Genetic studies of carbon dioxide fixation in the methane-producing archaeon Methanococcus maripaludis
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Methanococcus maripaludis is a facultative autotrophic methanogen that utilizes H2 + CO2 for carbon fixation and energy production. In CO2 fixation, acetyl-CoA is the precursor of cell carbon. To study autotrophic CO2 fixation pathways, acetate auxotrophs of M. maripaludis were generated by random mutagenesis. The mutagenesis was performed by disruptions of chromosomal genes with a plasmid library in the vector, pMEB.2, and screening by replica plating. JJ117 was one of the nine conditional acetate auxotrophs found and was auxotrophic for both acetate and cobalamin. However, the disrupted gene (ppm: phosphopentomutase) in JJ117 did not seem likely to be involved in the cobamide biosynthetic pathway. Cloning and sequencing of the downstream sequence of ppm found cbiJ, which is convergently transcribed. Thus, the cobamide auxotrophy may have been caused by an effect on cbiJ expression. The cbiJ was mutagenized by gene replacement, and the resulting mutant was an acetate and cobalamin auxotroph. To understand how acetate supports the growth of JJ117, the cobamide in wild type and JJ117 was quantified by a bioassay. The assay showed that JJ117 produced a small amount of cobamide that might be sufficient for cobamide – dependent activities that are essential for growth. To explain the acetate auxotrophy, acetate is hypothesized to spare the requirement of cobamide by its conversion to acetyl-CoA without cobamide. Previous research indicated the possible involvement of the act (pyruvate-formate lyase activating enzyme) or ehb (energy-conserving hydrogenase B) in the pathway of autotrophic CO2 fixation. These genes were mutagenized by gene replacement and the mutants were characterized. Growth of mutant strains, JJ310 and JJ320, which have mutations in act, was stimulated by acetate. However, more research is required to clarify the roles of the act. Mutagenesis of ehb in M. maripaludis generated an acetate auxotroph, S40, suggesting that the gene is involved in autotrophic CO2 fixation. In addition, methanogenesis from pyruvate was also inhibited in S40, indicating that the hydrogenase encoded by ehb may be linked to the activity of pyruvate oxidoreductase (POR). These data are consistent with previous reports that show that this hydrogenase is coupled to endergonic reactions by energy conservation.