The iron-sulfur cluster assembly genes iscS and iscU of Entamoeba histolytica were acquired by horizontal gene transfer
BMC Evolutionary Biology volume 4, Article number: 7 (2004)
Iron-sulfur (FeS) proteins are present in all living organisms and play important roles in electron transport and metalloenzyme catalysis. The maturation of FeS proteins in eukaryotes is an essential function of mitochondria, but little is known about this process in amitochondriate eukaryotes. Here we report on the identification and analysis of two genes encoding critical FeS cluster (Isc) biosynthetic proteins from the amitochondriate human pathogen Entamoeba histolytica.
E. histolytica IscU and IscS were found to contain all features considered essential for their biological activity, including amino acid residues involved in substrate and/or co-factor binding. The IscU protein differs significantly from other eukaryotic homologs and resembles the long type isoforms encountered in some bacteria. Phylogenetic analyses of E. histolytica IscS and IscU showed a close relationship with homologs from Helicobacter pylori and Campylobacter jejuni, to the exclusion of mitochondrial isoforms.
The bacterial-type FeS cluster assembly genes of E. histolytica suggest their lateral acquisition from epsilon proteobacteria. This is a clear example of horizontal gene transfer (HGT) from eubacteria to unicellular eukaryotic organisms, a phenomenon known to contribute significantly to the evolution of eukaryotic genomes.
After malaria, Entamoeba histolytica is the second leading cause of death due to parasitic disease in humans . E. histolytica has been cited as infecting one tenth of the world population, although it is now known that these infections are caused by two very similar species, E. histolytica and E. dispar. The former is the cause of all invasive disease, with an estimated 50,000 to 100,000 fatalities each year . This human parasite was traditionally considered a classic example of a primitive eukaryote due to its apparent lack of `typical' eukaryotic cell structures such as mitochondria, peroxisomes, Golgi apparatus and endoplasmic reticulum . The lack of morphologically identifiable mitochondria led to the suggestion that its ancestors predate the endosymbiotic acquisition of this organelle , despite the observation that Entamoeba branches after well established mitochondrial groups in ribosomal RNA phylogenies .
The "primitively amitochondrial" view was overturned by the discovery of genes encoding mitochondrial proteins (e.g., chaperonin 60 (Cpn60), mitochondrial-type Hsp70 (mtHsp70), pyridine nucleotide transhydrogenase (PNT)), and by the demonstration that mitochondrial remnant organelles (mitosomes) housing chaperonin Cpn60 have been retained in this organism [6–9]. Several lines of evidence support the mitochondrial ancestry of mitosomes: i) Cpn60 and mtHsp70 cluster with mitochondrial homologs to the exclusion of prokaryotic sequences in phylogenetic reconstructions; ii) Cpn60, mtHsp70 and PNT contain amino terminal regions rich in hydroxylated and positively charged amino acids, reminiscent of mitochondrial/hydrogenosomal targeting presequences; iii) Deletion of amino acids 2–15 from the putative targeting presequence of Cpn60 leads to an accumulation of the truncated protein in the cytosol, a phenotype that can be reversed by the addition of a functional mitochondrial targeting signal from Trypanosoma cruzi Hsp70 to the truncated protein .
Since the discovery of mitosomes in E. histolytica, mitochondrial remnant organelles have also been identified in the microsporidian Trachipleistophora hominis , the apicomplexan Cryptosporidium parvum  and, most recently, in the diplomonad Giardia intestinalis . Giardia mitosomes have been shown to function in FeS cluster biosynthesis and FeS protein maturation , essential mitochondrial functions of eukaryotic organisms . FeS proteins are involved in energy metabolism, DNA repair, transcriptional regulation, and biosynthesis of nucleotides and amino acids . The identification of genes encoding putative Isc proteins in the genomes of all amitochondrial protists sequenced so far [15–19] suggests that this mitochondrial function might have been retained in all amitochondrial protists and may be a general functional feature of all mitochondrion-derived organelles [12, 16, 20–22].
