温度影响新型极端微生物Anoxybacillus flavithermus subsp. yunnanensis有机溶剂耐受性的机理研究

Solvent-tolerant microorganisms are a relatively new group of extremophiles, which are capable of thriving in the presence of extremely toxic solvents on account of their unique adaptive mechanisms. Anoxybacillus flavithermus subsp. yunnanensis is the current only new species of thermophilic bacteria able to tolerate solvents, and is a typical representative of adapting to extreme environments. Experiments have indicated that temperature plays a very important role in the solvent tolerance of the new species, but the intrinsic mechanism remains unknown. In this project, the regular variations in protein expression level in response to temperature change will be assessed by comparative proteomic analysis and statistical methods. The functions of the differentially expressed proteins will be investigated via bioinformatics analysis, targeted gene disruption and other experimental techniques. The results will gain insights into the global mechanism underlying the effect of temperature on solvent tolerance. Then, several typical proteins with different functions will be picked and a comprehensive functional analysis will be performed, which provide a thorough understanding of the biological role of the candidate proteins in the mechanism. This is the first study of a relationship among biological function of proteins, temperature and solvent tolerance. This study will not only extend our current understanding for solvent-tolerant microorganisms, but also expand our horizons in complexity and diversity of life. The important proteins derived from the study may contribute to improve the adaptability of industrial bacteria in media with toxic solvents and high temperature.

有机溶剂耐受菌能够依赖某种独特的机制在强毒性的有机溶剂环境下正常生长，是一种新型的极端微生物。Anoxybacillus flavithermus subsp. yunnanensis是目前唯一具有有机溶剂耐受性的嗜热细菌新物种，是生命对环境极限适应能力的又一典型代表。已经证明温度对耐受性具有极其重要的影响，但是内在机制不清楚。本项目利用蛋白质组学和统计学方法遴选出差异表达强度随温度变化而规律性变化的蛋白，结合生物信息学和基因敲除等实验验证它们的生理功能，初步阐述温度影响有机溶剂耐受性的物质基础。然后，对几个代表性的重要蛋白进行全面的功能分析，深入理解它们在该机制中的贡献。这是首次将蛋白的生理功能、温度和有机溶剂三者联系起来研究。预计研究结果不仅拓宽了对有机溶剂耐受菌的认识，而且丰富了对生命多样性和复杂性的理解。获得的关键蛋白也可能对高温有毒环境下的工业菌的适应性改造具有重要意义。

有机溶剂耐受菌能够依赖某种独特的机制在强毒性的有机溶剂环境下正常生长，是一种新型的极端微生物。黄热芽孢杆菌云南亚种（Anoxybacillus flavithermus subsp. yunnanensis）是目前唯一具有有机溶剂耐受性的嗜热细菌新物种。本项目利用传统的2D-PAGE蛋白质组学方法分析了在45、52和60°C时，甲苯亚致死浓度下的差异蛋白。遴选出17个差异表达强度随温度和甲苯都变化的蛋白。结合生物信息学和基因敲除等实验验证了其中的3个蛋白进行了功能研究，确认了β-葡萄糖苷酶和脂肪酸合成调控因子FabT蛋白与有机溶剂耐受性相关。以往的认知中并不知道这两个蛋白对有机溶剂耐受性有贡献。饱和直链脂肪酸可能在抵御高温有机溶剂方面具有重要作用。由于蛋白质组学获得的差异蛋白数量不多，随后采用转录组技术重新分析了黄热芽孢杆菌云南亚种在三个温度下响应甲苯的情况。每个样本都获得了600多个差异表达基因，比较全面的调查了全细胞的变化情况。本研究结果拓宽了对有机溶剂耐受菌的认识，也丰富了对生命多样性和复杂性的理解。