Novel Responses to Oxygen by Anaerobic Microorganisms

描述(由申请人提供)：根据定义，无论是原核生物还是真核生物，都需要分子氧来保存能量。然而，这是喜忧参半的，因为在正常新陈代谢过程中会产生极具活性的氧衍生物，可能会损害所有细胞成分。这些所谓的活性氧物种(ROS)与多种慢性和传染性人类疾病有关，包括癌症、阿尔茨海默病、关节炎和艾滋病，但ROS也被用作抵抗病原体的防御系统和信号转导途径。了解微生物对氧气的反应对厌氧病原体引起的疾病的治疗具有直接的影响。1999年，我们提出厌氧菌对ROS有一种新的反应，其中一种名为超氧化物还原酶(SOR)的非血红素铁蛋白在其中发挥了关键作用。SOR的特征来自于高温厌氧菌--狂热杆菌，在之前的资助期间，它是使用结构和光谱方法建立的，SOR是唯一适合催化超氧化物还原的方法。利用DNA微阵列技术对呋喃假单胞菌全基因组中的所有2065个ORF进行分析，结果表明，在没有任何氧化休克的情况下，编码SOR和相关蛋白的基因都有显著水平的表达。因此，P.Furiosus一直处于“武装”状态，随时准备应对ROS的暴露。然而，这是第一道防线，因为DNA微阵列分析表明，对氧化应激的完整反应需要诱导大量新的蛋白质(由保守/假设基因编码)，其中一些蛋白质也是在次适温度下生长诱导的。在拟议的研究中，新的应激调节蛋白以及SOR和相关的还原酶和氧化酶，将利用免疫学、生化和结构分析来表征它们的调节、多蛋白复合体的形成和催化功能。各种互补的光谱技术，包括EPR、Endor、MCD、共振拉曼、FTIR和X射线吸收，将被用来探索与应力相关的特定成员的催化功能，特别是SOR。这些结果将为厌氧菌的应激反应提供全新的见解，并为确定通常占微生物基因组一半的未表征的假想基因的功能提供策略。

DESCRIPTION (provided by applicant): By definition, aerobic organisms, both prokaryotic and eukaryotic, require molecular oxygen for energy conservation. This is a mixed blessing, however, as extremely reactive oxygen derivatives are produced during normal metabolism that can damage all cellular components. These so-called reactive oxygen species (ROS) have been implicated in a wide variety of chronic and infectious human diseases, including cancer, Alzheimer's disease, arthritis and AIDS, yet ROS are also used as a defense system against pathogens and in signal transduction pathways. Understanding the responses of microbes to oxygen has direct ramifications for the treatment of diseases caused by anaerobic pathogens. In 1999 we proposed that anaerobes have a novel response to ROS in which a non-heme iron protein termed superoxide reductase (SOR) played a key role. SOR was characterized from the hyperthermophilic anaerobe, Pyrococcus furiosus, and over the prior funding period it has been established using structural and spectroscopic approaches that SOR is uniquely suited to catalyze superoxide reduction. Using DNA microarrays to all 2065 ORFs in the complete P. furiosus genome, it was shown that the genes encoding SOR and related proteins are all expressed at significant levels in the absence of any oxidative shock. P. furiosus is therefore continuously 'armed' and ready to deal with ROS exposure. This is a first line of defense, however, as DNA microarray analyses show that the complete response to oxidative stress requires the induction of a large number of novel proteins (encoded by conserved/hypothetical genes), some of which are also induced by growth at sub-optimal temperatures. In the proposed research, the novel stress-regulated proteins, together with SOR and related reductases and oxidases, will be characterized with respect to their regulation, multiprotein complex formation, and catalytic functions using immunological, biochemical and structural analyses. A variety of complementary spectroscopic techniques, including EPR, ENDOR, MCD, resonance Raman, FTIR and X-ray absorption, will be utilized to probe the catalytic function of specific members of the stress-related pathways, with particular emphasis on SOR. The results will provide completely new insights into the stress responses of anaerobes, and provide strategies for determining the function of uncharacterized hypothetic algenes that typically account for half of a microbial genome.