Here we report on the cloning, structural characterization and phylogenetic analysis of E. histolytica genes encoding Isc proteins. Both E. histolytica IscU and IscS homologs were found to contain all the structural features required for their biological activity, including substrate and co-factor binding sites, suggesting a fully operational FeS cluster biosynthetic pathway in E. histolytica. Phylogenetic analyses show that both Isc proteins have a different evolutionary history to that of mitochondrial homologs, indicating their lateral acquisition from bacteria. Moreover, the observation that both proteins seem to have been acquired from the same bacterial taxon might suggest a single transfer event of a small bacterial Isc operon.
Results and Discussion
Identification and primary sequence analyses of E. histolytica genes encoding the FeS assembly proteins IscS and IscU
BLAST searches of preliminary data generated by the E. histolytica genome-sequencing project revealed clones with extensive sequence similarity to the G. intestinalis iscS gene. PCR amplification of E. histolytica genomic DNA using primers based on these putative E. histolytica EhiscS sequences and on a putative EhiscU sequence (accession number: AY040613) generated products of the expected size. DNA sequencing confirmed the identity of the amplified clones. The 5' untranslated regions of EhiscU and EhiscS contain distinct putative promoter elements reported to be typical for E. histolytica . All three conserved regions are present in the first 40 bases upstream the initiation codon of iscU and iscS (Fig. 1), suggesting both genes are functional, although the GAAC-element is less well conserved in the iscU promoter region. The E. histolytica IscU protein is 348 amino acids in length and has a predicted molecular mass of 38.9 kDa and a predicted isoelectric point of 5.71. Its large size indicates it is a long-form IscU, similar to the one described for Azotobacter vinelandii , and not a short form as found in other eukaryotes (Fig. 2). For IscS these values are 390 amino acids, 42.8 kDa, and 5.92, respectively. The GC values for the iscU (iscS) genes are 33 % (32 %) for the coding region, 29 % (25 %) for the 5' untranslated region and 29 % (18 %) for the 3' untranslated region (250 bp each). These values are in agreement with GC values reported for other E. histolytica genes based on 75,615 codons analyzed . Codon usage is also similar to E. histolytica codon usage and no introns are present in either of these two genes.
Both E. histolytica IscU and IscS contain structural motifs typical of FeS assembly proteins. Pfam (PF01106, PF01592), PRODOM (PD002830), and InterProScan (IPR001075, IPR002871) motifs characteristic of IscU and NifU proteins are present in the E. histolytica homolog (Fig. 2A) [26–28]. E. histolytica IscS contains Pfam (PF00266), PROSITE (PS00595), and InterProScan (IPR000192) motifs that are normally associated with aminotransferase class V proteins, a subfamily of the aminotransferase proteins. IscS is one of the eight members of the class V subfamily (Fig. 2A). As indicated above, IscU has an extension at the carboxy-terminus relative to most IscU homologs. This extension is also present on the A. vinelandii NifU gene whose amino-terminal part is homologous to that of IscU. In addition, this C-terminal extension is similar to a completely different gene from Saccharomyces cerevisiae, Nfu1 (NifU-like in Fig. 2B). Since Nfu1- and Isu-like sequences are part of the same gene in Azotobacter, Campylobacter, Entamoeba and Helicobacter it could be inferred that both proteins interact with each other when found on two separate genes. Such informative fusion proteins (or Rosetta Stone sequences) indicate an interaction between protein pairs . The existence of long IscU isoforms would therefore suggest that the Nfu1 and Isu1/2 proteins do interact in yeast as postulated by Garland et al. .
Both proteins align along their whole length to homologous proteins from other organisms (Fig. 3). Residues implicated in function are conserved in both IscU and IscS proteins. The three cysteine residues that are conserved in Escherichia coli IscU which provide a scaffold for the assembly of iron-sulfur clusters  are conserved in the E. histolytica protein (Fig. 3A). In addition, in E. coli one of these IscU cysteines interacts with a conserved cysteine from IscS which is also present in the E. histolytica IscS (Figs. 3A and 3B). Most residues considered to be important for IscS function are also present on the E. histolytica protein (Fig. 3B). To test whether the E. histolytica IscS protein assumes a normal three-dimensional conformation, this protein was modeled on the solved NifS protein structure from Thermotoga maritima. The overall topology of both proteins is quite similar and the force field energy of the computed E. histolytica IscS model is -13,800 kJ/mol, indicating an energetically plausible model . The putative active site architecture of E. histolytica IscS and the solved active site of T. maritima NifS show similar structures (Fig. 4). The ring of the cofactor vitamin B6 (or pyridoxal-5'-phosphate; PLP) is sandwiched between EhHis106/TmHis99 and EhThr184/TmVal179 and further fixed by residues EhAsp182/TmAsp177 and EhGln185/TmGln180. The phosphate-group is anchored by six hydrogen bonds from EhThr76/TmThr71, EhHis207/TmHis202, EhThr198/TmSer200, and EhThr243/TmThr238 . The presence of all residues considered to be important for IscU and IscS activity on the E. histolytica proteins suggest that these proteins are indeed involved in FeS cluster assembly.
