Expanding the genetic toolbox for methanogenic archaea and investigating ferredoxin redox circuit formation in Methanosarcina acetivorans

Methane-producing archaea (methanogens) occupy a number of anoxic niches from soil sediments to the gastrointestinal tracts of numerous animals. In these anaerobic environments methanogens have specialized in converting simple substrates like carbon dioxide (CO2), formate, and acetate to methane (CH4). CH4 is an important component of the global carbon cycle but is also a potent greenhouse gas. While some of the CH4 evolved from methanogenesis is converted to CO2 by aerobic methane oxidizing organisms, the rest is released into the atmosphere, where it can have severe environmental impacts. Beyond these environmental consequences, methanogens inhabiting the human gastrointestinal tract have been linked to disease states including obesity and irritable bowel syndromes indicating methanogens may have important impacts on human health. Although much progress has been made to understand methanogen cell biology, genetics, physiology, and metabolism, much remains unknown about these fundamental attributes. The work presented in this Thesis addresses some of these aspects. In Chapter 2, I report the finding that a single ferredoxin is the primary electron donor for phospholipid saturation in Methanosarcina acetivorans, enhancing our understanding of ferredoxin dependent redox circuits and lipid biosynthesis in methanogens. In Chapter 3, I report the sat gene as a novel positive selectable marker for Methanosarcina spp. This work has expanded the genetic toolbox for Methanosarcina spp. and provides a novel selection system that may be used for the development of genetic tools for other methanogens as well. Finally, in Appendix A I report my progress towards developing tools for the human gut-associated methanogen M. smithii. Although we were not successful at constructing M. smithii mutants during the course of this work, our preliminary results could aid efforts to make this organism genetically tractable in future studies.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste