Analysis of Oxygen Adaptation in Methanogenic Archaea

描述(申请人提供)：拟议研究的长期目标是确定和表征厌氧产甲烷古细菌中与氧气适应相关的新蛋白质，以便为氧气解毒酶的功能和进化提供基础。这项研究之所以重要，是因为氧化物种的解毒是所有活细胞中的一种普遍防御机制，而且在病原体对宿主防御细胞产生的氧化猝发的反应中也是如此。此外，甲烷的产生与全球变暖问题有关，关于古生代的生理、生化和遗传学仍有许多需要了解的地方。这项研究建议的具体目的是：a)利用乙酰链球菌全基因组微阵列来鉴定在细胞暴露于氧气、超氧化物和过氧化氢时上调的基因，b)对先前发现的编码可能的氧化应激蛋白的基因簇进行转录分析和表达谱，包括超氧化物还原酶和红曲霉毒素：氧氧化还原酶(Roo)，c)从乙酰链霉菌中高效表达和纯化可能的Roo，以验证其在该生物体的生理学背景下的催化特性。

DESCRIPTION (provided by applicant): The long term objectives of the proposed research are to identify and characterize novel proteins associated with oxygen adaptation in anaerobic methane-producing Archaea in order to provide a foundation of the function and evolution of oxygen detoxification enzymes. This investigation is important due to the relevance of detoxification of oxidative species as a general defense mechanism in all living cells, but also in the response of pathogens to oxidative burst produced by host defense cells. In addition, methane production is relevant to the problems of global warming and there is still much to learn about the physiology, biochemistry, and genetics of Archaea. The specific aims of this research proposal are: a) utilize a Methanosarcina acetivorans complete genome microarray to identify genes up-regulated during exposure of cells to oxygen, superoxide, and hydrogen peroxide, b) conduct transcriptional analysis and expression profiling of a previously identified gene cluster encoding probable oxidative stress proteins, including a superoxide reductase and rubredoxin: oxygen oxidoreductase (ROO), c) overexpress and purify the putative ROO from M. acetivorans to verify its catalytic properties in the context of the physiology of this organism.