No N-terminal or C-terminal organelle targeting domains could be unambiguously identified in E. histolytica IscS/U proteins using subcellular localization and targeting prediction software (e.g., PSORT II , MitoProt , NNPSL ). The C-terminal signature motif which is considered to be characteristic of proteobacterial and eukaryotic IscS proteins  is not present in homologs from E. histolytica, Campylobacter or Azotobacter (Fig. 3B). Because these organisms all possess the long-type IscU isoforms, it is possible that the extended IscU protein might negate the need for the C-terminal signature residues on the interacting IscS protein. However, functional studies using deletion mutants are needed to confirm this hypothesis.
Phylogenetic analyses of the E. histolytica FeS cluster assembly proteins
Bayesian and maximum likelihood (ML) phylogenetic analyses of E. histolytica IscU and IscS protein sequences revealed that the Entamoeba Isc proteins form a well supported clade with Helicobacter pylori and Campylobacter jejuni – two bacteria encountered in the human digestive tract – to the exclusion of all other prokaryotic and eukaryotic homologs (Fig. 5). All three independent Bayesian analyses converged on the same tree with similar posterior probabilities. For IscU, the ML tree had a slightly better likelihood than the Bayesian tree, while for IscS both trees had similar likelihoods. The overall topologies of IscS and IscU phylogenetic trees are very similar to each other and major taxonomic clades like plants, animals, and fungi are well conserved. The position of the microsporidium Encephalitozoon cuniculi in the IscU tree is poorly resolved as indicated by the very low support for this node at the base of the metazoa, contrary to its well-documented association with fungi .
The position of Rickettsia prowazekii IscS basal to the eukaryotes suggests that eukaryotic IscS proteins originated from the mitochondrial endosymbiont, since this bacterium is considered to be a close relative to the mitochondrial ancestor. Indeed, the mitochondrial ancestry of E. cuniculi, T. vaginalis and G. intestinalis IscS proteins is strongly supported by their clustering with mitochondrial homologs [15, 20, 37]. For IscU, the base of the eukaryotic clade is not well resolved. Animals and plants cluster together with a proteobacterial sister clade containing the α-proteobacterium R. prowazekii, while fungi, G. intestinalis, and the alveolates are basal to this clade. However, the well-supported clustering of E. histolytica Isc proteins with homologs from the bacteria H. pylori and C. jejuni, to the exclusion of all other eukaryotes, suggests that E. histolytica acquired its isc genes laterally from ε-proteobacteria (Fig. 5). This suggestion is further supported by the fact that Campylobacter, Helicobacter and E. histolytica all possess long form IscU proteins to the exclusion of the short isoforms found in eukaryotic organisms and in many bacterial taxa (see orange branches in Fig. 5).
Mitochondrial-type IscS/U proteins have been identified in several amitochondrial eukaryotes including Giardia, Encephalitozoon, Trichomonas and Cryptosporidium, and there is significant direct and indirect evidence that these proteins are targeted into their highly derived mitochondrion-related organelles [12, 15, 16, 20]. Thus, E. histolytica appears to be unique amongst eukaryotic organisms that contain mitochondrion-related organelles in harbouring bacterial-type IscS/U proteins. That no mitochondrial-type IscS/U proteins have thus far been identified in E. histolytica would suggest that its original mitochondrial-type iscS/U genes were replaced during the course of evolution by the more recently acquired bacterial homologs. However, since the E. histolytica genome has not yet been fully sequenced, the possibility that mitochondrial type iscS/U genes might have escaped detection cannot be formally excluded.
