Isolation,Identification And Mechanistic Study Of MMAs Demethylation By Anaerobic Bacteria From Paddy Soil

Arsenic（As）is a toxic metalloid that is widely distributed in soil,atmosphere and water.Arsenic in the environment is mainly divided into inorganic arsenic and organic arsenic.Inorganic arsenic includes arsenite[As（Ⅲ）]and arsenate[As（Ⅴ）].Organic arsenic includes monomethylarsenic[MMAs],dimethylarsenic[DMAs],trimethylarsenic[TMAs],volatile organic arsenic[MMAs（Ⅲ）H]and others.The toxicity of arsenic compounds depends on their chemical species.The availability of arsenic is increased in paddy soils under the flooded conditions.Under aerobic conditions,As（Ⅴ）is the dominant As species in paddy soils.Upon flooding,As（Ⅴ）is reduced to As（Ⅲ）and released into the solution phase via iron-reducing bacteria and arsenate-reducing bacteria.As（Ⅲ）is the major As species under the flooded conditions.As（Ⅲ）can be methylated partially into MMAs,DMAs and TMAs by As methylators.In flooded paddy soil,methylated arsenic species are produced first and then disappear through demethylation.It has been reported that DMAs is mainly demethylated into MMAs by methanogenic archaea and then MMAs is further demethylated by some other microorganisms.However,the microbes mediating MMAs demethylation under anaerobic conditions in paddy soil are still unknown.In order to resolve this scientific question,we carried out soil incubation and anaerobic enrichment of anaerobes with the ability of MMAs demethylation from paddy soil.We further isolated and identified a single bacterial strain which could demethylate MMAs under anaerobic conditions Using this strain,we investigated the mechanism of MMAs demethylation.The main results are summarized as follows:1.Exogenously addition of nitrate enhanced MMAs demethylation in CZ paddy soils under anaerobic conditions.To investigate the role of denitrifiers in MMAs demethylation,MMAs（Ⅴ）and nitrate were added exogenously in CZ paddy soils and incubated under anaerobic condition.The results show that a part of MMAs（Ⅴ）was methylated to DMAs,the others were demethylated.Addition of nitrate significantly stimulated denitrifiers and MMAs（Ⅴ）disappearance without increase of DMAs.The results indicated that addition of nitrate enhance the demethylation of MMAs（Ⅴ）.The present study revealed the dynamics of MMAs（Ⅴ）in paddy soil under anaerobic condition.by anaerobic cultivation of the tested soil from Chenzhou and the addition of external sources of MMAs（Ⅴ）and NO₃^-.The results showed that MMAs showed a trend of dual conversion between methylation and demethylation during the culture cycle.With the addition of NO₃^-,the activity of denitrifying microorganisms was promoted,and the disappearance of MMAs（Ⅴ）was also promoted.Under these conditions,the production of DMAs did not increase.This result proved that NO₃^-promoted the demethylation of MMAs（Ⅴ）in the soil.2.An enrichment culture with the MMAs demethylation ability was obtained from the test soil under the anaerobic denitrification conditions.Both exogenously added MMAs（Ⅴ）and MMAs（Ⅲ）were demethylated by the enrichment culture.MMAs（Ⅲ）was detected during MMAs（Ⅴ）demethylation,suggesting that reduction of MMAs（Ⅴ）is necessarily prior to its demethylation.A single bacterial strain with the abilities of MMAs demethylation and denitrification was isolated from the enrichment culture.After identification,the strain was named as Bacillus sp.CZDM1.This strain could demethylate 75%of MMAs（Ⅲ）to As（Ⅲ）in 36 hours.3.From the draft genome of Bacillus sp.CZDM1,a homologous gene Bcars I that could potentially mediate the demethylation of MMAs（Ⅲ）was identified.To test whether Bc Ars I could catalyze MMAs（Ⅲ）demethylation under anaerobic denitrification conditions,the gene was heterologously expressed in E.coli AW3110.The results showed that E.coli AW3110expressing Bcars I was able to catalyze demethylation of MMAs（Ⅲ）under anaerobic denitrification conditions and also showed increased resistance to MMAs（Ⅲ）.In addition,five conserved cysteine residues in the Bc Ars I protein were mutated individually by site-directed mutagenesis.All mutants lost the ability to demethylate MMAs（Ⅲ）,indicating that the five cysteine residues play an important role in demethylation.4.We investigated whether Bc Ars I-catalyzed demethylation MMAs（Ⅲ）was coupled with denitrification in Bacillus sp.CZDM1 and E.coli AW3110（p ET29a-Bcars I）under anaerobic denitrification or fermentation conditions.The results showed that the Bcars I gene was expressed under both fermentation and denitrification conditions in strain Bacillus sp.CZDM1.However,it could demethylate MMAs（Ⅲ）only under the anaerobic denitrification conditions.Similarly,bearing Bcars I gene conferred the ability of MMAs（Ⅲ）demethylation to E.coli AW3110 only under the anaerobic denitrification conditions and not under the fermentative conditions.Moreover,the demethylation ability of MMAs（Ⅲ）could be restored when the strain Bacillus sp.CZDM1 was transferred to denitrification conditions from fermentation cultures.These results further proved that the demethylation process of MMAs（Ⅲ）catalyzed by Bc Ars I was coupled with nitrate reduction under anaerobic condition.Additionally,we heterologously expressed the Bzars I gene from the non-denitrifier strain Bacillus sp.CZ-2 into E.coli AW3110 under anaerobic condition.The experiment showed that Bzars I could still confer MMAs（Ⅲ）demethylation ability to E.coli AW3110.This result indicated that as long as the strain possesses the ars I gene and the denitrification ability,it could catalyze MMAs（Ⅲ）demethylation under anaerobic condition.5.The coexistence of the ars I gene and denitrification gene was analyzed by bioinformative analysis to verify the contribution of denitrifying microorganisms in the detoxification of MMAs（Ⅲ）in the anaerobic habitats.The results showed that,among 56bacterial species containing ars I,19 species also contain the dissimilation nitrate-reducing genes including nar and nap.The results indicate that denitrifying microorganisms containing ars I genes are widely distributed in the environment.These microorganisms may play an important role in anaerobic demethylation of MMAs（Ⅲ）under anaerobic condition.In summary,this study aims to reveal the process and mechanism of demethylation of organic arsenic regulated by microorganisms under anaerobic conditions,and to verify the contribution of denitrifying microorganisms to the detoxication of MMAs（Ⅲ）.Otherwise,this study can provide a theoretical basis to geobiochemical cycle of As and N.