C1-Oxidation Pathways in Proteobacteria: Function and Regulation

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This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Single carbon (C1) compounds commonly found in the environment include methane, methanol, methylated amines, halogenated methanes, and methylsulfonates. These compounds are of particular environmental concern as some of them (methane, methylsulfonates) directly contribute to the greenhouse effect, while others (methanol, and methylated amines) serve as precursors for methanogenesis. Microbial oxidation of methanol and methylated amines are important parts of environmental cycling of C and N as well as an essential step in preventing formation of greenhouse gases such as methane and N2O. While some of the pathways for primary oxidation of these C1 compounds are well established, others remain poorly characterized. The amino acid-mediated pathways for methylamine utilization were once discussed as an important part of both plant and microbial metabolism, however these pathways are still invisible for genome-based predictions. This research aims to obtain new genetic insights into the metabolic mechanisms of microbial utilization of methylamine and methanol. Uncovering these mechanisms will allow for development of probes for detecting these systems in the environment, and thus in turn will provide a more complete understanding of C1 cycling in the environment, in a global sense. The project will provide valuable insights into the fundamental understanding of metabolism of single carbon compounds, as well as the evolution of methylotrophy as a metabolic capacity. The purified and characterized enzyme will expand the database of enzymes for production of N-methyl amino acids (such as N-methyl-phenylalanine derivative and theanine) and could be further explored for industrial applications. Briefly, the specific aims include the following: Aim 1. Obtain draft sequence and annotate the genome of a novel methylotroph. Aim 2. Identify genes involved in methylamine and methanol oxidation via comparative gene expression studies and transposon mutagenesis. Aim 3. Identify proteins involved in primary oxidation of methylamine via protein purification and proteomic studies, complementing the data from Aim 2. Aim 4. Perform functional analysis of the genes of interest, using genetic and biochemical approaches. These analyses will complement the data from specific Aims 2 and 3 and ultimately confirm the activities and functions proposed as parts of Aims 2 and 3. Broader Impacts: This project will support extensive undergraduate research and will interface with existing outreach programs. This project will support the laboratory tradition of involving high school students in research projects, and provide them with opportunities to participate in biological discoveries. Graduate, undergraduate and high school students involved in the project will be encouraged to participate in two outreach programs at a local K-6 elementary school that include lectures describing the importance of bacteria for humans, plants and the environment as well as a "hands-on" science experience.