根癌农杆菌GW4在有氧和无氧条件下砷氧化与铬还原相关性的分子机制

Arsenite [As(III)]-oxidizing bacteria oxidize toxic trivalent arsenic to less toxic pentavalent arsenic [As(V)] and chromate [Cr(VI)]-reducing bacteria reduce toxic hexavalent chromium to less toxic trivalent chromium [Cr(III)], which can participate in autologous or environmental detoxification of As(III) and Cr(VI). Arsenical and chromium compounds often coexist in the environment, but whether there is a correlation between bacterial As(III) oxidation and Cr(VI) reduction is still unknown. Previously, we found that Agrobacterium tumefaciens GW4 was able to oxidize As(III) and reduce Cr(VI). Under aerobic conditions, the presence of As(III) could promote bacterial Cr(VI) reduction. However, under anaerobic conditions, strain GW4 showed the mutual promotion between As(III) oxidation and Cr(VI) reduction, revealing that electron may be transferred between these two processes. Besides, we found that arsenic resistant protein ArsH and ArsK, which were both induced by As(III), were participated in bacterial Cr(VI) reduction and resistance, revealing that they may have the function of Cr(VI) reductase or Cr(VI) efflux transporter, respectively. We will use gene knock-out and complementation, in vitro enzyme activity assays, report gene assays and electrophoretic mobility shift assays to identify the functions of ArsH and ArsK to bacterial Cr(VI) reduction and resistance. These results will reveal the molecular mechanism by which As(III) promotes the reduction of Cr(VI) underlying aerobic conditions. The differential proteomics and molecular genetic manipulation will be employed to identify the anaerobic Cr(VI) reductase and electron transport chain, and it will reveal the coupled mechanism between As(III) oxidation and Cr(VI) reduction under anaerobic conditions. The result has significant theoretical value and will provide theoretical foundation for the remediation of arsenic and chromium in the environment.

砷氧化细菌能将高毒性的三价砷氧化成低毒性的五价砷，铬还原细菌能够将高毒性的六价铬还原成低毒性的三价铬，具有自身和环境解毒意义。环境中砷与铬化合物常共存，但细菌砷氧化与铬还原是否存在相关性还未有报导。前期发现根癌农杆菌GW4具有砷氧化和铬还原能力。有氧条件下，三价砷能够促进细菌铬的还原；无氧条件下，细菌砷氧化与铬还原相互促进，预示着电子传递的偶联。此外，我们发现受三价砷诱导的砷抗性蛋白ArsH及ArsK参与细菌的铬还原及抗性，预示ArsH及ArsK可能具有铬还原及外排的功能。我们将通过基因敲除与互补、体外酶活、报告基因表达和凝胶迁移率实验等鉴定ArsH及ArsK对铬还原及抗性的作用，阐明有氧条件下三价砷促进铬还原与抗性的机制。通过蛋白质组和分子遗传操作等鉴定厌氧铬还原酶及电子传递链，阐明无氧条件下砷氧化与铬还原偶联的机制。其结果具有开创性理论价值并为环境砷铬污染的修复提供依据。

重金属铬(Cr)和砷(As)污染物威胁着全球居民的健康，环境中铬和砷的毒性与它们的氧化价态具有相关性，而将六价铬[Cr(VI)]还原和三价砷[As(III)]氧化是去除铬和砷重要的步骤。本研究中，我们分离得到一株肠杆菌Z1，该菌具有同时催化Cr(VI)还原和As(III)氧化的能力，此氧化与还原过程可在较高范围的温度和pH条件中进行。因此，我们以肠杆菌Z1为研究对象，揭示了其Cr(VI)还原和As(III)氧化的偶联机制，具体结果如下：(1)以细菌Z1制备的固定化小球处理污染废水，表现出稳定地Cr(VI)还原和As(III)氧化能力。微生物联合Ca(OH)2和FeCl3处理后的废水，溶液中铬和砷的含量符合国家污水排放标准。(2)蛋白质组学表明半胱氨酸代谢、硫和甲硫氨酸代谢、砷抗性和氧化还原酶受到Cr(VI)和As(III)]的诱导，额外添加半胱氨酸能显著提高细菌对培养基中Cr(VI)的还原。(3)蛋白质组学和荧光定量PCR分析表明nemA基因的表达受到Cr(VI)和有机三价砷的诱导，将nemA基因异源表达于大肠杆菌中，能提高大肠杆菌对Cr(VI)和有机三价砷的抗性。(4)将NemA蛋白纯化后，该蛋白能同时催化Cr(VI)还原和有机三价砷氧化，且Cr(VI)的添加能提高NemA氧化有机三价砷的能力。(5)通过凝胶迁移实验和荧光实验证明了调控蛋白NemR能结合有机三价砷，并确认了Cys21、Cys106和Cys116为保守功能位点。本项目的研究成果为修复环境中的铬和砷污染提供了理论依据和新的方法。