Environmental Microbiology (2005) 7(2), 153–164 doi:10.1111/j.1462-2920.2004.00670.x © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd et al . Received 17 February, 2004; revised 25 May, 2004; accepted 25 May, 2004. *For correspondence. E-mail cournoye@biomserv.univ- lyon1.fr; Tel. (+33) 4 72 43 14 95; Fax (+33) 4 72 43 12 23. † Present address: UMR de Microbiologie et Géochimie des Sols, INRA/CMSE, 17 rue de Sully, 21 065 Dijon, France. Freshwater selenium-methylating bacterial thiopurine methyltransferases: diversity and molecular phylogeny S. Favre-Bonté, L. Ranjard, † C. Colinon, C. Prigent-Combaret, S. Nazaret and B. Cournoyer* UMR CNRS UCBL 5557 Ecologie Microbienne (Center for Microbial Ecology), Research Group on Opportunistic Pathogens and Environment, Université Claude Bernard – Lyon 1, Mendel Bldg, 5th floor, 69622 Villeurbanne Cedex, France. Summary The diversity of bacterial thiopurine methyltrans- ferases (bTPMT) among five natural Se-methylating freshwaters was investigated by polymerase chain reaction (PCR) screenings and sequencings. DNA sequence analyses confirmed the cloned products’ identity and revealed a broad diversity of freshwater TPMTs. Neighbour-joining (NJ) phylogenetic analyses combining these sequences, all GenBank entries closely related to these sequences and deduced TPMTs obtained in this work from selected g- proteobacteria showed TPMTs to form a distinct radi- ation, closely related to UbiG methyltransferases. Inside the TPMT phylogenetic cluster, eukaryote sequences diverged early from the bacterial ones, and all the bacterial database entries belonged to a subgroup of g-proteobacteria, with an apparent lateral transfer of a particular allele to b-proteobacteria of Bordetella. The NJ phylogenetic tree revealed 22 bTPMT lineages, 10 of which harboured freshwater sequences. All lineages showed deep and long branches indicative of major genetic drifts outside regions encoding highly conserved domains. Selected residues among these highly variable domains could reflect adaptations for particular eco- logical niches. PCR lineage-specific primers differen- tiated Se-methylating freshwaters according to their ‘tpm lineage’ signatures. Most freshwater tpm alleles were found to be distinct from those available in the databases, but a group of tpm was found encoding TPMTs identical to an Aeromonas veronii TPMT char- acterized in this work. Introduction Selenium belongs to the VIA group of the Periodic Table. It is important for most living forms and essential for human and animal health. However, it can become toxic at high doses. Chronic selenium toxicity (selenosis) has been reported many times, and the main symptoms in human populations are hair and nail brittleness and loss, gastrointestinal disturbances, a garlic breath odour and nervous system abnormalities (Koller and Exon, 1986; Holness et al., 1989). In wildlife, exposure to high sele- nium concentrations can result in birth defects and abnormalities (Yang et al., 1983; Ohlendorf et al., 1986). The sensitivity of bacteria and fungi to inorganic sele- nium (selenite or selenate) seems to result from the oxi- dative properties of these molecules, which can lead to an oxidative burst (e.g. Bebien et al., 2001). Some micro- organisms can resist this oxidative stress through sele- nium reduction (Stolz and Oremland, 1999) or methylation processes (Gadd, 1993). Significant contri- butions to the understanding of the biochemistry of these bacterial processes were the findings of the Thauera sel- enatis dissimilatory selenate reductase enzyme complex (Schroeder et al., 1997), and the characterization of the selenium-methylating methyltransferase named bTPMT (standing for b acterial t hiop urine m ethylt ransferase) (Ranjard et al., 2002). The selenate reductase was shown to reduce selenate to selenite and to have a high affinity and turnover rate for selenate, much higher than that of the periplasmic nitrate reductase (Sabaty et al., 2001). T. selenatis was also shown to reduce selenite to elemental selenium by the nitrite respiratory system (in the presence of nitrate) (Demoll-Decker and Macy, 1993). T. selenatis was isolated from sediments of the San Joaquin Valley, and its selenate-reducing capacity was used recently for the bioremediation of selenium from agricultural drainage water (Cantafio et al., 1996). The bTPMT was shown to volatilize organic and inorganic selenium into dimethyl selenide (DMSe) and dimethyl diselenide (DMDSe) (Ranjard et al., 2002). It was origi- nally characterized from Pseudomonas syringae but was recently shown to be found among a freshwater Pseudomonas anguiliseptica-like strain of an aquifer showing reproducible emission of DMSe and DMDSe after spiking with various Se substrates (Ranjard et al., 2003). Microbial methylation of toxic Se oxyanions is a well-accepted bioremediation strategy for cleaning up