Three colonies from an M1 pure culture plate were initially vortexed with 50 μL lysing solution (0.05% sodium dodecyl sulfate; 30 mM NaOH). Following incubation for 15 min at 95 °C and brief centrifugation, the solution was diluted with 450 μL H2O and centrifuged for 15 min. BMN 673 mouse In addition to 2.0 μL alkaline lysis supernatant as the template, the 50-μL PCR mixture contained 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.2 mM MgCl2, each dNTP (0.1 mM each), 0.2 μM of each primer, and 0.625 U of Perpetual OptiTaq DNA polymerase (Roboklon, Berlin, Germany). Amplification was performed using the following conditions: 95 °C for 2 min, followed by 33 cycles of 95 °C for 20 s, 55 °C for 30 s, and 72 °C for 1.5 min, with a final step

of 10 min at 72 °C. The PCR product was sequenced by SMB (Berlin, Germany). Chromatogram sequences were trimmed using Chromas Lite (Technelysium Pty Ltd, Tewantin, Qld, Australia), alignments were performed using bioedit (Hall, 1999), and the phylogenetic tree was constructed using the neighbor-joining method in the arb program (Ludwig et al., 2004). Fe(II) oxidation experiments were performed using the bicarbonate-buffered, gradient-culture medium described above. To rule out the possibility that the growth of strain M1 was occurring on organic compounds in the agarose CHIR-99021 clinical trial gel,

we designed an experiment with three treatments (three replicates each). The first treatment utilized gradient vials with 50 mM FeCl2 in the lower layer. The second treatment excluded FeCl2 from the lower layer and the third treatment substituted 5 mM Na2S for FeCl2. The sulfide was used to establish a redox and O2 gradient in the vials in case the growth of M1 required microoxic conditions. In all cases, resazurin (0.0001%) was included in

the 15-mL, upper layer to allow visualization of the depth of O2 penetration. Inoculum was prepared by resuspending colonies from plates in 2 mL sterile Phosphatidylinositol diacylglycerol-lyase upper layer. Two 50-μL aliquots of the resultant suspension were used to inoculate gradient systems at a depth of about 1 cm below the upper-layer surface. Two additional vials containing FeCl2 in the lower layer were inoculated with only sterile upper layer and used as abiotic controls. All gradient vials were purged with N2 : CO2 for 10 s before closing the screw-caps to partially remove O2. Vials were incubated statically in the dark at room temperature. At the conclusion of the 8-day experiment, cells were counted by epifluorescence microscopic examination after staining cells with 4′,6-diamidino-2-phenylindole (DAPI) after fixation with 3.4% formaldehyde (Kepner & Pratt, 1994). Where necessary, iron oxide precipitates were removed before staining using an oxalate dissolution method as described elsewhere (Roden & Zachara, 1996). To determine the vertical distribution of cells in an iron-oxidizing, gradient-culture system, aliquots of an upper-layer suspension for DAPI counting were withdrawn at 5-mm depth intervals using a sterile syringe.