Study On The Arsenic Methylation Mechanism By Noviherbaspirillum Denitrificans

Arsenic(As)is a toxic element,which is ubiquitous in the environment.In recent years,As contamination has attracted widespread attention in the world,because As pollution frequently occurs in soil and water environment,causing a series of problems,including human health,and is considered as one of the environmental pollutants that need to be controlled urgently.There are many As species in the environment,microorganisms have evolved many different As transformation mechanisms,including arsenic oxidation,reduction,methylation and demethylation.The arsenic methylation process of microorganisms is an important part of the arsenic biogeochemical cycle,which transform inorganic As(Ⅲ)into mono-(MAs(Ⅲ)),di-(DMAs(Ⅲ))and tri-(TMAs(Ⅲ))methylarsenicals,can influence the species of As and toxicity.In general,the trend in toxicity to organisms(most to least)is MAs(Ⅲ)> DMAs(Ⅲ)>As(Ⅲ)>As(V)>DMAs(V)、MAs(V)>TMAs(V)O.It plays an important role in As detoxification.In this study,here we describe arsenic methylation in the siol microbe Noviherbaspirillum denitrificans HC18.The aim of this study is to provide insights into the roles of soil microorganism in As biogeochemistry and basis for the in-depth study of the mechanism of As methylation in soil microbes,with the main results are as following:The concentrations of As(Ⅲ)and MAs(Ⅲ)that caused 50% growth inhibition(EC50)were 337.0 μM and 2.2 μM,respectively.Strain HC18 was not able to methylate As(Ⅲ)under either oxic or anoxic conditions,catalyze MAs(Ⅲ)methylation under oxic or anoxic conditions.Strain HC18 was able to methylate MAs(Ⅲ)rapidly to dimethylarsenate(DMAs(V))and small amounts of trimethylarsenic oxide TMAs(V)O.Through the analysis of the draft genome of strain HC18,an As(Ⅲ)adenosylmethionine methyltransferase(Ars M)gene was identified and functionally characterized,To date,every characterized Ars M has four conserved cysteine residues.All four cysteines are required for methylation of As(Ⅲ)to MAs(Ⅲ).Quantitative transcription profiling of the ars M gene was done to determine if expression of ars M gene was arsenic dependent.The expression of ars M was low to As(Ⅲ).Expression with MAs(Ⅲ)was nearly 70-fold higher than in cells grown in the absence of arsenic.Heterologous expression of Ndars M in an As-sensitive strain of E.coli AW3110 could not confer As(Ⅲ)resistance and As(Ⅲ)methylation ability.But expression of Ndars M in AW3110 conferred MAs(Ⅲ)resistance and MAs(Ⅲ)methylation ability.In vitro enzyme activity studies showed that Nd Ars M methylates MAs(Ⅲ)but not As(Ⅲ),Purified Nd Ars M protein methylated MAs(Ⅲ)to dimethylarsenate as the main product in the medium and also produced dimethylarsine and trimethylarsine gases.Through multiple sequence alignment,we found that Nd Ars M contains four conserved Cystein(Cys32,Cys67,Cys155 and Cys205),corresponding to Cys44,Cys72,Cys172 and Cys224 in Cr Ars M.Althouge there are four conserved cysteine residues in Nd Ars M,Nd Ars M has the ability to methylate MAs(Ⅲ)but not As(Ⅲ).By the site-directed mutagenesis studies,only C155 S and C205 S lost their methylation ability,indicating that Nd Ars M requires two conserved Cys to catalyze MAs(Ⅲ).