Physiology and Mechanism of Archaeal Nitrate Respiration

反硝化是一种独特的原核呼吸途径，其中硝酸盐被还原为大气中的N2。这种呼吸方式在维持地球上的全球氮循环中起着至关重要的作用。本提案的目的是研究古细菌的反硝化作用，因为生物能量途径在这些微生物中还没有得到很好的理解。古细菌在代谢方面和细菌一样多样化，但由于它们生活在地球上的极端环境，它们也具有独特的生理特征。在极端嗜热环境中反硝化提出了几个挑战。首先，脱氮中间体由于温度高，毒性作用增强。其次，高温环境富含钨，这种金属通常会使含钼的硝酸还原酶失活，而硝酸还原酶是反硝化途径的关键酶。超嗜热古细菌嗜气焦杆菌将被用作模型生物来研究古细菌反硝化的独特生物化学和生物能量学。本项目将采用生化、生物物理和分子相结合的方法进一步表征含钼钨的硝酸还原酶。此外，还对N2O还原酶和甲酸脱氢酶进行了表征，N2O还原酶被预测为一种新型酶。由于钨在嗜气假单胞菌的反硝化生理中起着重要作用，因此将对嗜气假单胞菌的钨吸收系统进行研究。最后，对脱氮途径基因的操纵子结构和钨依赖基因调控进行了研究。更广泛的影响：将从加州大学洛杉矶分校关怀项目中招募未被充分代表的少数族裔学生，该项目为学术和技术领域未被充分代表的个人提供培训。PI定期参加加州大学洛杉矶分校的夏季项目，该项目的学生通常在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中招募的少数民族学生提供研究机会。

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.