Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic;
Rockefeller University, New York, NY, USA
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Abstract |
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Introduction |
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In spite of the importance of FeS proteins for all living cells, little is known of how and where FeS clusters are synthesized in vivo and which proteins are involved in their insertion into the apoproteins. The best-characterized enzyme participating in this process is a pyridoxal-5'-phosphate-dependent cysteine desulfurase which catalyzes the formation of L-alanine and elemental sulfur by using L-cysteine as substrate. Initially, the enzyme was described as NifS in Azotobacter vinelandii (Zheng et al. 1993
), in which it provides sulfur for FeS cluster formation in nitrogenase (Zheng and Dean 1994
). Later, a NifS homolog designated IscS (iron-sulfur cluster) was found in A. vinelandii, as well as in a number of nonnitrogen-fixing bacteria. It has been proposed that IscS plays a general role in the formation of FeS clusters or repair of FeS proteins with a housekeeping function (Zheng et al. 1998
). More recently, a major role of IscS in de novo FeS cluster synthesis has been demonstrated using an iscS deletion strain of Escherichia coli (Schwartz et al. 2000
). Importantly, IscS homologs have been identified in the genomes of diverse eukaryotes (Arabidopsis, Caenorhabditis, Drosophila, Homo, Mus, Saccharomyces), suggesting a general role for IscS in FeS cluster formation. In mice (Nakai et al. 1998
) and yeast (Strain et al. 1998
), IscS is localized in mitochondria. In human cells, the IscS homologs are targeted either to mitochondria or to the cytosol and nucleus (Land and Rouault 1998
). Mutation in an iscS-like gene in yeast (NFS1) caused reduction in the activities of the mitochondrial FeS proteins, aconitase and succinate dehydrogenase (Strain et al. 1998
). According to Kispal et al. (1999)
and Lill and Kispal (2000)
, mitochondria also play a crucial role in the FeS cluster formation of extramitochondrial FeS proteins. In addition, IscS homologs have been found to mediate several other functions that are independent of FeS cluster assembly but require IscS as a sulfur donor. Thus, IscS homologs are involved in biosynthesis of thiamin (Lauhon and Kambampati 2000
), NAD (Sun and Setlow 1993
), 4-thiouridine (Kambampati and Lauhon 1999
), and molybdopterin (Amrani et al. 2000
). Finally, IscS/NifS homologs mediate release of elemental selenium from L-selenocysteine (Mihara et al. 2000
), and they may participate in tRNA splicing (Kolman and Söll 1993
).
IscS has not been reported in amitochondriate eukaryotes, although FeS proteins are of particular importance for these organisms (Müller 1998
). Here we report the identification of genes encoding IscS in the type II organism Trichomonas vaginalis and the type I organism Giardia intestinalis. Phylogenetic analysis indicates the presence of a common mechanism for FeS cluster formation in mitochondria and hydrogenosomes, as well as in organisms that secondarily lost the mitochondrion/hydrogenosome-like organelles.
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Materials and Methods |
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Probe Preparation, Cloning, and Screening of the Genomic Library
To obtain probes for screening a T. vaginalis genomic library, two pairs of degenerate primers, a GC-rich one and an AT-rich one, were designed based on the conserved regions of IscS/NifS sequences in GenBank (National Center for Biomedical Information): EIIFTSGATE (GC-rich sense: 5'-GARATYATYTTCTCVTCHGGHGCHACHGAR-3'; AT-rich sense: 5'-GAAATWATWTTYACWWSWGGWGCWACWGAA) and HKIH/YGPKGV/IG (GC-rich antisense: 5'-CCRAYDCCYTTTGGDCCRTRRATYTTRTG-3'; AT-rich antisense: 5'-CCWAYWCCYTT TGGWCCRTRWATYTTRTG-3'). Corresponding fragments were amplified by PCR, purified with a gel extraction kit (Qiagen) and cloned into pCR 2.1 vector (TA cloning kit, Invitrogen). The inserts were excised from the vector, gel-purified, and labeled by means of a Random Primers DNA Labeling System (GIBCO/BRL) with -[32P]dATP. These probes were used for screening a genomic DNA library in
ZAP II vector (Stratagene). The sequences of positive clones were determined for both strands by primer walking.
