Identification And Function Study On Arsenic And Antimony Resistant Genes In Pseudaminobacter Sp. HC19

Arsenic（As）is a highly toxic metalloid and is widely distributed in the environment.The toxicity of arsenite[As（Ⅲ）]and methylated arsenite[MAs（Ⅲ）]are significantly stronger than its oxidation products.Antimony（Sb）is a metallic element with similar chemical properties to As.The biological toxicity of trivalent antimonite[Sb（Ⅲ）]is also stronger than that of antimonate[Sb（V）].Therefore,the oxidation of As（Ⅲ）and Sb（Ⅲ）is an important biological detoxification mechanism.Microorganisms play a key role in the biogeochemical cycle and biological detoxification of arsenic and antimony.In this study,a facultative anaerobic arsenic-resistant strain Pseudaminobacter sp.HC19 was ioslated from the arsenic-contaminated paddy soils in He Chi city,Guangxi province.Through physiological experiments,gene cluster analysis,gene heterologous expression,and enzyme activity analysis,arsenic and antimony resistance and oxidation characteristics by strain HC19 as well as the functional study of the MAs（Ⅲ）oxidase gene arsV in strain HC19.The main research results used are as follows:1.Pseudaminobacter sp.HC19 was resistant to As（Ⅲ）,MAs（Ⅲ）and Sb（Ⅲ）,and the EC₅₀ of these substrates are 627μM As（Ⅲ）,3.4μM MAs（Ⅲ）and 146μM Sb（Ⅲ）respectively.This strain was able to oxidize As（Ⅲ）to As（V）,MAs（Ⅲ）to MAs（V）,and Sb（Ⅲ）to Sb（V）.2.Genome analysis of strain HC19 showed that there are two arsenic resistance gene clusters in Pseudaminobacter sp.HC19,including arsR,aio A,and the new MAs（Ⅲ）oxidase gene arsV.The complete sequence of the HC19 includes two aio gene clusters and three arsoperons based on genome-mining analysis.The arsoperon（arsR-arsV-MFS）contains an arsR gene,an a gene encoding an methylarsenite（MAs（Ⅲ））oxidase（arsV）,and an unknown major facilitator superfamily（MFS）transporter gene.Transcription of three arsgenes（arsR,arsV,and MFS）in HC19responds to addition of As（Ⅲ）,MAs（Ⅲ）or Sb（Ⅲ）revealed that the three genes in the arsR-arsV-MFS gene cluster are induced by As（Ⅲ）,MAs（Ⅲ）and Sb（Ⅲ）,and the three genes are cotranscripted.3.It was found that the arsV can confer resistance to As（Ⅲ）,MAs（Ⅲ）and Sb（Ⅲ）in E.coli AW3110,and the simultaneous expression of arsV and MFS can confer even more resistance to MAs（Ⅲ）.While the expression of MFS gene alone cannot confer resistance to any of As（Ⅲ）,MAs（Ⅲ）and Sb（Ⅲ）in E.coli AW3110.In addition,the recombinant strain was able to oxidize MAs（Ⅲ）to MAs（V）and Sb（Ⅲ）to Sb（V）but not As（Ⅲ）.Subsequently,the purified Ars V protein has the ability to catalyze the oxidation of MAs（Ⅲ）and Sb（Ⅲ）.In summary,MAs（Ⅲ）oxidation possibly constitute the key mechanism of MAs（Ⅲ）detoxification in Pseudaminobacter sp.HC19.When MAs（Ⅲ）enters the cell,it will be oxidized to MAs（V）by Ars V and then be excreted by MFS.At the same time,when Sb（Ⅲ）is present in the cell,Ars V oxidizes it to Sb（V）which would be excreted through transporters.The results of this dissertation have made great contributions to the exploitation of arsenic-resistant microorganisms,the bacterial arsenic and antimony oxidation,and the molecular mechanisms of bacterial heavy metal transform and resistance.