Since both E. histolytica Isc proteins form a strongly supported clade with homologs from gut bacteria, we investigated whether other intestinal inhabitants would form part of this clade. The genomes of 23 bacterial and 2 eukaryotic inhabitants of the human gut were screened using E. histolytica IscU and IscS as query sequences, but no additional homologs were identified. Only a fraction of the estimated 400–500 bacteria species living in the human intestine  have been sequenced and therefore we may not have been able to identify any other members of this clade due to sampling limitations. Nevertheless, the most parsimonious explanation for the clustering of E. histolytica Isc proteins with those of bacteria is that E. histolytica, or its ancestors, acquired its iscS/U genes by horizontal gene transfer (HGT), a well-documented contributor to prokaryotic and eukaryotic genome evolution. In higher eukaryotes the most obvious example of HGT is the relocation of genes from endosymbiosis-derived organelles to the cell nucleus, which might be regarded as a special case of HGT. However, over the past few years evidence has accumulated of the frequent incorporation of genes into the genomes of microbial eukaryotes by HGT [39–47]. The transfer of bacterial genes into eukaryotes might occur in several possible ways. One hypothesis is the `you are what you eat' gene transfer ratchet of HGT which suggests that when a genome is continuously bombarded with DNA, some of these genes might eventually replace the host's own genes . Since both Helicobacter and Campylobacter occupy the same ecological niche as E. histolytica, an avid consumer of gut bacteria, HGT via this mechanism seems plausible. Establishing unequivocally the timing of HGT will be important to test this hypothesis.
Analysis of the organization of Isc/Nif loci on the genomes of several bacteria revealed the presence of a small Isc operon consisting exclusively of IscU and IscS in H. pylori and C. jejuni, whilst the well-studied E. coli and A. vinelandii isc operons contained several other genes involved in FeS cluster assembly (see Fig. 6). This observation provides a mechanistic explanation for the presence of two interacting proteins with similar ancestry in the genome of E. histolytica. It is possible that E. histolytica might have incorporated the entire isc operon from Helicobacter/Campylobacter, or from their ancestors, into its genome in a single transfer event. Once freed from the constraints of operon-type prokaryotic gene expression, the iscS/U genes might have become separated in the E. histolytica genome during the course of evolution.
E. histolytica or its ancestors appear to have acquired their iscS/U genes by HGT from ε-proteobacteria. The apparent absence of mitochondrial-type IscS/U proteins in an organism with mitochondrion-bearing ancestors such as E. histolytica suggests that its original mitochondrial iscS/U genes might have been replaced with the more recently acquired bacterial homologs. This finding, like several other recently reported cases of prokaryote to eukaryote gene transfers [39–47], highlights the important role played by HGT in protozoan genome evolution. Since no recent HGT events from prokaryotes to humans have been detected in the human genome , HGT from bacteria to protozoan parasites might have important implications for public health. Targeting enzymes or metabolic pathways of bacterial origin in human pathogens should have more severe consequences for the parasite than for its host, making these proteins promising targets for chemotherapy.
Organism and DNA isolation
E. histolytica HM-1:IMSS clone 9 was maintained axenically by subculture in YI-S medium with 15% adult bovine serum as described . Entamoeba genomic DNA was isolated using cetyltrimethylammonium bromide (CTAB) according to Clark .
Cloning and sequencing of the E. histolytica iscS and iscU genes
Standard recombinant DNA techniques were used as described elsewhere . PCR was performed on isolated E. histolytica genomic DNA. Primers were designed using Primer3 . The EhiscU gene was amplified using primers based on a NifU-like E. histolytica sequence (accession number AY040613). The primers were Eh_IscU_936F, 5'-CCA ACG TAT CGC CAC GAA AA-3' and Eh_IscU_2270R, 5'-GCA AAA CAA AGT ATG GCA GAA GCA-3' for forward and reverse primers, respectively. The EhiscS gene was identified on the E. histolytica genome by BLAST searches of preliminary data generated by the Entamoeba genome sequencing project  using G. intestinalis GiiscS (accession number AAK39427) as the query sequence. Putative EhiscS gene sequences (1000 bases up- and downstream of the ORF) were used for primer design. The EhiscS coding region was amplified using primers Eh_IscS_681F, 5'-CAA GTG CGA ATA CCC AAT TTG AA-3' and Eh_IscS_2515R, 5'-GGC TGA AGC CAT GAC ACC TC-3' (forward and reverse primers, respectively). The resulting PCR fragments were all cloned into pGEM-T-Easy (Promega) and sequenced to confirm their identity. The new E. histolytica IscS sequence has been deposited in Genbank (accession number AY277946).
