Iron-Dependent Hydrogenases of Entamoeba histolytica and Giardia lamblia: Activity of the Recombinant Entamoebic Enzyme and Evidence for Lateral Gene Transfer JULIE E. J. NIXON 1 , JESSICA FIELD 1 , ANDREW G. MCARTHUR 2 , MITCHELL L. SOGIN 2 , NIGEL YARLETT 3 , BRENDAN J. LOFTUS 4 , AND JOHN SAMUELSON 1, * 1 Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave., Boston, Massachusetts; 2 Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, Massachusetts; 3 Department of Biochemistry, Pace University, New York, New York; and 4 The Institute for Genomic Research, Rockville, Maryland Abstract. Entamoeba histolytica and Spironucleus barkhanus have genes that encode short iron-dependent hydrogenases (Fe-hydrogenases), even though these protists lack hydrogenosomes. To understand better the biochemis- try of the protist Fe-hydrogenases, we prepared a recombi- nant E. histolytica short Fe-hydrogenase and measured its activity in vitro. A Giardia lamblia gene encoding a short Fe-hydrogenase was identified from shotgun genomic se- quences, and RT-PCR showed that cultured entamoebas and giardias transcribe short Fe-hydrogenase mRNAs. A second E. histolytica gene, which encoded a long Fe-hydrogenase, was identified from shotgun genomic sequences. Phyloge- netic analyses suggested that the short Fe-hydrogenase genes of entamoeba and diplomonads share a common ancestor, while the long Fe-hydrogenase gene of entamoeba appears to have been laterally transferred from a bacterium. These results are discussed in the context of competing ideas for the origins of genes encoding fermentation en- zymes of these protists. Introduction One of the great recent discoveries in the cell biology of protists is that the hydrogenosome of Trichomonas vagina- lis, cause of vaginitis, is a modified mitochondrion, in which the enzymes of oxidative phosphorylation have been re- placed by fermentation enzymes that produce hydrogen gas (Mu ¨ller, 1993, 1998; Bui et al., 1996; Horner et al., 1996; Andersson and Kurland, 1999; Rotte et al., 2000). Proof of this idea includes the presence of mitochondrion-like chap- erones and a mitochondrion-like ATP/ADP transporter within the hydrogenosome, as well as organelle-targeting sequences at the N-termini of hydrogenosomal proteins that are encoded in the nucleus (Johnson et al., 1990; Hrdy and Mu ¨ller, 1995a, b; Bui et al., 1996; Bui and Johnson, 1996; Horner et al., 1996; Bradley et al., 1997; Dyall et al., 2000). The common origin of hydrogenosomes and mitochon- dria is included in a new biochemical explanation for the origin of mitochondria, called the hydrogen hypothesis (Martin and Mu ¨ller, 1998). The hydrogen hypothesis, a revision of the widely accepted endosymbiont hypothesis (Gray et al., 1999), suggests that the -proteobacterium, which became the mitochondrion, was a facultative anaer- obe that was selected for its ability to produce hydrogen in a methanogenic archaeal host. Consistent with this idea, multiple hydrogenosomal fermentation enzymes—includ- ing ferredoxin, succinyl-CoA synthetase, and malic en- zyme—resemble their counterparts in mitochondria (John- son et al., 1990; Hrdy and Mu ¨ller, 1995b). One alternative hypothesis suggests that the mitochondrial endosymbiont was selected for its ability to consume oxygen and thus protect the proto-eukaryote from oxidative damage (Andersson and Kurland, 1999). Another alternative hy- Received 20 June 2002; accepted 6 December 2002. * To whom correspondence should be addressed. Current address: De- partment of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, 715 Albany St., Boston, MA 02118. E-mail: jsamuels@bu.edu Reference: Biol. Bull. 204: 1–9. (February 2003) © 2003 Marine Biological Laboratory 1