Ch for homologous sequences employing BLAST (13). One of the most equivalent sequences were retrieved and aligned working with the ARB_EDIT4 tool in the ARB application package (14). A phylogenetic tree was constructed using neighbor-joining analysis (15), and the topology from the clustering was estimated with bootstrap sampling. Methanogen strains and cultivation. M. mazei GT was bought in the Japan Collection of Microorganisms (JCM) (Tsukuba, Japan). Strain zm-15 was isolated from the Zoige wetland soil in this study and deposited in the China Basic Microbiological Culture Collection Center (CGMCC) (Beijing, China) below accession number CGMCC 1.5193. For enrichment, soil samples were inoculated into basal medium supplemented with 20 mM (final concentration) methanol or acetate because the methanogenic substrate in an anaerobic chamber (Forma Anaerobic Technique 1029; Thermo Fisher Scientific, Waltham, MA, USA), as previously described (16). Total media had been dispensed into screw-cap serum bottles sealed with butyl rubber stoppers, with N2 as the gas phase at 101.three kPa. The IL-2 medchemexpress enriched samples have been incubated at 15 for about two weeks before colony isolation by means of the Hungate rolling-tube process (17). The roll tubes have been incubated at 15 till single colonies appeared. Single colonies were picked, along with the purified strain that made CH4 from methanol and acetate was designated strain zm-15. For identification of strain zm-15, the 16S rRNA gene was amplified together with the universal archaeal primer A2F along with the prokaryotic primer U1510R (see Table S1 inside the supplemental material), as previously described (18). Each strains G and zm-15 were grown beneath anaerobic conditions in 50 ml of DSMZ 120 medium, as previously described (4), within a 100-ml serum bottle containing methanol or acetate (20 mM final concentration). Strain zm-15 formed substantial multicellular aggregates when grown in the medium, plus the development of cultures was determined from measurements of CH4 production, as previously described (19). Cells at the exponential phase growing in acetate or methanol medium have been collected at five,000 g for 15 min in an anaerobic chamber. Soon after washing with prereduced phosphate buffer (1.7 mM cysteine-HCl H2O, 1.two mM Na2S 9H2O, 50 mM NaK-phosphate, pH 7.0; O2 was removed from the buffer by 8 Survivin Biological Activity cycles of evacuation and flushing with N2), the resting cells had been prepared. Methane determination. Methane production was measured having a Shimadzu GC 14B gas chromatograph (Shimadzu, Kyoto, Japan) with a flame ionization detector plus a C18 column as described previously (20). The temperature parameters have been set as follows: 50 for the column, 80 for the injector, and 130 for the detector. N2 was used as a carrier gas. Enzymatic assays. For the methanol-coenzyme M methyltransferase assay, strain zm-15 was cultured in 50 ml of DSM 120 medium with methanol because the sole carbon supply till mid-exponential phase (the CH4 concentration is about 4 mM, with methanol as the substrate), and after that cells were harvested anaerobically at 5,000 g for 15 min. The cell pellets had been resuspended with 20 ml of wash answer (38 mM NaCl, 20 mM NaHCO3, 9 mM NH4Cl, two mM MgCl2 6H2O, 1.7 mM CaCl2 2H2O, 50 mM MOPS [morpholinepropanesulfonic acid], pH 7.0), collected by centrifugation at 7,400 g for 15 min, and resuspended in 50 mM MOPS (pH 7.0). Each of the wash methods had been performed anaerobically at space temperature. Buffers had been prereduced before use. Cell extracts (CE) had been prepared by sonication on ice (one hundred W; 1-s sonic.