Nucleotide sequences of Escherichia coli and Saccharomyces cerevisiae IscS/NifS homolog genes were used to search the Giardia lamblia genome sequence database (http://www.mbl.edu/baypaul/Giardia-HTML/index2.html; McArthur et al. 2000
) with the BLAST program. Clones Ai0824 and Ki1686 contained sequences homologous to the N- and C-terminal ends of the bacterial and eukaryotic homologs. Based on the nucleotide sequence of these clones, we designed a pair of oligonuclueotide primers (sense: 5'-GATGACGAGCGTGCAAGGAAAGCTC-3'; antisense: 5'-GGTGACTACATGCGGATGCTCAGCC-3') located in the 5' and 3' untranslated regions of the putative G. intestinalis nifS homolog gene, respectively. PCR reactions, utilizing these oligonucleotides and G. intestinalis genomic DNA as template, amplified a 2.4-kb fragment that was purified, cloned into the pCR2.1 vector (Invitrogen), and sequenced.
Sequence Alignment
Nucleotide and protein database searches were performed at the National Center for Biomedical Information using the BLAST program (Altschul et al. 1997
). Sequences were extracted from databases using the BlastAli program (http://www.joern-lewin.de/). The IscS sequences of T. vaginalis and G. intestinalis were aligned to sequences from 64 taxa using ClustalX (Thompson et al. 2000
). The alignment was further edited visually with the use of the ED program of MUST (Philippe 1993
). The alignment of all 67 taxa resulted in 231 shared amino acid positions, while an alignment of 21 selected taxa consisted of 362 shared amino acid positions. The T. vaginalis TviscS-1, T. vaginalis TviscS-2, and G. intestinalis GiiscS sequences have been submitted to GenBank under accession numbers AF321005, AF321006, and AF311744, respectively.
Phylogenetic Analysis
Phylogenetic relationships were analyzed by means of the Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods using PHYLIP, version 3.6 (Felsenstein 1989
), and by the Maximum-Likelihood (ML) method using the PROTML program in MOLPHY, version 2.3 (Adachi and Hasegawa 1996
). The ML tree was constructed by local rearrangement of an NJ tree using the Jones-Taylor-Thornton model of amino acid substitutions with the F-option (JTT-F) to account for amino acid frequencies in the data set. User-defined trees were analyzed to compare alternative topologies (Kishino and Hasegawa 1989
). The local bootstrap proportion value was calculated for each internal branch of the ML tree using a local rearrangement option of the PROTML program. Bootstrap support for distance and parsimony analyses were based on 100 resampled data sets using SEQBOOT, PHYLIP, version 3.6.
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Results and Discussion |
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Amino acid sequences deduced from the Trichomonas and Giardia genes were compared with IscS/NifS homologs from 64 species, including bacteria, fungi, plants, invertebrates, and vertebrates (alignment available on request from J.T.). An alignment of selected sequences including that of eubacterial IscS from A. vinelandii and mitochondrial sequence from S. cerevisiae is shown in figure 1
. Both Trichomonas and Giardia sequences contained all conserved regions proposed to mediate the cysteine desulfurase activity in IscS/NifS-like proteins: (1) His111 (numbered according to TviscS-1), which is involved in initial deprotonation of the substrate (Kaiser et al. 2000
); (2) the pyridoxal-5'-phosphate-binding site with the Schiff base forming Lys222 residue and Asp187 and Gln190, which bind the pyridine nitrogen and the phenolate oxygen of PLP, respectively, and residues involved in forming an additional six hydrogen bonds anchoring the phosphate group: Thr82, His221, Ser/Thr219, and Thr250 (Zheng et al. 1993
); and (3) the substrate-binding site including Cys371, which provides a reactive cysteinyl residue (Zheng et al. 1994
), as well as Arg362, Asn162, and Asn 41, which anchor the cysteine with a salt bridge and hydrogen bond (Kaiser et al. 2000
).
