Functional Characterization Of S-Adenosylmethionine Arsenite Methyltransferase Of Pseudomonas Alcaligenes And The Construction Of Genetic Engineered Rhizobia For Arsenic Bioremediation

Arsenic(As)is highly toxic and widespread in the environment.As can be transformed into mono/di/trimethylarsenic derivatives by microorganisms.This process can directly influence the species,toxicity and mobility of As during the As biogeochemical cycle.Symbiosis rhizobia and leguminous plants may decrease the toxicity of As to plants through absorbing or transforming the species of arsenic.They can also promote the accumulation and recycling of nitrogen in soils,improve soil fertility and reduce fertilizer use.The potential of legume-rhizobium symbiosis in the remediation of As contaminated soils remains to be explored.The aims of this study were to characterize the function of PaarsM in Pseudomonas alcaligenes NBRC14159 and to genetically engineered Mesorhizobium sp.ACCC18001.A range of experiments were performed,including two parents conjuction,laboratory pot experiment test,plant axenic cultivation.Arsenic speciation was quantified using HPLC-ICP-MS(High performance liquid chromatography-Inductively coupled plasma mass spectrometry).In this study,arsenic biotransformation by Pseudomonas alcaligenes NBRC14159 was investigated.P.alcaligenes was demonstrated to have the ability of methylating arsenite to dimethylarsenate(DMAs(Ⅴ))and small amounts of trimethylarsenic oxide(TMAs(Ⅴ)O).An arsenite S-adenosylmethionine methyltransferase(arsM)gene,PaarsM was identified and functionally characterized.PaArsM shares low similarities with other reported ArsM enzymes(less than 55%).When PaarsM was disrupted,the mutant lost As methylation ability and became more sensitive to arsenite.PaarsM was expressed in the absence of As(Ⅲ)and the expression was further enhanced by arsenite exposure.Heterologous expression of PaarsM in an As-hypersensitive strain of Escherichia coli conferred arsenite resistance.Purified PaArsM protein methylated arsenite to dimethylarsenate as the main product in the medium and also produced dimethylarsine and trimethylarsine gases.The Chlamydomonas reinhardtii arsM gene(CrarsM)behind the kanamycin promoter was integrated into the chromosome of Mesorhizobium sp.ACCC18001,a microsymbiont of red clover which does not have an arsM gene and does not methylate arsenic.CrArsM was expressed in strain ACCC18001,was methylated by cells that expressed the CrarsM gene,whereas no arsenic methylation products were found in the control cells.The genetically engineered Mesorhizobium was able to form nodules in red clover roots and fix nitrogen in both axenic agar culture and vermiculite pot experiment.Furthermore,DMAs(V)was detected in the nodules and the shoots of red clover.In contrast,clover plants inoculated with control Mesorhizobium contained no methylated As species.The study demonstrates that rhizobium can be genetically engineered to transform toxic inorganic As species into less toxic organic As species.Further experiments are needed to test the bioremediation potential of the genetically engineered rhizobium-legume symbiosis system in arsenic contaminated soils.