Unlike its phylogenetic relatives GM1 was unable to grow with either cis-dichloroethene or naphthalene as sole carbon source (data not shown). Figure 2 16S rRNA phylogenetic tree of arsenite-oxidising strain GM1 and published Polaromonas species. GenBank accession numbers are in parentheses. Significant bootstrap values (per 100 trials) are shown. The tree is rooted with the 16S rRNA gene sequence of p38 MAPK pathway Alcaligenes find more faecalis (AY027506) (not shown). Growth of GM1 was tested at 4°C, 10°C and 20°C in a minimal

salts medium (MSM) with 0.04% (w/v) yeast extract in the presence and absence of 4 mM arsenite as described previously [15] (Note: GM1 was unable to grow chemolithoautotrophically with arsenite). Under all conditions arsenite was oxidised RG7112 nmr to arsenate and oxidation occurred in the early exponential phase of growth (Figure 3). The generation time of

GM1 was shorter in the absence of arsenite, and decreased with increasing temperature (without arsenite at 4°C, 10°C and 20°C: 19 h, 16.5 h and 7 h, respectively; with arsenite at 4°C, 10°C and 20°C: 21.5 h, 17.7 h and 8.5 h, respectively). GM1 did not grow above 25°C. To date, only one arsenite oxidiser has been demonstrated to grow below 20°C [16]. This organism, a chemolithoautotrophic arsenite oxidiser designated M14, is a member of the Alphaproteobacteria related to Sinorhizobium species. M14′s temperature range was between 10°C and 37°C with an optimum of 22°C [16]. GM1 is the first reported arsenite oxidiser capable of growth below 10°C. Figure 3 Growth curves of GM1 grown at 4°C, 10°C and 20°C in the Minimal Salts Medium (MSM) with 0.04% (w/v) yeast extract. With 4 mM arsenite, closed circle; without arsenite, open circle; arsenite concentration, closed square. Error bars are the standard deviation of multiple experiments. The arsenite-oxidising ability of GM1 was further confirmed by testing for arsenite oxidase (Aro) activity in cells grown in the MSM with 4 mM arsenite and 0.04% (w/v)

yeast extract. Aro activity was measured at room temperature (i.e. 24°C) in its Cetuximab optimal buffer, 50 mM 2-(N-Morpholino)ethanesulfonic acid (MES) (pH 5.5) (data not shown). Aro activity was higher when GM1 was grown at 10°C (0.334 U/mg) compared with growth at 4°C (0.247 U/mg) and 20°C (0.219 U/mg) which were comparable. In growth experiments although all the arsenite is oxidised to arsenate in the early exponential growth phase the highest Aro activity was observed in the stationary phase of growth (i.e. 0.334 U/mg compared with 0.236 U/mg at early exponential phase). In most cases, arsenite is required in the growth medium for arsenite oxidase gene expression [6]. There are two exceptions, Thiomonas sp. str. 3As and Agrobacterium tumefaciens str.