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A C-terminal sequence signature differentiates proteobacterial and eukaryotic IscS from homologs in all other organisms. TviscS-2 and GiiscS also contain this signature, which consists of 20 to 21 amino acids with consensus sequence SPL(W/Y)(E/D)(M/L)X(K/Q)XG(I/V)D(L/I)XX(I/V)XWXXX (fig. 1 ). NifS genes of nitrogen-fixing bacteria also possess a similar C-terminal extension that starts with the Ser-Pro motif, but the subsequent sequence is not conserved. The only sequence from a eukaryote that lacks this signature is TviscS-1. The lack of this signature and the "atypical" N-terminal extension might indicate that Tvisc-1 is a pseudogene or that its product has a different function or localization. Eukaryotic IscS is distinguished from all other organisms, including proteobacteria, by the invariable Cys113 in the substrate deprotonation region. This residue is present in both trichomonad iscS products as well as in GiiscS. Prokaryotes possess Ala, Ser, or Gly at this position. Interestingly, Giardia IscS possessed two unique highly hydrophilic inserts, Thr137-Glu145 and Glu300-Ser321, which are not present in any of the 66 other species. It will be of interest to determine the function of such inserts, which might be associated with a specific localization of the gene product in Giardia.
Our sequencing data provide a solid basis for the prediction of the function of the products of iscS genes in amitochondriate eukaryotes. However, further studies are required to confirm their physiological function and cell localization.
Phylogenetic Analysis of IscS/NifS-like Sequences
In all global phylogenetic reconstructions, IscS/NifS-like homologs formed two distinct groups that were previously designated groups I and II (Mihara et al. 1997
). The IscS sequences of Trichomonas and Giardia and those of the mitochondrial homologs in other eukaryotes formed a single clade (group I) with a high bootstrap value (99%) using the local rearrangement option of the PROTML program (fig. 3
). Within this clade, Trichomonas and Giardia formed a subgroup together with Plasmodium falciparum and Arabidopsis thaliana. The second eukaryotic subgroup consisted of metazoan IscS, and the third group comprised homologs in fungal mitochondria. The
-proteobacterium Rickettsia prowazeki, often considered a close relative to the mitochondrial ancestor, clustered together with metazoan mitochondrial IscS (Andersson and Kurland 1999
).
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Additional IscS/NifS-like homologs of eukaryotic organisms (Mus musculus and A. thaliana) also appeared in another two distinct clades. The mouse homolog was located in a heterogeneous eubacterial group (fig. 3
). This gene codes for the cytosolic pyridoxal-5'-phosphate-dependent selenocysteine lyase, which resembles NifS in primary structure as well as in catalytic function (Mihara et al. 2000
). If the partial sequence of a human counterpart was also included in the analysis (data not shown), it was a sister group to the mouse sequence. The function of bacterial gene products of this clade has not been studied except in Synechocystis sp. (S74526 corresponds to sll0704 in CyanoBase http://www.kauza.or.jp/cyano/ investigated by Kato et al. [2000]
) and Bacillus subtilis (AAA21613 corresponds to the nifS-like gene according to Sun and Setlow [1993]
). The product of the Synechocystis gene showed selenocysteine lyase activity, although it also acted on L-cysteine sulfinic acid and other substrates (Kato et al. 2000
). The B. subtilis gene product has been suggested to participate in NAD biosynthesis (Sun and Setlow 1993
). Thus, it is likely that other members of this clade also have biochemical functions that are different from those of NifS and IscS proteins. The topology of A. thaliana genes was of particular interest. While one gene was related to the subtree of genes coding for mitochondrial IscS in protists, a second gene was placed in group II together with Synechocystis S76601. Since cyanobacteria share a common ancestor with plastids, we analyzed the second A. thaliana sequence for its possible subcellular localization with the PSORT program. The analysis gave the highest score for a chloroplast stroma localization (certainty = 0.501) of the gene product. This analysis suggests that the second IscS homolog of A. thaliana may operate in the chloroplast. However, it is difficult to predict its possible function. Group II consists of the most divergent NifS/IscS homologs. Function has been established only for products of two E. coli genes, which encode cysteine sulfinate desulfinase (F65063; Mihara et al. 1997
), and for selenocysteine lyase (H64925; Fujii et al. 2000
). Thus, the A. thaliana IscS homolog, as well as other members of this heterogenous bacterial group, might have functions different from FeS cluster formation, and their distance from genes of group I might reflect different evolutionary pressures. In any case, further biochemical studies on the functions of group II members are required.
