Denitrification is a uniquely prokaryotic respiratory pathway, in which nitrate is reduced to atmospheric N2. This mode of respiration plays an essential role in the maintenance of the global nitrogen cycle on earth. The goal of this proposal is to examine denitrification in the archaea, since bioenergetic pathways are not well understood in these microbes. The archaea are metabolically as diverse as the bacteria, but also possess unique physiological features due to the extreme environments that they inhabit on Earth. Denitrification in extreme thermophilic environments presents several challenges. First, the toxic effect of the denitrification intermediates is enhanced because of the high temperature. Second, hot environments are enriched with tungsten, a metal that typically inactivates the molybdenum-containing nitrate reductase that is a key enzyme of the denitrification pathway.
The hyperthermophilic archaeon Pyrobaculum aerophilum will be used as a model organism to examine the unique biochemistry and bioenergetics of archaeal denitrification. In this project a combination of biochemical, biophysical and molecular approaches will be employed to further characterize the molybdenum and tungsten-containing nitrate reductase. In addition, the N2O reductase, which is predicted to be a novel enzyme, as well as the electron donating formate dehydrogenase will be characterized. Since tungsten plays a major role in the physiology of P. aerophilum denitrification, the tungsten-uptake system in P. aerophilum will be investigated. Finally, studies concerning operon structure and tungsten-dependent gene regulation of denitrification pathway genes are proposed.
Broader Impact: Underrepresented minority students will be recruited from the UCLA CARE program, which provides training for underrepresented individuals in academic and technological fields. The PI has routinely participated in the UCLA Care's summer program and students from this program typically continue their scientific training in the PI's laboratory. In addition, the PI will continue to provide research opportunities to minority students recruited from the large Introductory Microbiology class that she teaches yearly.
