William W. Metcalf
Genetic Analysis of Methanogenesis in Methanosarcina
Research in my laboratory involves the use of molecular, genetic, and biochemical approaches to study novel aspects of metabolism used by microorganisms involved in the global cycling of the carbon and phosphorus.
My primary research interest is in the development and application of methods for genetic analysis of the methanogenic Archaea. These organisms play a crucial role in the global carbon cycle by recycling organic carbon compounds from anaerobic environments into the atmosphere as methane gas. Further, their study is relevant to a number of human problems including; global warming by production of a key greenhouse gas, use for production of methane fuel from biological materials, and waste treatment.
Although the basic biochemical pathways for methanogenesis have now been elucidated, relatively little is known about other aspects of these unique and important prokaryotes. This has been due, in large part, to a lack of genetic tools for analysis of methanogenic Archaea. To address this deficiency we have developed a variety of genetic methods for use in the genus Methanosarcina. The most significant of these methods is the development of a broad host range, plasmid shuttle vector capable of replication in a number of Methanosarcina species and in Escherichia coli. Using this vector a highly efficient transformation protocol for Methanosarcina species has been developed. An efficient method for random mutagenesis of the Methanosarcina chromosome is under development. These achievements, along with other advances in the field, have paved the way for true genetic analysis of methanogenic Archaea.
These new genetic methods, as well as more traditional biochemical and molecular techniques are being used in our laboratory to study the novel aspects of the metabolism of methanogenic Archaea. Our target organism for these studies is Methanosarcina barkeri, because unlike the majority of methanogens, Methanosarcina species are capable of growth on a variety of substrates. This metabolic diversity makes them an especially attractive target for genetic analysis. Further, non-methanogenic growth has recently been demonstrated in Methanosarcina. Thus, with the development of workable genetic techniques for these organisms, wide areas of metabolism, including the methanogenic pathway itself, can be targets for mutagenic analyses. I am particularly interested in the role of gene regulation and structural elements in methanogenesis, biosynthesis of the unique enzyme cofactors used in methanogenesis, and the entry of various compounds into the methanogenic pathway.
My second research interest lies in the area of phosphorus metabolism: specifically the question of whether a biological redox cycle for this important nutrient exists, and, if so, the nature of the organisms responsible and the metabolic pathways involved. Our research clearly indicates that such a cycle exists. We have isolated numerous organisms that are capable of oxidizing reduced phosphorus compounds. These microorganisms were identified by standard biochemical means, and by sequence analysis of 16s rDNA. One of these organisms was shown to be a strain of Pseudomonas stutzeri. Others fall into the Rhizobium and Mycobacterium/Nocardia groups. The means by which P. stutzeri oxidizes the reduced phosphorus compounds phosphite and hypophosphite is currently under examination by cloning and mutant analysis of the required genes for both traits. This process appears to be surprisingly complex because the minimal plasmid clones encoding these traits are no less than 25 kilobase pairs in size. Our studies suggest that oxidation of hypophosphite to phosphate involves a phosphite intermediate. DNA sequence analysis of the genes required for phosphite oxidation has been completed, and biochemical analysis of the enzymes is in progress. In addition to these results, we, and others, have shown production of reduced phosphorus compounds by environmental samples. Preliminary experiments suggest that we may be able to isolate organisms capable of carrying out the reactions for these and other steps in the phosphorus redox cycle. These will certainly merit further study since they undoubtedly involve novel metabolism and biochemistry.