the HC cluster, which is otherwise unique to [Fe] hydrogenases (figs. 1 and 2
).
Phylogeny of [Fe] Hydrogenases
Since some of the analyzed sequences are very divergent, we investigated the level of potential saturation and other properties of the data prior to phylogenetic analysis. For the final alignment, pairwise identities at the amino acid level ranged from 35% (C. pasteurianum vs. E. histolytica) to 82% (C. acetobutylicum A vs. C. perfringens). Transition-versus-transversion plots for hydrogenase family DNA sequences revealed points resolved as distinct clusters (fig. 3A
). Comparisons involving eukaryote sequences formed a cluster outside the range of distribution of most intrabacterial comparisons. Furthermore, the coefficient of regression for a linear distribution of all points was low (r2 = 0.580). These observations suggest that there may be differences in the mode/rate of evolution of the different sequences and/or mutational saturation. To further investigate the latter possibility (Philippe 1993
), we made "saturation" plots (Philippe and Adoutte 1998
) of observed versus inferred substitutions (fig. 3B
) for all pairwise comparisons of amino acid sequences (inferred substitutions were calculated from a maximum-parsimony tree using the TREEPLOT program in the MUST package (Philippe 1993
). With the exception of some comparisons between closely related sequences, the ratio of inferred to observed substitutions was between 1.5 and 2.5, suggesting that mutational saturation may be a problem for some comparisons, particularly distant ones, within this data set. As these figures are derived from a maximum-parsimony tree, they likely represent a conservative estimate of the degree of mutational saturation for these sequences.
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The favored position for the Nyctotherus sequence in all of our analyses was as the sister group to the D. vulgaris [Fe] hydrogenase gamma. In LogDet/Paralinear distances bootstrap analyses, 58% of trees contained this relationship, while it was found in 48% of DNA ML bootstrap trees. This compares with less than 2% of bootstrap trees, for either analysis, where the Nyctotherus sequence clustered with any of the other eukaryote sequences.
The slightly higher support obtained using LogDet/Paralinear distances for the relationship between the Nyctotherus [Fe] hydrogenase and the D. vulgaris [Fe] hydrogenase gamma may be due in part to the ability of this method to better deal with changing nucleotide compositions across the tree (Lake 1994
; Lockhart et al. 1994
). This can be illustrated by examining the other bootstrap partitions (data not shown) involving the D. vulgaris [Fe] hydrogenase gamma sequence. A relationship between this sequence and the D. fructosovorans NAD reducing hydrogenase was the next best supported partition for the D. vulgaris hydrogenase gamma sequence in both bootstrap analyses (ML, 31% of trees; LogDet/Paralinear distances, 20% of trees). However, comparison of base compositions revealed that these two sequences also have the highest G+C% compositions (61% and 54% GC at variable positions, respectively) of the sequences analyzed. Furthermore, in "GC trees" based solely on the nucleotide composition of variable sites, these two sequences clustered together. This might suggest that part of the reason why these two sequences come together in our phylogenetic analyses is because they share a common compositional bias to which LogDet/Paralinear distances are less susceptible and hence give lower support. In contrast, the nucleotide composition of the Nyctotherus sequence is much lower, at 44% G+C, and it does not cluster with the D. vulgaris hydrogenase gamma sequence in "GC trees." Thus, positive support for this relationship in our phylogenetic analyses cannot be attributed to nucleotide composition bias, but the reduction in support in ML analyses might indeed be influenced by this problem.
Evaluation of the Relationships Between Eukaryotic [Fe] Hydrogenases Using Parametric Bootstrapping and a Likelihood Ratio Test of Monophyly
We used a likelihood ratio test (Goldman 1993
) to evaluate whether the GTR+
+I model plus the best tree we found could have plausibly given rise to the observed data. The null hypothesis was that the data arose from the tree under the model. The alternative unconstrained hypothesis was that the data are not related by a tree or a model, i.e., assuming a multinomial distribution for the frequencies of site patterns. The test statistic was L1 - L0, where L1 is the unconstrained likelihood and L0 is the likelihood under the tree + model (Goldman 1993
). When calculating the L0 of simulated data, the branch lengths and model parameters were optimized. In order to calculate the unconstrained likelihood L1, there can be no gaps or ambiguities in the alignment, and for this reason the two positions in the alignment which had gap characters were removed, leaving an alignment of 538 characters. Branch lengths and GTR+
+I model parameters were reoptimized with the new alignment of 538 characters. Data sets were first simulated on the ML tree + model, and the test statistic was calculated. The original data had a statistic of 4,083.39, compared with a critical 95% value estimated through simulation of 4,232.99. Therefore, there was no evidence to reject the null hypothesis, as the original data set is a plausible realization of the tree + model.
