Methanogenesis is critically important as a key step in the global carbon cycle and in the production of a significant greenhouse gas. It is also at the center of alternative fuel strategies and is essential in both agriculture and the waste treatment industry. Methanosarcina species are believed to be among the most significant methane producers, all of which are members of the so-called "third form of life" known as the Archaea. Despite their importance, relatively little is known about these unusual organisms, or about the process of methane production (methanogenesis). The aim of the proposed research is to use genetic analysis to investigate the metabolic processes involved in methanogenesis by members of the genus Methanosarcina. Such studies have not previously been possible due to a lack of robust genetic methods for these unique organisms; however, recently developed methods now make this possible. The application of genetic methodologies to the study of methanogenesis is likely to reveal novel aspects of the methanogenic process that have not been addressed by the biochemical methods used to study this process to date. By using a genetic approach, our analysis will identify not only the main methanogenic pathways, but also the metabolic, regulatory, and structural components that directly and indirectly effect methanogenesis. The experiments to be conducted will take advantage of the metabolic diversity of Methanosarcina species relative to other methanogenic organisms. This metabolic diversity should allow isolation of mutants that are incapable of methanogenesis fueled by certain substrates while retaining the ability to utilize others. Importantly, Methanosarcina species are the only genetically tractable methanogenic organisms for examining this metabolic diversity. Moreover, the tools we have developed should have broad application to the Archaea in general, and to more distantly related organisms for which methods of genetic analysis are lacking. The specific goals of the project are; 1) genetic analysis of Methanosarcina species using these recently developed, but proven, genetic methods, and 2) the refinement and modification of currently available genetic tools to simplify the execution of these experiments and to expand the range of the studies possible in this organism. This project is aimed at developing and utilizing genetic tools for examining the metabolic basis of methanogenesis in the archaeon Methanosarcina. This is an extremely important process about which little is known. The potential outcomes of this work are an understanding of the genetic basis of methanogenesis and the development of molecular genetic tools to examine other members of the phylogenetic domain.
