Physiology and Mechanism of Archaeal Nitrate Respiration

反硝化是一种独特的原核生物呼吸途径，在这个途径中硝酸盐被还原为大气中的氮气。这种呼吸模式对维持地球上的全球氮循环起着至关重要的作用。这项提议的目标是检查古生菌中的反硝化作用，因为这些微生物中的生物能量途径还没有被很好地理解。古生菌的新陈代谢与细菌一样多样，但由于它们居住在地球上的极端环境，它们也具有独特的生理特征。极端嗜热环境中的反硝化带来了几个挑战。首先，高温加剧了反硝化中间产物的毒性作用。其次，高温环境中富含钨，这种金属通常会使反硝化途径的关键酶-含钼硝酸还原酶失活。高温考古杆菌嗜氧高温杆菌将作为模式生物来研究古细菌反硝化作用的独特生物化学和生物能量学。在这个项目中，将使用生化、生物物理和分子方法相结合的方法来进一步表征含钼和钨的硝酸还原酶。此外，还将对被预测为新型酶的N2O还原酶和给电子体甲酸脱氢酶进行表征。由于钨在嗜氧假单胞菌反硝化的生理过程中起着重要作用，因此我们将对嗜氧假单胞菌中的钨吸收系统进行研究。最后，提出了关于反硝化途径基因操纵子结构和钨依赖基因调控的研究。广泛的影响：将从加州大学洛杉矶分校CARE计划招募代表不足的少数族裔学生，该计划为在学术和技术领域代表不足的个人提供培训。PI经常参加加州大学洛杉矶分校的暑期项目，该项目的学生通常会在PI的实验室继续他们的科学训练。此外，PI将继续为从她每年教授的大型微生物学入门课程招募的少数族裔学生提供研究机会。

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.