| Abstract |
Methylotrophic methanogenesis predominates at low temperatures in the cold Zoige wetland in Tibet. To elucidate the basis of cold-adapted methanogenesis in these habitats, Methanosarcina mazei zm-15 was isolated, and the molecular basis of its cold activity was studied. For this strain, aceticlastic methanogenesis was reduced 7.7-fold during growth at 15°C versus 30°C. Methanol-derived methanogenesis decreased only 3-fold under the same conditions, suggesting that it is more cold adaptive. Reverse transcription-quantitative PCR (RT-qPCR) detected <2-fold difference in the transcript abundances of mtaA1, mtaB1, and mtaC1, the methanol methyltransferase (Mta) genes, in 30°C versus 15°C culture, while ackA and pta mRNAs, encoding acetate kinase (Ack) and phosphotransacetylase (Pta) in aceticlastic methanogenesis, were 4.5- and 6.8-fold higher in 30°C culture than in 15°C culture. The in vivo half-lives of mtaA1 and mtaC1B1 mRNAs were similar in 30°C and 15°C cultures. However, the pta-ackA mRNA half-life was significantly reduced in 15°C culture compared to 30°C culture. Using circularized RNA RT-PCR, large 5' untranslated regions (UTRs) (270 nucleotides [nt] and 238 nt) were identified for mtaA1 and mtaC1B1 mRNAs, while only a 27-nt 5' UTR was present in the pta-ackA transcript. Removal of the 5' UTRs significantly reduced the in vitro half-lives of mtaA1 and mtaC1B1 mRNAs. Remarkably, fusion of the mtaA1 or mtaC1B1 5' UTRs to pta-ackA mRNA increased its in vitro half-life at both 30°C and 15°C. These results demonstrate that the large 5' UTRs significantly enhance the stability of the mRNAs involved in methanol-derived methanogenesis in the cold-adaptive M. mazei zm-15.
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