The likelihood ratio test of monophyly (Huelsenbeck, Hillis, and Nielsen 1996
) was used to test whether we could reject the null hypothesis of monophyly of eukaryotic [Fe] hydrogenases. The test statistic (
) was
= ln L1 - ln L0, and the null distribution of
was generated by parametric bootstrapping using the GTR+
+I model and the best eukaryote monophyly tree. If the likelihood ratio statistic
calculated from the real data is greater than the 95% confidence interval determined through simulation, the null hypothesis that the group in question is monophyletic can be rejected (Huelsenbeck, Hillis, and Nielsen 1996
). Figure 5 shows the distribution of
for 1,000 simulations. The observed
for the original data was 9.64 and thus was well outside of the critical 95% value determined through parametric bootstrapping. Thus, monophyly of eukaryote [Fe] hydrogenase DNA sequences can be rejected using this test.
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The 24-kDa component of complex I/NuoE family of genes is poorly conserved and could not be reliably aligned for phylogenetic analysis. However, the 51-kDa component of mitochondrial complex I, bacterial NuoF gene products, and bacterial [Ni-Fe] hydrogenase components are highly conserved and easy to align. The 51-kDa component of mitochondrial complex I formed a strongly supported monophyletic group with the NuoF sequences from -proteobacteria (fig. 6
). This topology is consistent with other data which suggest that the mitochondrion endosymbiont was a member of the
-proteobacteria (Yang et al. 1985
; Andersson et al. 1998
). However, the 51-kDa-like domain of the N. ovalis fusion protein was never part of this clade in our analyses. Thus, there is no support from our analyses for the hypothesis that the carboxyl terminus of the N. ovalis fusion protein is derived from the mitochondrial respiratory chain. In LogDet/Paralinear distances (fig. 6
) and protein ML trees, the 51-kDa-like domain of the N. ovalis fusion protein clustered with the carboxyl terminus components of NAD(H)-dependent [Ni-Fe] hydrogenases isolated from the ß-proteobacterium Alcaligenes eutrophus and the Gram-positive actinomycete Rhodococcus opacus. In DNA ML analyses, the 51-kDa-like domain of the N. ovalis fusion protein clustered with the
-proteobacteria Escherichia coli and Salmonella typhimurium in the ML tree (ln L = 5,413.6). However, this tree was no better, considering the estimated error, than the best ML tree we found (ln L = 5,414.6 ± 6.3), which clustered it with A. eutrophus and R. opacus.
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Discussion |
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The Entamoeba sequence is from cDNA, and it is incomplete at its 5' terminus, so we do not know whether it carries a targeting sequence or not. E. histolytica has recently been shown to contain an organelle, called a mitosome (Tovar, Fischer, and Clark 1999
) or crypton (Mai et al. 1999
), which likely shares common ancestry with the mitochondrion, since it contains a mitochondrial chaperonin 60, and it uses a mitochondrial-like cleavable N-terminal targeting motif in protein import (Clark and Roger 1995
; Roger et al. 1998
; Mai et al. 1999
; Tovar, Fischer, and Clark 1999
). Trichomonad hydrogenosomes use a similar import mechanism (Plümper, Bradley, and Johnson 1998
), and there are strong similarities between the sequences of some trichomonad targeting motifs and those for mitosome/crypton proteins (Mai et al. 1999
; Tovar, Fischer, and Clark 1999
). Because of these similarities, it has been suggested that Entamoeba might once have contained a hydrogenosome-like organelle (Tovar, Fischer, and Clark 1999
). Further work is required to investigate whether the novel [Fe] hydrogenase we have detected is targeted to the mitosome, but it may be relevant that in E. histolytica, PFO lacks an obvious targeting sequence and is apparently active in the cytoplasm (Takeuchi, Weinbach, and Diamond 1975
; Reeves et al. 1977
). In trichomonads, PFO is a key enzyme of the hydrogenosome organelle releasing reducing equivalents which are transferred to protons with the formation of hydrogen by hydrogenase (Müller 1993
).
