砷氧化根癌农杆菌中砷氧化与砷趋化的共调控机制

Arsenite-oxidizing bacteria oxidize toxic arsenite [As(III)] to less toxic arsenate [As(V)], which can participate in the environmental As(III) detoxification. So far, the As(III) chemotaxis has been found in As(III)-oxidizing bacteria, but the relevance and the regulation mechanism between As(III) oxidation and As(III) chemotaxis are still unknown. Previously, we found As(III)-oxidizing Agrobacterium tumefaciens GW4 has positive As(III) chemotaxis, and the coding gene of methyl-accepting chemotaxis protein (mcp) is located in the arsenic-resistance gene island. The disruption of mcp interrupted both As(III) oxidation and As(III) chemotaxis in strain GW4. Moreover, the reported As(III) oxidation regulator AioR was determined to be able to bind with the operon region of mcp gene. Based on these preliminary results, we predict that the presence of co-regulation mechanism between As(III) oxidation and As(III) chemotaxis in strain GW4. To confirm this hypothesis, we will use the gene knock-out and complementation, swimming plate method to analyze the relevance between As(III) oxidation and As(III) chemotaxis. The report gene assays and qRT-PCR will be employed to study the regulation of the genes involved in As(III) oxidation and As(III) chemotaxis. Meanwhile, tryptophan fluorescence spectrometry and gel chromatography will be used to test the bound between the methyl-accepting chemotaxis protein MCP and As(III), while protein-DNA in vitro interactions, phosphorylation Western blot, Phos-tag SDS-PAGE and P32 labeling will be employed to establish the co-regulation mechanism of As(III) oxidation and As(III) chemotaxis in strain GW4. The results of this project will reveal the co-regulation mechanism between As(III) oxidation and As(III) chemotaxis and make a significant contribution for deeply understanding the bacterial response on arsenic in the environment.

砷氧化细菌能将毒性强的亚砷酸盐氧化成毒性较弱的砷酸盐，具有环境解毒意义。目前虽已发现砷氧化菌的砷趋化现象，但砷氧化与砷趋化之间的关联及调控关系还未阐明。我们前期研究发现砷氧化根癌农杆菌GW4对砷正趋化，其砷抗性基因岛中有甲基趋化基因mcp。突变该基因会影响细菌对砷正趋化和砷氧化表型。且砷氧化调控蛋白AioR能与mcp基因的启动子区域结合。以上结果预示该菌中砷氧化与砷趋化间存在共调控机制。在此基础上，我们将通过砷氧化和砷趋化相关基因的缺失、互补、游动平板法、报告基因表达、qRT-PCR等阐明砷氧化与砷趋化的关联。利用色氨酸荧光光谱法和凝胶色谱法确定甲基趋化蛋白MCP结合砷。再使用蛋白-DNA体外互作，磷酸化Western blot，Phos-tag SDS-PAGE和同位素P32标记等建立细菌砷氧化与砷趋化的共调控机制。该研究将揭示砷氧化和砷趋化的共调控机制，深入阐述细菌对环境砷的响应过程。

砷氧化细菌Agrobacterium tumefaciens GW4能够将高毒性的亚砷酸盐[As(III)]氧化为低毒性的砷酸盐[As(V)]。在该细菌的基因组中存在一套完整的砷氧化酶编码基因及相关的调控蛋白编码基因。经过申请人前期的研究积累发现，砷氧化调控蛋白AioR不仅参与细菌的砷氧化酶编码基因的调控，还能够参与细菌的全局性调控。由此，本课题主要针对砷氧化细菌A. tumefaciens GW4中砷氧化调控蛋白AioR的调控机制进行深入的研究。主要研究内容为：1）砷氧化和砷趋化的相关性及共调控机制； 2）砷氧化调控蛋白AioR对细菌GW4中其他代谢系统相关基因的调控； 3）砷氧化调控蛋白与磷酸盐调控蛋白之间的交互调控机制； 4）新型电子传递蛋白AioE的鉴定。.本课题根据细菌GW4的基因组分析，发现其与多株砷氧化菌的砷抗性基因岛中都存在甲基趋化蛋白编码基因mcp，且在该mcp基因的启动子区域发现了砷氧化调控蛋白AioR的结合位点。以此为根据，通过基因缺失突变与互补，lacZ报告基因融合及RT-PCR的方法，蛋白质表达纯化及色氨酸荧光光谱法检测等方法证实砷氧化调控蛋白AioR参与了砷趋化相关基因的表达调控。本课题利用已构建好的aioR缺失突变株进行了比较蛋白质组学研究，发现与野生株相比，共有超过200个蛋白在aioR突变株中发生不同程度的上调和下调。由此证实砷氧化调控系统参与细菌代谢全局性调控。并且砷氧化调控蛋白AioSR与磷酸盐调控蛋白PhoBR之间存在交互调控，从而证实细菌砷磷代谢共调控的分子机理。此外，本课题在研究过程中，鉴定了新型的砷氧化电子传递蛋白AioE，解释了该细菌能够利用砷氧化产生的能量进行生长的电子传递路径。