EAGER: Redox Regulation of Methanogenesis: A Basic Microbial Process at the Interface of Climate Change and Renewable Energy

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Intellectual merit: Methane is an important fuel and a major greenhouse gas linked to global warming. The current biological production of methane in nature is solely due to the methanogens a diverse group of strictly anaerobic microorganisms belonging to the newly recognized domain of life, the archaea. By generating methane in a process known as methanogenesis, these organisms facilitate the anaerobic degradation of complex polymers such as cellulose and starch that are produced by plants and, in so doing, play a central role in the global carbon cycle. Further, one of the modes of methanogenesis in which carbon dioxide and hydrogen are converted to methane and water is considered one of the most ancient forms of respiration on Earth. Although much has been learned about the biochemical pathways of methane formation, little is known as to how the process is regulated in response to change in environmental conditions. Importantly, it is not known how methanogens cope with sporadic exposure to oxygen, a common occurrence in their natural habitats. It is possible that, under moderate oxygen contamination, the organisms modify their cellular machinery so that methanogenesis is sustained--an important capability since methane formation represents their only source of energy. Preliminary studies indicate that modification of key enzymes by thioredoxin (Trx) plays a role in this control strategy. Trx is a ubiquitous regulatory protein that acts in partnership with an enzyme, Trx reductase, in the fine control of metabolism. However, this system remains unexplored in the methanogens, despite the fact that every member of this group carries genes with the potential for encoding these proteins. In this project we will test the concept that Trx/Trx reductase-based control is a determinant for the biological production of methane. We will utilize two methanogenic archaea as models: (i) Methanocaldococcus jannaschii, a deeply rooted autotroph that inhabits deep-sea hydrothermal vents and produces methane exclusively from carbon dioxide and hydrogen, and (ii) Methanosarcina mazei, a late-evolving freshwater methanogen with more diverse methanogenic capability. The latter organism will be investigated in collaboration with the University of Kiel (Germany). The objectives of the study are: (i) To characterize the putative Trxs and Trx reductases of M. jannaschii and M. mazei using proteins over-produced in a methanogen host, thereby providing in vivo relevance to the investigation; (ii) To identify the enzymes and other proteins that Trx modifies in these organisms; and (iii) To determine the effect of Trx-based modification on the activity of select methanogenic enzymes. The work will provide an entrée to broader contemporary questions relating to the control of methanogenesis in natural habitats and to how redox-based control systems of bacteria, animals and plants evolved from those originating in the archaea.Broader impact: This research will elucidate how methane-producing microorganisms cope with the challenging effects of exposure to oxygen in their habitats. Consequently, it will have broad societal and scientific implications relating to energy production, recycling of carbon in the biosphere and global warming. Greater insight into the regulation of methane formation raises the possibility of controlling its production in a useful manner?e.g., in bioreactors and sewage digesters. The project will integrate research and education through the training of two graduate students, four undergraduates, and a high school student. Undergraduate students and high school teachers from a current NSF-REU program directed by the PI ("Microbiology in the Post-Genome Era") will have an opportunity to participate in this research. Every effort will be made to recruit from underrepresented minority groups at all levels. The collaborative training will broaden the research experience of graduate students in the participating laboratories at Virginia Tech, University of Kiel and University of California-Berkeley. Its scientific and societal relevance coupled with the participation of a diverse group of investigators at different career stages will enrich the project as well as the research experience of all participants.