Denitrification Linked to Methanol Oxidation in Methylotenera Mobilis: a New Paradigm of Methylotrophy

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Intellectual Merit:Methylotenera mobilis is a recently described bacterium that is a prominent member of the active methylotroph community in Lake Washington sediment. More recent experiments suggested that M. mobilis and related bacteria carry out denitrification linked to methanol oxidation, in the presence of oxygen, resulting in emission of N2O. This is a novel process that may be an important part of the nitrogen cycle in lakes. Genomic analysis of M. mobilis identified few traditional components of the classic denitrification pathway, suggesting involvement of alternative enzymes. The bacterium also lacks a traditional methanol dehydrogenase. This phenomenon is interesting for a number of reasons. First, this is the first case of denitrification by a member of the Methylophilaceae family; second, oxygen-dependent denitrification is unusual in the microbial world; third, denitrification by Methylotenera and related organisms may represent an important biogeochemical process that has been overlooked, accounting for a portion of N2O released into the atmosphere from the environments active in C1 metabolism, such as freshwater lakes. The major goal of this project is to understand the biochemistry and the physiology of denitrification by Methylotenera and to uncover the specific nature of coupling of denitrification to methanol oxidation. This work will present new paradigms of both methylotrophy and denitrification and will provide new insights into the connection between the global cycles of carbon and nitrogen. M. mobilis will be the model system. The specific objectives are 1) identification of genes, enzymes and pathways involved in denitrification and methanol oxidation, 2) testing the functions of the genes predicted to be involved in both methanol oxidation and denitrification, via mutation, and 3) purification and characterization of one or more key enzymes involved in denitrification by M. mobilis, the primary candidate being a novel periplasmic nitrate reductase. The intellectual merit of this proposal is in obtaining biochemical insights into methylotrophy linked to denitrification, transforming the paradigms of both processes and broadening the knowledge on energy-generating pathways in microbes in general. Broader Impacts:This project will involve graduate, undergraduate and high school students, with special emphasis on the members of underrepresented groups. Established partnerships with local schools and colleges will provide the public outreach. The data will be broadly disseminated via educational materials, conference presentations, scientific and popular publications and the dedicated web site. The societal impact will be in providing biochemical and genomic insights into a novel, previously overlooked biogeochemical process that potentially contributes to climate change.