根癌农杆菌GW4中磷酸盐转运调控因子PhoB介导的砷氧化调控机制

Some microorganisms could oxidize the more toxic As(III) to the less toxic As(V) by arsenite oxidase AioAB to relieve toxic effect. Since many of the similar physic-chemical properties between arsenic and phosphorus, there may be close relationship between metabolism of phosphorus and arsenic, and the co-regulation of arsenite oxidation and phosphorus metabolism is complex. Our previous studies found that Agrobacterium tumefaciens GW4 could obtain energy by oxidizing As(III), and the oxidation product As(V) was incorporated into cells by replacing partial phosphorus. In this study, we focused on the Agrobacterium tumefaciens GW4 which could oxidize arsenite to arsenate. By gene in frame deletion and Atomic fluorescence analysis form, it is found that the missing of phosphate transport system regulator phoB accelerated the arsenite oxidation, and resulted in improvement of arsenite resistance and significantly reduction of exopolysaccharides. The bioinformatic analysis suggested there were some potential PhoB-binding domain in the promoter of arsenic oxidation gene operon. That means phoB may be involved in the regulation of arsenite oxidation. The reduction of exopolysaccharides may allow much more arsenite to enter bacterial cells, and arsenite resistance was enhanced because of the more effective arsenic oxidation. To confirm our hypothesis, we will study the arsenite oxidation regulation mechanism of phoB by introducing comprehensive techniques, which including gene complementation, report gene fusion, qRT-PCR, in vivo/vitro interactions between proteins and DNA, DNase I footprinting, in vivo protein phosphorylation and its detection. The aim of this research is to clarify the function of phoB in arsenic resistance, arsenic oxidase expression regulation and biosynthesis of exopolysaccharides, establish the regulation model of arsenite oxidation mediated by phoB and the influences of exopolysaccharides to arsenite oxidation. The results of this project will give us a significant perspective to better understanding the regulation of arsenite oxidation and the co-regulation of arsenic-phosphorus metabolism, to provide new insights into the biological significances of bacterial exopolysaccharides, and to provide theoretical basis for microbial remediation of arsenic-pollution.

砷氧化细菌能将高毒性的As(III)氧化成低毒的(AsV)。砷磷具有很多相似的性质，细菌砷磷代谢及其调控之间可能关系密切。前期研究发现，根癌农杆菌GW4的砷氧化产能，氧化产物As(V)进入细胞并取代部分磷。我们近期发现磷酸盐转运调控基因phoB的缺失使砷氧化速率加快，砷抗性增强，且胞外多聚物减少。生物信息学分析表明砷氧化基因启动子区存在PhoB结合域，预示phoB可能参与了砷氧化的调控，胞外多聚物的屏障作用减弱可能加快砷氧化进而增强对砷的抗性。本研究在此基础上，将通过报告基因融合、qRT-PCR、蛋白质与DNA互作、DNase I足迹法、蛋白质磷酸化等技术研究phoB在砷氧化酶的表达调控及胞外多聚物合成中的作用，建立phoB砷氧化调控及胞外多聚物影响砷氧化的模型。该研究对于认识砷氧化调控及砷-磷共代谢具有重要作用，为理解胞外多糖的生物学意义提供新见解，为砷污染环境的微生物修复提供理论依据。

以GW4为研究对象，采用基因敲除与互补、荧光定量PCR、lacZ报告基因融合、iTRAQ比较蛋白质组学、蛋白质与DNA相互作用等方法深入研究了细菌砷代谢-磷酸盐转运系统之间的关联和交互调控机制。该菌两个磷酸盐转运系统Pst1和Pst2的蛋白均显著受到低磷的诱导，且Pst2表达量高于Pst1；Pst1受到As(III)的诱导，而Pst2受As(III)的抑制。对两个Pst系统中的调控基因phoB1、phoB2分别进行单敲除及双敲除，ΔphoB1生长未受到影响，且胞内APase活性没有变化；而ΔphoB2生长显著减弱，胞内APase活性升高；双敲除突变株ΔphoB1ΔphoB2生长最弱，APase活性降低。通过qRT-PCR及iTRAQ定量蛋白质组学方法检测基因/蛋白表达，结果表明，phoB1缺失突变对phoB2及pstS2没有显著影响，砷氧化酶基因aioB表达略有降低；而phoB2缺失突变导致phoB1和pstS1均显著，并且在加砷条件下促进了aioB的表达，导致砷氧化加快。表明phoB1、phoB2以直接或间接的方式参与了砷氧化酶的表达调控。通过生物信息学分析，在砷氧化酶AioBA操纵子的启动子区找到PhoB的潜在结合位点。经体内细菌单杂交及体外凝胶阻滞试验（EMSA）证实，PhoB1、PhoB2确实能与AioBA启动子结合。但是由于phoB2的表达在加砷条件下是被抑制的，所以只有phoB1真正参与了砷氧化酶的表达调控，这也进一步证实磷酸盐转运调控因子PhoB1对砷代谢有调控作用，Pst1与砷代谢更密切。砷氧化酶调控蛋白AioR能结合在Pst2操纵子的phoU2启动子区。当aioR突变以后，phoB2及pstS2的表达不再受到As(III)的抑制，表明了砷代谢对磷酸盐转运系统的调控作用。根据以上结果，提出根癌农杆菌GW4中砷-磷代谢的严谨型调控模型：在低磷环境中，菌株依靠Pst2磷酸盐转运系统吸收磷维持生长，Pst1以较低能耗工作；当有As(III)存在时，As(III)诱导砷氧化酶系统并促进phoB1 的表达及磷酸盐的吸收，同时由于phoB1的调控作用进一步促进砷氧化酶的表达（磷酸盐系统对砷代谢的调控），负责高效率的磷吸收及As(III)的氧化解毒。同时，被诱导的AioR抑制Pst2的表达（砷对磷酸盐转运的调控），减少能量消耗，实现砷代谢-磷转运的严谨节约型调控。