Physiology and Mechanism of Archaeal Nitrate Respiration

反硝化作用是一种独特的原核生物呼吸途径，其中硝酸盐被还原为大气N2。 这种呼吸模式在维持地球上的全球氮循环中起着至关重要的作用。这个提议的目的是研究古细菌中的反硝化作用，因为这些微生物中的生物能途径还不清楚。古生菌在代谢上与细菌一样多样，但由于它们在地球上居住的极端环境，它们也具有独特的生理特征。极端嗜热环境中的反硝化提出了几个挑战。 首先，由于高温，反硝化中间体的毒性作用增强。 第二，高温环境中富含钨，这种金属通常会使含钨的硝酸还原酶失活，而硝酸还原酶是反硝化途径的关键酶。超嗜热古菌Pyrobaculum aerophilum将被用作模式生物，以研究古菌反硝化的独特生物化学和生物能量学。在这个项目中，生物化学，生物物理和分子方法的组合将被用来进一步表征钼和含钨硝酸还原酶。 此外，N2O还原酶，这被预测为一种新的酶，以及电子供体甲酸脱氢酶的特点。由于钨在嗜氧假单胞菌反硝化生理中起着重要作用，因此将研究嗜氧假单胞菌中的钨吸收系统。最后，研究有关操纵子结构和钨依赖的基因调控的反硝化途径genes.Broader影响：代表性不足的少数民族学生将被招募从加州大学洛杉矶分校的关怀计划，这为代表性不足的个人在学术和技术领域提供培训。PI定期参加UCLA Care的夏季计划，该计划的学生通常在PI的实验室继续他们的科学培训。 此外，PI将继续提供研究机会，少数民族学生从大型微生物学导论类，她教每年招募。

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.