AA; methylcobalamin-coenzyme M methyltransferase), which catalyzes the transfer from the methyl group from MtaC to CoM. In the sequenced methanosarcinal genomes, three copies of mtaC and mtaB and two copies of mtaA are located (four). In aceticlastic methanogenesis, acetate is initial activated to acetyl-coenzyme A (CoA) by acetate kinase (Ack) and phosphotransacetylase (Pta). Acetyl-CoA is then cleaved into an enzyme-bound methyl group and CO2 by acetyl-CoA synthase (ACS)/CO dehydrogenase (CODH). The methyl carbon is then transferred to CoM by way of the C1 carrier tetrahydrosarcinapterin (five). Opulencia et al. (6) indicated that the mtaA and mtaCB transcripts exhibited distinct stabilities, implying posttranscriptional regulation. mRNA stability can be a main determinant of posttran-Rscriptional control of gene expression (7, eight) and plays important roles in cellular adaptation, as a result of its prompt response to environmental changes (9). To investigate the effect of mRNA stability on cold-active methanol-derived methanogenesis, within this study, a psychrotolerant Methanosarcina mazei strain, zm-15, which performs both methylotrophic and aceticlastic methanogenesis, was isolated from the cold Zoige wetland in Tibet. We located that within this coldadapted organism, methanol supported cold-active methanogenesis more than acetate, which was attributed, no less than partially, for the longer life span of your mRNAs from the essential enzymes.Supplies AND METHODSSoil sample collection. Soil covered by Eleocharis valleculosa at a depth of 10 to 30 cm was collected from the Zoige wetland (336=N, 1022=E; altitude, three,430 to 3,460 m), located around the Tibetan Plateau, in April 2007. The soil samples were stored in sterile serum bottles sealed with butyl rubber stoppers (with N2 as the gas phase) and kept in an ice-cold box during transportation to the laboratory. DNA extraction, 16S rRNA sequencing, and phylogenetic evaluation. Total DNA was extracted in the soil samples (roughly 5 g) and purified using a FastDNA Spin kit for Soil (MP Biomedicals, Solon, OH, USA). The purified DNA was stored at 20 . For PCR amplification of methanogenic 16S rRNA genes, the methanogen-specific primers Met83F and Met1340R (see Table S1 in the sup-Received 24 October 2013 Accepted two December 2013 Published ahead of print 6 December 2013 Address correspondence to Xiuzhu Dong, [email protected]. Supplemental material for this short Insulin Receptor list article may be discovered at http://dx.doi.org/10.1128 /AEM.03495-13. Copyright 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AEM.03495-February 2014 Volume 80 NumberApplied and Environmental Microbiologyp. 1291aem.asm.orgCao et al.plemental material) have been made use of (10) with Taq DNA polymerase (TaKaRa, Otsu, Japan). The PCR parameters used had been as follows: denaturation at 94 for 7 min, followed by 30 cycles of denaturation (94 for 1 min), annealing (50 for 1 min), and extension (72 for 1.5 min) as well as a final extension at 72 for ten min. The PCR items have been purified with a PCR purification kit (Axygen, Tewksbury, MA, USA) and Nav1.3 custom synthesis cloned into a pMD18-T vector (TaKaRa) to construct a methanogen 16S rRNA gene library. The clones were sequenced by BioSune Inc. (Beijing, China). The 16S rRNA gene sequences had been checked for chimeras with DECIPHER (11). Clones with 97 similarity had been assigned as an operational taxonomic unit (OTU) working with MOTHUR (12) based on the distance matrix. The methanogenic 16S rRNA gene sequences have been then submitted towards the GenBank database to sear.