The conceptually translated E. histolytica IscS and IscU amino acid sequences were aligned using Clustal W  to reference sequences from Genbank. The alignments were manually refined and only unambiguously aligned regions without gaps were used for phylogenetic analysis, leaving data sets of 28 taxa with 116 amino acid positions (IscU) and a similar taxon set consisting of 29 taxa with 326 amino acid positions (IscS). Likelihood searches were performed in a Bayesian framework under the JTT-f substitution model accommodating site rate variation (fraction of invariable sites plus four variable gamma rates) using the program MrBayes . All analyses started with randomly generated trees and ran for 200,000 generations, with sampling at intervals of 100 generations that produced 2,000 trees. To ensure that the analyses were not trapped on local optima, the data set was run three times independently, each run beginning with a different starting tree. The log-likelihood values of the 2,000 trees in each analysis were plotted against the generation time (not shown). Although the likelihood model stabilized very rapidly, only the last 1,500 trees in each of the three independent analyses were used to estimate separate 50% majority rule consensus trees for these. The frequency of any particular clade, among the individual trees contributing to the consensus tree, represents the posterior probability of that clade . For the maximum likelihood analyses, protein data sets were resampled 100 times using SEQBOOT from PHYLIP . These resampled datasets were analysed using PHYML  with alpha and invariant sites parameters optimized on the Bayesian tree in TREE-PUZZLE 5.0  with a mixed four-category discrete gamma plus invariable sites model of rate heterogeneity. The JTT substitution model was used in the protein analyses. Majority rule consensus trees were obtained from the resulting 100 trees using CONSENSE (PHYLIP).
Stanley SL: Amoebiasis. Lancet. 2003, 361: 1025-1034. 10.1016/S0140-6736(03)12830-9.
WHO: The world health report. 1995, Geneva: World Health Organization
Meza I: Entamoeba histolytica: phylogenetic considerations. Arch Med Res. 1992, 23: 1-5.
Hasegawa M, Hashimoto T, Adachi J, Iwabe N, Miyata T: Early branchings in the evolution of eukaryotes: ancient divergence of entamoeba that lacks mitochondria revealed by protein sequence data. J Mol Evol. 1993, 36: 380-388.
Sogin ML: Early evolution and the origin of eukaryotes. Curr Opin Genet Dev. 1991, 1: 457-463.
Bakatselou C, Kidgell C, Clark CG: A mitochondrial-type hsp70 gene of Entamoeba histolytica . Mol Biochem Parasitol. 2000, 110: 177-182. 10.1016/S0166-6851(00)00264-4.
Clark CG, Roger AJ: Direct evidence for secondary loss of mitochondria in Entamoeba histolytica . Proc Natl Acad Sci USA. 1995, 92: 6518-6521.
Tovar J, Fischer A, Clark CG: The mitosome, a novel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica . Mol Microbiol. 1999, 32: 1013-1021. 10.1046/j.1365-2958.1999.01414.x.
Mai Z, Ghosh S, Frisardi M, Rosenthal B, Rogers R, Samuelson J: Hsp60 is targeted to a cryptic mitochondrion-derived organelle ("crypton") in the microaerophilic protozoan parasite Entamoeba histolytica . Mol Cell Biol. 1999, 19: 2198-2205.
Williams BAP, Hirt RP, Lucocq JM, Embley TM: A mitochondrial remnant in the microsporidian Trachipleistophora hominis . Nature. 2002, 418: 865-869. 10.1038/nature00949.
Riordan CE, Ault J, Langreth SG, Keithly JS: Cryptosporidium parvum Cpn60 targets a relict organelle. Curr Genet. 2003, 44: 138-147. 10.1007/s00294-003-0432-1.
