Nitrate Respiration of the Hyperthermophile Pyrobaculum aerophilum

嗜热热杆菌的硝酸盐呼吸

• 批准号: 0091351
• 负责人: Imke Schroeder
• 金额: $ 33万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-15 至 2004-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0091351&HistoricalAwards=false
• 关键词: Nitrate; Respiration; Hyperthermophile; Pyrobaculum; aerophilum

SchroederHyperthermophlic archaea inhabit volcanic environments such as hydrothermal vents or hot springs that may resemble conditions when life on earth originated. The hyperthermophilic, marine Archaeon Pyrobaculum aerophilum belongs to a deep branch of the phylogenetic tree suggesting that this organism is relatively ancient. The objective of this research project is to study the mechanism of denitrification in the Archaeon P. aerophilum. Denitrification is a unique prokaryotic pathway that allows the microbe to convert nitrate into dinitrogen gas. It is the only pathway in nature that generates nitrogen gas from fixed N oxides and thus it is essential for the maintenance of the global nitrogen cycle on earth. Respiration of nitrate is coupled to the generation of energy that fuels cell propagation. The pathway is found in both the domain Bacteria and the domain Archaea suggesting that denitrification evolved most likely before the last common ancestor. P. aerophilum represents the oldest denitrifier isolated thus far and provides the opportunity to compare an ancient process with that found in today's modern microbes. The denitrification pathway is well described in Gram-negative bacteria, where two membrane-bound and two soluble enzyme complexes as well as several soluble electron mediating proteins are required. Based on previous results in the PI's laboratory a different mechanism for denitrification is proposed for P. aerophilum: Four membrane-bound enzyme complexes exist that all interact with a menaquinone pool to reduce nitrate to N2 gas. A combination of biochemical, biophysical and molecular techniques will be employed to test this hypothesis and further our understanding of the physiology of hyperthermophlic Archaea. The objectives of this project are to further characterize the nitrate reductase enzyme, which is interesting because of its novel membrane anchor. To obtain a more complete picture of the denitrification pathway, two additional denitrification pathway enzymes, the nitrite reductase and the NO reductase, will be purified and characterized. To facilitate future structural analysis of the nitrite and NO reductases the nir and nor genes will be cloned and overexpressed. Since the DNA sequence of P. aerophilum will be available in the near future a selected set of genes involved in the denitrification pathway will be analyzed for their differential expression in response to selected and defined environmental growth conditions. The results from this research will further our knowledge about respiratory processes that are essential functions of life. The study of an ancient Archaeon may give insight into how electron transfer reactions have evolved. The investigation of the denitrification process in diverse organisms will further our ability to understand, predict and deal with global environmental changes that include the nitrogen cycle.

超嗜热古细菌生活在火山环境中，如热液喷口或温泉，这些环境可能类似于地球上生命起源的条件。嗜热的海洋古细菌嗜气焦杆菌属于系统发育树的一个深分支，表明这种生物相对古老。本课题旨在研究嗜气古菌的反硝化作用机理。反硝化是一种独特的原核生物途径，允许微生物将硝酸盐转化为二氮气体。它是自然界中唯一由固定氮氧化物生成氮气的途径，因此它对维持地球上全球氮循环至关重要。硝酸盐的呼吸作用与细胞繁殖所需能量的产生有关。该途径在细菌和古生菌领域都被发现，这表明反硝化作用很可能在最后一个共同祖先之前进化。嗜气杆菌代表了迄今为止分离出的最古老的反硝化菌，并提供了将古代过程与现代微生物中发现的过程进行比较的机会。革兰氏阴性菌的反硝化途径得到了很好的描述，其中需要两种膜结合酶和两种可溶性酶复合物以及几种可溶性电子介导蛋白。根据PI实验室先前的结果，提出了嗜气假单胞菌反硝化的不同机制：存在四种膜结合酶复合物，它们都与甲基萘醌池相互作用，将硝酸盐还原为N2气体。我们将采用生物化学、生物物理和分子技术的结合来检验这一假设，并进一步了解嗜热古菌的生理学。该项目的目的是进一步表征硝酸还原酶，这是有趣的，因为它的新颖的膜锚。为了更全面地了解反硝化途径，我们将对另外两种反硝化途径酶——亚硝酸盐还原酶和NO还原酶进行纯化和表征。为了便于将来亚硝酸盐和NO还原酶的结构分析，nir和nor基因将被克隆并过表达。由于嗜气假单胞菌的DNA序列将在不久的将来可用，因此将对一组参与反硝化途径的基因进行分析，以确定其在选定和确定的环境生长条件下的差异表达。这项研究的结果将进一步加深我们对生命基本功能呼吸过程的认识。对古细菌的研究可以使我们深入了解电子转移反应是如何演变的。对不同生物的反硝化过程的研究将进一步提高我们理解、预测和处理包括氮循环在内的全球环境变化的能力。