贪铜菌CNP-8趋化响应卤代硝基酚污染物的分子机制

卤代硝基芳烃是一类人工合成的重要环境污染物，开展微生物对此类污染物的趋化响应机制研究，有助于认知微生物在环境中如何感应污染物及其在污染环境中的进化策略。目前，微生物趋化人工合成芳烃大多是趋化现象的观察，趋化响应这类污染物的机制尚不明确。申请人前期揭示了贪铜菌CNP-8降解多种人工合成卤代硝基酚的机理，并发现该菌株对这些污染物具有趋化响应现象。基于已有基础，本项目开展以下研究：鉴定CNP-8趋化感应卤代硝基酚的甲基受体趋化蛋白；表征趋化受体蛋白配体结合域与效应物的相互作用并鉴定其在趋化感应中的生理功能；解析配体结合域结构及其结合趋化信号分子的关键位点，阐明关键位点在细菌趋化中的作用，系统揭示细菌趋化响应卤代硝基芳烃的分子机制。项目成果对认知异生物质污染环境中微生物的生理学特性及其适应性进化机制、利用合成生物学构建微生物传感器用于环境监测或智能微生物用于污染环境修复具有重要的科学意义。

Halogenated nitroaromatic compounds are an important class of synthetic environmental pollutants. It is of significance to study the chemotactic response of microorganisms to these pollutants because this knowledge will help us to understand how microorganisms sense the pollutants, as well as the microbial evolutionary strategies in the polluted environment. To date, the microbial chemotaxis to synthetic aromatic pollutants is limited in reporting the chemotactic phenomenon, and the chemotactic mechanism is unknown. Cupriavidus sp. strain CNP-8 previously identified by us with the ability to degrades many synthetic nitroaromatic compounds. Recently, we have revealed the catabolic mechanism of several halogenated nitrophenols in this strain, and found that strain CNP-8 exhibits obvious chemotaxis towards these synthetic pollutants. . The project is proposed on the basis of our previous research to conduct a comprehensive and systematic research, principally aiming to reveal the mechanism of microbial chemotaxis to synthetic halogenated nitroaromatic compounds from molecular, biochemical, and genetic levels. To identify the methyl-accepting chemotaxis protein (MCP) triggering the chemotactic response to halogenated nitrophenols, an MCP-free and non-chemotactic mutant of CNP-8 will be constructed by gene knockout of all of the 17 putative chemoreceptor genes, followed by respective gene complementary and chemotaxis analysis. The ligand-binding domain (LBD) of MCP will be purified, followed by interaction analysis between LBD and effectors by isothermal titration calorimetry, and the physiological function of LBD will be verified by gene knockout. The structure of LBD will be resolved by homology modeling or crystallization, and the key roles of the ligand binding sites will be verified by site-directed mutagenesis in vitro and in vivo. . The results of this proposed project would help us to understand the microbial physiological characteristics and adaptive evolutionary mechanism in the xenobiotics-polluted environment. The proposal also has important scientific significance with respect to exploiting microbial sensors for environmental monitoring or intelligent microorganisms for polluted-environment remediation.