We also found evidence of short sequences (hereafter termed "Narf-like," after the human homolog) related to [Fe] hydrogenases on all of the well-surveyed eukaryotic genomes (see also Barton and Worman 1999
). The organisms comprise the fungi K. lactis, S. cerevisiae, and S. pombe, the metazoa C. elegans, D. rerio, D. melanogaster, and H. sapiens, and the plant A. thaliana. These sequences do not contain all of the residues which are thought to be important for the functioning of the C. pasteurianum [Fe] hydrogenase 1 (Peters et al. 1998
), and they also lack one of the iron sulfur clusters (FS4B) which is found on the short-form [Fe] hydrogenases found in Entamoeba, Spironucleus, and Trichomonas. They do contain the expected arrangement of cysteines for coordination of FS4a and also for the HC cluster, which is otherwise unique to [Fe] hydrogenases (Peters et al. 1998
). The paucity of complete Narf-like genes and their short length confounded our attempts to make phylogenetic inferences from these proteins. However, based on their similarities in structure, we speculate that the Narf-like genes are descended from an ancestral [Fe] hydrogenase which had lost some structural features, perhaps reducing its sensitivity to inactivation by oxygen, while retaining others.
It is clearly important to try and establish what the sequences related to [Fe] hydrogenases, which are found otherwise only in anaerobic prokaryotes and eukaryotes, are doing in such a diverse range of aerobic eukaryotes. The [Fe] hydrogenases from prokaryotes and eukaryotes which have been investigated are very oxygen-sensitive. For example, the [Fe] hydrogenase in the green alga Scenedesmus is only active after a period of anaerobic adaptation, and once exposed to aerobic conditions, hydrogen production rapidly stops (Urbig, Schulz, and Senger 1993
; Appel and Schulz 1998
). It is thus unlikely, given also the lack of reports of hydrogen production, that the new sequences encode proteins which are functioning as classic hydrogenases. The H. sapiens gene has already been the subject of investigation and apparently encodes a protein which interacts with prenylated prelamin A (Barton and Worman 1999
). The encoded protein was shown to localize to the nucleus of human cells and to interact with the C-terminal region of prenylated prelamin A, which is processed to become mature lamin A. The protein has thus been named nuclear prelamin A recognition factor (Narf), but its precise function is still unknown. In S. cerevisiae, deletion of the yeast Narf-like sequence has been shown to be lethal in a haploid background (Munich Information Centre for Protein Sequenceshttp://www.mips.biochem.mpg.de/). Interestingly, S. cerevisiae does not encode a homolog of lamin A, suggesting that the yeast Narf-like protein may not have the same function in yeast as in humans.
Iron sulfur proteins occur in all organisms and assume a diversity of functions. Iron sulfur clusters in these proteins may facilitate electron transfer, contribute to catalytic function, or help to maintain structural integrity (Roualt and Klausner 1996
). Other [Fe-S] centercontaining proteins have been implicated in biosensing of oxidative stress and redox-dependent gene regulation in prokaryotes and, potentially, eukaryotes (Zheng and Storz 2000). While some of these proteins do not contain [Fe-S] centers identical to those predicted to be present in the Narf-like proteins investigated here and elsewhere (Barton and Worman 1999
), the function of these sensory/regulatory molecules is believed to be dependent on the exquisite redox sensitivity of diverse types of [Fe-S] clusters (Zheng and Storz 2000). It is known that redox/nitric oxide sensing and processing/cleavage of nuclear lamins are key events in programmed cell death (apoptosis) of higher animal cells (Kluck et al. 1997
). It therefore seems reasonable to speculate that the features of eukaryotic Narf-like proteins might allow them to participate as sensory/regulatory proteins in such processes.
