S.A.). The 16S rRNA genes were amplified by PCR using the 27f:1492r primer pair [39]. A 743 nt-long fragment of the rpoA gene of each organism was amplified using the rpoAf2a:rpoAr2a primer pair (GGBGTGSTCCACGARTAY and GCRAGSACTTCCTTRATYTC, respectively). The aoxAf:aoxABr primer pair (TGYACCCAYATGGGMTGYCC and CSATGGCTTGTTCRGTSASGTA, respectively) were used to amplify 1451

nt of the aoxA and aoxB genes, including the short (~27 nt) intragenic region. The generic arsBf:arsBr primer pair (GGTGTGGAACATCGTCTGGAAYGCNAC and CAGGCCGTACACCACCAGRTACATNCC, respectively) were designed to amplify between 740 and 760 bp of both copies of the arsB gene in all Thiomonas strains. Following subsequent analysis, arsB1- and arsB2-specific internal forward and reverse primers were designed. The

arsB1i2f:arsB1i2r primer pair (TGGCGTTCGTGATGGCNTGCGG and CACCGGAACACCAGCGSRTCYTTRAT, respectively) amplified 268 bp GW-572016 purchase ROCK inhibitor of the arsB1 gene, whereas the arsB2i2f:arsB2i1r primer pair (TGGCCGTGGCCTGTTYGCNTTYYT and ACCCAGCCAATACGAAAGGTNGCNGGRTC, respectively) amplified 417 bp of the arsB2 gene. Virtual digestions of the arsB1 and arsB2 genes of strain 3As suggested that the two genes should be differentiated by restriction fragment length polymorphism (RFLP) analysis using the restriction enzyme RsaI. Phylogenetic analysis Sequences were aligned using the ClustalX alignment programme [40]. SuperGene analysis was performed by concatenating the 16S rRNA and rpoA

gene sequences of each organism, to improve the phylogenetic analysis as proposed recently [41]. Neighbour-Joining trees were eFT-508 order constructed using ClustalX, with bootstrap values determined from 1000 replications. Maximum likelihood (ML) trees were constructed using the PhyML algorithm [42]. The ModelGenerator programme [43] was used to select the optimal nucleotide substitution model for ML analysis. Bootstrap values were determined from 500 replications. A list of sequences generated during this study Adenylyl cyclase and their GenBank Accession IDs can be found in Table 3. Table 3 PCR target and GenBank Accession IDs for strains used in this study. Strain 16S rpoA aoxAB arsB1 arsB2 3As AM492684a EU339226 EU339209 EU339214 EU339217 Ynys1 AF387302a EU339223 n/d EU339216 n/d WJ68 AY455805a EU339224 EU339213 n/s n/d T. arsenivorans AY950676a EU339231 EU304260a n/d EU339222 T. perometabolis AY455808a EU339230 n/d EU339215 n/d a Accession IDs from other studies; n/d, no data; n/s, sequence not submitted: the arsB1 and arsB2 sequences obtained with the internal primers were short and therefore were not submitted to the GenBank sequence repository. Acknowledgements T. perometabolis was obtained from the Pasteur Institute, Paris, France. The authors would like to thank Dr Violaine Bonnefoy and Dr Kevin Hallberg for providing the Thiomonas strains and their invaluable advice on all things Thiomonas and Dr Catherine Joulian for her help with functional gene primer design.