Nitrate Respiration of the Hyperthermophile Pyrobaculum aerophilum

Schroeder超嗜热古菌生活在火山环境中，如热液喷口或温泉，可能类似于地球生命起源时的条件。 超嗜热的海洋细菌Pyrobaculum aerophilum属于系统发育树的深分支，表明这种生物相对古老。 本研究的目的是研究嗜氧假单胞菌的反硝化机理。反硝化作用是一种独特的原核途径，允许微生物将硝酸盐转化为双氮气体。它是自然界中从固定的氮氧化物产生氮气的唯一途径，因此对于维持地球上的全球氮循环至关重要。 硝酸盐的呼吸与细胞繁殖所需的能量的产生有关。 该途径在细菌和细菌中都有发现，这表明反硝化作用最有可能在最后一个共同祖先之前进化。 P. aerophilum代表了迄今为止分离出的最古老的微生物，并提供了将古代过程与当今现代微生物中发现的过程进行比较的机会。 反硝化途径在革兰氏阴性菌中得到了很好的描述，其中需要两种膜结合的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，提出了嗜氧假单胞菌的不同脱氮机制：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。 生物化学，生物物理和分子技术的结合将被用来测试这一假设，并进一步我们的了解hyperthermophlic的生理。该项目的目标是进一步表征硝酸还原酶，这是有趣的，因为它的新的膜锚。 为了获得更完整的反硝化途径的图片，两个额外的反硝化途径酶，亚硝酸还原酶和NO还原酶，将被纯化和表征。 为了便于将来亚硝酸盐和NO还原酶的结构分析，将克隆和过表达nir和nor基因。 由于嗜气假单胞菌的DNA序列将在不久的将来可用，因此将分析参与反硝化途径的选定基因组的响应于选定和限定的环境生长条件的差异表达。这项研究的结果将进一步加深我们对呼吸过程的了解，呼吸过程是生命的基本功能。对一个古老的电子元的研究可能会让我们深入了解电子转移反应是如何演变的。对不同生物体中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。

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.