The Effect Of Glycerol Facilitator Protein GlpF On Arsenic Methylation Of Microorganisms By Dual Fluorescence Analysis

The health risks caused by arsenic pollution have become a global problem.Microbial arsenic adsorption and arsenic methylation methods are considered to be one of the potential effective ways to repair environmental arsenic pollution.However,due to the influence of genetic factors and environmental factors,the arsenic adsorption and volatilization capacity of most microorganisms is low.Bacteria glycerol facilitator protein GlpF is the channel protein of As(?)entering the cell.Studies have shown that the expression of glpF can improve the arsenic adsorption capacity of the strain.No report.Therefore,exploring the As(?)absorption activity of GlpF and its effect on arsenic volatilization efficiency and its mechanism of action are of great significance for improving the arsenic adsorption and volatilization capacity of the strain.Based on this,this study established a dual fluorescent biosensor analysis method to monitor the expression of functional genes and respond to intracellular arsenic concentration.The glpF gene from different strains was genetically engineered to construct a dual fluorescent arsenic biosensor system,combining arsenic resistance,Intracellular arsenic content and arsenic volatility and other indicators were measured,and the effects of different Glp F on As(?)adsorption and arsenic volatility were compared.The main findings are as follows:1.Research on the construction and characteristics of dual fluorescent reporter plasmids.Using green(egfp)and red(mrfp1)fluorescent protein genes as reporter genes for inducible expression elements and constitutive expression elements,respectively,a dual fluorescent recombinant plasmid(pUC19-ars-egfp-arsR-T7-mrpf1)was constructed with arsenic sensitive strain E.coli AW3110(DE3)was used as the host to construct an arsenic biosensor system to monitor functional gene expression and respond to intracellular arsenic concentration,and explore the response relationship between the system and As(?)concentration.The results showed that the excitation wavelengths were set to 479 nm and 564 nm respectively,and the fluorescence signals of EGFP and mRFP1 protein could be detected at 511 nm and 607 nm,respectively,indicating that the recombinant plasmid can be correctly expressed;the results of arsenic resistance showed that In the 0.1~5.0 mg/L As(?)concentration range,its growth is negatively correlated with the arsenic concentration;the response ratio of the fluorescence intensity of EGFP to mRFP1 is linear in the concentration range of 0.00~60.00 ?g/L As(?).2.Analyze and compare the arsenic absorption characteristics of four kinds of GlpF by dual fluorescence method.Connect the glpF gene from four bacteria to the downstream of the mrpf1 gene to construct four recombinant strains of E.coli AW3110(glpFn,n=1,2,3,4),and take the recombinant bacteria without glpF gene as a reference control,Comparison of As(?)absorption activity of four different GlpF.The higher the GlpF activity and the higher the arsenic content in the cell,the higher the fluorescence intensity response ratio.The results showed that the capacity of the four recombinant bacteria to absorb arsenic was E.coli AW3110(glpF1),E.coli AW3110(glpF4),E.coli AW3110(glpF0),E.coli AW3110(glpF3)and E.coli AW3110(glpF2),which is consistent with the experimental results of arsenic resistance and intracellular arsenic content.E.coli AW3110(glpF3)and E.coli AW3110(glpF2)have lower arsenic absorption capacity than the control strain.It is preliminarily speculated that GlpF2 and GlpF3 may have the function of efflux As(?).3.The effect of water-glycerol channel protein gene expression on microbial volatilization.The arsM from Rhodopseudanonas palustris CGA009 strain was connected downstream of the dual fluorescent recombinant plasmid glpF to construct four glpF-arsM co-expressing recombinant bacteria E.coli AW3110(glpFn-M),and the recombinant bacteria The half-growth inhibition rate(IC50),fluorescence response ratio,intracellular arsenic content and arsenic volatilization,the effect of different GlpF on the microbial arsenic volatilization ability was studied.The results showed that the coexpressed recombinant E.coli AW3110(glpFn-M)had a higher IC50 value,a lower fluorescence response ratio,and a lower intracellular arsenic accumulation.Compared with the arsenic volatility(29.58%)without glpF gene,the arsenic volatility of the four co-expressing recombinant bacteria were 36.70%,18.37%,20.96% and 35.90%,respectively.It can be seen that when glpF coexists with arsM,when GlpF absorbs extracellular As(?),it can enhance the microbial arsenic volatilization ability.Conversely,when GlpF drains As(?)out,it will also restrict the microbial arsenic volatilization ability.In summary,this study established a dual-fluorescence analysis method for monitoring functional gene expression and responding to intracellular As(?)arsenic biosensors,which was applied for the first time to absorb As(?)activity of microbial glycerol facilitator protein GlpF Compared with the effect of microbial arsenic volatilization,compared with the traditional arsenic resistance and arsenic content determination method,the dual fluorescence analysis method is simple to operate,the amount of arsenic is less,and the operation is safer.In addition,the higher the GlpF activity,the higher the arsenic volatilization efficiency of the recombinant bacteria,which provides a reference for further construction of strains with high activity arsenic volatilization capacity.