CALIFORNIA INSTITUTE OF TECHNOLOGY

Although small in scale, microorganisms have a profound and fundamental role in the cycling of carbon and energy on our planet, Integral to the success of these diverse and abundant forms of life are complex microbial interactions and syntrophic associations, occurring on the scale of micrometers. We are only now beginning to fully appreciate the diversity and pervasiveness of syntrophy in nature, the majority of these intimate metabolic interactions occurring between microbial taxa that canno t be grown in the laboratory and thus require the use of new culture-independent molecular approaches. Syntrophic relations are central to the biogeochemical cycling of carbon in anoxic environments, including the regulation of methane emissions from continental margin sediments. Molecular and isotopic investigations of methane-rich anoxic environments have characterized unique lineages of uncultured archaea (ANME's) in association with distinct lineages of sulfate-reducing bacteria, wh o together mediate sulfate-coupled methane oxidation (AOM). Despite their global importance, our knowledge of their function and capabilities in nature is limited. To advance our understanding of the genetic, phenotypic, and metabolic differences underlying these as yet uncultured methanotrophs and their syntrophic bacterial partners, we propose to use a multi-disciplinary methodological approach, that uses culture-independent molecular and metagenomic analyses, integrating a recently develope d technique called Magneto-FISH, that enables the selective enrichment of intact methanotrophic archaea and their physically associated microbial partners from methane-rich sediments, combined with high resolution isotopic and elemental analysis using nanometer secondary ion mass spectrometry (nanoSIMS) allowing for quantitative isotopic and elemental analysis of single microbial cells. A major goal of this work is the optimization of methods that directly couple molecular visualization methods including phylogenetic 'stains' (e.g., rRNA targeted fluorescence in situ hybridization, FISH) and immunocytochemical localization of expressed metabolic proteins (for example methyl coenzyme M reductase; dissimilatory (bi) sulfate reductase; and nitrogenase) with stable isotope anaylsis of individual microorganisms by FISH-nanoSIMS while maintaining critical spatial information on the structural organization of AOM consortia. Through the development and application of these compli mentary and state-of-the-art methodologies, our broad research objectives for this proposal include: (1) Characterizing the diversity and metabolic potential of physically-associated microbial partners believed to be capable of syntrophically mediated anaerobic methane oxidation using Magneto-FISH and targeted metagenomics. (2) Testing hypotheses regarding the metabolism, interspecies interactions and environmental adaptation of structurally and phylogenetically diverse AOM consortia using sta ble isotope labeling experiments coupled with nanoSIMS ion mapping of syntrophic consortia. Results form this work have the potential to advance our understanding of syntrophic partnerships at the level of single cells and, more specifically, will provide fundamental information on the diversity and metabolic properties of methane-oxidizing syntrophic microorganisms that are necessary for refining predictive models of biological methane cycling.

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