Tovar J, León-Avila G, Sánchez L, Sutak R, Tachezy J, van der Giezen M, Hernández M, Müller M, Lucocq JM: Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation. Nature. 2003, 426: 172-176. 10.1038/nature01945.
Lill R, Kispal G: Maturation of cellular Fe-S proteins: an essential function of mitochondria. Trends Biochem Sci. 2000, 25: 352-356. 10.1016/S0968-0004(00)01589-9.
Kato S, Mihara H, Kurihara T, Takahashi Y, Tokumoto U, Yoshimura T, Esaki N: Cys-328 of IscS and Cys-63 of IscU are the sites of disulfide bridge formation in a covalently bound IscS/IscU complex: implications for the mechanism of iron-sulfur cluster assembly. Proc Natl Acad Sci USA. 2002, 99: 5948-5952. 10.1073/pnas.082123599.
Katinka MD, Duprat S, Cornillot E, Metenier G, Thomarat F, Prensier G, Barbe V, Peyretaillade E, Brottier P, Wincker P, Delbac F, El Alaoui H, Peyret P, Saurin W, Gouy M, Weissenbach J, Vivares CP: Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi . Nature. 2001, 414: 450-453. 10.1038/35106579.
LaGier MJ, Tachezy J, Stejskal F, Kutisova K, Keithly JS: Mitochondrial-type iron-sulfur cluster biosynthesis genes (IscS and IscU) in the apicomplexan Cryptosporidium parvum . Microbiology. 2003, 149: 3519-3530. 10.1099/mic.0.26365-0.
Mann BJ: Entamoeba histolytica Genome Project: an update. Trends Parasitol. 2002, 18: 147-148. 10.1016/S1471-4922(01)02231-0.
Bankier AT, Spriggs HF, Fartmann B, Konfortov BA, Madera M, Vogel C, Teichmann SA, Ivens A, Dear PH: Integrated mapping, chromosomal sequencing and sequence analysis of Cryptosporidium parvum . Genome Res. 2003, 13: 1787-1799.
McArthur AG, Morrison HG, Nixon JE, Passamaneck NQ, Kim U, Hinkle G, Crocker MK, Holder ME, Farr R, Reich CI, Olsen GE, Aley SB, Adam RD, Gillin FD, Sogin ML: The Giardia genome project database. FEMS Microbiol Lett. 2000, 189: 271-273. 10.1016/S0378-1097(00)00299-8.
Tachezy J, Sánchez LB, Müller M: Mitochondrial type iron-sulfur cluster assembly in the amitochondriate eukaryotes Trichomonas vaginalis and Giardia intestinalis, as indicated by the phylogeny of IscS. Mol Biol Evol. 2001, 18: 1919-1928.
Embley TM, van der Giezen M, Horner DS, Dyal PL, Foster P: Mitochondria and hydrogenosomes are two forms of the same fundamental organelle. Phil Trans R Soc Lond. 2003, 358: 191-204. 10.1098/rstb.2002.1190.
van der Giezen M, Tovar J: Hydrogenosomes, mitosomes and mitochondria; variations on a theme?. In: Organelles, Genomes and Eukaryote Phylogeny: An Evolutionary Synthesis in the Age of Genomics. Edited by: Horner DS, Hirt RP. 2004, CRC Press
Purdy JE, Pho LT, Mann BJ, Petri WA: Upstream regulatory elements controlling expression of the Entamoeba histolytica lectin. Mol Biochem Parasitol. 1996, 78: 91-103. 10.1016/S0166-6851(96)02614-X.
Zheng L, Cash VL, Flint DH, Dean DR: Assembly of iron-sulfur clusters. Identification of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii . J Biol Chem. 1998, 273: 13264-13272. 10.1074/jbc.273.21.13264.
Nakamura Y, Gojobori T, Ikemura T: Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucl Acids Res. 2000, 28: 292-10.1093/nar/28.1.292.
Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy SR, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer ELL: The Pfam Protein Families Database. Nucl Acids Res. 2002, 30: 276-280. 10.1093/nar/30.1.276.
