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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.
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