Regulatory Interplay between Nitrogen and Methane Metabolism: New Frontiers in Product Discovery, Physiology and Ecology of Methanotrophic Bacteria

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Metabolism of single-carbon compounds is an ancient and widespread biological function, yet only methylotrophic bacteria exclusively utilize single-carbon compounds as sources of energy and carbon. Methanotrophic (i.e. methane-eating) methylotrophs are essential as the Earth’s biological methane sink, which is becoming critical to understand as greenhouse gases continue to accumulate during this period of rapid global change. Comparison of over 30 methanotroph genome sequences revealed a rich diversity of inventory for nitrogen metabolism that is intimately interwoven with carbon metabolism. The overarching hypothesis of the proposed research is that nitrogen source and availability predictably control carbon metabolism in methanotrophic bacteria, regardless of genomic variability, enabling students in my laboratory to: 1) determine how nitrogen metabolism regulates carbon transformations into value-added end products for industrial exploitation, 2) characterize enzymes, functionality, and regulation for our recently discovered pathway of methane-dependent denitrification, and 3) develop discrete molecular markers to determine the presence, strength, and relevance of methane-dependent denitrifiers in regulating greenhouse gas fluxes in diverse environments. Our laboratory-based approaches utilize a large genome-sequenced strain collection that we curate and maintain along with a suite of analytical tools to measure cell growth in batch and continuous culture, real-time trace gas measurements, gene expression levels, and metabolite and protein production. Our goal in Objective 1 is to determine how N-source and availability gate carbon into molecules of industrial relevance like fatty acids, isoprenoids, N-storage polymers, C-storage polymers, and organic acids. Growth kinetics and analysis of nucleic acids, proteins, and metabolites from cultures grown with variable combinations of nitrogen and carbon sources will enable discovery of products for industrial exploitation. Objective 2 will determine the molecular regulation of methane-dependent denitrification from nitrate, a novel physiology discovered in my laboratory, that connects the metabolism of two potent greenhouse gases (methane and nitrous oxide) and a major environmental pollutant (nitrate) within a single organism. Using physiological, transcriptomic and proteomic tools, our aim is to fully characterize the enzymatic diversity and physiological benefit of this activity and to predict, based on gene content and regulatory features, whether a particular strain should have this physiology. Objective 3 will reveal the ecological relevance of methane-dependent denitrification and the microorganisms that perform it in diverse environments from cow rumen to thawing permafrost. Denitrifying methanotrophs possess phylogenetically distinct genes that we aim to find in environments where nitrous oxide production at the expense of methane consumption has been noted. Together, the proposed research fully employs our genome-sequenced collection of methanotrophic bacteria and analytical capacity to a) interface with the energy sector in Canada by converting industrial waste products into value-added products, b) characterize the enzymatic diversity and function of a novel physiology in methanotrophic bacteria, and c) provide tools to assess the ecological significance of methane-dependent denitrification as a novel linkage between the carbon and nitrogen biogeochemical cycles. In addition to developing a potentially lucrative and ecologically important area of research for Canada and the broader scientific community, this work offers a unique training experience for students of microbiology and biotechnology.