The Microbiological Process And Molecular Mechanism Of Dimethylarsenate Methylation And Reductive Volatilization

Dimethylarsenic(DMAs(V))is a common representative of methyl arsenic compounds and is widely distributed in natural environments.The methylation process of microbiological process is the main source of DMAs(V)in the environment,and at the same time as the main product of microbial arsenic-methylation in the soil,DMAs(V)pollution can induce rice straight-head disease,resulting in a serious reduction in rice production and destroying the soil ecological environment.The use of microbial DMAs(V)volatilization to reduce the DMAs(V)residues in the rice soil environment has the potential to prevent rice straight-head disease.It is of great significance to explore the microbiological process and molecular mechanism of dimethylarsenate methylation and reductive volatilization,which lays the foundation for the construction of engineering bacteria to reduce soil environment DMAs(V)pollution in the future.In this study,the conserved cysteine residues of arsenic methyltransferase Ar Ars M were subjected to site-directed mutations to determine the function of each conserved cysteine site in the microbial dimethylarsenic methylation pathway.We found that the key point of C155 S Ar Ars M unmethylated As(III)forms DMAs(V)but can methylate DMAs(V)to produce volatile gaseous arsine TMA(III).At the same time,the strain Alcaligenes sp.SQHF-1 with strong DMAs(V)reductive volatilization ability was screened in the soil with the incidence of rice straight-head disease in Suqian,Jiangsu.This strain can reductive volatilize DMAs(V)into gaseous arsine DMA(III)H.This provides usable strains and genetic material for exploring the molecular mechanism of microbial reductive volatilization of DMAs(V).The main results of this research are as follows:One of the pathways of DMAs(V)volatilization is DMAs methylation forming TMA(III).Through studying Ar Ars M from the As(III)methylation strain Arsenicibacter rosenii SM-1,we found that : A recombinant large intestine expressing the Arars M gene heterologously E.coil AW3110(p ET29a-Arars M)and BL21(p ET29a-Arars M)can be endowed with the ability to transform DMAs(V);strain BL21(p ET29a-Arars M)produces TMAs(III)15-50 times that of AW3110(p ET29a-Arars M);therefore,E.coli strain BL21(DE3)is more suitable as a host for the Arsenicibacter rosenii SM-1 arsenic methyltransferase gene Arars M.Ar Ars M protein consists of 307 amino acid residues and a total of four conserved cysteine(Cys)residues were found in its sequence,located at positions 30,67,155,and 205 in the sequence.Based on this,we constructed their single mutants(C30S,C67 S,C155S and C205S);recombinant strains expressing wild-type protein can further convert As(III)and MAs(III)into the final product pentavalent trimethylarsine oxide TMAs(V)O and DMAs(V);recombinant strains expressing mutations of protein C155 S and C205 S have completely lost the activity of methylating As(III);compared with the recombinant strain expressing wild-type protein,the recombinant strain expressing mutant protein C205 S produced less volatile gaseous arsine,but the expression of the other three mutant proteins basically does not affect the DMAs(V)transformation ability of the recombinant strain.We explored an enzymatic material C155 S,and the strain expressing the protein mutant lost the activity of methylated As(III),but DMAs(V)can still be further transformed.The discovery of this material can provide a reference for the subsequent development of genetically engineered bacteria to reduce DMAs(V)pollution in the environment.Another way of DMAs(V)volatilization is the reductive volatilization of DMAs(V).We found that the formation of DMA(III)H was detected in 6 soils with strong arsenic methylation ability,and DMAs(V)reduction and volatilization ability of the soil culture solutions of Sihong,Siyang,Xuyi and Suqian was significantly stronger than that of the soil culture solutions of Long’an and Tancheng.A bacteria with strong ability of DMAs(V)reductive volatilization was selected from the soil culture solution of Suqian.The amount of gaseous arsenic produced is about 4 times that of the reported Bacillus sp.CZ-2.The 16 S r RNA gene sequence analysis identified this strain as an Alcaligenes sp.and we named it Alcaligenes sp.SQHF-1;the strain SQHF-1 is a yellow,transparent,gram-negative bacterium with neat edges.The cells are spherical with many flagella all over the body.The optimum growth temperature is 37 ℃,and the optimum environment p H value is 9.0.Strain SQHF-1 does not have the ability to transform As(III)and MAs(V),but can reduce a small amount of As(V)to As(III),but cannot use As(V)as a substrate to produce volatilization.The reductive volatilization ability of strain SQHF-1 increases significantly with the increase of the concentration of substrate DMAs(V).The ratio of the amount of volatile gaseous arsine produced in the medium containing 1 m M DMAs(V)were 498 and 34 times higher than the yields of the 10 μM and 100 μM treatment groups,respectively;the addition of molybdate can significantly increase the DMAs(V)reductive volatilization ability of the strain SQHF-1.40 m M and 100 m M molybdic acid were added to the system can increase the reductive volatilization ability of strain SQHF-1 by 1.4 times and 2.6 times,respectively;through the genome analysis of strain SQHF-1,a total of four groups of gene clusters related to molybdenum pterin synthesis and transport have been identified,namely molybdenum butterfly the protein-encoding genes moa A-mob A-moe A-moa BEDC and mog A as well as the transport channel protein encoding genes mod ABD and mod ABE of molybdoxin synthesis related protein;strain SQHF-1 can tolerate ampicillin(Amp),bleomycin(Zeo)and Gentamicin(Gm)stressness,but it is sensitive to kanamycin sulfate(Km)and chloramphenicol(Cm).At the same time,competent cells have the highest electroporation transformation efficiency at 2.3-2.4 KV,which is the construction gene knockout Provide a theoretical basis for the addition of mutants.