Arsenic Biotransformation By Spirulina Platensis FACHB-439 And Characterization Of The Arsenite Methyltransferase(ArsM) From S. Platensis FACHB-439

Arsenic is an important environment pollutant,which toxicity to organism is depend on its concentration and species.Inorganic As is the main form in aquatic environments,and it can be transformed into organic forms by several microorganisms.On the other hand,organic As could be degraded into inorganic As under the action of other microbes.Thus,microorganisms,such as plankton and bacterium,have a crucial influence on As species and distribution in aquatic environments,and play an important role in the biogeochemical cycling of As.In this study we investigated the effect of Spirulina platensis on As transformation.Based on the tolerance of S.platensis to As(?)and its ability of As biotransformation.The associated gene of As was identified,and further cloned,expressed,and purified.In addition,the function of arsM gene was investigated in vivo and in vitro.This study present detailed information about the molecular mechanism of As biotransformation by S.platensis,and provided a theoretical basis for using S.platensis to As contaminated environment remediation.The main results were described below:1.The main As species in medicinal Spirulina supplements are arsenite[As(?)]and arsenate[As(?)].In addition,a small amount of unknown As species were found in two brands of Spirulina(Yunnan Baiyao and Xiuzheng Pharmaceutical Group).The tolerance experiment of S.platensis to As(?)was conducted in laboratory conditions,the results showed that the lag phase of S.platensis was approximately 8 d,and then turned into a logarithmic growth.Growth of the cyanobacterium was not inhibited by As(?)concentrations of 10 and 100 ?M,and it could tolerate As(?)concentration of up to 500 ?M.After exposed to different concentrations of As(?)and As(?)for 15 d,S.platensis showed a difference As accumulation.The total As in cellular treated with As(?)or As(?)was 4.14 mg kg-1 and 4.31 mg kg-1.The main As species in cells was As(?).Additionally,0.62-38.65%of dimethylarsenic[DMA(?)]and 0.32-7.74%of monomethylarsine[MMA(?)]were detected in cells.These results indicated that S.platensis has the ability of As methylation.When exposed to series concentration(0.1 to 100 pM)As(?)or As(?),S.platensis can methylate inorganic As.The percentage of methylated As was high in low As concentration treatments.As methylation experiment by S.platensis showed that the percentage of methylated As in cellular was rise in the initial stage and then decreased,and the percentage of methylated As in the treatment of As(?)was higher than As(?).2.SparsMin S.platensis was cloned and expressed in coli SpArsM showed a low identity compared with previous reported ArsM enzymes,and the highest identity was 39%.The E.coli AW3110 bearing SparsM gene resulted in the resistance to As(?)and the ability of As methylation.As(?)was transformed into DMA(?)by E.coli AW3110 with SparsM gene.In vitro assay showed that purified SpArsM could methylate As(?)at pH between 2.5 and 9.5,with highest methylation activity at pH 7.5.DMA(?)concentration was increased with the decrease of As(?).DMA(?)and a small amount of MMA(?)were detected in the reaction system within 0.5 h.The C-terminal truncated SpArsM34 possessed the same function with the full-length SpArsM.In addition,the site-directed matagenesis results showed that any one conserved cysteine residues(Cys59,Cys186,Cys238)mutation will significantly affect the ability of As methylation.These results indicated that these three conserved cysteine residues were necessary for As(?)methylation.As methylation is an important As detoxification pathway in S.platensis.This study broadens the knowledge of As biotransformation by microorganisms in the aquatic environment,and provides theoretical basis for biological As remediation.