The global gene tree showed that amitochondrial and mitochondrial IscSs share a common eubacterial ancestor, suggesting a common biosynthetic mechanism for FeS proteins. The tree also showed that both eukaryotes and bacteria possess several paralogous or orthologous IscS/NifS-like genes. The topology of these gene trees possibly reflects their specialized function or cell localization more than their large-scale phylogenetic relationships. Therefore, in a subsequent analysis we restricted the data set to eukaryotic and proteobacterial IscSs. The trees constructed by the ML, MP, and NJ methods confirmed the close relationship between amitochondrial and mitochondrial IscS with high bootstrap support (fig. 4
). The robustness of the relationship within the eukaryotic group was further assessed through the analysis of alternative tree topologies. We defined six branches on the tree: (1) amitochondriates and Plasmodium, (2) Arabidopsis, (3) Fungi, (4) Metazoa, (5) Rickettsia, and (6) Proteobacteria. Evaluation of the 105 alternative trees confirmed a common ancestry of genes from amitochondriate and mitochondriate organisms. However, the branching order within the eukaryotic clade was not resolved, as several alternative positions for eukaryotic subtrees with comparable significance were found (table 1
). We further suspected that the subtree of protists could be affected by long-branch attraction in spite of high bootstrap support. Thus, the positions of the Trichomonas and Giardia genes were tested using a data set from which we removed the most divergent sequences of A. thaliana and P. falciparum. The analysis clearly showed the instability of the protist group. Although NJ and MP reconstruction placed Trichomonas and Giardia as sister taxa with bootstrap support of 85% and 87%, respectively, ML constraint analysis gave comparable support to several alternative hypotheses. In the best tree topology and in four other tree topologies with ln L <1 SE from the best tree, Trichomonas and Giardia were not placed as sister taxa (table 1
). Nevertheless, in all alternative trees, both amitochondriates were part of the eukaryotic clade.
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A common origin of FeS cluster formation in mitochondriate and amitochondriate eukaryotes could be explained by the recently proposed hydrogen hypothesis of eukaryotic origin (Martin and Müller 1998
). The hypothesis assumes that all eukaryotes, including contemporary amitochondrial organisms, once harbored the mitochondrion/hydrogenosome-like organelle derived from a proteobacterial endosymbiont. The ancestral endosymbiont is viewed as a facultatively anaerobic proteobacterium which possessed both anaerobic and aerobic metabolic machineries for electron transportlinked ATP production, including "aerobic" and "anaerobic" types of FeS proteins. A possible scenario is that the "anaerobic" set of FeS proteins was preserved in hydogenosomes, whereas the "aerobic" set was preserved in mitochondria. Both organelles inherited the common mechanism of the FeS cluster assembly. This scenario is supported by the close phylogenetic relationship between eukaryotic and proteobacterial IscS proteins. Our hypothesis is also congruent with a common origin of mitochondria and Trichomonas hydrogenosomes, as well as a secondary loss of the mitochondrion/hydrogenosome-like organelle in Giardia. We cannot, however, rule out alternative explanations for the origin of hydrogenosomes and biochemistry of amitochondrial eukaryotes, including the mechanism of FeS cluster assembly. Indeed, independent lateral gene transfers (Doolittle 1998
) or preservation of certain biochemical pathways from an anaerobic past of eukaryotic evolution might be involved. Nevertheless, comparative analysis of mechanisms responsible for the formation of FeS clusters, which are considered to be among the most ancient biologically active metal cofactors (Cammack 1996
), appears to be a promising tool for tracing eukaryotic history.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: ML, maximum likelihood; MP, maximum parsimony; NJ, neighbor-joining; PLP, pyridoxal-5'-phosphate.
2 Keywords: IscS
iron-sulfur cluster
hydrogenosome
Trichomonas vaginalis
Giardia intestinalis
3 Address for correspondence and reprints: Jan Tachezy, Department of Parasitology, Faculty of Science, Charles University, Vininá 7, Prague 128 44, Czech Republic. tachezy{at}natur.cuni.cz
.
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