丛毛单胞菌等对芳环类污染物的趋化、降解及其在生物修复中的作用

Aromatic pollutants can be natural or xenobiotic compounds. Microbial degradation of aromatic pollutants are the key process for bioremediation processes.Both the metabolic diverisity and chemotaxis of microbes are important for the peformance of microbial bioremediation. Chmeostaxis is defined as the responses of microbial cells towards or away from chemicals. The chemicals that attract microbial cells are called attractants. Microbial chemotaxis to aromatic compounds is important for microbes in natural and polluted enviroments, as this chemotaxis may determine the availability of aromatic compounds to microbial cells. This is particular true when the aromatic compounds are hydrophobic such as polyaromatic hydrocarbons. This proposal aims to disclose the molecular pathway of microbial chemotaxis to aromatic compounds..In our previous studies, a metabolically diverse Comamonas sp. strain CNB-1 was isolated and characterized. This strain CNB-1 is able to degrade chloronitrobenzene and many other aromatic compounds. The genome of CNB-1 has been sequenced and 29 putative methyl-accepting proteins (MCPs) as well as many putative signal transducing proteins were annotated. Recently, we observed strain CNB-1 exhibited chemotaxis towards phenol, aminophenol, benzoate, hydroxylated benzoates, gentisate, protocatechuate etc. This proposal will focus on identification of the genes and proteins involving in biodegradation and chemotaxis to aromatic compounds by strain CNB-1. . Nitrazine-pollutants (such as herbicides) are serious pollutants in soil from chemical plants and agriculture.This proposal will also work on cultivation of microbes that degrade and behave chemotaxis to these chemicals. Tailor-made chips with microfluidics will be designed and applied for enrichment of such microbial cells, and those cells possess degrading ability and chemotaxis will be cultivated. The biodegradation and chemotaxis of the newly cultivated bacterial strains to nitrazines will be evaluated. Application of newly isolated bacterial strains in lab-scale bioremediation of nitrazine-polluted soil will be conducted.

芳环类化合物是环境中重要的一类污染物，开展微生物对芳环类化合物的降解和趋化性研究，对认知污染环境中微生物的行为、发挥其降解功能、提高生物修复效率，具有重要意义。本项目包括两部分内容：第一部分以丛毛单胞菌CNB-1为研究对象，研究丛毛单胞菌对氨基酚、苯甲酸、苯酚、龙胆酸、原儿茶酸等趋化和降解的过程和机理，重点鉴定趋化过程中甲基化受体蛋白，表征甲基化受体蛋白与效应物的相互作用，揭示丛毛单胞菌对芳环类化合物降解和趋化的分子途径；第二部分针对三嗪类污染物，收集具有降解作用和趋化性微生物资源。首先是基于微生物趋化运动、设计和加工微流控富集芯片，在芯片富集的基础上优化分离培养方案，获得兼有趋化性和降解功能的微生物菌种；然后，选择具有特色的菌种开展对三嗪类化合物污染物降解途径、趋化机理、及其在生物修复中作用的研究。

芳环类化合物是环境中重要的一类污染物，开展微生物对芳环类化合物的降解和趋化性研究，对认知污染环境中微生物的行为、发挥其降解功能、提高生物修复效率，具有重要意义。本项目首先以睾丸酮丛毛单胞菌CNB-1 菌株为对象，对苯甲酸的降解过程进行了研究。1)通过对CNB-1 菌株基因组数据的挖掘，发现CNB-1 菌株中苯甲酸降解基因簇（box 基因簇）,证明在CNB-1 菌株中，苯甲酸是经由依赖于辅酶A 的环氧化物途径进行好氧降解的。2)研究表明CNB-1中龙胆酸降解途径是通过龙胆酸1,2-双加氧酶、马来酰丙酮酸异构酶和富马酰丙酮酸水解酶依次作用，最终代谢为反式丁烯二酸（富马酸或延胡索酸）和丙酮酸进入三羧酸循环；MarR型的转录调控蛋白GenR在龙胆酸代谢基因簇的转录中发挥负调控作用，芳香族辅酶A硫酯物是一类新的MarR型调控蛋白的效应物分子。3)分析发现CNB-1 菌株降解香草酸、3-羟基苯甲酸及4－羟基苯甲酸都通过原儿茶酸4，5-开环途径，烯醇式OMA是 PmdE的唯一底物。4)确定了参与对芳香族化合物趋化的甲基趋化受体蛋白MCP-2201的功能；同时，发现了一类通过感应TCA循环中间化合物实现对芳香族化合物响应的完全不依赖于电子传递链的全新通用感应机制；发现了MCP2983可以识别乌头酸，是一种新的TCA循环中间产物识别机制；发现MCP2901能够介导菌株对苯甲酸、4-羟基苯甲酸、原儿茶酸和龙胆酸等多种芳香族化合物的趋化，该蛋白是报道的能够直接结合芳香化合物的第一个甲基受体趋化蛋白。.研究进一步1）设计和开发了基于层流效应物扩散的Y型通道微生物趋化分离系统, 实现了趋化微生物单细胞微流控液滴包裹及趋化菌株的高效分离和培养；2）开发了一种基于浓度梯度扩散的趋化性微生物分离分析滑动芯片（SlipChip）, 实现了对三个微坑中趋化细菌选择性地收集，而无需染色及标记。3）利用微液滴芯片从施用除草剂的土壤和多环芳烃污染的土壤中分离获得了三嗪类污染物除草剂及多环芳烃趋化降解菌，并解析了其降解途径。