Date of Graduation
Doctor of Philosophy in Cell & Molecular Biology (PhD)
Second Committee Member
Third Committee Member
Fourth Committee Member
Nitrogenase is the metalloenzyme only found in bacteria and archaea that is essential for biological nitrogen fixation (diazotrophy), but it can also serve as a catalyst in biofuel production. All diazotrophs contain a molybdenum (Mo) nitrogenase, while some species contain additional alternative nitrogenases where either vanadium (V) or iron (Fe) replace Mo in the active site cofactor. Nitrogen fixation by bacteria has been extensively studied. The limited investigation of nitrogen fixation in methanogenic archaea (methanogens) indicates production of nitrogenase is simpler than in bacteria and methanogen nitrogenase has different biochemical properties. Thus, methanogen nitrogenases provide a promising alternative for genetic engineering of nitrogen fixation in in heterologous hosts (e.g., crop plants) and in biofuel production. To exploit the use of methanogen nitrogenases in biotechnological applications, it is necessary to fully understand nitrogenase regulation, maturation, and catalysis. This dissertation investigates nitrogenase regulation in Methanosarcina acetivorans, a methanogen that contains all three nitrogenase isozymes. Expression studies demonstrate that nitrogenase regulation in M. acetivorans is unique; all three nitrogenases can be produced at once, whereas other diazotrophs typically produce one nitrogenase at a time based on metal availability. Results from mutational studies reveal that the production of Mo-nitrogenase is required for expression of V- and Fe-nitrogenases in the absence of Mo, despite Mo-nitrogenase unable to support nitrogen fixation without Mo. Additional mutational and expression studies identified ModE as the Mo-dependent repressor of vnf and anf operons encoding the V- and Fe-nitrogenases, respectively. Lastly, the physiological role of uncharacterized nitrogenase-like proteins MA2032-33 and MA1631-33 was examined, pointing to potential functions in nitrogen fixation and carbon specific methanogenesis. Overall, these results provide substantial insight into the regulation of M. acetivorans nitrogenases, revealing previously unknown complexity.
Chanderban, M. (2022). Molecular Characterization of Nitrogenase Regulation in Methanosarcina acetivorans. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/4763