Servant F, Bru C, Carrere S, Courcelle E, Gouzy J, Peyruc D, Kahn D: ProDom: automated clustering of homologous domains. Brief Bioinform. 2002, 3: 246-251.
Zdobnov EM, Apweiler R: InterProScan – an integration platform for the signature-recognition methods in InterPro. Bioinformatics. 2001, 17: 847-848. 10.1093/bioinformatics/17.9.847.
Marcotte EM, Pellegrini M, Ng HL, Rice DW, Yeates TO, Eisenberg D: Detecting protein function and protein-protein interactions from genome sequences. Science. 1999, 285: 751-753. 10.1126/science.285.5428.751.
Garland SA, Hoff K, Vickery LE, Culotta VC: Saccharomyces cerevisiae ISU1 and ISU2: members of a well-conserved gene family for iron-sulfur cluster assembly. J Mol Biol. 1999, 294: 897-907. 10.1006/jmbi.1999.3294.
Manzetti S: Structural genomics and molecular visualization – part 2; Basic homology modelling of proteins.: Proinformatix.com. 2001
Kaiser JT, Clausen T, Bourenkow GP, Bartunik HD, Steinbacher S, Huber R: Crystal structure of a NifS-like protein from Thermotoga maritima: implications for iron sulphur cluster assembly. J Mol Biol. 2000, 297: 451-464. 10.1006/jmbi.2000.3581.
Nakai K, Horton P: PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci. 1999, 24: 34-36. 10.1016/S0968-0004(98)01336-X.
Claros MG, Vincens P: Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur J Biochem. 1996, 241: 779-786.
Reinhardt A, Hubbard T: Using neural networks for prediction of the subcellular location of proteins. Nucleic Acids Res. 1998, 26: 2230-2236. 10.1093/nar/26.9.2230.
Van de Peer Y, Ben Ali A, Meyer A: Microsporidia: accumulating molecular evidence that a group of amitochondriate and suspectedly primitive eukaryotes are just curious fungi. Gene. 2000, 246: 1-8. 10.1016/S0378-1119(00)00063-9.
Emelyanov VV: Phylogenetic affinity of a Giardia lamblia cysteine desulfurase conforms to canonical pattern of mitochondrial ancestry. FEMS Microbiol Lett. 2003, 226: 257-266. 10.1016/S0378-1097(03)00598-6.
Berg RD: The indigenous gastrointestinal microflora. Trends Microbiol. 1996, 4: 430-435. 10.1016/0966-842X(96)10057-3.
Andersson JO, Roger AJ: Evolutionary analyses of the small subunit of glutamate synthase: gene order conservation, gene fusions, and prokaryote-to-eukaryote lateral gene transfers. Eukaryot Cell. 2002, 1: 304-310. 10.1128/EC.1.2.304-310.2002.
Andersson JO, Sjogren AM, Davis LA, Embley TM, Roger AJ: Phylogenetic analyses of diplomonad genes reveal frequent lateral gene transfers affecting eukaryotes. Curr Biol. 2003, 13: 94-104. 10.1016/S0960-9822(03)00003-4.
Field J, Rosenthal B, Samuelson J: Early lateral transfer of genes encoding malic enzyme, acetyl-CoA synthetase and alcohol dehydrogenases from anaerobic prokaryotes to Entamoeba histolytica . Mol Microbiol. 2000, 38: 446-455. 10.1046/j.1365-2958.2000.02143.x.
Rosenthal B, Mai Z, Caplivski D, Ghosh S, de la Vega H, Graf T, Samuelson J: Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica . J Bacteriol. 1997, 179: 3736-3745.
Boucher Y, Doolittle WF: The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways. Mol Microbiol. 2000, 37: 703-716. 10.1046/j.1365-2958.2000.02004.x.
Sanchez L, Horner D, Moore D, Henze K, Embley T, Müller M: Fructose-1,6-bisphosphate aldolases in amitochondriate protists constitute a single protein subfamily with eubacterial relationships. Gene. 2002, 295: 51-10.1016/S0378-1119(02)00804-1.
Henze K, Horner DS, Suguri S, Moore DV, Sánchez LB, Müller M, Embley TM: Unique phylogenetic relationships of glucokinase and glucosephosphate isomerase of the amitochondriate eukaryotes Giardia intestinalis, Spironucleus barkhanus and Trichomonas vaginalis. Gene. 2001, 281: 123-131. 10.1016/S0378-1119(01)00773-9.
de Koning AP, Brinkman FS, Jones SJ, Keeling PJ: Lateral gene transfer and metabolic adaptation in the human parasite Trichomonas vaginalis . Mol Biol Evol. 2000, 17: 1769-1773.
Suguri S, Henze K, Sánchez LB, Moore DV, Müller M: Archaebacterial relationships of the phosphoenolpyruvate carboxykinase gene reveal mosaicism of Giardia intestinalis core metabolism. J Eukaryot Microbiol. 2001, 48: 493-497.
Doolittle WF: You are what you eat: a gene transfer ratchet could account for bacterial genes in eukaryotic nuclear genomes. Trends Genet. 1998, 14: 307-311. 10.1016/S0168-9525(98)01494-2.
Stanhope MJ, Lupas A, Italia MJ, Koretke KK, Volker C, Brown JR: Phylogenetic analyses do not support horizontal gene transfers from bacteria to vertebrates. Nature. 2001, 411: 940-944. 10.1038/35082058.
Clark CG, Diamond LS: Methods for cultivation of luminal parasitic protists of clinical importance. Clin Microbiol Rev. 2002, 15: 329-341. 10.1128/CMR.15.3.329-341.2002.
Clark CG: DNA purification from polysaccharide-rich cells. In: Protocols in Protozoology. Edited by: Lee JJ, Soldo AT. 1992, Lawrence, Kansas: Allen Press, 1: D3.1-D3.2.
Sambrook J, Fritsch E, Maniatis T: Molecular cloning, a laboratory manual. 1989, New York, USA: Cold Spring Harbor Laboratory Press
Rozen S, Skaletsky HJ: Primer3 on the WWW for general users and for biologist programmers. In: Bioinformatics Methods and Protocols: Methods in Molecular Biology. Edited by: Krawetz S, Misener S. 2000, Totowa, NJ: Humana Press, 365-386.
Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Acids Res. 1994, 22: 4673-4680.
Huelsenbeck JP, Ronquist F: MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 2001, 17: 754-755. 10.1093/bioinformatics/17.8.754.
Felsenstein J: PHYLIP (Phylogeny Inference Package). 1993, Felsenstein, Department of Genetics, University of Washington, Seattle
Guindon S, Gascuel O: A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003, 52: 696-704.
Schmidt HA, Strimmer K, Vingron M, von Haeseler A: TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics. 2002, 18: 502-504. 10.1093/bioinformatics/18.3.502.
Schwartz RM, Dayhoff MO: Origins of prokaryotes, eukaryotes, mitochondria, and chloroplasts. Science. 1978, 199: 395-403.
Guex N, Peitsch MC: SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis. 1997, 18: 2714-2723.
We wish to thank Drs Salvador Carranza (University of Barcelona, Spain) and David S. Horner (University of Milan, Italy) for advice and help with the phylogenetic analyses and Dr. Hermie Harmsen (University of Groningen, The Netherlands) for compiling a list of gut bacteria currently being sequenced. Preliminary sequence data for E. histolytica is deposited regularly into the GSS division of GenBank. The Sequencing effort is part of the International E. histolytica Genome Sequencing Project and is supported by an award from the National Institute of Allergy and Infectious Diseases, National Institutes of Health. SC was supported by an undergraduate research bursary from the Nuffield Foundation (URB/00970/G). This work was supported by a grant from the BBSRC (111/C13820) to JT.
MvdG designed and coordinated the molecular genetic studies, carried out the homology modelling, phylogenetic analyses and drafted the manuscript. SC carried out the molecular genetic studies and participated in the sequence alignment. JT edited the manuscript and participated in the design and coordination of the study. All authors read and approved the final manuscript.
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van der Giezen, M., Cox, S. & Tovar, J. The iron-sulfur cluster assembly genes iscS and iscU of Entamoeba histolytica were acquired by horizontal gene transfer. BMC Evol Biol 4, 7 (2004). https://doi.org/10.1186/1471-2148-4-7