The Relatedness Between Arsenite Oxidation And Arsenite Chemotaxis And The Regulation Roles Of AioR In Agrobacterium Tumefaciens GW4

Arsenic(As)is a metalloid which widely distributed in the environment.The most common arsenic compounds are arsenite [As(III)] and arsenate [As(V)].Microbial As(III)oxidation transforms the more toxic As(III)to the less toxic As(V),and such process is catalyzed by As(III)oxidase Aio BA In most As(III)-oxidizing bacteria,the expression of aio BA is regulated by three component system Aio XSR,and AioR is the regulator.Some As(III)-oxidizing bacteria could use As(III)oxidation as an energy source and exhibit positive chemotaxis towards As(III),however,the related As(III)chemoreceptor and the regulatory mechanism remain unknown.Besides,the related metabolic pathways mediated by As(III)oxidation and whether AioR could regulate other cellular responses to As(III)are also unclear.In this study,we analyzed an As(III)-oxidizing bacterium Agrobacterium tumefaciens GW4 isolated from As-enriched groundwater sediments.Strain GW4 showed high As(III)resistance and As(III)oxidation could enhance its growth.Strain GW4 also displayed positive chemotaxis towards As(III).A total of twelve genes named as putative chemoreceptor-encoding gene were found in GW4 genome,and one of them was located in the arsenic gene island.The expression of mcp and other chemotaxis related genes(che A,che Y2 and fli G)was inducible by As(III),and a predicted promoter binding site for AioR was found in the mcp regulatory region by MEME analysis.Capillary assays showed that the wild type GW4 could display positive chemotaxis towards As(III)under the concentration from 0.5 mmol/L to 2 mmol/L.However,aio R and mcp mutants lost the ability of chemotaxis and the complemented strains recovered chemotaxis towards As(III).These results indicated that mcp and aio R genes were necessary to bacterial As(III)chemotaxis.Intrinsic tryptophan fluorescence spectra analysis showed that Mcp could specifically bind As(III)but not As(V).In combination with these two results,we concluded that Mcp is the As(III)chemoreceptor.Reporter gene assays showed that mcp gene could not be induced by As(III)in aio R mutant.A bacterial one-hybrid system technique,EMSA and q RT-PCR showed that AioR could interact with the mcp regulatory region to activate its expression,and the precise AioR binding site was confirmed by DNase I footprinting assay.Additionally,the disrupting of mcp gene affected bacterial As(III)oxidation rate,growth and As(III)resistance.Taken together,these results indicated that mcp is essential for As(III)oxidation,and AioR could regulate As(III)oxidase Aio BA and As(III)chemoreceptor Mcp.There is some sort of mutually reinforcing connection between As(III)oxidation and As(III)chemotaxis(Fig.A).Since we had proved that AioR could regulate bacterial As(III)oxidation and As(III)chemotaxis,we speculated that AioR may regulate other functions.In order to determine whether AioR could regulate other cellular responses to As(III),isobaric tags for relative and absolute quantitation(i TRAQ)was performed in four treatments: GW4(+As III)/GW4(-As III),GW4-?aio R(+As III)/GW4-?aio R(-As III),GW4-?aio R(-As III)/GW4(-As III)and GW4-?aio R(+As III)/GW4(+As III).A total of 41,71,82 and 168 differentially expressed proteins were identified,respectively.EMSA assays showed that 12 genes/operons could be interacted with AioR.In combination with the comparative proteomics results and q RT-PCR results,we concluded that:(A)AioR is the main regulator for bacterial As(III)oxidation,but it could also affect several cellular processes related to As(III)oxidation.(B)In order to adapt the limitation of phosphate,AioR could regulate phosphate metabolism(Pst/Pho)to economically transport As(V)and Pi.(C)AioR could regulate the expression of As(III)resistance genes(ars C and acr3),peptidoglycan recycling gene anm K,proline synthesis gene pro C and repair protein gene PIMT to enhance bacterial As(III)resistance.(D)AioR could also regulate TCA cycle and strain GW4 may use As(III)oxidation as the main energy resource(Fig.B).Taken together,this study successfully identified the first As(III)chemoreceptor Mcp,and proved that AioR could regulate bacterial As(III)oxidation and As(III)chemotaxis.This study also revealed that there is some sort of mutually reinforcing connection between As(III)oxidation and As(III)chemotaxis.For the first time,we illuminated the metabolic pathways in a strain representing heterotrophic As(III)-oxidizing bacteria,which could use As(III)oxidation to generate energy.Besides,we also proved that AioR is the main regulator for As(III)oxidation but it could also regulate the expression of some other metabolic pathways related to As(III).This study enhances our understanding of bacterial As(III)oxidation mechanism and has an important scientific value.