The Effect Of Arsenic Transporter Gene Expression On Arsenic Methylation Of Microorganisms

Arsenic?As?contamination has become a global problem;many new innovative methods to lower As exposure are currently explored.As methylation can convert highly toxic arsenite[As?III?]to low-toxic volatile trimethylarsine[TMAs?III?],which is regarded as an effective strategy for As removal.However,the majority of microorganisms exhibited a relatively limited ability to volatilize As,making it difficult to achieve the application.The reason may be that As transporters in microorganisms reduce intracellular As content and competitively limit the As volatilization efficiency of microorganisms.However,little information is available about the As transporter that limits microbial As methylation efficiency.Rhodopseudomonas palustris CGA009 has been well characterized and it has been regarded a good model organism for studying As detoxification;however,it showed a very low ability to methylate and volatilize As,which may be caused by As efflux transporters pump As out of cells,lowering intracellular As concentration,which may limit As methylation and subsequent volatilization.To test this hypothesis,this study established a biosensor responding to intracellular As concentration to investigate the rate of As efflux by As efflux transporters and determine the effect of As efflux on microbial As methylation;the effect of As efflux transporters derived from CGA009strain on As methylation was further investigated by gene knockout.The main results obtained are as follows:?1?Establishment of plug-in biosensor and comparison of As efflux transporters activity.Based on the ars operon?Pars,arsR,arsB and arsC?of Escherichia coli BL21?DE3?,green fluorescent protein gene?gfp?was used as the reporter gene,and the As?III?-hypersensitive E.coli AW3110?DE3?as the host to construct a plug-in As biosensor system?referred to as B system?in response to intracellular As concentration.The activity of As efflux transporters(ArsB_RP/Acr3_RP)derived from CGA009 was compared to the ArsB_ECC from E.coli BL21?DE3?.The results showed that the fluorescence intensity was in a linear correlation in the range of 0 to 2.0?mol/L As?III?.The higher the activity of the As efflux transporter,the lower the As?III?concentration in the cell,the lower the fluorescence intensity;and the As efflux capacity of As efflux transporters displayed the order:ArsB_RP>ArsB_EC>ACR3_RP,which were consistent with the results of the As resistance assays.This study provided a method to compare the activity of As efflux transporters,which has better linear relationship,higher sensitivity,less As dosage and safer operation than the method of classical resistance determination.?2?Arsenic efflux transporters limit microbial As methylation.The As?III?S-adenosylmethionine methyltransferase gene?arsM?from CGA009 strain was co-expressed with As efflux gene in B system to construct an As biosensor?BM system?.The effect of different As efflux transporters on the microbial As methylation was investigated by As?III?resistance assay,fluorescence detection,As speciation analysis by HPLC-ICP-MS and total As content determination.The results showed that the higher the activity of As efflux transporter,the higher the microbial resistance to As,the lower the fluorescence intensity of GFP,the lower the microbial As methylation and As volatilization efficiency.When incubated at 100.0?mol/L As?III?for 12 h,the As volatilization rate of the recombinant strains coexpressing arsB_RP,arsB_ECC and acr3_RPP with arsM was 3.99%,5.65%and 8.20%,respectively,compared with that of the recombinant strain without As efflux gene?10.01%?.Therefore,the As efflux gene and arsM are co-expressed in microorganisms,the As efflux transporter excretes intracellular arsenic,which limits microbial As methylation.?3?Effects of As efflux genes knockout on microbial As methylation.Two mutants of?arsB_RPP and?acr3_RPP were obtained by knocking out arsB_RPP and acr3_RPP in CGA009 strain,respectively.The order of resistance to As?III?and As?V?was wild type>?arsB_RP>?acr3_RP.After incubation for 12 h at 100?mol/L As?III?,the order of As volatilization rate was wild type?0.49%?<?arsB_RP?3.61%?<?acr3_RP?5.13%?,while that of wild type?0.59%?<?arsB_RP?4.25%?<?acr3_RP?6.08%?after 12 h of incubation at25?mol/L As?V?.When exposed to 25.0?mol/L As?III?for 24 h,both wild type and mutants detected low doses of MAs?V?and DMAs?V?,and residual As?III?in the medium was wild type>?arsB_RP>?acr3_RP.Thus,As efflux transporter limits the As methylation of microorganisms,and knocking out the As efflux gene can improve the efficiency of As methylation and volatilization in microorganisms,and a mutant with high As volatilization rate(?acr3_RP)was obtained.The activity of As efflux transporters in CGA009 was Acr3_RP>ArsB_RP,which was inconsistent with the order of activity studied by As biosensors?B system?,which may be caused by the difference in the expression of arsB_RPP and acr3_RPP locating in different ars operons in CGA009 strain.Collectively,this study developed a plug-in biosensor for responding to changes in intracellular As concentration,and As efflux activities of different As efflux transporters and the effect of As efflux transporters on microbial As methylation were investigated for the first time.As efflux transporters were shown to be a remarkable intrinsic factor limiting As methylation and volatilization efficiency,and As volatilization rate could be significantly improved by deleting genes encoding microbial As efflux transporters.This study provided an explanation for the low rate of microbial As methylation and an effective strategy for screening microorganisms with high As volatilization.