Additional studies are also required to investigate the functions of the proteins encoded by the E. histolytica and S. barkhanus [Fe] hydrogenase gene sequences. These sequences closely resemble in structure the two short-form [Fe] hydrogenases which are targeted to hydrogenosomes in T. vaginalis (Bui and Johnson 1996
). However, it is still not known if the short-form Trichomonas hydrogenases are actually responsible for hydrogen production from Trichomonas hydrogenosomes. For example, Payne, Chapman, and Cammack (1993)
found hydrogenase activity purified from T. vaginalis to be associated with a polypeptide of approximately 64 kDa in size, which is larger than predicted (ca. 50 kDa) for the published sequences. In the present investigation, we isolated an almost complete coding sequence for a [Fe] hydrogenase of approximately the same size as the polypeptide discovered by Payne, Chapman, and Cammack (1993)
. The new T. vaginalis [Fe] hydrogenase contains all of the key residues known to be important for the function of the C. pasteurianum hydrogenase 1 (Peters et al. 1998
). The new T. vaginalis gene apparently contains an N-terminal extension which is similar to characterized Trichomonas hydrogenosome-targeting motifs (Plümper, Bradley, and Johnson 1998
), suggesting that the encoded protein is also targeted to the hydrogenosome. Thus, the currently available data suggest that there may be at least three [Fe] hydrogenases, possibly doing different things, within the hydrogenosomes of T. vaginalis. Moreover, phylogenetic analyses suggest that the observed structural diversity of Trichomonas [Fe] hydrogenases arose within the Trichomonas lineage.
The data presented here demonstrate that sequences related to [Fe] hydrogenases are more widely distributed on the genomes of eukaryotes than previously thought. According to published hypotheses, this distribution might result from vertical inheritance of ancestral genes via speciation (orthology), from one or more horizontal transfers (xenology), or from a mixture of both (Whatley, John, and Whatley 1979
; Müller 1980
; Embley, Horner, and Hirt 1997
; Akhmanova et al. 1998
; Martin and Müller 1998
). An additional complication when trying to interpret patterns of gene history is the potential of confusing orthologs with paralogs arising via gene duplication. It is already clear from available data that multiple [Fe] hydrogenases exist on some prokaryotic and eukaryotic genomes (fig. 4
). Limited and/or skewed sampling of taxa or genomes may further compound difficulties of interpretation and limit inferences.
ML analyses support monophyly of Entamoeba, Spironucleus, and Trichomonas [Fe] hydrogenases, with the Entamoeba and Spironucleus sequences forming a strongly supported sister group in all analyses. Current ideas about species relationships between microbial eukaryotes are in a state of flux, with the relationships between parasitic anaerobes such as the taxa investigated here being considered particularly problematic (Embley and Hirt 1998
; Hirt et al. 1999
; Roger 1999
). However, there are no published data to suggest that Entamoeba, Spironucleus, and Trichomonas form a monophyletic group to the exclusion of other eukaryotes. Thus, the hypothesis that at least one [Fe] hydrogenase gene was present on the genome of a common ancestor shared by these species and other eukaryotes is not excluded by the data.
Monophyly of all of the eukaryote sequences was never observed in unconstrained analyses and, indeed, was rarely recovered among nonparametric bootstrap replicates. Testing the robustness of phylogenetic hypotheses can be problematic, because the statistical basis of some tests is poorly understood (Huelsenbeck and Rannala 1997
). For example, the most common likelihood-based statistical test for comparing two topologies is probably the Kishino-Hasegawa test (Kishino and Hasegawa 1989
), but the validity of this test has recently been questioned for its most common usage, that of testing the ML tree against another topology (Shimodaira and Hasegawa 1999
; Goldman, Anderson, and Rodrigo 2000
). Here, we used parametric bootstrapping and a likelihood ratio test of monophyly (Huelsenbeck, Hillis, and Nielsen 1996
) to investigate if the [Fe] hydrogenase data could reject trees where eukaryote sequences were constrained to be monophyletic. We made no other assumptions regarding the internal relationships among eukaryote sequences relying on our search strategy to find the optimal arrangement for each data set. Likelihood ratio tests coupled with parametric bootstrapping are among the most powerful approaches for hypothesis testing (Huelsenbeck and Rannala 1997
), especially when it can be shown, as here, that the model and tree which are used for simulations also fit the original data. The likelihood ratio test of monophyly strongly rejected the hypothesis that eukaryote [Fe] hydrogenase sequences formed a monophyletic group.
