| Methane is one of the most important greenhouse gases and methanogenic archaea living in anoxic rice soils are major contributors. Methanocellales are widespread in paddy field soils and play the key role in methane production. These methanogens feature largely in their adaptation to low H2and syntrophic growth with anaerobic fatty acids oxidizers. The adaptive mechanisms, however, remain unknown. Therefore, we chose Methanocella conradii HZ254, a strain recently isolated from paddy field soil as a model organism and construted syntrophic cocultures with Pelotomaculum thermopropionicum SI (propionate syntroph) and Syntrophothermus lipocalidus TGB-C1(butyrate syntroph). By employing qRT-PCR and strand specific RNA-Seq, we explored the reponse of M. conradii to low H2and syntrophic growth at the transcriptional level. The main results are as followings:(1) Syntrohic coculturs between M.conradii and P. thermopropionicum and S. lipocalidus were successfully constructed and the growth of them were stable. Comparison of the relative transcript abundances showed that three hydrogenase encoding genes and all methanogenesis related genes tested were up-regulated in cocultures relative to monoculture. The genes encoding for bifurcation mediating complex Mvh/Hdr/Fwd and the membrane-bound energy converting hydrogenase (Ech) were the most up-regulated among the evaluated genes. The co-expression of Mvh/Hdr/Fwd conding gene cluster was also determined by intergenic region amplification. The expression of formate dehydrogenase (Fdh) encoding gene was also significantly up-regulated. By contrast, acetate assimilation gene was down-regulated in cocultures.(2) Transcriptome analysis of syntrophic propionate coculture by RNA-Seq revealed that M. conradii and P. thermopropionicum enhanced the expression of energy conservation related pathways under syntrophic conditions. The results of M. conradii between qRT-PCR and RNA-Seq were the same. Besides, we also found the up-regualtion of A-type ATP synthase and a novel Ech-like/Hdr’ complex which is unique to Methanocella species. IGV view of mapping data confirmed the gene expression pattern and gene structure of Mvh/Hdr/Fwd and Ech-like/Hdr’. P. thermopropionicum remarkably up-regualted the expression of its central catabolic-pathway (MMC pathway). Due to the thermodynamic limits, the growth of M. conradii and P. thermopropionicum were slower under coculture conditions. Accordingly, the genes related to anabolism and biosynthesis were significantly down-regulated in M. conradii and P. thermopropionicum. M. conradii possessed complete oxidative branch of TCA cycle and related genes were also significantly down-regulated.(3) Formate played the major role in syntrophic cocultures. Besides the significantly up-regulation of M.conradii Fdh, the markedly up-regulation of Fdh in P. thermopropionicum also proved this. The utilisation of formate by M.conradii in the presence of H2, the inhibition of syntrophic propionate oxidation by formate but not by H2all confirmed the major role of formate during syntrophic propionate oxidation.(4) RNA-Seq revealed multiple interactions between M. conradii and P. thermopropionicum. The up-regulation of ABC type ammonia acid transporters and flagellum encoding genes in P. thermopropionicum and active expression of ammonia acid transporters in M. conradii indicated the possible of metabolite transfer other than formate or H2and direct signal transduction.It was the first time that gene expression study was conducted for Methanocella spp. isolates. Our results revealed responses of M. conradii and P. thermopropionicum to low H2and syntrophic growth at the transcriptional levels and multiple interactions between M. conradii and P. thermopropionicum. These results broaden and deepen our understanding of adaptive mechanisms of Methanocella spp. to their natural niches. |
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