Genetic Analysis of Methylotrophic Methanogenesis in Methanosarcina Species

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The long-term goal of the research is to expand our knowledge regarding the physiology, metabolism and evolutionary adaptation of the methane-producing Methanosarcina species. Although the primary emphasis of these studies will be the identification and characterization of genes that directly and indirectly effect the utilization of methanol as a growth substrate (methylotrophic methanogenesis), the results are expected to have broad implications with respect to Methanosarcina and other methanogenic organisms. In particular, it is expected that these studies will reveal not only novel aspects of the methylotrophic pathway, but also how methanogenesis is integrated into the overall metabolism of the organism. Further, it is expected that a clearer understanding of archaeal transcriptional control will result from examination of the highly regulated methylotrophic genes. The experiments will be primarily based on recently developed methods for genetic analysis of Methanosarcina species; however, physiological, biochemical and molecular approaches will also be utilized to complement the genetic studies. This approach is expected to generate data that are both novel and complementary to the results of preceding studies. To achieve these aims Methanosarcina strains with mutations in a number of genes believed to be involved in methylotrophic methanogenesis will be constructed. These mutant strains will then be characterized with respect to a variety of physiological and biochemical traits. Random mutagenesis and proteomics approaches will be used to identify new genes that are specifically required for growth on methylotrophic substrates. Separate studies will examine the regulation of genes known to be involved and attempt to identify the genes that mediate this regulation. Finally, during the course of this work additional methods will be developed for genetic analysis of Methanosarcina species.