In most analyses, the [Fe] hydrogenase module of the N. ovalis fusion protein formed a sister group relationship with the D. vulgaris [Fe] hydrogenase gamma sequence (Stokkermans et al. 1989
), albeit with rather low support from nonparametric bootstrapping. One possible explanation of this topology is that horizontal gene transfer has occurred between the ancestors of these two organisms. An alternative hypothesis is one of hidden paralogy whereby the Nyctotherus and D. vulgaris sequences are paralogous to the other eukaryote and prokaryote sequences in the tree. To discriminate between these possibilities, we need more data than we have here. To make a strong case for horizontal transfer, sequences should ideally be available for a good taxonomic sample of organisms, and the tree relating the sequences should correspond to a conventional phylogenyexcept that one or more sequences appears in a radically unexpected position (Smith, Feng, and Doolittle 1992
) and preferably nested with strong support in an otherwise accepted monophyletic group.
It has been suggested that the N. ovalis [Fe] fusion protein contains components derived from the mitochondrial respiratory (complex I modules) chain (Andersson and Kurland 1999
; Hackstein et al. 1999
). Our analyses provide no support for this hypothesis. The 51-kDa component of mitochondrial complex I formed a strongly supported monophyletic group with the NuoF sequences from
-proteobacteria, in agreement with other data for mitochondrial origins (Gray et al. 1998
; Andersson and Kurland 1999
). The 51-kDa subunitlike component of the Nyctotherus fusion protein was never part of this clade, but its relationships to other proteins were not strongly resolved. In most analyses, the Nyctotherus sequence clustered with the 51-kDa-like component of the NAD(H)-dependent [Ni-Fe] hydrogenases isolated from the ß-proteobacterium A. eutrophus and the Gram-positive actinomycete R. opacus. This result is interesting because, like the C-terminal domain of the N. ovalis fusion protein, these subunits are part of a fusion comprising 24-kDa/NuoElike components and 51-kDa/NuoFlike components. The overall homologies between components of respiratory chain complex I and hydrogenase components has previously been noted (Pilkington et al. 1991
; Albracht, Mariette, and de Jong 1997
).
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Conclusions |
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Two recently published hypotheses have postulated that eukaryotic cells originated through a symbiosis between two prokaryotes, and each posits that interspecies hydrogen transfer was the key to binding one partner to the other. In the hydrogen hypothesis, the prokaryotes comprise an anaerobic hydrogen-consuming archaebacterium (the host) and a hydrogen-producing facultative anaerobic -proteobacterium (the symbiont), with the
-proteobacterium subsequently becoming the mitochondrion (Martin and Müller 1998
). The syntrophic hypothesis posits that eukaryotes arose from a primary symbiosis between an anaerobic hydrogen-consuming methanogenic archaebacterium which became the nucleus and an anaerobic hydrogen-producing
-proteobacterium which became the host, with mitochondria originating from a later symbiosis involving a facultatively anaerobic methane-consuming
- proteobacterium (Moreira and Lopez-Garcia 1998
). While they differ in key features, both hypotheses can be interpreted to predict that PFO and hydrogenase were present early in eukaryotic evolution, a prediction for which our present and previous analyses provide some support. However, our analyses failed to identify a eubacterial sister group for either enzyme and thus cannot discriminate between the hydrogen and syntrophic hypotheses regarding the identity of a potential eubacterial donor for eukaryotic enzymes. This situation may improve with more samplingparticularly of
- and
-proteobacterial genes. However, unless the trees for PFO and [Fe] hydrogenase can be rooted, it may remain difficult to identify a potential source for ancestral enzymes.
Trees where the [Fe] hydrogenase from the hydrogenosomal ciliate N. ovalis was constrained to be monophyletic with the other eukaryote sequences were rejected by our analyses. The favored position for the Nyctotherus sequence was as the sister group to a [Fe] hydrogenase on the genome of the eubacterium D. vulgaris. Thus, as previously suggested (Akhmanova et al. 1998
) it seems possible that Nyctotherus has obtained its hydrogenosomal [Fe] hydrogenase from a different source (either from a different paralog or via horizontal transfer) from Trichomonas for its hydrogenosomes.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: HC, hydrogenase catalytic FeS cluster; PFO, pyruvate : ferredoxin oxidoreductase.
2 Keywords: hydrogenase
hydrogenosomes
eukaryote origins
Entamoeba,
Spironucleus,
Trichomonas.
3 Address for correspondence and reprints: T. Martin Embley, Department of Zoology, Natural History Museum, London SW7 5BD, United Kingdom. E-mail: tme{at}nhm.ac.uk
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