【作者】 赵凯；

【导师】 王革娇；

【作者基本信息】 华中农业大学 ， 微生物学， 2010， 硕士

【摘要】 微生物在砷循环过程中起着重要的作用,三价砷氧化菌作为其中的一类微生物,通过将三价砷氧化为五价砷降低了环境中砷的毒性,并且在微生物环境治理方面也具有潜力,因此得到了人们的关注。目前已知aoxAB基因编码三价砷氧化过程中的关键酶,然而对于该过程中的调控机制研究的还不够深入。食酸菌属Acidovorax作为一类重要的三价砷氧化菌,对该属的三价砷氧化机制报道却很少。本文以该属的两株三价砷氧化菌为研究对象,对三价砷氧化菌的三价砷氧化酶及其调控方面进行了研究。本研究的两株菌Acidovorax sp. GW2和Acidovorax sp. NO1是分别分离于山西省山阴县古城村砷地下水污染区土样和湖北省黄石市大冶金矿土壤的三价砷氧化细菌。通过构建GW2的fosmid文库,分离得到了GW2菌的As(Ⅲ)氧化酶基因簇和砷抗性基因,包括aoxRSXABCD和arsBCR共10个基因,分别预测编码双组分信号传导系统转录调控子AoxR,周质感应组氨酸激酶AoxS,周质结合蛋白AoxX,三价砷氧化酶AoxAB,硝基还原酶AoxC,细胞色素c AoxD,三价砷转运蛋白ArsB,五价砷还原酶ArsC和砷抗性转录调控子ArsR。通过反向PCR和Tail-PCR分离得到了NO1菌的As(Ⅲ)氧化酶基因簇,该基因簇基因种类和GW2菌基本相同。和GW2菌不同的是NO1菌的aoxD和aoxC基因的排列顺序正好与GW2菌相反。GW2菌反转录PCR结果显示,构成双组分系统的aoxRS基因共转录,而与之转录方向相反的结构基因aoxABCD处于同一个操纵子中,aoxX基因和aoxRS基因不在同一操纵子之中。通过二亲本杂交的方法,分别对GW2菌的aoxS, aoxX, aoxD基因和NO1菌的aoxC基因进行敲除。GW2菌的aoxS和aoxX基因的敲除使其三价砷氧化性丧失。对于两个突变株分别进行aoxS和aoxX基因的互补实验,表型得到了回复,证实了并非发生极性效应使得其它基因表达受到影响而产生三价砷氧化表型丧失的现象。aoxS和aoxX基因为GW2菌三价砷氧化的必须基因。GW2菌的aoxD基因的功能丧失减慢了三价砷氧化速率,但不是影响三价砷氧化的关键基因。NO1菌的aoxC基因行敲除后仍然具有三价砷的氧化能力,并不是其三价砷氧化所必须的基因。本研究对三价砷的氧化调控机理进行了初步的研究,并且首次证明了aoxX基因的的作用。预示细菌的砷感应调控除了受到感应蛋白和调控蛋白双组分系统作用之外还可能有一个砷结合蛋白的参与。更多还原

【Abstract】 Microorganisms play an important role in the process of arsenic cycle. Arsenite-oxidizing bacteria as one kind of microorganisms in this cycle have already been given more and more attention since transformation of arsenite to arsenate reduces the toxicity of arsenic in environments which showed the protential in environmental control. AoxAB has been known as the key enzyme in arsenite oxidition, however the mechanism in that process was not well identified. As one significant genus of arsenite-oxidizing bacteria, the mechanism of arsenite-oxidation of Acidovorax is rarely reported. This study focused on two Acidovorax strains to study the arsenite oxidase and its regulation.Strain Acidovorax sp. GW2 and Acidovorax sp. NO1 are the arsenite-oxidizing bacteria isolated from arsenic-contaminated sediment near groundwater in Gucheng, Shanyin, Shanxi province and gold mine soil in Daye, Huangshi, Hubei province, respectively. The arsenite oxidase gene cluster and arsenic resistant genes of strain GW2 were isolated by constructing a fosmid library. There are ten genes including aoxRSXABCD and arsBCR, and predictively encoding the transcriptional regulator AoxR of a two-component signal transduction system, a periplasmic sensor histidine kinase AoxS, a periplasmic binding protein AoxX, arsenite oxidase AoxAB, nitroreductase AoxC, cytochrome c AoxD, arsenite transporter ArsB, arsenate reductase ArsC and arsenic resistance regulatory protein ArsR, respectively.Using Reverse-PCR and Tail-PCR the arsenite oxidase gene cluster was isolated from strain NO1, which is similar to that of strain GW2. However, the order of aoxD and aoxC in strain NO1’s gene cluster opposite to strain GW2.According to the reverse transcriptase PCR experiments of strain GW2, aoxR and aoxS encoding for a two-component system are co-transcribed and opposite to structural genes aoxABCD. aoxX and aoxRS are not in the same operon. By the method of biparental mating, we knocked out the aoxS, aoxX, aoxD of strain GW2 and aoxC of strain NO1, respectively. Deletions of aoxS and aoxX in strain GW2 caused the losses of arsenite oxidation ability. Both of the mutants’phenotypes were recovered through the complementary assaies of aoxS gene and aoxX gene respectively, which indicated that there is no polar effect to affect downstream genes generating the phenomenon of arsenite oxidation ability loss. aoxS and aoxX are the essential genes in arsenite oxidation of strain GW2. The loss of aoxD in strain GW2 did not show significant effects on arsenite oxidation and just slowed down the velocity of arsenite oxidation. By knocking out aoxC in strain NO1, the ability of arsenite oxidation still remained, which demonstrates that this gene is not essential for arsenite oxidizion of strain NO 1.In this research, the mechanism of arsenite oxidizing regulation was preliminarily studied, and for the first time, the function of aoxX gene was found to be related to bacterial arsenite oxidation. It indicated that there may be another arsenite-binding protein involved in bacterial sensing and regulating besides the two-component signaling system containing sensor proteins and regulated protein.更多还原